# PHYSIOLOGICAL ADAPTATIONS OF INSECTS EXPOSED TO DIFFERENT STRESS CONDITIONS

EDITED BY : Bin Tang, Su Wang, Nicolas Desneux and Antonio Biondi PUBLISHED IN : Frontiers in Physiology

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ISSN 1664-8714 ISBN 978-2-88966-224-1 DOI 10.3389/978-2-88966-224-1

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# PHYSIOLOGICAL ADAPTATIONS OF INSECTS EXPOSED TO DIFFERENT STRESS CONDITIONS

Topic Editors:

Bin Tang, Hangzhou Normal University, China Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China Nicolas Desneux, Institut National de la Recherche Agronomique (INRA), France Antonio Biondi, University of Catania, Italy

Citation: Tang, B., Wang, S., Desneux, N., Biondi, A., eds. (2020). Physiological Adaptations of Insects Exposed to Different Stress Conditions. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-224-1

# Table of Contents


Yiou Pan, Fayi Tian, Xiang Wei, Yongqiang Wu, Xiwu Gao, Jinghui Xi and Qingli Shang


Balachandar Balakrishnan, Sha Su, Kang Wang, Ruizheng Tian and Maohua Chen

*49 Heritability and Evolutionary Potential Drive Cold Hardiness in the Overwintering* Ophraella communa *Beetles*

Chenchen Zhao, Fangzhou Ma, Hongsong Chen, Fanghao Wan, Jianying Guo and Zhongshi Zhou

*58 Specific Binding Protein ABCC1 is Associated With Cry2Ab Toxicity in*  Helicoverpa armigera

Lin Chen, Jizhen Wei, Chen Liu, Wanna Zhang, Bingjie Wang, LinLin Niu and Gemei Liang


Qiong Yao, Shu Xu, Yizhi Dong, Yinli Que, Linfa Quan and Bingxu Chen


Shoulin Jiang, Yang Dai, Yongqing Lu, Shuqin Fan, Yanmin Liu, Muhammad Adnan Bodlah, Megha N. Parajulee and Fajun Chen


Weixing Zhang, Wenfeng Chen, Zhenfang Li, Lanting Ma, Jing Yu, Hongfang Wang, Zhenguo Liu and Baohua Xu

*232 FoxO Transcription Factor Regulate Hormone Mediated Signaling on Nymphal Diapause*

Zhen-Juan Yin, Xiao-Lin Dong, Kui Kang, Hao Chen, Xiao-Yan Dai, Guang-An Wu, Li Zheng, Yi Yu and Yi-Fan Zhai

*242 Transcriptomic Responses to Different Cry1Ac Selection Stresses in*  Helicoverpa armigera

Jizhen Wei, Shuo Yang, Lin Chen, Xiaoguang Liu, Mengfang Du, Shiheng An and Gemei Liang

*259 Adipokinetic Hormone Receptor Mediates Lipid Mobilization to Regulate Starvation Resistance in the Brown Planthopper,* Nilaparvata lugens Kai Lu, Xinyu Zhang, Xia Chen, Yue Li, Wenru Li, Yibei Cheng, Jinming Zhou, Keke You and Qiang Zhou

	- *354 RNA-Seq Analyses of Midgut and Fat Body Tissues Reveal the Molecular Mechanism Underlying* Spodoptera litura *Resistance to Tomatine* Qilin Li, Zhongxiang Sun, Qi Shi, Rumeng Wang, Cuicui Xu, Huanhuan Wang, Yuanyuan Song and Rensen Zeng
	- *366 Effects of Fungicide Propiconazole on the Yeast-Like Symbiotes in Brown Planthopper (BPH,* Nilaparvata lugens *Stål) and Its Role in Controlling BPH Infestation*

Xuping Shentu, Xiaolong Wang, Yin Xiao and Xiaoping Yu

*376 Insect Behavior and Physiological Adaptation Mechanisms Under Starvation Stress*

Dao-Wei Zhang, Zhong-Jiu Xiao, Bo-Ping Zeng, Kun Li and Yan-Long Tang

*384 Starvation Stress Causes Body Color Change and Pigment Degradation in*  Acyrthosiphon pisum

Xing-Xing Wang, Zhan-Sheng Chen, Zhu-Jun Feng, Jing-Yun Zhu, Yi Zhang and Tong-Xian Liu

*397 Role of Modified Atmosphere in Pest Control and Mechanism of Its Effect on Insects*

Yu Cao, Kangkang Xu, Xiaoye Zhu, Yu Bai, Wenjia Yang and Can Li

*405 Plant Defense Responses Induced by Two Herbivores and Consequences for Whitefly* Bemisia tabaci

Dan Lin, Yonghua Xu, Huiming Wu, Xunyue Liu, Li Zhang, Jirui Wang and Qiong Rao

*414 Microorganism-Based Larval Diets Affect Mosquito Development, Size and Nutritional Reserves in the Yellow Fever Mosquito* Aedes aegypti *(Diptera: Culicidae)*

Raquel Santos Souza, Flavia Virginio, Thaís Irene Souza Riback, Lincoln Suesdek, José Bonomi Barufi and Fernando Ariel Genta

*438 Dietary Stress From Plant Secondary Metabolites Contributes to Grasshopper (*Oedaleus asiaticus*) Migration or Plague by Regulating Insect Insulin-Like Signaling Pathway*

Shuang Li, Xunbing Huang, Mark Richard McNeill, Wen Liu, Xiongbing Tu, Jingchuan Ma, Shenjin Lv and Zehua Zhang

# Behavioral, Morphological, and Gene Expression Changes Induced by <sup>60</sup>Co-γ Ray Irradiation in *Bactrocera tau* (Walker)

Jun Cai 1,2†, Hongxia Yang3†, Song Shi 1,2, Guohua Zhong1,2 \* and Xin Yi 1,2 \*

<sup>1</sup> Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture, South China Agricultural University, Guangzhou, China, <sup>2</sup> Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, China, <sup>3</sup> Guangzhou Entry-Exit Inspection and Quarantine Bureau, Guangzhou, China

#### *Edited by:*

Bin Tang, Hangzhou Normal University, China

#### *Reviewed by:*

Muhammad Rizwan Ul Haq, King Faisal University, Saudi Arabia Xiaoling Tan, Chinese Academy of Agricultural Sciences, China Genevieve Lanouette, Université de Montréal, Canada

*\*Correspondence:*

Xin Yi yixin423@126.com Guohua Zhong guohuazhong@scau.edu.cn

† These authors have contributed equally to this work.

#### *Specialty section:*

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

*Received:* 07 November 2017 *Accepted:* 05 February 2018 *Published:* 20 February 2018

#### *Citation:*

Cai J, Yang H, Shi S, Zhong G and Yi X (2018) Behavioral, Morphological, and Gene Expression Changes Induced by <sup>60</sup>Co-γ Ray Irradiation in Bactrocera tau (Walker). Front. Physiol. 9:118. doi: 10.3389/fphys.2018.00118 The sterile insect technique (SIT) may reduce pest populations by allowing sufficient amount of irradiation-induced sterile males to mate with wild females whilst maintaining mating ability comparable to wild males. Although the SIT methods are well understood, the optimal sterilizing dose and processing development stage for application vary among species. To ensure effective pest control programs, effects of irradiation on physiology, behavior, and gene function in the target species should be defined, however, little is known about irradiation effects in Bactrocera tau. Here, the effects of irradiation on rates of fecundity, egg hatch, eclosion, mating competitiveness, flight capability, morphology of reproductive organs, and yolk protein (YP) gene expression were studied. The results showed that rates of female fecundity and egg hatch decreased significantly (51 ± 19 to 0.06 ± 0.06 and 98.90 ± 1.01 to 0, respectively) when pupae were treated with >150 Gy irradiation. Flight capability and mating competitiveness were not significantly influenced at doses < 250 Gy. Ovaries and fallopian tubes became smaller after irradiation, but there was no change in testes size. Finally, we found that expression of the YP gene was up-regulated by irradiation at 30 and 45 days post-emergence, but the mechanisms were unclear. Our study provides information on the determination of the optimal irradiation sterilizing dose in B. tau, and the effects of irradiation on physiology, morphology and gene expression that will facilitate an understanding of sub-lethal impacts of the SIT and expand its use to the control of other species.

Keywords: *Bactrocera tau*, sterile insect technique, optimal dose, yolk polypeptides, morphological changes

# INTRODUCTION

The Bactrocera (Diptera: tephritidae) comprises one of the major pests of tropical fruits (Jamnongluk et al., 2003), and as a result of its euryphagy on many agricultural crops and rapid range expansion, the pumpkin fruit fly [Bactrocera tau (Walker)] has become a focus of global quarantine and control programs (Yan et al., 2015). B. tau occurs over a wide geographical range and causes significant damage to crop plants that results in reduced quality and yield of vegetable and melon crops (Li et al., 2017). Reductions in fruit quality and yield are caused by larvae, which emerge from eggs laid on the fruit surface and feed inside the fruit (Li et al., 2017). As a technique with proven high specificity and persistence in controlling other species of Diptera pests, the sterile insect technique (SIT) may be a leading method to reduce populations of B. tau.

**7**

The SIT is species specific, has no negative non-target effects, and successfully eradicated tephritid fruit flies (Pereira et al., 2013). SITs could reduce pest populations by decreasing the hatch rate of eggs through releasing large amounts of irradiationinduced sterile males, which have a comparable competitive mating ability to wild males, to mate with wild females. Insect irradiation is an ideal "genetic" treatment, because the efficacy doses against most insects and mites do not affect the quality of the protected commodities (Follett, 2004). The International Consultative Group was the first group to formally recommend genetic treatment of insect pests, and, based on irradiation data for many tephritid fruit fly species and a limited number of other insect pests, the group proposed a dose of gamma irradiation of 150 Gy for fruit flies and 300 Gy for other insects (Follett, 2004). However, subsequent work has shown that effective doses of irradiation vary among species. For example, 232 Gy is recommended for oriental fruit moth (Grapholita molesta) (Hallman, 2004), whereas, a dose of 100 Gy has been shown to provide a high level of quarantine security against Ceratitis capitata, by preventing the emergence of adult (Torres-Rivera and Hallman, 2007). A minimum dose of 85 Gy has been suggested for the treatment of B. tau in fruit and vegetables (Zhan et al., 2015), where adult eclosion is prevented. An effective sterilizing dose for B. tau has not yet been defined. It is important that the mating rates of wild female B. tau with irradiated and control males are not skewed, because reductions in the mating fitness of irradiated males compared with wild males would require larger numbers of insects to be released to avoid failure of a control program (Kean et al., 2011).

Irradiation may induce sub-lethal molecular or biochemical impacts that cause a cascade of physiological changes. Variations in gene and protein levels in response to radiation have been found to contribute to radioresistance or radiosensitivity (Suman et al., 2015), for example, heat shock protein is a potential molecular marker due to its high levels of response to irradiation (Shim et al., 2009). It has been suggested that irradiation on pupae at a sterilizing dose could influence expression levels of the responsive genes in the subsequent adult age (Chang et al., 2015). Following X-ray irradiation on B. dorsalis pupae, some of the alter proteins act in central energy-generating and in pheromone-signal processing pathways that may contribute to an overall reduction in survival and mating ability (Chang et al., 2015). During the period of vitellogenesis, insect oocytes are packed with yolk proteins that are vital for the development of embryo through the provision of nutrients (Hansen et al., 2014). It is likely that variation in expression of yolk protein following irradiation was closely related to the reproductive ability in insects. Therefore, it is important to understand these, currently unknown, effects of irradiation on B. tau behavior, physiology and morphology prior to undertaking a SIT program.

Our study showed that the dose of 150 Gy (or higher) irradiation on the final stage of female pupae could lead to significant decrease in fecundity and hatch rate. When such treatment was applied to male pupae, the hatch rate also decreased after they had mated with untreated females, achieving sterilization. As long as the applied dose was under 250 Gy, there were no effects on the ability of flight capability and mating competitiveness. When irradiation was conducted on pupae, the reproductive organs of female adult flies appeared to be shrink, compared with control, while the morphology of male flies did not show any obvious change. This study could provide information for the validation of sterilizing doses and processing stages in B. tau using irradiation. Potential sublethal impacts following irradiation treatment were also recorded that could provide a feasible technology package for control of B. tau.

#### MATERIALS AND METHODS

#### Insect Rearing

Pumpkin fruit flies were provided by the Department of Entomology at South China Agricultural University and kept in insect cages (30 × 30 × 35 cm) at 25◦C and 75% relative humidity, with a 12 h: 12 h (light:dark) photoperiod. An artificial diet of sugar:yeast powder (1:1) and water was provided, and the diet was renewed every 3 days. When the flies reached sexual maturity at 25–30 days post-emergence, they were fed pumpkin and the adult rearing process followed that described (Shen et al., 2014).

# <sup>60</sup>Coγ Ray Irradiation Treatments

The irradiation treatments were conducted at Guangzhou Furui High-Energy Technology Co., Ltd. When the irradiation was applied to adult flies, the newly emerged flies (<24 h after emergence) were examined, and divided into groups comprising 200 flies that were irradiated with <sup>60</sup>Coγ at 400 Gy. Three independent groups of flies were irradiated. The female and male flies were irradiated separately. When the treatment was conducted on pupae, three replicates of three groups of 200 pupae were irradiated with 150, 200, or 250 Gy at 2 days before emergence, respectively. Following irradiation, the flies continued to be reared under normal conditions until they reached sexual maturity, when three replicate group of 50 irradiated flies of both sexes from each irradiated group were chosen at random to mate with 50 untreated flies (50 flies from every independent irradiated group were selected for one testing group). Untreated adults (50 males + 50 female flies) were the control. For recording the amount of laid eggs, an artificial instrument was used to collect eggs was placed from 9 a.m. to 6 p.m. As the peak period for copulating could last for 15 days (25–40 days post-emergence), the egg collecting process was repeated every 3 days until the copulating peak ends, to improve accuracy. Fecundity rate and hatch rates were calculated as described previously (Aye et al., 2008).

We assessed flying capability and eclosion rate of 100 irradiated flies as measures of physiological damage. Following irradiation on pupae at different doses, pupae were placed in plastic cylinders (15 cm in height, 6 cm with an upper diameter of 8.5 cm) that were coated with talcum powder on the inside. After the flies were emerged to adult, the rates of eclosion and flight from the cylinder (flight capability) were calculated for females and males separately. Each group was replicated three times to improve accuracy and the untreated flies were set as control group.

We used a high dose (200 Gy) of irradiation to investigate its effects on mating competitiveness and reproductive organs, which represented the highest irradiation level that induced a reduction in fertility and hatch rate, but maintained similar flying capability as wild flies (see section Physiological Changes after Irradiation). For assessing mating competitiveness, after irradiation on pupae, we divided flies into three groups of 75 flies (25 untreated male flies + 25 untreated female flies + 25 irradiated male flies or 25 untreated female flies + 25 untreated male flies + 25 irradiated female flies). For better observation, the treated flies were marked with solution of 0.25% basic fuchsin and 70% ethanol solution prior mating. Mating activity was recorded between 9 p.m. and 11 p.m., and mated pairs were immediately removed. This experiment was performed three times.

#### Morphological Observation of Reproductive Organs

For assessing effects of irradiation on reproductive organs, we irradiated 50 pupae with 200 Gy and maintained them until they reached sexually maturity. Once mature, at least 5 female and male flies were selected for dissection to observe the reproductive organs using microscopy (OPTPro2008 Digital microscope imaging system) following methods described by Yi et al. (2014a). Flies were prepared for assessment using SEM by fixing in 2.5% glutaraldehyde mixed with phosphate buffer solution (pH 7.4) followed by incubation at 4◦C for 24 h. Flies were then placed in 1% osmium tetroxide for 2 h, washed three times in double-distilled water and then dehydrated in a critical point dryer for 15 min at each graded concentration of alcohol (30, 50, 70, 80, and 90%). The flies were coated with gold prior to assessment by adhering to carbon double sided tape. Reproductive organs were assessed using an FEI-XL30 SEM operated at 20 kV.

#### Cloning of *B. tauYP*

The full sequence of the yolk protein (YP) gene was cloned using the total RNA extracting from B. tau, as described by Yi et al. (2014b). The first strand cDNA was transcribed using the isolated RNA, following the manufacturer's instructions (TaKaRa, China), and the final product was stored at −20◦C prior to analysis. The full sequence of the YP gene was derived using homologous cloning, where the partial sequence was amplified using the degenerate primers yp-F1, yp-F2, and yp-R1, ypR2 (**Table 1**). Rapid-amplification of cDNA ends (RACE) was used to obtain the full length of the YP gene by following the instructions of GeneRacer Kit (Clontech, US), using the primers listed in **Table 1**. The amplification process was carried out according to the manufacturer's protocol and sequence verification was carried out by cloning the sequence into a pMD20-T vector (TaKaRa, China) and sequenced completely in both directions.

#### Quantitative Real-Time (qRT)PCR

The gene expression levels of B. tau YP at different developmental stages were investigated following the irradiation treatment on pupae. The samples of RNA were isolated from flies at different developmental stages, including pupae and adults at 7, 15, 30, and 45 days post-emergence. Each treatment had three biological replicates and was transcribed to firststrand cDNA. SYBR green dye (TaRaKa, China) and the primers listed below were used in the qRT-PCR to examine the levels of gene expression. To improve the accuracy, for each biological sample, we performed three times of amplifications to create three technical replicates. For analysis, the method described as Livak was used (Livak and Schmittgen, 2001).

#### Data Analysis

We tested for differences using one-way and two-way analysis of variance (ANOVA) and t-tests. All statistical analyses were computed in SPSS 17.0 software (IBM Corporation, Somers, NY). Data are presented as means ± S.E.Ms.

#### RESULTS

#### Physiological Changes after Irradiation

There were no effects of irradiation on fecundity rate when 400 Gy was applied to male flies mated with untreated female flies, however fecundity was lower for irradiated females mated with untreated males compared with control. There were no differences in egg hatch rate between either of the two kinds of treatments and the control (**Table 2**).

There were no effects of irradiation dose on the number of eggs oviposited by untreated females that were mated



Product size refers to the number after removing primers and ambiguous end positions.

TABLE 2 | Effects of 400 Gy irradiation of adults on fecundity and egg hatch rates.


Data are means ± S.E.M. Different letters indicate differences compared to control group (t-test, p < 0.05).

with irradiated males compared with the control group, when irradiation was applied at the pupal stage. However, there were dose effects for irradiated females that were mated with untreated males, where there was a significant decrease in the amount of eggs laid by treated female flies (**Table 3**). The results indicated that fecundity decreased significantly only when female flies had been irradiated. There were effects of irradiation at all three doses (150, 200, and 250 Gy) on hatch rates from the two treatment groups (irradiated male + untreated female and irradiated female + untreated male), where fewer eggs hatched from both of the treated groups compared with control (**Table 3**). The results indicated that irradiation of >150 Gy at the pupal stage could induce significant decreases in hatch rate in the subsequent stage of development.

There were no differences in eclosion and flying capability of adult flies that had been irradiated as pupae with doses < 200 Gy compared with control group, however, both rates (eclosion rate and flying capability) were lower in flies that were irradiated with 250 Gy as pupae (**Figure 1**). Mating competitiveness of males and females was not associated with irradiation at 200 Gy (χ <sup>2</sup> = 0.027, P = 0.869 and χ <sup>2</sup> = 2.199, P = 0.138, respectively, **Table 4**).

#### Morphological Changes of Reproductive Organs after Irradiation

We found that ovaries in the untreated (control) females were well-stocked with eggs that were covered by a transparent membrane, however following irradiation as pupae at 200 Gy, the sizes of ovaries and fallopian tubes were smaller than those of untreated flies (**Figures 2A,B**). The testes tended to be hippocampus-shaped, yellow in color, and covered with a transparent membrane (**Figures 2C,D**). SEM imaging of the ovaries (**Figures 3A,B**) showed they were round in shape and covered with reticular membrane. In the control group, ovaries were dense and well arranged, and separated by ovarioles (**Figure 3C**), however following irradiation, the shape became flattened, separation by ovarioles was indistinct, and the reticular membrane partially disappeared (**Figure 3D**). There was no clear difference in shape of testes between the control and irradiated flies (**Figures 3E,F**).

#### Sequencing and Expression of *B. tauYP*

The full-length cDNA of the YP gene consisted of 1,296 nucleotides (GenBank accession no. KC985244.1), with an

#### TABLE 3 | Response of adult fecundity and egg hatch rates to different doses of irradiation applied to pupae.


Data are means ± SEMs. Different letters within a column indicate differences at P < 0.05.



CK♂ a /CK♀ a represented the corrected value of the marked mating adults in treated group.

open reading frame encoding 432 amino acids (**Figure 4**), and exhibited high similarity to the YP gene of B. dorsalis (88%) and moderate similarity to A. suspensa (75%), Lucilia cuprina (60%), Neobellieria bullata (61%), D. melanogaster (56%), and Musca domestica (51%), as shown in NCBI BLAST (**Figure S1**). Phylogenetic analysis showed that the B. tau YP had high homology with B. dorsalis and A. suspense in the Diptera tephritidae. The scale showed the estimates of occurring amino acid site mutations in single locus of each branch (**Figure S2**).

The irradiation treatment applied to the stage of pupae could increase significantly the expression level of the YP gene at the 45 days post-emergence (**Figure 5**). Prior to this time, the level of the YP gene expression in treated flies showed no significant change compared with control. When the irradiation treatment was applied to newly emerged adults, the YP gene expression level only showed a significant increase at the 30 and 45 days post-emergence compared with control (**Figure 5**).

# DISCUSSION

Irradiation dose and processing stage of SIT programs are essential for determining the sterile effect (Ramírez-Santos et al., 2017). Optimizing the irradiation dose for inducing sterility is the most important factor for ensuring an effective control program, because insects may not be adequately sterile at low doses and yet less competitive at high doses (Parker and Mehta, 2007). Irradiation at 250 Gy for control of all arthropods in mango and papaya has been recommended and approved for commercial trade (Hallman and Loaharanu, 2002), and a dose of 250 Gy is supported by studies of 34 species of Lepidopteran from 11 families (Hallman et al., 2013). There appear to be variations in response to irradiation doses among Bactrocera species. For example, Collins et al. suggested that 70–75 Gy could be used as the lowest practical dose rate for B. tryoni (Collins et al., 2008), while 125 Gy irradiation applied to late third instars of B. dorsalis could result in no survival to adult stage (Follett and Armstrong, 2004), and a dose of 85 Gy could be applied to late

third instars of B. tau to prevent adult eclosion (Zhan et al., 2015). Our investigations on the effects of irradiation were initiated to determine whether the treatment doses could cause sterilization and minimize any adverse effects on quality. Irradiated males competition with wild males for females was not reduced. In our study, we showed that male flies irradiated with 150 Gy as pupae were sterilized (the eggs did not hatch after irradiated male mated with untreated female) and their flight capability and mating competitiveness were not significantly affected. We found that irradiation at 400 Gy of newly-emerged adult males did not reduce fecundity or egg hatch rate (**Table 2**). These results suggest that irradiation of adult males may not lead to complete sterility, which is consistent with a study that showed tolerance to irradiation increased with age and developmental stage (Zhan et al., 2015). Generally, irradiation should be applied at later developmental stages to ensure sterility, such as late stage of pupae, before the somatic cells become fully developed, because the reproductive organs are more sensitive to radiation than other tissues. Another advantage of applying irradiation at the pupae stage is ease of transport. There has been a great deal of research and application of irradiation at the pupae stage due to the convenience of operation and transportation, such as the investigations on Aedes albopictus (Oliva et al., 2013), Melolontha vulgaris (Oliva et al., 2012), and especially in dipteran insects (Follett and Armstrong, 2004; Zhao et al., 2017). While accounting for factors, such as mating competitiveness, flight capability and the quality of population, this laboratory study indicates that the application of gamma irradiation at doses


FIGURE 4 | Full length sequence of the Btau YP gene. Underline: initiation codon, tailing signal, and termination codon, framed: glycosylation site, and dashed area: predicted phosphorylation sites.

between 150 and 200 Gy to the pupae stage of B. tau could ensure sterilization in a control program.

We observed significant morphological changes in the ovaries following 200 Gy irradiation, but not in the testes. These results are supported by previous reports that also showed irradiation did not consistently cause differences in morphological features (Kheirallah et al., 2017). The reduction in the size and functionality of the female reproductive organs observed in this

study would probably lead to reductions in overall fecundity and fertility (Sachdev et al., 2017; Stringer et al., in review). Consistent with the results of male flies in our study, Paoli et al. showed that there were no differences in the ultrastructure of non-irradiated and irradiated Rhynchophorus ferrugineus Oliv sperm, except for some abnormalities in maturing spermatids (Paoli et al., 2014). The results from this study suggest that 200 Gy-irradiation may not induce significant morphological damage in male reproductive organs, but cause abnormalities in female reproductive organs that may lead to a decrease in oviposition activities. We were unable to establish the impact on fertilization of irradiation using morphology assessments, so we recommend further work on the effects of irradiation on the reproductive system in this species, particularly, the variations in the number, viability, and motility of sperm.

We hypothesized that irradiation on pupae would elicit sublethal effects that would be unobservable at the behavioral level, but would be detectable in mRNA level, which may lead to changes in protein function and abnormal behavior. We found that the YP gene from B. tau was conserved among B. dorsalis, A. suspensa and N. bullata. Large amounts of YP gene expression following a blood meal reflect large scale protein synthesis to provide essential nutrients required for embryonic development in mosquitoes and reveal the vital role of YP in the development of vitellogenesis (Hansen et al., 2014). In adult female D. melanogaster, there is an increase in the synthesis and secretion of yolk polypeptides occurring during the first 24 h following eclosion (Jowett and Postlethwait, 1980). After it is synthesized in fat body, YP protein is secreted into the circulatory system and forms oligomers that are transported into the egg, by endocytosis, to form yolk granules, and then hydrolyzed to amino acids or small peptides by protease (Hung and Wensink, 1983; Raikhel and Dhadialla, 1992). In this study, YP gene expression increased significantly at 30 and 45 day post-emergence when irradiation had been applied to pupae, compared with control group (**Figure 5**). Previous work has shown that 100 Gy of Xray irradiation also led to a 3.4-fold increase in the expression of profilin, a protein involved in spermatogenesis (Chang et al., 2015). Despite the higher transcription level of YP gene compared with control, in this study, there was a decrease in the fecundity that may have been caused by lower amounts of active YP protein or the inhibited effects on oocyte maturation by the irradiation (Rondaldson, 1995). Such hypothesis needs further demonstration by proteinomics study. Moreover, many researches pointed that the poor correlation between mRNA and protein is not unusual, and the transcription and translation process are no where close to linear (Maier et al., 2009; Chang et al., 2015). As many factors during the process could have influences on the efficiencies of translation of proteins, including RNA secondary structure, condon bias and ribosome occupancy (Maier et al., 2009).

In this study, we investigated the optimal sterilizing dose and processing stage of irradiation in B. tau by evaluating rates of fecundity, egg hatch, eclosion, mating competitiveness and flight capability. We then assessed the effects of irradiation on morphology of reproductive organs and found that while there were changes in the ovaries of irradiated females, there were no effects on the testes of irradiated males. We found expression of the YP gene increased in response to irradiation at specific growth stages, but the mechanisms were unclear. Overall results showed the influence of irradiation on physiology, morphology and gene expression, but additional efforts should be made to finally confirm the effective sterilizing dose for B. tau.

#### AUTHOR CONTRIBUTIONS

JC and SS performed the experiments; HY analyzed the data, and XY and GZ wrote and revised the manuscript.

#### ACKNOWLEDGMENTS

We thank Prof. Xiaolin Dong, for gene cloning and data analysis. This work was supported by the grants from National Natural Science Foundation of China (No. 31572335 and No. 31101439), and Science and Technology Planning Project of Guangdong Province (No. 2016A020210090 and No. 2017A010105023).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00118/full#supplementary-material

Figure S1 | Homologous comparison of YP deduced amino acid sequences between B. tau and other flies. The sequences were obtained from GenBank. Bd-yp (B. dorsalis, AAM00372.1), As-yp (A. suspense, AAC01961.1), Lc-yp (L. cuprina, ACY56509.1), Nb-yp (N. bullata, AAS75325.1), Dm-yp (D. melanogaster, AAL68367.1), and Md-yp (M. domestica, CAA65731.1).

Figure S2 | Phylogenetic tree of B. tau YP. Accession numbers are D. primaeva AAB06034.1, D. truncipenna AAC47253.1, Calliphora vicina CAA50066.1, and Glossina morsitans AAP84615.1.

### REFERENCES


protein 1 from Plutella xylostella L. Int. J. Biol. Macromol. 63, 233–239. doi: 10.1016/j.ijbiomac.2013.09.037


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Cai, Yang, Shi, Zhong and Yi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Thiamethoxam Resistance in *Aphis gossypii* Glover Relies on Multiple UDP-Glucuronosyltransferases

Yiou Pan1†, Fayi Tian1†, Xiang Wei <sup>1</sup> , Yongqiang Wu<sup>1</sup> , Xiwu Gao<sup>2</sup> , Jinghui Xi <sup>1</sup> and Qingli Shang<sup>1</sup> \*

*<sup>1</sup> College of Plant Science, Jilin University, Changchun, China, <sup>2</sup> Department of Entomology, China Agricultural University, Beijing, China*

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II enzymes that conjugate a variety of small lipophilic molecules with UDP sugars and alter them into more water-soluble metabolites. Therefore, glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. In this study, two inhibitors of UGT enzymes, sulfinpyrazone and 5-nitrouracil, significantly increased the toxicity of thiamethoxam against the resistant strain of *Aphis gossypii*, which indicates that UGTs are involved in thiamethoxam resistance in the cotton aphid. Based on transcriptome data, 31 *A. gossypii UGTs* belonging to 11 families (UGT329, UGT330, UGT341, UGT342, UGT343, UGT344, UGT345, UGT348, UGT349, UGT350, and UGT351) were identified. Compared with the thiamethoxam-susceptible strain, the transcripts of 23 *UGTs* were elevated, and the transcripts of 13 *UGTs* (*UGT344J2*, *UGT348A2*, *UGT344D4*, *UGT341A4*, *UGT343B2*, *UGT342B2*, *UGT350C3*, *UGT344N2*, *UGT344A14*, *UGT344B4*, *UGT351A4*, *UGT344A11,* and *UGT349A2*) were increased by approximately 2.0-fold in the resistant cotton aphid. The suppression of selected *UGTs* significantly increased the insensitivity of resistant aphids to thiamethoxam, suggesting that the up-regulated *UGTs* might be associated with thiamethoxam tolerance. This study provides an overall view of the possible metabolic factor *UGTs* that are relevant to the development of insecticide resistance. The results might facilitate further work to validate the roles of these *UGTs* in thiamethoxam resistance.

#### *Edited by:*

*Bin Tang, Hangzhou Normal University, China*

#### *Reviewed by:*

*Guorui Yuan, Southwest University, China Muthugounder S. Shivakumar, Periyar University, India*

> *\*Correspondence: Qingli Shang shangqingli@163.com*

> > *†Co-fist authors.*

#### *Specialty section:*

*This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology*

*Received: 04 February 2018 Accepted: 15 March 2018 Published: 03 April 2018*

#### *Citation:*

*Pan Y, Tian F, Wei X, Wu Y, Gao X, Xi J and Shang Q (2018) Thiamethoxam Resistance in Aphis gossypii Glover Relies on Multiple UDP-Glucuronosyltransferases. Front. Physiol. 9:322. doi: 10.3389/fphys.2018.00322* Keywords: UDP-glucuronosyltransferase, insecticide resistance, *Aphis gossypii*, RNAi, thiamethoxam

# INTRODUCTION

Uridine diphosphate (UDP)-glycosyltransferases (UGTs, EC 2.4.1.17) catalyze the conjugation of a range of small lipophilic compounds with sugars to produce glycosides, which are soluble in water and can be efficiently excreted (Mackenzie et al., 1997). The protein structure of UGTs is divided into two main parts: the aglycone substrate-binding domain at the N-terminus and the UDP sugar donor-binding domain at the C-terminus (Magdalou et al., 2010). Glycoside conjugation is one of the most important metabolic pathways for the biotransformation of a number of lipophilic endogenous and exogenous compounds of xenobiotics and endobiotics (Bock, 2003, 2016; Bowles et al., 2005). Therefore, the glycosylation of toxins by UGTs is a particularly important detoxification mechanism (Heckel, 2014; Heidel-Fischer and Vogel, 2015). Long-term evolution has led to the development of sophisticated detoxification systems that

**17**

allow organisms to resist various harmful substances occurring in the external environment. Insects use UDP-glucose as an activated sugar donor that is then transferred to UGTs, which are anchored in the endoplasmic reticulum (ER) (Ahn et al., 2012). Previous studies have indicated the involvement of insect UGTs in the detoxification of plant secondary xenobiotics in Manduca sexta (Ahmad and Hopkins, 1992), Helicoverpa assulta (Ahn et al., 2011a,b), Spodoptera littoralis (Wouters et al., 2014), Helicoverpa armigera, and Heliothis virescens (Krempl et al., 2016). In addition to plant secondary xenobiotic tolerance, insect UGTs might be involved in insecticide detoxification. Recent studies have demonstrated that the overexpression of UGT2B17 (renamed UGT33AA4) is associated with chlorantraniliprole resistance in Plutella xylostella (Li et al., 2017a,b), and many studies have reported on cytochrome P450 monooxygenase-mediated insecticides resistance (Scott, 2008; Feyereisen, 2015). However, few studies have described the involvement of UGTs in the detoxification of insecticide resistance.

The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is one of the most economically important insect pests in agriculture and has developed different levels of resistance to broad-spectrum insecticides, including organophosphates, pyrethroids, carbamates, and neonicotinoids (Denholm and Rowland, 1992; Shang et al., 2012; Chen et al., 2017). Thiamethoxam, a second-generation neonicotinoid insecticide that irreversibly binds to the nicotinic acetylcholine receptors (nAChR) of cells in the nervous system and interferes with the transmission of nerve impulses in insects (Casida and Durkin, 2013), and is effective for controlling resistant A. gossypii (Elbert et al., 2008). Research studies have indicated that enhanced detoxification caused by P450 gene overexpression accounts for neonicotinoids in Bemisia tabaci, Myzus persicae, and Nilaparvata lugens (Karunker et al., 2008, 2009; Puinean et al., 2010; Bao et al., 2016; Zhang et al., 2016). Consistent with these reports, our previous synergism analysis demonstrated that P450s are also involved in thiamethoxam resistance in A. gossypii (Wei et al., 2017). Whether UGTs are involved in insecticide resistance as well as P450-mediated resistance in A. gossypii has not been determined. The results of a synergism study illustrate that UGTs might be involved in the resistance present in thiamethoxam-resistant A. gossypii.

In this study, to clarify the potent roles of UGTs in thiamethoxam resistance in cotton aphids, (1) the UGT genes in the A. gossypii transcriptome were identified, and the phylogenetic relationships between these genes and their homologs in two other insects were analyzed; (2) the expression profiles of these UGTs in thiamethoxam-susceptible and thiamethoxam-resistant strains were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR); and (3) the involvement of overexpressed UGTs in resistance was functionally tested by RNA interference (RNAi). Our data provide preliminary insights into the dynamic changes in the gene expression of UGTs and their involvement in thiamethoxam resistance. The results might facilitate further study of the functions in UGTs in the insecticide resistance of A. gossypii.

# MATERIALS AND METHODS

#### Insects

Two cotton aphid (A. gossypii) strains were used in this study. One strain was resistant to thiamethoxam (ThR), and the other was susceptible to thiamethoxam (SS) (Pan et al., 2015). The SS strain was collected in 2008 from Jilin Province of China, where limited insecticides have been applied. The aphid species has been maintained without any insecticide treatment since its collection. The ThR strain was established from the SS population via consecutive selection with increased concentrations of thiamethoxam (LC30) via the leaf-dipping method. Both the resistant and susceptible strains were reared on cotton plants [Gossypium hirsutum (L.)] in the laboratory at 20–23◦C with a photoperiod of 16:8 h (light:dark).

#### Chemicals

Sulfinpyrazone (Sul) and 5-nitrouracil (5-Nul) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Thiamethoxam (25% WDG) was purchased from Syngenta (Switzerland). The PrimeScriptTM First-Strand cDNA Synthesis kit, SYBR <sup>R</sup> Premix Ex TaqTM II (Tli RNaseH Plus), oligo(dT)18, Ex Taq DNA polymerase, RNase-free DNase I, RNase Inhibitor, DNA Marker DL2000, and agarose were purchased from TaKaRa (Dalian, China). The pGEM-T vector and the T7 RiboMAXTM Express RNAi System were purchased from Promega (USA). All the reagents were of the highest purity available.

#### Bioassays

The synergistic effects of two UGT inhibitors, 5-nitrouracil (5- Nul) and sulfinpyrazone (Sul), on the toxicity of thiamethoxam to the SS and ThR strains were tested using a leaf dipping method, as described by Peng et al. (2016a) and Wei et al. (2017) with some modifications. The maximum sublethal doses of 5-Nul and Sul for the SS strain were determined using the bioassay method described by Wei et al. (2017). 5-Nul and Sul were used to prepare a series of concentrations (six or seven concentrations) with distilled water containing 0.05% (v/v) Triton X-100. The leaves were dipped for 15 s in the required concentration of insecticide or into 0.05% (v/v) Triton X-100 water (as the control treatment) and then placed in the shade and allowed to air dry. Bioassays were conducted by transferring at least 30 apterous adult aphids onto the treated cotton leaves obtained from each whole seedling. The bioassay samples were maintained in the laboratory at 20–23◦C with a photoperiod of 16:8 h (light:dark). Three replicates were performed for each concentration, and the mortality was assessed after 3 days. The maximum dose that led to zero mortality in the SS strain was adopted as the maximum sublethal concentration in our study, and the maximum sublethal concentrations of 5-Nul and Sul were 400 mg/L. For synergism bioassays, apterous adult aphids were exposed to cotton leaves that were treated with the mixture of thiamethoxam with 5-Nul or Sul (final concentration of 400 mg/L). Three replicates were performed for each concentration, and the mortality was assessed after 3 days and used to estimate the synergistic effects of 5-Nul or Sul with thiamethoxam in both strains. The synergistic ratio was calculated using the conventional approach, which divides the LC<sup>50</sup> without the synergist by the LC<sup>50</sup> with the synergist. A probit analysis was conducted using POLO software (LeOra Software Inc., Berkeley, CA, USA).

#### Nomenclature and Phylogenetic Analysis

The UGT sequences were derived from the transcriptome (the clean reads have been submitted to and are available from the National Center for Biotechnology Information (NCBI)/SRA database with SRA experiment accession number SRX683625) (Pan et al., 2015). These UGT sequences were named by the UGT Nomenclature Committee guidelines (Dr. Michael H. Court, Department of Veterinary Clinical Sciences, Washington State University, USA) using the following criteria: the gene symbol UGT, a family number, a subfamily letter, and an individual gene number. UGT families are defined at 40% amino acid sequence identity, and subfamilies are defined at 60% amino acid identity or greater (Mackenzie et al., 1997, 2005). Names were assigned to the A. gossypii sequences on this basis. The UGT predicted protein sequences from H. armigera and Bombyx mori were extracted from the UGT Nomenclature Committee (http:// prime.vetmed.wsu.edu/resources/udp-glucuronsyltransferase-

homepage)and analyzed with the A. gossypii UGTs via ClustalW alignment using MEGA 7 software (http://www.megasoftware. net/). The alignment results were used to build a consensus phylogenetic tree using the neighbor-joining method. Pairwise and multiple alignments were performed with a gap opening penalty of 10 and a gap extension penalty left of 0.2. A total of 1,000 bootstrap replications were performed, and branches with bootstrap values above 50% are indicated.

#### Protein Structure Prediction

Multiple alignments of 10 overexpressed representative protein sequences from the A. gossypii UGTs were obtained via ClustalW, and the structural domains, such as the UGT signature motif, were detected by comparison with other sequences for which primary structures have been characterized. The signal peptides were predicted by SignalP 4.1 on the CBS Prediction Servers (http://www.cbs.dtu.dk/services/SignalP/). The C-terminal transmembrane domain was searched using TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM).

#### Total RNA Isolation and cDNA Synthesis

The total RNA from apterous adult aphids was extracted using TRIzol (Invitrogen, USA) according to the manufacturer's instructions and then treated with RNase-free DNase I (TaKaRa, Japan). The RNA samples were quantified by measuring the absorbance at 260 nm, and the quality was assessed via agarose gel electrophoresis. First-strand cDNA was synthesized from the total RNA using a PrimeScriptTM First-Strand cDNA Synthesis kit (TaKaRa, Japan) with oligo (dT)<sup>18</sup> as a primer.

# Quantitative Real-Time PCR and Data Analysis

Quantitative real-time PCR was performed on an ABI 7500 system (Applied Biosystems) using SYBR <sup>R</sup> Premix Ex TaqTM II (Tli RNaseH Plus; TaKaRa, Japan) (Wei et al., 2017). Gene-specific primers for real-time PCR (**Supplementary Table 1**) were designed based on the UGT sequences (**Supplementary Data 1**) and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The thermal cycling protocol included an initial denaturation at 95◦C for 30 s followed by 40 cycles of 95◦C for 5 s and 60◦C for 34 s. The fluorescence signal was measured at the end of each extension step at 60◦C. After the amplification, a dissociation step consisting of 95◦C for 15 s, 60◦C for 1 min and 95◦C for 15 s was performed to confirm that only specific products were amplified. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1-alpha (EF1a) were used as internal reference genes for A. gossypii (Peng et al., 2016a,b). Relative gene expression was calculated using the 2−11CT method (Pfaffl, 2001). The experiment was independently performed three times for each strain. Significant differences were analyzed using GraphPad InStat3 statistical software (GraphPad Software, 2000, http://www.apponic.com/publisher/graphpad-software-18307/ top-downloads/).

# Rearing on Artificial Diet and dsRNA Feeding

The dsRNA design and synthesis methods were previously described by Peng et al. (2016a). Based on the UGT sequences (**Supplementary Data 1**) and the possible interference sites predicted with online prediction software (http://www.dkfz. de/signaling/e-rnai3/), we designed specific primers using DNAMAN 6.0 software (http://www.lynnon.com/dnaman. html). The gene fragments were amplified from cDNA and cloned into pGEM-T (Promega, USA). The purified plasmids served as templates for RNA synthesis using the T7 RiboMAXTM Express RNAi System (Promega, USA). ECFP dsRNA was used as the control and synthesized under the same conditions as the primers (**Supplementary Table 1**). The artificial diet and the rearing method used in this study were previously reported by Peng et al. (2016a,b). The diet was prepared in DEPC-treated water to ensure the absence of RNase activity. For the dsRNA feeding experiments, dsRNA was added to the artificial diet at a concentration of 100 ng/µL. An artificial diet containing dsRNA-ECFP was used as a control. Sixty adult apterous thiamethoxam-resistant A. gossypii were transferred onto the artificial diet for rearing. To analyze the efficiency of dsRNA knockdown on UGT expression, the aphids were fed an artificial diet containing dsRNA (100 ng/µL) for 48 h and then subjected to RT-qPCR. To assess the sensitivity of the cotton aphids to thiamethoxam after RNAi of UGT, 80 resistant adult aphids were transferred to the artificial diet containing thiamethoxam (1.0 mg/L) mixed with dsRNA-UGT (100 ng/µL), and dsRNA-ECFP was used as the control. The mortality of the cotton aphids was recorded after 48 h. Each treatment included three replicates (80 aphids were used in each replication).

# RESULTS

# Thiamethoxam Toxicity and Synergism Bioassays

The probit analyses of thiamethoxam toxicity and synergism bioassays of A. gossypii are summarized in **Table 1**. 5-Nul and


TABLE 1 | Synergistic effects of Sul and 5-Nul on the toxicity of thiamethoxam in the SS and ThR strains.

\**Data obtained from Pan et al. (2015). <sup>a</sup>Probit model fitted using POLO-PC (LeOra Software, 1987). <sup>b</sup>Confidence limits. <sup>c</sup>SR (synergism ratio)* = *LC*<sup>50</sup> *of thiamethoxam/LC*<sup>50</sup> *of thiamethoxam with synergist.*

Sul increased the thiamethoxam toxicity in the ThR strain by 9.38- and 10.31-fold, respectively. These results indicate that UDP-glucuronosyltransferases are involved in thiamethoxam resistance in the ThR strain at the observed resistance state.

#### Identification and Phylogenetic Analysis of the *A. gossypii* UGTs

Based on the transcriptome data from A. gossypii, the full lengths of 31 UGT genes were identified, and these UGT genes were named by the UGT Nomenclature Committee (Dr. Michael H. Court, Department of Veterinary Clinical Sciences, Washington State University, USA). The GenBank accession numbers are listed in **Supplementary Data 1**. To construct a phylogenetic tree (**Figure 1**), a total of 83 UGT gene sequences of H. armigera and B. mori from the UGT Nomenclature Committee (**Supplementary Data 1**) and 31 UGT gene sequences from our cotton aphid transcriptome were used in the phylogenetic tree. ClustalW alignments performed using MEGA 7 software (http://www.megasoftware.net/) were used to align the amino acid sequences, and the neighbor-joining method with 1,000 bootstrap replicates was used to construct the phylogenetic trees.

The 31 A. gossypii UGTs were distributed into 11 families, namely, the UGT344 (12 UGTs), UGT350 (4), UGT342 (3), UGT343 (3), UGT329 (2), UGT351 (2), UGT330 (1), UGT341 (1), UGT345 (1), UGT348 (1), and UGT349 (1) families. The number of UGT genes in A. gossypii (31 UGTs) was less than that in H. armigera and B. mori, and this finding is related to the lack of a complete genome sequence for A. gossypii.

# Structural Motifs of the *A. gossypii* UGT Proteins

Multiple alignments of eight representative A. gossypii UGT amino acid sequences revealed two major domains: the highly variable N-terminal substrate-binding domain and the conserved C-terminal sugar donor-binding domain (**Figure 2**) (Ahn et al., 2012). All A. gossypii UGTs consisted of a different length amino acid signal peptide found at the N-terminal end, which is presumably cleaved after integration into the ER compartment. The two predicted sugar donor-binding regions (DBR1 and DBR2), important residues interacting with the sugar donor, and catalytic residues were conserved. The UGT motif signature sequences [also called the plant secondary product glycosyltransferase (PSPG) motif in plants], (FVA)-(LIVMF)-(TS)-(HQ)-(SGAC)-G-X(2)-(STG)-X(2)-(DE)- X(6)-P-(LIVMFA)-(LIVMFA)-X(2)-P-(LMVFIQ)-X(2)-(DE)-Q (where X is any amino acid), was found in the middle of the C-terminal domain, which shows higher conservation

FIGURE 2 | Alignment of the amino acid sequences of eight UGT genes from *A. gossypii*. The predicted signal peptides are underlined, and the UGT signature motif is boxed. The transmembrane domains and cytoplasmic tails in the C-terminus are underlined by black and gray lines above the alignment, respectively. The conserved catalytic residues, H and D, are indicated by \* above the alignment. DBR refers to the donor-binding region, and several important residues interacting with the sugar donor are indicated (a, b, or c) above the alignment.

(Mackenzie et al., 1997; Ahn et al., 2012). The alignment data suggested that most A. gossypii UGTs were active proteins.

# Expression Profiling of *A. gossypii UGT* Genes in the Resistant and Susceptible Strains

The quantitative real-time PCR results indicated that the transcripts of 23 UGT genes of the 27 determinate genes were elevated, and the transcripts of 13 UGT genes were increased by approximately 2.0-fold or greater in the thiamethoxamresistant cotton aphid compared with the susceptible aphids. Specifically, the mRNA levels of UGT344J2, UGT348A2, UGT344D4, UGT341A4, UGT343B2, UGT342B2, UGT350C3, UGT344N2, UGT344A14, UGT344B4, UGT351A4, UGT344A11, and UGT349A2 were increased to 4.96, 3.95, 3.64, 2.98, 2.54, 2.22, 2.14, 2.14, 2.18, 2.05, 2.00, 1.96, and 1.94-fold, respectively. In contrast, the transcripts of UGT344D6, UGT329A3, UGT344B5, and UGT351A3 were down-regulated in the ThR strain compared with the SS strain, and the UGT351A3 level was decreased by 0.29-fold (**Figure 3**).

# Suppression of *UGT* Transcripts Increases Thiamethoxam Toxicity

An orally delivered dsRNA method for RNAi was performed to elucidate the relationship between the overexpression of massive UGT genes and thiamethoxam resistance. Because many UGT genes were up-regulated in the ThR strain, we chose five UGT genes for the RNAi experiment. Under the RNAi treatments, the expression levels of UGT342C2, UGT344B4, UGT344J2, UGT348A2, and UGT349A2 were reduced to 0.65-, 0.72-, 0.67-, 0.65-, 0.69-, and 0.86-fold in the corresponding dsRNA-UGTtreated (100 ng/µL) aphids after 48 h of treatment compared with the control expression levels (**Figure 4**). The mortality increased from 50.46% in the control to 55.03, 60.74, 57.66, 61.79, 52.38, and 63.27% in the aphids fed dsRNA-UGT342C2, dsRNA-UGT344B4, dsRNA-UGT344J2, dsRNA-UGT348A2, dsRNA-UGT349A2, and dsRNA-Mix (the ratio of six dsRNA-UGT is 1:1:1:1:1:1:1) under the 1.0 mg/L thiamethoxam treatments, respectively (**Figure 5**).

# DISCUSSION

Due to the extensive use of the neonicotinoid insecticide imidacloprid for controlling cotton aphids in the field, the resistance to imidacloprid ranged from 1.48 to >1,200-fold among different A. gossypii populations collected from various Bt cotton planting areas in China in 2014, and the LC50 value of imidacloprid was >5,000 mg/L in the population from Yuncheng of Shanxi Province (Chen et al., 2017). Thiamethoxam has been used as an alternative neonicotinoid insecticide for the control of cotton aphids. Our previous synergism assay showed that phase I enzyme P450s (acting directly on the toxin molecule) are involved in thiamethoxam resistance (Wei et al., 2017). In addition to P450-mediated detoxification resistance,

the roles of the phase II enzyme UGTs (which conjugate endogenous molecules to the toxins) in thiamethoxam resistance remains unknown. To clarify the roles of UGTs in thiamethoxam tolerance, the UGT inhibitors 5-nitrouracil and sulfinpyrazone were used in a synergism assay, and the results illustrated that these two UGT inhibitors significantly increased thiamethoxam toxicity in the ThR strain (**Table 1**), suggesting that UGTs, in addition to P450, are involved in thiamethoxam resistance. Therefore, glycosylation by UGTs might play an important role in the detoxification of xenobiotics in the cotton aphid. Notably, information regarding UGTs in the cotton aphid has not been available until now.

In this study, 31 UGT genes were identified from the transcriptome data of A. gossypii (Pan et al., 2015). The A. gossypii UGTs were distributed into 11 families: UGT329, UGT330, UGT341, UGT342, UGT343, UGT344, UGT345, UGT348, UGT349, UGT350, and UGT351 (**Figure 1**). The fewer number of UGT genes identified in A. gossypii (31 UGTs) compared with those found in H. armigera and B. mori might be due to the lack of a complete genome sequence for A. gossypii. The UGT protein structure is divided into two main parts: the aglycone substrate-binding domain in the N-terminus and the UDP sugar donor-binding domain in the C-terminus (Meech and Mackenzie, 1997; Meech et al., 2012). Alignments of the A. gossypii UGT amino acid sequences showed conserved domains, including the sugar donor-binding region (DBR1 and DBR2), important residues interacting with the sugar donor and catalytic residues, and the UGT motif signature sequences [(FVA)-(LIVMF)-(TS)-(HQ)-(SGAC)-G-X(2)-(STG)-X(2)-

(DE)-X(6)-P-(LIVMFA)-(LIVMFA)-X(2)-P-(LMVFIQ)-X(2)-

(DE)-Q] (**Figure 2**) (Mackenzie et al., 1997; Ahn et al., 2012). These findings suggested that A. gossypii UGTs were likely active proteins, which is similar to observations in mammals. Insect UGTs are capable of detoxifying plant secondary compounds. For example, the stereoselective reglucosylation of benzoxazinoid by UGT represents a detoxification strategy in S. littoralis (Wouters et al., 2014). UGTs are capable of glycosylating gossypol primarily to the diglycosylated gossypol isomer 5, which is a crucial step in gossypol detoxification in H. armigera (Krempl et al., 2016). Insect UGTs mediate plant xenobiotic tolerance and have also been reported to be involved in insecticide resistance. The ingestion of dsRNA, which successfully silences overexpressed UGTs, significantly increases the susceptibility of resistant Leptinotarsa decemlineata to imidacloprid (Kaplanoglu et al., 2017). In P. xylostella, the overexpression of UGTs is associated with chlorantraniliprole resistance (Li et al., 2017a,b). In this study, a screening of the expression profile of UGTs revealed that the expression of 13 UGT genes was increased by nearly 2-fold or more in the ThR strain compared with the SS strain (**Figure 3**), suggesting that multiple up-regulated UGTs might be associated with thiamethoxam resistance. To verify the influence of UGT gene overexpression on the susceptibility to thiamethoxam in A. gossypii, we performed RNAi via a dsRNA oral feeding method (Peng et al., 2016a,b) and found that the RNAi of the overexpressed UGT genes could have resulted in increased thiamethoxam susceptibility in the resistant cotton aphids (**Figure 5**). This result further confirmed that enzymes encoded by these overexpressed UGTs might contribute to the detoxification of thiamethoxam by glycosylation in A. gossypii. In conclusion, this study provides insights into the potential roles of UGTs in thiamethoxam resistance. These results should be useful for understanding thiamethoxam resistance mechanisms.

#### AUTHOR CONTRIBUTIONS

All the authors listed have made a substantial, direct and intellectual contribution to the work and have approved its publication. YP and FT: designed and performed most of the experiments. XW and YW: performed the RNAi assays. XG and

#### REFERENCES


JX: revised the manuscript. QS: designed the experiments and wrote the manuscript.

#### ACKNOWLEDGMENTS

This work was sponsored by the National Natural Science Foundation of China (31772188, 31301728). The authors thank Prof. Michael H. Court of Washington State University for his kind help in renaming the UGTs identified in this study on behalf of the UGT nomenclature committee.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00322/full#supplementary-material

Supplementary Table 1 | Primers used in the experiments. dsRNA, double-stranded RNA; EF1a, elongation factor 1-alpha; F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ORF, open reading frame; R, reverse. The lowercase letters indicate the T7 RNA polymerase promoter.

Supplementary Data 1 | Gene sequences of *A. gossypii UGTs* and other insect orthologs.


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Pan, Tian, Wei, Wu, Gao, Xi and Shang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

#### Edited by:

*Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China*

#### Reviewed by:

*Pin-Jun Wan, China National Rice Research Institute (CAAS), China Abid Ali, University of Agriculture Faisalabad, Pakistan*

> \*Correspondence: *Guohua Zhong guohuazhong@scau.edu.cn*

*†These authors have contributed equally to this work.*

#### Specialty section:

*This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology*

Received: *10 January 2018* Accepted: *27 March 2018* Published: *11 April 2018*

#### Citation:

*Shu B, Zhang J, Cui G, Sun R, Sethuraman V, Yi X and Zhong G (2018) Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions. Front. Physiol. 9:372. doi: 10.3389/fphys.2018.00372* Benshui Shu† , Jingjing Zhang† , Gaofeng Cui, Ranran Sun, Veeran Sethuraman, Xin Yi and Guohua Zhong\*

*Key Laboratory of Crop Integrated Pest Management in South China, Ministry of Agriculture, Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, China*

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest *Spodoptera litura*. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in *S. litura* and ensure the data more accurate for the mechanism analysis of azadirachtin.

Keywords: Spodoptera litura, azadirachtin, RT-qPCR, reference gene, stability

# INTRODUCTION

Real-time quantitative polymerase chain reaction (RT-qPCR) is considered to be the reliable and effective method for the quantitative analysis of candidate genes expression level and the verification of transcriptomic analysis, especially in species which lacking the genomic information, due to the advantages of high sensitivity and specificity, more convenience and good reproducibility (Ibanez and Tamborindeguy, 2016; Zhang et al., 2017). However, some factors including RNA

**26**

integrity and quality, reverse transcription efficiency and primer amplification efficiency could interfere and influence the accuracy and reliability of RT-qPCR. Therefore, the reference genes attracted attention and were used for the precise normalization (Zhang et al., 2015; Arya et al., 2017). The idealized reference genes which defined as the "constitutively expressed to maintain cellular function" should have the relatively stable expression under various tissues and physiological conditions (Pan et al., 2015; Chen et al., 2016). Simultaneously, the expression levels of reference genes might vary under different experimental conditions. Selection of appropriate reference genes is the prerequisite to ensure the accuracy of experimental results (Nagy et al., 2017). Of course, standardizing experimental results with two or more reference genes could also improve the accuracy and be recommended (Shi et al., 2016).

Previous studies have shown that the basic metabolism genes including actin, tubulin, ribosomal protein, glyceraldehydes 3 phosphate dehydrogenase, elongation factor have been used as the reference genes for RT-qPCR (Płachetka-Bozek and Augustyniak, 2017; Wan et al., 2017). However, recent reports indicated that reference genes also have independent functions and were involved in various physiological and pathological processes, such as GAPDH and 18S rRNA (Nicholls et al., 2012; Kozera and Rapacz, 2013). Azadirachtin, a proverbial tetranortriterpenoid, was considered to be the most promising botanical pesticide for pest control with the effect of antifeedant, insect growth and development inhibition (Shu et al., 2015; Wang et al., 2015). It was demonstrated that some basic metabolism genes were affected by azadirachtin. In Drosophila melanogaster, immunohistochemistry and in silico analysis confirmed that azadrachtin bind to actin and inhibited its polymerization, which indicated that actin could be act as the putative target of azadirachtin (Anuradha et al., 2007; Pravin Kumar et al., 2007). Simultaneously, azadrachtin bind to actin was verified in Plutella (Anuradha and Annadurai, 2008). Further report showed that the cytoskeletal function was also influenced by azadirachtin (Huang et al., 2010).

As the most serious polyphagous insect pest, Spodoptera litura has the characteristics of high fertility, short life cycle, abundant host plants, devastating for many economic crops and widely distributed in the tropical and subtropical Asia (Bano and Muqarab, 2017; Feng et al., 2017; Kaur et al., 2017). Besides, the problems of pest resistance produced by frequent and irrational use of chemical insecticides are also more prominent (Sang et al., 2016; Lin et al., 2017). Azadirachtin has the significant antifeedant and growth inhibitory action against S. litura (Jeyasankar et al., 2011). Recently, the discovery and publication of S. litura genome data have provided new insights into many biological problems including the mechanisms of evolution and resistance, the specialization of host plants and ecological adaptation (Cheng et al., 2017). It also renewed interests in interpreting the mechanisms of azadirachtin against S. litura at the molecular level.

In this study, in order to verify the suitable reference genes of S. litura for RT-qPCR under azadirachtin treatments, eight candidate reference genes actin (Actin), elongation factor 1alpha (EF-1α), elongation factor 2 (EF2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L7A (RPL7A), ribosomal protein L13A (RPL13A), alpha-tubulin (αtubulin), beta-tubulin (β-tubulin) were identified and cloned from S. litura. The expression stabilities of eight reference genes in larva, cuticle, fat body, and midgut samples from larvae of control and different concentrations of azadirachtin treatments were measured by five programs (NormFinder, BestKeeper, Delta Ct method, geNorm, and RefFinder). This study could potentially reveal the expression variations of reference genes in response to azadirachtin, which could provide some foundations for RT-qPCR analysis of S. litura in the future.

#### MATERIALS AND METHODS

#### Insects

The third-instar larvae of S. litura fed with a standard artificial diet were used as control group and the azadirachtin-treatment group were the third-instar larvae fed with the diet added azadirachtin (1.0, 2.5, and 5.0 mg/g) for 7 d. All the larvae were kept in the conditions of 25 ± 1 ◦C, 60–70% relative humidity and a 16:8 h light: dark cycle in Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University.

# Sample Collection

After feeding with artificial diet for 7 d, some of the larvae with different treatments (n = 5) were collected and kept in −80◦C and some were dissected (n = 20). The cuticle, fat body and midgut were separated and washed in cold phosphate buffered saline (PBS), then collected and kept in −80◦C. For the tissues or larva samples, three biological replications of each treatment were collected.

# Total RNA Isolation and cDNA Synthesis

All the samples were ground into powder by liquid nitrogen and 1 ml RNAiso plus (Takara, Japan) was added for total RNA isolation following the experimental procedures. Samples were mixed well and lysed for 5 min at room temperature, 200 µL chloroform was added and shaken for 15 s. The mixture was incubated for 3 min at room temperature. After centrifuged with 12,000 rpm for 10 min at 4◦C, 500 µL supernatant was separated and mixed with 500 µL isopropanol, incubated for 10 min at room temperature, then centrifuged with 12,000 rpm for 10 min at 4◦C. Removed the supernatant and 75% ethanol was added and washed the precipitate. After centrifuged, the ethanol was removed and the precipitate was dried at room temperature for 3 min, an appropriate amount of DEPC water was added to dissolve the precipitate. The concentration and purity of total RNA were measured by a NanoDrop <sup>R</sup> spectrophotometer (Thermo Fisher, MA, USA).

Qualified RNA (1 µg) was used for cDNA synthesis by PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan) following the manufacturer's instructions. A total 10 µL reaction system contained 1 µg total RNA, 2 µL 5 × gDNA Eraser Buffer and 1 µL gDNA Eraser was incubated for 2 min at 42◦C, and then kept at 4◦C. Another 10 µL reaction solution were prepared with 4 µL RNase free water, 4 µL 5 × PrimeScript Buffer 2, 1 µL RT Primer Mix and 1 µL PrimeScript RT Enzyme Mix I and then mixed with the solution as above, the admixture was incubated at 42◦C for 15 min, 85◦C for 5 s and stored at −20◦C.

#### RT-PCR and RT-qPCR Analysis

According to the transcriptome of S. litura (GenBank number: GBBY00000000) (Gong et al., 2015), eight genes (Actin, EF-1α, EF2, GAPDH, RPL7A, RPL13A, α-tubulin, β-tubulin) were selected as candidate reference genes. The RT-qPCR primers of those genes were designed by Primer Premier 5.0 (Premier, Canada). The primers were used to clone the fragments with LA Taq (Takara, Japan). The amplification was fulfilled by denaturing at 95◦C for 3 min, followed with 32 cycles of 95◦C for 30 s, 60◦C for 15 s, and a 10 min extension at 72◦C. The PCR products were checked by 1.5% agarose gel electrophoresis. PCR products were ligated with pMD-19T and transformed into Escherichia coli DH5α. Plasmids were extracted by TIANprep Mini Plasmid kit (TIANGEN, China) and used as the templates for standard curve of reference genes. RT-qPCR was performed by iTaqTM Universal SYBR <sup>R</sup> Green Supermix (BIO-RAD, USA) in the CFX ConnectTM Real-Time System (BIO-RAD, USA). The reaction procedure was consists of the following steps: a denaturation step at 95◦C for 3 min, followed with 95◦C 10 s; 60◦C 10 s; 72◦C 15 s for 40 cycles, and ended with a melting-curve analysis. For the PCR efficiency, a series of 10-fold serially diluted plasmids were used as templates and the procedure for RT-qPCR was performed as above.

# Data Analysis

All the Ct-values from RT-qPCR were collected and the stability of reference genes were analyzed by the software of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. NormFinder could identify the optimal normalization reference genes according to the direct measure to estimate expression variation and verify the expression stability (Andersen et al., 2004). BestKeeper is another algorithm for stability detection of reference genes and it could calculate by the amplification efficiency of primers and the Ct-values of reference genes (Pfaffl et al., 2004). The Delta 1CT method evaluated the stability of reference genes by comparing the relative expression of pairwise genes within each sample (Silver et al., 2006). geNorm was used to evaluate the stability of candidate reference genes and determine the appropriate reference gene number in RT-qPCR. The lowest Ct value in the reference gene is very important for this algorithm and the stability value (M) calculated by geNorm was used to assess the stability of reference genes. Furthermore, the average geNorm M ≤ 0.2 was considered to be a reference for high reference genes stability. Besides, pair-wise variation value (V) calculated by geNorm was used for determine the optimal normalization factor number and geNorm V < 0.15 could be the standard for better normalization (Vandesompele et al., 2002). Furthermore, the powerful web-based analysis tool, RefFinder (http://www.leonxie.com/referencegene.php), was used to get the comprehensive ranking results for the stability assessment of reference genes by integrating the results of four software we used as above.

# RESULTS

# Transcriptional Profiling of Candidate Reference Genes

Before the expression stability evaluation, eight candidate reference genes belong to four functional groups including 3 of the structure-related genes (Actin, α-Tubulin and β-Tubulin), 2 of the ribosomal protein (RPL7A and RPL13A), 2 of the protein factor (EF-1α and EF2), and one metabolism-related gene (GAPDH) were cloned and identified by sequencing. All the PCR products amplified by the primers were detected with 1.5% agarose gel and the single band was observed in each PCR product with the expected strip size ranged from 106 to 222 bp (**Figure 1A**). All the gene primers were validated before the reference genes selected for normalization and the primer efficiency and correlation coefficient (R 2 ) were calculated and showed in **Table 1**. The amplification efficiencies of primers met the standard requirement of conventional RT-qPCR, the primer efficiency was ranged from 92.4 to 101.1% and almost all the R 2 of standard curve line were more than 0.995.

The raw Ct-values of eight candidate reference genes were detected by RT-qPCR and ranged from 14.31 (RPL7A) to 28.15 (α-Tubulin). The average Ct-values of Actin, EF-1α, EF2, GAPDH, RPL7A, RPL13A, α-Tubulin and β-Tubulin were 16.76 ± 1.08, 16.27 ± 0.50, 17.94 ± 0.49, 19.20 ± 0.76, 16.41 ± 0.50, 17.21 ± 0.49, 24.53 ± 1.89, and 18.40 ± 0.41, respectively (**Figures 1B–F**). All the threshold fluorescence peak of candidate reference genes were fall in the cycles of 15–30 and α-Tubulin has the lowest expression level.

# Identification of Best Reference Gene for RT-qPCR

In this study, the stability of eight candidate reference genes was analyzed by five programs NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder in different samples.

# Normfinder Analysis

The stability of eight candidate reference genes evaluated by NormFinder software were depended on the stability value and the stability of reference genes was negatively correlated with the stability values. As shown in **Figure 2**, EF-1α, RPL7A, and EF2 were regarded as the most stable genes in the tissues of cuticle, fat body, and midgut, and the least steady genes in these tissues was GAPDH. In addition, EF-1α was the ideal reference gene and followed with EF2 and Actin for larva. For all the samples, the order of reference gene stability was: RPL13A > RPL7A > EF-1α > EF2 > β-Tubulin > GAPDH > Actin > α-Tubulin.

# BestKeeper Analysis

BestKeeper determines the stability of reference genes based on the standard deviation (SD) value, the coefficient of variation (CV) and correlation coefficient (r). The reference gene with high stability has the lower SD value and CV, and higher correlation coefficient. Besides, the candidate gene with SD > 1 indicated unstable and could not be used as the reference. As shown in **Table 2**, the most and least stable reference genes in cuticle and larva was EF2 and GAPDH, respectively. Furthermore, Actin

could be the most stable reference gene with the lowest SD value in the tissue of fat body and followed by β-Tubulin and EF2. In addition, the reference gene β-Tubulin was considered to be the most stable gene for the midgut samples. The reference gene stability arrangement in all samples was showed as followed: β-Tubulin > RPL13A > RPL7A > EF-1α > EF2 > GAPDH > Actin > α-Tubulin. These results indicated that single reference gene could not be the ideal reference for all organizations.

#### Delta CT Analysis

According to the results of Delta CT analysis, EF2 with the lowest STDEV was regarded as the most sable gene for the samples of midgut and larva. Simultaneously, the genes with the most stable expression in cuticle and fat body were EF-1α and RPL7A, respectively. For all the samples, RPL13A, EF2, and EF-1α could be considered as the most stable internal reference gene because the minimal STDEV difference was existed among three genes (**Figure 3**).

#### geNorm Analysis

The M value calculated by geNorm software is an important index to evaluate the stability of reference genes and the reference gene with high stability has the lower M value. As shown in **Figure 4**, RPL7A, β-Tubulin, RPL7A, and EF-1α were evaluated as the most stable reference genes in cuticle, fat body, midgut, and larvae. geNorm also has the function to determine the optimal


number of reference genes required for the analysis. As shown in **Figures 4B,D,F,G**, the pair-wise variation (V2/V3) in all the tissues and larvae were <0.15, this results revealed that two of the reference genes were required for the reliable normalization of all samples. Therefore, the best combination of reference genes in different samples were: RPL7A and RPL13A for cuticle, β-Tubulin and EF2 for fat body, RPL7A and EF-1α for midgut, EF-1α and RPL13A for larvae.

#### RefFinder Analysis

The stability results of reference genes were also assessed by RefFinder which integrates major computational programs currently available. The comprehensive ranking of reference genes for different tissues were showed in **Figure 5**. In cuticle samples, EF-1α and EF2 were ranked as the most and second stable reference genes. In fat body samples, β-Tubulin was evaluated as the best reference gene. In midgut and larva samples, EF2 was ranked in the first place. Furthermore, RLP13A was the most stable genes for all the samples and followed with RPL7A and EF-1α.

# DISCUSSION

Compared to some other quantitative methods including Northern blotting, in situ hybridization, cDNA arrays, RT-qPCR was more convenience, efficient and widely used. The selection of reference genes could be the critical step to eliminate the variations and get the accurate RT-qPCR results. It was reported that the expression of reference genes is not static in different species, tissues or experimental conditions (Lu et al., 2013), so the stability verifications of reference genes are necessary before evaluating target gene expression levels by RT-qPCR. Recently, more and more research considered this and the stability verifications were also performed in many Lepidoptera insects, for example: Plutella xylostella (Fu et al., 2013), Danaus plexippus (Pan et al., 2015), Sesamia inferens (Lu et al., 2015), Heliconius numata (Piron Prunier et al., 2016), Thitarodes armoricanus (Liu et al., 2016), Bombyx mori (Guo et al., 2016), Helicoverpa armigera (Chandra et al., 2017), Grapholita molesta (Wang et al., 2017), Chilo suppressalis (Xu et al., 2017), Spodoptera exigua (Płachetka-Bozek and Augustyniak, 2017), and grassland caterpillars (Zhang et al., 2017). As the important agricultural pest, the identification and validation of reference genes in S. litura with three biotic factors and four abiotic treatments were also investigated (Lu et al., 2013).

Azadirachtin is the most efficient and environmentally friendly botanical insecticide for contemporary society which emphasizes green and organic agriculture. It also had significant effects on S. litura, including antifeedant, growth inhibitory and so on. Cuticle is the largest organ in insect and azadirachtin has the effect of changing the cuticular protein levels (Yooboon et al., 2015). The midgut was function for food digestion and nutritional absorption and azadirachtin also had many effects on it, including reduced the activities of digestion enzymes and induced apoptosis (Nathan et al., 2005; Shu et al., 2018). In addition, the fat body was also an important organ for metabolic activities and immune response, which speculated it could be involved in the metabolism of azadirachtin (Sun et al., 2017). In this study, these three important tissues or organs were chosen for expression stability

validation of eight candidate reference genes after S. litura long-time exposure to azadirachtin by NormFinder, BestKeeper, geNorm, Delta CT, and RefFinder. The results indicated that the expression stability obtained by different algorithms were different. Simultaneously, no single internal reference gene showed the most stable expression under different tissues or processing conditions, this was similar to the previous results of S. litura (Lu et al., 2013). Therefore, the selection of suitable reference genes should consider all the results of these algorithms.


Ribosomal proteins are abundant in eukaryotic ribosome and function for protein biosynthesis and ribosome assembly, they also play crucial roles in various physiological processes, including cell proliferation and growth (Ladror et al., 2014; Zhou et al., 2015). According to the previous studies, ribosomal proteins were identified as the stable internal reference genes for RT-qPCR. For example, RP49 was considered to be the most stable in B. mori and D. plexippus for different conditions (Wang et al., 2008; Pan et al., 2015). In addition, RPS18 exhibited the stable expression for most treatments in Lipaphis erysimi and Bactericera cockerelli (Ibanez and Tamborindeguy, 2016; Koramutla et al., 2016). Other ribosomal proteins were also verified as the stable genes, such as RPL3 for wing discs of H. numata (Piron Prunier et al., 2016); RPS13 for P. xylostella in different developmental stages (Fu et al., 2013); RPS15 and RPL32 for H. armigera in different tissues and treatments (Zhang et al., 2015). In addition, RPL10 was confirmed to be the stable gene in tissues and different populations and RPS3 for the starvation condition of S. litura (Lu et al., 2013). Furthermore, RLP13A was determined as the steady in L. erysimi, T. armoricanus, and S. exigua (Koramutla et al., 2016; Liu et al., 2016; Płachetka-Bozek and Augustyniak, 2017). Simultaneously, RPL7A was also the steady in S. exigua and Lethrus apterus (Nagy et al., 2017; Płachetka-Bozek and Augustyniak, 2017). In this research, consistent with the above results, we found that RLP13A and RPL7A performed relative higher stable ranking in most samples of S. litura under azadirachtin treatments.

Ribosomal translation factors are highly conserved in organisms and response for the protein synthesis by delivering the aminoacyl-tRNAs to ribosome (Mateyak and Kinzy, 2010; Jank et al., 2017). It was reported that elongation factors were suitable as reference genes in different species, tissues, and treatments. Such as, EF1α was recommended for normalization in different tissues, developmental stages, and Bt toxins of Diabrotica virgifera virgifera (Rodrigues et al., 2014), the adult tissues of H. armigera (Zhang et al., 2015), tissues of Myzus persicae (Kang et al., 2016) and most conditions of D. plexippus (Pan et al., 2015). Besides, it was evaluated as the normalization gene in different tissues and temperature-stressed samples of S. litura. EF2 was also regard as the most stable reference in males of L. apterus (Nagy et al., 2017) and all tissue samples of S. exigua (Zhu et al., 2014). In our present study, EF1α and EF2 were ranked as the most stable genes in most samples of S. litura, which indicated that these two could be used for normalization of RT-qPCR.

In contrast, we found two categories of internal reference genes (skeleton and metabolism-related proteins) which were widely used for RT-qPCR showed instability in most samples, although these genes had been reported as being stable in other species or treatment samples. The skeleton proteins (actin, α-Tubulin and β-Tubulin) could be acknowledged as the target of azadirachtin and the mRNA transcript profiles were affected, which could be the reason for the unstable of these three genes in samples of S. litura after azadirachtin treatments (Anuradha and Annadurai, 2008). The metabolism-related protein GAPDH was ranked the least stable genes under azadirachtin, which was consistent with the result of P. xylostella for insecticide

TABLE

2


Expression

stability

of

the

eight

candidate

reference

genes

in

*S.*

*litura*

by

BestKeeper

algorithm.

susceptibility (Fu et al., 2013). The above results indicated that not all housekeeper genes could be used as the reference in a variety of different experimental conditions, and it also highlights the importance for the selection of appropriate reference gene.

In most cases, a single reference gene was used for standardization in RT-qPCR and unsuitable internal reference gene selection could interfere with the accuracy of experimental results. In this study, the software geNorm was used for determining the optimal number of reference genes and we found the index for judging of minimum number V in all the samples were <0.15, which means two reference genes with less expressional variation were need for normalization in S. litura under azadirachtin treatments. Therefore, multiple reference genes could be evaluated and used for RT-qPCR to ensure the accuracy of target gene expression.

In summary, eight candidate reference genes of S. litura were cloned and identified in this study. In addition, the expression stably of candidate reference genes under azadirachtin treatments were systematically evaluated by five software: geNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. Furthermore, the optimal number of reference genes was also determined. Our results indicated that the best reference genes

for RT-qPCR in S. litura under azadirachtin treatments could be as followed: EF-1α and EF2 for cuticle samples, β-Tubulin and RPL7A for fat body samples, EF-1α and Actin for midgut, EF2 and RPL13A for lavra and RPL13A and RPL7A for all the samples. Our results not only confirmed the stability of reference genes in S. litura, but also provide a basis for the accuracy of target genes and the toxicological mechanism of azadirachtin in insect at the transcriptional level.

#### AUTHOR CONTRIBUTIONS

BS and GZ: Conceived and designed the experiments; BS and JZ: Performed the experiments; BS, GC, and XY: Analyzed the data;

#### REFERENCES


BS, JZ, RS, and GC: Contributed reagents, materials, and analysis tools; BS: Drafted the manuscript; VS, XY, and GZ: Revised the draft. All authors reviewed the manuscript.

#### ACKNOWLEDGMENTS

The work was financially supported through grants from the National Nature Science Foundation of China (Grant No. 31572335), Public welfare industry (Agriculture) scientific research special fund project, China (Grant No. 201303017) and Guangdong Nature Science Foundation (Grant No. 2014A030313461).


Spodoptera litura Fab. (Lepidoptera: Noctuidae). Pestic. Biochem. Phys. 83, 46–57. doi: 10.1016/j.pestbp.2005.03.009


by geometric averaging of multiple internal control genes. Genome Biol. 3:RESEARCH0034. doi: 10.1186/gb-2002-3-7-research0034


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Shu, Zhang, Cui, Sun, Sethuraman, Yi and Zhong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Identification, Expression, and Regulation of an Omega Class Glutathione S-transferase in Rhopalosiphum padi (L.) (Hemiptera: Aphididae) Under Insecticide Stress

Balachandar Balakrishnan, Sha Su, Kang Wang, Ruizheng Tian and Maohua Chen\*

State Key Laboratory of Crop Stress Biology for Arid Areas and Key Laboratory of Crop Pest Integrated Pest Management on the Loess Plateau of Ministry of Agriculture, Northwest A&F University, Yangling, China

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Wei Dou, Southwest University, China Rakesh Kumar Seth, University of Delhi, India

#### \*Correspondence:

Maohua Chen maohua.chen@nwsuaf.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 17 January 2018 Accepted: 05 April 2018 Published: 20 April 2018

#### Citation:

Balakrishnan B, Su S, Wang K, Tian R and Chen M (2018) Identification, Expression, and Regulation of an Omega Class Glutathione S-transferase in Rhopalosiphum padi (L.) (Hemiptera: Aphididae) Under Insecticide Stress. Front. Physiol. 9:427. doi: 10.3389/fphys.2018.00427 Glutathione S-transferases (GSTs) play an essential role in the detoxification of xenobiotic toxins in insects, including insecticides. However, few data are available for the bird cherry-oat aphid, Rhopalosiphum padi (L.). In this study, we cloned and sequenced the full-length cDNA of an omega GST gene (RpGSTO1) from R. padi, which contains 720 bp in length and encodes 239 amino acids. A phylogenetic analysis revealed that RpGSTO1 belongs to the omega class of insect GSTs. RpGSTO1 gene was highly expressed in transformed Escherichia coli and the protein was purified by affinity chromatography. The recombinant RpGSTO1 displayed reduced glutathione (GSH) dependent conjugating activity toward the substrate 1-chloro-2, 4-dinitrobenzene (CDNB) substrate. The recombinant RpGSTO1 protein exhibited optimal activity at pH 7.0 and 30◦C. In addition, a disk diffusion assay showed that E. coli overexpressing RpGSTO1 increased resistance to cumene hydroperoxide-induced oxidative stress. Real-time quantitative PCR analysis showed that the relative expression level of RpGSTO1 was different in response to different insecticides, suggesting that the enzyme could contribute to insecticide metabolism in R. padi. These findings indicate that RpGSTO1 may play a crucial role in counteracting oxidative stress and detoxifying the insecticides. The results of our study contribute to a better understanding the mechanisms of insecticide detoxification and resistance in R. padi.

Keywords: glutathione S-transferase, Rhopalosiphum padi, insecticide detoxification, omega class, gene expression

#### INTRODUCTION

Glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of multifunctional phase II enzymes that play a crucial role in the detoxification of many exogenous and endogenous xenobiotics compounds and have been widely found in almost all living organisms (prokaryotic and eukaryotic) (Booth et al., 1961; Tu and Akgül, 2005; Li et al., 2007). The enhanced metabolic capability of detoxification enzymes, such as carboxylesterase (CarE), cytochrome P450 monooxygenases (P450) and GSTs are important for insecticide resistance (Rufingier et al., 1999; Puinean et al., 2010; Cui et al., 2015). The major function of GSTs is conjugation of electrophilic

**38**

compounds with the thiol group of reduced glutathione (GSH), thus making them less toxic, more soluble and easier to excrete from the cell (Enayati et al., 2005; Ketterman et al., 2011). Cytosolic insect GSTs can be classified into six major classes: delta, epsilon, omega, sigma, theta, and zeta; there are also several unclassified genes (Ranson et al., 2001). Different classes of GSTs can be distinguished based on their primary amino acid sequences; identity is approximately 50% within a class and less than 30% among different classes (Sheehan et al., 2001; Mannervik et al., 2005). The omega class of GSTs (GSTO) is one of the largest GST subfamilies, with multiple functions identified in various species. GSTOs have unique structures and play essential physiological roles that differ from other GST classes (Meng et al., 2014). GSTOs are ubiquitous across taxa and play an essential physiological role including detoxifying insecticides (Chen and Zhang, 2015; Wu and Hoy, 2016). The recent studies indicate that GSTOs are also involved in oxidative response (Meng et al., 2014). However, the mechanisms involved the GSTOs effect still need further clarification. The first GSTO was identified through a bioinformatics analysis of expressed sequence tags in humans (Board et al., 2000). GSTOs have since been found in plants, yeast, bacteria and insects (Dixon et al., 2002; Garcerá et al., 2006; Walters et al., 2009; Xun et al., 2010). In GSTOs, a novel cysteine residue (Cys) is present in the active site, whereas GSTs from other classes have canonical serine and tyrosine residues (Caccuri et al., 2002). Insect GSTs display different substrate specificities, catalytic activities and have unique N-terminal and C-terminal extensions that are not observed in the other GST classes (Board, 2011). As GSTs can play roles in detoxification of various insecticides, a change in the GST activity is one mechanism of metabolic resistance to insecticides (Ranson and Hemingway, 2005; Li et al., 2007).

Aphids are common phloem-feeding pests found worldwide, and they damage plants by removing nutrients (Rabbinge et al., 1981). The bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), is a serious wheat pest in China (Wang et al., 2006). It can significantly reduce grain yields (Triticum aestivum L.) (Kieckhefer and Gellner, 1992; Blackman and Eastop, 2000) and is also an important vector for the barley yellow dwarf virus, which infects and damages wheat crops (Watson and Mulligan, 1960). Insecticides are stress factors that can affect many physical and biochemical process in insects. Insect populations have increased over time due to acquisition of insecticide resistance (Bass et al., 2014).

Here, we report the identification and classification of an omega class GST gene (RpGSTO1) from R. padi. The recombinant protein, RpGSTO1, was expressed in Escherichia coli cells. The biochemical properties of the purified recombinant GST protein were characterized. The transcriptional patterns of RpGSTO1 following exposure to various concentrations of β-cypermethrin, isoprocarb, malathion, and sulfoxaflor were analyzed. The potential roles of the RpGSTO1 fusion protein in antioxidant defense were also investigated. Our results provide valuable insight into the function of RpGSTO1 in the stress response to insecticides.

# MATERIALS AND METHODS

#### Insects

Rhopalosiphum padi was collected from a wheat field in Gansu Province of China in 2013, and used to establish a colony on seedlings of wheat (cultivar "Xiaoyan 22") in mesh cages (41 cm × 41 cm × 41 cm) in the laboratory. The colony was reared under regulated conditions (23 ± 1 ◦C temperature, 70% relative humidity and 16 h light/8 h dark photoperiod) without microbial infection and without insecticide exposure (Wang et al., 2016).

# RNA Extraction and cDNA Synthesis

Total RNA was extracted from 15 apterous adult aphids using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer's instructions and treated with RNase-Free DNaseI (Takara, Kyoto, Japan) to remove genomic DNA contamination. The purity of the extracted RNA was determined by agarose gel electrophoresis, and the concentration was checked using a biophotometer (Eppendorf Biophotometer Plus, Eppendorf, Germany). First-strand complementary DNA (cDNA) was synthesized from 2 µg total RNA using M-MLV reverse transcriptase cDNA Synthesis Kit (Promega, Madison, WI, United States) according to the manufacturer's instructions. The cDNA was stored at −80◦C prior to use as the template for PCR in subsequent gene cloning procedures.

#### Identification and Gene Cloning of Omega Glutathione S-Transferase Gene From R. padi

Using the published R. padi transcriptome data (Duan et al., 2017), sense and antisense primers were designed using Lasergene Primerselect (DNASTAR Inc, Madison, WI, United States) to amplify the full-length coding region for the omega GST gene, RpGSTO1. The amplification reaction mix contained 4 mM MgCl2, 100 µM dNTPs, 0.4 µM of forward and reverse primers, 2 units of Taq DNA polymerase (5 U/µL, Sangon Biotech Co., Ltd., Shanghai, China) and 1 µL of template DNA. Amplification occurred under the PCR conditions of 95◦C for 3 min followed by 35 cycles of 95◦C for 30 s, 55◦C for 30 s, 72◦C for 45 s and a final 5 min at 72◦C. The PCR product was verified on 1% (w/ν) agarose gel and visualized after staining with SYBR green using an imaging instrument (Sagecreation Science Co., Beijing, China). The target GST gene product was purified using gel extraction kit (Promega, Madison, WI, United States). The purified PCR product was then ligated to the pGEM-T Easy Vector (Promega, Madison, WI, United States) and transformed into Escherichia coli DH5α competent cells (Takara, Kyoto, Japan). The transformants were selected on LB agar plates containing 50 µg/mL kanamycin grown overnight at 37◦C. Five independent colonies were sequenced in both directions using an Applied Biosystems 3730 automated sequencer (Applied Biosystems, Foster City, CA, United States) at Sangon Biotech Co., Ltd. (Shanghai, China).

# Sequence Identity and Phylogenetic Analysis

The deduced amino acid sequence for RpGSTO1 was determined using the NCBI open reading frame (ORF) finder website<sup>1</sup> . The ExPASy tool<sup>2</sup> was used to predict the theoretical isoelectric point (pI) and molecular weight of the predicted protein. Sequence similarity was determined by aligning sequences with ClustalX (Chenna et al., 2003), and the file was converted for analysis using Molecular Evolutionary Genetic Analysis (MEGA) version 7.0 (Kumar et al., 2016). The phylogenetic tree was constructed using the neighbor-joining (NJ) method with pairwise deletion options, and the branch of the tree was evaluated using 1000 bootstrap replicates.

# Plasmid Construction and Recombinant Protein Expression

The RpGSTO1 was amplified using a pair of primers containing restriction enzymes BamHI and HindIII. The BamHI restriction site was incorporated to sense primer, and HindIII restriction site was incorporated to antisense primer for double restriction digestion reaction. PCR fragments were purified using a gel extraction kit (Promega, Madison, WI, United States), cloned into the pGEM-T Easy vector and then digested with BamHI and HindIII. The digested fragments were purified and ligated into the prokaryotic expression vector, pET-28a (Novagen, Merck, Germany), using a quick ligation kit (TaKaRa, Kyoto, Japan). The expression plasmid was transformed into E. coli BL-21 (DE-3) competent cells (Takara, Kyoto, Japan). The transformed cells were cultured in Luria-Bertani media containing 50 µL/mL kanamycin at 37◦C with 220 rpm shaking until the OD<sup>600</sup> reached 0.7. Then, isopropyl 1-thio-β-Dgalactopyranoside (IPTG) was added to a final concentration of 1 mM and the culture was shifted to 30◦C to induce the production of RpGSTO1. After incubation for 3 h, the cells were harvested by centrifugation at 10,000 rpm for 3 min. The cell pellet was washed with sterile water and then resuspended in 20 mM Tris-HCL buffer (pH 8.0) containing 0.5 M NaCl, 1 mg/mL of lysozyme, and 1 mM phenylmethanesulfonyl fluoride (PMSF). The expressed recombinant protein was analyzed by 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using a standard protein marker (PageRulerTM Prestained protein ladder). Protein bands were visualized by Coomassie Brilliant Blue R250 staining.

#### Recombinant Protein Purification and Western Blot Analysis

The recombinant RpGSTO1 cells were grown at 37◦C in 100 mL Luria-Bertani media containing 50 µg/mL kanamycin until the optical density (OD) reached 0.8. Then, 0.5 mM IPTG was added and cells were grown at 25◦C overnight with shaking at 180 rpm. The cells were harvested by centrifugation at 12,000 rpm for 3 min. The cell pellet was resuspended in lysis buffer (20 mM Tris-HCL, pH 7.4, 500 mM NaCl, 15% glycerol, and 1 mM PMSF). The cell lysate was subjected to centrifugation at 12,000 rpm for 10 min at 4◦C to remove the cellular debris, and the supernatant was passed through a 0.45-nM syringe filter. The filtered protein extract was loaded onto a cOmplete His-Tag purification resin affinity chromatographic column (Roche Diagnostics GmbH, Mannheim, Germany). Non-target protein in the supernatant was removed with wash buffer (50 mM NaH2PO4, 300 mM NaCl and 20 mM imidazole, pH 8.0). The protein was eluted with a linear imidazole gradient of 50–250 mM in buffer. The eluted samples were desalted using a dialysis membrane in 50 mM sodium phosphate buffer, pH 7.4 for 24 h at 4◦C. The protein purity was checked by 12% (w/v) SDS-PAGE and stained with Coomassie Brilliant Blue R250. The concentration of protein was measured using a BCA protein assay kit (Cwbiotech, Beijing, China), with bovine serum albumin as the standard.

After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane (PVDF) by immune blotting. After blotting, the membrane was blocked by incubation for 2 h at room temperature in a 5% bovine serum albumin (BSA) solution. Then, membrane was incubated overnight with primary 6-His monoclonal antibody (1:2000, v/v) at 4◦C, and then membrane was washed in TBST [10 mM Tris-HCL, pH 8.0, 100 mM NaCl and 0.1% (w/v) Tween 20]. The membrane was then incubated with 1:5000 (v/v) horseradish peroxidaseconjugated anti-mouse IgG. After repeated washing with TBST, the membrane immersed with ECL detection reagents (BioRad, Hercules, CA, United States).

#### Measurements of Enzyme Activity

RpGSTO1 activity was determined spectrophotometrically using 1–chloro-2, 4-dinitrobenzene (CDNB) and reduced glutathione (GSH) as standard substrates (Habig et al., 1974). Enzymatic activity is expressed as mol CDNB conjugated with GSH per min per mg of protein. The stock solution of CDNB was prepared in ethanol, and GSH was dissolved in 0.1 M sodium phosphate buffer. Each 300-µL reaction mixture contained 100 ng of RpGSTO1, 0.5 mM CDNB, 5 mM GSH in 0.1 M phosphate buffer. The optimum pH for RpGSTO1 activity was determined at 30◦C, with pH at 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. The thermostability of RpGSTO1 was determined by preincubation of the enzyme solution at 10, 20, 30, 40, 50, 60, or 70◦C for 30 min prior to performing a residual activity assay at pH 7.0. These optimal pH and temperature experiments were conducted with fixed concentrations of CDNB (0.5 mM) and GSH (5 mM). The reaction was monitored by measuring absorbance at 340 nm with 15 s intervals using a TECANTM Infinite <sup>R</sup> 200 PRO multimode micro-plate reader (ε340 = 9600 M−<sup>1</sup> cm−<sup>1</sup> ). The reduced GSH concentration was held at 5 mM, while CDNB concentration was varied from 0.02 to 0.14 mM. The kinetic parameters (K<sup>m</sup> and Vmax) were determined by linear regression of double reciprocal plot. All assays were performed in quadruplicate and repeated three times with non-enzymatic controls for reference blanks.

#### Disk Diffusion Assay

A disk diffusion assay was performed in according to Yan et al. (2013). The E. coli culture containing overexpressed

<sup>1</sup>http://www.ncbi.nlm.nih.gov/gorf.html

<sup>2</sup>http://web.expasy.org/compute\_pi/

RpGSTO1 was plated on Luria-Bertani agar plates and incubated at 37◦C for 1 h. Cells with the pET-28a (+) were used as the controls and treated with the same conditions. Filter disks (6-mm diameter) were soaked with different concentrations of cumene hydroperoxide (0, 30, 50, 100, and 200 mM). All the disks were placed on the surface of the agar plates and incubated at 37◦C for 24 h. The inhibition zones around the disks were measured. The assay was repeated three times, and statistical significance of the inhibition zone was calculated using the program JMP13 (SAS Institute-9.3, Cary, NC, United States).

#### Real-Time qPCR Analysis of RpGSTO1 Expression Under Different Insecticide Stress

The β-cypermethrin, isoprocarb, malathion, and sulfoxaflor (Yancheng Nongbo Bio-technology co., Ltd., Jiangsu, China) used in this study were of technical grade. Based on our previous bioassay results (Wang et al., 2017), two concentrations (LC<sup>25</sup> and LC50) of each insecticide were used. The LC<sup>25</sup> and LC<sup>50</sup> concentrations were 0.7671 mg/L and 1.3082 mg/L for β-cypermethrin, 0.0372 mg/L and 0.0618 mg/L for isoprocarb, 1.4230 mg/L and 2.7048 mg/L for malathion, and 0.0674 mg/L and 0.1217 mg/L for sulfoxaflor, respectively. A previously reported leaf-dipping method was adopted for insecticide stress treatment (Wang et al., 2016). Wheat leaves with 50–60 apterous adult aphids were dipped in the two concentrations (LC<sup>25</sup> and LC50) of each chemical for 10–15 s and then dried with the help of filter papers. Wheat leaves treated with solution in the absence of insecticide were used as the control. Three replicates were maintained at a constant temperature of 23 ± 1 ◦C and photoperiod of 16:8 (L:D) h both during and after treatment, and the live aphids were collected at 12, 24, or 36 h posttreatment.

Total RNA was isolated from the live aphids (5 mg) collected at each treatment, and expression of RpGSTO1 was analyzed. Total RNA extraction and cDNA synthesis were performed as described above. The real-time quantitative PCR (qPCR) reactions were conducted in a Rotor Gene Q Real Time Thermal Cycler (Qiagen, Hilden, Germany) using SYBR Green to detect the amplification signals. Primers for qPCR are listed in **Table 1**. The β-Actin and EF-1α (elongation factor 1α) genes were used as internal references to normalize target gene expression (Wang et al., 2016; Li et al., 2017). The reaction mixture consisted of 1 µL cDNA template, 0.8 µL of 10 µM forward/reverse primers, 10 µL 2X FastStart Essential DNA Green MasterTM (Roche, Shanghai, China) and 7.4 µL RNase-free water. Thermal conditions were as follows: initial denaturation at 95◦C for 10 min, followed by 40 cycles of denaturation at 95◦C for 15 s, annealing 58◦C for 30 s and elongation for 72◦C for 30 s. The real-time data were acquired by raising the temperature from 65◦C to 95◦C for 10 s at 0.5◦C increments. Reactions for all samples were performed independently repeated triplicates. The relative expression levels were calculated using the comparative CT method (2−11C<sup>T</sup> ) (Livak and Schmittgen, 2001).

# Statistical Analysis

All statistical analyses were performed using SAS JMP13 (SAS Institute-9.3, Cary, NC, United States). The results are presented as the mean ± standard error from triplicate experiments, and data were analyzed using Student's t-test for comparison of two means or one-way analysis of variance followed by Tukey's test. The level of significance was set at p < 0.05 for all treatments. All the graphs were created using Prism 6.0 for windows (GraphPad, La Jolla, CA, United States)<sup>3</sup> .

#### RESULTS

#### Identification and Characterization of RpGSTO1 Gene

The full-length cDNA sequence of RpGSTO1 gene was obtained from R. padi and deposited in GenBank (Accession Number: MG709032). The cDNA sequence of RpGSTO1 is 785 bp long, which contains a 31-bp 5<sup>0</sup> untranslated region (UTR), and a 34-bp 3<sup>0</sup> UTR. The full length open reading frame (ORF) is 720 bp in length, encoding a 239-amino acid protein with a predicted molecular mass of 27.469 kDa and a theoretical pI of 6.13 (**Figure 1**).

### Phylogenetic Analysis of RpGSTO1 and Other Insect GSTs

The amino acid sequence of RpGSTO1 has high identity with omega class GSTs from other insect species such as Acyrthosiphon pisum GSTO1 (GenBank: NP\_001155757, 85% identity), Bemisia tabaci GSTO1 (GenBank: AST11637, 54% identity), Sogatella furcifera GSTO1 (GenBank: AFJ75814, 51% identity) and Apis dorsata (GenBank: XP\_006623084, 45% identity) (**Figure 2**). A domain analysis revealed that the RpGSTO1 monomer includes 9 α-helics and 4 β-strands. The conserved residues of the insect cytosolic GSTs N-terminal and C-terminal domains were similar, and G-site implied common GSH-binding characteristics. RpGSTO1 shared the highest similarity with the pea aphid A. pisum GSTO1. A neighbor-joining phylogenetic tree

<sup>3</sup>www.graphpad.com

TABLE 1 | Oligonucleotide primer pairs used in this study.


conserved binding residues are highlighted in gray.

was constructed using the MEGA tool with sequences of other insect cytosolic GSTs. The phylogenetic relationship analysis revealed that RpGSTO1 clustered together with the omega class GSTs. The GSTs from other classes (delta, epsilon, theta, omega, zeta, and sigma class) were generally clustered together in the tree (**Figure 3**).

# Expression and Purification of RpGSTO1

Recombinant RpGSTO1 protein was successfully overexpressed in E. coli, as confirmed by SDS PAGE (**Figure 4**). The recombinant RpGSTO1 was in a soluble form and purified to homogeneity by His-Tag resin affinity chromatography and gel filtration. The purified protein (>95% purity) showed a single band on the gel with a molecular weight of approximately 27 kDa, similar to the calculated molecular weight of 33 kDa (the pET-28a His-tag is approximately 3 kDa). The expressed recombinant protein was detected by western blot using a 6× His mouse monoclonal antibody (**Figure 4**).

# GST Activity and Properties of RpGSTO1

The enzymatic properties of RpGSTO1 were investigated using purified recombinant RpGSTO1 with CDNB and reduced GSH

as substrates. The recombinant RpGSTO1 exhibited optimum catalytic activity toward CDNB with the pH at approximately 7.0 (**Figure 5A**). The thermostability of RpGSTO1 was analyzed by measuring residual activity after incubation for 30 min at pH 7.0 and varying temperatures. The purified GST enzyme had relatively higher activity during incubation at 30◦C (**Figure 5B**). Steady-state kinetic analysis was performed with 5 mM GSH and different CDNB concentrations at pH 7.0, and K<sup>m</sup> and Vmax were determined. Recombinant RpGSTO1 showed a K<sup>m</sup> of 0.120 mM and a Vmax of 2.906 µmol/mg/min (**Figure 5C**).

#### Disk Diffusion Assay Performed Under Cumene Hydroperoxide Stress

A disk diffusion assay was used to provide direct evidence that RpGSTO1 is capable of providing protective antioxidant activity. E. coli cells overexpressing RpGSTO1 were exposed to oxidative stress by treatment with cumene hydroperoxide (Burmeister et al., 2008; Liu et al., 2016). Following overnight exposure, the zones of inhibition around the cumene hydroperoxide soaked filters of the E. coli expressing RpGSTO1 were found to be much smaller than the control, which were transfected with the vector. The halo diameter sizes were reduced by 30% for bacteria expressing RpGSTO1 (**Figure 6**).

# Expression Profiles of RpGSTO1 After Exposure to Different Insecticides

The relative expression level of RpGSTO1 was investigated by RT-qPCR after exposure to LC<sup>25</sup> and LC<sup>50</sup> concentrations of β-cypermethrin, isoprocarb, sulfoxaflor and malathion (**Figure 7**). The R. padi were treated with LC<sup>25</sup> and LC<sup>50</sup> concentrations of different insecticides and the time-dependent relative expression of RpGSTO1 normalized to their reference genes were quantified. Expression levels of RpGSTO1 were significantly up-regulated (2.15-fold and 1.45-fold) 12 h postexposure to the LC<sup>50</sup> and LC<sup>25</sup> concentrations of β-cypermethrin, respectively, compared with the untreated insect regimen. Expression levels of RpGSTO1 were significantly lower than that of the control at 24 h and 36 h post-exposure to the LC<sup>25</sup> and LC<sup>50</sup> concentrations of β-cypermethrin, and the expression levels of the gene within these insecticide-treated samples were different but not statistically significant. The mRNA levels of RpGSTO1 were significantly higher at 12 h post-exposure to the LC<sup>25</sup> and LC<sup>50</sup> concentrations of isoprocarb than at 24 or 36 h post-exposure. The transcription levels of the RpGSTO1 were significantly lower at 12 h post-exposure to LC<sup>25</sup> isoprocarb than that of 12 h post-exposure to LC<sup>50</sup> isoprocarb. RpGSTO1 expression was increased 4.46-fold at 24 h post-exposure to LC<sup>50</sup> malathion and 3.88-fold to LC<sup>25</sup> malathion, which were both significantly higher than that of 12 h and 36 h post-exposure to malathion. The mRNA level was significantly increased at 12 h post-exposure (2.49-fold) and significantly decreased at 36 h post-exposure (0.73-fold) to LC<sup>50</sup> malathion, while LC<sup>25</sup> doses of malathion significantly increased the expression of the gene at 36 h post-exposure. RpGSTO1 mRNA expression level was highest 12 h post-exposure to LC<sup>50</sup> and LC<sup>25</sup> concentrations of sulfoxaflor. Compared to untreated insect regimen, the respective expression level of RpGSTO1 was 2.53-fold, 2.07-fold and 1.58 fold less at 12, 24, and 36 h post-exposure to LC<sup>50</sup> concentrations of sulfoxaflor, and 1.98-fold, 1.51-fold, and 0.58-fold less at 12, 24, and 36 h post-exposure to LC<sup>25</sup> concentration, respectively.

neighbor-joining and bootstrap support values based on 1000 replicates by MEGA 7.0. RpGSTO1 is marked with a solid black circle.

# DISCUSSION

Glutathione S-transferases are multifunctional enzymes that play a central role in the detoxification of both endogenous and xenobiotic compounds. The different classes of GST enzymes are found in a variety of insect species (Booth et al., 1961; Tu and Akgül, 2005; Li et al., 2007). The omega class of GSTs (GSTO) is a class of cytosolic GSTs with structure and characteristics that differ from other GST class (Whitbread et al., 2005; Burmeister et al., 2008). In this study, a novel GST gene of the omega class (RpGSTO1) was identified from the bird cherry-oat aphid R. padi, a serious winter wheat pest in China. A phylogenetic analysis comparing RpGSTO1 to GSTs from different classes and insects revealed that belongs into the omega class. RpGSTO1

has high identity with the GSTO1 from pea aphid A. pisum (**Figure 3**). The deduced protein sequence of RpGSTO1 includes conserved functional domains, including the G-site and H-site, which were highly conserved and located at the C-terminal region and N-terminal region.

The most important function of GSTs is to catalyze the conjugation of GSH to various endogenous and exogenous compounds (Hayes et al., 2005). The synthetic substrate CDNB is commonly used in GST activity assays (Ketterman et al., 2011). We observed the ability of recombinant RpGSTO1 to catalyze CDNB substrate in the presence of reduced GSH. GSTs from different insects showed high activity at different temperatures and pH values (**Figure 5**). We determined that the recombinant RpGSTO1 enzyme had optimal activity at a pH of 7.0 and a temperature of 30◦C. In previous studies, the enzyme activity was stable, and high enzyme activity was observed at pH 5.0 to 8.0 from different insect GSTs (Samra et al., 2012; Yamamoto et al., 2013; Wan et al., 2016). GSTs from insects had an optimal activity at a temperature range between 25◦C to 40◦C (Samra et al., 2012; Zhang et al., 2013; Tan et al., 2014; Wan et al., 2016; Liu et al., 2017).

We investigated the involvement of RpGSTO1 in the oxidative stress response. To perform disk diffusion assay, we cultured E. coli with recombinant RpGSTO1 and the vector for a control to achieve the same cell density. Cumene hydroperoxide is a known oxidative stress inducer (Burmeister et al., 2008; Yan et al., 2013; Meng et al., 2014; Chen et al., 2015). Inhibition of the growth of the bacteria was observed following overnight exposure to cumene hydroperoxide. GSTs have a key functional role in the detoxification process involved in intracellular

transport, synthesis of bio-hormones, and protection against oxidative stress of both endogenous and xenobiotic compounds (Armstrong, 1997; Enayati et al., 2005). Previous studies indicated that GSTO1 was involved in antioxidant defense (Burmeister et al., 2008; Wan et al., 2009; Yamamoto et al., 2011; Zhang et al., 2016). In this study, cumene hydroperoxide induced oxidative stress in cells expressing recombinant RpGSTO1 but showed the zone was decreased compared to cells expressing the vector (**Figure 6**). Our results provide evidence that RpGSTO1 is an antioxidant enzyme that protects cells from oxidative stress.

FIGURE 6 | (A) The resistance of bacteria cells overexpressing RpGSTO1 to cumene hydroperoxide. The labels 0, 1, 2, 3, and 4 on filter disks represent different concentrations of cumene hydroperoxide (0, 30, 50, 100, and 200, respectively). (B) The halo diameters of the killing zones were compared in the histograms. The data are the mean ± SE of three replicates.

different among treatments by Tukey's test. Asterisks above bars indicate significant differences between the treatment and the corresponding control (one-way ANOVA with Tukey's HSD test, p < 0.05).

Insect GST can detoxify many synthetic insecticides and plant allelochemicals (Li et al., 2007). Synthetic insecticides can cause physiological changes in insects. Currently, R. padi has developed resistance against various insecticides (Zuo et al., 2016). To explore and characterize the putative roles that RpGSTO1 might play, we analyzed the expression patterns of the gene under different insecticide treatments (**Figure 7**). We treated insects with the pyrethroids β-cypermethrin, carbamate isoprocarb, organophosphorus malathion, and neonicotinoids sulfofoxaflor and then measured the mRNA expression level of RpGSTO1. The relative expression of RpGSTO1 was affected by these insecticides, and the pattern varied among the different insecticide treatments. An omega class GST gene in B. mori has been reported to be induced by treatment with various environmental stresses, such as diazinon, permethrin, imidacloprid, ultra violet-B (UV-B), and bacteria (Yamamoto et al., 2011). The relative expression level of RpGSTO1 at 12 h post-exposure to LC<sup>50</sup> concentrations of β-cypermethrin, sulfoxaflor and malathion were significantly higher than the respective expression level at 12 h post-exposure to LC<sup>25</sup> concentrations of each chemical, however, RpGSTO1 expression at 12 h post-exposure to LC<sup>50</sup> concentrations to isoprocarb was significantly lower that at 12 h post-exposure to LC<sup>25</sup> concentrations to isoprocarb, indicating the same GSTO varied at the responses to different types of

insecticides which could possibly be caused by different binding pattern of the enzyme to the chemicals. This result suggests that RpGSTO1 may play a significant role in detoxifying various groups of insecticides in R. padi. In previous reports, up-regulation of GST genes following exposure to pyrethroid, organophosphate, carbamate and neonicotinoid were found in insecticide-resistant strains (Hemingway et al., 1991; Yang et al., 2013; Wei et al., 2015). Down-regulation of GSTOs were reported in Cnaphalocrocis medinalis exposed to chlorpyrifos (Liu et al., 2015). GSTO gene expression was induced by different stress conditions, such as different temperature, UV, H2O2, cyhalothrin, phoxim, pyridaben, and paraquat in Apis cerana (Zhang et al., 2013). In this study, the mRNA level of RpGSTO1 responded to different insecticide challenges, and the responses maybe associated with the oxidative stress caused by insecticide treatment, which were positively correlated with the previous studies, including that omega GSTs can be induced by insecticides and could play a part in detoxification of insecticides in R. padi.

#### CONCLUSION

Our study demonstrated the unique functional characterization, expression pattern, and physiological roles of a novel GSTO

#### REFERENCES


gene from R. padi. To our knowledge, this is first time that an omega class GST has been cloned and characterized from the bird cherry-oat aphid. This study also revealed that recombinant RpGSTO1 possesses antioxidant activity in response to oxidative stress. The expression level of R. padi RpGSTO1 can be induced under the stresses caused by different insecticides. Our findings provide valuable insight into the functions of the GSTO in this serious pest.

#### AUTHOR CONTRIBUTIONS

BB and MC: conceived and designed the experiments. BB: performed the experiments. BB, KW, and MC: analyzed the data. SS and RT: contributed reagents/materials/analysis tools. BB and MC: wrote the paper.

# FUNDING

This work was funded by National Natural Science Foundation of China (Grant Nos. 31772160 and 31471766).



environmental stress. Comp. Biochem. Physiol. C 150, 558–568. doi: 10.1016/ j.cbpc.2009.08.003


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Balakrishnan, Su, Wang, Tian and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Heritability and Evolutionary Potential Drive Cold Hardiness in the Overwintering Ophraella communa Beetles

Chenchen Zhao<sup>1</sup>† , Fangzhou Ma2,3† , Hongsong Chen<sup>4</sup> , Fanghao Wan<sup>1</sup> , Jianying Guo<sup>1</sup> and Zhongshi Zhou<sup>1</sup> \*

<sup>1</sup> State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, <sup>2</sup> Key Laboratory of Biosafety, Ministry of Environmental Protection, Nanjing, China, <sup>3</sup> Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing, China, <sup>4</sup> Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Institute of Plant Protection, Guangxi Academy of Agricultural Sciences, Nanning, China

#### Edited by:

Su Wang, Beijing Academy of Agriculture and Forestry Sciences, China

#### Reviewed by:

Dandan Wei, Southwest University, China Shannon Bryn Olsson, National Centre for Biological Sciences, India

#### \*Correspondence:

Zhongshi Zhou zhongshizhou@yahoo.com

†These authors have contributed equally to this work as joint first authors.

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 07 March 2018 Accepted: 14 May 2018 Published: 05 June 2018

#### Citation:

Zhao C, Ma F, Chen H, Wan F, Guo J and Zhou Z (2018) Heritability and Evolutionary Potential Drive Cold Hardiness in the Overwintering Ophraella communa Beetles. Front. Physiol. 9:666. doi: 10.3389/fphys.2018.00666 Chill tolerance plays a crucial role that allows insect species to adapt to cold environments. Two Chinese geographical populations (Laibin and Yangzhou populations) were selected to understand the chill resistance and evolutionary potential in the Ophraella communa, a biological control agent of the invasive common ragweed, Ambrosia artemisiifolia. Super-cooling point assays, knockdown tests under static lowtemperature conditions and determination of glycerol content were studied. ANOVAs indicated significant differences regarding chill coma recovery time, super-cooling point, and glycerol content across populations and sexes. The narrow-sense heritability (h 2 ) estimates of cold resistance based on a parental half-sibling breeding design ranged from 0.39 to 0.53, and the h 2 value was significantly higher in the Yangzhou population than in the Laibin population. Additive genetic variances were significantly different from zero for cold tolerance. The Yangzhou population of O. communa has a strong capability to quickly gain resistance to cold. We conclude that the O. communa beetle has a plasticity that can provide cold resistance in the changing climate conditions.

Keywords: Ophraella communa, heritability, cold hardiness, super cooling point, glycerol content, chill coma recovery time

# INTRODUCTION

Poikilothermic character is a crucial and widely recognized factor affecting insect development. Fluctuations in environmental temperature can affect the survival, development, and establishment of an insect in the field. Many insect species are currently experiencing habitat destruction due to climate change (Hoffmann et al., 2003), and some are facing previously unencountered low temperatures during the winter months. Cold climates often impede the establishment and persistence of insect populations in temperate regions. Across the long process of evolution, many types of insects have undergone several physiological changes that improved their coldhardiness (Danks, 1996; Bale, 2002; Chen and Kang, 2002; Wang and Kang, 2005). Coldhardiness may be a coping strategy that would allow insects to successfully migrate and establish themselves under new environmental conditions (Hoffmann, 2003; Chen et al., 2011;

**49**

Kellermann et al., 2012a; Findsen et al., 2013). Due to a plastic cold-hardiness, insects can often survive at subzero temperatures during cold winter months (Salt, 1961; Kostal et al., 1998; Overgaard et al., 2007). Many previous studies have suggested that cold tolerance differs significantly across various geographical populations of an insect species mainly due to acclimation to different climatic conditions (Chen and Kang, 2002; Worland et al., 2004; Regniere and Bentz, 2007; Zhou et al., 2011b).

Adaptive phenotypic plasticity and genetic adaptation are the main ways in which natural populations respond to strong environmental stresses. Generally, natural selection can lead to micro-evolutionary changes in some particular traits of wild populations of insects (Merilä et al., 2001; Roff, 2003; Kellermann et al., 2009; Ma et al., 2014). Species distribution is often closely related to the appropriate genetic variation in the key traits which is necessary for adaptation to the changing environmental conditions (Merilä and Crnokrak, 2001). In general, evolutionary change requires that the traits subject to selection will be heritable (Roff, 2003), and thus the key traits for adapting a new environment are often coupled with genetic variation. A rapid evolutionary change in natural populations has provided the evidence for the abundance of genetic variation (Reznick et al., 1997; Hendry and Kinnison, 1999; Kellermann et al., 2009; Ma et al., 2014). Pliability of cold tolerances in insects is also closely associated with the genetic variation to overcome the cold spell in winter months (Gibert et al., 2001; Kimura, 2004; Kellermann et al., 2009; Hoffmann, 2010; Arboleda-Bustos and Segarra, 2011). Nevertheless, the evolution of plasticity, such as cold tolerances for example, under extreme conditions has become increasingly important for natural predators of specific pests increasingly exposed to colder envirenment, and there is an urgent need to excavate empirical information for evolved adaptive phenotypic plasticity.

Ophraella communa LeSage (Coleoptera: Chrysomelidae), is native to North America (Futuyma, 1990; Palmer and Goeden, 1991), and it has been considered as an important biological control agent of invasive common ragweed, Ambrosia artemisiifolia L. (Asterales: Asteraceae) in China (Chen et al., 2009; Guo et al., 2011; Zhou et al., 2011c, 2014). A. artemisiifolia has caused serious economic, ecological, and health problems (Zhou et al., 2011a), and it has extensively invaded and established itself in over 21 provinces in China (Zhou et al., 2011a). A. artemisiifolia ages and dies after the end of October, and its seeds in soil germinate and grow new seedlings from late April to early June next year. O. communa adults often hibernate in soil from the end of October to early May or June of the following year. In general, air temperatures differ significantly with latitudes. The hibernating O. communa adults from different latitudes often need to overcome different low temperatures in the cold winter months. Our previous studies have demonstrated that hibernating O. communa use freeze avoidance to survive winter cold and geographically separated populations of O. communa are diverging with respect to baseline cold hardiness because of the differing severity of low temperatures experienced during the coldest winter season in each locality (Zhou et al., 2011a). The Yangzhou population may be one of the most northern populations of this species which ranges from Laibin, Guangxi Province, north to the border of Yangzhou City, Jiangsu Province. Moreover, significant differences were observed in cold hardiness across different seasonal populations of O. communa have been reported (Zhou et al., 2011b). This indicates the cold-hardiness plasticity of O. communa plays an important role in adapting to seasonal and geographic changes in air temperatures.

As A. artemisiifolia gradually expands northward, O. communa encounters a colder environment, and new selection pressures may result in genetic variation within the species and may even result in the rapid adaptive microevolution of chill tolerance traits. Short-term evolutionary potential depends on the intra-specific additive genetic variance. The additive variance is often measured as heritability, the fraction of the total phenotypic variance that is additive. In this way, heritability is a common measure of evolutionary potential. In general, the proportion of intra-population phenotypic variance (VP) due to additive genetic variation, termed narrow-sense heritability (h2), may be the most fundamental aspect of a trait's genetic architecture, and has evolutionary significance (Lynch and Walsh, 1998; Visscher et al., 2008; Hoffmann and Sgro, 2011). In this study, coefficient of additive genetic variance (CVA) served as a measure of evolvability, because its numerical value has a more direct interpretation as the expected relative change in a trait under a unit strength of selection (Houle, 1992; Sgro and Hoffmann, 1998; Hansen et al., 2003). Few empirical studies have clearly demonstrated how the transgenerational effects contribute to insects' adaptability to cold conditions. Previous studies in Drosophila melanogaster and Bemisia tabaci have produced evidence of adaptive cross-generation plasticity, which has suggested that variance in evaluability is greater when insects are exposed to a changing temperature stress (Cui et al., 2008; Mitchell and Hoffmann, 2010; Ma et al., 2014). Species with adequate genetic variability are often able to make rapid evolutionary adjustments in nature. In this way, temperature resistance plasticity of insects may be heritable (Ma et al., 2014). Therefore, understanding the evolutionary potential in cold resistance plasticity can help predict the future responses of O. communa to climate change.

In the present study, we hypothesize that the cold-hardiness plasticity of overwintering O. communa adults should be heritable in geographically separated cold climates in the winter months. Two geographical populations (Laibin and Yangzhou) were selected to estimate specific parameters crucial to cold tolerance, including super cooling point (Lee, 1989; Chen and Kang, 2002; Andreadis and Athanassiou, 2017), glycerol content (Krunic and Salt, 1971; Minder et al., 1984), and chill coma recovery time (Findsen et al., 2013) of the adults. The purpose of this study was to compare the evolutionary potential of the baseline cold hardiness of hibernating O. communa populations from two different geographical regions, and so provide insight into the potential of this beetle to range further into China. To this end, we used laboratory measurements to compare the heritability of cold hardiness in overwintering O. communa adults from the two geographical populations. Based on estimation of the heritability of cold hardiness, we will explain how O. communa establishes its populations successfully to survive cool climates during winter months in different latitude localities of China.

#### MATERIALS AND METHODS

#### Host Plants and Insects

fphys-09-00666 June 2, 2018 Time: 20:58 # 3

The beetle Ophraella communa LeSage can survive throughout the year on the common ragweed (Ambrosia artemisiifolia L.) from May to November in the open fields and hibernate in the soil during the winter months.

The overwintering O. communa adults were collected from naturally grown ragweed, in the fields of Laibin (Guangxi Zhuang Autonomous Region, South China) and Yangzhou (Jiangsu Province, East China) when they emerged from the soils in the spring 2014. The adults were maintained (two geographical populations were reared separately) in the laboratory at a temperature of 27 ± 1 ◦C, a relative humidity of 70 ± 5% RH, and a light dark photoperiod of 14:10 h. The beetles were bred gregariously in insect rearing cages (40 cm × 60 cm). The cages contained ragweed plants for eating, climbing, molting, and hiding. The moisture levels (15.5–18.5%) were maintained along with sterilized soil to facilitate ragweed growth.

Laibin belongs to subtropical monsoon climate and its location is 23◦ 430 7.4700N, 109◦ 240 54.1000E, where the average lowest temperatures in November, December, January, and February were 6.7◦C, 3.2◦C, 2.6◦C, and 4.7◦C, respectively, and the average low temperatures in November, December, January, and February were 14.1◦C, 10.3◦C, 8.2◦C, and 12.1◦C, respectively. Yangzhou also belongs to subtropical monsoon climate and its location is 32◦ 160 12.5300N, 119◦ 100 31.9900E, where the average lowest temperatures in November, December, January, and February were −0.4◦C, −5.4◦C, −6.7◦C, and −4.6◦C, respectively, and the average low temperatures in November, December, January, and February were 7.7◦C, 1.7◦C, −0.5◦C, and 2.1◦C, respectively. Although Laibin and Yangzhou both belong to subtropical monsoon climate, Laibin have higher temperature all year-round, especially in winter (**Figure 1**). The data were provided by the China meteorological data service center (CMDC)<sup>1</sup> .

#### Experimental Design and Data Collection

After a generation, male and female beetles' generation 1 (G1) were transferred to separate plastic boxes (24 cm × 16 cm × 9 cm) covered with organdy mesh fabric. These were placed in a temperature-regulated laboratory within 24 h, and then maintained for 2 days on common ragweed plants in insect rearing cages. The two geographical populations were reared for one generation to ensure a large, effective population size.

#### Paternal Half-Sibling Breeding Design

When the adult beetles of generation 2 (G2) were 3 days old (sexual maturity), we randomly assigned one male (sire) to mate with five virgin females (dams) in turn, in a transparent plastic

box containing a glass vial (3 cm × 8 cm) with a ragweed branch from each geographical population (N = 30 for one population). The ragweed branch was for mating, oviposition, climbing, molting, and hiding. After mating was complete, we gently removed each female to a new "house," numbered them by mating pair, then placed another female in the old "house." After all pairs had finished mating, we checked females' oviposition daily. When the female laid her eggs, was removed from the vial by aspiration immediately, left the eggs behind. Then we observed the eggs until they hatched.

<sup>1</sup>http://data.cma.cn/

When their offspring, generation 3 (G3) emerged, 10 females of each combination were collected, reared on ragweed plants, mated with males, and scored for cold tolerance traits (two females for cold stress, two females for testing of super-cooling point and glycerol content separately) within 3 h of eclosion. The breeding experiment was performed in the laboratory at 27◦C, at humidity of 65–75% RH under a photoperiod of 14L: 10D cycle with light coming on at 08:00.

# Chill Coma Recovery Time-TRC

To compare the cold resistance between the populations and sexes, 400 individuals were randomly selected from each mass bred population and scored for knockdown resistance (100 females and 100 males per cold stress experiment) on the third day after eclosion.

Five adults were randomly selected from every replication, the centrifuge tubes were paced (one adult/tube) in temperature controller (−10 ± 0.1◦C). The centrifuge tubes were removed, immediately rubbed dry, and placed on a piece of paper, where they were allowed to recover at room temperature (25◦C) without any mechanical stimuli. Then we started a timer observed their activity, noted the recovery time, measured as period from placement on the paper to the beetle reaching an upright position.

#### Super-Cooling Point

We randomly selected 150 males and 150 females from both geographical populations, a thermocouple was used to test the super-cooling point, we cut one 2 mm aperture of the pipet tips, then placed the probe of thermo detector in said pipet tips. Connect the probe with the insect body, then put it in a low temperature refrigerator (−20◦C and −30◦C). Insects obtain marked cooling at an average rate of about 0.5◦C/min, when the body fluids starts turning to ice, latent heat is released within the insect, producing a large bending curve in the recorder, distinguished the super-cooling point (when the curve rose to a certain value, and began to decline; we recorded the freezing point) numbered and recorded the data.

#### Determination of Glycerol Content

Glycerol contents were estimated from 100 randomly selected males and 100 randomly selected females from both geographical populations (400 specimens in total). Experimental procedures were conducted according to the kit instructions (provided by Nanjing Jiancheng Bioengineering Institute) and data were recorded.

#### Statistical Analysis

Data were analyzed using mixed modeling within a restricted maximum likelihood framework implemented in Proc Mixed in SAS (version 9.2. SAS Institute Inc., Cary, NC, United States) with following the model:

$$\mathbf{y}^{\;} = \alpha + X\mathbf{B} + Z\_{\mathbf{s}}\mathbf{d}\_{\mathbf{s}} + Z\_{\mathbf{d}}\mathbf{d}\_{\mathbf{d}} + \mathbf{e} \tag{1}$$

Here the constant (α) was ensemble average of each protocol. A previous study suggested that observer error may affect the estimation of variance components for thermal tolerance traits (Castaneda et al., 2012). All three protocols, (knockdown test, super-cooling point assays, and determination of glycerol content), were performed by a single person. We here considered protocol run (B) as a fixed effect, and three random effects were fit: sire (ds), dam nested within sire (dd), and the residual (e.).

The total phenotypic variance (σ 2 P ) for the breeding design for estimating genetic parameters was represented as follows:

$$
\sigma\_\mathrm{p}^2 = \sigma\_\mathrm{S}^2 + \sigma\_\mathrm{D}^2 + \sigma\_\mathrm{W}^2(2)
$$

Here σ 2 S , σ 2 D , and σ 2 <sup>W</sup>, are the sire, dam, and intra-group level variance components, respectively. Because we used a half-sib breeding design, the sire variance, σ 2 S , is one-fourth of the additive genetic variance (VA) (McGuigan and Blows, 2010). Thus, to estimate VA, we multiplied the sire variance by four.

The additive genetic variance for each trait was first estimated using a univariate model. Log likelihood ratio tests were performed, where the final model for each trait was compared to a model in which <sup>σ</sup> 2 S was set to zero to determine whether the levels of additive genetic variance for each trait were significantly different from zero (de Assis et al., 2010; McGuigan and Blows, 2010; Simonsen and Stinchcombe, 2010). The phenotypic variance (VP) in knockdown resistance was computed using all known relationships among individuals. We then estimated the narrow-sense heritability for both traits. Narrow-sense heritability for each trait was estimated as the additive genetic variance (VA) divided by the total phenotypic variance (VP) (Frankham, 1996). We conducted Student's t-tests to determine whether the variance components and heritability estimates differed significantly. Estimates of evolvability, IA, and the additive genetic coefficient of variance,

$$I\_{\mathcal{A}} = V\_{\mathcal{A}} / \bar{X}^2$$

where − X is the trait mean (Hansen et al., 2011), were also computed for both traits.

For the population and sexual comparisons (G3), two-way ANOVA was performed to evaluate the differences in the indexes, followed by the Tukey multiple comparison test. The population (Laibin and Yangzhou) and sex (female and male) were used as the two factors. P < 0.05 was considered statistically significant.

#### RESULTS

#### Differences in Cold Resistance Between the Populations and Sexes

**Figure 2** showed the mean value of Trc, SCP, and GC in two populations of O. communa beetles (G3). An ANOVA indicated significant differences between the two populations (F3,<sup>178</sup> = 74.99, P < 0.0001) and between males and females (F3,<sup>178</sup> = 19.55, P < 0.0001) in Trc (**Figure 2A**). Similar results were observed for SCP of population (F3,<sup>199</sup> = 20.77, P < 0.0001)


TABLE 1 | Narrow-sense heritability (h 2 ), variance, and coefficient of variation components for chill coma recovery time (Trc), super-cooling point (SCP), and glycerol content (GC) (Laibin and Yangzhou populations) of Ophraella communa beetles.

Additive genetic variance (VA), environmental variance (VE), phenotypic variance (VP), narrow-sense heritability (h<sup>2</sup> ), evolvability (I<sup>A</sup> × 100), coefficient of additive genetic variance (CVA), and coefficient of environmental variance (CVE) for chill coma recovery time, super-cooling point and glycerol content. N = sample size. <sup>1</sup>P < 0.05 for log likelihood ratio test of significant differences from zero; <sup>∗</sup>P < 0.05.

and sex (F3,<sup>199</sup> = 23.13, P < 0.0001), and GC of population (F3,<sup>397</sup> = 10.41, P = 0.0014) and sex (F3,<sup>397</sup> = 9.01, P = 0.0029). Generally females showed markedly more cold resistance than males.

#### Heritability and Genetic Variance Components

As shown in **Table 1**, the estimates of genetic variance differed statistically significantly between the two populations, Narrowsense heritability estimates were significantly different for the chill coma recovery times of both the Laibin and Yangzhou populations, at values of 0.39 ± 0.01<sup>∗</sup> and 0.53 ± 0.01<sup>∗</sup> , respectively. Significant levels of additive genetic variance were detected in chill coma recovery time (2.15 ± 0.01) of the Laibin population and in chill coma recovery time (5.44 ± 0.02) and super-cooling point (1.67 ± 0.22) of the Yangzhou population. For chill coma recovery time, the narrowsense heritability estimates were significant for both the Laibin (0.39 ± 0.01<sup>∗</sup> ) and Yangzhou populations (0.53 ± 0.01<sup>∗</sup> ). The coefficient of environmental variation was slightly larger (CV<sup>E</sup> of glycerol content, 30.34 > 27.37) for the Laibin population and that of and additive genetic variation was slightly smaller (CV<sup>A</sup> of chill coma recovery time, 0.539 < 1.111; CV<sup>A</sup> of super-cooling point, 0.181 < 0.393; CV<sup>A</sup> of glycerol content, 0.231 < 0.250).

#### DISCUSSION

Different populations react differently to cold stress. What one population finds very stressful another may not (Overgaard et al., 2011; Hoffmann, 2013). Temperature has an important influence on insect population distribution, life history, behavior, and species abundance (Dahlgaard et al., 2001; Kimura, 2004; Hoffmann, 2010). For many insects, chill tolerance is crucial to the ability to persist in cold environments (Findsen et al., 2013). Cold resistance is associated with the distributions of many insect species, particularly in temperate regions. Insects have low levels of resistance. A. artemisiifolia has become distributed across over 21 provinces from Southern to Northern China, covering a range of different climatic temperatures (Guo et al., 2011), its seeds grow new seedlings from late April to early June next year (Teshler et al., 2001). O. communa can still survive the cold winter months in the field (Zhou et al., 2011b). In the research studies conducted, we found that the hibernation survival range of beetle population in Langfang City, Hebei Province (39◦ 300 3800N, 116◦ 360 2 <sup>00</sup>E) (Zhou, unpublished data), is still smaller than the range of ragweed. Previous studies have suggested there may be significantly differences in cold hardiness among different seasonal and geographic populations of O. communa (Zhou et al., 2011a,b), and it is important to determine whether cold tolerance can be inherited by different geographic populations.

In our study, we have further documented genetic differences in cold tolerance between populations of O. communa. The Yangzhou population was found to be more cold-tolerant than in the Laibin population. This indicates a significant difference in the adaptive strategies to cold climates between the Laibin and Yangzhou populations of O. communa. As shown, the Yangzhou population can survive in a colder climatic environment than the Laibin population. In general, adapting and shifting their geographical distributions rendered some species better adapted to their environment (Kirkpatrick and Peischl, 2012). Previous studies have suggested that conspecific populations of the fruit fly Drosophila melanogaster from different environments may vary substantially in stress resistance because of acclimation to local conditions (Hoffmann et al., 2005). This is particularly evident when exploring the relationship between cold tolerance and minimal environmental temperature, such that strong correlations exist between cold tolerance and environmental distribution (Chen et al., 2011; Kellermann et al., 2012b).

Given adequate genetic variability, insect species are often able to make rapid evolutionary adjustments. Under pressure of the global climate warming, the majority of studies on insect adaptation have focused on the heat resistance of poikilotherms.

Estimates of heritability in D. melanogaster based on knockdown time experiments at 39◦C suggest values ranging from 0.03 to 0.28 (Huey et al., 1992; McColl et al., 1996), whereas experiments at 38◦C suggest values ranging from 0.14 to 0.22 (Mitchell and Hoffmann, 2010). These estimates might not be directly comparable to those obtained here, which ranged from 0.47 to 0.51 at 45◦C, due to the difference in the temperature used to assess the trait and to the different species examined. Nevertheless, the values obtained here showed that a substantial proportion of the total phenotypic variation in heat resistance was caused by additive genetic variation. Few studies have examined the genetic variance of cold resistance (Huey et al., 1992; Charo-Karisa et al., 2005; Kellermann et al., 2009). Estimates of heritability on recovery from a chill coma at 0◦C suggest values ranging from 0.01 to 0.38 (Charo-Karisa et al., 2005). Our estimates of heritability for O. communa based on chill coma recovery time at −10◦C (0.39–0.53) also indicated that a substantial proportion of the total phenotypic variation was caused by additive genetic variation.

Insects typically need to overcome cold conditions in order to become established in a given habitat (Bale, 1996). Cold acclimation, heritability and evolutionary potential could all drive cold hardiness (Sinclair et al., 2003). Cold acclimation was found to have enormous benefits at low temperatures, such that only cold-acclimated flies were able to find resource when they were released into cold environments (Kristensen et al., 2008). Because the insects that prey on pest species tend to follow insect pests into new areas, cold resistance is an important part of their arsenal. A. artemisiifolia has spread from south to north in China, so O. communa needs to adapt more colder winter climates if it is to suppress the population of A. artemisiifolia

#### REFERENCES


in colder areas in the northern subtropics or even in temperate regions. Based on the results of our study, we found O. communa populations to have considerable evolutionary potential with respect to cold tolerance in colder areas, and its heritability and evolutionary potential drive cold hardiness. This indicates that the beetle has a good plasticity of cold tolerance in nature, so it may be able to overcome the cold climate in Northern China whether the beetle experiences further artificial or natural cold acclimation.

#### AUTHOR CONTRIBUTIONS

ZZ conceived and designed the work, and also edited the manuscript. CZ and FM performed the experiments and wrote the manuscript. HC and JG helped with the theoretical analysis. FW helped to revise the manuscript.

#### FUNDING

This work was supported by the National Natural Science Foundation of China for Excellent Young Scholars (No. 31322046) and National Natural Science Foundation of China (No. 31672089).

#### ACKNOWLEDGMENTS

We would like to thank Yao Wang (Institute of Plant Protection, Chinese Academy of Agricultural Sciences) for help during the experiment. We also thank LetPub for editing the English.


Ambrosia artemisiifolia, at different growing stages. Biocontrol Sci. Technol. 21, 1049–1063. doi: 10.1080/09583157.2011.603823



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhao, Ma, Chen, Wan, Guo and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in Helicoverpa armigera

Lin Chen<sup>1</sup> , Jizhen Wei<sup>2</sup> , Chen Liu<sup>1</sup> , Wanna Zhang<sup>1</sup> , Bingjie Wang<sup>1</sup> , LinLin Niu<sup>1</sup> and Gemei Liang<sup>1</sup> \*

<sup>1</sup> State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, <sup>2</sup> College of Plant Protection, Henan Agricultural University, Zhengzhou, China

A pyramid strategy combining the crystal (Cry) 1A and 2A toxins in Bacillus thuringiensis (Bt) crops are active against many species of insects and nematode larvae. It has been widely used to delay pest adaption to genetically modified plants and broaden the insecticidal spectrum in many countries. Unfortunately, Cry2A can also bind with the specific receptor proteins of Cry1A. ATP-binding cassette (ABC) transporters can interact with Cry1A toxins as receptors in the insect midgut, and ABC transporter mutations result in resistance to Bt proteins. However, there is limited knowledge of the ABC transporters that specifically bind to Cry2Ab. Here, we cloned the ABCC1 gene in Helicoverpa armigera, which expressed at all larval stages and in nine different tissues. Expression levels were particularly high in fifth-instar larvae and Malpighian tubules. The two heterologously expressed HaABCC1 transmembrane domain peptides could specifically bind to Cry2Ab with high affinity levels. Moreover, transfecting HaABCC1 into the Spodoptera frugiperda nine insect cell significantly increased its mortality when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference significantly reduced the mortality of larvae exposed to Cry2Ab in vivo. Altogether current results suggest that HaABCC1 serves as a functional receptor for Cry2Ab.

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Yuan-Xi Li, Nanjing Agricultural University, China Muthugounder S. Shivakumar, Periyar University, India

> \*Correspondence: Gemei Liang gmliang@ippcaas.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 19 March 2018 Accepted: 28 May 2018 Published: 19 June 2018

#### Citation:

Chen L, Wei J, Liu C, Zhang W, Wang B, Niu L and Liang G (2018) Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in Helicoverpa armigera. Front. Physiol. 9:745. doi: 10.3389/fphys.2018.00745 Keywords: Helicoverpa armigera, HaABCC1, functional receptors, binding, RNAi

# INTRODUCTION

The crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are specifically toxic to some insect pests, such as Lepidoptera, Diptera, and Coleoptera, while they are almost harmless to nontarget organisms (Sanahuja et al., 2011; Pardo-López et al., 2013; Comas et al., 2014; Nicolia et al., 2014). To reduce the use of chemical insecticides, Bt proteins, such as Cry1Ac and Cry2Ab, have been used worldwide as bio-pesticide sprays or expressed in genetically modified (GM) plants to control certain insect pests (Sanahuja et al., 2011; James, 2016). The hectares of Bt crops worldwide increased from 1.1 million in 1996 to 98.5 million in 2016, Bt corn, cotton, and soybean accounted for >99% of this total amount, with a cumulative total of more than 830 million (James, 2016).

The high selection pressure of Bt could lead to the rapid evolution of insect resistance. Cases of pest resistance to Bt proteins produced by GM crops increased from 3 in 2005 to 16 in 2016 (Pardo-López et al., 2013; Jakka et al., 2016; Tabashnik and Carrière, 2017).

Bt cotton (expressing the Cry1Ac protein) has been planted in China since 1997, and recent bioassay data showed that the percentage of resistant Helicoverpa armigera collected from fields in north China increased from 0.93% (2010) to 5.5% (2013) (Jin et al., 2015). To delay pest adaption to GM crops, some different resistance management strategies have been used, including trait pyramiding (Xue et al., 2008; Brévault et al., 2013; Jin et al., 2015). The "pyramid" strategy has been widely adopted to replace the first generation Bt crops. For example, the transgenic cotton which can produce Cry1Ac and Cry2Ab is the only type of Bt cotton grown in Australia. And it's also the predominant type of Bt cotton grown in India and the United States (Tabashnik et al., 2013a; Fabrick et al., 2014).

However, the occurrence of cross-resistance sometimes weakens these advantageous characteristics of Cry1A + Cry2A "pyramid" strategy (Tabashnik et al., 2013b; Wei et al., 2015; Welch et al., 2015). Although Cry1A and Cry2A were predicted to have different structures and different mode of actions in the target lepidopteran pests because of their low amino acid homology (Hernández-Rodríguez et al., 2008; Caccia et al., 2010), but studies have indicated that they share some of the same receptors. Several important functional Cry1A receptors, such as cadherin (CAD), amionpeptidase-N (APN), or alkaline phosphatase (ALP), they have been identified as binding protein or reported to play vital functional role in the toxicity of Cry2A (Onofre et al., 2017; Yuan et al., 2017; Zhao et al., 2017). Further research showed although CAD was a functional receptor for both the Cry2Aa and Cry1Ac toxins in Spodoptera exigua, but Cry1Ac and Cry2Aa toxins did not compete for the same binding sites and may bind to diverse CAD protein epitopes (Qiu et al., 2015). Thus, the same receptors but different binding sites may associate with the asymmetrical cross-resistance between Cry1A and Cry2A (Tabashnik et al., 2009; Wei et al., 2015). So, it is urgent to find the receptor of Cry2Ab.

Recently, some ATP-binding cassette (ABC) transporters are involved in the resistance of insect to Bt toxins. For example, ABCC2 mutations cause the resistance of H. armigera and Heliothis virescen to Cry1Ac (Gahan et al., 2010; Xiao et al., 2014). The Cry1Ac-resistance of Plutella xylostella is closely related to the reduced expression of PxABCC2, PxABCC3, and PxABCG1 in the midgut (Guo et al., 2015a,b). Meanwhile, high levels of resistance to Bt Cry2Ab toxin have been verified to be genetically linked with loss of function mutations of an ABC transporter gene (ABCA2) in two Lepidopteran insects, H. armigera and H. punctigera (Tay et al., 2015). Moreover, two HaABCA2 knockout strains created from the susceptible SCD strain by using the CRISPR/Cas9 genome editing system display high levels of resistance to Cry2Ab (>100-fold) compared with the original SCD strain (Wang et al., 2017). In addition, the binding experiments showed there is more than one receptor of Cry2Ab in insect midgut (Wei et al., 2016). Functional ABC transporter family proteins contain four core domains: two membrane-spanning domains (transmembrane domains, TMs), each built from six membrane-spanning α-helices, alternating with two nucleotide-binding domains (NBDs) located on the cytosolic side (Dassa and Bouige, 2001; Linton, 2007). All of these indicated that other ABC transporters may also involve in the mode of action of Cry2Ab.

ABCC1 has four core domains like other ABC transporter proteins. Additionally, ABCC1 involved in the toxicity of Cry2Ab to H. armigera by itraq data (unpublished data). However, whether ABCC1 serves as a functional receptor for Cry2Ab in H. armigera remained unclear. Here, we discovered Cry2Ab could bind to the heterologously expressed peptides with high affinity levels, transfecting HaABCC1 into the Sf9 cell line significantly increased its mortality rate when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference (RNAi) significantly reduced the mortality rates of larvae exposed to Cry2Ab in vivo. Based on our results, we propose that ABCC1 in H. armigera is involved in the action mode of Cry2Ab.

# MATERIALS AND METHODS

# Insect Rearing and Tissue Sampling

Susceptible H. armigera (96S strain) was collected from the cotton fields in Xinxiang County, Henan Province, China in 1996. The larvae of this colony were reared in the laboratory on an artificial diet without exposure to any Bt toxins or insecticides (Liang et al., 1999).

We collected samples from nine developmental stages of H. armigera: egg, first- to sixth-instar larva, pupa, adult (male and female). For each biological replicate, we collected samples from 200 eggs and 5–20 individuals from other developmental stages. The following different tissues: head, foregut, midgut, hindgut, Malpighian tubule, peritrophic membrane, hemolymph, fat body, and cuticle from fifth-instar larvae were collected, and tissues dissected from 25 larvae served as one biological replication. All collected samples were quickly frozen in liquid nitrogen and stored at −80◦C for subsequent RNA extraction. Four biological replicates were prepared for each treatment.

# Prepared Cry2Ab and Biotinylated Cry2Ab

The activated Bt toxin Cry2Ab was purchased from Envirologix Inc. (Portland, ME, United States). Activated Cry2Ab protein was biotinylated using the EZ-Link Sulfo N-hydroxysuccinimide Liquid Chromatography (LC) Biotinylation Kit (Pierce, FL, United States) with a 1:20 molar ratio (Cry protein:biotin) following the manufacturer's instructions. Biotinylated Cry2Ab proteins were separated on a 4–20% SDS–polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Billerica, MA, United States) (150 mA, 1 h) and were treated in 5% (w/v) bovine serum albumin (BSA) diluted in phosphate buffered saline (PBS; 135 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, and 1.7 mM KH2PO4, pH 7.4) containing 0.5% (v/v) Tween-20 (PBST) at 4◦C overnight. The membranes were washed three times with PBST, and streptavidin horseradish peroxidase was used to detect biotinylated toxins. The blots were developed with the Easysee Western Blot Kit (Transgen, Beijing, China) and observed in a multifunction laser imager (TYPhoon 9410, GE Healthcare, United States).

# Cloning, Expression, and Purification of HaABBC1

#### Total RNA Extraction and First-Strand cDNA Synthesis

TRIzol reagent (Invitrogen, CA, United States) was used to extract total RNA from the midguts of 25 fifth-instar larvae of H. armigera according to the manufacturer's instructions. Then, contaminating genomic DNA was removed by treating with DNase I (TakaRa, Japan). The 260/280 and 260/230 ratios measured using a NanoDrop 3300 (Thermo Fisher, MA, United States) were used to evaluate the purity of the total RNA, and a 1% agarose gel was used to determine the integrity. Then, the first-strand cDNA was synthesized immediately from 1 µg of total RNA using a SuperScriptTM III First-Strand Synthesis Kit (Invitrogen) following the manufacturer's instructions and stored at −20◦C for further use.

#### Cloning and Sequence Analysis

Parts of the ABCC1 gene were obtained from the previous transcriptome sequencing data in our laboratory. Rapid amplification of cDNA ends (RACE) was used to obtain the full-length cDNA. RACE-ready cDNAs were amplified using a SMARTScribeTM RACE cDNA Amplification Kit (Clontech, Mountain View, CA, United States) according to the manufacturer's instructions. Gene-specific primers were designed based on the fragments of the ABCC1 gene. The primers were designed by Primer Premier 5.0, and UPM was used as the universal primer (Supplementary Table S1). RACE PCR was used under the following conditions: Initial denaturation at 94◦C for 4 min, followed by five cycles at 94◦C for 30 s and 72◦C for 2.5 min, followed by another five cycles at 94◦C, for 30 s, 70◦C for 30 s, and 72◦C for 2.5 min. Subsequently, 25 cycles were performed at 94◦C for 30 s, 68◦C for 30 s, and 72◦C for 2.5 min, followed by 72◦C for 10 min. To ensure the entire open reading frame (ORF) was amplified, specific primers that included initiation and stop codons were designed and used to amplify the entire ORF sequence. PCR products were separated by 1% agarose gel electrophoresis, and the expected bands were gel-purified and cloned into the pEasy-T3 vector (TransGen). The cloned fragments were then sequenced. The full sequence was obtained and submitted to GenBank (accession no. KY796050).

The NCBI BLAST database was used to analyze the homology of the ABCC1 gene with other ABC sequences (Supplementary Table S2). The molecular weights and isometric points of the proteins were predicted using the ExPaSy proteomics server website<sup>1</sup> . The TM helices were analyzed using TMHMM Server v.2.0<sup>2</sup> . The protein domains were predicted using the ExPaSy-PROSITE<sup>3</sup> . The N-terminal signal peptide positions were determined using SignalP 4.1 Server<sup>4</sup> . NetNGlyc was analyzed using NetNGlyc 1.0 Server<sup>5</sup> . The NetOGlyc were analyzed using NetOGlyc 4.0 Server<sup>6</sup> . The percentage of amino acid sequence identity was calculated using ClustalW, and the phylogenetic tree was constructed in MEGA 7.0, using the neighbor-joining method (Tamura et al., 2013).

#### Cloning, Protein Expression, and Purification of Two HaABCC1 Protein Fragments

Based on the nucleotide sequence of the gene encoding HaABCC1, we designed two pairs of primers with EcoRV restriction sites contained in forward primers and HindIII restriction sites contained in reverse primers, to clone two fragments of HaABCC1 that incorporated the potential toxin-binding regions, which we termed TMD1 and TMD2, respectively. The primers used for the PCR-amplification of TMD1 and TMD2 fragment were TM1-EcoRV-F and TM1- HindIII-R, and TM2-EcoRV-F and TM2-HindIII–R, respectively (Supplementary Table S4).

Then, the synthesized cDNA was used as the template to amplify the two partial fragments by PCR. The PCR program included denaturation at 95◦C for 3 min, 35 cycles of denaturation at 95◦C for 30 s, annealing at 53◦C for 30 s, and extension at 72◦C for 90 s, and final extension at 72◦C for 10 min. After purified by AxyPrep DNA Gel Extraction Kit (Axygen Scientific, CA, United States), the PCR products were cloned into the pEASY-T3 vector (TransGen Biotech), and then transformed into competent cell, Escherichia coli Trans1-T1 cells (TransGen Biotech). The recombinant plasmids were doubledigested with EcoRV and HindIII (TaKaRa, Dalian, China) for 3 h at 37◦C, and the products were subcloned into the expression vector pET32a (Novagen, United States), which was digested with the same restriction enzymes to generate the two pETHaABCC1 recombinant plasmids, which were transfected into E. coli BL21 (DE3) cells (Tiangen, Beijing, China) for protein expression.

The two HaABCC1 proteins fragments were tested by gradient SDS–polyacrylamide gel electrophoresis (PAGE) 4– 20% (Genscript Biology Co., China). Then, the two HaABCC1 proteins fragments were identified by LC-MS/MS (Q-TOF) at the Beijing Protein Institute (Beijing, China).

The expressed HaABCC1 protein fragments were purified using a micro-protein PAGE recovery kit (Sangon Biotech., Shanghai, China). The two HaABCC1 fragments were tested for purity using a 4–20% gradient SDS–PAGE (Genscript Biology Co., NJ, United States).

#### Ligand Blot Analyses

For the ligand blot analysis, 10 µg purified HaABCC1 fragments were separated using 4–20% gradient SDS-PAGE gels (Genscript Biology Co., NJ, United States) and then electro-transferred onto PVDF filters (Millipore Corp.). After blocking, the membrane was incubated with biotinylated Cry2Ab toxin (5 µg/ml) in blocking buffer for 1 h at room temperature and then washed three times (15 min each) with PBST. Then, the PVDF membrane was incubated with horseradish peroxidase-conjugated antirabbit IgG (ZSGB-BIO, Beijing, China) as a secondary antibody in blocking buffer (1:10,000) for 1 h at room temperature.

<sup>1</sup>http://www.expasy.org

<sup>2</sup>http://www.cbs.dtu.dk/services/TMHMM/

<sup>3</sup>http://prosite.expasy.org/

<sup>4</sup>http://www.cbs.dtu.dk/services/SignalP/

<sup>5</sup>http://www.cbs.dtu.dk/services/NetNGlyc/

<sup>6</sup>http://www.cbs.dtu.dk/services/NetOGlyc/

After additional washing, the membrane was developed using an EasySee Western Blot Kit (TransGen) and exposed on an ImageQuant LAS4000mini system (GE Healthcare, Japan).

#### Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA extraction and cDNA synthesis of samples from different tissues and developmental stages were performed as described above. The relative mRNA expression levels of the ABCC1 gene were analyzed by qRT-PCR. The primers can be found in Supplementary Table S3. Each TaqMan qRT-PCR (Tiangen) reaction was performed in a total volume of 20 µl containing the following components: 1 µl template cDNA, 10 µl 2 × SuperReal PreMix (probe), 0.6 µl each 10 µM primer, 0.4 µl 10 µM probe, 0.2 µl 50 × ROX Reference Dye and 7.2 µl RNase-Free ddH2O. All qRT-PCR reactions were performed in 96-well optical plates in an ABI 7500 Real-time PCR System (Applied Biosystems). qRT-PCR was performed for 40 cycles of 95◦C for 3 s and 60◦C for 32 s. Both β-actin (GenBank EU527017) and GAPDH (GenBank JF417983) genes were used internal references, and the expression level of the target gene (ABCC1) for each treatment was normalized with the geometric mean of the expression of the two reference genes (β-actin and GAPDH) (Liu et al., 2015; Zhang et al., 2016). To check reproducibility, six biological replicates were analyzed with each biological replicate consisting of three technical repeats.

# Transient Transfection and Cell Bioassay

The Spodoptera frugiperda 9 (Sf9) cell line was cultured in Sf-900 II SFM medium (GIBCO/BRL/Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone-QB perbio, Logan, UT, United States), 50 U/ml penicillin, 50 mg/ml streptomycin, and 12 mg/ml gentamycin (Invitrogen) in an incubator at 28◦C (Cordova et al., 2006).

The whole ORF of ABCC1 was cloned into pAc5.1b vector (Huayueyang Biotechnology Co., Ltd, Beijing, China). First, we designed gene-specific primers that contained two restriction enzyme sites. The primers for the PCR-amplification of the ABCC1 ORF were ORF-PmeI-F and ORF-StuI-R (Supplementary Table S4). The PCR reaction and conditions were described in Section 2.4.2. PCR products were cloned into pEasy-T3 vector (TransGen), followed by restriction enzyme digestion with PmeI and StuI (TaKaRa). The pAc5.1b vector was also digested with PmeI and StuI for 3 h at 37◦C. The double-enzyme digestion products were electrophoresed on a 1% low-melting point agarose gel (Invitrogen) and the PCR products were purified by AxyPrep DNA gel extraction kit. Then, the ABCC1 gene was ligated into the pAc5.1b vector for 3 min at 22◦C using T4 DNA ligase (Thermo Fisher). The ligation product was transfected into E. coli Trans1-T1 cells (TransGen), and individual clones were selected from an LB plate.

First, the activated Cry2Ab was precipitated by adding 4 M acetic acid dropwise to the activation reaction tube to adjust the pH to 8.0. Then, the tube was incubated at 4◦C for 30 min and

centrifuged at 12,000 × g for 20 min. The activated Cry2Ab pellets were washed three times using 40 ml ice cold ddH2O, dissolved in 15 ml Sf-900 II SFM media (GIBCO/BRL/Life Technologies) and centrifuged at 12,000 × g for 5 min. The upper supernatant was transferred into a clean tube and used as the stock solution for cytotoxicity assays. Finally, the concentrations of activated Cry2Ab toxin in the stock solution were estimated by electrophoresis of 15 µl 200 µg/ml BSA solution as well as 15 µl of Cry2Ab stock solution using SDS–PAGE, and the intensity of the corresponding bands was quantified with Image J software (NIH, v1.46).

Sf9 cells were seeded onto a 12-well plate (∼9 × 10<sup>5</sup> cells/well), allowing cells to attach overnight (12 h). Then, cells were transiently (5.5 h) transfected with (∼2 mg/well) pAc-ABCC1 plasmid using Cellfectin (Invitrogen; 8 µl per well). The empty pAc5.1b vector was used as the control. The transfection mixture was removed and replaced with 1.5 ml of supplemented medium (Sf-900 II SFM media containing antibiotics and serum). The cells were incubated at 28◦C for 60 h. Then, the cells were collected and the cell concentration was measured with a hemocytometer using Trypan blue. In a 96-well micro-plate, 100 µL of cells (∼10,000 cells) were reseeded allowing cells to attach for at least 2.5 h. Using activated Cry2Ab toxin (0.0091 mg/ml) to treat the cells, the mortality was calculated after 5 h (Wei et al., 2016).

# Silencing of HaABCC1 by RNAi

RNA interference with small interfering RNA (siRNA) was conducted by microinjection to study the role of HaABCC1 in the susceptibility of H. armigera larvae to the Bt toxin Cry2Ab. The HaABCC1 siRNA and enhanced green fluorescent protein (EGFP) siRNA (negative control) used were sequence specific and custom synthesized (Invitrogen). The sequences 5'-GGAUGUACCUGGUGGGCAUTT-3' and 5'-GCGUUGGGAAGUCAAGUUUTT-3'designed based on the gene-specific TMD region to avoid potential off-target effects were used as siHaABCC1 and siEGFP, respectively.

Two microgram siRNA (siHaABCC1 and siEGFP) by 5 µlmicrosyringe (Hamilton, Bonaduz, Switzerland) or 2 µg DEPC water (blank control) was microinjected in the abdomen of the newly emerged third-instar larvae. The injection point was

means and standard errors. Different letters indicate significant expression differences among different tissues or developmental stages based on three biological

sealed immediately with geoline. In addition, a third parallel non-treated control was performed. Each treatment included 24 individuals and replicated three times. To calculate the RNAi efficiency by qRT-PCR as the above described, 10 larvae were randomly selected for testing at 48 h after the injection.

replications and four technical repeats (p < 0.05).

To evaluate the susceptibility of H. armigera to Cry2Ab after RNAi, diet overlay bioassays were used (Hernández-Rodríguez et al., 2008). The toxin was dissolved and diluted in PBS. At the beginning, we distributed 1 ml liquid artificial diet into each well of 24-well plates (TianJin Xiangyushun Co., TianJin, China). After the diet was solidified, 75 µl solution of Bt toxin Cry2Ab (105 µg/cm<sup>2</sup> ) and PBS (control) were overlaid on the surface of the artificial diet on two 24-well plates, respectively. Finally, within 48 h of microinjection, 24 third-instar larvae were placed on the surface of the dried diet in the 24-well plates. Each treatment included 24 larvae, and there were three replicates. After 5 days, the numbers of dead larvae were recorded (Zhou et al., 2010).

#### Data Analysis

Cell mortality was calculated as the description in Wei et al. (2016). Significant differences among the different treatments were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's honestly significance difference (HSD) test for mean comparison. All statistical analysis was performed with SPSS v.18.0 (SPSS Inc., Chicago, IL, United States) at P < 0.05 level of significance.

#### RESULTS

#### Cloning, Sequencing, and Phylogenetic Analysis of HaABCC1

The ORF of HaABCC1 (GenBank KY796050) was 4,545 bp, and it encoded predicted proteins of 1,515 amino acid, with a predicted molecular masses of 169.75 kDa. It predicted isoelectric point was 6.68. The protein sequence analysis revealed that

HaABCC1 had no signal peptide. Additionally, it had 14 TM helices, 14 NetNGlyc sites, 16 NetOGlyc sites, and four domains for HaABCC1 (**Figure 1**). In total, 33 ABCC1–3 sequences from various species, including those from the current study and those available in GenBank, were aligned and used to construct a phylogenetic tree (**Figure 2**). The phylogenetic tree showed that the ABCC1, ABCC2, and ABCC3 proteins were closely related in different lepidopteran insects. HaABCC1 was most closely to SlitABCC1, AtraABCC1, PpolABCC1, and PxutABCC1.

# Spatio-Temporal Expression Pattern of the HaABCC1 Gene

HaABCC1 was expressed in all developmental stages but peaked in the fourth- and fifth-instar larvae (p < 0.001) (**Figure 3A**). Meanwhile, Expression levels of HaABCC1 were markedly different among the tissues (p < 0.001) (**Figure 3B**). The results showed it expressed in midgut, but the highest expression was detected in the Malpighian tubules, for other tissues, the expression levels of HaABCC1 were relatively low and not significantly different.

# HaABCC1 Expression, Purification and Ligand Blotting

Two cDNA fragments, TMD1 and TMD2, of the H. armigera gene HaABCC1 (924-bp and 882-bp) were cloned and expressed in E. coli BL21 (DE3) cells. In prediction, these cDNA fragments were translated to peptides of 308 and 294 amino acid residues, respectively, of which the molecular weights were 34 and 33 kDa, respectively. All proteins extracted from E. coli BL21 (DE3) cells were confirmed by SDS–PAGE (**Figure 4A**), and the recombinant proteins (HaABCC1-pET32a construct) were present in the pelleted inclusion bodies. The molecular masses of these proteins were consistent with the predicted molecular weights. The expressed HaABCC1 protein fragments were purified, and the LC-MS/MS (Q-TOF) indicated that they were parts of the HaABCC1 protein. To test for specific interactions between the Cry2Ab toxins and HaABCC1, a ligand blot analysis was conducted, and the two expressed HaABCC1 fragments were bound to the activated Cry2Ab. TMD2 had a higher binding affinity than TMD1 (**Figure 4B**).

# Transient Transfection and Cell Bioassay

HaABCC1 was expressed after be transfected into Sf9 cells (**Figure 5A**). The additional expression of HaABCC1 led to the Cry2Ab susceptibility significantly increasing, with 41.25% cells dying after being treated by 0.0091 mg/ml Cry2Ab. The mortality rate was significantly greater than that of the control (2.35%) (**Figure 5B**).

# Knockdown of HaABCC1 in Vivo

To further explore the potential function of the HaABCC1 gene in the action mode of Cry2Ab against H. armigera, we used in vivo RNAi to knockdown HaABCC1 expression by injecting siABCC1 into early third-instar larvae. The siABCC1 sequence was complementary to the internal gene-specific TMD region of

the HaABCC1 mRNA. The qPCR analysis showed that there were no significant differences among controls (injections of DEPC water and siEGFP, and the non-treated control). Additionally, the injection of siABCC1 into larvae significantly reduced HaABCC1 transcript levels by 54.0, 49.4, and 52.2% relative to the nontreated, DEPC water- and siEGFP-injected larvae, respectively (p < 0.001) (**Figure 6A**). When larvae pretreated with siRNA of HaABCC1 for 2 days were transferred to a diet containing Cry2Ab, the larval mortality rate of the Cry2Ab-treated group decreased by 70.0, 70.7, and 65.7% relative to non-treated, DEPC water- and siEGFP-injected larvae, respectively. Moreover, there were no significant differences among the three controls (p > 0.05) (**Figure 6B**).

#### DISCUSSION

Recently, ABC transporters, especially members of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies, have become a focus of research in arthropods because of their important roles in xenobiotic transport and insecticide resistance (Dermauw and Van Leeuwen, 2014; Merzendorfer, 2014; Xiao et al., 2014; Guo et al., 2015a,b; Tay et al., 2015). The HaABCC1 in our study had a structure similar to those of other ABC transporters, containing two extracellular domains that were present as long loops between helices TM I and TM II, 14 NetNGlyc sites and 16 NetOGlyc sites (**Figure 1**). Moreover, its sequence was similar to SlitABCC1, AtraABCC1, PpolABCC1, and PxutABCC1 (**Figure 2**). HaABCC1 expression was widespread in the H. armigera larvae (**Figures 3**, **4**), with the highest expression level detected in fourth- and fifth-instar larvae, and in Malpighian tubules, which are part of the excretory and osmoregulatory systems in insect larvae. It was confirmed that ABCC1 was expressed highest in Malpighian tubules in H. armigera larvae (Bretschneider et al., 2016). Thus, HaABCC1 may have functions similar to those of ABC transporters involved in Bt intoxication.

For Cry1A toxins, a sequential mode of action had been proposed, and some specific and saturable binding membrane targets on the midgut are important for the toxicity (Bravo et al., 2011; Heckel, 2012). The toxin first binds to membrane-bound glycosylated proteins, such as aminopeptidases, ALP, and other glycoproteins, and then binds to the 12-CAD domain protein, resulting in processing and accelerated oligomerization (Bravo et al., 2011; Vachon et al., 2012). Cry2A proteins have three domain structures comparable to those of Cry1A toxins (Pigott and Ellar, 2007; Hernández-Rodríguez et al., 2008), making them likely to act in similar ways as pore-forming toxins. Upon activation, Cry2A toxins can bind to the glycosylated loops of TMD1 and/or TMD2 in ABC transporters of Helicoverpa species, and this binding can form the basis of oligomerization and bring the pre-pore structures close to the TMDs for pore insertion (Hernández-Rodríguez et al., 2008; Gahan et al., 2010). Additionally, other proteins may be involved in Cry2Ab binding and pore formation, particularly because mammalian ABCs may

occur in multi-protein complexes in the membrane (Kaminski et al., 2006). In our study, we also found the two expressed HaABCC1 fragments (TMD1 and TMD2) could bind with the activated Cry2Ab as assessed by ligand blotting experiments (**Figure 4B**). We hypothesized that ABCC1 may also provide binding and pore insertion functions in the action mode of Cry2Ab, like other ABC transporters.

In fact, some ABC transporters, like ABCC2, ABCC3, and ABCG1 etc. have been proved as functional receptors for Cry1A (Xiao et al., 2014; Guo et al., 2015a,b; Tanaka et al., 2016). For example, the over-expression of SlABCC3 strongly increases the susceptibility of Trichoplusia ni Hi5 cells to Cry1A, which suggested that ABCC3 was also a functional receptor of Cry1A toxins (Chen et al., 2015). Additionally, the ABC transporter mutations have been identified as being associated with Bt resistance. ABCC2 in H. virescens were first reported as being involved in Cry1Ac resistance based on quantitative trait locus (QTL) mapping results that investigated the insertion of a premature stop codon in ABCC2 (Gahan et al., 2010). Then, different deletions, point mutations, truncations and spliceosome variants in ABC transporter orthologs were subsequently reported as being associated with the resistance of P. xylostella, T. ni, and H. armigera to Cry1Ac or Cry2Ab (Baxter et al., 2011; Xiao et al., 2014; Tay et al., 2015), Bombyx mori to Cry1Ab (Atsumi et al., 2012), and Ostrinia nubilalis survival on transgenic Cry1Fa maize (Coates and Siegfried, 2015). Furthermore, the changes in ABCC2, ABCC3, or ABCG1 transcript levels or their down-regulation were also linked to Cry1A or Cry1Ca toxinresistance in S. exigua, P. xylostella, and Ostrinia furnacalis (Park et al., 2014; Guo et al., 2015a,b; Zhang et al., 2017). Here, we provided evidences that HaABCC1 encoded a functional receptor for Cry2Ab in H. armigera. We tested the susceptibility changes in in vitro experiments after transfecting HaABCC1 into Sf9 cells, and the transfected cells were more susceptible to Cry2Ab (**Figure 5B**). To confirm this view, we knocked down HaABCC1 expression using RNAi technology, and the larval mortality significantly decreased (**Figure 6B**). The specific and saturable binding to membranes in Helicoverpa species has been shown for Cry2Ab (Hernández-Rodríguez et al., 2008, 2013), and the resistance to Cry2Ab is also associated with a loss of binding

between receptors and Cry2Ab (Caccia et al., 2010). However, the function of ABCC1 in the resistance evolution of H. armigera to Bt requires further research. Meanwhile, ABCC1 and ABCC2 are belonging to ABC transporter C family members. ABCC2 is a functional receptor of Cry1Ac and the mutations of it caused the resistance to Cry1Ac. But here we first report ABCC1 is a functional receptor of Cry2Ab. So, whether ABCC1 (or ABCC2) have function in mode of action of Cry1Ac (or Cry2Ab), and caused the cross-resistance between Cry1Ac and Cry2Ab, then reduced the benefit of Cry1A + Cry2A "pyramid" strategy, which are also requires further study.

# CONCLUSION

This is the first report to demonstrate that ABCC1 is associated with Cry2Ab toxicity in H. armigera. The results presented here indicated that HaABCC1 could bind with the Cry2Ab toxin and had important roles in the action modes of Cry2Ab. We suggested that ABCC1 serves as a functional receptor in H. armigera for Cry2Ab. Our findings provide mechanistic insights into the interactions between ABCC1 and Cry toxins.

# REFERENCES


# AUTHOR CONTRIBUTIONS

GL and LC designed the study. LC prepared experimental materials, performed the experiments, and analyzed the data. GL, LC, and JW wrote the manuscript. CL, WZ, BW, and LN contributed to completing the experimental contents. All authors have read and approved the manuscript for publication.

#### FUNDING

This research was supported by the Key Project for Breeding Genetically Modified Organisms (Grant No. 2016ZX08011– 002), the National Natural Science Funds of China (Grant No. 31621064), and the State Key Laboratory for Biology of Plant Diseases and Insect Pests (Grant No. SKLOF201708).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00745/full#supplementary-material


Guo, Z., Kang, S., Zhu, X., Xia, J., Wu, Q., Wang, S., et al. (2015b). The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management. Sci. Rep. 5:13728. doi: 10.1038/srep13728



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chen, Wei, Liu, Zhang, Wang, Niu and Liang. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Antioxidant Responses of Ragweed Leaf Beetle Ophraella communa (Coleoptera: Chrysomelidae) Exposed to Thermal Stress

Hongsong Chen1,2, Ghulam Sarwar Solangi<sup>3</sup> , Jianying Guo<sup>1</sup> , Fanghao Wan<sup>1</sup> \* and Zhongshi Zhou<sup>1</sup> \*

<sup>1</sup> State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, <sup>2</sup> Guangxi Key Laboratory for Biology of Crop Diseases and Insect Pests, Institute of Plant Protection, Guangxi Academy of Agricultural Sciences, Nanning, China, <sup>3</sup> Department of Entomology, Sindh Agriculture University Subcampus, Umerkot, Pakistan

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

David Rivers, Loyola University Maryland, United States Tong-Xian Liu, Northwest A&F University, China

\*Correspondence:

Fanghao Wan wanfanghao@caas.cn Zhongshi Zhou zhongshizhou@yahoo.com

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 11 February 2018 Accepted: 08 June 2018 Published: 06 July 2018

#### Citation:

Chen H, Solangi GS, Guo J, Wan F and Zhou Z (2018) Antioxidant Responses of Ragweed Leaf Beetle Ophraella communa (Coleoptera: Chrysomelidae) Exposed to Thermal Stress. Front. Physiol. 9:808. doi: 10.3389/fphys.2018.00808 Ophraella communa LeSage is an effective biological control agent of common ragweed, Ambrosia artemisiifolia L., which competes with crops and causes allergic rhinitis and asthma. However, thermal stress negatively affects the developmental fitness and body size of this beetle. High temperatures cause a variety of physiological stress responses in insects, which can cause oxidative damage. We investigated the total protein content and activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidases (PODs) in O. communa adults when its different developmental stages were exposed to high temperatures (40, 42, and 44◦C) for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), and a whole generation to high temperatures (40, 42, and 44◦C) for 3 h each day. A control group was reared at 28 ± 2 ◦C. Under short-term daily phasic high-temperature stress, total protein contents were close to the control as a whole; overall, SOD activities increased significantly, CAT activities were closer to or even higher than the control, POD activities increased at 40◦C, decreased at 42 or 44◦C; stage-specific response was also observed. Under long-term daily phasic high-temperature stress, total protein content increased significantly at 44◦C, SOD activities increased at higher temperatures, decreased at 44◦C; CAT activities of females increased at ≤42◦C, and decreased at 44◦C, CAT activities of males decreased significantly; POD activities of females increased at 40◦C, decreased at ≥42◦C, POD activities of males decreased at 44◦C; and antioxidant enzymes activities in females were significantly higher than those in males. Antioxidative enzymes protect O. communa from oxidative damage caused by thermal stress.

Keywords: Ophraella communa, thermal stress, total protein, antioxidant enzymes activity, biological control

# INTRODUCTION

Temperature is one of the most important environmental factors affecting life history, behavioral and physiological traits, population structure, and community composition in insects (Zhang et al., 2015b; Esperk et al., 2016). Insects have an optimal temperature range to which their biological functions are best adapted; over this range, insects might suffer physiological costs and

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fitness decrease (Jerbi-Elayed et al., 2015). Climate change has led to an increase in the frequency, intensity, and length of extreme high temperatures around the globe and this trend is expected to continue (Ma et al., 2018). In many parts of China, summer daily maximum temperatures in the field often exceed 40◦C for several hours, and the number of such hot days has also increased in the last few years (Zhang et al., 2015b). Small insect herbivores often have short life cycles leading to overlapping generations, so any life stage may experience heat stress and the effects of heat stress also depend on heat stress (Zhao et al., 2017).

Thermal stress can result in oxidative damage and oxidative stress (Lopez-Martinez et al., 2008; Zhang et al., 2014). Oxidative stress refers to elevated intracellular levels of reactive oxygen species (ROS) that cause damage to lipids, proteins, and DNA (Schieber and Chandel, 2014). To prevent damage from ROS, insects have developed antioxidant defense mechanisms and these systems have both enzymatic and non-enzymatic components (Felton and Summers, 1995); antioxidant enzymes are key components in the regulation of intracellular ROS balance (Wang et al., 2012; Jia et al., 2014; Li et al., 2017). Major antioxidative enzymes in insects include superoxide dismutase (SOD), catalase (CAT), and peroxidases (POD) which are reported to be involved in insect defense systems (Zhang et al., 2015a; Li et al., 2017). SOD converts superoxide anion (O<sup>2</sup> <sup>−</sup>) into oxygen (O2) and hydrogen peroxide (H2O2), and CAT and POD break down hydrogen peroxide (H2O2) into oxygen and water (Covarrubias et al., 2008; Li et al., 2017), which protect insects from oxidative damage.

Ophraella communa LeSage (Coleoptera: Chrysomelidae) is the best biological control agent of common ragweed, Ambrosia artemisiifolia L. (Asterales: Asteraceae) (Zhou et al., 2017). The larvae and adults of this leaf beetle feed on A. artemisiifolia leaves, destroying plants when its adults and larvae reach a high density, thus it performs a good control efficacy on A. artemisiifolia (Zhou et al., 2017). A. artemisiifolia is one of the most noxious weeds in agriculture around the world (Wan and Wang, 1988), due to its significantly negative effects on agricultural production, allergic rhinitis and asthma to human and impacts on biodiversity (Zhou et al., 2017). Research on O. communa has provided important insights into temperature. At constant temperature in the laboratory, the optimum developmental temperature for O. communa ranges from 25 to 28◦C, and temperatures not far beyond this range are harmful (Zhou et al., 2010); at ≥36◦C, all first-instar larvae are dead within 24 h, and the survival of other instars and female fecundity decreases significantly (Zhou et al., 2010). Under brief heat stress, the pre-adult development and survival, adult survival, longevity, and fecundity of O. communa were all adversely affected by 2 h at ≥35◦C (Zhou et al., 2011) or 3 h at ≥40◦C (Chen et al. unpublished data), the body size of O. communa adults is also adversely affected after exposure of immature stages to 3 h at ≥40◦C (Chen et al., 2014). However, the over 50% survival rates of eggs, pupae, and adults of O. communa exposure to 44◦C for 3 h (Chen et al. unpublished data), the effective control of O. communa and Epiblema strenuana on A. artemisiifolia in the field in summer (Zhou et al., 2014) both indicate that O. communa has a large thermal tolerance plasticity. To date, the response of antioxidant enzymes in O. communa exposed to thermal stress has not been reported. The aim of the present study was to determine the effects of short-term and long-term phasic high-temperature exposure on the anti-oxidant enzymes activities of O. communa and identify the physiological repair responses to hot summer in the field.

#### MATERIALS AND METHODS

# Host Plants

Ambrosia artemisiifolia seeds were collected from more than 10,000 plants from a field (28◦ 560 2600N, 113◦ 14'3800E) near the town of Dajing, in Miluo county, Yueyang city, Hunan province, China, during late October 2010. The seeds were then stored at 4◦C. Adequately stored seeds were sown in a greenhouse at 28 ± 2 ◦C under natural light at the Institute of Plant Protection, Hunan Academy of Agricultural Sciences (25◦ 210 1800N, 114◦ 330 4000E), Changsha, Hunan province, China, in late March 2011. When seedlings reached a height of approximately 15 cm, some of them were used in O. communa adult heat treatments. Apical buds of the remaining seedlings were removed to prevent apical dominance, and the seedlings were transplanted into pots (21 cm × 17 cm) containing soil with one seedling per pot, a total of 1,000 common ragweed seedlings were prepared. All the plants were watered every day and fertilizer was applied (N:P:K = 13:7:15) twice per month to maintain growth (Zhou et al., 2010). When the plants were approximately 40 cm high, approximately 400 of the potted plants were used for heat treatments of eggs, larvae, and pupae.

#### Insect Culture

More than 1,000 O. communa adults were collected from A. artemisiifolia plants in the same place for the plants in the previous subsection on June 24, 2011. Colonies of the beetle were maintained on A. artemisiifolia plants under natural light in the same greenhouse for the seedlings in the previous subsection.

Twelve pairs of O. communa adults were randomly collected from the rearing colony, and each individual was placed with the aid of a fine brush (size 0) onto a fresh common ragweed plant in a pot, which was then covered with nylon gauze (40 mesh size). After allowing two days for oviposition, the adult beetles were removed, the plants with newly eggs were placed in the greenhouse for normal growth until lots of the needed life stage emergence (such as eggs ≤ 12-h old, first-instar larvae ≤ 24-h old, pupae ≤ 24-h old, adults ≤ 12-h old).

#### Heat Treatment Intensities and Durations

The durations and intensities of our heat stress treatments were based on the duration and intensity of the highest temperatures in summer, which in central China is usually a few hours on any 1 day (maximum temperature of 44◦C for approximately 3 h per day; Chen et al., 2014). Therefore, 40, 42, and 44◦C for 3 h per day were selected, the treatment of 28◦C was considered as control (Zhou et al., 2010). The exposure periods continue for 3–5 (one developmental stage) or over 20 (one generation) days based on the developmental periods of different developmental stages of O. communa (4.0 days for the egg, 7.6 days for the

larva, and 6.0 days for the pupa) earlier recorded at a constant high temperature (32◦C) in laboratory (Zhou et al., 2010) and the number of hot days (daily maximum temperature ≥ 40◦C) at present and in the near future in China (Zhang et al., 2015b; Ma et al., 2018). The high-temperature exposures for each treatment were performed separately in environmental chambers (PRX-450D, Ningbo Haishu Safe Experimental Equipment Co., Ltd., Zhejiang, China) at 28 (control), 40, 42, or 44 ± 1 ◦C, with a relative humidity of 70 ± 5%, a photoperiod of 14:10 (L:D) h (Zhou et al., 2010), and a light intensity of 12,000 lx for 3 h daily. Each treatment was repeated three times.

# Short-Term Phasic Thermal Stress on Eggs, Larvae, Pupae, and Adults of O. communa

The experiments were started in early July 2011. One hundred eggs ≤ 12-h old, 100 first-instar larvae ≤ 24-h old, and 50 pupae ≤ 24-h old were separately retained on three potted plants in the greenhouse. Ten ragweed plants were randomly selected for each developmental stage, and they were then exposed to high temperatures for 3 h daily for 3, 5, and 5 consecutive days for eggs, larvae, and pupae in environmental chambers, respectively (by stage), after which the infested potted plants were kept in the greenhouse. A total of 120 ragweed plants were used.

Following high-temperature stress, treated pupae were collected by detaching the leaves they were on and placing the individual leaves into open transparent plastic boxes (19 cm × 12 cm × 6 cm) in an unsealed cuvette plastic tube covered with nylon gauze (60 mesh size) in the laboratory at 28 ± 2 ◦C and 70 ± 5% relative humidity, where pupae were checked daily for adult emergence. The treated eggs and larvae were kept in the greenhouse until they reached the pupal stage. The process for these pupae was the same as that for the treated pupae following high-temperature stress. The sex of each newly emerged adult was determined with the help of stereo microscope. These adults were kept in the laboratory at 28 ± 2 ◦C with relative humidity of 70 ± 5% for 5 days.

Newly emerged adults ≤ 12-h old (45 pairs) in the greenhouse culture were randomly selected for phasic high-temperature exposures. Fifteen pairs were placed on each of three fresh common ragweed seedlings (15 cm height) potted in a plastic box (19 cm × 12 cm × 6 cm) with a hole (15 cm × 4 cm) and covered with nylon gauze (60 mesh size), and this constituted one replicate. Ragweed seedlings containing these adults were exposed to high temperatures in environmental chambers for 3 h daily for 5 consecutive days, after which the infested potted plants were kept in a greenhouse, and a total of 36 ragweed plants were used.

#### Long-Term Phasic Thermal Stress on O. communa

The experiments were also started in early July 2011. Approximately 1,000 eggs ≤ 12-h old in the greenhouse were retained on one potted plant. Ten potted plants were selected for each treatment temperature, and they were then exposed to the high-temperature treatments in environmental chambers for 3 h daily until the emergence of the adult. After which the infested potted plants were kept in a greenhouse, and a total of 40 ragweed plants were used. Fifteen pairs of newly emerged adults ≤ 12-h old were placed on three fresh common ragweed seedlings (15 cm height) potted in a plastic box (19 cm × 12 cm × 6 cm) with a hole (15 cm × 4 cm) and covered with nylon gauze (60 mesh size), and this constituted one replicate. The O. communa adults on ragweed seedlings were then exposed to high temperatures in the environmental chambers for 3 h daily for 5 consecutive days, after which the infested potted plants were kept in a greenhouse, and a total of 36 ragweed plants were used.

#### Total Protein Content and Enzymes Activity Assays of O. communa

After exposure to high temperatures, the total protein content and antioxidant enzyme activity in subsequent adults were determined. The protein extraction protocols were carried out according to a total protein quantitative assay (A045-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein concentrations were determined according to the Bradford (1976) method with bovine serum albumin as the standard.

The activities of SOD, CAT, and POD were examined using commercially available assay kits (A001-1-1, A007-1-1, A084- 1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's protocols.

Superoxide dismutase activity was measured spectrophotometrically at 550 nm by xanthine and xanthine oxidase systems. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the xanthine oxidase system reaction in 1 ml enzyme extract with 1 mg protein (U mg−<sup>1</sup> protein). CAT activity was determined spectrophotometrically at 405 nm by measuring the decrease of H2O<sup>2</sup> due to hydrogen peroxide decomposition. One unit of CAT activity was defined as the amount that decomposes 1 µmol of H2O<sup>2</sup> per second per mg protein (U mg−<sup>1</sup> protein). POD activity was determined at 420 nm by catalyzing the oxidation of a substrate in the presence of H2O2. One unit of POD activity was defined as the amount that catalyzes 1 µg substrate per minute per mg protein (U mg−<sup>1</sup> protein; Jia et al., 2011).

#### Statistical Analyses

All data were analyzed using SPSS 21.0 (SPSS Inc., Chicago, IL, United States). Means were separated using Tukey's honestly significant difference (HSD) test (one-way ANOVA) when significant differences were found at P < 0.05 and were denoted as the means ± SE (standard error of the mean).

# RESULTS

#### Total Protein Content

First, in order to assay the antioxidant enzymes activities after exposure of different developmental stages or whole generation to phasic high temperatures, we evaluated the total protein content in subsequent adults. Total protein contents

in O. communa females were significantly affected by the previous exposure of larvae (F3,<sup>8</sup> = 52.31, P < 0.0001), pupae (F3,<sup>8</sup> = 85.78, P < 0.0001), and adults (F3,<sup>8</sup> = 434.51, P = 0.0007) to phasic high temperatures, except for eggs (F3,<sup>8</sup> = 3.83, P = 0.0571); the total protein contents were stage-specific when different developmental stages were exposed to any phasic high temperature compared to the control (**Table 1**). Total protein contents in O. communa males were also significantly affected by the previous exposure of eggs (F3,<sup>8</sup> = 53.87, P < 0.0001), larvae (F3,<sup>8</sup> = 48.83, P < 0.0001), pupae (F3,<sup>8</sup> = 5.85, P = 0.0205), and adults (F3,<sup>8</sup> = 32.49, P = 0.0001) to phasic high temperatures; the total protein contents were stage-specific when different developmental stages were exposed to 40 or 42◦C for 3 h compared to the control and 44◦C (**Table 2**).

After long-term phasic thermal stress, the total protein contents of O. communa female (F3,<sup>8</sup> = 10.83, P = 0.0007) and male (F3,<sup>8</sup> = 20.50, P = 0.0004) adults were significantly affected (**Figure 1A**). Total protein content of female adults significantly increased when exposed to 44◦C, and male adults showed significantly increased content at 42 and 44◦C, compared with the controls. No significant difference was observed in female adults between the 42◦C and control conditions, or in male adults between the 40◦C and control conditions (**Figure 1A**).

Taken together, these results suggest total protein contents in subsequent adults were significantly affected by the previous exposure of different developmental stages or whole generation to brief high temperatures.

#### Antioxidative Enzymes Activity

Next, we measured the antioxidant enzymes activities of O. communa adults after exposure of different developmental stages or whole generation to short- and long-term phasic high temperatures. In total, SOD activities of O. communa females increased after exposure of different developmental stages to phasic high temperatures (P < 0.05) compared to the control; the SOD activities were also stage-specific when different developmental stages were exposed to any phasic high temperature (P < 0.05) compared to the control (**Table 3**). The SOD activities of O. communa males also increased significantly by the previous exposure of eggs (F3,<sup>8</sup> = 33.2, P < 0.0001), larvae (F3,<sup>8</sup> = 240.02, P < 0.0001), and adults (F3,<sup>8</sup> = 23.07, P = 0.0003) to phasic high temperatures, but the result for pupae is opposite (F3,<sup>8</sup> = 7.91, P = 0.0089); the stage-specific responses were also observed when different developmental stages were exposed to any phasic high temperature compared to the control (**Table 4**).

After long-term phasic thermal stress, the SOD activities of both O. communa female and male adults increased at higher temperatures (females at 40 or 42◦C, males at 40◦C), decreased at the highest temperature (**Figure 1B**). SOD activities of female adults were significantly higher than those of males both at high temperatures and 28◦C (**Figure 1B**).

Overall, the CAT activities in O. communa adults were closer to or even higher than the control after exposure of different developmental stages to phasic high temperatures; the stage-specific responses were also observed when different developmental stages were exposed to any phasic high temperature compared to the control (**Tables 5**, **6**).

After long-term phasic thermal stress, the CAT activities of O. communa female adults increased at 40 and 42◦C, decreased at 44◦C (F3,<sup>8</sup> = 34.45, P < 0.0001); the CAT activities of male adults decreased significantly at high temperatures compared to the control (F3,<sup>8</sup> = 5.52, P = 0.0238); higher CAT activities were observed in female adults than those of males both at high temperatures and control (**Figure 1C**).

TABLE 1 | Mean (±SE) protein content of O. communa female adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

TABLE 2 | Mean (±SE) protein content of O. communa male adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

FIGURE 1 | Effect of long-term phasic thermal stress (12-h-old eggs to 5-days-old adults) on protein content and antioxidant enzyme activities of O. communa adults. The temperature 28◦C served as a control. Each value represents the mean (±SE) in adult females (black bars) and males (white bars). Different lowercase letters indicate significant differences among heat treatments for the same gender. Different uppercase letters indicate significant differences between males and females at the same temperature (Tukey's HSD test, P < 0.05). (A) Protein content. (B) Superoxide dismutase (SOD) activity. (C) Catalase (CAT) activity. (D) Peroxidase (POD) activity.

TABLE 3 | Mean (±SE) SOD activity of O. communa female adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

Overall, the POD activities of both O. communa female and male adults increased at 40◦C, decreased at 42 or 44◦C; the stage-specific responses were also observed when different developmental stages were exposed to any phasic high temperature compared to the control (**Tables 7**, **8**).

After long-term phasic thermal stress, the POD activities of O. communa female adults increased at 40◦C, decreased at 42 and 44◦C (F3,<sup>8</sup> = 14.49, P = 0.0013); the POD activities of male adults were close to the control at 40 and 42◦C, decreased at 44◦C (F3,<sup>8</sup> = 4.78, P = 0.0341); higher POD activities were also observed in female adults than those of males both at high temperatures and control (**Figure 1D**).

Taken together, antioxidant enzymes (SOD, CAT, and POD) activities were observed for the first time to be

TABLE 4 | Mean (±SE) SOD activity of O. communa male adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

TABLE 5 | Mean (±SE) CAT activity of O. communa female adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

TABLE 6 | Mean (±SE) CAT activity of O. communa male adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

TABLE 7 | Mean (±SE) POD activity of O. communa female adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.


Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

sex-dependent – with few exceptions, enzyme activities were significantly higher in female adults than in male adults.

#### DISCUSSION

The effects of heat stress on insects depend on the frequency, amplitude, and duration of the stress (Ma et al., 2018), and the sex and developmental stage of the insect (Enriquez and Colinet, 2017). It is reported that the frequency, intensity, and length of heat hot days will increase in the short- and in the longterm (Ma et al., 2018). As an overlapping-generation species with a relatively short generation period (Zhou et al., 2010), any developmental stage or the whole generation of O. communa might encounter phasic high-temperature stress. The level of SOD, CAT, and POD activity in O. communa adults by the previous exposure of different developmental stages or whole generation to brief high temperatures increased, suggesting the defensive function of these enzymes in abating the adverse effect of ROS generated by the heat stress.


TABLE 8 | Mean ( ± SE) POD activity of O. communa male adults when eggs, larvae, pupae, and adults were exposed for 3 h each day for 3, 5, 5, and 5 days, respectively (by stage), to 28 (control), 40, 42, and 44◦C.

Values within the same column followed by different lowercase and uppercase letters within the same row are significantly different (Tukey's HSD test, P < 0.05).

In the present study, we explored the effects of phasic and long-term daily thermal stress on total protein content and enzymatic antioxidant defense systems (SOD, CAT, and POD) of the common ragweed beetle, O. communa. Our results indicate that these parameters change significantly under different levels of phasic high temperature for 3–5 days, and under long-term high-temperature stress. Protein is reported as one of the major constituents imparting heat tolerance in the red flour beetle, Tribolium castaneum (Swetaleena et al., 2013). Overall, the total protein contents in O. communa adults at high temperatures similar with control (28◦C), under long-term thermal stress at 44◦C even significantly higher than control (28◦C), which suggests that proteins protect O. communa from heat stress damage. The total protein content was found to increase with thermal stress, and this result is in accordance with a study conducted on T. castaneum (Swetaleena et al., 2013). By contrast, a study involving high-temperature treatments of 31◦C reported that heat stress decreased protein contents in the termite Coptotermes formosanus (Tarver et al., 2012).

Superoxide dismutase is the most important antioxidant enzyme defense system against ROS, which catalyzes the breakdown of superoxide anions and transforms them into hydrogen peroxide and oxygen (Zelko et al., 2002). In the present study, the SOD activity in O. communa adult males and females increased significantly as a whole under short-term daily phasic high-temperature stress. Under long-term daily phasic hightemperature stress, the SOD activity in O. communa adult males and females increased at higher temperatures, decreased at 44◦C. These results suggest that the activity of SOD might be an adaptive response of different developmental stages O. communa to overcome high temperature ≤ 44◦C induced ROS toxicity, using other superoxide anion scavenging pathways. A previous study indicated that SOD has an important role in reducing the high level of superoxide radicals induced by low or high temperatures (Celino et al., 2011). The increased activities of SOD under high temperature in our results are in accordance with studies of the role of SOD, in the antioxidant responses to thermal stress in the wolf spider, Xerolycosa nemoralis (Wilczek et al., 2013), cucumeris mite, Neoseiulus cucumeris (Zhang et al., 2014), and Propylaea japonica (Zhang et al., 2015a). SOD activity increased significantly at 39◦C, and markedly decreased at 41◦C after exposure of P. japonica adults to heat stress for 1 h compared with control (Zhang et al., 2015a), which indicated that the activity of SOD was induced by moderate heat stress. A significant increase in SOD activities at 36 and 39◦C for 1 h compared with control in Chilo suppressalis larvae was observed (Cui et al., 2011). SOD activity in the predatory mite N. cucumeris was significantly increased compared to the control at 35 and 38◦C for 1-3 h (Zhang et al., 2014). Decreased SOD activity could also impair the O2-scavenging ability of the cells, thus favoring the accumulation of O<sup>2</sup> and H2O<sup>2</sup> (Jaleel et al., 2008), as SOD is the first and most important defense against ROS (Feng et al., 2015). Therefore, we hypothesized that SOD also plays a key role in the response of O. communa to short-term or long-term phasic thermal stress.

Catalase removes H2O<sup>2</sup> only at high cellular concentrations and is inefficient at low concentrations (Yang et al., 2010). In this study, the CAT activities in O. communa adults were closer to or even higher than the control under short-term daily phasic high-temperature stress. Under long-term daily phasic hightemperature stress, the CAT activities of female O. communa adults increased at ≤42◦C, decreased at 44◦C, CAT activities of male O. communa adults decreased significantly. The present results are in accordance with a study which reported that CAT activity significantly increases at 35–41◦C for 1 h in ladybeetle P. japonica adults (Zhang et al., 2015a). Increased CAT activity under thermal stress has also been reported in the oriental fruit fly Bactrocera dorsalis adults (Jia et al., 2011), and the fifth instar silkworm Bombyx mori (Nabizadeh and Kumar, 2011), the rice stem borer C. suppressalis larvae (Cui et al., 2011; Lu et al., 2017). These results suggest that CAT provides protection of O. communa under short-term or long-term phasic thermal stress ≤42◦C.

Peroxidase plays an important role in scavenging H2O2. Under short-term daily phasic high-temperature stress, POD activities increased at 40◦C, decreased at 42 or 44◦C. Under long-term daily phasic high-temperature stress, POD activities of female O. communa adults increased at 40◦C, decreased at ≥ 42◦C, POD activities of male communa adults decreased at 44◦C. These results are in accordance with POD activity increase in P. japonica adults at 41◦C for 1 h (Zhang et al., 2015a), and B. dorsalis adult at 35–40◦C for 3–6 h (Jia et al., 2011). In previous study, POD activity was significantly increased in N. cucumeris after heat shock for 1–2 h and decreased with the duration of exposure (Zhang et al., 2014). POD had an important role in the antioxidant response to thermal stress in P. japonica, and no significant difference in POD activity was found from 35 to 39◦C, whereas at 41◦C a remarkable increase was observed, compared to the control (Zhang et al., 2015a). We hypothesized that POD also provides protection of O. communa under short-term or long-term phasic thermal stress ≤ 40◦C.

In general, antioxidative enzymes (SOD, CAT, and POD) activity was found to be sex-dependent – it was higher in females than males. Overall, the higher antioxidative enzymes activity of O. communa females was obtained in the present study, which indicates higher thermal tolerance of female O. communa. A high body weight, large size, and high survival rate of O. communa females (Chen et al., 2014; Chen et al. unpublished data) may be closely related to the high antioxidative enzyme activity level of females under heat stress. The stage-specific thermal tolerance is very common in insects (Zhao et al., 2017), the stage-specific antioxidative enzyme activity was reported in C. suppressalis (Li et al., 2017), and the stage-specific antioxidative enzyme activity of O. communa may be related to the sensitivity of stages to heat stress (Chen et al. unpublished data).

In conclusion, thermal stress is one of the factors that can generate oxidative stress products in O. communa. Hightemperature exposures cause oxidative stress at 44◦C and changes in antioxidant enzymes (SOD, CAT, and POD) play an important part in reducing oxidative damage in O. communa up to 42◦C, the increased antioxidant defense systems of SOD, CAT, and POD may be an adaptive response of O. communa to avoid oxidative stress during exposure to high-temperature stress.

#### REFERENCES


#### AUTHOR CONTRIBUTIONS

ZZ and FW conceived and designed the work. GS and JG contributed to the revision of the manuscript. HC performed the experiments and wrote the manuscript.

#### FUNDING

This work was supported by the National Natural Science Foundation of China for Excellent Young Scholars (No. 31322046) and National Natural Science Foundation of China (No. 31171908).

#### ACKNOWLEDGMENTS

We thank Wanmei Yang and Tianang Lei (Hunan Agricultural University) for their help during the experimental work. We also thank Springer Nature Author Services for editing the English of the manuscript.


and their involvement in response to ultraviolet-A stress. J. Insect Physiol. 58, 1250–1258. doi: 10.1016/j.jinsphys.2012.06.012


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chen, Solangi, Guo, Wan and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Induction of Resistance Against Plutella xylostella (L.) (Lep.: Plutellidae) by Jasmonic Acid and Mealy Cabbage Aphid Feeding in Brassica napus L.

Gadir Nouri-Ganbalani<sup>1</sup> \*, Ehsan Borzoui<sup>1</sup> , Maryam Shahnavazi<sup>2</sup> and Alireza Nouri<sup>3</sup>

<sup>1</sup> Department of Plant Protection, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran, <sup>2</sup> Department of Oral and Maxillofacial Radiology, Faculty of Density, AJA University of Medical Sciences, Tehran, Iran, 3 Institute of Higher Education of Sabalan Ardabil, Ardabil, Iran

#### Edited by:

Nicolas Desneux, Institut National de la Recherche Agronomique (INRA), France

#### Reviewed by:

Xiaoling Tan, Chinese Academy of Agricultural Sciences, China Dandan Wei, Southwest University, China

> \*Correspondence: Gadir Nouri-Ganbalani gnouri@uma.ac.ir

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 15 January 2018 Accepted: 15 June 2018 Published: 10 July 2018

#### Citation:

Nouri-Ganbalani G, Borzoui E, Shahnavazi M and Nouri A (2018) Induction of Resistance Against Plutella xylostella (L.) (Lep.: Plutellidae) by Jasmonic Acid and Mealy Cabbage Aphid Feeding in Brassica napus L. Front. Physiol. 9:859. doi: 10.3389/fphys.2018.00859 The diamondback moth, Plutella xylostella (L.), has become the most destructive insect pest of cruciferous plants, such as B. napus throughout the world including Iran. In this study, the induction of resistance was activated in oilseed rape plants (Brassica napus L.) using foliar application of jasmonic acid (JA) and mealy cabbage aphid either individually or in combination against diamondback moth. Induced resistance by inducers significantly reduced the population growth parameters, as well as the survival rate of immature P. xylostella. Also, the nutritional indices of P. xylostella were studied to evaluate the potential impact of induced resistance on the insect feeding behavior. The values of the efficiency of conversion of ingested food, the efficiency of conversion of digested food, relative consumption rate, and relative growth rate of P. xylostella on JA-treated plants were significantly reduced compared to control. These are because glucosinolates and proteinase inhibitors are induced following treatment of plants. Also, we found a significantly higher glucose oxidase activity in the salivary gland extracts of larvae fed on JA treatment. These results express that JA and/or Aphid application induces systemic defenses in oilseed rape that have a negative effect on P. xylostella fitness. These findings develop our knowledge the effects of induced defenses on P. xylostella.

Keywords: induced resistance, growth indices, nutritional indices, glucosinolate, trypsin inhibitors

# INTRODUCTION

Oilseed rape (Brassica napus L.; Brassicaceae) is a major oilseed crop in Iran and throughout the world. It is most important vegetable oil source with an annual growth rate exceeds that of palm (FAO, 2011). Also, the seeds of B. napus are the important source of dietary protein for humans globally (Alizadeh et al., 2015). This plant is attacked by several insect pests in the field that often necessitate control measures by the growers to protect the crop (Alford et al., 2003).

In recent years, the diamondback moth, Plutella xylostella (L.) (Lep.: Plutellidae), has become the most destructive insect pest of cruciferous plants including the oilseed rape (Soufbaf et al., 2010; Furlong et al., 2013). The larvae feed on host plants' leaves, causing substantial crop losses

(Akandeh et al., 2016). Currently, the chemical control is the primary tactic used to control the diamondback moth. Since frequent application of insecticide is required per season to obtain satisfactory control; therefore, the pest has become resistant to most of the registered insecticides (Shelton et al., 1993). Also, pesticide use has had adverse effects on the human health and environment (Saldo and Szpyrka, 2009). To avoid such undesirable consequences, many scientists have focused their attention on the use of less hazardous practices and/or methods to protect the crops.

One of the most environmentally sound and economically feasible insect control methods is the use of resistant cultivars and/or reinforcement of plant defense system. Various defense traits of host plants affect the fitness of herbivore insects (Rask et al., 2000; Harvey et al., 2007; Mithöfer and Boland, 2012; Soufbaf et al., 2012). The plant chemicals may act as repellents, deterrents, antinutrients, and antidigestive compounds that interfere with the physiology of the herbivore and reduce its developmental and survival rate (Kessler and Baldwin, 2001; Franceschi et al., 2005; Karban, 2011; War et al., 2011; Lu et al., 2014; Nikooei et al., 2015).

The induction of plant defenses by insect feeding is regulated via multiple signaling cascades. In cruciferous plants, defense systems against herbivores are induced by the jasmonic acid (JA) and salicylic acid (SA) pathways (de Vos et al., 2005; Hopkins et al., 2009), which can be induced by synthetic JA (War et al., 2011) and by phloem-feeding insects (Zhu-Salzman et al., 2004). These signaling pathways cross talk and may act antagonistically or synergistically (Zarate et al., 2007; Koornneef and Pieterse, 2008). It is reported that the profile and concentration of glucosinolates and myrosinase in cruciferous plants have significant effects on the fitness of their insect pests (Martin and Müller, 2007; Verkerk et al., 2009; Angelino et al., 2015; Popova et al., 2017).

Synthetic JA is not directly toxic or inhibitory to the herbivores (Kagale et al., 2004); but, it induces toxic chemicals (alkaloids, phenolics, and terpenoids) in the plant organs that can limit the attack of herbivores. The induced defenses may augment the plant's constitutive defenses against subsequent attackers (Howe and Schaller, 2008; Poelman et al., 2008). Sharma et al. (2009) reported the high amounts of polyphenols and tannins in resistant wild relatives of pigeonpea as compared to the cultivated pigeonpea and introduced these compounds as plant resistance factors to Helicoverpa armigera (Hübner) (Lep.: Noctuidae). Mouttet et al. (2011) assessed the influence of pre-infestation by a first attacker on the performance of a second attacker. They resulted that the induction of plant defense reactions lead to the production of secondary metabolites and in this way, the first attack enhances the plant's ability to resist the second attacker and reduction in its performance.

The individual effects of JA and herbivory in the induction of resistant in plants and their effect on subsequent herbivory have been well documented. Despite this abundance of researches, there exists a lack of studies to characterize the effects of JA and prior herbivory induced resistance in oilseed rape plants on the diamondback moth. Therefore, the aim of this research has been to study the induction of resistance on oilseed rape plants treated with JA and mealy cabbage aphid either individually or in combination against diamondback moth through the assessment of life table parameters, growth indices (GI), nutritional indices, and glucose oxidase activity (GOX) of the pest. This study provides an insight into the mode of action of JA- and herbivorydependent defenses against subsequent pest and hence can help to find a solution to pest control management.

# MATERIALS AND METHODS

#### Chemicals

Substrates, synthetic JA, o-dianisidine, proteinase K, D-glucose, trypsin enzyme, and trichloroacetic acid (TCA) were purchased from Sigma Chemical Co. (St. Louis, MO). Horseradish peroxidase was purchased from Roche Co. (Grenzach-Wyhlen, Germany) and Tris, acetone, and potassium phosphate was purchased from Merck Co. (Darmstadt, Germany).

#### Plant

Seeds of B. napus cultivar RGS003, as a semi-resistance cultivar, were obtained from the Plant and Seed Improvement Research Institute (Karaj, Iran). Plants were grown in 10 L plastic pots filled with a mixture of autoclaved soil (with the ratio of 2:1:1 field soil, sand, and vermicompost, respectively) and protected by 100-mesh muslin to prevent insect infestation. The pots were arranged in a randomized block design within the research greenhouse of the University of Mohaghegh Ardabili (Ardabil, Iran), set at 25◦C with a natural photoperiod. Thirty-five-daysold plants were used for the experiments.

#### Insects

A colony of P. xylostella used in the experiments was obtained from a cabbage field in Karaj (Iran), in September 2015. The colony has been maintained for about 15 months in the Laboratory of Entomology, University of Mohaghegh Ardabili, Ardabil, Iran, without any exposure to insecticide. They were reared on oilseed rape leaves inside a growth chamber that was set at 25◦C, 60% relative humidity, and a photoperiod of 16:8 (L:D).

A colony of Brevicoryne brassicae L. (Hem.: Aphididae) used in the experiment was originally obtained from kohlrabi fields in Ardabil (Iran), in August 2016. The insects have been maintained for more than 4 months in the greenhouse without any exposure to insecticide. To maintain the aphid colony every 2 weeks, 10–15 apterous aphids were transferred from infected plants to healthy plants.

#### Experiments

All following experiments including induction of resistance, life table parameters, GI, and nutritional indices were carried out under laboratory conditions inside a growth chamber, that was set at 25◦C, 60% relative humidity, and a photoperiod of 16:8 (L:D).

#### Jasmonic Acid Preparation

Synthetic JA was dissolved in acetone at a rate of 1 g ml−<sup>1</sup> and dispersed in an appropriate volume of water to achieve 5 mM JA

solution (Thaler et al., 1999). The control solution consisted of only acetone dissolved in water.

#### Treatments Application

Resistance was artificially induced on oilseed rape by the foliar application of JA (5 mM), Aphid (B. brassicae), and JA plus Aphid.

In order to determine the induced resistant by JA, 30 oilseed rape plants were sprayed with JA and control solutions (15 plants for each solution). Total of 15 ml of solution was applied to each plant by a hand pressure sprayer. Plants treated with JA and control solutions were separated by using different cages to prevent volatiles induced by JA from eliciting defenses in control plants. After 2 days, the surface of plants was cleaned with water. Then, the treated and control plants were used for the bioassays or extractions.

In order to determine the induced resistant by B. brassicae, apterous adults (within 24 h) from the stock colony were transferred on central leaves of 15 caged oilseed rape plants at the rate of 20 females per plant. Fifteen plants were kept as a control without the prior release of aphids before P. xylostella. Aphids were removed from plants after being allowed to feed for 2 days. Then, the treated and control plants were used for the bioassay or extraction. During infestation with aphid, offspring were removed from the plants on a 12 h basis.

In order to determine the induced resistant JA plus Aphid, 15 plants were sprayed with JA solution. After that, B. brassicae adults (within 24 h) from the stock colony were transferred on central leaves of 15 caged oilseed rape plants at the rate of 20 females per plant. Treated and control plants were separated by using different cages to prevent volatiles induced by JA and Aphid from eliciting defenses in control plants. After 2 days, Aphids were removed from plants and the surface of plants was cleaned with water. Then, the treated and control plants were used for the bioassays or extractions.

#### Life Table Parameters

To obtain P. xylostella eggs of the same age, 20 male–female pairs of the newly emerged moths from the stock colony were transferred to oviposition plastic cages (diameter 30 cm, depth 30 cm) covered with 100-mesh screen net for ventilation. Male and female adults were distinguished based on their abdomen; the tip of the abdomen in female moths is slightly swollen and in male moths is slender and elongated. After 12 h, laid eggs were collected and placed in the incubator until hatching. Once hatching, 60 P. xylostella newly hatched larvae (within 12 h) were released at the rate of 6 larvae per plant on 10 caged oilseed rape plants. Larvae were also released on caged oilseed rape plants that were not treated by JA and/or aphid, to be kept as controls. The plants were monitored daily until the immature stages of P. xylostella completed their development or died.

After eclusion, a pair of newly emerged adults (one male and one female) was transferred to plastic oviposition containers (diameter 11 cm, depth 12 cm); the containers were closed at the top with a 100-mesh screen net for ventilation. The number of pairs of tested moths for each host plant depended on their survival from the previous stage and ranged from 18 to 44 couples. A small cotton wick soaked in 10% honey solution was placed in each oviposition container to supply a source of carbohydrate to the adult feeding insects. Leaves from treated and control plants were replaced with fresh leaves every day, and the number of deposited eggs was recorded until the female's death. The eggs were maintained for 10 days to estimate the percentage of hatched eggs (fertility).

The development time, immature survival rate, and fecundity were used to the calculation of the life table parameters. Calculations were made for age-stage survival rate (sxj) of P. xylostella on different treatments based on the method of Chi and Su (2006). Estimates for net reproductive rate (R0), the intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T) of P. xylostella on different treatments were calculated based on Huang and Chi (2013).

# Ovipositional Preference Experiment

In order to determine the ovipositional preference of P. xylostella in a free-choice situation, the control, and treated plants were arranged in a randomized complete block design with five replications inside a metal cage (length 200 cm, width 180 cm, height 120 cm) covered by 100-mesh muslin screen net. Ten pairs of 24- to 48-h-old moths were randomly collected from the rearing chamber, released inside each cage, and provided a 10% sterile honey solution for adult feeding. The number of deposited eggs by females on each plant was counted and recorded 24 h after moth releasing.

#### Growth Indices

Growth indices of P. xylostella were determined as described by Itoyama et al. (1999). To estimate the GI, we used growth and survival data from life table parameters experiment. Also, pupae obtained from the life table parameters experiment were individually weighed 24 h after pupation. In this study, GI of P. xylostella fed on control and treated plans were calculated using the following formulae:

Immature GI = (immature survival rate)/(immature duration)

Standardized insect − growth index (SII) = (pupal wt.)/ (larval period)

Fitness index (FI) = (percentage of pupation × pupal wt.)/ (immature duration)

where immature refer to larvae and pupae stages.

#### Nutritional Indices

Nutritional indices were determined as described by Waldbauer (1968), Manuwoto and Scriber (1982), and Farrar et al. (1989) with some modifications by Dastranj et al. (2017). Initially, neonates were reared on the treated and control plants until the unset of fourth instar. This experiment carried out with seven replications (40 larvae in each) for each treatment. Nutritional

indices were determined using the fourth instar larvae. Seven groups of 10 larvae each were prepared, weighted, and transferred into glass Petri dishes (12 cm diameter and 1.5 cm depth) containing the fresh oilseed rape leaf of each treatment or control. The petioles of the leaves were inserted into cotton ball soaked in water to maintain freshness. For 4 days, the initial fresh food, food remnant, and feces remaining at the end of each experiment were weighed daily. Also, the larvae in each Petri dish were checked daily for mortality or ecdysis. To establish the percentage of dry weight of the food, larvae, and feces, 20 specimens for each were weighed, oven-dried (48 h at 60◦C), and subsequently reweighed. Nutritional indices were calculated using the following formulas, based on dry weights:

> Efficiency of conversion of ingested food (ECI) = [(insect wt. gain)/(wt. food eaten)] × 100

Efficiency of conversion of digested food (ECD) = [(insect wt. gain)/(wt. food eaten – wt. frass)] × 100.

Relative consumption rate (RCR) = (wt. food eaten)/ (insect wt. at beginning of trial) (time)

Relative growth rate (RGR) = (insect wt. gain)/ (insect wt. at beginning of trial) (time)

#### Preparation of Extract From the Whole Body of Larvae

To investigate GOX, neonates were reared on the treated and control plants until the beginning of fourth instar. In fourth instar, groups of 15 larvae (24–48 h old) were pooled in a precooled Teflon pestle and immediately homogenized in Nathathan's saline (Christensen et al., 1991), containing proteinase inhibitor (PI) to inhibit digestive proteases in the saliva and cellular proteases released during homogenization, and used in whole body GOX experiments. The homogenate was centrifuged at 12,000 g for 15 min (4◦C). The supernatant was used as the salivary gland extract.

#### Glucose Oxidase Activity

Glucose oxidase activity was determined by the method of Kelley and Reddy (1988), with slight modification. To assay the GOX, the reaction mixture containing 0.17 mM o-dianisidine-HCl in potassium phosphate buffer (0.1 M; pH 7.0), 95 mM D-glucose, and 60 U ml−<sup>1</sup> horseradish peroxidase was incubated at 37◦C and saturated with oxygen. Then, 100 µl salivary gland extracts were added and, over 15 min, the change in absorbance at 460 nm min−<sup>1</sup> was calculated to obtain the slope of the linear portion. For the control, 100 µl potassium phosphate buffer (0.1 M, pH 7.0) was added instead of the salivary gland extract. This experiment was repeated five times for each treatment and control.

#### Extraction of Oilseed Rape Leaves

Initially, each extract of the leaves was prepared using liquid nitrogen to homogenize the whole leaf in a Tris–HCl buffer (50 mM; pH 8). A volume (1 ml) of homogenate was removed and placed in a 1.7-ml centrifuge tube. The tubes were vortexed and centrifuged at 8,000 g for 10 min at 4◦C. The supernatant was taken and used as a source of inhibitor for the inhibition assays.

# Inhibitory Assay of Oilseed Rape Leaf Extract

The ability of oilseed rape leaf extract inhibitors to inhibit trypsin activity was determined by incubating a mixture of 40 µl trypsin (1 mg trypsin/10 ml 1 mM HCl), 100 µl of supernatant of leaf extract of plants containing trypsin inhibitors, and 160 µl Tris– HCl buffer (pH 8) for 30 min at 37◦C. A control containing no enzyme extract with buffer was run simultaneously with the reaction mixture. Ten additional tubes (five without enzyme blanks, and five with enzyme but without leaf extract) were also prepared to determine the maximum enzyme activity. The reaction was terminated by adding 200 µl of TCA (10% w/v H2O), continued by cooling at 4◦C for 30 min and centrifuging at 15,000 g for 10 min (4◦C). One hundred microliters of supernatant were added to 100 µl of 2 M NaOH and the absorbance was read at 450 nm. This experiment was replicated five times.

#### Glucosinolate Analysis

The collected leaves that originated from 24 plants (6 plants for each control and treatment) were freeze-dried and ground to a fine powder. Fifty grams of ground leaf material per sample was dissolved in methanol. The extract was analyzed as desulpho derivatives using the HPLC method described by van Dam et al. (2004). Results are expressed in micromoles of glucosinolate per gram dry mass.

# Data Analysis

All data calculated for each individual were subjected to the bootstrap method with 500 resampling for estimating the means, variances, and standard errors of population parameters. Difference between treatments was then compared by using the paired bootstrap test (Efron and Tibshirani, 1993; Akköprü et al., 2015). One-way ANOVA was used to compare the effects of the induced resistant on the GI, nutritional indices, and GOX activity of P. xylostella fed on control and treated plants and also enzyme inhibition by plant extracts. Means were compared at the P < 0.05 and Tukey's HSD method using SAS 9.2 software (PROC GLM; SAS Institute, 2009). Correlation analysis of the life table parameters, GI, nutritional indices, and GOX activity of P. xylostella fed on control and treated plans with glucosinolate level of plants and leaf extract inhibitors was performed using SPSS 16.0.

# RESULTS

Controls were compared in the experiments and, because there was no significant difference, mean of data from controls were presented in the tables and figures.

# Individual and Combined Effect of JA and Aphid on Life Table Parameters

The age-stage specific survival rate (sxj) of P. xylostella on control and treated plants indicates the probability that a newborn

survival to age x and develop to stage j (**Figure 1**). The immature developmental time was longer and the survival rate was lower on JA-treated plants. The survival rate of immature stages was approximate to 80% on the control plants. The survival rate of immature stages on JA-treated plants was 40% lower than those reared on the control plants.

The population growth parameters of P. xylostella fed on control and treated plants are presented in **Table 1**. The population fed on control plants had a higher net reproductive rate (R0; 63.73) and those reared on JA treatment had lowest R<sup>0</sup> value (16.35). The difference in the intrinsic rate of increase (r) was statistically significant in control and P. xylostella fed on treated plants. The population fed on control plants had a much higher r value (0.1831 d−<sup>1</sup> ) than those on JA-treated plants (0.1070 d−<sup>1</sup> ). The finite rate of increase (λ) for P. xylostella populations varied from 1.1130 d−<sup>1</sup> on JA-treated plants to 1.2010 d−<sup>1</sup> on control plants. The mean generation time (T) was also different in control and P. xylostella fed on treated plants with the control plants promoting the fastest generation times (23.82 d).

# Individual and Combined Effect of JA and Aphid on Ovipositional Preference

The number of deposited eggs by P. xylostella females significantly differed among control and treatments (F3,<sup>16</sup> = 5.60; P = 0.0081). In 24 h, the numbers of deposited eggs on JA-treated plants exceeded other treatments and control (**Figure 2**). Females laid 2.0-, 1.5-, and 1.8-fold more eggs on JA-treated plants than on control, Aphid-treated plants, and JA + Aphid-treated plants, respectively.

#### Individual and Combined Effect of JA and Aphid on Growth Indices

Plutella xylostella that fed on oilseed rape plants treated with JA, Aphid, or JA + Aphid showed statistically significant reductions in the immature GI, SII, and FI in comparison to those insects that fed on control plants (**Table 2**). The insects fed on control had the highest immature growth index (0.0528), while the lowest was on JA + Aphid treatment (0.0214). The SII of P. xylostella was 0.634 mg d−<sup>1</sup> on control and 0.279 mg d−<sup>1</sup> on JA treatment.



<sup>1</sup>The plants were treated with jasmonic acid (5 mM for 2 days) and/or aphid (20 adults for 2 days) prior to determining nutritional indices of P. xylostella. Means in a row followed by different letters are significantly different at P < 0.05 by using paired bootstrap test. R0, net reproductive rate; r, intrinsic rate of increase; λ, finite rate of increase; T, mean generation time. JA, jasmonic acid.

The mean FI was 10.1, 10.5, and 21.8 mg d−<sup>1</sup> on plants treated with JA, JA + Aphid, and Aphid, respectively. The control P. xylostella FI was 34.8 mg d−<sup>1</sup> , a value which was significantly higher than the FI of insects on treated plants.

#### Individual and Combined Effect of JA and Aphid on Nutritional Indices

In general, the nutritional indices (ECI, ECD, RCR, and RGR) of fourth instar P. xylostella that fed on oilseed rape plants treated with JA, Aphid, or JA + Aphid were significantly lower than those fed on control plants (**Table 3**). The ECI of larvae varied from 3.49 to 4.45%, with the minimum on JA treatment and the maximum on control. Similarly, the ECD was the highest in control (5.77%) and the lowest on JA treatment (4.30%). The data revealed that the highest RCR was recorded for larvae fed on control plants (2.83 mg mg−<sup>1</sup> d −1 ), while the lowest was on JA-treated plants (1.88 mg mg−<sup>1</sup> d −1 ). The RGR was highest for larvae fed on control plants (0.126 mg mg−<sup>1</sup> d −1 ), at an intermediate level on JA + Aphid-treated plants (0.091 mg mg−<sup>1</sup> d −1 ), and the lowest on JA-treated plants (0.066 mg mg−<sup>1</sup> d −1 ).

#### Glucose Oxidase Activity

Glucose oxidase activity of salivary gland of P. xylostella larvae fed on control and treated oilseed rape plants is presented in **Figure 3** (F3,<sup>16</sup> = 35.98, P < 0.01). Quantitative estimation of GOX activity per individual revealed significantly higher enzyme activity in the salivary gland extracts of larvae fed on JA treatment (0.77 U 10 individuals−<sup>1</sup> ). In contrast, control plants fed larvae (0.21 U 10 individuals−<sup>1</sup> ) possessed the lowest level of GOX activity.

# Trypsin Inhibitor Activity of Control and Treated Plants

The effect of leaf extract inhibitors from control and treated plants on trypsin activity is shown in **Figure 4** (F3,<sup>16</sup> = 63.21, P < 0.01). The inhibitors of control plants and JA, Aphid, JA + Aphid treatments inhibited 20.0, 39.3, 46.3, and 71.6% of enzyme activity, respectively.

#### Glucosinolate Contents

Nine main glucosinolates were detected in treated and control B. napus plants belonging to the three chemical classes: six aliphatics (3-Methyl sulfinyl propyl, 2-OH-3-butenyl, 2-propenyl, 2-OH-4-pentenyl, 3-Butenyl, and 4-pentenyl), two indoles (3-indolyl, 1-Methoxy-3-indolylmethyl), and one aromatic (2-Phenylethyl). Total glucosinolate concentration was higher in the treated plants compared to control. In response to application of JA, there were more increases in the concentrations of 2-OH-3-butenyl, 4-pentenyl, and 3-indolyl, being 19.4, 6.6, and 8.3 µmol g−<sup>1</sup> dw, respectively. Treatment with Aphid resulted in more accumulation of 2-phenylethyl, being 12.3 µmol g−<sup>1</sup> dw, in plants. The concentrations of other glucosinolates were also increased by treatments, but to a smaller extent (**Figure 5**).

#### Correlation Analysis

The analysis of correlation coefficients of the examined biological and physiological characteristics of P. xylostella fed on control and treated plants with total glucosinolate content and inhibition

.



<sup>1</sup>The plants were treated with jasmonic acid (5 mM for 2 days) and/or aphid (20 adults for 2 days) prior to determining nutritional indices of P. xylostella. Mean values followed by different letters in the same column are significantly different (Tukey's test, P < 0.05). GI, immature growth indices; SII, standardized insect-growth index; FI, fitness index; JA, jasmonic acid.

TABLE 3 | Nutritional indices of fourth instar larvae of Plutella xylostella fed on oilseed rape plants treated with jasmonic acid and/or aphid<sup>1</sup>


<sup>1</sup>The plants were treated with jasmonic acid (5 mM for 2 days) and/or aphid (20 adults for 2 days) prior to determining nutritional indices of P. xylostella. This experiment was replicated seven times. Mean values followed by different letters in the same column are significantly different (Tukey's test, P < 0.05). ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate; JA, jasmonic acid.

of trypsin is shown in **Table 4**. The results of this study revealed that high correlations existed between r and FI on one side and total glucosinolate content and inhibition of trypsin on the other. Very high negative correlations were also found between RCR (r = 0.982) and RGR (r = 0.996) with inhibition of trypsin activity.

# DISCUSSION

Resistance induced in many plant species is known to influence the fitness and performance of insect pests (Mithöfer and Boland, 2012 and references therein). However, the induced resistance by elicitors did not produce any phytotoxicity or negative effect on natural enemies (Bruce, 2014). Our results showed that both JA and Aphid treatments, as resistance inducers in B. napus, had detrimental effects upon P. xylostella growth and development, and JA proved to be the most detrimental to this insect pest at the concentration tested. Of course, we did not found positive interactions in the reduction survivorship and life table parameters of P. xylostella that was fed on the plants treated with JA + Aphid; probably because of interference in signaling events related to induce resistance by these treatments (Pontoppidan et al., 2003; Cipollini et al., 2004; Ehlting et al., 2008; Zhang et al., 2013). It is reported that inducible defenses in plants against insect herbivores can be strongly influenced by the mix of signals generated by external biotic and abiotic factors (Bostock, 1999; Kranthi et al., 2003; Lambdon and Hassall, 2005). The interactions among signaling cascades triggered by two or several inducers could be synergies and/or antagonisms (Zarate et al., 2007; Koornneef and Pieterse, 2008).

FIGURE 4 | Mean (±SE) percentage inhibition of trypsin activity by leaf extract inhibitors of oilseed rape plants treated with jasmonic acid and/or aphid. Each point is average of five replications. Mean values followed by different letters are significantly different (Tukey's test, P < 0.05).

In our study, artificial induction of defense in oilseed rape plants reduced the survivorship of immature stages of P. xylostella. Probably, the reduction in the survival rate of P. xylostella was because of the increase of inhibitor level in treated plants that affect the feeding, growth, and survival of the immature (Dastranj et al., 2017). Also, inducers are TABLE 4 | Correlation coefficients (r) of some biological and physiological characteristics of Plutella xylostella fed on oilseed rape plants treated with jasmonic acid and/or aphid<sup>1</sup> with total glucosinolate content and inhibition of trypsin.


<sup>1</sup>The plants were treated with jasmonic acid (5 mM for 2 days) and/or aphid (20 adults for 2 days) prior to determining nutritional indices of P. xylostella. FI, fitness index; RCR, relative consumption rate; RGR, relative growth rate.

responsible for producing certain secondary metabolites which decrease the survival of herbivores (Thaler et al., 2001). This was also evidenced in Spodoptera exigua (Hübner) (Lep.: Noctuidae), feeding on induced foliage (Stout and Duffey, 1996). Correlation coefficients are given in **Table 4** clearly indicate that glucosinolates and trypsin inhibitors have reduced survival of P. xylostella.

The slowest population development of the pest was observed on JA treatment; mainly due to the longer development time, the higher morality of immature stages, low fecundity, and a later peak in reproduction. The r value was moderate for insect fed on Aphid-treated plants when compared to control and JA treatment. HPLC analysis of plant extracts revealed that total glucosinolate level was 50% higher in JA-treated plants compared to control. It is highly probable that P. xylostella performance is most likely driven directly by glucosinolate level which in turn was significantly influenced by the type of treatments (**Figure 5**). The correlation coefficient between r and total glucosinolate level support this viewpoint (**Table 4**). Similarly, Bartlet et al. (1999) reported that an increase in the concentration of indole glucosinolates are being induced by the JA treatment and having negative effects on subsequent herbivory. Also, Mouttet et al. (2013) showed that previous feeding by whiteflies negatively affected the performance of leaf miners in locally damaged plants.

Careful examination of the signaling events related to systemic induced resistance has revealed that chemical cascades involving JA are involved in many induced responses against herbivore attack (War et al., 2012). Our results are in compliance with previous studies on chewing pests in this regards. For example, on tomato, foliar JA application significantly inhibited the damage of some leaf feeding caterpillars and flea beetles (Thaler, 1999). Besides glucosinolate, other secondary metabolites and digestive enzyme inhibitors might have changed and influenced the r of P. xylostella; as its correlation is shown in **Table 4**. Dastranj et al. (2017) reported that plant inhibitors reduced survival and delayed growth and development of the P. xylostella larvae.

The GI are considered to be the most important determining factors of immature performance that show whether a host is suitable or unsuitable for feeding insect (Setamou et al., 1999). We found that when P. xylostella larvae fed on high-quality

host their survival rate increased (**Figure 1**) and completed developmental time faster (data not shown) compared to those insects that fed on a low-quality host. In the present study, it was found that the GI, SII, and FI of P. xylostella on control plants were nearly twofold to threefold higher than those on treated plants. According to the correlation analysis, there was a significant negative correlation between the FI of lepidopteran pest and the levels of glucosinolate of plants (**Table 4**). Therefore, the observed effects of individual and combined applications of JA and Aphid on P. xylostella GI are likely to be due to these anti-nutritive and/or toxic compounds (Zhang et al., 1992). Our results agreed with those achieved by Tan et al. (2012), who reported exogenous MeJA can elevate the activity of defensive enzymes, like PIs, and reduce the growth rate of H. armigera

There was a significant reduction in ECI and ECD, a significant decrease in RCR, and generally lower RGR for P. xylostella fed on JA-treated plants compared to control plants. The results of inhibitory assay showed that the higher impairment of the digestive process of trypsin occurs after incubation with the extracts of treated plants (**Figure 4**). Inhibition of digestive enzymes by proteinaceous inhibitors results in micronutrient deficiencies that negatively affect the food intake of the herbivore (Borzoui et al., 2017), which was also proven in our experiment. Also, glucosinolates may target digestive process of P. xylostella (Li et al., 2000). Our results are in agreement with previous findings that the treatment of plants by inducers had disruptive effects on feeding performance of herbivores (Lin and Kogan, 1990; Gill et al., 2003; Rayapuram and Baldwin, 2006).

Our results are an additional example of induced changes in the chemical composition and inhibitors level of plants by different treatments. Bartlet et al. (1999) reported woundinduced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by Psylliodes chrysocephala (L.) (Coleoptera: Chrysomelidae). Tan et al. (2012) showed that jasmonate induces trypsin inhibitors in tomato leaves. Lu et al. (2014) observed a contrasting effect of ethylene biosynthesis on induced plant resistance against the striped stem borer and the brown planthopper in rice.

In the present study, where the GOX was assayed, we concluded that saliva of P. xylostella may play an important

#### REFERENCES


role in counteracting the larvae with the induction of secondary metabolites and probably PIs. P. xylostella that fed upon treated plants elevated the saliva GOX activity in comparison to those on control plants. Hu et al. (2008) reported that sugars and secondary metabolites are the possible causes of induction of GOX activity. Furthermore, Musser et al. (2002) found that glucose oxidase was the principal salivary enzyme responsible for suppressing the induction of nicotine in wounded tobacco plants.

#### CONCLUSION

Our study has shown that JA and/or prior herbivory by the mealy cabbage aphid could be useful elicitors to elevate oilseed rape plant's defense against P. xylostella. The induction by these elicitors has significantly decreased the life table parameters, GI and nutritional indices of this insect pest. The reason for these effects could be the induction of glucosinolates and PIs following treatment of plants. Also, we found that JA-treated plants were the most preferred host for oviposition by P. xylostella; therefore, these plants can be used as trap crops that lure P. xylostella adults away from the main crop by providing an alternative site for oviposition. It is hoped that these findings could contribute to a better utilization of inducer-dependent defenses into integrated pest management P. xylostella. However, further studies are needed to understand the ecological role of induced defenses in plant interactions with herbivores and their natural enemies.

#### AUTHOR CONTRIBUTIONS

GN-G and EB conceived and designed the research. EB and MS conducted the experiments and analyzed the data. GN-G, EB, MS, and AN contributed analytical tools and wrote the manuscript.

#### ACKNOWLEDGMENTS

The work received financial support from the University of Mohaghegh Ardabili (Ardabil, Iran) which is greatly appreciated.




**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Nouri-Ganbalani, Borzoui, Shahnavazi and Nouri. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Physiological and Evolutionary Changes in a Biological Control Agent During Prey Shifts Over Several Generations

Mei-Lan Chen† , Tao Wang† , Yu-Hao Huang, Bo-Yuan Qiu, Hao-Sen Li\* and Hong Pang\*

State Key Laboratory of Biocontrol, Ecology and Evolution, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Guanyang Zhang, University of Florida, United States Aram Megighian, Università degli Studi di Padova, Italy Yanyan Li, Chinese Academy of Agricultural Sciences, China Lisheng Zhang, Institute of Plant Protection (CAAS), China

#### \*Correspondence:

Hao-Sen Li lihaosen3@mail.sysu.edu.cn Hong Pang lsshpang@mail.sysu.edu.cn †These authors have contributed

equally to this work.

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 18 April 2018 Accepted: 02 July 2018 Published: 19 July 2018

#### Citation:

Chen M-L, Wang T, Huang Y-H, Qiu B-Y, Li H-S and Pang H (2018) Physiological and Evolutionary Changes in a Biological Control Agent During Prey Shifts Over Several Generations. Front. Physiol. 9:971. doi: 10.3389/fphys.2018.00971 Biological control agents usually suffer from a shortage of target prey or hosts in their post-release stage. Some predatory agents turn to attacking other prey organisms, which may induce physiological and evolutionary changes. In this study, we investigated life history traits, gene expression and genotype frequency in the predatory ladybird beetle Cryptolaemus montrouzieri during experimental prey shifts. C. montrouzieri were either continuously fed on aphids Megoura japonica as an alternative prey for four generations or were shifted back to the initial prey mealybugs Planococcus citri in each generation. In general, the utilization of aphids resulted in reduced performance and severe physiological adjustments, indicated by significant changes in development and fecundity traits and a large number of differentially expressed genes between the two offering setup prey treatments. Within the aphid-fed lines, performance regarding the developmental time, the adult weight and the survival rate recovered to some level in subsequent generations, possibly as a result of adaptive evolution. In particular, we found that a shift back to mealybugs caused a gradual increase in fecundity. Accordingly, a genotype of the fecundity-related gene vitellogenin, of which there were several minor alleles in the initial population, became the main genotype within four generations. The present study explored the short-term experimental evolution of a so-call specialist predator under prey shift conditions. This potential rapid adaptation of biological control agents to novel prey will increase environmental risks associated with non-target effects.

Keywords: Cryptolaemus montrouzieri, biological control, non-target effects, prey shifts, life history traits, gene expression, genotype frequency

# INTRODUCTION

In biological control programs, natural enemies of pests are translocated, mass-reared and introduced as biological control agents. Following the release of these agents in their new ranges, their populations are subjected to new environmental conditions, including novel potential prey or hosts. In the post-release stage, with the decline of pest populations, the released biological control agents will suffer from a shortage of target prey or hosts. Some will therefore attack nontarget organisms, which sustain the populations of agents and sometimes expand the range of controlled pests. On the other hand, there is growing concern about the side-effects of their prey

**89**

or host expansion (Louda et al., 2003; van Lenteren et al., 2003; De Clercq et al., 2011). Empirical evidence has shown that the nontarget effects of biological control agents have threatened complex biological communities and led to a negative impact on local environments [e.g., Harmonia axyridis (Koch, 2003) in Europe and North America and Cactoblastis cactorum (Zimmermann et al., 2004), and Compsilura concinnata (Elkinton and Boettner, 2012) in North America]. Tests of host or prey range are therefore among the key procedures currently used to evaluate the potential environmental risks of an introduced agent in the pre-release stage (van Lenteren et al., 2003). In this context, the release of most generalists is now restricted, while specialists are still widely used as environmentally safe agents.

In the context of evolution, utilization of alternative food resources may act as an evolutionary driver in insects. The physiological systems of insects are confronted with various chemical components from their novel diets. Consequently, dietary shifts may impose new selection pressures, driving early physiological plasticity and subsequent evolutionary changes (Vogel and Musser, 2014; Hoang et al., 2015). Thus, it is hypothetically possible that specialists are still capable of utilizing non-target prey or hosts through an adaptation process. However, there is still little evidence of adaptive prey or host expansion to non-target organisms among specialist biological control agents (Wright and Bennett, 2017).

Numerous lines of evidence have supported the idea that herbivorous arthropods evolve in association with their host plants. On a macro-evolutionary scale, the reconstructed phylogenetic trees of such species provide histories of insectplant co-evolution [e.g., in butterflies (Janz et al., 2006), weevils (McKenna et al., 2009), and mites (Li et al., 2016a)]. On a micro-evolutionary scale, some host-associated populations are deeply divergent according to genetic marker analyses, indicating rapid evolutionary changes caused by host shifts [e.g., in thrips (Brunner et al., 2004), wasps (Stireman et al., 2005), and mites (Li et al., 2014)]. The physiological changes in host shifts reflected by expression profiling have generally involved detoxification (as reviewed in Li et al., 2007; Vogel and Musser, 2014). Accordingly, some detoxification-related genes have evolved during host adaptation [e.g., glutathione S-transferase in fruit flies (Matzkin, 2008), nitrile-specifier protein in butterflies (Wheat et al., 2007) and cytochrome P450 in fruit flies (Bono et al., 2008), and aphids (Bass et al., 2013)]. Thus far, studies on the evolutionary changes caused by dietary shifts have been restricted to herbivorous arthropods. In contrast, the evolution of carnivorous arthropods due to dietary shifts has seldom been considered (Grenier and De Clercq, 2003). After being sustained by artificial diets over a long period, carnivorous biological control might lose the ability to capture and kill live prey, although this conjecture has not been supported by any published research (Riddick, 2008). Thus, the evolutionary potential and patterns of carnivorous arthropods during prey shifts remain largely unclear, which hampers the environmental risk assessment of specialist predators used in biological control programs.

Cryptolaemus montrouzieri (well known as the mealybug destroyer) is native to Australia, and is now used worldwide as a specialist predator of mealybugs in biological control (Slipi ´ nski, 2007 ´ ). It can feed on quite a broad range of mealybug species (Kairo et al., 2013). And no significant change in developmental traits was observed in the use of different mealybug species (Qin et al., 2014). However, it can also feed on aphids, whiteflies and the eggs of moths or other ladybirds under laboratory conditions (Maes et al., 2014), suggesting potential non-target effects in its field use. Some non-target diets can sustain a complete life history (e.g., Ephestia kuehniella eggs) but are overall less suitable for survival, development, and reproduction (Maes et al., 2014), suggesting that a further adaptation process occurs when these diets are used continuously. Moreover, the macro-evolutionary pattern of the ladybird family Coccinellidae based on molecular phylogeny supports the idea that dietary shifts have played an important role in species diversification (Giorgi et al., 2009; Escalona et al., 2017). To predict the potential for and consequences of nontarget effects of C. montrouzieri, we previously examined its response to the novel prey species Megoura japonica, a common aphid pest in China, and detected reduced performance and expression of genes related to biochemical transport, metabolism, and detoxification (Li et al., 2016b). However, the question of whether these physiological changes in response to alternative prey were simply plastic or had further consequences remained unsolved.

In the present study, we test whether evolution occurs associated with prey shift of a predatory biological control agent. We used an experimental evolutionary approach (Kuhnle and Muller, 2011; Muller et al., 2017) to test the potential consequences of prey shifts in the use of C. montrouzieri for biological control. An initial population was either continuously fed alternative prey for four generations or shifted back to the initial prey in each generation. Life history traits and gene expression were investigated in each generation/prey treatment. In particular, due to pronounced changes in fecundity traits, we also examined the changes in the genotype frequencies of fecundity-related genes across generations.

#### MATERIALS AND METHODS

#### Insect Rearing

The ladybird C. montrouzieri used in this study were obtained from a population at Sun Yat-sen University, Guangzhou, China, that has been maintained under laboratory conditions with mealybugs as prey for more than 10 years. The initial prey of C. montrouzieri, the citrus mealybug Planococcus citri, was maintained on fruits of the pumpkin Cucurbita moschata. The alternative prey aphid M. japonica was maintained on plants of the horse bean Vicia faba. In the experimental stages, all insects and plants were kept in climate chambers set at 25 ± 1 ◦C with 75 ± 5% relative humidity (RH) and a 14:10 (L:D) h photoperiod.

#### Experimental Prey Shifts

A prey shift during the biological control release of C. montrouzieri was simulated under laboratory conditions

as shown in **Figure 1**. In detail, first-instar larva from the original population of C. montrouzieri (<24 h old) were distributed into two different lines, which were then either maintained on mealybugs (designated "F1M") or shifted to aphids (first generation reared on aphids, designated "F1A"). Freshly hatched larvae from F1A eggs were again randomly distributed on the initial prey mealybugs (F2M) or reared on aphids (F2A). The F3M, F3A, F4M, and F4A lines were generated similarly, resulting in a total of eight lines.

# Comparison of Life History Traits

The life history traits of the eight lines were investigated during the experimental prey shifts. Developmental traits including the survival rate and the development time of larvae as well as the adult weight and the sex ratio were investigated with 85 individuals for F1A, 135 for F2A, 93 for F3A, 96 for F4A, 91 for F1M, 63 for F2M, 83 for F3M, and 90 for F4M. Freshly hatched larvae were placed individually in plastic Petri dishes (3.5 cm diameter, 1.2 cm height), and their prey were offered ad libitum and replenished daily. The survival and the developmental time of the ladybird larvae were monitored daily. Newly emerged adults (no food or water was provided) were weighed and sexed based on the color of forelegs (Babu and Azam, 1987).

Fecundity traits, including the pre-oviposition time and the number of deposited eggs, were also surveyed, with 11 adult pairs for F1A, 10 for F2A, 11 for F3A, 14 for F4A, 12 for F1M, 12 for F2M, 14 for F3M, and 14 for F4M. Newly emerged males and females were paired in plastic Petri dishes (5 cm diameter, 2 cm height) and continuously fed by aphids or mealybugs. A piece of cotton (∼1 cm × 1 cm) was provided as an oviposition substrate and was checked daily for eggs to determine the pre-oviposition period. Once the first egg was laid, the substrates were replaced, and the number of eggs was monitored three times a week for a total period of 30 days.

Variations of all life history traits were analyzed using SPSS 20.0 (SPSS Inc.). The Kolmogorov–Smirnov test indicated that the adult weight of both males and females was normally distributed and was therefore analyzed using one-way analysis of variance (ANOVA). Because the Levene test indicated homoscedasticity, the means were separated using Tukey tests. According to the Kolmogorov–Smirnov test, the developmental time, the pre-oviposition time and the number of deposited eggs were not normally distributed. Therefore, we used the non-parametric Kruskal–Wallis H test, followed by the Mann– Whiney U test. The significance level of all tests was set at p < 0.05.

# Comparison of Gene Expression

Genome-wide expression profiling based on transcriptome sequencing was performed to screen for regulation of expression in response to the experimental prey shifts. Two females from each line were collected on the 30th day of the oviposition period for transcriptome sequencing (designated F1A1, F1A2, F2A1, F2A2, F3A1, F3A2, F4A1, F4A2, F1M1, F1M2, F2M1, F2M2, F3M1, F3M2, F4M1, and F4M2). RNA was extracted from the whole individual after 12 h of starvation. RNA extraction, RNA-seq analysis, data quality control, de novo assembly and unigene annotation followed Li et al. (2016b). Specifically, we used the FPKM (fragments per kilobase of transcript per million mapped reads) method to normalize gene expression (Trapnell et al., 2010). We removed the genes showing low expression, with an FPKM < 1, from further analysis. The regulation of gene expression in each pair of lines was tested using DESeq (Anders and Huber, 2010), employing a fold change >2 or <0.5 and a false discovery rate (FDR) < 0.05 were the criteria for defining differentially expressed genes (DEGs). To further characterize these DEGs, the number of DEGs in each pair of lines and their distribution according to EuKaryotic Orthologous Groups (KOG) classification were calculated. To visualize the expression profiles, heatmaps and clustering of the normalized expression of DEGs were generated using R (R Development Core Team, 2013).

generated in a similar way.

Since we focused on fecundity changes under prey shifts in this study, potential fecundity-related genes of C. montrouzieri were selected based on a comprehensive literature review and a recent insect fecundity study (Gilbert et al., 2005; Sun et al., 2015). A total of 10 selected genes were detected in the transcriptome data obtained in this study: vitellogenin (Vg), vitellogenin receptor (VgR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), angiotensin converting enzyme gene (ACE), Fizzy, sex-lethal (Sxl), heat shock protein 70 (HSP70), Hunchback, heat shock protein 90 (HSP90), and bicaudal D (BicD). The detail functions of these genes can be found in Sun et al. (2015). Heatmaps of the normalized expression of these genes were generated.

#### Allele Frequencies of Fecundity-Related Genes

The transcriptome analysis also enabled us to initially detect changes in allele frequencies of unigenes. First, single nucleotide polymorphisms (SNPs) of unigenes in the transcriptome were screened using GATK (McKenna et al., 2010). Then, the transcriptome data of four individuals within each generation were grouped. For example, F1A1, F1A2, F1M1, and F1M2 were grouped as F1. The allele frequencies and coding changes in the SNPs of 10 fecundity-related genes among F1–F4 were counted.

We further validated the pattern of allele frequency changes in Vg, a fecundity-related gene, in a larger sample. We partially repeated the experiment using the prey shift system described above, where a new subset of the original population was continuously fed with the alternative prey, M. japonica aphids, for five generations to generate new F1–F5 populations. Two pairs of primers were designed and used to amplify two Vg fragments that included all SNP loci (Supplementary Table 1). Twentyeight to thirty individuals from each generation were randomly selected. DNA was then extracted, and polymerase chain reaction (PCR) and sequencing of the products were then performed as described in Li et al. (2015). The allele frequencies in the SNPs were subsequently counted, and the haplotype networks of the two PCR fragments were drawn in Network 5.0.0.3 using a median-joining method (Bandelt et al., 1999).

#### RESULTS

#### Life History Traits

The developmental traits of each line, including the development time, the adult weight, the female ratio and the mortality, are shown in **Figure 2**. In comparison with the mealybug-fed lines, the aphid-fed lines always exhibited significantly longer larval developmental times (**Figure 2A**) and lower weights of adult males and females (**Figures 2C,D**). When four generations were continuously fed aphids, the larval developmental time decreased from F2A onward (**Figure 2A**), and the adult weight increased in F4A (**Figures 2C,D**). The overall mortality among developmental stages was increased in F2A compared with F1A but decreased greatly in F3A and F4A (**Figure 2F**). The shift back to mealybugs led to an increase in larval developmental time in F4M (**Figure 2A**) and a decrease in pupal developmental time from F2M onward (**Figure 2B**). In addition, it appeared that there was no strong effect of the prey shift on the female ratio (**Figure 2E**).

The fecundity traits of each line, including the pre-oviposition time and the number of eggs deposited within 30 days, are shown in **Figure 3**. The pre-oviposition time of the aphid-fed lines was usually significantly longer than that of the mealybug-fed lines (**Figure 3A**). However, the number of deposited eggs in F1M was not significantly higher than that in F1A (**Figure 3B**). The number of deposited eggs did not significantly change between generations in the aphid-fed lines but gradually increased from the first to the fourth generation in the lines shifted back to mealybugs (**Figure 3B**).

#### Gene Expression

The transcriptomes of all eight lines and 16 female individuals were sequenced. Each of the sequenced samples exhibited 23–34 million high-quality reads, comprised of 5–11 billion base pairs (bp). A total of 162 genes were considered DEGs across the treatments experiment-wide. The KOG classification of these DEGs showed that most were distributed among carbohydrate, amino acid, lipid transport and metabolism, energy production and conversion, and signal transduction mechanisms (Supplementary Figure 1). Among these DEGs identified experiment-wide, an average of 19.33 DEGs were identified between pairs of aphid-fed lines, 32.50 between pairs of mealybug-fed lines, and 47.38 between pairs of aphid- and mealybug-fed lines (Supplementary Table 2). The heatmaps of the expression of all DEGs showed that their expression depended on not only their prey species but also their generation (Supplementary Figure 2).

Due to the pronounced change observed in fecundity, we further focused on potential genetic changes associated with fecundity under prey shifts. Among the 10 selected fecundityrelated genes, none was a DEG. Vg was the only gene that was always expressed at a lower level in aphid-fed lines than in mealybug-fed lines (**Figure 4**).

#### Allele Frequencies of Fecundity-Related Genes

A total of 93 SNPs in fecundity-related genes were detected in the transcriptome data (Supplementary Table 3). Among these SNPs, 4/7 were involved in non-synonymous changes in Vg, 9/22 in VgR, 0/9 in HMGCR, 1/17 in ACE, 2/11 in Fizzy, 0/6 in Sxl, 0/11 in HSP70, 0/5 in HSP90, and 1/7 in DisC. Among these non-synonymous SNPs, those in Vg exhibited the greatest allele frequency changes from F1 (or the initial population) to F4, ranging from 0.375 to 1 (**Table 1**).

The repeated rearing scheme and Sanger sequencing-based validation showed that the frequencies of these four minor alleles in Vg involving non-synonymous changes ranged from 0.133 to 0.533 in F1 (or the initial population). The frequencies of these alleles did not change greatly in F2 and F3 but increased to 0.679– 0.821 in F4, and these alleles were the only alleles detected in F5 (**Table 1**). Additionally, the haplotype network of the two PCR fragments suggested that a rare haplotype in F1-F3 became the main haplotype in F4 and F5 (Supplementary Figure 3).

# DISCUSSION

#### Physiological Changes During Feeding on Non-target Prey

Diet is one of the major determinants of physiological performance. When their diet changes, both herbivores and carnivores usually require physiological adjustments to meet their nutritional requirements and cope with new toxins from their new diet (Glendinning, 2007; Vogel and Musser, 2014). In the present study, the initial prey (mealybugs) and the alternative prey (aphids) exhibited different biochemical compositions (Brown, 1975) and should present differences in nutritional value or toxins, with which C. montrouzieri would have to cope. Based on the evidence regarding life history traits and gene expression,

we found that both the shift to aphids and the shift back to mealybugs led to severe physiological changes in C. montrouzieri. The shift to aphids always led to reduced performance, as inferred by the significant extension of development and preoviposition times and the decreases in adult weight and the number of deposited eggs. Furthermore, the whole-genome expression profiles after prey shifts mainly showed modulation of the expression of genes related to biochemical transport and metabolism. This transcriptional plasticity is expected be a response to the change in the biochemical composition of the prey. These findings regarding physiological changes were in line with those from previous studies in which C. montrouzieri was


TABLE 1 | Single nucleotide polymorphisms (SNPs) in vitellogenin (Vg) and their allele frequencies from F1 to F4 (or F5).

switched to aphid prey for one generation (Maes et al., 2014; Li et al., 2016b).

#### Short-Term Adaptation Under Prey Shifts

Because C. montrouzieri experienced performance reductions and physiological adjustments when feeding on aphids, we assumed that further adaptation occurred when the ladybirds were continuously maintained on this alternative prey. Several reports of experimental evolution in herbivorous arthropods support the idea that dietary shifts impose strong selection pressure, forcing herbivores to evolve within several generations (Sezer and Butlin, 1998; Warbrick-Smith et al., 2006; Kuhnle and Muller, 2011). In this study, variation of life history traits and gene expression was also detected in lines within the same prey treatment. Within the aphid-fed lines, performance recovered to a certain degree in subsequent generations, as inferred by the larval developmental time (recovered from F2), the female and male weight (recovered from F4) and the mortality (recovered from F3). It appeared that the physiological plasticity of the initial population was already sufficient for utilization of the alternative prey. However, performance under this adverse condition was reduced, and positive selection is therefore expected to further alter performance in the direction of the optimum (Crispo, 2007; Hoang et al., 2015). In addition to positive selection, this prey adaptation might be a result of genetic drift or a maternal effect.

#### Change in Fecundity and Its Potential Genetic Basis

In this study, the change in fecundity was the most pronounced physiological change observed experiment-wide. In the lines shifted back to mealybugs from aphids, the younger generations always exhibited a significantly greater number of deposited eggs than the older generations. To explore the potential genetic basis of this change in fecundity, we first selected candidate genes from 10 fecundity-related genes based on the regulation of their expression under prey shifts. Among these candidate genes, only the expression of Vg in females was consistently altered in response to prey type. Similarly, in a previous study, we found that when C. montrouzieri was switched to aphids, Vg was significantly downregulated, while other fecundity-related genes were not differentially expressed (Li et al., 2016b). Vg encodes the major egg yolk protein precursor in insects, and its expression generally corresponds to fecundity in females (Tufail et al., 2014). In this study, however, the increase in fecundity was not based on increased expression of Vg; rather, it is expected to be a consequence of genetic changes.

Therefore, we further tested the potential genetic changes in fecundity-related genes in response to this change in fecundity. Again, Vg exhibited the greatest change in allele frequencies in non-synonymous SNPs in this prey shift experiment. In this process, a genotype that contained several minor alleles in the initial population became the main genotype in F2 and the only genotype in F3. Replication of this prey shift experiment showed that this minor genotype became the main genotype in F4 and the only genotype in F5. Based on the results of these two replicate experiments, we suggested that positive selection, not random genetic drift, led to this change in genotype frequency.

Increased fecundity is considered to be adaptative (Stearns and Hoekstra, 2003). It has been reported that the fecundity of insects can be affected by several mutations in specific genes (Sun et al., 2015), and a host shift of insects may lead to the evolution of genes related to reproduction (Guillen et al., 2014). In this study, we explored the downregulation of Vg during the utilization of alternative prey as well as the relationship between the Vg genotype frequency and fecundity in lines that were shifted back to the initial prey. However, the changes in Vg genotype frequency neither increased the fecundity of the aphid-fed lines nor affected Vg expression to a significant level. We suggest that this lack of change was due to the limitation of fecundity by insufficient nutritional conditions associated with the alternative prey, even though a selected genotype presents the potential for higher fecundity. On the other hand, the selection pressure exerted by the alternative prey might be exerted directly on Vg or indirectly on other genes linked to Vg.

#### Conclusion and Implications for Biological Control

This study explored the short-term physiological and evolutionary changes in a so-call specialist biological control agent during experimental prey shifts over several generations. For better explaining this prey shift adaptation, other factors such as behavior and gut microbiota needed to be considered in the further. This is the first report to address the experimental evolution of carnivorous arthropods and biological control

agents under prey shifts. This potential rapid adaptation of biological control agents to novel prey will increase environmental risks associated with non-target effects. Hence, the current environmental risk assessment strategy based on the host ranges of biological control agents (van Lenteren et al., 2003) should also include evolutionary considerations.

#### DATA AVAILABILITY STATEMENT

All raw sequence data generated and analyzed for this study can be found in the NCBI Short Read Archive (SRA) under BioProject ID PRJNA449583 (BioSample ID: SAMN08912452– SAMN08912467).

#### AUTHOR CONTRIBUTIONS

M-LC, Y-HH, and B-YQ performed the experiments. M-LC and H-SL analyzed the data. M-LC, TW, H-SL, and HP wrote and revised the manuscript.

#### REFERENCES


#### FUNDING

This work was supported by the National Natural Science Foundation of China (Grant Nos. 31572052 and 31702012), the Science and Technology Planning Project of Guangdong Province, China (Grant No. 2017B020202006), and the National Key R&D Program of China (Grant No. 2017YFD0201000).

#### ACKNOWLEDGMENTS

We would like to thank Li-Jun Ma, Zhan Ren, Pei-Tao Chen, and Si-Wen Wu of Sun Yat-sen University for assistance with the experiments.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00971/full#supplementary-material



exceptions? Evidence from a goldenrod-insect community. Evolution 59, 2573– 2587. doi: 10.1554/05-222.1


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chen, Wang, Huang, Qiu, Li and Pang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Functional Analysis of a Carboxylesterase Gene Associated With Isoprocarb and Cyhalothrin Resistance in Rhopalosiphum padi (L.)

#### Kang Wang, Yanna Huang, Xinyu Li and Maohua Chen\*

State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Crop Pest Integrated Pest Management on the Loess Plateau of Ministry of Agriculture, Northwest A&F University, Yangling, China

#### Edited by:

Su Wang, Beijing Academy of Agriculture and Forestry Sciences, China

#### Reviewed by:

Bruno Gomes, Liverpool School of Tropical Medicine, United Kingdom Pin-Jun Wan, China National Rice Research Institute (CAAS), China

> \*Correspondence: Maohua Chen maohua.chen@nwsuaf.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 03 April 2018 Accepted: 06 July 2018 Published: 25 July 2018

#### Citation:

Wang K, Huang Y, Li X and Chen M (2018) Functional Analysis of a Carboxylesterase Gene Associated With Isoprocarb and Cyhalothrin Resistance in Rhopalosiphum padi (L.). Front. Physiol. 9:992. doi: 10.3389/fphys.2018.00992 Carboxylesterase (CarE) is an important class of detoxification enzymes involved in insecticide resistance. However, the molecular mechanism of CarE-mediated insecticide resistance in Rhopalosiphum padi, a problematic agricultural pest, remains largely unknown. In the present study, an isoprocarb-resistant (IS-R) strain and a cyhalothrinresistant (CY-R) strain were successively selected from a susceptible (SS) strain of R. padi. The enzyme activity indicated that enhanced carboxylesterase activity contributes to isoprocarb and cyhalothrin resistance. The expression levels of putative CarE genes were examined and compared among IS-R, CY-R, and SS strains, and only the R. padi carboxylesterase gene (RpCarE) was significantly over expressed in both the IS-R and CY-R strains compared to the SS strain. The coding region of the RpCarE gene was cloned and expressed in Escherichia coli. The purified RpCarE protein was able to catalyze the model substrate, α-naphtyl acetate (Kcat = 5.50 s−<sup>1</sup> ; Km = 42.98 µM). HPLC assay showed that the recombinant protein had hydrolase activity against isoprocarb and cyhalothrin. The modeling and docking analyses consistently indicated these two insecticide molecules fit snugly into the catalytic pocket of RpCarE. Taken together, these findings suggest that RpCarE plays an important role in metabolic resistance to carbamates and pyrethroids in R. padi.

Keywords: carboxylesterase, Rhopalosiphum padi, isoprocarb, pyrethroid, metabolism resistance

# INTRODUCTION

The bird cherry-oat aphid, Rhopalosiphum padi, is a serious worldwide wheat pest. R. padi causes serious damage to wheat by directing extracting nourishment and transmitting barley yellow dwarf virus (Du et al., 2007; Wang et al., 2016). In China, aphid control is dependent primarily on the application of chemical insecticides, including carbamates, and pyrethroids. Unfortunately, the extensive application of insecticides has resulted in the development of resistance in field populations of R. padi (Chen et al., 2007; Zuo et al., 2016). Thus, uncovering the resistance mechanism and its molecular basis is essential to better control this important pest.

**98**

The two main mechanisms predominantly responsible for insecticide resistance are target site insensitivity and metabolic resistance due to elevated levels of insecticide detoxifying enzymes (Li et al., 2007; Liu, 2015; Barres et al., 2016). Biochemical assays have demonstrated the resistance to carbamates and organophosphates caused by insensitive acetylcholinesterase (ACE) in a lot of insect species (Hemingway et al., 2004; Bass et al., 2014). A S431F substitution of ACE-1 in Myzus persicae resistant clones was correlated with the resistance of the species to pirimicarb (Nabeshima et al., 2003). The A302S in ACE-1 was found to be associated with organophosphates resistance in Aphis gossypii (Li and Han, 2004). The substitutions in ACE disturbed both the space and hydrophobicity, thus, preventing pirimicarb from interacting with the active site in the resistant M. persicae and A. gossypii (Nabeshima et al., 2003; Andrews et al., 2004). Mutations in the voltage-gated sodium channel, a trans-membrane ion channel that plays an essential role in the initiation and propagation of action potentials in neurons, are involved in resistance to pyrethroids (Zlotkin, 1999). L1014F and M918T initially identified in the housefly and, respectively, referred as kdr and super-kdr mutations are two most common mutations in the voltage-gated sodium channel (Williamson et al., 1996; Franck et al., 2012). The kdr mutation can cause moderate resistance to DTT and pyrethroids, whereas the super-kdr mutation is usually linked to the kdr and shown to significantly enhance the resistance phenotypic expression due to kdr (Vais et al., 2000; Eleftherianos et al., 2008; Franck et al., 2012). The kdr or super-kdr mutations have been indentified in the pyrethroid resistant strains of several aphid species (Eleftherianos et al., 2008; Marshall et al., 2012; Foster et al., 2014). The metabolic resistance has evolved by the amplification, overexpression and coding sequence variation of three major detoxification enzymes, i.e., cytochrome P450 monooxygenases (P450s), carboxylesterases (CarE), and glutathione-S-transferases (GSTs) (Hemingway and Ranson, 2000; Hemingway et al., 2004; Li et al., 2007; Ramsey et al., 2010).

In recent years, R. padi had developed resistance to some insecticides (Zuo et al., 2016; Zhang et al., 2017). Regional susceptibility analyses of 12 R. padi field populations has shown that the populations varied in their resistance levels to the tested insecticides, with the highest resistant ratio of 13.6, 18.2, 13.1, and 12.1 to imidacloprid, bifenthrin, decamethrin, and abamectin, respectively (Zuo et al., 2016). Both insecticide target site mutations and metabolic resistance were found in this aphid. Acetylcholinesterase gene mutations might contribute to the resistance of this pest to organophosphate and carbamate insecticides (Chen et al., 2007; Bettaibi et al., 2016). Significant higher GST activity was found in two field populations collected in China (Zhang et al., 2017). In the imidacloprid resistant strain of R. padi, the CYP6CY3-1 and CYP6CY3-2 were significantly overexpressed (Wang et al., 2018). So far, information about CarE from R. padi and its role in insecticide resistance is still unavailable.

Carboxylesterases (CarEs, EC 3.1.1.1) are a superfamily of metabolic enzymes that hydrolyse carboxylic ester bonds with the addition of water and are thought to play important physiological roles in xenobiotic metabolism (Campbell et al., 2003; Wheelock et al., 2005; Birner-Gruenberger et al., 2012). The overexpression of CarEs has been associated with carbamate and pyrethroid resistance in several insects (Wheelock et al., 2005). Carboxylesterase E4, which is produced by resistant M. persicae, both hydrolyses and sequesters the insecticide, leading to carbamate and pyrethriod resistance in these aphids (Devonshire and Moores, 1982; Lan et al., 2005). Elevated esterase hydrolysis activity is related to cyhalothrin resistance in Aphis glycines (Xi et al., 2015).

In the present work, isoprocarb-resistant (IS-R) and cyhalothrin-resistant (CY-R) strains and a relatively susceptible (SS) strain were obtained from the same field population by successive selection with or without insecticide. CarE activity was evaluated among the IS-R, CY-R, and SS strains, and the expression patterns of seven putative CarE or CarE-likes genes were studied by RT-PCR, with the target gene (RpCarE) being confirmed and cloned. We heterologously expressed RpCarE in Escherichia coli cells and purified the fusion protein. Moreover, we measured the activity of the fusion protein against the standard substrate (α-naphthyl acetate) and examined the hydrolase activity against isoprocarb and cyhalothrin. Homology modeling and insecticide docking studies were also conducted to interpret the substrate metabolic detoxification. The current results will contribute to understanding of the mechanism of insecticide resistance mediated by RpCarE.

# MATERIALS AND METHODS

#### Insects

The susceptible strain (SS) originated from a field R. padi population collected in 2013 from Gansu Province, China. The isoprocarb resistant strain (IS-R) and cyhalothrin resistant strain (CY-R) were successively selected by exposing isoprocarb or cyhalothrin for more than 60 generations. The IS-R strain showed an LC<sup>50</sup> of 33.436 mg L−<sup>1</sup> for isoprocarb, with an ∼32.4-fold increased resistance compared with the SS (LC<sup>50</sup> of 1.032 mg L−<sup>1</sup> for isoprocarb). The CY-R strain (LC<sup>50</sup> of 8.858 mg L −1 for cyhalothrin) displayed ∼27.8-fold increased resistance compared to the SS (LC<sup>50</sup> of 0.319 mg L−<sup>1</sup> for cyhalothrin) (Supplementary Table S1). During the insecticide selection, the toxicity of insecticides was evaluated every four generations, and the evaluated LC<sup>50</sup> values were used as the selection concentration for the following four generations. Resistance ratio = LC<sup>50</sup> of resistant strain/LC<sup>50</sup> of susceptible strain. All insects were reared on seedlings of wheat (cultivar "Xiaoyan 22") in mesh cages (41 cm × 41 cm × 41 cm) in the laboratory at 23 ± 1 ◦C, 70% relative humidity and a photoperiod of L16:D8.

#### Chemicals

The insecticides used for bioassays included isoprocarb (95% purity, Anhui Huaxing Chemical industry Co. Ltd., China) and cyhalothrin (96% purity, Yancheng Nongbo Bio-technology Co. Ltd., China).

α-naphthol (α-N) and fast blue RR salt were products of Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). α-naphthyl acetate (α-NA) was purchased from Solarbio (Beijing,

China). All other chemicals were of analytical grade and purchased from commercial suppliers.

#### Carboxylesterase Activity Assays

Carboxylesterase (CarE) activity was determined by the method of Han et al. (2012) and Li et al. (2016) with modification. All aphids tested were fed on seedlings (three-leaf stage) of wheat cultivar "Xiaoyan 22" under the aforementioned condition, and did not contact with any insecticides from the first instar to the adult before enzyme activity assays. Ten 9-days old apterous adult specimens were taken from each of the three strains (IS-R, CY-R, and SS), homogenized on ice in 1 mL of pre-chilled PBS (0.1 mol L−<sup>1</sup> , pH 7.5, containing 1.0 × 10−<sup>3</sup> mol L−<sup>1</sup> EDTA) and centrifuged at 4◦C and 12,000 × g for 10 min. The enzyme source was supernatant of the homegenized specimens. Supernatants were used for testing. Protein concentrations were determined using the Bradford method with bovine serum albumin as the standard (Bradford, 1976). The assay mixture contained 100 µL of substrate solution (10 mM α-NA and 3 mM Fast Blue RR salt, pH 6.0) and 100 µL of enzyme solution. After incubation at 30◦C for 10 min, assays were conducted at 30◦C in 96-well microplates, and absorbance was measured at 450 nm in a microplate reader (M200 PRO, Tecan, Männedorf, Switzerland). The experiment was repeated three times.

#### Screening of R. padi CarE Genes Associated With Insecticide Resistance

Putative CarE genes were systematically searched based on R. padi transcriptome data (Duan et al., 2017) and resequenced via PCR amplification. Sequences of the seven CarE genes obtained were deposited in GenBank database. The GenBank accession numbers for each sequence are: CL2012, MH561903; CL3077, MH561904; CL869, MH561905; U11937, MH561906; U14486, MH561907; U4474, MH561908; and U6896, MH561909. Ten apterous adult aphids from each of the three strains (IS-R, CY-R, and SS) were subjected to total RNA extraction using Trizol (Invitrogen, Carlsbad, CA, United States) and Direct-zol RNA miniprep kit (Zymo, Irvine, CA, United States) according to the manufacturer's protocol, including a DNAse treatment. First-strand cDNA was synthesized using the M-MLV reverse transcriptase cDNA Synthesis Kit (Promega, Madison, WI, United States). RT-qPCR was performed in a final volume of 20 µL, including 10 µL of FastStart Essential DNA Green Master (Roche, NJ, United States), 0.8 µL of each specific primer (**Table 1**), 2 µL of cDNA template, and 6.4 µL of RNase-free water. The reaction was performed with the thermal cycler program: 95◦C for 10 min, followed by 40 cycles of 95◦C for 10 s, 58◦C for 20 s, and 72◦C for 20 s. A melting curve was determined (ramping from 55◦C to 95◦C by 0.5◦C every 5 s) to confirm the amplification of specific PCR products. The R. padi β-actin and EF-1α (elongation factor 1α) genes were used as the internal control (**Table 1**). RT-qPCR was performed on a LightCycler Nano System (Roche, Mannheim, Germany), and the relative expression level was calculated using the 2−11Ct method (Pfaffl, 2001). qPCR experiments were repeated using three biological replicates using aphids from the TABLE 1 | Primers used for qRT-PCR, RACE, cloning and protein expression.


same generation, and each replicate was performed at least three times.

# Cloning of Carboxylesterase From R. padi

Based on the above-mentioned screening of CarE genes from R. padi transcriptome data (Duan et al., 2017), a carboxylesterase gene (CL2012, hereinafter referred as RpCarE)) which was overexpreesed in the resistant strains was confirmed by qPCR, and the coding region was cloned by RT-PCR. Pre-analysis showed the 5<sup>0</sup> sequences of RpCarE from R. padi transcriptome were sufficient for protein expression. Gene-specific primes were used for 3<sup>0</sup> -RACE (RpCarE-3R1 and RpCarE-3R2) to clone the 3<sup>0</sup> sequences of the gene. To confirm the accuracy of the RpCarE linked from the 3<sup>0</sup> -RACE results, a specific primer pair (RpCarE-CF and RpCarE-CR) was designed to amplify the full coding region of the gene. The primers used for RACE are shown in **Table 1**. The amplification reaction mix contained 2 units of Taq DNA polymerase (5 U/µL, Sangon Biotech Co. Ltd., Shanghai, China), 100 µM dNTPs, 4 mM MgCl2, 0.4 µM of forward and reverse primers, and 1 µL of template DNA. All purified PCR products were cloned into pGEM-T easy vectors (Promega, Madison, WI, United States) and transformed into Escherichia coli DH5α competent cells (Takara, Kyoto, Japan). To ensure that the correct cDNA sequences were obtained, three positive clones from each sample were randomly chosen for bidirectional sequencing on an Applied Biosystems 3730 automated sequencer (Applied Biosystems, Foster City, CA, United States).

#### Sequence Analysis

fphys-09-00992 July 23, 2018 Time: 15:53 # 4

Sequence identification and similarities were analyzed using BLAST<sup>1</sup> . Amino acid sequences of RpCarE and homologs from other insect species were aligned using ClustalW2 software (Larkin et al., 2007). The molecular weight (MW) and theoretical isoelectric point (pI) of RpCarE were calculated using the ExPASy (Gasteiger et al., 2003). Signal peptides were predicted using the Signal P4.1 software (Petersen et al., 2011).

# Protein Expression/Purification and Western Blot Analysis

The encoding region of RpCarE was amplified from the pGEM-T/RpCarE plasmid with gene specific primers (RpCarE - BamHI: CGCGGATCCATGGAAGTGGTCATCGAACAAGGT; RpCarE-HindIII: CCCAAGCTTTTAAACAATGGATTCTTTT ATTAA) introducing BamHI and HindIII restriction sites (underlined). Amplicon was purified and digested with corresponding restriction enzymes and subcloned into pET-28a (Novagen, Merck, Germany). The plasmid was verified by restriction digestion and nucleotide sequencing. The expression of RpCarE in the E. coli BL-21 (DE-3) strain (Takara, Kyoto, Japan) was induced using Isopropyl β-D-thiogalactoside (IPTG, with final concentration of 0.4 mM). Cultured cells were harvested by centrifugation (6,000 × g, 20 min, 4◦C). The resulting pellet was re-suspended in 50 mM Tris-HCl (pH 8.0) and lysed by sonication (Sonics Vibra-Cell, 130 w, 30% power). Cell lysate was centrifuged at 12,000 × g for 30 min, and the supernatant was incubated with 2 mL of cOmplete His-Tag Purification Resin (Roche) for 1 h with shaking, and protein was eluted using lysis buffer supplemented with 50 mM imidazole. Protein concentrations were determined using the Bradford method with bovine serum albumin as the standard (Bradford, 1976).

Recombinant RpCarE was analyzed using 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. For western blot, separated proteins were electrotransferred to a PVDF membrane (Millipore), and the membrane was subsequently blocked with 5% skim milk powder in TBST for 1 h. Target proteins were verified with mouse anti-His mouse monoclonal antibodies (CWBIO) followed by staining with goat-anti-mouse IgG (CWBIO). Blots were visualized using WesternBright ECL kit (Advasta), and signal was detected using a chemiluminescence imaging system (Clinx Science Instruments, Shanghai, China).

#### Determination of Enzymatic Activity

The kinetics of purified RpCarE protein against α-NA was determined using the method of Li et al. (2016) with modification. Reactions were carried out in a 96-well microplate, with each well containing 100 µL of appropriately diluted purified protein in Tris-HCl buffer and 100 µL of α-NA in buffer (3 mM Fast Blue RR salt and increasing concentrations of α-NA). The formation of α-N was recorded at 450 nm for 5 min in a microplate reader (M200 PRO, Tecan, Männedorf, Switzerland) and quantified using α-N standard curves. The values for kcat and Km were estimated using the "Hyper32" hyperbolic regression software (Xie et al., 2014).

#### HPLC Analysis of Insecticide Metabolism

Assays for the hydrolysis of isoprocarb and cyhalothrin were carried out by monitoring substrate loss with a Hitachi D-2000 Elite HPLC system (Hitachi High-Technologies Corporation, Tokyo, Japan) as described by Li et al. (2016), with some modifications. First, 100 µL of isoprocarb and cyhalothrin (each in 100 µM) was incubated separately with 100 µL of recombinant RpCarE protein (0.2 mg mL−<sup>1</sup> ). Insecticides with heat-inactivated enzyme served as controls. Incubation reactions were carried out at 30◦C for 1 h and stopped by addition of 100 µL acetonitrile. Samples were centrifuged at 12,000 × g for 10 min before transferring the supernatant to HPLC vials. Ten microliter of the supernatant were injected onto a reverse-phase Symmetry C18 column (250 mm × 4.6 mm, 5 µm, Waters Crop., Milford, MA, United States) with a flow rate of 1 mL min−<sup>1</sup> at 30◦C for 30 min. Reactions were run with a mobile phase (80% acetonitrile: 20% water) and monitored at 215 and 230 nm for isoprocarb and cyhalothrin, respectively.

#### RpCarE Modeling and Substrate Docking

The molecular model of RpCarE was created as described by Zhang (2008) and Elzaki et al. (2017) using the I-TASSER on-line server<sup>2</sup> . Isoprocarb and cyhalothrin molecules were docked into the active site of RpCarE using the Surflex-Dock (SFXC) function in the SYBYLx2.0 software (Tripos, St. Louis, MO, United States). Final figures were prepared using the PyMOL program (DeLano, 2002).

#### Statistical Analysis

The significance of the differences in mRNA levels and enzyme activities was determined by non-parametric Mann–Whitney Utest with the level of significance at P < 0.05. Data analyses were performed using SPSS Version 21.0 software (SPSS Inc., Chicago, IL, United States).

# RESULTS

#### Determination of CarE Activity

Carboxylesterase activities of different strains were determined using α-NA as a substrate. IS-R and CY-R strains exhibited significantly higher carboxylesterase activity (2.20- and 2.05-fold) compared with the susceptible strain (P < 0.05) (**Figure 1** and Supplementary Table S2).

<sup>1</sup> blast.ncbi.nlm.nih.gov/blast/

<sup>2</sup>https://zhanglab.ccmb.med.umich.edu/I-TASSER/

#### CarE Gene mRNA Level Determination

Seven putative carboxylesterase genes were obtained from the transcriptome database of R. padi (**Table 1**), and compared among susceptible and resistant strains. Relative expression levels of CL2102 were 4.99- and 2.73-fold higher in IS-R and CY-R than in the susceptible strain, respectively (**Figure 2**). Relative expression analysis of the seven carboxylesterase genes revealed that CL2102 was moderately abundant in the isoprocarb resistant and cyhalothrin resistant strains of R. padi, whereas the other six carboxylesterase genes was lowly abundant in the two resistant strains.

#### cDNA Amplification

To obtain the complete sequence, gene-specific primers for CL2102 were designed for amplification of the 3<sup>0</sup> cDNA ends. The resulting carboxylesterase cDNA sequence contains an open reading frame (ORF) of 1,581 bp that encodes a putative protein containing 526 amino acid residues. Its calculated molecular weight is 59.22 kDa, and the predicted isoelectric point is 6.61. No signal peptide was detected upon amino acid sequencing. Homology analysis against the already published genes in GenBank indicated that carboxylesterase shares high identity with M. persicae FE4-like esterase (XP\_022165140; 80% identity), Acyrthosiphon pisum FE4 esterase (XP\_001951456; 80% identity), A. glycines carboxylesterase (AEI70326; 78% identity), and A. gossypii carboxylesterase (BAE66715; 78% identity). The alignment shows that this R. padi carboxylesterase has highly conserved residues that form an atalytic triad (S185-H432-E312) (**Figure 3**).

#### Expression of RpCarE in E. coli and Verification via Western Blot

To functionally express RpCarE, its coding sequence was inserted into the pET-28a expression vector and expressed in the BL-21 (DE-3) E. coli strain. Cells harvested 12 h after IPTG induction were considered to be the most suitable for protein expression and isolation based on the Coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) images (data not shown). Immunoreactivity of the target proteins for His-mouse antibodies in the western blot are shown in **Figure 4**. The fusion protein with a His6 tag migrated as a single band with a molecular mass of ∼60 kDa, which matched the

FIGURE 2 | Comparison of carboxylesterase gene mRNA levels among the isoprocarb resistant (IS-R), cyhalothrin resistant (CY-R), and susceptible (SS) strains. Values are plotted as the mean ± SE of three repeats. Vertical bars represent the standard error of the mean of three independent replicates. Asterisk indicates significant differences by Mann–Whitney U-test (∗P < 0.05, ∗∗P < 0.01).

calculated molecular mass of 59.22 kDa. These results indicate that RpCarE was successfully expressed in E. coli.

# RpCarE Activity for α-Naphthyl Acetate

The activity of the purified recombinant enzyme toward α-NA is shown in **Table 2**. RpCarE exhibited high catalytic efficiency with a Km of 42.98 µM, a Kcat of 5.50 s−<sup>1</sup> and a specific activity of 4.43 µM s−<sup>1</sup> (µM−<sup>1</sup> ·protein). These results suggest that the purified recombinant protein (RpCarE) has a high affinity and turnover of substrate.

# RpCarE Metabolism of Isoprocarb and Cyhalothrin

Specific activities of the purified recombinant enzyme against isoprocarb and cyhalothrin were assessed by measuring substrate depletion. As shown in **Figure 5**, the fusion protein exhibited significant activity toward both isoprocarb and cyhalothrin.

#### The Binding Mode of Insecticides to RpCarE

To better understand the underlying mechanism causing RpCarE to metabolize insecticides, a molecular docking simulation was conducted using the homology mode of RpCarE with insecticides, including isoprocarb and cyhalothrin. The docking experiments were conducted using the Surflex-Dock (SFXC) program from the SYBYL X2.0 software. The RpCarE protein model was generated based on the crystal structure of Lucilia cuprina α-E7. RpCarE contains several conserved CarE characteristics, such as the canonical catalytic triad (serine, glutamate, and histidine) and the oxyanion hole (alanine and glycine) (**Figure 3**). Modeling and docking analyses showed that isoprocarb and cyhalothrin fit snugly into the catalytic pocket of RpCarE (**Figure 6**). The Asn-108 residue was predicted to anchor isoprocarb by hydrogen donors (**Figure 6A**). Tyr-109 and Glu-116 were the major determinants in cyhalothrin binding, positioning the molecule close to catalytic triads (Ser185-His432- Glu312) (**Figure 6B**). These results indicate that the active site pocket of RpCarE is ideally shaped for isoprocarb and cyhalothrin and is able to effectively metabolize these insecticides.

# DISCUSSION

Carboxylesterases, an important detoxification enzyme family, are involved in mediating the metabolic resistance to carbamate, organophosphate and pyrethroid insecticides in many insect species (Wheelock et al., 2005). To reveal the CarE-mediated metabolism of isoprocarb and cyhalothrin in R. padi, one isoprocarb resistance strain (IS-R), and one cyhalothrin resistance strain (CY-R) were obtained by successive selection with insecticides. CarE activities were measured in IS-R, CY-R and susceptible strains of R. padi, yielding significantly higher activities in the IS-R and CY-R strains than in the SS strain (**Figure 1**), suggesting a correlation between CarE and isoprocarb

and cyhalothrin resistance. Furthermore, the hydrolytic activity of a R. padi CarE (RpCarE) to isoprocarb and cyhalothrin suggest that RpCarE is involved in resistance to the chemicals. In the early 1970s, resistant strains of M. persicae were shown to possess improved hydrolytic activity against the esterase standard substrate α-NA (Needham and Sawicki, 1971). Further study revealed that EF4 accounts for the broad spectrum of resistance to carbamate and pyrethroid insecticides (Devonshire and Moores, 1982). CarE activity in A. gossypii was determined using several standard substrates and was shown to be significantly higher in omethoate, deltamethrin, and malathion resistant strains than in susceptible strains (Cao et al., 2008a,b; Pan et al., 2009). There was a significant difference in carboxylesterase activity between a beta-cyperthrin-resistant strain (4,419-fold) and a susceptible strain of Musca domestica (Zhang et al., 2007).

Based on sequence alignment and conserved motifs, seven carboxylesterase and carboxylesterase-like genes were identified. The relative expression patterns of candidate genes were determined in the insecticide resistant and susceptible strains. Among the seven CarE genes, only CL2012 (RpCarE) was found to have significantly increased expression levels in both the IS-R and CY-R compared to the SS. A homology search of CL2012 showed maximum similarity (86%) to A. gossypii carboxylesterase. Therefore, we proposed that the CL2012 gene, as the carboxylesterase gene of R. padi, might participate in isoprocarb and cyhalothrin resistance. Twenty-eight CarE and CarE-like genes were identified in the transcriptome of Laodelphax striatellus, and LsCarE1 was significantly overexpressed in the chlorpyifos-resistant and chlorpyifos-relaxed selection strains by 32.06- and 8.6-fold, respectively. Additionally, overexpressed LsCarE1 mediated chlorpyifos resistance in L. striatellus (Zhang et al., 2012). In the deltamethrin-resistant strain of A. gossypii, the transcript levels of carboxylesterase were significantly enhanced, and this up-regulation was responsible for the development of resistance to deltamethrin (Cao et al., 2008a). The enhanced activity of carboxylesterases of M. persicae confers broad spectrum resistance to organophosphate, carbamate and pyrethroid (Bass et al., 2014). The M. persicae field population resistant to methomyl and omethoate exhibited a higher carboxylesterases activity compared to the laboratory susceptible strain (Tang et al., 2017). Three populations of the cowpea aphid (Aphis craccivora) collected in Egypt showed higher carboxylesterases which may cause the resistance of the species to organophosphates, carbamates, and neonicotinoids (Fouad et al., 2016). CarE enzyme activity were positively correlated with resistance level to chlorpyrifos, deltamethrin, and methomyl in the field populations of Sitobion avenae collect in wheat fields (Zhang et al., 2017).

There are a conserved catalytic triad (Ser, His, and Glu) and an oxyanion hole for carboxylesterases (Stok et al., 2004; Wheelock et al., 2005). During hydrolysis of carboxylesterases, a nucleophilic attack occurs by Ser on the carbon of the carbonyl group, which is next transferred to the His from the catalytic Ser. The protonated His is, in turn, stabilized via a hydrogen bond to the Glu (Stok et al., 2004). The Ser nucleophile attacks the substrate and forms the first tetrahedral intermediate, which is stabilized by two Gly residues in the oxyanion hole (Wheelock et al., 2005). The intermediate rapidly collapses to produce the acyl-enzyme complex, which then undergoes attacks by an His-activated water molecule and forms the second tetrahedral intermediate. The acid component of the substrate is released after the rapid rearrangement (Stok et al., 2004; Wheelock et al., 2005). To further investigate the role of RpCarE in resistance to isoprocarb and cyhalothrin in R. padi, the full coding region was cloned and characterized. The deduced amino acid sequence of RpCarE was found to exhibit striking homology to CarEs in other insects, including the highly conserved catalytic triad (Ser, His, and Glu) and an oxyanion hole consisting of backbone amide group of Ala, Gly, and Gly which indicate that RpCarE can function as an active esterase (Tsubota and Shiotsuki, 2010; Jackson et al., 2013).

RpCarE was cloned into pET-28a and expressed the in E. coli. The molecular mass was similar to the carboxylesterase E4 in M. persicae (60 kDa) (Lan et al., 2005) and differed from that of A. gossypii (65 kDa) (Gong et al., 2017). The purified fusion protein displayed significant hydrolase activity against the model substrate α-naphthyl acetate with a Kcat of 5.50 s−<sup>1</sup> , suggesting RpCarE was active and successfully expressed in the E. coli strain. Fusion proteins of carboxylesterase 001D from Helicoverpa armigera expressed in E. coli showed

TABLE 2 | Kinetic parameters for the purified R. padi carboxylesterase toward the α-naphthyl acetate.


Kcat, catalytic constant; Km, Michaelis constant; Vmax, Maximum Velocity. Units: Km: µM; Kcat: s−<sup>1</sup> ; Kcat/Km: 1/(s × µM). The results are shown as the mean ± SE (n = 3).

enzyme activities against α-NA with a Kcat between 0.35 and 2.29 s−<sup>1</sup> (Li et al., 2016). HPLC showed that purified RpCarE can metabolize the two insecticide substrates, isoprocarb and cyhalothrin, in vitro, indicating that the carboxylesterase is involved in the detoxification of isoprocarb and cyhalothrin in R. padi. Many studies have demonstrated that carboxylesterases can metabolize organophosphorus, carbamate and pyrethroid insecticides in pest insects (Bass and Field, 2011; Coppin et al., 2012; Li et al., 2013). The E4 carboxylesterase degraded 64% of carbaryl and 80% of malathion within 2.5 and 1.25 h, respectively (Lan et al., 2005). All recombinant expressed wild type and mutant A. gossypii carboxylesterases can hydrolyse paraoxon and parathion to varying degrees (Gong et al., 2017). Li et al. (2016) showed that the H. armigera carboxylesterase 001D exhibited low but measurable hydrolase activity toward β-cypermethrin and fenvalerate. Fifty percent of malathion and 89% of malathion were hydrolysed by recombinant D1CarE5 within 25 and 100 min, respectively, (Xie et al., 2013).

Our modeling and docking data were in accordance with our metabolism analyses. Some residues created a hydrophobic interface at the binding cavity, and critical residues anchored the insecticide molecule. In our study, isoprocarb and cyhalothrin were docked into the predicted active site pocket and were further stabilized by hydrogen bonds with Asn-108, Tyr-109, and Glu-116. These analyses predicted that RpCarE is capable of hydrolysing isoprocarb and cyhalothrin. L. cuprina α-E (LcαE7),

FIGURE 5 | Hydrolytic activity of purified fusion RpCarE protein against isoprocarb (A) and cyhalothrin (B). Insecticide metabolism was analyzed using a Hitachi D-2000 Elite HPLC system. Isoprocarb and cyhalothrin were monitored via their absorption at 215 and 230 nm, respectively, and quantified via their peak integration. A control was prepared using heat-inactivated RpCarE. Values are shown as the mean ± SE of three repeats. Vertical bars represent the standard error of the mean of three independent replicates. Asterisk indicates significant differences by Mann–Whitney U-test (P < 0.05).

sticks. Residues of the catalytic triad (serine, glutamate, and histidine) are represented as yellow sticks. Other residues that interact with the substrate are shaded green.

the template for RpCarE model building, showed a strong hydrophobic binding ability. Molecular modeling of LcαE7 indicated that some mutations in the enzyme were responsible for organophosphates resistance of L. cuprina (Yan et al., 2009). LcαE7 could sequester the insecticide molecule and slowly detoxify it; this high affinity gave LcαE7 an important function in insecticide resistance (Jackson et al., 2013). The A. gossypii CarE protein models based on the crystal structure of LcαE7 showed that some of non-synonymous mutations (H104R, A128V, T333P, and K484R) affected the active site pocket and binding energy, resulted in different binding affinity between A. gossypii CarE with insecticide compounds (Gong et al., 2017).

In summary, we cloned a carboxylesterase gene from R. padi and overexpressed it in both isoprocarb-resistant and cyhalothrin-resistant strains. RpCarE was successfully expressed in E. coli and exhibited hydrolytic activity toward the model substrate α-NA and to isoprocarb and cyhalothrin. These results suggest that RpCarE is involved in resistance to isoprocarb and cyhalothrin in R. padi. Further studies will be required to determine the detailed role of RpCarE as well as to clarify whether other metabolic enzymes (e.g., P450s and GST) are involved in

#### REFERENCES


metabolic resistance or whether target site mutations can play a role in the insecticide resistance of R. padi resistant strains.

#### AUTHOR CONTRIBUTIONS

KW and MC designed the research and wrote the paper. KW, YH, and XL performed research. KW analyzed data.

# FUNDING

This work was funded by the National Natural Science Foundation of China (Grants # 31772160 and 31471766).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00992/full#supplementary-material



Zuo, Y., Wang, K., Zhang, M., Peng, X., Piñero, J. C., and Chen, M. (2016). Regional susceptibilities of Rhopalosiphum padi (Hemiptera: Aphididae) to ten insecticides. Fla. Entomol. 99, 269–275. doi: 10.1653/024.099.0217

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Wang, Huang, Li and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Characterization of Vitellogenin and Vitellogenin Receptor of Conopomorpha sinensis Bradley and Their Responses to Sublethal Concentrations of Insecticide

Qiong Yao, Shu Xu, Yizhi Dong, Yinli Que, Linfa Quan and Bingxu Chen\*

Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China

#### Edited by:

Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China

#### Reviewed by:

Zhaojiang Guo, Chinese Academy of Agricultural Sciences, China Wen-Jia Yang, Guiyang University, China

> \*Correspondence: Bingxu Chen gzchenbx@163.com

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 22 June 2018 Accepted: 20 August 2018 Published: 11 September 2018

#### Citation:

Yao Q, Xu S, Dong Y, Que Y, Quan L and Chen B (2018) Characterization of Vitellogenin and Vitellogenin Receptor of Conopomorpha sinensis Bradley and Their Responses to Sublethal Concentrations of Insecticide. Front. Physiol. 9:1250. doi: 10.3389/fphys.2018.01250 Conopomorpha sinensis Bradley is the dominant borer pest of Litchi chinesis and Euphoria longan. Current management of C. sinensis relies upon insecticide application to adult moths. In addition to the direct mortality induced by insecticides, a sublethal dose of insecticides also affects growth, survival, and reproduction in the exposed insects. Vitellogenin (Vg) and vitellogenin receptor (VgR) are normally identified as essential reproduction-related proteins in insects. In this study, we characterized these two genes from C. sinensis, and investigated their differential responses to sublethal concentrations of insecticide. Cloned CsVg and CsVgR consist of 5391 and 5424-bp open reading frames, which encode proteins of 1796 and 1807 amino acid residues, respectively. The CsVg protein contains the typical vitellogenin, DUF1943 and VWFD domains as other reported lepidopteran Vgs. The CsVgR was characterized as a typical low density lipoprotein receptor with two highly conserved LBD and EGF precursor domains, one hydrophobic transmembrane domain, one cytoplasmic domain, and 13 putative N-glycosylation sites. We next assessed the sublethal effect of four major insecticides on egg-laying in C. sinensis. The toxicity against C. sinensis varied among the insecticides tested, with LC<sup>50</sup> values ranging from 0.23 ppm for chlorpyrifos to 20.00 ppm for β-cypermethrin, among which emamectin benzoate (EB) showed a significant negative impact on egg-laying, survival rate, ovarian development, and mating rate of C. sinensis at LC<sup>30</sup> doses. Further investigation showed that the transcriptional level of CsVg and CsVgR were impaired in different way at 24, 48, and 72 h after EB exposure, and this result was in agreement with the diminished egg-laying of C. sinensis in the sublethal concentration EB-treated group. A repressed transcription level of CsVgR was observed at 48 h after treatment, suggesting that EB elicits a delayed response in the abundance of CsVgR. These results established different roles of CsVg and CsVgR in response to the sublethal effect of insecticides. CsVg might be a better parameter than CsVgR for assessing the effect of sublethal insecticides on reproduction in C. sinensis.

Keywords: Conopomorpha sinensis Bradley, vitellogenin, vitellogenin receptor, insecticides, sublethal effects

# INTRODUCTION

fphys-09-01250 September 8, 2018 Time: 18:36 # 2

Conopomorpha sinensis Bradley (Lepidoptera: Gracilariidae) is the most destructive borer pest of Litchi chinesis and Euphoria longan, and causes severe economic loss in litchi and longan cultivated areas, including India, Nepal, Thailand, Vietnam, and China (Menzel, 2002; Schulte et al., 2007). Immediately after egg hatching, C. sinensis larvae bore tunnels into the center of tender shoots, flowers and fruits, and spend their larval stage inside host plants (Thanh et al., 2006; Dong et al., 2018). Thus, control of C. sinensis larvae is hampered due to its cryptic life habit and overlapping of generations in orchards. To date, frequent application of insecticides to adult C. sinensis is the most effective strategy for this borer pest, since the egg laying amount can be effectively reduced by decreasing the density of adult C. sinensis in orchards.

In addition to the direct mortality due to acute toxicity (lethal effect) after initial insecticide application, insects are exposed to low-lethal or sublethal doses of insecticides for a long period in fields (Biondi et al., 2013). Investigations of the acute toxicity and persistence of sublethal effects of insecticides are both needed for the assessment of insecticide efficiency. Sublethal doses of insecticides may affect various the physiological, biochemical, and behavioral traits of exposed insects (He et al., 2013; Abdu-Allah and Pittendrigh, 2018). The sublethal effects of insecticides on beneficial arthropods have received considerable attention, while their impacts on pest insects are poorly studied (Desneux et al., 2007; Liu et al., 2016). Actually, identifying the sublethal effects of insecticides on pest insects could help us better understand the overall insecticide efficacy in controlling the insect population, thereby optimizing the insecticide usage and in turn delaying resistance in pest insects.

Insecticide sublethal effects on reproduction of insects are traditionally been the most sublethal parameter studied for decades (Stark and Banks, 2003). LC25 of clothianidin could cause reduce mortality and reduction of egg-laying in Bemisia tabaci (Hemiptera: Aleyrodidae), but have no effect on oviposition duration and egg hatching rate of target pest insect (Fang et al., 2018). LC<sup>30</sup> of buprofezin could significantly decrease the fecundity, longevity and egg hatchability in Sogatella furcifera (Hemiptera: Delphacidae) (Ali et al., 2017). While, LC<sup>20</sup> of Cycloxaprid had no impact on net reproductive rage in Aphis gossypii (Hemiptera: Aphididae) (Cui et al., 2018). Thus, as a crucial parameter, the obtained reproduction related results are of primary importance for studies of insecticide sublethal effects on target pest insects. But limited literature had aimed to assessing insecticide sublethal effects on reproduction-related molecules of target insects. Vitellogenin (Vg) and vitellogenin receptor (VgR) are normally identified as two of the most important reproduction-related proteins in insects (Roth and Khalaila, 2012; Lee et al., 2017). Vg is the precursor reserve protein of the main yolk protein vitellin (Vn) in all oviparous species, including insects. The synthesis of Vg is extraovarian in origin with a stage-specific manner in the fat body of female insects. After releasing into the hemolymph and transportation to the ovaries, Vg is selectively accumulated by the terminal oocytes via a receptor-mediated endocytosis process. The receptor responsible for vitellogenin uptake is a membrane-bound protein (VgR) that is a member of the low density lipoprotein receptor (LDLR) family (Sappington and Raikhel, 1998; Roth and Khalaila, 2012). In addition to endocrine control, expression of Vgs and VgRs could be disturbed by several extraneous factors, including metal contamination stress, nutritional conditions, infection and chemical exposure. Previtellogenic nutrition alters the expression of VgR in Aedes aegypti (Hymenoptera: Apidae) (Clifton and Noriega, 2012). Both transcription and protein levels of Vg and VgR were down-regulated by bacterial infection in Apis mellifera (Hymenoptera: Apidae) (Abbo et al., 2017). Intriguingly, the expression of Vgs is dynamic and varies after exposure to different insecticides; e.g., Vg was decreased in a chlorpyrifosresistant strain and identified as a novel potential resistancerelated protein in Frankliniella occidentalis (Thysanoptera: Thripidae), while a drastic increase in the abundance of Vg was observed after exposure of sublethal concentrations of triazophos and deltamethrin in Cyrtorhinus lividipennis Ruter (Hemiptera: Miridae) (Yan et al., 2015; Lu et al., 2017). Hence, the identification and characterization of Vg and VgR, as well as the determination of their expression after exposure to sublethal doses of different insecticides, need to be established in each species to better understand the molecular mechanism of the sublethal effects of insecticides on reproduction in insects.

Here, we report the first molecular information on Vg and VgR in C. sinensis, the most destructive pest in the litchi and longan industry in China. In addition, the sublethal effects of different insecticides on egg-laying in C. sinensis were analyzed. Further, the transcript abundance of CsVg and CsVgR, as well as the survival rate, egg hatchability, ovary development, and mating rate of C. sinensis were investigated after treatment with different sublethal concentrations of emamectin benzoate (EB). This work aimed to provide richer molecular information for insect reproduction-related proteins, as well as to provide a more comprehensive picture of the relationship between sublethal effects of insecticides and reproduction in insects.

#### MATERIALS AND METHODS

#### Insect Rearing and Collection

Conopomorpha sinensis pupae were collected as described earlier (Yao et al., 2016). One day-old female and male adult moths were raised separately for later use. The rearing condition were: constant-temperature incubation at 26 ± 1 ◦C (temperature), 65– 85% RH (relative humidity), 14:10 h L: D photoperiod and 20% (v: v) diluted honey.

#### Identification of the Vitellogenin (CsVg) and Vitellogenin Receptor (CsVgR) Gene in Conopomorpha sinensis

Total RNA was extracted from five female adults of C. sinensis using the E.Z.N.A. Total RNA kit I (Omega Bio-tek, Norcross, GA, United States) and treated with DNase I (Omega). The RNA sample was dissolved in diethylpyrocarbonate (DEPC)-treated H2O and the RNA integrity was confirmed using agarose gel

electrophoresis. First-strand cDNA was synthesized from 1 µg of total RNA in a 20 µl reaction mixture using the GoScript Reverse Transcriptase kit (Promega, Madison, WI, United States).

Four pairs of degenerate primers (CsVg-F1/CsVg-R1, CsVg-F2/CsVg-R2, CsVgR-F1/CsVgR-R1, and CsVgR-F2/CsVgR-R2) (**Supplementary Table 2**) were designed on the basis of the conserved Vg and VgR cDNA sequences of other Lepidoptera insects. PCR was performed to obtain partial cDNA sequences using TransTaq DNA Polymerase High Fidelity (Transgene Biotech, Beijing, China). PCR amplification was carried out as follows: 94◦C for 5 min; five cycles of 94◦C for 40 s, 48◦C for 1 min and 72◦C for 40 s; 25 cycles of 94◦C for 40 s, 53◦C for 1 min and 72◦C for 40 s; with a final extension at 72◦C for 6 min. The amplified products were separated on agarose gels and purified using a Gel Extraction kit (Axygen Biosciences, Union City, CA, United States). The purified PCR products were sub-cloned into the pGEM-T Easy Vector (Promega, Tokyo, Japan) and transformed into Escherichia coli DH5α-competent cells (Tiangen, Beijing, China). Positive clones were confirmed by PCR and automated sequencing [The Beijing Genomics Institute (BGI), China].

To obtain the full-length CsVg and CsVgR coding regions, nested gene-specific primers for CsVg and CsVgR (**Supplementary Table 2**) were designed based on the partial cDNA sequence obtained as described above. Following the instructions of the SMARTTM RACE (rapid amplification of cDNA ends) cDNA Amplification kit (Clontech, Mountain View, CA, United States), 5<sup>0</sup> -RACE and 3<sup>0</sup> -RACE were performed using gene-specific primers and universal anchor primers (Universal Primer Mix/UPM and Nested Universal Primer A/NUP, Clontech). The RACE products were confirmed using agarose gel electrophoresis and purified, sub-cloned into vectors and sequenced as described above. The overlapping sequences of the PCR fragments were assembled to obtain the full-length CsVg and CsVgR cDNA. Each kit was used according to the manufacturer's instructions.

#### Characterization of CsVg and CsVgR

The open reading frames of the CsVg and CsVgR genes were obtained using ORF finder<sup>1</sup> , and the amino acid sequences were deduced from the corresponding cDNA sequences using the translation tool on the ExPASy Proteomics website<sup>2</sup> . Various physical and chemical parameters for the CsVg and CsVgR proteins (such as predictions of the theoretical isoelectric point, molecular weight, theoretical isoionic point) were performed with analysis tools from the ExPASy ProtParam tool<sup>3</sup> . The signal peptide cleavage site was predicted using SignalP 4.1 Server<sup>4</sup> . The transmembrane helices were analyzed by TMHMM Server v.2.0<sup>5</sup> . Cellular localization was predicted by PSORT II6 . The N-glycosylation site prediction was performed using

<sup>6</sup>http://psort.hgc.jp/form2.html

the NetNglyc webserver<sup>7</sup> . The GPP prediction server was used to predict potential O-linked glycosylation sites<sup>8</sup> . The domain architecture and conserved domains were identified using Scan-Prosite<sup>9</sup> , SMART<sup>10</sup> and InterProScan<sup>11</sup> online tools. On the National Centre for Biotechnology Information (NCBI) website, the sequence of the CsVg and CsVgR cDNAs were individually compared with other available Lepidoptera vitellogenin and vitellogenin receptor sequences deposited in GenBank using the BLAST-X tool. Multiple sequence alignments of the deduced CsVg and CsVgR amino acid sequences were made using Multiple Alignment software<sup>12</sup>. Phylogenic and evolutionary analyses were conducted using Molecular Evolutionary Genetics Analysis (MEGA) software v.5.05 by a neighbor-joining (NJ) method with bootstrap of 1000 replicates after removing the highly divergent signal peptide sequences at the N-terminus.

#### Sublethal Effect of Insecticides on Egg-Laying in C. sinensis Sublethal Toxicity Test

A glass-vial bioassay was modified based on the scintillation glass-vial bioassay described in Snodgrass (Snodgrass, 1996) and was used in sublethal toxicity tests on adult C. sinensis in this study. This method allows for rapid dosing of large numbers of insects using small amounts of insecticides. Stock solutions of the four most common insecticides used in C. sinensis control, including chlorpyrifos, emamectin benzoate (EB), triazophos and β-cypermethrin (Sigma-Aldrich, St. Louis, MO, United States and Siminuo, Beijing, China), were obtained by dissolving these insecticides in acetone. Aliquots of these stock solutions were diluted with acetone to yield the desired concentrations for the bioassay. The concentration range of the test solutions varied for each insecticide, as follows: chlorpyrifos, 0.75– 12.00 mg/kg; EB, 0.50–8.00 mg/kg; triazophos, 0.04–25.00 mg/kg, and β-cypermethrin, 0.13–18.00.

The insecticides were applied by pipetting 1 ml of acetone containing the test solutions into 500 ml beaker flasks, while the control breaker flask received only 1 ml of acetone. The beaker flask was rolled on its side until its interior surface was coated with an even layer of insecticide solution and dried at room temperature before treatment. Groups of 30 4-day-old adult C. sinensis (15 females and 15 males) were placed in each insecticide-treated and control beaker flasks. The bottleneck of the beaker flask was covered with medical gauze, and food for adult C. sinensis was provided by adding a cotton ball dipped in diluted honey. Beaker flasks were stored upright in the laboratory, and mortality was determined after 24 h. All bioassays were repeated three times. Mortality for the treated group was corrected for natural mortality in the control group using Abbott's formula. All data were subjected to Probit

<sup>1</sup>https://www.ncbi.nlm.nih.gov/orffinder/

<sup>2</sup>http://expasy.org/tools/dna.html

<sup>3</sup>https://web.expasy.org/protparam/

<sup>4</sup>http://www.cbs.dtu.dk/services/SignalP/

<sup>5</sup>http://www.cbs.dtu.dk/services/TMHMM/

<sup>7</sup>http://www.cbs.dtu.dk/services

<sup>8</sup>http://comp.chem.nottingham.ac.uk/glyco/

<sup>9</sup>http://prosite.expasy.org/scanprosite/

<sup>10</sup>http://smart.embl-heidelberg.de/

<sup>11</sup>http://www.ebi.ac.uk/Tools/pfa/iprscan/

<sup>12</sup>http://www.ebi.ac.uk/clustalw/index.html

analysis using PROC PROBIT (SAS Institute, 2008), generating a concentration-mortality regression line for each chemical.

#### Egg-Laying

Based on the regression equations obtained in this study, the LC<sup>10</sup> and LC<sup>30</sup> values of the four insecticides were used to evaluate their sublethal effects on egg-laying in C. sinensis (**Table 1**). Fourday-old C. sinensis females and males were selected randomly to be exposed to LC<sup>10</sup> and LC<sup>30</sup> values of the tested insecticides using the modified glass-vial bioassay described above. Ten pairs of surviving C. sinensis females and males were transferred from beaker flasks into insect rearing cages after 6 h. Each cage was considered to be a replicate and each treatment had three replicated cages. The replacement of oviposition stimulants (i.e., fresh litchi fruits), collection of eggs, and removal of dead C. sinensis were carried out every 24 h for 3 days.

#### Fecundity Analysis After Sublethal EB Exposure in C. sinensis Survival Rate

Based on the effect of the tested insecticides on fecundity of C. sinensis, EB was selected to investigate the sublethal effect of the insecticide on the survival rate of C. sinensis. Briefly, Sixty 4 day-old C. sinensis adults were exposed to LC<sup>10</sup> and LC<sup>30</sup> doses of EB. Dead C. sinensis male and females were removed and counted every 24 h for 7 days.

#### Egg Hatchability

Eggs from each treatment were collected and kept separately for hatching assessment. Hatched larvae were counted daily until no larvae hatched for at least 48 h.

#### Ovary Dissection

To verify the influence of sublethal concentrations of EB on the ovary development of C. sinensis, ovaries of females were dissected in phosphate buffered saline (PBS) at 72 h after sublethal EB exposure. Dissected ovaries were washed and photographed.

#### Mating Rate

Ten females were dissected at 72 h after sublethal EB exposure. Mating rate was calculated based on the form of bursa copulatrix. Small and wizened bursa copulatrix indicates unmated female, while big and plump bursa copulatrix indicates mated female. Each treatment had three replicates.

#### Gene Expression

Quantitative real-time PCR (qRT-PCR) analysis was performed to determine the expression of CsVg and CsVgR in C. sinensis after exposure to sublethal doses of EB. Four-day-old C. sinensis adults were exposed to LC<sup>10</sup> and LC<sup>30</sup> doses of EB using the modified glass-vial bioassay described above. Sixty surviving C. sinensis were transferred from beaker flasks into insect rearing cages after 6 h. Five females were selected randomly from the treatment and control groups every 24 h for 3 days. All bioassays were repeated three times. Total RNA was isolated from total insects of individual samples (5 females/sample) and treated with DNase I (Omega). cDNA was synthesized from 1 µg of total RNA in a 20 µl reaction mixture using the GoScript Reverse Transcriptase kit (Promega, Madison, WI, United States) according to the manufacturer's protocol. The mRNA transcripts of CsVg and CsVgR were assessed using the GoTaq qPCR Master Mix (Promega, Madison, WI, United States) with the specific primers described in **Supplementary Table 2**. PCR was performed using the specific primers mentioned in **Supplementary Table 2**. The PCR conditions were hot-start activation at 95◦C for 2 min; 40 cycles of denaturation at 95◦C for 15 s and extension at 60◦C for 1 min; followed by a final dissociation at 72◦C. The specificity of the reaction was checked by analyzing the melting curve of the final amplified product. β-actin (KF598848) was chosen as a suitable housekeeping gene, and the housekeeping gene and target genes from each sample were run in triplicate on the same PCR plate. The relative expression levels were calculated using a modified comparative Ct method (Schmittgen and Livak, 2008), and the relative expression levels of the CsVg and CsVgR genes were calculated by normalization to β-actin.

#### Statistical Analysis

The LC<sup>50</sup> ratio for each insecticide was tested for significance according to Robertson and Preisler (1992) to determine differences at P > 0.05, which was achieved by calculating the corresponding 95% confidence intervals (Robertson and Preisler, 1992). The daily fecundity for each egg collection time was analyzed separately using one-way analysis of variance (ANOVA) with the type of insecticide as the independent variable. Then, multiple comparison procedures were performed by Tukey's test when significant differences were found (P < 0.05). For all experimental data, statistical analyses were performed by ANOVA followed by Tukey's test for multiple comparisons. Significant differences were considered at P < 0.05.

TABLE 1 | LC<sup>50</sup> values (with corresponding 95% confidence intervals) for Conopomorpha sinensis adults after 24 h of exposure to insecticides.


The results are presented as regression equations, degree of freedom (df), LC50, corresponding 95% confidence intervals (CI), estimated LC<sup>30</sup> and LC10. Low X<sup>2</sup> value (<11.00) indicates the data adequacy to the probit model used to estimate the mortality curves.

type D domain; SP, signal peptide.

#### RESULTS

#### Sequence and Structural Analysis of CsVg

Cloning of CsVg was accomplished by RT-PCR using degenerate primers (CsVg-F1, CsVg-R1, CsVg-F2, and CsVg-R2; sequences in **Supplementary Table 2**) designed based on the conserved amino acid regions of other lepidopteran Vg genes. Two cDNA fragments of 834 and 1075 bp were identified, and one fulllength cDNA was obtained with a combination of 3<sup>0</sup> and 5<sup>0</sup> RACE technologies using two pairs of nested gene-specific primers based on the cDNA fragments mentioned above. The deduced protein of CsVg is composed of 1796 amino acids and had a signal peptide (MKVLVLAALLAAASC) at the N-terminus. The deduced CsVg protein is predicted to be an unstable protein with a calculated molecular mass of 205.8 kDa and a pI value of 7.99 (**Supplementary Table 1**).

Three domains and several exposed functional residues were identified in CsVg, which are highly conserved in the sequenced Vgs of Lepidoptera. Domain architecture analysis by the Scan-prosite and InterProScan server confidently predicted the presence of three functional domains in CsVg (**Figure 1**). The Vitellogenin domain (PS51211), also called the lipoprotein amino-terminal region or LPD\_N, is shown to span amino acids 40–752. The DUF1943 domain, which was rarely studied and of unknown function, is in the middle of CsVg (spanning amino acids 784–1062). The Von Willebrand Factor type D domain (VWFD domain, PS51233) is positioned near the C-terminus of the CsVg, from amino acids (aa) 1447 to 1627. Several tetra residue motifs, R/KXXR/K, are present in CsVg at the N-terminus and in the middle of CsVg (**Supplementary File 1**). In addition, a highly conserved GL/ICG motif and five cysteine residues are located at conserved positions, as in other reported insect Vgs.

The deduced CsVg was aligned with corresponding amino acid sequences of other lepidopteran Vgs by NCBI protein BLAST. The results revealed that CsVg had low a degree of conservation with other lepidopteran Vgs, with the overall identity ranked from 47 to 37%. The evolutionary relationship of 21 Vgs derived from lepidopteran insects was evaluated after sequence alignment and phylogenetic tree construction. The monophyly of nine families was well-supported by an NJ tree with high values, with CsVg belonging to Gracillariidae located in a separated branch.

#### Sequence and Structural Analysis of CsVgR

Several vitellogenin receptors (VgRs) were identified from different families of lepidopteran insects, such as Bombyxi mori (Lepidoptera: bombycidae), Spodoptera exigua (Lepidoptera: noctuidae), and others. However, the VgR protein from gracilariidae was not previously characterized. Therefore, we cloned the gene encoding the VgR protein from C. sinensis using a similar strategy as that used for CsVg cloning. Two

CsVgR fragments of 1221 and 525 bp were generated from adult C. sinensis cDNA by RT-PCR using two pairs of degenerate primers (CsVgR-F1, CsVgR-R1, CsVgR-F2, and CsVgR-R2), and then full-length cDNA of CsVgR was obtained using two pairs of specific primers (sequences in **Supplementary Table 2**). The deduced protein of CsVgR is composed of 1807 amino acids with a predicted molecular mass of 201.2 kDa, a pI value of 5.57, and the highest aa composition of serine (**Supplementary Table 1** and **Supplementary File 2**). Analysis of the deduced amino acid sequence revealed a signal peptide with 20 aa residues (MSNKWLVTMITVSLCGVAWA) located at the N-terminus of CsVgR, which was presumed to be cytoplasmic in nature, as detected by SignalP 4.1 and PSORT II Server.

Analysis of the CsVgR protein sequence indicated that it contained all of the typical features of the LDLR family. The structural organization of mature insect VgRs consists of (i) two ligand-binding domains with LDL-receptor class A (LDLRA) repeats, (ii) two epidermal growth factor (EGF) precursor domains with EGF-like repeats and LDL-receptor class B (LDLRB) repeats, (iii) an O-linked sugar domain, (iv) a transmembrane domain, and (v) a cytoplasmic domain (Tufail and Takeda, 2009). In the architecture analysis of the mature CsVgR protein, we observed 11 cysteine-rich LDLRA repeats in two patches, 7 cysteine-rich LDLRB repeats in three patches, 6 EGF-like repeats, 2 calcium binding EGFlike repeats, one hydrophobic transmembrane domain, one cytoplasmic domain with two sequence motifs (NPLF at residues 1748–1751 and LL at residues 1756–1757) as potential receptor internalization signals, and 13 putative N-glycosylation sites characterized by the consensus sequence NXS/T (**Figure 2** and **Supplementary Table 3**). The deduced amino acid sequence of CsVgR was aligned with other lepidopteran VgRs by a blastp search of the NCBI database. The CsVgR protein sequence was most similar to those of VgR from S. exigua and Papilio xuthus (Lepidoptera: Papiloinidae) (42% overall identity), followed by Helicoverpa armigera (Lepidoptera: Noctuidae) and Papilio machaon (Lepidoptera: Papiloinidae) (41%) (**Figure 2**).

#### Sublethal Effect of Insecticides on Egg-Laying in C. sinensis

Probit analyses of concentration-mortality data showed that after 24 h of exposure to chlorpyrifos, EB, triazophos and β-cypermethrin, the LC<sup>50</sup> values were estimated to be 0.23, 1.88, 2.11, and 20.00 ppm, respectively (**Table 1**). These results revealed that chlorpyrifos had the highest toxicity to C. sinensis, followed by EB and triazophos, whose LC<sup>50</sup> values were more than 10 times higher than that of β-cypermethrin. The LC<sup>10</sup> and LC<sup>30</sup> values of each insecticide to adult C. sinensis were estimated based on the regression equations of the four insecticides (**Table 1**). The egg-laying of C. sinensis females after exposure to sublethal insecticides was determined. C. sinensis females in the EB-treated group laid the fewest eggs among the four insecticide-treated groups, followed by the triazophos-treated group. The other two insecticides showed little to no effect on egg-laying in C. sinensis (**Figure 3**). Moreover, the average number of eggs laid per female in control group was five times and 14 times higher than that of the LC<sup>30</sup> EB-treated group at 48 and 72 h after chemical exposure, respectively. Therefore, taking the results of toxicity and impact on oviposition in C. sinensis into account, the most detrimental chemical is with EB. Thus, EB was selected to further investigate the sublethal effect of insecticide on the fecundity of C. sinensis.

#### Sublethal Effects of EB on Fecundity in C. sinensis

The two sublethal concentrations (LC<sup>10</sup> and LC30) of EB caused diminished survival rates of adult C. sinensis males and females that were significantly different from the survival rates recorded for the control group (**Figure 4A**). The survival rates of adult

C. sinensis at both LC<sup>10</sup> and LC<sup>30</sup> of the treatments exhibited a similar trend and had a sharp decline 2–3 days after EB exposure. Only 21.87% of females and 9.82% of male at LC<sup>30</sup> concentration, and 23.79% of female and 19.12% of males at LC<sup>10</sup> concentration were able to survive for 3 days post-chemical exposure. The survival rates of adult C. sinensis females were higher than those of males, indicating that female moths were more tolerant to EB. The mating rate was remarkably decreased relative to the control and L<sup>10</sup> EB treatment group when C. sinensis adults were exposed to LC<sup>30</sup> EB (**Figure 4C**). By contrast, egg hatchability was unaffected by EB exposure (**Figure 4B**). In the morphological photographs of ovaries of the C. sinensis females at 72 h after EB treatment, no significant difference was observed between the LC<sup>10</sup> EB treatment and control groups, while ovaries with fewer eggs or undeveloped ovaries with small and wizened bursa copulatrix were observed after the LC<sup>30</sup> EB exposure (**Figure 4D**).

#### Sublethal Effects of EB on Transcription Level of CsVg and CsVgR in C. sinensis

To address the impact of sublethal concentrations of EB on gene expression of reproduction-related proteins in C. sinensis, the relative mRNA expressions of of CsVg and CsVgR were determined. Exposure of LC<sup>10</sup> and LC<sup>30</sup> EB to adult C. sinensis resulted in a significantly diminished transcriptional abundance of CsVg and CsVgR. The CsVg mRNA levels in insects from the LC<sup>30</sup> EB-treated group stayed very low at 48 and 72 h after treatment, less than 30% of that in insects from the LC<sup>10</sup> EB-treated group (**Figure 5A**). By contrast, no significant differences in the transcriptional abundances of CsVgR were observed at 48 and 72 h after the LC<sup>10</sup> and LC<sup>30</sup> EB treatment, indicating that EB had a lower adverse impact on CsVgR expression than CsVg in C. sinensis (**Figure 5**). It is interesting to note that a significantly decreased transcription level of CsVg was observed at 24 h after treatment, while a repressed transcription level of CsVgR was observed at 48 h after treatment.

# DISCUSSION

Vgs and VgRs have been extensively identified in different species of vertebrates and invertebrates, including insects. To date, 20 Vg and 9 VgR sequences from lepidopteran are available in the GenBank database, but no information has been reported for Vg and its receptor from gracillariiae insects. In this study, we identified the sequences of Vg and VgR in C. sinensis (lepidoptera: gracillariiae), which are the first full-length sequences of Vg and VgR from the gracillariidae insects, and provided basic information for their functional analysis. The evolutionary relationship of CsVg and CsVgR with other lepidoteran insect Vgs and VgRs was inferred by constructing two phylogenetic trees. CsVg and CsVgR were separate from other lepidoteran insects and formed a single clade of gracillariidae, as expected.

Similar to other reported insect Vgs, CsVg led to a 205.8 kDa precursor protein with three functional domains, including highly a conserved vitellogenin domain and VWFD domain (Upadhyay et al., 2016). The DUF1943 domain, which is rarely detected in insects and is of unknown function, is present in all lepidopteran Vgs characterized to date (Robertson and Preisler, 1992; Thompson and Banaszak, 2002) (**Figure 1**). In the meantime, the CsVg protein sequence also contained different features from other insects. (1) The consensus cleavage sites of the R/KXXR/K tetra-residue motif are near the N-terminus of most insect Vg proteins (Tufail and Takeda, 2008). However, multiple potential R/KXXR/K sequence motifs are found at the N-terminus, center and C-terminus of Vg in C. sinensis. (2) Most insect Vgs characterized to date are heavily phosphorylated, especially at serine regions. Polyserine tracts containing tandem serine repeats are mostly present at both of the termini in insect Vgs (Tufail and Takeda, 2008). These polyserine tracts are expected to serve as good phosphorylation sites and may contribute to the interaction between Vg and VgR during endocytosis (Goulas et al., 1996; Upadhyay et al., 2016). Although 315 putative phosphorylated residues (S = 149, Y = 103, and T = 99) were predicted in CsVg, polyserine tracts were not observed in CsVg as well as missing polyserine tracts

reported in S. exigua, indicating that there may be different mechanism for Vg and VgR binding on the oocyte surface in some insects, such as C. sinensis and S. exigua (Zhao et al., 2016). (3) In most of the insect Vg sequences, DGXR motif, conserved cysteine residues, and GI/LCG motif occur at highly conserved locations near the C-terminus, and the DGXR motif is normally located 17–19 residues upstream of the GI/LCG motif (Sun et al., 2016). It is proposed that they may form a structure that is necessary for the proper function of insect Vgs during embryogenesis. However, no DGXR motif was

P < 0.05).

detected upstream of the GLCG motif in CsVg, as reported previously for L. maderae Vg, indicating the alternation of the DGXR motif in some insect species (Tufail and Takeda, 2008).

Analysis of the CsVgR sequence showed that it was composed of multiple conserved modular elements, similar to other insect VgRs and was a typical member of the LDLR superfamily (Sappington and Raikhel, 1998). A striking characteristic of lepidopteran VgRs is the existence of 11 cysteine-rich LDLRA repeats in two LBD domains, which are four and seven repeats in the first and second LBD domain, respectively. However, the number of LDLRA repeats and arrangement are quite different from those other insect orders, There are five- and eight- LDLRA repeats in Blattaria and Diptera, two/four- and eight- repeats in Hymenoptera, and eight-repeats in Coleoptera (Cong et al., 2015; Zhang et al., 2016). Moreover, we found comparable patterns in each lepidoteran VgR, although the numbers of conserved modular elements were variable. Lepidoteran VgRs all contain two ligand binding domain (LBD) domains and two EGF precursor domains that are responsible for ligand binding and acid-dependent dissociation; each LBD domain is followed by an EGF precursor domain (Tufail and Takeda, 2009). However, compared with other lepidopteran VgRs, the number and arrangement of EGF/Calcium-binding-like repeats in the two EGF precursor domains are different in C. sinensis. In common, two/three EGF-like repeats were presented in the first EGF precursor domain, but an extra repeat was observed in the C. sinensis VgR. In addition, the numbers of EGF/Calciumbinding EGF-like repeats in the second EGF precursor domain in lepidoteran VgRs varied substantially (**Figure 2**). Another intriguing difference among lepidopteran VgRs is the existence of the O-linked sugar domain, which is a short serine and threonine enriched region at the C-terminus of some insect LDLRs. It is proposed that the O-linked sugar domain is important for VgR stability and regulation of the signal pathway (Willnow, 1999; Tufail and Takeda, 2009). However, the CsVgR do not contain an O-linked sugar domain, as in Antheraea pernyi (Lepidoptera: Saturniidae), which is different from the VgRs of Actias selene (Lepidoptera: Saturniidae) and Bombyx mori (Lepidoptera: Saturniidae) (**Figure 2**). These results indicate that the presence of the O-linked sugar domain is not universal even among the same insect family.

Emamectin benzoate (EB) is a macrocyclic lactone insecticide and acts by disrupting the nervous system, inhibiting muscle contraction, damaging the detoxic ability, and thereby leading to changes in metabolism and behavior of pest insects (Isaac et al., 2002; Luan et al., 2017). With long residual ingestion activity on target arthropods and low toxicity to beneficial arthropods, EB is widely used for control of pest insects (López et al., 2010). In previous research, EB exhibited ovicidal activity against Cydia molesta (Busck) (Lepidoptera: Tortricideae), larvicidal activity against Culex quinquefasciatus say (Diptera: Culicidae), and adulticidal activity against Cydia pomonella (L.) (lepidoptera: Tortricidae) (Ioriatti et al., 2009; Wu et al., 2015; Shah et al., 2016). However, EB was reported to be a harmless insecticide for adults of Adalia bipunctata (L.) (Coleoptera: Coccinellidae), Coccinella transversalis (F.) (Coleoptera: Coccinellidae), and Macrolophus pygmaeus (Hemiptera: Miridae) (Cole et al., 2010; Martinou et al., 2014; Depalo et al., 2017). Thus, the effectiveness of EB against insects is species- and phase- dependent. In the current study, the sublethal concentration of EB on the survival rate of adult C. sinensis was evaluated under laboratory conditions. The LC<sup>30</sup> concentration of EB had a long-lasting toxic activity and reduced the survival rate by ∼50% in adult C. sinensis 2 to 4 days after treatment. These results are consistent with the field application of EB in litchi and longan orchards; i.e., high mortality of C. sinensis moths is observed 3– 4 days after EB spraying, and the application of EB decreases the pest population and achieves a long-term pest population decrease (Shu Xu, unpublished). These results demonstrated that EB was a long-lived insecticide for the adult C. sinensis control. Insecticides, as an environmental hazard, can affect insect reproduction by directly and indirectly linking with insect population via physiological and biochemical pathways (He et al., 2013; Roditakis et al., 2013). Among the four tested insecticides, the sublethal concentrations of EB showed a significant negative

impact on egg-laying in C. sinensis at 48 and 72 h after chemical exposure. Moreover, ovarian development was disturbed and mating rate of C. sinensis was decreased to 56.67% after LC<sup>30</sup> EB exposure. Likewise, a notable reduction in the mating frequency in female Helicoverpa zea (Lepidoptera: Noctiudae) was observed after treatment with sublethal concentrations of EB (López et al., 2010). Therefore, the influence of sublethal concentrations of EB on insect mating behavior could be a possible explanation available for the impact of this hazardous chemical on oviposition in C. sinensis.

Among all insect reproduction-related proteins, Vg and VgR have traditionally been used as adequate parameters for assessing female fertility (Lee et al., 2017; Seixas et al., 2018). In our study, the transcriptional abundance of CsVg and CsVgR in insects from the control group was increased at 6 and 7 days after eclosion of C. sinensis (sampling time of 48 and 72 h in the control group, **Figure 5**). These results are in agreement with the readiness of the female C. sinensis for mating and oviposition after preoviposition period for 5 days in our previous study (Dong et al., 2015). On the contrary, the transcript levels of CsVg and CsVgR were generally decreased in different ways at 48 and 72 h after EB exposure, and this result was coincident with the diminished egg-laying of C. sinensis in the LC<sup>10</sup> and LC<sup>30</sup> EB-treated groups. Interestingly, EB down regulated the expression CsVg, but left the transcriptional level of CsVgR undisturbed in the initial 24 h after EB exposure. In previous studies, exposure of 3rd instar larvae to sublethal concentrations of chlorantraniliprole resulted in decreased fecundity and Vg expression in adult Chilo suppressalis (Lepidoptera: Crambidae) females; exposure to sublethal doses of both fipronil and deltamethrin did not affect Vg expression in Apis mellifera (Hymenoptera: Apidae) while induced Vg expression was observed in Nilaparvata lugens (Hemiptera: Delphacidae) after a sublethal deltamethrin treatment (Ge et al., 2010; Li et al., 2016; Bordier et al., 2017). Therefore the expression of Vg in different species of insects varied substantially after sublethal insecticide exposure. Our findings provide evidence that EB elicited an important response in adult C. sinensis females by modulating the expression of CsVg and CsVgR.

#### CONCLUSION

This study molecularly characterized CsVg and CsVgR in C. sinensis, and is the first report of Vg and its receptor in gracillariiaes insects. In addition, the primary toxicity and fecundity regulation of insecticides on C. sinensis and

#### REFERENCES


expression response of CsVg and CsVgR to EB exposure were also investigated. Thereby further our understanding of the role of the Vg and VgR genes in insecticide-responsive gene expression. Its more timely response and more drastic reduction in the abundance of CsVg than CsVgR indicated that CsVg might be a better parameter for the assessment of sublethal insecticide impacts on reproduction in target insects. Our results demonstrated that EB could be considered an effective insecticide with high persistence for controlling C. sinensis. However, more detailed studies of non-target beneficial arthropods, and adaptation and resistance to long-term EB exposure need to be carried out for a comprehensive understanding of the influence of EB application in litchi and longan orchards.

#### AUTHOR CONTRIBUTIONS

QY, SX, and BC conceived the study. QY conducted the experiments and drafted the preliminary manuscript. QY, YQ, YD, and LQ interpreted the results. SX and BC refined and approved the final manuscript.

#### FUNDING

This study received financial support from the National Natural Science Foundation of China (Grant No. 31801800), the Pearl River S and T Nova Program of Guangzhou (Grant No. 201710010180), the Natural Science Foundation of Guangdong Province (Grant No. 2017A030310095), and China Litchi and Longan Research System Foundation (Award No. CARS-32-12).

#### ACKNOWLEDGMENTS

We are grateful to Dr. Wenqing Zhang (Sun Yat-sen University) for the help with manuscript correction. We thank Ribi Feng, Wenjing Li, and Shaoyuan Lin for their excellent technical assistance, Haiming Xu and Yanyan Chi for their suggestions for the data analysis.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01250/full#supplementary-material


physiology and energetic metabolism. J. Insect Physiol. 98, 47–54. doi: 10.1016/ j.jinsphys.2016.11.013


fphys-09-01250 September 8, 2018 Time: 18:36 # 11


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yao, Xu, Dong, Que, Quan and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

#### Edited by:

Su Wang, Beijing Academy of Agriculture and Forestry Sciences, China

#### Reviewed by:

Christen Kerry Mirth, Monash University, Australia Angelique Christine Paulk, Harvard Medical School, United States

#### \*Correspondence:

Federica Calevro federica.calevro@insa-lyon.fr Stefano Colella stefano.colella@inra.fr

†These authors are joint first authors

#### ‡Present address:

Stefano Colella, LSTM, Laboratoire des Symbioses Tropicales et Méditerranéennes, INRA, IRD, CIRAD, SupAgro, Université de Montpellier, Montpellier, France Yvan Rahbé, UMR5240, Microbiologie, Adaptation et Pathogénie, Université de Lyon CNRS, Villeurbanne, France

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 23 May 2018 Accepted: 04 October 2018 Published: 25 October 2018

#### Citation:

Colella S, Parisot N, Simonet P, Gaget K, Duport G, Baa-Puyoulet P, Rahbé Y, Charles H, Febvay G, Callaerts P and Calevro F (2018) Bacteriocyte Reprogramming to Cope With Nutritional Stress in a Phloem Sap Feeding Hemipteran, the Pea Aphid Acyrthosiphon pisum. Front. Physiol. 9:1498. doi: 10.3389/fphys.2018.01498

# Bacteriocyte Reprogramming to Cope With Nutritional Stress in a Phloem Sap Feeding Hemipteran, the Pea Aphid Acyrthosiphon pisum

Stefano Colella<sup>1</sup> \* †‡, Nicolas Parisot<sup>1</sup>† , Pierre Simonet<sup>1</sup> , Karen Gaget<sup>1</sup> , Gabrielle Duport<sup>1</sup> , Patrice Baa-Puyoulet<sup>1</sup> , Yvan Rahbé<sup>1</sup>‡ , Hubert Charles<sup>1</sup> , Gérard Febvay<sup>1</sup> , Patrick Callaerts<sup>2</sup> and Federica Calevro<sup>1</sup> \*

<sup>1</sup> Univ Lyon, INSA-Lyon, INRA, BF2I, UMR0203, F-69621, Villeurbanne, France, <sup>2</sup> Laboratory of Behavioral and Developmental Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium

Nutritional symbioses play a central role in the ability of insects to thrive on unbalanced diets and in ensuring their evolutionary success. A genomic model for nutritional symbiosis comprises the hemipteran Acyrthosiphon pisum, and the gamma-3-proteobacterium, Buchnera aphidicola, with genomes encoding highly integrated metabolic pathways. A. pisum feeds exclusively on plant phloem sap, a nutritionally unbalanced diet highly variable in composition, thus raising the question of how this symbiotic system responds to nutritional stress. We addressed this by combining transcriptomic, phenotypic and life history trait analyses to determine the organismal impact of deprivation of tyrosine and phenylalanine. These two aromatic amino acids are essential for aphid development, are synthesized in a metabolic pathway for which the aphid host and the endosymbiont are interdependent, and their concentration can be highly variable in plant phloem sap. We found that this nutritional challenge does not have major phenotypic effects on the pea aphid, except for a limited weight reduction and a 2-day delay in onset of nymph laying. Transcriptomic analyses through aphid development showed a prominent response in bacteriocytes (the core symbiotic tissue which houses the symbionts), but not in gut, thus highlighting the role of bacteriocytes as major modulators of this homeostasis. This response does not involve a direct regulation of tyrosine and phenylalanine biosynthetic pathway and transporter genes. Instead, we observed an extensive transcriptional reprogramming of the bacteriocyte with a rapid down-regulation of genes encoding sugar transporters and genes required for sugar metabolism. Consistently, we observed continued overexpression of the A. pisum homolog of RRAD, a small GTPase implicated in repressing aerobic glycolysis. In addition, we found increased transcription of genes involved in proliferation, cell size control and signaling. We experimentally confirmed the significance of these gene expression changes detecting an increase in bacteriocyte number and cell size in vivo under tyrosine and phenylalanine depletion. Our results

**121**

support a central role of bacteriocytes in the aphid response to amino acid deprivation: their transcriptional and cellular responses fine-tune host physiology providing the host insect with an effective way to cope with the challenges posed by the variability in composition of phloem sap.

Keywords: pea aphid, symbiosis, bacteriocyte, amino acid stress, phenylalanine and tyrosine pathway, transcriptome profiling

#### INTRODUCTION

fphys-09-01498 October 23, 2018 Time: 14:25 # 2

Hemiptera are a major group of insects occupying a diverse range of ecological niches. Phloem-feeding hemipterans are among the most destructive insect pests of agriculture and forest crops due to their wide host range, rapid reproduction and ability to vector numerous phytopathogens (Girousse et al., 2005; Brault et al., 2010; Perilla-Henao and Casteel, 2016). These insects are the only group of animals using phloem sap, a nutritionally unbalanced diet rich in carbohydrates (e.g., sucrose) but poor in essential amino acids (Hayashi and Chino, 1986; Douglas, 1993; Sandström and Pettersson, 1994; Karley et al., 2002; Dinant et al., 2010), as their predominant or sole food source (Douglas, 2006). All phloem-feeding hemipterans are capable of utilizing phloem sap thanks to their intimate association with symbiotic bacteria which furnish them with (or cooperate with them for the production of) vitamins and/or amino acids otherwise lacking in this food source (Akman Gündüz and Douglas, 2009). In addition to being nutritionally unbalanced, plant phloem sap shows considerable spatial and temporal variability in composition dependent on, e.g., plant species, environment, age of the plant and position of the insect on the plant, all making this a challenging food source for these insects (Sharkey and Pate, 1976; Smith and Milburn, 1980; Hayashi and Chino, 1990; Winter et al., 1992; Corbesier et al., 2001; Douglas, 2003, 2006; Gholami, 2004). Despite their ecological and agronomical importance, how hemipterans and their symbiotic bacteria cope with changes in phloem sap composition and the nature of the molecular responses to these changes are poorly understood.

The pea aphid Acyrthosiphon pisum is an excellent hemipteran model to study the interactions between animals and their resident microorganisms. First, its parthenogenetic reproduction allows studying large numbers of individuals of a single genotype. Furthermore, the pea aphid primary symbiont, the gamma-3-proteobacterium, Buchnera aphidicola, which has coevolved with its host for over 150 million years, is one of the best characterized symbiotic bacteria (Moran et al., 1993; Moran and Baumann, 1994; Von Dohlen and Moran, 2000; Tagu et al., 2010).

The genomes of the pea aphid and its primary symbiont have both been sequenced and their analysis revealed that these two obligate mutualists are fully interdependent for the biosynthesis of amino acids with the two genomes forming a highly integrated metabolic network (Shigenobu et al., 2000; International Aphid Genomics Consortium, 2010). The amino acid biosynthetic pathways illustrate the metabolic interdependence of host and endosymbionts. Both partners participate in the biosynthesis of the 10 amino acids: arginine, cysteine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine. Histidine and tryptophan are synthesized exclusively by Buchnera, which in turn is auxotrophic for the biosynthesis of alanine, asparagine, aspartate, glutamine, glutamate, proline, serine, and tyrosine (Wilson et al., 2010; Hansen and Moran, 2011; Poliakov et al., 2011; Wilson, 2011; Wilson and Duncan, 2015).

The tyrosine (Tyr) and phenylalanine (Phe) biosynthetic pathway is one of the most integrated, consisting of a network of genes encoded by the genomes of host and primary symbiont. B. aphidicola produces all the necessary precursors to synthesize Phe and Tyr, but has lost the terminal biosynthetic enzymes of the pathway and fully relies on the host insect for their biosynthesis (International Aphid Genomics Consortium, 2010; Wilson et al., 2010). The exchange of precursors and terminal products between the host and the symbiont is often referred to as the aromatic shuttle (Rahbé et al., 2002), where (i) the endosymbiont provides the host with phenylpyruvate the direct precursor of Phe, (ii) the aphid synthesizes Phe from this precursor, and subsequently Tyr from Phe, and (iii) the aphid supplies both its own cells and B. aphidicola with Phe and Tyr (Wilson et al., 2010; Simonet et al., 2016b). Several studies have demonstrated the importance of Tyr and Phe for aphid performance, and embryonic development (Douglas, 1996; Wilkinson and Ishikawa, 1999, 2000; Bermingham and Wilkinson, 2010). The Tyr/Phe pathway is activated during pea aphid parthenogenetic development, with several enzyme-encoding genes being up-regulated in the late phases of embryonic development and at the beginning of nymphal development (Rabatel et al., 2013). Recently, Simonet et al. (2016b) confirmed the functional importance of this pathway in aphid development through the disruption of phenylalanine hydroxylase (ApPAH, EC:1.14.16.1). This enzyme catalyzes the conversion of Phe to Tyr, and is only encoded by the host genome. ApPAH gene inactivation shortened the adult aphid lifespan and considerably affected fecundity by diminishing the number of produced nymphs and impairing embryonic development, with severe morphological defects in the progeny.

In apparent contrast to the fact that these aromatic amino acids are important for aphid development and growth, Tyr and Phe have been reported to be in low abundance in plant sap (from trace amounts to 0.5–4%) (Hayashi and Chino, 1990; Karley et al., 2002; Douglas, 2006). This thus raises the important question of how this symbiotic system copes with fluctuations in the concentrations of these two amino acids.

Organisms facing environmental constraints often display extensive transcriptional plasticity (modulation of gene expression). However, similar to other symbiotic bacteria,

the B. aphidicola genome has undergone drastic size reduction (Gil et al., 2002) and most of the classical bacterial gene expression regulatory networks are missing [reviewed in Brinza et al. (2009)]. Several studies have indicated the lack of a strong and specific transcriptional response of this bacterium following a stress applied to the aphid host (Moran et al., 2005; Wilson et al., 2006). Furthermore, Reymond et al. (2006) demonstrated that the transcriptional response of B. aphidicola from aphids reared on a Tyr/Phe depleted diet was not specifically oriented toward aphid needs. These results led us to hypothesize that the host regulates the response to nutrient stress.

To test this hypothesis and uncover the aphid response at the molecular level we used a chemically defined artificial diet to characterize the impact of deprivation of Tyr and Phe on A. pisum transcriptome and life history traits. The complete control diet (AP3) supports normal development, feeding and reproduction (Febvay et al., 1988). Selective removal of Tyr and Phe (YFØ diet) has the advantage to create a framework to identify the cellular and molecular basis of the response of A. pisum/B. aphidicola to naturally occurring fluctuations in phloem sap composition. We focused on gut and bacteriocytes because the gut epithelium is known to act as a regulatory hub in insect nutrition (Huang et al., 2015) while bacteriocytes represent the core symbiotic tissue, as they house the symbiotic bacteria (Buchner, 1965; Baumann et al., 1995). Our analysis revealed that the pea aphid does not show major phenotypic differences under this nutritional challenge. Moreover, the gut transcriptome is not significantly altered under nutrient stress. By contrast, the major response to this nutritional constraint occurs at the level of the bacteriocyte cell. Surprisingly, these alterations do not involve a modulation of the tyrosine and phenylalanine biosynthetic pathways to directly compensate for the depletion of these two amino acids from the aphid diet. Instead, we discovered a transcriptional reprogramming of bacteriocyte cells including a reduction in expression of genes required for sugar metabolism, a modulation of the transport function, and increased transcription of genes related to cell growth, proliferation and associated signaling pathways. We experimentally validated these observations in vivo detecting a significant increase in bacteriocyte number and size. Our findings are indicative of the transcriptional and phenotypic plasticity of bacteriocyte cells. Given the universal presence of bacteriocytes in hemipterans and other symbiotic insects, and their central role in the interactions of these insects with their symbionts, we anticipate that equivalent mechanisms may be present in other symbiotic systems that live and reproduce on nutritionally unbalanced diets.

#### MATERIALS AND METHODS

#### Aphid Rearing and Performance Measures

A long-established parthenogenetic clone (LL01) of A. pisum (Harris), devoid of secondary symbionts, was maintained on young broad bean plants (Vicia faba, L. cv. Aquadulce), at 21◦C, with a 16 h photoperiod. In order to obtain a source of synchronized apterous parthenogenetic aphids, winged adults were left on seedlings, to allow them to produce nymphs, and were removed after 24 h. Synchronized N1 nymphal instars were then transferred to two artificial diets differing only in their amino acid composition: the AP3 and the YFØ diets (see section "Artificial Diets" for details). N1 nymphs (30 for the AP3 and 30 for the YFØ diets, respectively) were left to develop for 30 days and were checked daily for survival and the presence of possible different phenotypic effects. Aphids were weighed at Day 7, the time point of transition to adulthood in aphids reared on plants. Ten aphids per diet were isolated and followed individually for fecundity: the number of newborn nymphs was counted, their size measured with a Leica MZFLIII (Leica, Wetzlar, Germany) microscope using an F-view camera link to the CellF software (Soft Imaging 197 System, Tokyo, Japan) and they were checked for any visible morphological phenotype.

#### Artificial Diets

The composition of the artificial diet (AP3) was as originally defined by us combining data on the amino acid composition in both the phloem sap of leguminous plants and aphid hemolymph (Febvay et al., 1988). The YFØ diet is the same as AP3 minus tyrosine (Y) and phenylalanine (F). Following the preparation of new aliquots of the artificial diets, actual amino acid concentrations are determined by HPLC analysis (Febvay et al., 1999). Furthermore, for the AP3 control diet, aphid life history traits, survival and honeydew production are measured prior to use in experiments (see also the section "Results"). On this diet, aphid survival is standardly comparable to that of aphids reared on plants.

# Sampling of Aphid Tissues for RNA Extraction

This study represents the first time-course analysis of A. pisum gene expression on a genome-wide scale (see **Figure 1** for the experimental design). Aphids were dissected in ice-cold isoosmotic buffer A (pH 7.5, 0.025 M KCl, 0.01 M MgCl2, 0.25 M Sucrose, and 0.035 M Tris–HCl) under 25–40× magnification with an MDG-17 stereomicroscope (Leica) and two tissues were isolated: the gut and the bacteriocytes. All collected nymphs were randomly selected from the synchronized source population. Gut samples were isolated at seven distinct time points following the transfer on the two artificial diets after 12 h (D0), 1 day (D1), 2 days (D2), 3 days (D3), 4 days (D4), 5 days (D5), and 7 days (D7) (**Figure 1** and **Supplementary Table S1**). Three biological replicates were collected per time point (seven) and per diet (two), with each biological replicate consisting of 30 guts. The total number of sample replicates for the gut was 42. Bacteriocyte samples were collected at D3, D4, D5, and D7. Earlier stages did not yield sufficient RNA for subsequent processing and microarrays. Three biological replicates were collected per time point (four) and per diet (two) with each biological replicate containing between 800 and 1000 bacteriocytes. The total number of sample replicates for the bacteriocytes was 24 (**Figure 1** and **Supplementary Table S1**). All dissected tissues were placed in RNAlater <sup>R</sup> (Thermo Fisher Scientific, Waltham, MA, United

States) and stored at −80◦C. Further details on sample numbers and size are provided in **Supplementary Table S1**.

#### RNA Extraction

Total RNA was prepared using the RNeasy Mini kit (Qiagen, Hilden, Germany). Total RNA concentration and quality were initially checked using the NanoDrop <sup>R</sup> ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, United States) and samples had to meet the following quality parameters: A260/A280 ≥ 1.8 and A260/A230 ≥ 1.8, in order to be used in the subsequent analysis. The integrity of the RNA samples was checked using the Agilent RNA 6000 Nano Kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). Only good quality samples responding to the described criteria were used for subsequent analyses.

# Amplification of mRNA and cDNA Synthesis

Microarrays require RNA quantities that exceed what can be isolated from 30 guts or 800–1000 bacteriocytes. Therefore, we applied a commonly used preparatory step for microarrays, i.e., linear amplification of RNA. Linear amplification maintains the relative amounts of existing mRNAs in a cell or tissue without skewing the representation of individual mRNAs in a complex mixture (Schneider et al., 2004). The MessageAmpTM II aRNA Amplification kit (Thermo Fisher Scientific) was used following the manufacturer's instructions. Based on the total RNA quantification profiles, amplifications of the bacteriocyte samples were conducted on RNA quantities two times greater than for gut samples (0.5 µg) to compensate for the twofold higher prokaryotic rRNA concentrations (16S/23S rRNA peaks) relative to eukaryotic rRNA (18S/28S) in bacteriocytes.

The amplified RNA (aRNA) was used to prepare double stranded cDNA with the Superscript II kit (Thermo Fisher Scientific), as recommended by NimbleGen in the NimbleChipTM Arrays User's Guide for gene expression analysis. Starting with 5 µg of aRNA, the samples were processed according to the manufacturer's instructions, including these four steps: (i) initial cDNA synthesis using random primers, (ii) second strand synthesis, (iii) RNase A clean-up, and (iv) cDNA precipitation. For each sample, the integrity of the aRNA and cDNA was checked for possible degradation using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer. Only good quality samples were retained for the microarray

experiments performed by Roche NimbleGen (Madison, WI, United States).

# Microarray Experiments and Data Collection

The "INRA-BF2I\_A.pisum\_Nimblegen-ACYPI\_4x72k\_v1" microarray for the pea aphid was developed in collaboration with Roche NimbleGen using the pea aphid genome v1.0 assembly (International Aphid Genomics Consortium, 2010). This NimbleGen 385K 4-plex (4 × 72 000 probes) high-density array can accommodate 4 samples that are hybridized onto a section of the array containing 72 000 60-mers oligonucleotide probes, representing 24 011 pea aphid transcripts (corresponding to 23855 genes) as described in Rabatel et al. (2013). The microarray design can be found in the ArrayExpress database (Accession No. A-MEXP-1999). Labeling (using the NimbleGen One-Color DNA Labeling Kits and Cy3 Random Nonamers), hybridization on the arrays (at 42◦C for 16–20 h) and scanning (using MS 200 Microarray Scanner and the MS 200 Data Collection Software) were carried out by Roche NimbleGen, as described in the NimbleGen arrays user's guide for gene expression arrays. All the transcriptomic data obtained are available in the ArrayExpress database (Accession No. E-MTAB-4456).

#### Microarray Data Analysis: Quality Control

A first global data analysis using principal component analysis (PCA) revealed that one of the three gut replicates from aphids reared on the AP3 diet for 3 days (Gut-AP3\_D3-1) was aberrant (**Supplementary Figure S1**). This replicate did not cluster with either gut or bacteriocyte samples, and is most likely an incorrectly isolated tissue or a contaminant. It was therefore removed from the subsequent analyses. In order to keep a symmetric design for analysis purposes, one replicate at the same time point from gut in aphids reared on YFØ diet was also removed (Gut-YFØ\_D3-1). Note that this sample did cluster with other gut samples (**Supplementary Figure S1**). Thus, in the final differential expression analysis for the time-point D3, for gut samples, two replicates were used both on the control (AP3) and Tyr/Phe depleted diet (YFØ) samples. 40 gut samples and 24 bacteriocyte samples (total 64) were used for further analyses.

# Microarray Data Analysis: Differential Expression

Microarray data were normalized, using the RMA method (Irizarry et al., 2003), and then transformed into log<sup>2</sup> for subsequent analyses. Differentially expressed genes (DEGs) were predicted using one-way between groups ANOVA analyses [Limma package (Ritchie et al., 2015) from the R Bioconductor project v3.2]. Moderated t-test P-values were adjusted using a false discovery rate [FDR, (Dudoit et al., 2003)] threshold of 0.05. Tissue-enriched genes were detected using the control diet (AP3) samples and only the genes harboring a |log2(Fold-Change)| > 2 were kept for further analyses. Pairwise comparisons between the two diets at each time point and for each tissue were also performed to identify the DEGs in response to a Tyr and Phe depleted diet. For this contrast, no additional filtering was performed. Interactive graphs were obtained through Cytoscape v3.5.0 (Shannon et al., 2003). All PCA were performed using the R ADE4 package (Dray and Dufour, 2007).

# Microarray Data Analysis: Functional Annotation Analysis

As the microarray was originally designed on the v1.0 of the pea aphid A. pisum genome, we filtered the dataset based on the recently released pea aphid genome v2.0 assembly to exclude both outdated genes and genes with cross-hybridizing probes using PatMaN (Prüfer et al., 2008) with a threshold of three mismatches. Using this filter, 13 668 genes were excluded among which 13 646 (99.8%) that were unannotated. Only the remaining genes (10 343) were taken into account for the functional annotation analysis. The Gene Ontology (GO) analysis was first performed using the pea aphid annotation v2.1b. In order to have more detailed results and given the more extensive functional annotation of genes in Drosophila melanogaster, we decided to perform a second GO analysis using the corresponding D. melanogaster genes for each differentially expressed A. pisum gene. For simplicity's sake, we refer to these genes as "putative homologs" throughout the text. To perform this analysis, we retrieved putative D. melanogaster homologs for each differentially expressed A. pisum gene using BLASTP (Altschul et al., 1990) and we carried out a GO analysis based on the annotations collected in FlyBase version FB2017\_01 (Gramates et al., 2017). Enrichment analysis of the DEG sets was performed using BINGO v3.0.3 (Maere et al., 2005) with an FDR threshold of 0.01. Lists of enriched GO terms were further refined using REVIGO (Supek et al., 2011) with a similarity threshold of 0.5 and the D. melanogaster database.

# Microarray Data Validation

To validate the transcriptional differences identified with the microarray analyses, we conducted quantitative reverse transcription-PCR (qRT-PCR) experiments for a selection of genes (seven) corresponding to different functional classes and spanning a broad range of expression differences as determined with the microarrays. Three genes were differentially expressed in the gut samples (ACYPI000653, ACYPI001701, and ACYPI010105), three genes were differentially expressed in the bacteriocytes (ACYPI003338, ACYPI004647, and ACYPI006800), and one gene was differentially expressed in both tissues (ACYPI001281). Expression of these seven genes was assessed for all the time points used in the microarray experiment (a total number of 35 sample replicates for the guts and 16 sample replicates for the bacteriocytes). Primers to target transcripts (**Supplementary Table S2**) were designed with the Oligo6 software (Rychlik, 2007). For data normalization, two genes were tested in the different tissues and time-points analyzed: rpl7 (ACYPI010200) and rpl32 (ACYPI000074). Real-time RT-PCR data were analyzed using the BestKeeper© software tool (Pfaffl et al., 2004) and the rpl7 gene was retained as the best candidate for data normalization.

Internal standard curves were generated for each gene using serial dilutions (from 2000 to 0.002 fg/µl) of purified PCR

products amplified from a pool of cDNA. The PCR reaction to prepare the control sample for the standard curve was carried out starting from 1 µl of reverse transcription product using UptiTherm DNA Polymerase (Interchim, Montluçon, France), according to the manufacturer's instructions.

qRT-PCR analyses were done following essentially the same strategy described in Dallas et al. (2005). aRNA samples were treated with DNase I (Promega, Madison, WI, United States) and reverse-transcribed in cDNA using the SuperScriptTM III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific), with random primers and oligodT, according to the manufacturer's instructions. RT-PCR was performed with a LightCycler 480 Instrument (Roche Diagnostics, Basel, Switzerland) using either 2.5 µl of cDNA (at around 1.5 µg/µl), diluted at 1/5, or water (for negative control reactions) in a total PCR reaction final volume of 10 µl. Quantitative RT-PCR data were analyzed using the REST software<sup>1</sup> (Pfaffl et al., 2002).

The correlation analysis between microarray and qRT-PCR data was done with (i) a regression analysis using a linear model (R-squared of 0.8299; F = 210.8; d.f. = 42; P < 2.2 × 10–16), and (ii) a Pearson's coefficient of determination.

#### Counting and Size Determination of Aphid Bacteriocytes

For cell counting, bacteriocytes were surgically isolated from the abdomen of each individual aphid and counted at 25–40× magnification with an MDG-17 stereomicroscope (Leica). For each time point and each artificial diet, a total number of 10 aphids were analyzed. To determine their size, bacteriocytes were collected with a micropipette and mounted on glass slides. Double spacers made from microscope coverslips, with a thickness of 170 µm each, were used to mount bacteriocytes following the procedure we recently developed (Simonet et al., 2016a; 2018). The total space of 340 µm between microscope slides and the coverslip covering the preparation exceeds the maximal diameter of bacteriocytes (ranging from 40 to 120 µm) thereby preventing any physical damage or deformation of bacteriocytes. Bacteriocyte images were acquired and volumes were calculated as previously described (Simonet et al., 2016a). At least seven aphids were analyzed for each time point and each artificial diet. A total of 700 bacteriocytes were analyzed. To avoid bias, bacteriocyte counting and size determination were performed by three researchers in a blinded fashion.

#### RESULTS

#### Life History Traits of Aphids Reared on a Tyr/Phe Depleted Artificial Diet

Different parameters of A. pisum performance were recorded as metrics of aphid fitness: survival, adult weight and morphology, number of nymphs produced by the treated parthenogenetic mothers, timing of nymph production and nymph length and morphology. Despite slight differences, aphid survival was not significantly different when aphids are reared on a Tyr/Phe depleted diet (YFØ) compared to the standard artificial diet AP3 (**Figure 2A**, P = 0.156, Log Rank test). No remarkable difference in aphid size or morphology was detected. The lack of Tyr/Phe in the aphid diet did, however, induce a clear difference in aphid weight 7 days after the transfer on artificial diet (**Figure 2B**, P < 0.0001, two-sample Welch's t-test) indicative of an impact on aphid physiology. This difference was also reflected in the fecundity of the parthenogenetic mothers (**Figure 2C**). Even if there was no significant difference in number of produced nymphs observed at the end of the laying period between aphids reared on the two diets (PD24 = 0.6212, Mann–Whitney U test), we observed that YFØ aphids started their laying period (Day 12) 2 days later than AP3 aphids (Day 10). This resulted in a lower number of nymphs produced by the YFØ aphids from Day 10–13 (PD10 = 0.032, PD11 = 0.015, PD12 = 0.021, PD13 = 0.014, Mann–Whitney U tests). However, YFØ aphids rapidly reduced the gap with AP3 aphids in the following days. Adult weight differences are probably due to the fact that 7-day-old AP3 aphids hold older (and thus more advanced and bigger) parthenogenetic embryos than YFØ aphids since the former are closer to starting their laying period. Hence, YFØ aphids physiologically require additional time to start parthenogenetic embryo production, but once the laying period has started there is no difference with AP3 aphids in reproduction. Furthermore, all progeny is normal, devoid of morphological defects and with body lengths of nymphs of both conditions not significantly different (**Figure 2D**, P = 0.544, two-sample Student's t-test).

In summary, despite variations in aphid weight and onset of laying period, the overall fitness of A. pisum reared on the YFØ diet does not differ substantially from aphids reared on a complete artificial diet. In addition, the observation that the physiological response to Tyr/Phe nutritional stress happens rapidly suggests the presence of robust cellular and molecular strategies to control metabolic homeostasis of the pea aphid and its endosymbiont when facing a nutritional constraint.

#### Bacteriocytes but Not Gut Show a Prominent Transcriptional Response to Nutritional Stress

In a time course design, aphids were collected at seven time points following their transfer on the AP3 and YFØ artificial diets. We isolated the two tissues and analyzed transcriptomes of the gut for the seven time points and of the bacteriocytes for the last four time points, due to technical limitations preventing the analysis on the three earlier time points (**Figure 1**).

To check overall data quality, we first performed a PCA of normalized expression values for all the 24 011 genes of each sample in all tissues and diets. Using this approach, we were able to clearly classify the data in the two tissues analyzed (**Supplementary Figure S2**) thereby confirming the quality of the data. In addition, the expression of seven A. pisum genes belonging to different functional classes was monitored using qRT-PCR to validate the microarray data (**Supplementary Table S2**). qRT-PCR assays were performed on the same RNA

<sup>1</sup>http://rest.gene-quantification.info/

Frontiers in Physiology | www.frontiersin.org

FIGURE 2 | Impact of the tyrosine and phenylalanine depleted diet on aphid performances. (A) Survival analysis of A. pisum reared on two artificial diets: a complete diet (AP3, blue) and a Tyr/Phe depleted diet (YFØ, yellow). Each diet group was composed of 30 aphids. Data were analyzed using Log Rank Test. (B) Effects of the YFØ diet on aphid weight 7 days after the transfer on artificial diets. Each diet group was composed of 30 aphids. Data were analyzed using Welch's t-test and significant differences are indicated with asterisks (∗∗∗∗P ≤ 0.0001). (C) Cumulative number of progeny laid per aphid reared on the YFØ and AP3 diets. Results are reported as means (±SD) of 10 isolated individuals per diet. Data were analyzed by a Welch's t-test and significant differences are indicated with asterisks ( <sup>∗</sup>P ≤ 0.05). (D) Effects of the YFØ diet on the size of produced nymphs. A total of 43 and 29 nymphs were measured for the AP3 and YFØ diet, respectively. Data were analyzed using Student's t-test (ns, not significant).

samples used for the microarray experiment (all time points from the gut and bacteriocytes tissues of AP3 and YFØ aphids). A Pearson's coefficient of determination of 0.91 (P < 2.2 × 10−16) was observed between the qRT-PCR and microarray datasets showing a high degree of reliability for the microarray results (**Supplementary Figure S3** and **Supplementary Table S3**).

We next identified gut- and bacteriocyte-enriched genes using data from all time points of aphid samples reared on the complete AP3 artificial diet. Genes were considered tissue-enriched using a fourfold change threshold in the gut/bacteriocyte contrast. We identified 1 079 genes mainly expressed in the gut while 638 were categorized as bacteriocyte-enriched (**Supplementary Table S4**). Analysis of the potential function of these genes is represented using the eggNOG classification (Huerta-Cepas et al., 2016) in **Figure 3**. Based on this classification, we observed that bacteriocyte-enriched genes were mainly involved in transcriptional activity, signal transduction mechanisms, and carbohydrate, amino acid and lipid transport and metabolism.

The latter highlighting the pivotal role of the bacteriocytes in the symbiotic relationship between pea aphids and their endosymbiotic bacteria Buchnera. Transcriptional profiling of aphid gut identified genes associated with energy production and conversion, cytoskeleton, intracellular trafficking, secretion and vesicular transport are in line with its role in nutrient absorption. Note that a large proportion of genes for the two tissues are poorly annotated and were therefore categorized in a class named "function unknown."

Differentially expressed genes from YFØ aphids compared to AP3 aphids were identified using a one-way between groups ANOVA (Limma) applied at each time point independently. Of the 10 343 transcripts analyzed, 857 (8.29%) were identified as being significantly differentially expressed during nymphal development of the pea aphid reared on a Tyr/Phe depleted diet. Strikingly, most of the transcriptional changes induced by Tyr/Phe nutritional stress occurred in bacteriocytes (771 DEGs) whereas limited transcriptomic changes were observed in gut samples (89 DEGs) (**Table 1** and **Supplementary Table S5**). These differences in transcriptional response to nutritional stress define the bacteriocytes as a prominent mediator of metabolic homeostasis.

To characterize the role of the two tissues in the response to the nutritional stress and provide insight into the functional roles of each list of DEGs, an enrichment analysis of the functional gene classes based on the GO annotation was performed. We first performed the GO analysis using the pea aphid annotation. However, given the low number of DEGs associated with a GO term, only few and mostly high-level GO terms were identified (**Supplementary Table S6**). This is a problem frequently encountered with ontology-based enrichment studies in arthropods due to the large number of proteins without homologs and/or of unknown function [discussed in Wybouw et al. (2015)]. Thus, given the more extensive functional


TABLE 1 | Number of differentially expressed genes in aphids reared on a Tyr/Phe depleted diet (YFØ), in comparison to the control diet (AP3), for each tissue and each time-point.

annotation of genes in D. melanogaster, the GO analysis was carried out using the putative D. melanogaster homologs for each differentially expressed A. pisum gene (**Supplementary Tables S7**, **S8**). While the GO analysis on the pea aphid annotation retrieved only 372 unique GO terms from the 857 different DEGs, the D. melanogaster GO analysis encompassed 1 978 unique GO terms. This analysis of the functional gene classes revealed that no significant GO term related to biological process, molecular function or cellular component was enriched for any of the time points sampled from the gut tissue (**Supplementary Table S8**) thus confirming the limited role of this tissue under Tyr/Phe nutritional stress. Conversely, this analysis showed the importance of the bacteriocytes with a total of 98 significantly enriched GO terms (**Supplementary Table S8**).

# Tyr/Phe Biosynthesis Is Not Regulated in the Bacteriocytes

Given that bacteriocytes together with B. aphidicola are essential for the production of tyrosine and phenylalanine, we first hypothesized that the expression of the genes involved in their biosynthesis would be altered to accommodate for the lack of the amino acids. To test this hypothesis, we analyzed all identified bacteriocyte DEGs using the functional annotation available in the ArthropodaCyc database (Baa-Puyoulet et al., 2016), which also contains the global reconstruction of the metabolic network of the pea aphid.

The most striking result was the absence of genes related to Tyr and Phe biosynthesis among the bacteriocyte DEGs (**Table 2**), despite the tyrosine biosynthetic pathway being crucial for the symbiotic metabolism of the pea aphid and B. aphidicola (Wilson et al., 2010) and for normal parthenogenetic development of A. pisum (Rabatel et al., 2013; Simonet et al., 2016b). Additionally, none of the amino acid transporters expected to have an important functional role in bacteriocytes (Price et al., 2014) was differentially regulated (**Table 2**). The fact that none of the genes encoding for the Tyr/Phe biosynthetic enzymes and amino acid transporters was differentially expressed in the bacteriocytes, suggests that A. pisum uses an alternative strategy to adapt its physiology to this nutritional constraint.

# Transcriptional Reprogramming of the Bacteriocytes

In bacteriocytes, the main transcriptional changes (574 DEGs, 74.4%) were observed 3 days after the transfer on the artificial diet whereas only 6 (0.78%), 164 (21.3%), and 65 (8.43%) were detected at Day 4, 5, and 7, respectively (**Table 1**). Among these DEGs, only 34 were shared between two or three time points, whereas no DEG was shared between all four time points (**Supplementary Table S5**).

Interestingly, the GO enrichment analysis of the bacteriocyte DEGs revealed a temporal sequence of transcriptional events allowing the A. pisum/B. aphidicola symbiotic system to cope with Tyr/Phe deprivation in the food.

The large proportion (28.6%) of up-regulated genes related to "chromatin remodeling," "histone modifications," and "transcription factors" at Day 3 is consistent with the large number of DEGs at this time point (**Figure 4A** and **Supplementary Tables S7**, **S8**). Chromatin remodelers are known to regulate nucleosome dynamics to gate access to the underlying DNA for replication, repair and transcription (Petty and Pillus, 2013). Among the up-regulated genes involved in chromatin remodeling, putative A. pisum homologs (ACYPI004047 and ACYPI008655) of the ISWI D. melanogaster gene were identified as well as of the maleless (ACYPI002202) and msl-3 (ACYPI000966) genes (**Table 3**). The ISWI gene encodes a subunit of the nucleosome remodeling factor (Tsukiyama et al., 1995). The maleless and msl-3 genes were first characterized as core members of the chromatin remodeling male specific lethal (MSL) complex (Kuroda et al., 1991; Sural et al., 2008), but maleless is also known to play a role in other chromatin remodeling pathways (Cugusi et al., 2015). Consistent with chromatin remodeling, several "histone modification"-related genes were up-regulated at Day 3 (**Table 3**) among which three putative homologs (ACYPI003204, ACYPI006180, and ACYPI007884) of the histone deacetylase 1 (HDAC1). Histone acetylation and deacetylation are dynamic processes with a key role in gene expression regulation by relaxing or compacting chromatin structure (Haberland et al., 2009). Together with chromatin remodeling and histone modification-associated genes, 23 transcription factors were differentially expressed at Day 3 (**Table 3**) suggesting that distinct transcriptional programs are activated following Tyr/Phe stress.

#### Aphids Cope With Tyr/Phe Deprivation by Increasing Bacteriocyte Number and Changing Their Phenotype

Among the differentially expressed transcription factors, three putative homologs (ACYPI008477, ACYPI009169, and ACYPI39352) of the putzig D. melanogaster gene were identified (**Table 3**). The putzig gene encodes a Zn-finger protein


TABLE 2 | Differential expression in bacteriocytes of selected genes involved in Tyr/Phe biosynthesis and amino acids transport between aphids reared on a depleted Tyr/Phe artificial diet (YFØ) and aphids reared on a complete artificial diet (AP3).

The full list of differentially expressed genes can be retrieved from Supplementary Table S5.

(Kugler and Nagel, 2007) belonging to a large multi-protein complex that includes the TATA-box-binding-protein-related factor 2 (TRF2) and the DNA-replication related element binding factor (DREF) (Hochheimer et al., 2002). The TRF2/DREF complex has been associated with the transcriptional regulation of replication-related genes and consists of more than a dozen proteins including several known chromatin-remodeling components such as the ISWI protein (Hochheimer et al., 2002). Accordingly, putzig acts as a key regulator of cell proliferation through the positive regulation of cell cycle and

TABLE 3 | Differential expression in bacteriocytes at Day 3 of selected genes likely involved in chromatin remodeling, histone modifications, and regulation of gene expression.


The full list of differentially expressed genes can be retrieved from Supplementary Tables S7, S8.

replication-related genes (Kugler and Nagel, 2007). The presence of up-regulated genes associated with cell proliferation in YFØ aphids after 3 days on artificial diet is consistent with several other overrepresented GO terms at this time point which were related to the cell cycle including DNA replication, cell growth and cytoskeleton reorganization (**Figure 4A** and **Supplementary Table S8**). Consistent with the cell proliferation activation, we identified, at Day 3, several up-regulated helicases involved in DNA replication and associated DNA repair processes (**Table 4**) such as the mcm3 (ACYPI005644) as well as mcm4 (ACYPI004569) genes belonging to the minichromosome maintenance (MCM) complex (Su et al., 1996). Furthermore, the mus301 (ACYPI004507), mus304 (ACYPI008670), and blm/mus309 (ACYPI005125) genes involved in the DNA damage checkpoint pathways (Brodsky et al., 2000; Adams et al., 2003; McCaffrey et al., 2006) were overexpressed (**Table 4**).

In addition to cell cycle and proliferation-associated genes, we also obtained further evidence indicative of cell proliferation and growth. Three genes belonging to the Hippo pathway were up-regulated in YFØ aphids at Day 3 (**Table 4**). Specifically, we have identified the putative A. pisum homologs of the D. melanogaster genes: fat (ACYPI006463), a receptor of the Hippo signaling pathway (Feng and Irvine, 2007), jub (ACYPI005965), which inhibits activation of this pathway (Thakur et al., 2010), and brahma (ACYPI004206), a major subunit of a chromatin-remodeling complex promoting the transcription of cell proliferation related genes (Zhu et al., 2015).


TABLE 4 | Differential expression in bacteriocytes at Day 3 of selected genes likely involved in DNA replication, cell growth and proliferation.

The full list of differentially expressed genes can be retrieved from Supplementary Tables S7, S8.

Furthermore, 14 genes associated with actin cytoskeleton organization were differentially expressed in YFØ aphids at Day 3 (**Figure 4A**, **Table 4**, and **Supplementary Table S8**), suggestive of cytoskeletal reorganization as is observed with cell proliferation and growth. Specifically, we have identified the putative A. pisum homolog of slik (ACYPI002580) which has been demonstrated to stimulate cell proliferation in Drosophila (Hipfner and Cohen, 2003).

At Day 5 after transfer on artificial diet, the GO terms "decapentaplegic signaling pathway," "unidimensional cell growth," and "maintenance of cell number" were also significantly enriched (**Figure 4B** and **Supplementary Table S8**) thus providing further support for cell proliferation and growth being part of the A. pisum response to accommodate for a Tyr/Phe depleted food source. Therefore, to provide further experimental support for this notion, we determined number and volume of bacteriocytes from AP3 and YFØ aphids. In line with the GO analysis, we observed a significantly increased number of bacteriocytes in YFØ aphids compared to AP3 aphids from Day 3 to Day 11 (**Figure 5A**) coupled with a greater volume (**Figure 5B**).

### The Transcriptional Response of Bacteriocytes to Tyr/Phe Deprivation Is Indicative of Stress Response, Metabolic Slowdown and Adjusted Transport of Biomolecules

In addition to changes in cell proliferation and size, the transcriptional reprogramming of the bacteriocytes also involves additional processes (**Figure 4** and **Supplementary Table S8**). The "response to stress" GO term encompasses 36 up-regulated genes at Day 3 belonging to the previously identified signaling pathways as well as the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway with the presence of the domeless gene (ACYPI40957), the transmembrane receptor for signaling ligands in the cytokine family (**Table 5**). The conserved JAK/STAT signaling pathway transmits information received from extracellular polypeptide signals such as growth factors and cytokines, through specific transmembrane receptors, directly to target gene promoters in the nucleus, providing a mechanism for transcriptional regulation without second messengers (Aaronson and Horvath, 2002). JAK/STAT is known

Welch's t-test and significant differences are indicated with asterisks (∗P ≤ 0.05, ∗∗∗∗P ≤ 0.0001).

to regulate growth and the competitive status of proliferating cells (Mukherjee et al., 2005; Rodrigues et al., 2012) which is consistent with the previous observations.

Additionally, 3 days after transfer on artificial diet, eight carbohydrate transporters were down-regulated in YFØ aphids (**Figure 4A**, **Table 5**, and **Supplementary Tables S7**, **S8**). We also observed a continued overexpression of the putative A. pisum homolog (ACYPI001449) of RRAD, a small GTPase belonging to the RGK family. It has been demonstrated that this GTPase inhibits aerobic glycolysis in human cells (Wang et al., 2014; Shang et al., 2016). In addition to the carbohydrate transporters, the most enriched GO term among the down-regulated genes was also related to transport function (**Figure 4A** and **Supplementary Table S8**) suggesting a fine regulation of bacteriocyte transporter expression in YFØ aphids.

# DISCUSSION

Nutritional symbiosis is an important factor allowing insects to feed on specialized diets and underpinning their evolutionary and ecological success. In phloem-feeding hemipterans, the close interaction with their symbiotic partners allows these insects to thrive in ecological niches associated with an unbalanced diet of plant phloem sap. Besides its nutritionally unbalanced composition, the amino acid profile of phloem sap also shows considerable spatial and temporal variability thus presenting important challenges to sap-sucking insects. This study aimed to improve our understanding of how the hemipteran A. pisum and its mutualist endosymbiont accommodate to this ever-changing dietary resource.

First, our results revealed remarkable physiological plasticity of the A. pisum/B. aphidicola symbiotic system to cope with changes in food source, i.e., with Tyr and Phe deprivation. This nutritional stress did not affect aphid survival and we only observed limited weight reduction and a 2-day delay in onset of laying without effect on fecundity (**Figures 2**, **6**). This is not a general effect of lack of dietary amino acids, as depletion of other amino acids (e.g., leucine) from the AP3 diet can result in stunted aphid growth (80% reduction in size) (Viñuelas et al., 2011). Based on this, we conclude that dietary Tyr and Phe are not important for aphid survival, that they are required for normal growth but less so than Leu, and that they are essential to attain normal reproductive performance. Second, we identified the mechanisms that could be responsible for the tolerance of


TABLE 5 | Differential expression in bacteriocytes of selected genes likely involved in stress response and sugar metabolism.

The full list of differentially expressed genes can be retrieved from Supplementary Tables S7, S8.

aphids to the depletion of these two amino acids (summarized in **Figure 6**).

By means of a detailed time-course analysis of A. pisum gene expression on a genome-wide scale, we found that bacteriocytes show very prominent changes in gene expression upon Tyr/Phe depletion, contrary to the gut where only minor changes are seen. Interestingly, neither bacteriocytes nor gut showed transcriptional changes in Tyr/Phe biosynthetic pathway or amino acid transporter genes to compensate for the deprivation of these two amino acids. Even though we cannot exclude changes of expression in these pathways in other aphid tissues, the major changes were expected in the gut, which acts as a metabolic hub in insects, and in the bacteriocytes, which have a central role in housing and maintaining endosymbiotic bacteria (Douglas, 2014) and in symbiotic amino acid metabolism and transport (Nakabachi et al., 2005; Price et al., 2014). We also cannot completely exclude that the observed lack of induction of Tyr/Phe biosynthetic pathway or amino acid transporter genes is due to the early sampling of these two tissues (collected 1–7 days after the beginning of the nutritional stress) and higher induction being possible at later stages of aphid development. However, we believe this is less likely because the highest needs for phenylalanine and tyrosine have been demonstrated to be situated during aphid nymphal development, which is almost complete at Day 7 in our experimental conditions (Rabatel et al., 2013; Simonet et al., 2016b). Finally, it is unlikely that the differences observed in bacteriocyte transcriptomes are caused indirectly by the observed reduced growth and delay in fecundity for the following reasons: (i) we observed developmental stage transitions at the same moments in our time course analysis for both AP3 and YFØ aphids; (ii) differences in developmental timing would also be expected to show in the gut, which we did not observe. Therefore, we argue that the differences seen in bacteriocyte gene expression are due to the nutritional stress imposed by the absence of Tyr/Phe in the artificial diet. The observed bacteriocyte response to metabolic changes with massive transcriptional reprogramming points to a more complex physiological role of these specialized cells central to symbiosis.

Our results indicate that bacteriocytes use alternative strategies to cope with metabolic stress different from the classic autophagy-based ones (Mizushima and Klionsky, 2007). Instead of triggering cell death (our dataset does not show induction of apoptotic and autophagy-related pathways under nutritional stress), we discovered that aphids display a defined temporal sequence of molecular events that includes an extensive transcriptional reprogramming of the bacteriocyte cells through chromatin remodeling. This involves an increased transcription of genes related to cell growth, proliferation and associated signaling pathways. Consistent with this, we observed increases in bacteriocyte cell size and in bacteriocyte cell number. We had previously shown that bacteriocyte cell numbers show dynamic changes that are coordinated with symbiont numbers throughout the aphid life cycle (Simonet et al., 2016a). Furthermore, Douglas and Dixon (1987) have shown that the number of bacteriocytes is less fixed than was initially suggested, showing that it can vary among different aphid species, and also among different morphs of the same aphid species. Finally, Hongoh and Ishikawa (1994) described an increase in bacteriocyte number in starved adult aphids provided with food. In our study, these changes in number and size of bacteriocytes would allow the aphid host to accommodate and support more endosymbionts and it could therefore represent a strategy to indirectly produce more tyrosine and phenylalanine using the precursors furnished by B. aphidicola to the A. pisum/B. aphidicola symbiotic system. Such strategy is consistent with previous studies that have demonstrated variations in B. aphidicola densities in different aphid species depending on the host plant or the availability of dietary nitrogen (Wilkinson et al., 2001, 2007; Zhang et al., 2016). We propose that the observed bacteriocyte-dependent

responses are part of a dynamic response to changes in nutrition and contribute to the ecological success of these insect pests.

In parallel, we found a rapid down-regulation of genes encoding sugar transporters and genes required for sugar metabolism. Consistently, we found continued overexpression of the putative A. pisum homolog of RRAD, a small GTPase implicated in repressing aerobic glycolysis. The up-regulation of this gene in YFØ aphids suggests that overall sugar metabolism is repressed. Since phloem sap is a food source rich in carbohydrates, this metabolic slow-down could represent a strategy for aphids facing reduced amino acid availability in their food source to reallocate energy, through the transcriptional reprogramming previously observed, in other metabolisms and/or pathways to cope with Tyr/Phe deprivation. Furthermore, we also observed changes in the expression of other transporter genes: such transport modulation could be an additional way to change the balance of precursors in order to optimize Tyr and Phe biosynthesis and distribution in the context of a nutritional stress. It is worth noting that, as often in non-model insect genomes, numerous genes among the significantly differentially expressed lack full functional annotation thus limiting the interpretation of the results. Nonetheless, their expression pattern (e.g., enriched in bacteriocytes) suggests that (some of) these genes may play a thus far unrecognized, novel role in the response to nutritional stress, a possibility that will be explored in future work.

Altogether these results shed light on unknown aspects of the transcriptional and cellular plasticity of insect bacteriocytes. They suggest that this plasticity is essential to a phloem-feeding insect like the pea aphid to cope with the ever-changing composition of the plant phloem sap. These findings are consistent with the previously described lack of response of the symbiotic bacteria and instead show that the host, i.e., the bacteriocytes, responds in a complex manner to the deprivation of some amino acids (i.e., Tyr and Phe) in its food source. Indeed, this nutritional stress induces a rapid and extensive transcriptional reprogramming of bacteriocytes that fine-tunes physiology of these cells and leads to changes in cell size that can possibly be involved in accommodating larger numbers of endosymbionts. By extension, equivalent responses may be present in other biological systems that depend on nutritionally unbalanced diets for their growth and reproduction, a hypothesis that remains to be tested in future research. Finally, our results on the A. pisum/B. aphidicola system raise further interesting future research perspectives that include (i) the bacteriocyte responses to deprivation of other amino acids and other nutritional/environmental challenges, (ii) the impact of additional endosymbionts on this process in multi-partner symbiotic relationships widely found in nature, and (iii) the role of bacteriocytes in nutritional homeostasis mechanisms in other aphids feeding on different host plants, in other hemipteran species (e.g., whiteflies and psyllids), and finally in symbiosis in other insect orders.

#### DATA AVAILABILITY

fphys-09-01498 October 23, 2018 Time: 14:25 # 16

The datasets generated for this study can be found in the ArrayExpress database (Accession No. E-MTAB-4456).

#### AUTHOR CONTRIBUTIONS

SC, YR, HC, GF, and FC conceived and designed the study. SC, PS, KG, GD, and FC performed the experiments. SC, NP, PS, PB-P, PC, and FC performed the data analysis. SC, NP, PS, GF, PC, and FC interpreted the results. SC, NP, PC, and FC wrote the paper with contributions from KG. All authors revised and approved the manuscript.

#### REFERENCES


#### FUNDING

This work was supported by INRA, INSA-Lyon, INSA-Lyon BQR program grant to SC, the French ANR-13-BSV7-0016-03 (IMetSym) program grant, and the French Ministry of Research that awarded a Ph.D. fellowship to PS. PC was supported by an Invited Professor Grant of INSA-Lyon.

#### ACKNOWLEDGMENTS

RT-qPCR analyses were carried out in the DTAMB (University of Lyon). The authors would like to thank Julia Gilhodes for her help at the beginning of the project.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01498/full#supplementary-material



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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Colella, Parisot, Simonet, Gaget, Duport, Baa-Puyoulet, Rahbé, Charles, Febvay, Callaerts and Calevro. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Molecular Identification of Two Thioredoxin Genes From Grapholita molesta and Their Function in Resistance to Emamectin Benzoate

Zhong-Jian Shen, Yan-Jun Liu, Xu-Hui Gao, Xiao-Ming Liu, Song-Dou Zhang, Zhen Li, Qing-Wen Zhang and Xiao-Xia Liu\*

Department of Entomology, MOA Key Laboratory of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, China

Thioredoxins (Trxs), a member of the thioredoxin system, play crucial roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative stress. In this study, we cloned and characterized two genes, GmTrx2 and GmTrx-like1, from Grapholita molesta. Sequence analysis showed that GmTrx2 and GmTrx-like1 had highly conserved active sites CGPC and CXXC motif, respectively, and shared high sequence identity with selected insect species. The quantitative real-time polymerase chain reaction results revealed that GmTrx2 was mainly detected at first instar, whereas GmTrx-like1 was highly concentrated at prepupa day. The transcripts of GmTrx2 and GmTrx-like1 were both highly expressed in the head and salivary glands. The expression levels of GmTrx2 and GmTrx-like1 were induced by low or high temperature, E. coli, M. anisopliae, H2O2, and pesticides (emamectin benzoate). We further detected interference efficiency of GmTrx2 and GmTrx-like1 in G. molesta larvae and found that peroxidase capacity, hydrogen peroxide content, and ascorbate content all increased after knockdown of GmTrx2 or GmTrx-like1. Furthermore, the hydrogen peroxide concentration was increased by emamectin benzoate and the sensitivity for larvae to emamectin benzoate was improved after GmTrx2 or GmTrx-like1 was silenced. Our results indicated that GmTrx2 and GmTrx-like1 played vital roles in protecting G. molesta against oxidative damage and also provided the theoretical basis for understanding the antioxidant defense mechanisms of the Trx system in insects.

Keywords: Grapholita molesta, Thioredoxins, RNA interference, oxidative stress, antioxidant defense

# INTRODUCTION

Reactive oxygen species (ROS) is a collective term for hydrogen peroxide, superoxide anion, and hydroxyl radical. It can be generated during metabolic activities and induced by external factors such as pro-oxidants, heavy metals, pesticides, and other adverse factors (Imlay, 2003). Excessive ROS can disrupt the balance of intracellular redox homeostasis (Droge, 2002) and cause oxidative damage to proteins, lipids, and nucleic acids (Wu et al., 2011). For protecting against the toxicity of excessive ROS, aerobic organisms have developed complex antioxidant enzymatic systems to maintain the intracellular ROS at proper levels (Kobayashi-Miura et al., 2007). The thioredoxin

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Aram Megighian, Università degli Studi di Padova, Italy Zhaojiang Guo, Chinese Academy of Agricultural Sciences, China

> \*Correspondence: Xiao-Xia Liu liuxiaoxia611@cau.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 18 June 2018 Accepted: 18 September 2018 Published: 25 October 2018

#### Citation:

Shen Z-J, Liu Y-J, Gao X-H, Liu X-M, Zhang S-D, Li Z, Zhang Q-W and Liu X-X (2018) Molecular Identification of Two Thioredoxin Genes From Grapholita molesta and Their Function in Resistance to Emamectin Benzoate. Front. Physiol. 9:1421. doi: 10.3389/fphys.2018.01421

**139**

(Trx) is an important part of thioredoxin systems that play important roles in redox-regulatory processes (Lu and Holmgren, 2014).

Thioredoxins (Trxs) are the family of soluble proteins involved in cellular dithiol-disulfide redox of organisms (Rietsch and Beckwith, 1998). The Trxs can protect cells from the damage of ROS generated under stressful conditions by acting as disulfide reductases or electron donors in the reduction of disulfide and dithiol (Stryer et al., 1967; Holmgren, 1979; Nakamura et al., 1997; Kalinina et al., 2008; Mahmood et al., 2012). It plays a crucial role in the maintenance of cellular thiol redox balance (Arnér and Holmgren, 2000; Chen et al., 2002; Myers et al., 2008).

In insects, research on Trx has been focused on a few species of insects, for example, Drosophila melanogaster, Bombyx mori, and Apis cerana cerana. In D. melanogaster, three Trx genes (Trx1, Trx2, and TrxT) have been identified (Kanzok et al., 2001; Bauer et al., 2002; Svensson et al., 2003), and DmTrx2 played important roles in redox regulation, the oxidative defense system, and modulating the lifespan of flies (Bauer et al., 2002; Svensson and Larsson, 2007). In B. mori, BmTrx has been shown to have a major role in resisting oxidative stress caused by extreme temperatures and microbial infection (Kim et al., 2007). In Apis mellifera, AmTrx1 was found in the mitochondria, which is an organelle responsible for aerobic respiration, suggesting AmTrx1 might be involved in scavenging ROS in mitochondria (Corona and Robinson, 2006). In A. cerana cerana, some Trxs, including AccTrx-like1, AccTrx1, and AccTrx2 have been demonstrated to participate in antioxidant defense (Lu et al., 2012; Yao et al., 2013, 2014). In Lepidoptera, SlTrx1 and SlTrx2 from Spodoptera litura and HaTrx2 from Helicoverpa armigera have been identified and demonstrated to participate in antioxidant defense (Kang et al., 2015; Zhang et al., 2015). These studies suggested that Trxs play important roles in maintaining redox homeostasis and resisting adverse circumstances in insects. However, there are no relevant reports in Grapholita molesta.

The G. molesta (Busck) is a worldwide pest of stone and pome fruits in most temperate fruit-growing regions (Kirk et al., 2013). Nowadays, due to the larvae's habits of drilling, insecticides used for egg and neonate are the main means to control G. molesta. Unfortunately, organophosphate insecticide resistance has occurred in G. molesta (Kanga et al., 2003). It is crucial to understand which genes are involved in the defense mechanism of insects. According to previous studies, Trxs play an important role in elucidating the ROS induced by extreme environments. We hypothesize that Trx2 and Trx-like1 can help to resist the insecticides in G. molesta. To elucidate the functions of Trx system genes in G. molesta, we have identified Trx2 and Trx-like1 and analyzed their spatio-temporal expression patterns. Moreover, the transcript levels of GmTrx2 and GmTrx-like1 were also assessed after various types of stress treatments (low or high temperatures, exposure to Escherichia coli, Metarhizium anisopliae, H2O2, and emamectin benzoate infection). After GmTrx2 or GmTrx-like1 knockdown, we further examined antioxidant enzyme activities and metabolite amounts. Finally, we detected the content of hydrogen peroxide caused by emamectin benzoate and examined the susceptibility of larvae to emamectin benzoate after GmTrx2 or GmTrx-like1 silencing.

# MATERIALS AND METHODS

# Ethics Statement

In this study, the larvae of oriental fruit moth G. molesta were originally collected in the Institute of Pomology in Liaoning province, China. No permissions were required for the insect collection, as the orchards are experimental plots belonging to the Institute of Pomology in Liaoning province. The "List of Protected Animals in China" excludes insects.

#### Insect Rearing

The G. molesta used in this study were reared on fresh apples and an artificial diet at a constant temperature of 25 ± 1 ◦C at 70 ± 10% RH and with a 15 L: 9 D light regime in our laboratory. Adults were reared in beakers (1 L in volume) with fresh apples inside for egg laying and fed with 10% honey solution. The fifth instars larvae picked out from rotten apples were transferred to finger-shaped glass tube (5.5 cm in length × 2.2 cm in diameter) on artificial diet until the pupation.

# Total RNA Extraction and cDNA Synthesis

The TRIzol reagent (TaKaRa, Kyoto, Japan) was used to extract total RNA according to the instructions. The quality and quantity of the extracted RNA products were determined by using an ultraviolet spectrophotometer (Abs260) and a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan); the first-strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA according to the manufacturer's instructions.

#### Primer Design and Quantitative Real-Time Polymerase Chain Reaction Amplification Conditions

The DNAClub and DNAman software were used to design GmTrx2 and GmTrx-like1 primers for reverse transcription PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), and dsRNA synthesis. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH, KJ094948.1) and actin (actin, KF022227.1) were used as internal reference genes for the qRT-PCR to normalize target gene expression. The primers were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China).

The qRT-PCR was conducted in a 20 µl reaction volume comprising 1 µl cDNA, 10 µl 2 × SYBR Green Supermix (TaKaRa, Kyoto, Japan), 2 µl qRT-PCR primers, and 7 µl ddH2O on a Bio-Rad CFX Connect Real-Time PCR Detection System (Hercules, CA, United States). The amplification was performed following the program as: 95◦C for 30 s, followed by 40 cycles at 95◦C for 5 s, and 60◦C for 30 s. The 2−11CT method was used for the quantitative analysis (Livak and Schmittgen, 2001).

# Cloning, Sequencing, and Sequence Analysis of GmTrx2 and GmTrx-like1

The genes of GmTrx2 and GmTrx-like1 were obtained from G. molesta transcriptome. The full-length cDNA sequences

of both Trx genes were amplified with specific primers (**Supplementary Table S1**) and G. molesta cDNA. According to the manufacturer's instructions of the DNA fragment purification kit (TaKaRa, Kyoto, Japan) and the pMDTM18-T Vector Cloning Kit (TaKaRa, Kyoto, Japan), the PCR products were sequentially purified and cloned into the PMD 18-T vector. The recombinant plasmid extracted was sequenced by Sangon Biotechnology Co., Ltd. (Shanghai, China).

The online bioinformatics ProtParam tool<sup>1</sup> was used to analyze the physicochemical properties of GmTrx2 and GmTrxlike1. The related homologous protein sequences from various species were obtained from NCBI database and analyzed using DNAman 6.0.3 software. The conserved domains in GmTrx2 and GmTrx-like1 were detected using bioinformatics tools available on the NCBI server<sup>2</sup> . The phylogenetic trees based on amino acid sequence were constructed by using the neighbor-joining method with poisson model, uniform rates, and complete deletion in MEGA 5.10 software. To ensure the accuracy of the tree structure, the tree was created with 1000 replicates.

### Developmental Analysis and Tissue Distribution of GmTrx2 and GmTrx-like1

To determine the spatio-temporal expression pattern of GmTrx2 and GmTrx-like1, samples from different developmental stages and different tissues of larvae were collected. The different developmental stages included eggs, first, second, third, fourth, fifth instar, and prepupa; first, third, fifth, and seventh day pupae; and first and third day adults. The stages of pupae and adults were divided into male and female. The different tissues including the head, epidermis, fat body, midgut, Malpighian tubules, and salivary glands were collected from larvae of the fifth instar. All samples were immediately stored at −80◦C for total RNA extraction and cDNA synthesis.

#### Effect of Different Types of Stress on the Expression of GmTrx2 and GmTrx-like1

To clarify the effect of various stress on GmTrx2 and GmTrx-like1 expression, fifth instar larvae were treated by low temperature (15◦C) or high temperature (35◦C), E. coli, M. anisopliae, H2O2, and emamectin benzoate. In the temperature treatments, the larvae were exposed to 15 and 35◦C and larvae in 25◦C were as a control; For E. coli, M. anisopliae, and H2O<sup>2</sup> treatments, the larvae were injected with 1 µL of 1.0 × 105 E. coli cells, M. anisopliae (1.0 cfu/mL × 105 cfu/mL), and hydrogen peroxide (concentration of 100 mM), respectively; control larvae were injected with an equal volume of phosphate buffered saline. The samples from each group were collected at 0, 6, 12, and 24 h after injection. In insecticide treatments, larvae were soaked in emamectin benzoate (active ingredient concentration: 16.42 mg/L), purchased from Hansen Biologic Science Co., Ltd. (Qingdao, China) for 5 s and samples were collected at 2, 4, and 8 h. The larvae soaked in water

<sup>1</sup>http://web.expasy.org/protparam/

<sup>2</sup>https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

were used as the controls. There were 13 larvae in each treatment group and 10 larvae in the control group in one replicate and three replicates were used for each treatment. All samples were immediately stored at −80◦C for later total RNA extraction.

#### Synthesis of dsRNA and Detecting of RNAi Efficiency

The DNA fragment purification kit (TaKaRa Biotechnology, Dalian, China) was used to purify PCR products, which were synthesized with the primers containing a T7 polymerase promoter sequence (**Supplementary Table S1**) and cDNA of G. molesta. The purified PCR products were used as templates and MEGAscript RNAi Kit (Ambion) was used to synthesize the dsRNA of EGFP, GmTrx2, and GmTrx-like1, according to the manufacturer's instructions. The synthesized dsRNA products were purified by using MEGAclear columns (Ambion) and redissolved with diethyl pyrocarbonate-treated nuclease-free water. The purity and concentration of dsRNA were measured with ultraviolet spectrophotometry and gel electrophoresis. To determine the interference efficiency of GmTrx2 and GmTrxlike1, fifth instar larvae were injected with approximately 3 µg of dsRNA into proleg using capillary microsyringe. The larvae were injected with dsEGFP as a control. One replicate of the treatments injected with dsRNA of GmTrx2 or GmTrx-like1 contained 13 larvae, while the treatments injected with dsEGFP had 10 larvae. Three replicates were used for each treatment. All samples were collected at 24, 48, and 72 h and stored at −80◦C for later detection of gene expression.

#### Analysis of Enzymatic Activity and Metabolite Content After Knockdown of GmTrx2 and GmTrx-like1

The samples were collected at 48 and 72 h after dsRNA injection of EGFP, GmTrx2, and GmTrx-like1. The BCA Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to extract and quantify total protein. The capacity of superoxide dismutase and peroxidase were assayed by using superoxide dismutase test kit and peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively. The hydrogen peroxide, and ascorbate test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to quantify the contents of hydrogen peroxide and ascorbate, respectively, according to the manufacturer's protocols.

# Determination of H2O<sup>2</sup> Contents and Survival Assay

Whole body larval samples were collected at 4 and 8 h after soaking in the emamectin benzoate (active ingredient concentration: 16.42 mg/L) for 5 s. The method for detecting hydrogen peroxide concentration was the same as earlier. In the survival assay, after injecting the 3 µg dsRNA of GmTrx2 or GmTrx-like1 for 12 h, the larvae were immersed in emamectin benzoate (active ingredient concentration: 1.642 mg/L) for 5 s. Non-injected and injection of dsEGFP larvae were as the controls.

species. Black represents 100% identity, red represents ≥75% identity, green represents ≥50% identity, and white represents <50% identity. The conserved CGPC motif is boxed and the active sites are marked by ↑. GmTrx2 (Grapholita molesta, MH443001), PrTrx2 (Pieris rapae, XP\_022125058.1), PpTrx2 (Papilio polytes, NP\_001298667.1), HaTrx2 (Helicoverpa armigera, XP\_021198961.1), SlTrx2 (Spodoptera litura, XP\_022828547.1), PxTrx2 (Papilio xuthus, NP\_001299755.1), DvTrx2 (Drosophila virilis, XP\_002057775.1), DmTrx2 (Drosophila mojavensis, XP\_002002787.1), BtTrx2 (Bombus terrestris, XP\_012164907.1), and AccTrx2 (Apis cerana cerana, AFU83101.1). (B) Phylogenetic tree analysis of GmTrx2 and its homologs in insects.

The number of dead larvae was recorded in each group for 96 h. The tested insects used for the groups of emamectin benzoate, emamectin benzoate + dsEGFP, emamectin benzoate + dsTrx2, and emamectin benzoate + dsTrx-like1 were 75, 73, 69, and 70, respectively.

#### Statistical Analyses

The data are presented as means ± standard error (SE) with three independent replicates. Statistically significant differences in gene expression, the results of enzymatic activity, and metabolite contents were evaluated with pair-wise Student's t-test analysis using SPSS 17.0 software and denoted by ∗ (0.01 < P < 0.05) and ∗∗(P < 0.01). The data for spatiotemporal expression pattern and mortality were analyzed using ANOVA, followed by a Tukey's HSD multiple comparison test, and the letters were used to indicate significant differences at P < 0.05. Survival curves were analyzed by the method of Kaplan-Meier and statistical significance between survival curves

was determined using the log-rank test, when P-values were <0.05.

#### RESULTS

#### Sequence Analysis of GmTrx2 and GmTrx-like1

Sequence analysis showed the GmTrx2 gene was 321 bp in length and encoded 106 amino acids with a predicted molecular weight of 11.8 kDa and an isoelectric point of 4.74. Multiple amino acid alignments showed GmTrx2 had 64–84% identity with homologous sequences of selected species (**Figure 1A**). The N-terminal portion of the GmTrx2 had a highly conserved active site sequence of CGPC among all of the selected insect species (**Figure 1A**). The phylogenetic tree indicated that GmTrx2 was most closely related to genes from Pieris rapae (**Figure 1B**), which was also consistent with the results of multiple amino acid alignments. The GmTrx-like1 had an open reading frame of 861 bp and encoded 286 amino acids. The predicted weight of

protein was 31.4 kDa and the isoelectric point was 5.48. Multiple alignment analysis of the amino acid sequence showed that GmTrx-like1 shared amino acid identity (61–88%) with Trx-like1 sequences from selected insect species and the high conserved CXXC motif was found in N-terminal portion (**Figure 2A**). Phylogenetic analysis showed that GmTrx-like1 was more closely related to the SlTrx-like1 and HaTrx-like1 homolog (**Figure 2B**).

# Temporal and Spatial Expression Profiles of GmTrx2 and GmTrx-like1

The GmTrx2 was detected in all developmental stages and mainly expressed at stage L1 (first larval day) and stage P1 in female (first day of female pupa) compared to other stages (**Figure 3A**). For spatial expression, GmTrx2 had the highest levels of expression in the salivary glands, followed by the head and Malpighian tubules (**Figure 3B**). The mRNA expression of GmTrx-like1 was highest at stage prep (prepupa day) and the spatial expression profiles revealed GmTrx-like1 transcripts were expressed the highest in the head and salivary glands (**Figures 3C,D**).

#### Expression Profiles of GmTrx2 and GmTrx-like1 Under Various Oxidative Stresses

The results of qRT-PCR revealed that GmTrx2 was obviously induced by 15 and 35◦C, E. coli, M. anisopliae, and H2O<sup>2</sup> treatments at 6, 12, and 24 h, and then being markedly upregulated at 2, 4, and 8 h after emamectin benzoate immersion (**Figure 4**). The expression of GmTrx-like1 was significantly induced at 6 h after 15◦C and H2O<sup>2</sup> exposure treatments (**Figures 5A,E**). At 35◦C, GmTrx-like1 was markedly increased at 6 and 12 h, whereas was suppressed at 24 h (**Figure 5B**). Under stress of E. coli, GmTrx-like1 transcription was dramatically induced at 12 and 24 h, in addition to being signally enhanced at 6 and 12 h after M. anisopliae exposure treatment and obviously induced at 2, 4, and 8 h in response to emamectin benzoate treatment (**Figures 5C,D,F**).

#### Knockdown of GmTrx2 and GmTrx-like1 and Effects on Enzymatic Activities and Metabolite Contents After Silencing of the Two Genes

The GmTrx2 and GmTrx-like1 expressions were significantly inhibited at 24, 48, and 72 h when both the genes were knocked down compared to with EGFP dsRNA injection (**Figure 6**). The interference efficiency of GmTrx2 was 53.55, 39.06, and 32.06% at 24, 48, and 72 h (**Figure 6A**), while GmTrx-like1 was 46.93, 43.57, and 81% at 24, 48, and 72 h (**Figure 6B**) after the injection of GmTrx2 or GmTrx-like1 dsRNA, respectively.

The peroxidase capacity after silencing of GmTrx2 or GmTrxlike1 was higher than that in the control groups (**Figure 7A**),

while there was no significant change in enzymatic activities of superoxide dismutase (**Figure 7B**). The contents of hydrogen peroxide and ascorbate were increased at the measured time after knockdown of GmTrx2 or GmTrx-like1, compared with control groups (**Figures 7C,D**).

#### Assay of Oxidant Status in vivo Under Emamectin Benzoate and Survival Assay

1To further determine the functions of GmTrx2 and GmTrx-like1, we detected the contents of H2O<sup>2</sup> under emamectin benzoate and the effects of GmTrx2 or GmTrx-like1 knockdown on the susceptibility of G. molesta larvae exposed to emamectin benzoate. The results showed that H2O<sup>2</sup> concentration was dramatically increased under emamectin benzoate (**Figure 8A**). Survival curves revealed that GmTrx2 or GmTrx-like1 knockdown also obviously promoted susceptibility for G. molesta larvae to emamectin benzoate, compared to EGFP dsRNA injection (**Figure 8B**). The mortality at 96 h of emamectin benzoate (37.26%) and emamectin benzoate + dsEGFP (35.50%) larvae was significantly lower than in emamectin benzoate + dsGmTrx2 (62.49%) or emamectin benzoate + dsGmTrx-like1 (67.94%) larvae (P < 0.05, **Figure 8C**).

# DISCUSSION

Oxidative stress is involved in many different disease processes and causes alterations in the cellular redox state (Jiménez et al., 2006). The Trxs have functions in resisting oxidative damage caused by ROS and maintaining cellular redox homeostasis

(Holmgren, 1985; Arnér and Holmgren, 2000; Myers et al., 2008). Previous studies have focused on model insects, for example, B. mori (Kim et al., 2007) and Apis cerana cerana (Yao et al., 2013). In this study, we focused on the important fruits pest, G. molesta. We have identified and characterized GmTrx2 and GmTrx-like1 from G. molesta. Domain analysis showed that GmTrx2 and GmTrx-like1 possessed highly conserved CGPC and CXXC sequences, respectively, suggesting that the two genes belonged to the Trx families.

Temporal expression profiles showed that both GmTrx2 and GmTrx-like1 expressions appeared as large fluctuations at the physiological processes of incubation, pupation, and emergence whether male or female. These results suggested that the two genes may play important roles in antioxidant defense in these stages, because these periods of intense physical activity may cause excessive accumulation of ROS. Our transcriptional analysis revealed that the GmTrx2 and GmTrx-like1 were expressed at higher levels in the head and salivary glands than other larval tissues. In other insects, BmTrx exhibited higher expression in the fat body and silk gland (Kim et al., 2007); AccTrx1 was mainly expressed in the epidermis (Yao et al., 2014); HaTrx2 gene was expressed at higher levels in the head and epidermis (Zhang et al., 2015). It revealed that the expression of Trx exhibits a tissue-specific pattern. In addition, the brain tissue was very sensitive to oxidative stress (Rival et al., 2004; Ament et al., 2008) and AccTrx2 and AccTrx-like1 had a higher transcript in brain (Lu et al., 2012; Yao et al., 2013), implying that GmTrx2 and GmTrx-like1 may play vital functions in the head.

Previous studies have reported that environmental conditions, such as ultraviolet radiation, pesticides, temperature, and heavy metals, can induce oxidative stress (Lushchak, 2011; Kottuparambil et al., 2012). The Trx is a stress-inducible protein and plays significant roles in the scavenging or quenching of oxidants. In D. melanogaster, Trx2 has been proved to be

closely associated with resistance to oxidative stress (Svensson and Larsson, 2007). The cumene hydroperoxide, indoxacarb, and metaflumizone could stimulate the expression levels of SlTrx1 and SlTrx2 in S. litura (Kang et al., 2015). The AccTrx1 and AccTrx2 from A. cerana cerana were upregulated by low or high temperatures, H2O2, and pesticides (acaricide, paraquat, cyhalothrin, and phoxime) treatments (Yao et al., 2013, 2014). In H. armigera, the transcript of HaTrx2 was

GmTrx2 or GmTrx-like1 knockdown. The data represent the mean ± SE of three biological samples. <sup>∗</sup>0.01 < P < 0.05; ∗∗P < 0.01. a and b: signification difference,

significantly stimulated by low or high temperatures, UV light, mechanical injury, microorganism [E. coli, M. anisopliae, and nucleopolyhedrovirus (NPV)] (Zhang et al., 2015). In this study, GmTrx2 and GmTrx-like1 were obviously induced by 15 and 35◦C, E. coli, M. anisopliae, H2O2, and pesticides-emamectin benzoate treatments, suggesting that GmTrx2 and GmTrx-like1 may play critical roles in resisting oxidative stress caused by these adverse conditions.

It was demonstrated that antioxidant enzymes (such as Grx, Trx, and POD) and antioxidant substances [such as ascorbate, protein carbonyl, and glutathione (GSH)] were used to protect organisms by scavenging excess ROS (Meister, 1994; Corona and Robinson, 2006). The changes in antioxidant enzyme activity and metabolite concentrations after gene silencing were also used to demonstrate the function of genes in resisting oxidative stress. For example, after knockdown of HaGrx, HaGrx3, and HaGrx5 in H. armigera and AccTrx1 in A. cerana cerana, the enzymatic activities of peroxidase, the amount of hydrogen peroxide, and the amount of ascorbate all increased (Yao et al., 2014; Zhang et al., 2016), implying these genes play important roles in resisting oxidative stress. In our study, RNAi mediated knockdown of GmTrx2, and GmTrx-like1 increased the enzymatic activities of POD and the metabolite contents of hydrogen peroxide and ascorbate. It suggested that larvae were exposed to higher oxidative stress after GmTrx2 and GmTrx-like1 were silenced, and the genes of GmTrx2 and GmTrx-like1 may be involved in protecting G. molesta against oxidative stress.

In A. cerana cerana, the H2O<sup>2</sup> concentration and the transcripts of AccTpx5 were induced by phoxim and pyriproxyfen, indicating that oxidative stress might be associated

P < 0.05.

with alterations in AccTpx5 expression (Yan et al., 2014). In H. armigera, the larvae injected by NPV increased ROS production and the lipid damage (Zhang et al., 2015). In this study, emamectin benzoate could dramatically increase the content of hydrogen peroxide and the expressions of GmTrx2 and GmTrx-like1. It revealed that the larvae were exposed to higher oxidative stress and the two genes may be involved in the clearance of hydrogen peroxide caused by insecticide. In order to determine the roles of GmTrx2 and GmTrx-like1 involved in protecting organisms from oxidative stress caused by emamectin benzoate, we further examined effects of GmTrx2 or GmTrx-like1 interference on the survival curves of emamectin benzoate to G. molesta and found that the silencing of the two genes increased the sensitivity of larvae to emamectin benzoate and significantly improved mortality at 96 h, suggesting GmTrx2 and GmTrxlike1 were essential in the removal of excessive ROS to protect organisms. At present, the resistance of pests to insecticides was mainly concentrated on important detoxification enzymes such as cytochrome P450. But some antioxidant genes also played an important role in the antagonism of insects, for example, SlTpx inhibited Nomuraea rileyi infection in S. litura (Chen et al., 2014); HaTrx2 was involved in the defense of larvae against NPV infections in H. armigera (Zhang et al., 2015); GmTrx2 and GmTrx-like1 protected larvae against emamectin benzoate in our study. Therefore, some antioxidant genes, included GmTrx2 and GmTrx-like1, may become potential targets for insecticide synergists against G. molesta.

The Trxs are ubiquitously distributed from Archaea to humans (Lu and Holmgren, 2014). Although it has a conserved domain in all species, Trxs from Archaea to humans have only 27–69% sequence identity to that of E. coli Trx1 (Eklund et al., 1991). The sequence inconsistency in a few residues at the amino and carboxyl ends (Eklund et al., 1991) and different mechanisms for reducing Trx (Gleason and Holmgren, 1988) may be responsible for that the physiological functions of Trxs in different types of organisms have evolved from a common fundamental reaction to a large number of different specialized functions. For example, E. coli Trx stabilized complexes of bacteriophage T7 DNA polymerase and primed templates (Huber et al., 1987). In bacteria and yeast, Trx served as electron donors of 30-phosphoadenylsulfate (PAPS) reductase (Schwenn et al., 1988; Lillig et al., 1999). In plants, Trx was involved in regulation of chloroplast photosynthetic enzymes (Buchanan, 1991). In mammals, Trx played an important role in the regulation of redox regulation of transcription factors (Schenk et al., 1994), regulation of apoptosis (Saitoh et al., 1998), and immune regulation (Silberstein et al., 1993; Nakamura et al., 1997; Bertini

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et al., 1999). Therefore, it is very necessary and useful to further study the protein structures, mechanisms, and specific active sites of Trxs in G. molesta for finding a suitable way to control pests without affecting other organisms.

# CONCLUSION

We have characterized two genes, GmTrx2 and GmTrx-like1, and determined their temporal-spatial expression profiles. The expression levels of GmTrx2 and GmTrx-like1 were induced by low or high temperature, E. coli, M. anisopliae, H2O2, and pesticides such as emamectin benzoate. After knockdown of GmTrx2 or GmTrx-like1, the enzymatic activities of POD and the metabolite contents of hydrogen peroxide and ascorbate all increased. These results revealed that GmTrx2 and GmTrxlike1 may play important roles in resistance to excessive ROS. Emamectin benzoate increased the H2O<sup>2</sup> concentration and GmTrx2 or GmTrx-like1 were silenced, which improved the sensitivity of larvae to insecticide-emamectin benzoate, further indicating that GmTrx2 and GmTrx-like1 play vital roles in protecting G. molesta against oxidative damage. These findings may be useful for understanding the antioxidant defense mechanisms of the Trx system in insects.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work. Z-JS and X-XL conceived and designed the experiments. Z-JS and Y-JL performed the experiments. Z-JS, X-ML, S-DZ, and Y-JL analyzed the data. Z-JS, X-ML, S-DZ, and X-HG contributed to reagents, materials, and analysis tools. Z-JS, ZL, Q-WZ, and X-XL wrote the paper.

# FUNDING

This study was supported by the China Modern Agro-industry Technology Research System (Grant no. CARS-28-17).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01421/full#supplementary-material



30-phosphoadenylsulfate reductase. J. Biol. Chem. 274, 7695–7698. doi: 10. 1074/jbc.274.12.7695


gene (AccTrx2) in Apis cerana cerana. Gene 527, 33–41. doi: 10.1016/j.gene. 2013.05.062


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Shen, Liu, Gao, Liu, Zhang, Li, Zhang and Liu. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cold Acclimation of Trogoderma granarium Everts Is Tightly Linked to Regulation of Enzyme Activity, Energy Content, and Ion Concentration

#### Mozhgan Mohammadzadeh\* and Hamzeh Izadi

Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran

In this study, cold hardiness and some physiological characteristics of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae) larvae, were investigated under different thermal regimes, i.e., control, cold-acclimated (CA), fluctuatingacclimated (FA), and rapid cold-hardened (RCH). In all the regimes, the larval survival rate decreased with a decrease in temperature. CA larvae showed the highest cold hardiness following 24 h exposure at −15 and −20◦C. Control larvae had the highest glycogen content (34.4 ± 2.3 µg/dry weight). In contrast, CA larvae had the lowest glycogen content (23.0 ± 1.6 µg/dry weight). Change in trehalose content was reversely proportional to changes in glycogen content. The highest myo-inositol and glucose contents were detected in CA larvae (10.7 ± 0.4 µg/dry weight) and control (0.49 ± 0.03 µg/dry weight), respectively. In control and treated larvae, [Na+] decreased, though [K+] increased, with increasing exposure time. The shape of the thermal reaction curve of AMP-depended protein kinase and protein phosphatase 2C followed the same norm, which was different from protein phosphatase 1 and protein phosphatase 2A. Protein phosphatase 2A and 2C showed a complete difference in thermal reaction norms. Indeed, thermal fluctuation caused the highest changes in the activity of the enzymes, whereas the RCH showed the lowest changes in the activity of the enzymes. Our results showed a significant enhancement of larval cold tolerance under CA regime, which is related to the high levels of low molecular weight carbohydrates under this regime. Our results showed that among the different thermal regimes tested, the CA larvae had the lowest supercooling point (about −22◦C) and the highest cold hardiness following 24 h exposure at −15 and −20◦C.

Keywords: cold-acclimated, fluctuating-acclimated, rapid cold-hardened, enzyme activity, ion concentration, Khapra beetle

# INTRODUCTION

The Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), is an important and destructive insect pest of stored products. The native distribution of this pest is not known for certain, but this beetle is found in hot dry areas, and it is believed that this pest originated from the Indian subcontinent (Banks, 1977). This pest causes economic losses, particularly in tropical and

#### Edited by:

Antonio Biondi, Università degli Studi di Catania, Italy

#### Reviewed by:

Najmeh Sahebzadeh, Zabol University, Iran Aram Megighian, Università degli Studi di Padova, Italy Jose Eduardo Serrão, Universidade Federal de Viçosa, Brazil

> \*Correspondence: Mozhgan Mohammadzadeh m.mohammadzadeh@stu.vru.ac.ir

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 24 May 2018 Accepted: 20 September 2018 Published: 30 October 2018

#### Citation:

Mohammadzadeh M and Izadi H (2018) Cold Acclimation of Trogoderma granarium Everts Is Tightly Linked to Regulation of Enzyme Activity, Energy Content, and Ion Concentration. Front. Physiol. 9:1427. doi: 10.3389/fphys.2018.01427

**152**

subtropical regions of Asia and Africa (Burges, 2008). Infestation of seeds and food commodities by T. granarium larvae and their cast skins and hairs cause loss of biomass and food quality of stored products. In response to food shortage and unfavorable conditions (i.e., temperatures below 30◦C), the larvae enter diapause, remain relatively inactive, and rarely feed. Diapausing larvae tend to leave the food and aggregate in crevices of buildings (Burges, 1962). Based on our previous study, larvae of T. granarium are freeze-avoidant or freeze-intolerant. Freezeavoiding species accumulate polyol cryoprotectants in response to harsh environmental conditions. In these species, polyols permit colligative suppression of supercooling point (SCP) to prevent body freezing (Mohammadzadeh and Izadi, 2018).

To overcome adverse effects of low temperature, several physiological mechanisms have been developed in insects of cold and temperate zones. Three main groups of these mechanisms are: (1) physiological, biochemical, and metabolic alterations [cryoprotectant synthesis and synthesis of antifreeze proteins (AFPs) and/or ice-nucleating agents (INAs)], (2) change in cell function (modification of membranes, regulation of ionhomeostasis, and mobilization of cryoprotectants), and (3) alternation in gene expression (upregulation of stress-related genes) (Overgaard et al., 2007, 2014, 2015; Teets and Denlinger, 2013).

Cold hardiness or cold tolerance is the capacity of an organism to survive long- or short-term exposure to low-temperature levels. This capacity highly depends on developmental stage, genetic potential, season, duration of exposure, and nutritional status of the species. The best known and extensively researched mechanisms of insect cold hardiness are by means of carbohydrate cryoprotectants, antifreeze proteins (AFPs), and ice-nucleating agents (INAs) or ice-nucleating proteins (INPs). All contribute to protective mechanisms that deal with problems of ice formation at subzero temperatures (Storey and Storey, 2012; Sinclair et al., 2015; Andreadis and Athanassiou, 2017). The SCP, the temperature at which freezing of a cell initiates, is experimentally determined by detecting the released latent heat of fusion as body water freezes. Insect cold-tolerance strategies are usually determined on the basis of the SCP (Bemani et al., 2012; Sinclair et al., 2015; Mohammadzadeh and Izadi, 2018). Low molecular weight carbohydrates or sugar alcohols as cryoprotectants play an important role in enhancement of insect's cold hardiness (Storey and Storey, 2012). Acclimation usually has a trend toward higher levels of cryoprotectants, lower SCP, and subsequently higher survival rates. So, in the coldacclimated (CA) insects, elevation of cold hardiness may be a function of a decrease in SCP and an increase in cryoprotectants synthesis and accumulation. Activation of the intermediary signal transduction enzymes is a key component of the induction and regulation of insect cold hardiness (Pfister and Storey, 2002a). The cyclic AMP-activated protein kinase (cAMPK) is proving to be a major regulator of catabolic vs. anabolic phase in cells, its actions favoring the former and inhibiting the latter. The AMPK was first discovered as a protein kinase that was allosterically activated by cAMP accumulation under low-energy conditions (e.g., hypoxia) and it is often called the energy sensor or the fuel gauge of the cell (Hardie, 2007; Hue and Rider, 2007). The best-known action of AMPK is phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), which inhibits lipogenesis and promotes fatty acid oxidation under energylimiting conditions. The AMPK activation also exerts inhibitory control over carbohydrate storage (by inhibiting glycogen synthase) and protein synthesis [by activating the protein kinase that inactivates the ribosomal eukaryotic elongation factor-2 (eEF2)]. A series of recent studies have consistently shown AMPK activation in animals transitioning into hypometabolic states (e.g., frog freeze tolerance, turtle, and fish anaerobiosis, nematode dauer) (Rider et al., 2011). Protein phosphatases are a group of signal transducing enzymes that catalyze phospho-ester bond hydrolysis of phosphorylated proteins resulting in dephosphorylation of cellular phosphoproteins (Barford, 1995). Four major subunits of serine/threonine-specific protein phosphatase are protein phosphatase 1 (PP1), PP2A, PP2B, and PP2C (Cohen, 1989). The critical role of PP1 in the control of glycogen phosphorylase (GP) and eventually low-temperature-triggered activation of glycogen breakdown for polyol synthesis have already been identified, but roles of other protein phosphatases in insect cold hardiness have not been demonstrated so far (Hayakawa, 1985; Pfister and Storey, 2002a).

Electrolyte (e.g., sodium, potassium, and chloride) balance in hemolymph inside and outside of the cell membrane regulates nerve and muscle function and maintains acid-base and water homeostasis. Sodium as the main extracellular cation and potassium as the main intracellular cation are responsible for osmotic pressure gradient between the interior and exterior of a cell membrane.

In our study, we hypothesize that variation in cold tolerance in T. granarium larvae acclimated with low temperature arises from variation in the ability to change cAMPK and protein phosphatases activities, maintain ion balance, and increase the concentration of cryoprotectant contents in the cold. We thus predict that if acclimated with low-temperature conditions, coldtolerant T. granarium would: (1) have its enzyme activities vary according to the thermal regimes, (2) maintain [Na+] and [K+] balance in their hemolymph fluid, and (3) allow changes in enzyme activity and ion balance to increase the concentration of cryoprotectant contents and improve cold tolerance.

# MATERIALS AND METHODS

#### Chemicals

All chemicals used for analysis were purchased from Sigma-Aldrich (St. Louis, MO, United States).

#### Insect Rearing

The T. granarium population used for the experiments was obtained from cultures that had been originated from stored rice seeds from Karaj (Iran) and maintained for 2 years in the Laboratory of Entomology, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran. The insects were fed on broken wheat seeds (Triticum aestivum L.) under a controlled environmental chamber at 33 ± 1 ◦C with 65 ± 5% RH (by using saturated salt solution) and a photoperiod of 14:10 h (L:D), as described by Nouri-Ganbalani and Borzoui (2017).

# Acclimation Treatments

fphys-09-01427 October 26, 2018 Time: 16:8 # 3

Beetles were raised from egg to the fourth instar in translucent plastic containers (diameter 15 cm, depth 6 cm) with a hole covered by a 50 mesh net for ventilation, containing broken wheat seeds. T. granarium fourth instar larvae were divided into four groups: control, CA, fluctuating-acclimated (FA), and rapid cold-hardened (RCH). For control treatment, 100 individuals were put in translucent plastic containers containing food and kept in standard rearing conditions. For CA treatment (Jakobs et al., 2015; with some modification in temperatures and exposing times), 100 individuals were put in translucent plastic containers containing food, cooled in a programmable refrigerator from rearing conditions to 15◦C at a rate of 0.5◦C min−<sup>1</sup> and kept at this temperature at 65 ± 5% RH with a 14:10 h (L:D) light cycle for the 10 days. Thereafter, the temperature was lowered to 5◦C at the same rate and the larvae were kept at this temperature at 65 ± 5% RH with a 14:10 h (L:D) light cycle for 10 days. For FA treatment (Bale et al., 2001; with some modification in temperatures and exposing times), one hundred individuals were put in translucent plastic cups containing food, cooled in a programmable refrigerator from rearing conditions as explained in the cycle: 240 min at 5◦C followed by 20 min at −10◦C followed by 240 min at 5◦C followed by 940 min at 33◦C, at 65 ± 5% RH with a 14:10 h (L:D) light cycle. This cycle was repeated for 10 consecutive days. For RCH treatment (Wang et al., 2011), the larvae were transferred from their rearing conditions to a programmable refrigerator at 0◦C for 4 h. After the treatment period, larvae that survived were used for subsequent experiments. The larvae that were able to walk were counted as alive and larvae that were either not showing any movement in their appendages or were moving, but unable to walk, were counted as dead.

#### Enzymes Preparation and Assay

The whole body of acclimated larvae of T. granarium was used; it was not feasible to separate out individual tissues. For all enzymes, activities were expressed as Unit per gram wet mass. All assays were repeated five times.

#### AMPK

Individuals were rapidly weighed, chilled, and homogenized 1:10 (w/v), with a few crystals of phenylmethylsulfonyl fluoride (PMSF) added, using a precooled homogenizer (Teflon pestle) in ice-cold potassium phosphate buffer (20 mM; pH 6.8), 2-mercaptoethanol (15 mM), and ethylenediaminetetraacetic acid (EDTA) (2 mM). The homogenates were centrifuged at 13000 g for 3 min (5◦C). Following centrifugation, the supernatant was pooled and stored on ice for subsequent use. The activity of AMPK was assayed by the procedure of Pfister and Storey (2002a). In brief, <sup>32</sup>P from <sup>32</sup>P-ATP was incorporated onto Kemptide (LRRASLG), a synthetic phosphate-accepting peptide, in the presence of 0.1 mM adenosine 30,50-cyclic monophosphate. One unit of AMPK activity is defined as the amount of enzyme required to catalyze the incorporation of 1 nmol <sup>32</sup>P onto the substrate per minute at 23◦C.

#### PP1

Individuals were rapidly weighed, chilled, and homogenized 1:3 (w/v) using a precooled homogenizer (Teflon pestle) in icecold buffer A [Tris–HCl (20 mM; pH 7.4), EDTA (2 mM), ethylene glycol-bis(β-aminoethyl ether)-N,N,N<sup>0</sup> ,N0 -tetraacetic acid (EGTA) (2 mM), X-mercaptoethanol (15 mM)] containing the protease inhibitors: PMSF (1 mM), tosyl phenylalanyl chloromethyl ketone (TPCK) (0.1 mM), aprotinin (1 mg/ml), and benzamidine (5 mM). The homogenates were centrifuged at 1000 g for 3 min (5◦C). Following centrifugation, the supernatant was carefully collected and assayed immediately for active PP1. Estimates of PP1 activities at physiological levels of modulating proteins and other factors were done based on assays of concentrated extracts. The PP1 activity was estimated at 23◦C by monitoring <sup>32</sup>P cleavage from <sup>32</sup>P-labeled phosphorylase (Pfister and Storey, 2002b). One unit of PP1 activity is defined as the amount of enzyme required to releases 1 nmol of phosphate per minute at 23◦C.

#### PP2

Individuals were extracted as for PP1 except for a 1:10 (w/v) dilution. The homogenates were centrifuged at 13000 g for 20 min (5◦C). Following centrifugation, the supernatant was carefully collected and desalted by centrifugation at low speed for 1 min (at room temperature) through 5 ml Sephadex G-25 columns equilibrated in ice-cold Buffer A. The eluant was collected, passed through a second, fresh column, and stored on ice for subsequent use. The activities of PP2A and PP2C were assayed by the procedure of Cowan et al. (2000). PP2A activity was measured as the difference in activity in the presence (blank) versus absence of okadaic acid (2.5 nM). To assess the PP2A activity, the reaction mixture containing peptide RRA(pT)VA (150 mM), EGTA (0.2 mM), X-mercaptoethanol (0.02%), and imidazole (50 mM), pH 7.2, and 10 µl of enzyme extract were incubated for 40 min. The reaction was terminated by adding 50 ml of malachite green dye solution [ammonium molybdate (10%) and malachite green dye (2%), both in HCl (4 N) mixed 1:3 v/v and diluted 2:3 v/v with distilled, deionized water, Tween 20 (0.05%), and Triton-X-100(0.05%)] (Ekman and Jaeger, 1993). Reactions were run in 96-well microplates and the absorbance was read at 595 nm. Appropriate blanks, to which TCA had been added prior to the substrate, were prepared for each treatment. The activity of PP2C was assayed as the same except for the presence of okadaic acid (2.5 nM) and incubation of the reaction mixture for 90 min; PP2C was detected as the difference in activity in the absence versus presence of MgCl<sup>2</sup> (10 mM) (Cowan et al., 2000).

#### Ion Concentration

Ion concentrations were measured in the hemolymph (n = 5) as previously described by MacMillan et al. (2015b) with some modification. The [K+] and [Na+] were measured in the hemolymph at 0, 1, 2, 4, 6, and 12 h after exposure to −10◦C in individuals treated with different thermal regimes (100

individuals from each treatment and time point). Hemolymph was sampled from 200 fourth instar larvae (and weighed, to estimate volume), using a micropipette, from an incision made at the coxal joint of a hind leg while applying gentle pressure to the abdomen to allow the hemolymph to flow into the tube. Then, the hemolymph was transferred to a 0.5 ml Eppendorf tube, which was placed in a microcentrifuge (DW-41-230, Radiometer A/S, Brønshøj, Denmark) and spun for 15 s to separate hemolymph from debris. Afterward, 1–5 µl sample of hemolymph was transferred by pipette to a 2 ml buffer solution containing 100 ppm lithium salt. After the preparation was made as described earlier, the [Na+] and [K+] were measured from the hemolymph using an atomic absorption spectrometry (AAS; Model iCE 3300, Thermo Scientific, Waltham MA, United States) and comparisons to standard curves.

#### Whole-Body Glycogen and Sugar Alcohols Quantification

The whole-body glycogen and polyol profiles of acclimated larvae of T. granarium were repeated with five replicates (one individual from each treatment and time point) for each experiment at the end of the thermal regimes. All concentrations are expressed as microgram per dry weight.

#### Glycogen

The glycogen content of the larvae was estimated using the modified anthrone method as described by Heydari and Izadi (2014). The larvae were weighed and homogenized in 200 µl of 2% Na2SO4. Thereafter, 1300 µl chloroform-methanol (1:2) was added to the homogenate. The homogenates were centrifuged for 10 min at 7150 g and the supernatant was removed. The pellet was washed in 400 µl of 80% methanol and 250 µl distilled water was added before the heating for 5 min at 70◦C. Subsequently, 200 µl of the solution was incubated with 1 ml of anthrone for 10 min at 90◦C. After cooling at room temperature, the absorbance of the solution was measured at 630 nm. The glycogen content was determined by comparison to a standard curve that was prepared using glycogen.

#### Sugar Alcohols

The extraction, derivatization, and analytical procedures (gas chromatography coupled to mass spectrometry) were similar to those described by Khani et al. (2007). After the weighing and homogenization of individual larvae in 1.5 ml of 80% ethanol and centrifugation (twice repeated), the supernatant (20 ml) was run along with standards of polyols from 1500 to 5500 ppm. Trehalose, sorbitol, myo-inositol, and glucose were analyzed by HPLC (Knauer, Berlin, Germany) using a carbohydrate column with 4 µm particle size (250 mm × 4.6 mm, I.D., Waters, Ireland) (Khani et al., 2007).

#### Cold-Tolerance Assays

In total, two separate experiments were done to study cold tolerance: (1) an acute cold-tolerance assay at subzero temperature for 1 h and (2) an experiment measuring coldtolerance at −5, −10, −15, and −20 for 24 h. Five replicates and 15 larvae for each replicate were used at each treatment and temperature point. To estimate the acute cold exposure, the larvae treated with different thermal regimes were exposed to acute low temperatures (−5 to −20 ◦C). Survival rate was assessed after 1 h. Finally, LT80-1 h was calculated as the lowest temperature at which 80% of the larvae died after 1 h exposure (Sinclair and Rajamohan, 2008). To estimate the cold tolerance, the larvae treated with different thermal regimes were kept in a programmable refrigerated test chamber, where temperature was lowered slowly (0.5◦C min−<sup>1</sup> ) from experimental conditions to the desired treatment temperature (−5, −10, −15 and −20 ± 0.5◦C) and held at each temperature for 24 h. The mortality of larvae was recorded via direct observation. The larvae showing no movement in their appendages were judged to be dead (Mohammadzadeh and Izadi, 2016).

#### Determination of SCP

The SCP was determined for the acclimated larvae of T. granarium (n = 15). To determine SCP, individual larvae were placed on a thermocouple (NiCr–Ni probe) connected to an automatic temperature recorder (Testo 177-T4, Testo, Germany) within a programmable refrigerated test chamber. The temperature of the refrigerated test chamber was reduced from experimental conditions to −30◦C, at a rate of 0.5◦C min−<sup>1</sup> . The lowest temperature reached before an exothermic event that occurred caused by the release of latent heat was taken as the SCP of the individual (Mohammadzadeh and Izadi, 2016).

#### Statistical Analysis

Data were initially tested for normality (Kolmogorov–Smirnov test) and homoscedasticity (Levene's test) before subjecting them to ANOVA. All the data were analyzed using SAS ver.9.2 program (PROC GLM; SAS, 2009). Statistical analyses were performed, based on a completely randomized design, using oneway analysis of variance (ANOVA) followed by a post hoc Tukey's test at α = 0.05.

# RESULTS

#### Effect of Thermal Regimes on Enzymes Activity

Profiles of enzymes activities in T. granarium under different thermal regimes are shown in **Figure 1**. Based on the results of this study, the highest and lowest activities of AMPK (57 and 26 units/gram wet mass, respectively) and PP2C (27 and 14 units/gram wet mass, respectively) were observed at CA and FA treatments, respectively. No significant differences were observed in the activities of these two enzymes between control and RCH. Although the activities of these two enzymes changed in the same norm in different regimes, AMPK was found to be much more active than PP2C. In control, the activity of AMPK was about 33 units/gram wet mass, whereas the activity of PP2C was about 19 units/gram. The activity of both enzymes increased by CA and reached to the highest levels of 55 and 27 units/gram, respectively. The activity of PP1 and PP2A also showed more

significantly different (Turkey's test, P < 0.05).

or less the same norm under different regimes. These norms were completely different from those of the two other enzymes. The highest (28 units/gram wet mass) and lowest (12 units/gram wet mass) activities of PP1 were observed at control and CA treatments, respectively. In the case of PP2A, the highest level of activity was recorded for control and FT treatments (1.1 and 1.2 units/gram wet mass, respectively), whereas the lowest level of activity was shown in CA and RCH (0.5 and 0.6 units/gram, respectively). In PP1, the highest (28 units/gram wet mass) and lowest (13 units/gram wet mass) levels of activity were observed in control and CA regimes, respectively.

# Effects of Thermal Regimes on Hemolymph Na<sup>+</sup> and K<sup>+</sup> Concentrations

Our results showed that in all the regimes as well as in the control, the concentration of Na<sup>+</sup> decreased, whereas the concentration of K<sup>+</sup> increased with an increase in exposure time of the larvae at −10◦C (**Figure 2**). In all the regimes, at the beginning time of exposure (0 h at −10◦C), the concentration of Na<sup>+</sup> was about 70 mM. The concentration of Na<sup>+</sup> increased and reached to the highest level after 1 h exposure at −10◦C. Then, the concentration of this ion in control and the steady state at different thermal regimes decreased with increasing exposure time. The sodium/potassium ratio decreased with an increase in exposure time. During different times of exposure at −10◦C, the highest and lowest levels of Na<sup>+</sup> and K<sup>+</sup> were recorded for control and CA treatment, respectively. In addition, at the highest levels, the concentration of Na<sup>+</sup> was about four times more that of K+.

#### Effects of Thermal Regimes on Carbohydrate Contents

Glycogen content in control larvae with 34.4 mg/dry weight was at the highest level and reached the lowest level of 23.0 in CA larvae. No significant differences were observed between glycogen contents in FT, RCH, and control (**Table 1**). Trehalose

TABLE 1 | Carbohydrate contents (n = 5) of Trogoderma granarium fourth instar larvae following different thermal regimes.


larvae following different thermal regimes. Each point is an average of five replications. The means followed by different letters are significantly different (Turkey's test,

The means followed by different letters in the same columns are significantly different (Turkey's test, P < 0.05).

and myo-inositol were found to be the predominant low molecular weight of carbohydrates in all the regimes. Changes in low molecular weight carbohydrate contents were reversely proportional to change in glycogen content. The highest and lowest contents of trehalose and myo-inositol were observed in CA (16.5 mg/dry weight) and control (9.9 mg/dry weight), respectively. No significant differences were observed in sorbitol contents in control at different thermal regimes. The highest and lowest glucose contents were observed in control (0.49 mg/dry weight) and CA (0.14 mg/dry weight), respectively.

P < 0.05).

#### Effect of Thermal Regimes on SCP and Survival of the Larvae

Data in **Table 2** showed that under CA and FT thermal regimes, SCPs of the larvae decreased to the lowest level (about −22◦C), which were significantly lower than SCPs of control and RCH regimes. No significant difference was observed between SCPs of control and RCH.

Our results also showed a profound effect of CA on the LT<sup>80</sup> value of the larvae (**Figure 3**). The LT80s of the larvae at control, RCH, FT, and CA regimes were calculated as 11, −14, −19, and −21◦C, respectively. In CA larvae, the temperature required for 80% mortality decreased by about 10◦C compared with control larvae. In addition, the temperature necessary for the beginning of mortality in the CA regime was 10◦C lower than that of control. In all the regimes, the survival rate of the larvae decreased with a decrease in the temperature and increase in the exposure time. CA larvae showed the highest cold hardiness in the −15 and −20◦C range. In control, RCH, FT, and CA, larval mortality began at −20, −25, −27, and −30◦C, respectively.

#### DISCUSSION

In this study, three different thermal regimes (CA, FA, and RCH) were examined. Out of these thermal regimes, substantial effect of CA on physiological adaptations (i.e., cryoprotectant accumulation and enzyme activity) and cold tolerance of the last instar larvae of the Khapra beetle, T. granarium, is highly obvious. In addition, results of the current study demonstrated a strong correlation between carbohydrate contents and cold tolerance of the larvae. The CA showed the highest impact on physiological adaptations and subsequently the survival rate of the larvae. In CA larvae, glycogen and SCP were at the lowest levels, whereas low molecular weight carbohydrates (e.g., trehalose), AMPK activity, and survival rates were at the highest levels. The decrease in SCP was proportional to an increase in cold hardiness of the larvae, which, in turn, was associated with an increase in the enzyme activity and cryoprotectant accumulation. Thus, a strong relation between enhanced cold hardiness, elevated enzyme activities, accumulated cryoprotectants, and decreased SCP of the Khapra beetle CA larvae can be concluded from our results. On the other hand, in the CA larvae, enhancement of cold hardiness is a function of both a decrease in SCP and an increase in trehalose synthesis and accumulation. Hiiesaar et al. (2001) showed that the mean SCP of Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae) decreased from −10.5 in non-acclimated to −17.5◦C in CA adult beetles. In Hermetia illucens (L.) (Dip.: Stratiomyidae) prepupae, the SCP was unaffected by cold acclimation, but cold hardiness increased in comparison to control (Spranghers et al., 2017). Insects also have the capability to improve cold tolerance and survival rate in a short period, called rapid cold-hardening (RCH) (Teets and Denlinger, 2013). In the current study, RCH had no significant effects on enzyme activities, survival rates, and cryoprotectant accumulations. This correlates well with the previous studies. Overgaard et al. (2014) in adults of Drosophila melanogaster Meigen (Dip.: Drosophilidae) reported no effect of RCH on the activity of GP. They found a small increase in glucose content, whereas, trehalose content remained unchanged following RCH. Kelty and Lee (2001) in RCH adults of D. melanogaster found no change in the levels of Hsp70 and carbohydrate cryoprotectants. In disagreement with our results, Lee et al. (2006) reported that

TABLE 2 | Relationship between low temperature survival rate (n = 5) and supercooling points (n = 15) of Trogoderma granarium fourth instar larvae following different thermal regimes.


The means followed by different letters in the same columns are significantly different (Turkey's test, P < 0.05).

RCH significantly increased survival of Belgica antarctica Jacobs (Dip.: Chironomidae) larvae. Lee et al. (2006) determined that RCH increased membrane fluidity of fat body cells of Sarcophaga bullata (Parker) (Dip.: Sarcophagidae) adult flies. They suggested that "membrane characteristics may be modified very rapidly to protect cells against cold-shock injury". In adults of Thrips palmi Karny (Thysan.: Thripidae), RCH caused accumulation of cryoprotectants mainly trehalose (Park et al., 2014). So, based on our results and results of other researchers, it is reasonable to conclude that insects may become cold hardy by RCH if RCH participates in the accumulation of cryoprotectants (polyols and sugar alcohols). In our study, no accumulation of cryoprotectants and consequently no cold hardiness were determined in RCH regime. Jakobs et al. (2017) found no evidence that acute cold tolerance of Drosophila suzukii larvae could be improved by RCH.

The AMPK as a downstream component of a kinase cascade and a key component of energy homeostasis has several functions (regulation of glycogen, sugar, and lipid metabolism) and cellular targets. This enzyme can be regulated by a growing number of hormones (e.g., leptin and adiponectin, insulin, interleukin-6, resistin, TNF-alpha, and ghrelin) and cytokines (Dzamko and Steinberg, 2009; Hardie, 2010; Lee et al., 2010). Our findings showed that AMPK is a predominant signal transduction enzyme of T. granarium last instar larvae. Activities of the tested enzymes could be rated as AMPK > PP1 > PP2C > PP2A. The results of some previous studies support the findings of the current study. Pfister and Storey (2006a) demonstrated changes in the activities of AMPK, PP1, PP2A, and PP2C in a freeze-avoiding insect, Epiblema scudderiana (Clemens) (Lep.: Olethreutidae) in winter and during exposure of the larvae to subzero temperatures. They demonstrated a limited change and the role for AMPK in overwintering larvae, but the activities of PP2A and PP2C increased when larvae were exposed to −20◦C. In another research, Pfister and Storey (2006b) studied changes in the activities of the same enzymes of the goldenrod gall fly, Eurosta solidaginis Fitch (Dip.: Tephritidae). They showed increases in AMPK and a decrease in PP1 activity over the winter season and/or at subzero temperature. However, the findings of the present research revealed that the shape of the thermal reaction curve in AMPK and PP2C follows the same norm, which is different from those of PP1 and PP2A. The PP2A and 2C showed opposite trends in activity and different thermal reaction norms. Indeed, thermal fluctuation caused the highest changes in the enzyme activities, whereas larvae of RCH treatment showed the lowest level of the enzyme activities. Decrease in glycogen content and increase in activity of AMPK and cryoprotectant contents in CA larvae of T. granarium suggest a role for coarse control of AMPK in the conversion of glycogen reserves into cryoprotectant synthesis and accumulation. Increase in cryoprotectant contents results in enhancement of cold tolerance and survival of the CA larvae. Our previous study has shown that larvae of T. granarium are freeze-avoidant or freeze-intolerant (Mohammadzadeh and Izadi, 2018). In these freeze-intolerant larvae, several metabolic adaptations, including synthesis of polyols and low molecular weight carbohydrates (as cryoprotectant), have been developed for survival at subzero temperatures and harsh environmental conditions. The results of the current study indicated a significant enhancement in larval survival and cold tolerance under CA regime. The results of our study also revealed the profound impact of CA on carbohydrate contents of the larvae. In the CA larvae, cold hardiness of the larvae was at the highest value and major cryoprotectants such as trehalose and myoinositol were at the highest levels, but glycogen reached the lowest concentration. So, it could be concluded from these results that cold acclimation is important in the conversion of glycogen to low molecular weight carbohydrates, which act as a cryoprotectant to enhance cold hardiness of the larvae. High level of cryoprotectants (e.g., trehalose) is essential for reduction of supercooling in freeze-avoidant species or to prevent intracellular ice formation in freeze-tolerant insects. The induction of insect cold hardiness and related adaptations require the intermediary action of signal transduction enzymes (Pfister and Storey, 2006b). In agreement with these aspects, in the current study, the activity of AMPK, as a signal transduction enzyme in the CA larvae, increased and reached the highest level. An increase in the activity of this enzyme was coincident with the increase of cold hardiness, cryoprotectants concentration, and survival rate. These findings strongly support a regulatory role for AMPK and PP1 in the synthesis of cryoprotectants from glycogen. In the CA larvae, glycogen content decreased with the increase in trehalose content and AMPK activity. So, it is reasonable to conclude that AMPK may be responsible for shutting down glycogen synthesis and activating conversion of glycogen to trehalose. For cryoprotectants synthesis, conversion of glycogen to polyols or sugar alcohols is necessary (Storey and Storey, 2012). In this process, the role of PP2C is much more limited than AMPK and there is no role for PP1 and PP2A. In overwintering larvae of E. scudderiana, PP1 was found to be responsible for shutting off glycogenolysis, whereas a limited role was attributed to PKA. In this moth, the activity of PP2A and PP2C increased by exposing the larvae to −20◦C (Pfister and Storey, 2006a). Some recent studies suggest a big role for AMPK in insect cold hardiness and diapause. Rider et al. (2011) showed a twofold higher activity of AMPK in winter larvae of E. solidaginis and E. scudderiana in comparison to summer ones. Joanisse and Storey (1995) found an increase in activities of glycogenolytic and hexose monophosphate shunt enzymes in CA E. scudderiana larvae, which resulted in the conversion of glycogen into glycerol as a cryoprotectant. In E. solidaginis CA larvae, an increase in activity of GP with a decrease in activity of glycolytic enzymes may be responsible for the temperaturedependent switch from glycerol to sorbitol synthesis. In our study, trehalose content was at the highest level in CA larvae. Trehalose, as a cryoprotectant, contributes to stabilizing the lipid bilayer of the cell membrane (Crowe et al., 1992; Storey and Storey, 2012). The results of this study are in agreement with the results of Pfister and Storey (2006b). They showed several metabolic adaptations in freeze-tolerant larvae of the goldenrod gall fly for subzero survival. Several other studies reported this sugar as a cryoprotectant in cold hardy insect species (Behroozi et al., 2012; Bemani et al., 2012; Sadeghi et al., 2012; Heydari and Izadi, 2014; Mohammadzadeh and Izadi, 2016). In agreement with our results, Mohammadzadeh et al. (2017) showed that high cold tolerance of larvae of

Eurytoma plotnikovi (Hym.: Eurytomidae) was not associated with accumulation of cryoprotectants during overwintering. Enhancement of cold tolerance in insects mostly relies on colligative effects through accumulation of high concentration of cryoprotectants such as trehalose and glycerol to depress SCP (Bemani et al., 2012; Hayward et al., 2014; Heydari and Izadi, 2014). In some insects, if cold exposure induces accumulation of cryoprotectants and subsequently elevates cold hardiness of the insect, SCP remains unchanged. In these insects, cold hardiness enhances through a non-colligative mechanism (Kostal et al., 2001). Cold-induced gene recognition usually consists of two steps: identification of metabolic adaptations that support cold hardiness (e.g., cryoprotectant synthesis) and determination of induced or upregulated proteins/enzymes which support this function (Storey and Storey, 2001).

Chilling insect at low temperature causes a loss of extracellular ions and water homeostasis (MacMillan et al., 2015a). In a recent experiment, MacMillan et al. (2015a) examined the capacity of chill susceptible Drosophila species malpighian tubules (MT) and demonstrated that MT lost [Na+] and [K+] selectivity at low temperatures, which participate in a loss of Na<sup>+</sup> and water balance and an increase in extracellular [K+]. These findings strongly support the results of the current study. Based on our results, exposure of the larvae to low temperature caused a substantial decrease in [Na+] and an increase in [K+]. This finding strongly supports the concept that low temperature reduced [Na+] and [K+] selectivity of MT, which contributed to a decrease in [Na+], a harmful increase in [K+], and consequently, an accumulation in chill injuries. Insect cold hardiness is strongly associated with the ability of MT to retain ions (particularly, K<sup>+</sup> and Na+) and water balance during cold exposure (MacMillan et al., 2015a; Andersen et al., 2017). MacMillan et al. (2015a) concluded that chill-tolerant Drosophila species maintained K<sup>+</sup> secretion better than chill-susceptible species and suppressed K<sup>+</sup> reabsorption during cold exposure. These data, therefore, are strongly in agreement with our findings. As our results show, at the beginning of the cold exposure, hemolymph [Na+] is at the highest level, whereas; [K+] is at the lowest level. By increasing exposure time, [Na+] decreased and [K+] increased. In most insects, hemolymph [Na+] is significantly higher than [K+]. So, Na<sup>+</sup> ions tend to leak into the gut lumen while K<sup>+</sup> ions tend to leak into the hemolymph. At normal temperature, passive ion movements from the MT lumen to the hemolymph and from the hemolymph to the MT lumen are regulated by the energydemanding proton pump located at the apical membrane MT epithelial cells. At low temperature, [Na+] leak away from the hemolymph to the MT lumen and so, its concentration reduces in hemolymph leading to increasing [K+] in the hemolymph. Increase in hemolymph [K+] causes depolarization of cell resting potential and this depolarization may be a primary reason of cold-induced injury (Kostal et al., 2004; MacMillan et al., 2014,

#### REFERENCES

Andersen, M. K., MacMillan, H. A., Donini, A., and Overgaard, J. (2017). Cold tolerance of Drosophila species is tightly linked to epithelial K + transport capacity of the Malpighian tubules MacMillan et al., 2015; Andersen et al., 2017). We can conclude from our results that the gene expression of ion homeostasis and consequently water balance have altered CA larvae of the Khapra beetle. The same results have been reported by Gerken et al. (2015); MacMillan et al. (2015a), and Des Marteaux (2017).

From a practical viewpoint, control of stored product pests relies mostly on the use of fumigants, e.g., methyl bromide. This pesticide is an ozone−depleting fumigant and its application has been restricted worldwide. Thus, there is an increasing interest to find new alternatives of control methods, including the use of low temperatures (Wilches et al., 2017). Cooling of the seeds and commodities near the SCP of the pests for a specific period of time may be an appropriate method for controlling of the P. interpunctella and E. ceratoniae (Phillips and Throne, 2010; Hagstrum and Phillips, 2017). Hence, the results of the current study strongly suggest phytosanitary temperature treatments as an alternative for methyl bromide and other fumigant pesticides in the control of stored product pests.

### CONCLUSION

Our results showed that a significant enhancement of larval cold tolerance under CA regime is related to the elevated level of low molecular weight carbohydrates, e.g., trehalose and protein kinase, and phosphatases activities, and hemolymph ion concentrations. This study provides support for the use of phytosanitary temperature treatments as a potential alternative to fumigant insecticide in the control of this stored product beetle. It also highlights the physiological changes that the insect makes to overcome low temperatures.

#### AUTHOR CONTRIBUTIONS

MM and HI conceived and designed the research, conducted the experiments, contributed the analytical tools and analyzed the data, and wrote the manuscript.

# FUNDING

This research was supported in part by Vali-e-Asr University of Rafsanjan (Rafsanjan, Iran).

# ACKNOWLEDGMENTS

The authors thank the Vali-e-Asr University of Rafsanjan (Rafsanjan, Iran), for cooperation by support for the experiment. Also, they thank the bioRxiv for release this manuscript as a preprint.

and rectal pads. J. Exp. Biol. 220, 4261–4269. doi: 10.1242/jeb. 168518

Andreadis, S. S., and Athanassiou, C. G. (2017). A review of insect cold hardiness and its potential in stored product insect control. Crop Prot. 91, 93–99. doi: 10.1016/j.cropro.2016.08.013



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Mohammadzadeh and Izadi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cold Acclimation Favors Metabolic Stability in Drosophila suzukii

#### Thomas Enriquez<sup>1</sup> , David Renault1,2, Maryvonne Charrier<sup>1</sup> and Hervé Colinet<sup>1</sup> \*

<sup>1</sup> ECOBIO – UMR 6553, Université de Rennes 1, CNRS, Rennes, France, <sup>2</sup> Institut Universitaire de France, Paris, France

The invasive fruit fly pest, Drosophila suzukii, is a chill susceptible species, yet it is capable of overwintering in rather cold climates, such as North America and North Europe, probably thanks to a high cold tolerance plasticity. Little is known about the mechanisms underlying cold tolerance acquisition in D. suzukii. In this study, we compared the effect of different forms of cold acclimation (at juvenile or at adult stage) on subsequent cold tolerance. Combining developmental and adult cold acclimation resulted in a particularly high expression of cold tolerance. As found in other species, we expected that cold-acclimated flies would accumulate cryoprotectants and would be able to maintain metabolic homeostasis following cold stress. We used quantitative target GC-MS profiling to explore metabolic changes in four different phenotypes: control, cold acclimated during development or at adult stage or during both phases. We also performed a time-series GC-MS analysis to monitor metabolic homeostasis status during stress and recovery. The different thermal treatments resulted in highly distinct metabolic phenotypes. Flies submitted to both developmental and adult acclimation were characterized by accumulation of cryoprotectants (carbohydrates and amino acids), although concentrations changes remained of low magnitude. After cold shock, non-acclimated chill-susceptible phenotype displayed a symptomatic loss of metabolic homeostasis, correlated with erratic changes in the amino acids pool. On the other hand, the most cold-tolerant phenotype was able to maintain metabolic homeostasis after cold stress. These results indicate that cold tolerance acquisition of D. suzukii depends on physiological strategies similar to other drosophilids: moderate changes in cryoprotective substances and metabolic robustness. In addition, the results add to the body of evidence supporting that mechanisms underlying the different forms of acclimation are distinct.

Keywords: spotted wing drosophila, cold tolerance, cold shock, homeostasis, recovery, metabolites, metabotype

# INTRODUCTION

Extreme temperatures often negatively affect survival of ectothermic animals as well as their biological functions such as reproduction, respiration, digestion, or excretion (Chown and Nicolson, 2004; Angilletta, 2009). In order to reduce the negative effects of temperature on their performances, ectotherms are capable of modulating thermal tolerance during their lifetime using a range of physiological adjustments that take place after pre-exposure to sub-lethal temperatures, a phenomenon referred to as thermal acclimation (Angilletta, 2009; Colinet and Hoffmann, 2012). The degree of tolerance acquisition directly depends on the thermal history of individuals, more

#### Edited by:

Antonio Biondi, Università degli Studi di Catania, Italy

#### Reviewed by:

Leigh Boardman, University of Florida, United States Pablo E. Schilman, Universidad de Buenos Aires, Argentina

> \*Correspondence: Hervé Colinet herve.colinet@univ-rennes1.fr

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 21 July 2018 Accepted: 08 October 2018 Published: 01 November 2018

#### Citation:

Enriquez T, Renault D, Charrier M and Colinet H (2018) Cold Acclimation Favors Metabolic Stability in Drosophila suzukii. Front. Physiol. 9:1506. doi: 10.3389/fphys.2018.01506

**163**

particularly on the timing and length of the pre-exposure (Chown and Nicolson, 2004). The capacity to plastically deal with thermal stress is also believed to be a key factor in the success of exotic invasive species (Davidson et al., 2011; Renault et al., 2018).

Developmental plasticity can irreversibly alter some phenotypic traits, such as morphology (Piersma and Drent, 2003). For example, insects developing at low temperature are characterized by a larger body size and darker cuticle pigmentation of adults that remains throughout their whole life (Gibert et al., 2000, 2007). However, physiological adjustments occurring during development, like those related to acquired cold tolerance, are not necessarily everlasting (Piersma and Drent, 2003). For instance, cold tolerance acquired during development is readily adjusted to the prevailing conditions during adult acclimation without a detectable developmental constraint (Slotsbo et al., 2016). The different forms of acclimation probably lie along a continuum of shared common mechanisms; however, several lines of evidence suggest that physiological underpinnings of each acclimation form show some specificity (Colinet and Hoffmann, 2012; Teets and Denlinger, 2013; Gerken et al., 2015).

Stressful low temperatures compromise cells' integrity by altering cytoskeleton structures and membranes' functions (Cottam et al., 2006; Lee et al., 2006; Denlinger and Lee, 2010; Des Marteaux et al., 2017). Cold stress also induces central nervous system shutdown and loss of ions and water homeostasis that result in coma and neuromuscular impairments (Koštál et al., 2004; MacMillan and Sinclair, 2011; Andersen et al., 2016). Alteration of metabolic homeostasis is another symptom of cold stress, likely resulting from downstream consequences such as loss of function of membranes and enzymes (Overgaard et al., 2007; Teets et al., 2012; Williams et al., 2014, 2016; Colinet et al., 2016; Koštál et al., 2016b; Colinet and Renault, 2018). Thermal acclimation likely depends on many concomitant physiological adjustments such as changes in membrane fluidity (e.g., Overgaard et al., 2005; Lee et al., 2006; Koštál et al., 2011a; Williams et al., 2014), preservation of membrane potential and ion balance (Andersen et al., 2016; Overgaard and MacMillan, 2016), maintenance of metabolic homeostasis (e.g., Malmendal et al., 2006; Colinet et al., 2012; Teets et al., 2012), altered expression of heat shock proteins (Colinet and Hoffmann, 2012), and accumulation of substances with cryoprotective functions, such as sugars, polyols and amino acids (Koštál et al., 2011a; Vesala et al., 2012; Foray et al., 2013; MacMillan et al., 2016). Cryoprotective solutes can have beneficial effects at high concentration, by decreasing hemolymph freezing temperature (colligative effect) (Zachariassen, 1985; Storey and Storey, 1991), but also at low concentration, by stabilizing membranes and protein structures (Carpenter and Crowe, 1988; Crowe et al., 1988; Yancey, 2005; Cacela and Hincha, 2006).

The spotted wing drosophila, Drosophila suzukii, is an invasive species that is now spread in West and East Europe (Calabria et al., 2012; Lavrinienko et al., 2017) as well as in North and South America (Hauser, 2011; Lavagnino et al., 2018; see also Asplen et al., 2015 for a review). Contrary to other drosophilids, D. suzukii females lay eggs in mature fruits. Larvae consume these fruits, causing important damages and economic losses to a wide range of fruit crops (Goodhue et al., 2011; Walsh et al., 2011). This fly is highly polyphagous and is therefore able to develop on a wide range of wild fruits in addition to those that are cultivated (Poyet et al., 2015; Kenis et al., 2016). This fly is chill susceptible, succumbing to temperatures well above 0◦C (Kimura, 2004; Dalton et al., 2011; Ryan et al., 2016; Enriquez and Colinet, 2017). Yet, D. suzukii is capable of overwintering in rather cold climates, such as in Northern America and Europe, probably by using various strategies such as migration to favorable microhabitats (Zerulla et al., 2015; Rossi-Stacconi et al., 2016; Tonina et al., 2016; Thistlewood et al., 2018) and/or high cold tolerance plasticity. These strategies allow maintenance of the populations in invaded areas, even if the number of adults is drastically reduced during winter (Mazzetto et al., 2015; Arnó et al., 2016; Wang et al., 2016).

Drosophila suzukii is capable of enhancing cold tolerance via a range of acclimation responses (Hamby et al., 2016). Jakobs et al. (2015) found that exposure at 0◦C for 1 h did not trigger a rapid cold hardening response in D. suzukii. Conversely, certain lines or populations of D. suzukii actually exhibit a typical rapid cold hardening response. Toxopeus et al. (2016) showed that flies acclimated during development could show a rapid cold hardening response after 1 or 2 h at 0◦C. Everman et al. (2018) found a similar rapid cold-hardening response in non-acclimated flies exposed at 4◦C for 2 h. This fly is also capable of acquiring cold tolerance via acclimation at adult stage (Jakobs et al., 2015; Wallingford and Loeb, 2016), or via developmental acclimation (Toxopeus et al., 2016; Wallingford and Loeb, 2016). In D. suzukii, developmental acclimation at temperatures below 12◦C (combined or not with short photoperiod) results in a phenotype showing increased body size, dark pigmentation, reproductive arrest, and enhanced cold tolerance; this phenotype, referred to as "winter morph", is supposed to be the overwintering form of D. suzukii (Stephens et al., 2015; Shearer et al., 2016; Toxopeus et al., 2016; Wallingford and Loeb, 2016; Everman et al., 2018). Effect of the different forms of acclimation on subsequent cold tolerance has been rather well described in D. suzukii, but surprisingly, the mechanisms underlying cold tolerance acquisition through acclimation in this species are still poorly understood. In order to better appreciate and predict overwintering strategies of D. suzukii, knowledge about its thermal biology, and particularly cold stress physiology, is urgently needed (Asplen et al., 2015; Hamby et al., 2016).

A recent transcriptomic study suggested that cold tolerance of winter morphs is associated with an upregulation of genes involved in carbohydrates' metabolism (Shearer et al., 2016). However, it is still not known whether cold-hardy D. suzukii flies use any specific cryoprotective arsenal. In this study we proposed the first characterization of metabolic adaptations linked to cold acclimation in D. suzukii. First, we aimed at assessing the impact of different forms of acclimation on cold tolerance in this species. To do so, we subjected flies to developmental acclimation, adult acclimation or a combination of both acclimation forms, and then assessed subsequent cold tolerance of adults. We expected that (i) each cold acclimation form would promote cold tolerance, and that (ii) combining cold acclimation during both development and

adult stage would further promote cold tolerance. Second, we intended to explore underlying mechanisms of cold acclimation in D. suzukii. Specifically, we performed quantitative target gas chromatography–mass spectrometry (GC-MS) profiling to explore metabolic changes, including accumulation of cryoprotectants, in four different phenotypes resulting from different thermal treatments. We also performed a timeseries GC-MS analysis to monitor metabolic homeostasis status during cold stress and recovery. We expected that, as other Drosophila species (Overgaard et al., 2007; Koštál et al., 2011a; Colinet et al., 2012; Vesala et al., 2012; MacMillan et al., 2016), cold acclimated D. suzukii would be characterized by increased concentrations of some cryoprotectants (sugars, polyols, or amino acids) and would be able to maintain metabolic homeostasis after cold shock contrary to chill susceptible phenotypes.

#### MATERIALS AND METHODS

#### Flies Rearing and Thermal Treatments

In this work, we used a wild-type D. suzukii population coming from the Vigalzano station of the Edmund Mach Foundation (Italy; 46.042574N, 11.135245E). This line was established in 2011 in Italy, and was sent to our laboratory (Rennes, France) in 2016. For the experimentations, D. suzukii was reared in glass bottle (100 mL) supplied with a carrot-based food (agar: 15 g, sucrose: 50 g, carrot powder: 50 g, brewer yeast: 30 g, cornmeal: 20 g, Nipagin: 8 mL, tap water: 1 L). Flies were maintained at 25◦C, 60% RH, 12 L:12 D into incubators (Model MIR-154-PE; PANASONIC, Healthcare Co., Ltd., Gunma, Japan). At least 12 bottles (each containing 100–300 flies) were used to continuously maintain the line at Rennes, and flies from different bottles were crossed every generation to limit inbreeding.

To generate flies for the experiments, groups of approximately 100 mature (7 days old) males and females were placed in 100 mL bottles containing food medium, and females were allowed to lay eggs during 48 h at 25◦C. Flies were then removed and bottles with eggs were randomly placed under the different thermal treatments to continue development (**Figure 1**). Bottles containing eggs were directly placed either at 25◦C (12 L:12 D) [no acclimation] or cold acclimated at 10◦C (10 L:14 D) [developmental acclimation] (**Figure 1**). Flies took about 10 and 60 days to start emergence at 25 and 10◦C, respectively. At emergence, adults from both developmental conditions were directly placed either at 25◦C (12 L:12 D) [control, no acclimation] or cold acclimated at 10◦C (10 L:14 D) [adult acclimation] with fresh food for seven consecutive days. Flies were transferred into new bottles containing fresh food every 2 days. Thereby, four different phenotypes that experienced four different thermal treatments were generated: non-acclimated control, adult acclimation, developmental acclimation and combined developmental and adult acclimation hereafter referred to as combined acclimation (**Figure 1**). All experiments were conducted on males, which were separated from females visually (with an aspirator) without CO<sup>2</sup> to avoid stress due to anesthesia (Colinet and Renault, 2012).

#### Recovery From Acute Cold Stress

Males randomly taken from each treatment group were distributed in 10 replicates of 10 individuals and were subjected to an acute cold stress using 10 glass vials that were directly immersed in a glycol solution cooled by a cryostat (Cryostat Lauda ECO RE 630) at −5 ◦C for 100 min. In a previous experiment, this combination of temperature and duration induced 40% survival in non-acclimated D. suzukii (Enriquez and Colinet, 2017). After exposure, flies were directly transferred in 40 mL food vials and allowed to recover at 25◦C (12 L: 12 D). Survival was assessed by counting the number of dead and living individuals in each vial 4, 24, and 48 h after the acute cold stress.

# Critical Thermal Minimum (Ctmin)

To measure Ctmin, we used a glass knockdown column that consisted of a vertical jacketed glass column (52 × 4.7 cm) containing several cleats to help flies not fall out the column while still awake. In order to regulate the temperature, the column was connected to a cryostat (Cryostat Lauda ECO RE 630), and temperature was checked into the column using a thermocouple K connected to a Comark Tempscan C8600 scanning thermometer (Comark Instruments, Norwich, United Kingdom). Thermocouple was positioned at the column center, at mid height through a tiny insulated hole. For each condition, approximately 60 flies were introduced at the top of the column. Flies were allowed to equilibrate in the device for few minutes, and then the temperature was decreased to −5 ◦C at 0.5◦C/min. For each individual fly falling out of the column, the Ctmin ( ◦C) was recorded.

#### Chill Coma Recovery Time

Chill coma recovery time (CCRT) is defined as the resurgence time of motor activity after a cold knockdown (David et al., 1998). In order to induce coma, 40 males of each experimental condition were subjected to 0◦C for 12 h, using a vial placed directly in a cooled-incubator. We choose this temperature and duration because in a previous experiment we showed that 12 h at 0 ◦C induced a relatively small mortality level in non-acclimated males (Enriquez and Colinet, 2017). Upon removal, adults were positioned supinely on a table in a thermally controlled room (25 ± 1 ◦C), using a fine paintbrush, and the time to regain the ability to stand (i.e., CCRT) was monitored individually. Experimentation lasted 60 min, and flies that did not recover by that time were marked as not recovered (censored).

#### GC-MS Metabolic Profiling

To assess the effect of the different thermal treatments on metabolic profiles, we sampled flies at the end of each thermal treatment period (i.e., before the stress exposure) (**Figure 1**). Then, males randomly taken from each treatment group were cold-stressed at −5 ◦C for 100 min (as explained above), and these stressed flies were then sampled during the recovery period after 15 min, 4, 8, and 12 h (**Figure 1**). For each time-point, seven replicates, each consisting of a pool of 10–12 living flies, were snap-frozen in liquid N<sup>2</sup> and stored at −80◦C. Fresh mass of

each sample was measured prior to metabolites' extraction using a microbalance (Mettler Toledo UMX2, accuracy 0.001 mg).

Samples were homogenized using a bead-beating device (Retsch MM301, Retsch GbmH, Haan, Germany) at 20 beats per second during 90 s in 600 µL of a cold solution of methanol-chloroform (2:1) with a tungsten grinding ball. Samples were then kept at −20◦C for 30 min. A volume of 400 µL of ultrapure water was added to each tube, and samples were vortexed. After centrifugation at 4000 g for 10 min at 4◦C, 120 µL aliquots of the upper aqueous phase containing polar metabolites were transferred to microtubes and vacuum-dried (SpeedVac Concentrator, miVac, Genevac Ltd., Ipswich, England). The dried polar phase aliquots were resuspended in 30 µL of 20 mg mL−<sup>1</sup> methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, United States) in pyridine, and incubated under orbital shaking at 40◦C for 60 min. Afterward, 30 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA; Sigma-Aldrich) was added, and derivatization was performed at 40◦C for 60 min under continuous agitation.

Gas chromatography/mass spectrometry was used to quantify primary metabolites (non-structural carbohydrates, polyols, amino and organic acids), as described in Colinet et al. (2016). Briefly, GC-MS consisted of a CTC CombiPal autosampler (CTC Analytics AG, Zwingen, Switzerland), a Trace GC Ultra chromatograph, and a Trace DSQII quadrupole mass spectrometer (Thermo Fischer Scientific Inc., Waltham, MA, United States). The autosampler enabled online derivatization and standardization of the preparation process: each sample was automatically prepared during GC analysis of the preceding sample, ensuring the highest possible throughput of the system, and resulting in equal derivatization duration for each compound prior to injection. For each sample, 1 µL was injected in the oven using the split mode (25:1; temperature of the injector: 250◦C). The oven temperature ranged from 70 to 147◦C at 9◦C min−<sup>1</sup> , from 147 to 158◦C at 0.5◦C min−<sup>1</sup> , from 158 to 310◦C at 5 ◦C min−<sup>1</sup> and held at 320◦C for 4 min. Helium was the gas carrier (1 mL min−<sup>1</sup> ) and MS detection was achieved using electron impact. All samples were run under the SIM mode (electron energy: −70 eV), thereby, we only screened for the 59 pure reference compounds included in our spectral database. GC-MS peaks were accurately annotated using both mass spectra (two specific ions), and retention index specific to each compound. Calibration curve samples for 59 pure reference compounds at 10, 20, 50, 100, 200, 500, 700, and 1000 µM concentrations were run. Chromatograms were deconvoluted using XCalibur 2.0.7, and metabolite levels were quantified using the quadratic calibration curves for each reference compound and concentration. Concentrations were then normalized with the sample weight.

#### Statistical Analyses

All analyses (except survival analyses of CCRT curves) were conducted using R (version 3.4.3; R Core Team, 2016). We modeled survival of flies exposed at −5 ◦C by specifying a generalized linear mixed-effects model (GLMM) with logit link function for proportions outcome (i.e., number of dead/alive flies per vial). The response variable was dependent on the developmental temperature (10 vs. 25◦C), the temperature at adult stage (10 vs. 25◦C), the time to survival measurement and the interaction among terms. Vial number was considered as

random effect. We analyzed the effect of each variable through an analysis of deviance ("Anova" function in "car" package, Fox and Weisberg, 2011). Differences among treatment groups were analyzed by comparing least-squares means using the "emmeans" package (Russell, 2018).

Ctmin data were analyzed using a two-way ANOVA, dependent on developmental (10 vs. 25◦C) and adult temperatures (10 vs. 25◦C), and the interaction between these two factors. Then, a Tukey HSD comparison was used to identify differences between the interaction of these two factors.

CCRT data were analyzed using survival analyses with the software GraphPad Prism5. We compared recovery curves using Gehan-Breslow-Wilcoxon Tests. We adjusted the alpha level of significance thanks to Bonferroni correction (α = 0.008).

Concentrations of each metabolite after the four thermal treatments have been checked for normality: for some metabolites, normality was not respected, but total number of individuals and sample size were sufficient to use oneway ANOVAs (Blanca et al., 2017). Tukey HSD comparisons were then used to identify differences between thermal treatments. Metabolite concentrations were also pooled in groups corresponding to their respective biochemical families (i.e., amino acids, carbohydrates, polyols, etc.). Similarly, concentrations of biochemical families were analyzed using one-way ANOVAs followed by Tukey tests.

If, as speculated, cold acclimated phenotypes are characterized by a post-stress metabolic inertia, while non-acclimated counterparts loose metabolic homeostasis, then, different temporal patterns should be observed during recovery period among phenotypic groups. This should result in significant time × treatment interactions, at both univariate and multivariate levels. Temporal changes in the concentrations of metabolites and biochemical families were analyzed using GLMs with Gaussian link function, and the effect of the interaction between the thermal treatments and time after the cold stress were analyzed through an analysis of deviance ("Anova" function in "car" package, Fox and Weisberg, 2011).

Data resulting from quantitative GC-MS analysis were also analyzed using several multivariate tests. The metabolites' compositions resulting from each phenotype (i.e., the metabotype) were compared using between-class principal component analysis (PCA) (Dolédec and Chessel, 1991) in the "ade4" package in R (Dray and Dufour, 2007). A first PCA was performed using metabolic datasets of the four treatments before stress exposure (see **Figure 1**), in order to identify main patterns and clustering resulting from the different thermal conditions. A second PCA was performed on the time-series datasets of the four phenotypes in order to monitor metabolic homeostasis status during recovery period (see **Figure 1**). Monte Carlo tests were performed to examine the significance of the difference among the classes (based on 1000 simulations). To identify the variables (i.e., metabolites) contributing the most to the PCA structure separation, the correlations to the principal components (PCs) were extracted and ranked. Data were scaled and mean-centered prior to the PCAs. In addition to the classical PCA, an ASCA was performed to further analyze the time-series datasets. The ASCA (ANOVA-Simultaneous Component Analysis) is a multivariate approach ideal for the analysis of time-series metabolomic studies (Smilde et al., 2005). ASCA applies a PCA to the estimated parameters for each source of variation of the model and tests the main effects (i.e., thermal treatment and time) as well as the interaction. If acclimated phenotypes have a distinct and more efficient homeostatic response than control chill-susceptible group, then different temporal metabolic trajectories should be observed, and this should result in a significant interaction effect in the ASCA. The ASCA was performed using the temporal analysis module of MetaboAnalyst 4.0 (Chong et al., 2018). This pipeline tests the main and the interaction effects via permutation tests.

# RESULTS

#### Cold Tolerance Survival to Acute Cold Stress

**Figure 2A** illustrates survival probability of the flies 4, 24, and 48 h after a cold shock (−5 ◦C) in the four phenotypes. Flies from combined acclimation were the only showing 100% survival, at 4 and 24 h post stress. GLMM model showed an effect of developmental conditions and rearing temperature at adult stage on cold survival (χ <sup>2</sup> = 40.41, df = 1; p-value < 0.001; χ <sup>2</sup> = 107.36, df = 1, p-value < 0.001, respectively). Survival decreased with increased time of assessment after cold exposure, particularly in control and developmental acclimation (χ <sup>2</sup> = 84.98, df = 1, p-value < 0.001). Outcomes from least-square means comparisons are available on **Figure 2A**: globally, flies from combined acclimation and adult acclimation showed the highest survival rates (97 and 96% after 48 h, respectively), followed by flies submitted to developmental acclimation and control group that showed the lowest survival rate (52 and 16% after 48 h, respectively).

#### Ctmin

Ctmin values of the four phenotypes are illustrated in **Figure 2B**. Ctmin was affected by developmental conditions and temperature at adult stage [F(1,215) = 26.56, p-value < 0.001; F(1,215) = 230.20, p-value < 0.001, respectively]. Outcomes from Tukey HSD comparisons are available on **Figure 2B**: Ctmin values of the four phenotypes were all significantly different, flies from combined acclimation had the lowest Ctmin (0.89◦C), followed by flies from adult acclimation (2.16◦C), developmental acclimation (4.70◦C), and finally control flies which had the highest Ctmin (5.95◦C).

#### CCRT

CCRT curves are displayed in **Figure 2C**. In flies from combined acclimation group, it took no more than two minutes for the flies to recover, in the other groups recovery took much longer. Consequently, flies from combined acclimation were the very first to recover, followed by flies submitted to adult acclimation and developmental acclimation, and finally controls flies took the longer time to recover: after one hour of recovery, 20% of

the control flies were still in coma. Outcomes from the Gehan-Breslow-Wilcoxon tests are available on **Figure 2C**.

# Metabolic Characterization of Acclimation

#### Metabolite Concentrations After Acclimation

Among the 59 metabolites included in our spectral library, 45 were identified and quantified (i.e., absolute quantification). The list of identified metabolites, their respective abbreviations and their biochemical families are presented in **Supplementary Table S1**. **Figure 3** presents the variation patterns by biochemical classes at the end of thermal treatments. Outcomes resulting from Tukey tests on the different biochemical families according to thermal treatments are available on **Figure 3**. Except for phosphorylated compounds, mean concentrations of all biochemical families varied according to thermal conditions experienced during development and as adult (**Figure 3**). The most striking changes were observed in flies submitted to combined acclimation that were characterized by significant increases in free amino acids, carbohydrates and tricarboxylic acid (TCA) intermediates in comparison to control flies (1.15-, 2-, and 6-fold change, respectively; **Figure 3**). Flies from adult acclimation displayed a significant decrease in amines and increased levels of TCA intermediates relative to control (0.03 and 1.5-fold change, respectively; **Figure 3**). These global patterns were driven by concentration changes in individual's metabolites that are all available in the panel **Supplementary Figures S1– S7**. Flies from the combined acclimation showed high amounts of several free amino acids (Ala, Glu, Ile, Leu, Phe, Ser, Val; **Supplementary Figure S1**), carbohydrates (Fru, Mal, Man, Suc, Tre; **Supplementary Figure S4**), TCA intermediates (citrate, malate, succinate; **Supplementary Figure S5**), and the other compounds GABA and GDL (**Supplementary Figure S7**). In this phenotypic group, a decrease of F6P and G6P was observed compared to control (**Supplementary Figure S6**). In comparison to control group, flies submitted to adult acclimation were characterized by an accumulation of several polyols (glycerol, mannitol, and xylitol; **Supplementary Figure S3**) and two TCA intermediates (malate, citrate; **Supplementary Figure S5**) and a decrease in spermine, F6P and G6P (**Supplementary Figures S2**, **S6**). Patterns of flies submitted to developmental

acclimation were relatively similar to those of control flies, only two amino acids had increased amounts (Ser and Thr), while three compounds had decreased concentrations (Ala, Cit, and Phe) (**Supplementary Figure S1**).

#### Multivariate Analysis on Metabotypes After Acclimation

Global metabolic changes according to thermal treatments were also characterized using a PCA, and the ordination of classes within the first plane is presented in **Figure 4A**. This multivariate analysis revealed a clear-cut non-overlapping separation among the four thermal treatments (**Figure 4A**). The metabotype reflecting combined acclimation was the most divergent and was opposed to the other metabotypes on the first axis of the PCA (PC1, 60.53% of total inertia). The three other metabotypes separated along the second axis (PC2, 31.19% of total inertia). Thus, PC1 and PC2 supported 91.72% of total inertia. The Monte-Carlo randomizations corroborated the significance of the differences among classes (i.e., treatments; observed p-value < 0.001). The projection of the variables of the PCA (i.e., metabolites) on the correlation circle are shown in panel **Figure 4B** and mirror concentration changes of individual metabolites (**Supplementary Figures S1–S7**).

#### Temporal Patterns of Metabolic Profiles During the Recovery From Acute Cold Stress

#### Variation of Metabotypes During Recovery From Acute Cold Stress

Metabolic compositions of the four thermal treatments were monitored before the stress, and during the post-stress recovery period. **Figure 5** displays temporal changes of concentrations of metabolites' biochemical families. Univariate statistics from GLMs describing the effects of recovery time, thermal treatments and their interaction are available in **Table 1**. Interactions (time × treatment) were found for all the biochemical families (**Figure 5** and **Table 1**) suggesting divergent homeostatic trajectories among thermal treatments. This was reflected for instance in the pool of free amino acids or polyols that remained relatively stable in flies submitted to combined acclimation, whereas it increased in other phenotypes (**Figure 5**). Flies from combined acclimation were the only to show temporal decrease in global concentration of carbohydrates (0.75-fold change) and TCA intermediates dropped from 7 to 5 nmol/mg after 12 h of recovery (**Figure 5**). These global temporal patterns were driven by concentration changes in individual's metabolites that are all available in the panel **Supplementary Figures S8–S14**, together with the statistics showing the significance of treatment x time interactions in **Supplementary Table S2**. Most of the metabolites showed significant treatment × time interaction (**Supplementary Table S2**). During the recovery from cold stress, control flies were characterized by increased concentrations in most metabolites, including free amino acids (Cit, Glu, Ile, Leu, Lys, Orn, Phe, Pro, Thr, and Val; **Supplementary Figure S8**), polyols (adonitol, erythritol, glycerol, sorbitol, and xylitol; **Supplementary Figure S10**), TEA, one TCA intermediate (citrate), PO4, galacturonate and GDL (**Supplementary Figures S9**, **S12–S14**). Flies from developmental acclimation also showed temporal increase in the concentrations of free amino acids (e.g., Ala, Glu, Ile, Leu, Phe, Pro, Thr, and Val; **Supplementary Figure S8**). Most of the quantified polyols accumulated at 4 or 8 h post-stress and returned to their initial concentrations after 12 h of recovery (**Supplementary Figure S10**). Furthermore, these

and PC2, respectively. Ctrl, control flies; DA, developmental acclimation; AA, adult acclimation; DA + AA, combined developmental and adult acclimation.

flies showed a temporal accumulation of several carbohydrates (Fru, Man, and Tre; **Supplementary Figure S11**) and GDL (**Supplementary Figure S14**), and a light decrease in the level of phosphorylated compounds (F6P and G6P; **Supplementary Figure S13**). Flies from the adult acclimation showed temporal variations in the level of several free amino acids (decrease of Ala, increase of Asp, Ile, Leu, Pro, Ser, and Val; **Supplementary Figure S8**), increased amounts of amines (TEA and spermine; **Supplementary Figure S9**), succinate and a decrease of the concentration of malate (**Supplementary Figure S12**). The concentration of several polyols (adonitol, erythritol, mannitol, and xylitol; **Supplementary Figure S10**), carbohydrates (Fru, Glc, Man, and Tre; **Supplementary Figure S11**), organic acids (galacturonate, lactate, and quinate) and GABA and GDL (**Supplementary Figure S14**) showed a similar temporal pattern: the concentration increased 4 h after the cold stress and returned to initial concentration within 8 or 12 h after the stress. Finally, flies submitted to combined acclimation showed a decrease in Ala and Phe concentration during recovery from cold stress (**Supplementary Figure S8**). Other free amino acids (Gly, Ile, Leu, Ser, and Val; **Supplementary Figure S8**) showed increased levels during the recovery period but returned to initial concentrations after 12 h of recovery. The level of carbohydrates (Fru, Glc, Mal, Man, and Tre; **Supplementary Figure S11**) and TCA intermediates (succinate and malate; **Supplementary Figure S12**) moderately decreased with recovery time, while lactate accumulated after the cold stress (**Supplementary Figure S12**).

#### PCA on Metabolite Compositions During Recovery From the Acute Cold Stress

A PCA was made on metabolic patterns across the different sampling times (**Figure 6A**). Temporal changes among metabotypes were mainly reflected along PC1 (29.99% of total inertia), while differences between thermal treatments were associated with PC2 (24.06% of total inertia; PC1 + PC2 = 54.05% of total inertia). Metabotypes for control showed a marked temporal deviation along PC1. Metabotypes for adult and developmental acclimation also showed temporal deviations from initial status (especially after 8 h of recovery), but less pronounced than in the control group. Metabotypes for combined acclimation were again clearly distinct from the other groups on PC2 and showed a temporal deviation of metabolic status after 4 and 8 h of recovery, but after 12 h of recovery the metabolic profiles had returned to initial state on PC1 (**Figure 6A**). These differences among metabotypes were validated by Monte Carlo randomizations (observed p-value < 0.001). The temporal changes of these metabotypes can be observed in **Figure 6B**, which represents the projections of the centroid scores on PC1. From these, it can clearly be discerned that flies from control and developmental acclimation showed the most intense temporal deviations from initial states, while counterparts from combined acclimation showed much moderate deviation, and a returned to the initial metabolic state within 12 h. Flies from adult acclimation showed an intermediate response. Correlation of metabolites with PC1 and PC2 are available in **Figures 6C,D**, respectively. A large number of free amino acids (e.g., Pro, Thr, and Val) and polyols (e.g., erythritol, inositol, and sorbitol) were positively correlated to PC1 (**Figure 6C**), and these changes reflected temporal increase in metabolites' concentrations, mainly in control flies (see **Figure 5** and **Supplementary Figure S2**). Several carbohydrates (e.g., Fru, Man, Suc, and Mal) were positively correlated with PC2 (**Figure 6D**), revealing relative higher amounts of these metabolites in flies from combined acclimation compared to control flies (see also **Figure 5** and **Supplementary Figure S2**).

#### ASCA on Metabolite Compositions During Recovery From Acute Cold Stress

The results of ASCA are presented in panel **Supplementary Figure S15**. The **Supplementary Figure S15A** shows the major patterns associated with the factor "treatment" (i.e., thermal treatments) of the ASCA. A decreasing trend was detected, with flies from combined acclimation showing positive score. Control flies were at the opposite, with a negative score and flies from developmental and adult acclimation were intermediate (**Supplementary Figure S15A**). This pattern mirrored the PCA results shown in **Figure 6A**. The **Supplementary Figure S15B** shows the major patterns associated with the factor "time". The scores was initially negative (before the stress) and remained stable after the stress (0 h), then a marked increase occurred at 4 h of recovery (time 4), when the score became positive, after which the scores remained positive at 8 and 12 h of recovery. These results are also consistent with PCA results shown in **Figures 6A,B**, in which the metabotypes of flies showing the lowest cold tolerance (control and developmental acclimation mainly) shifted in the same direction on PC1 (toward positive scores) over the time-course of the experiment. Interestingly, the major patterns associated with interaction terms suggested that flies from combined and adult acclimation had rather similar temporal patterns that differed from that of flies from developmental acclimation and control (**Supplementary Figure S15C**), suggesting a major importance of adult acclimation. Finally, the observed statistics with permutation tests were significant for both main factors (observed p-value < 0.05 for both "treatment" and "time"), confirming the clear differentiation of metabotypes according to both "treatment" and "time" (**Supplementary Figure S15D**). More interestingly, the permutation test for "treatment × time" interaction was also significant (observed p-value < 0.05), demonstrating that temporal metabolic trajectories significantly differed according to thermal treatments (**Supplementary Figure S15D**).

# DISCUSSION

#### Impact of Thermal Acclimation on D. suzukii Cold Tolerance

In this study, we subjected D. suzukii flies to four different thermal treatments to estimate the effect of developmental and adult acclimation or the combination of these two forms of acclimation on cold tolerance of adults. As expected, combining developmental and adult acclimation led to the highest cold tolerance of flies, based on three different measures of cold tolerance (survival, Ctmin, CCRT; **Figure 2**). These results confirm previous observations of Stephens et al. (2015),

Shearer et al. (2016), or Toxopeus et al. (2016), who showed that combination of developmental and adult acclimation leads to a highly cold tolerant phenotype in D. suzukii. In Drosophila melanogaster, the combination of developmental and adult acclimation also resulted in cumulative effects (Colinet and Hoffmann, 2012; Slotsbo et al., 2016), but this pattern is not a general rule in insects (Terblanche and Chown, 2006). The fact that acclimation benefits cumulated in D. suzukii further adds to the widely accepted vision that physiological underpinnings of the different forms of acclimation are somewhat specific (Colinet and Hoffmann, 2012; Teets and Denlinger, 2013; Gerken et al., 2015).

Both developmental and adult acclimations, when applied alone, increased cold tolerance of D. suzukii. However, the promoting effect was higher when these two acclimation forms were combined. Developmental and adult acclimations are well known to increase cold tolerance of D. melanogaster (Sinclair and Roberts, 2005; Rako and Hoffmann, 2006; Overgaard et al., 2008; Koštál et al., 2011a; Colinet and Hoffmann, 2012) and D. suzukii (Jakobs et al., 2015). Here, we found that adult acclimation improved both survival and Ctmin more intensely than developmental acclimation, though these two acclimation treatments resulted in similar CCRT. It is assumed that the thermal conditions experienced just before a stress mainly determine subsequent cold tolerance (Geister and Fischer, 2007; Fischer et al., 2010; Colinet and Hoffmann, 2012). Thus, the higher effect of adult acclimation on cold tolerance could be due to its "temporal proximity" with the acute cold stress, whereas



Treatment, acclimation treatment; Time, recovery time.

seven days at 25◦C separated developmental acclimation from the cold shock (**Figure 1**). It is also possible that flies from developmental acclimation deacclimated during this period.

In this study, light cycles were not strictly identical among the experimental treatments. However, we reasoned that temperature is the main driver of the observed phenotypic changes (cold tolerance). Bauerfeind et al. (2014) reported that stress resistance traits (cold, heat, starvation, and desiccation resistance) are predominantly affected by temperature and not by photoperiod in D. melanogaster. Likewise, in D. suzukii, delayed reproductive maturity (i.e., the reproductive diapause syndrome) seems temperature-dependent and not regulated by photoperiod (Toxopeus et al., 2016). We therefore assumed that effects observed here on cold tolerance and physiology were primarily due to temperature.

#### Acclimation Triggers Metabolic Changes in D. suzukii

One aim of this study was to identify metabolic changes resulting from different forms of cold acclimation in D. suzukii. We expected that the most cold-tolerant phenotypes would be characterized by accumulation of cryoprotectant molecules, such as carbohydrates, polyols or amino acids. GC-MS analysis revealed four non-overlapping metabotypes, suggesting different metabolic profiles among the four phenotypic groups (**Figure 4**). Amounts of several carbohydrates, such as Fru, Mal, Man, Suc, and Tre, increased in flies submitted to combined acclimation compare to controls (**Supplementary Figure S4**). Carbohydrates are known to be involved in cold tolerance of several insect species (Baust and Edwards, 1979; Kimura, 1982; Fields et al., 1998; Zeng et al., 2008; Ditrich and Koštál, 2011). In D. melanogaster, cold acclimation and rapid cold hardening increase the level of Fru, Glc, Mal, Suc, and Tre (Overgaard et al., 2007; Koštál et al., 2011a; Colinet et al., 2012). Even if caution should be exercised in designating a function to any upregulated or downregulated metabolite, as flux and pathways are unknown, the increased sugar level after combined acclimation in D. suzukii reverberates the results of Shearer et al. (2016). These authors found upregulations of gene clusters involved in the carbohydrates' metabolism in winter morphs of D. suzukii. In this species, the cold-hardy "winter morph" is generated using a combination of developmental and adult acclimation (Stephens et al., 2015; Shearer et al., 2016; Toxopeus et al., 2016; Wallingford and Loeb, 2016). Unlike proper cold tolerant overwintering species that accumulate several hundred mmol L−<sup>1</sup> of cryoprotectants (Salt, 1961), carbohydrate accumulation in cold acclimated drosophilids is of rather low magnitude, suggesting a non-colligatively contribution to cold hardiness (Koštál et al., 2011a; Colinet et al., 2012), for instance by stabilizing macromolecule structures (Arakawa and Timasheff, 1982; Crowe et al., 1988; Cacela and Hincha, 2006).

In comparison to controls, flies from the adult acclimation had increased concentrations of several polyols (glycerol, mannitol, and xylitol; **Supplementary Figure S3**). However, as for sugars, the concentrations and magnitude of changes remained too low to consider this pattern as a proper cryoprotective and

combined developmental and adult acclimation.

colligative response. Polyols, and especially glycerol, are common cryoprotectants in cold-adapted insects (Sømme, 1982; Storey and Storey, 2005; Denlinger and Lee, 2010). Accumulation of massive concentrations of polyol are linked to freezeprotective functions such as the diminution of the supercooling point (Crosthwaite et al., 2011). On the other hand, at low concentrations, polyols may play other protective functions, likely through a "preferential" exclusion of solutes from proteins, which help to stabilize their structures (Gekko and Timasheff, 1981; Koštál et al., 2011a). In drosophilids, however, there is no clear evidence for substantial polyols accumulation correlated with cold hardiness (Kimura, 1982; Kelty and Lee, 2001; Overgaard et al., 2007; Koštál et al., 2011a; Colinet et al., 2012).

Flies subjected to adult acclimation showed a low level of spermine (**Supplementary Figure S2**). Polyamines are involved in stress tolerance in plants (Gill and Tuteja, 2010) and putatively in insects (Michaud et al., 2008). Previous reports have detected increase in some polyamines (putrescine and cadaverine) in cold acclimated flies (Koštál et al., 2011a; Colinet et al., 2012). However, there is no report on variations of spermine levels in response to acclimation. Spermine is a polyamine formed from spermidine, and it has been shown to mediate stress resistance in Drosophila (Minois et al., 2012). It remains unclear why spermine was specifically at low levels in adult acclimated flies. Polyamine metabolism is very dynamic and low level of spermine may be due to synthesis of spermidine. Unfortunately, we could not detect this latter metabolite.

Flies from combined acclimation were also characterized by accumulation of several amino acids (Ala, Ile, Leu, Phe, Ser, and Val; **Supplementary Figure S1**) in comparison to control. Amino acids are known to possess cryoprotective properties. For example, Pro is responsible of increasing cold and freeze tolerance in D. melanogaster (Koštál et al., 2012), and it allows larvae from the fly Chymomyza costata to survive exposure to liquid nitrogen (Koštál et al., 2011c). Here, the concentration of Pro did not change dramatically in response to combined acclimation, while it increased in response to acclimation in D. melanogaster (Koštál et al., 2011a). The increased concentrations of the other amino acids were relatively small compared to changes observed with Pro in C. costata by Koštál et al. (2011c). In addition to Pro, many other free amino acids (mainly Arg, but also Ile, Leu, Val, and Ala) can promote flies' cold tolerance when supplemented in food (Koštál et al., 2016a). Although with different protocols, cold acclimation in D. melanogaster also triggered the accumulation of various amino acids in both adults (e.g., Val, Leu, Ser, Thr, Ile) (Colinet et al., 2012) and larvae (e.g., Pro, Asn, His, Glu) (Koštál et al., 2011a). In other insect species, cold acclimation or rapid cold hardening has consistently been correlated with the accumulation of various amino acids, among which Ala was often represented (Morgan and Chippendale, 1983; Storey, 1983; Hanzal and Jegorov, 1991; Fields et al., 1998; Michaud and Denlinger, 2007; Li et al., 2015). Here, Ala was the only amino acid showing the highest concentration after adult or combined acclimation (**Supplementary Figure S1**). The functional reasons for these amino acids accumulations in cold-resistant phenotypes are not clearly understood, but it might relate to metabolic protection or stabilization of macromolecules (Yancey, 2005). Cold-protective mechanisms of free amino acids (against freezing mainly) have been extensively discussed by Koštál et al. (2016a) who suggested a range of different interactions, such as preferential exclusion, protection of native protein structure, binding of partially unfolded proteins, stabilization of membrane structure and vitrification. Apart from these cold-protective functions, many amino acids are components of biosynthetic pathways linked to glycolysis and TCA cycle; therefore, these changes may also reflect consequences of perturbations of the metabolic rate that often characterize acclimation (Lu et al., 2014; Li et al., 2015). Similarly, Koštál et al. (2011b) observed a moderate accumulation of amino acids, such as Pro, Gln, and Ala, in overwintering Pyrrhocoris apterus, which probably resulted from a decrease of TCA cycle turnover rate, due to metabolic alteration induced by cold temperatures.

Here, we found that flies submitted to adult and combined acclimation were characterized by higher amounts of TCA intermediates than control flies or flies acclimated during development only (**Figure 3**). This could be linked to changes in metabolic rate. Indeed, metabolic cold adaptation or temperature compensation theories presume that at a same temperature, cold hardy insects showed similar or even higher metabolic rate than cold sensitive individuals (Hazel and Prosser, 1974; Chown and Gaston, 1999). Even if these theories are not always supported, several studies showed that cold acclimated insects had higher metabolic rate than warm acclimated ones (Terblanche et al., 2005; Isobe et al., 2013) and this may explain, at least in part, the changes in levels of TCA intermediates.

Despite showing unique metabotype, flies from developmental acclimation did not show drastic changes in their metabolite composition as observed in flies from combined and adult acclimation. Cold acclimation is a reversible trait (Slotsbo et al., 2016), therefore the moderate difference between flies from developmental acclimation and controls may be due to deacclimation. Despite these light metabotype divergences, our data showed that flies from developmental acclimation were more cold tolerant than control, based on all tested proxies. This suggests that physiological changes occurring during development, not only metabolic adjustments, persists and even carried over in adult stage, even if temperature returns permissive at adult stage.

#### Temporal Trajectories of Metabotypes After Acute Cold Stress

Maintenance of metabolic homeostasis has been repeatedly associated with thermal tolerance in D. melanogaster (Malmendal et al., 2006; Colinet et al., 2012; Williams et al., 2014), although the functional mechanisms behind this correlated pattern are unknown and likely highly complex. We thus expected signs of metabolic inertia in cold acclimated phenotypes and signs of metabolic deregulation in chill-susceptible flies. Temporal analysis of metabotypes revealed different temporal

patterns and mirrored cold tolerance of the four phenotypes (**Figure 6**). Control flies (the least cold tolerant) described a large and persistent metabolic deviation from initial state, suggesting a loss of metabolic homeostasis. Conversely, the most cold tolerant flies (flies from combined acclimation) showed only light initial deviation of metabolic trajectories followed by a rapid return to initial state during recovery period, suggesting a metabolic robustness and an efficient homeostatic response. Flies submitted to developmental and adult acclimation had intermediate cold tolerance and showed intermediate temporal metabolic response: initial deviation followed by incomplete return to initial metabolic state. Loss of homeostasis is likely related to development of thermal injuries (Malmendal et al., 2006) and several studies reported similar chill-induced homeostatic disruption in insects (Overgaard et al., 2007; Colinet et al., 2012; Teets et al., 2012; Williams et al., 2014).

Effects of chilling injuries can be immediate, accumulative or latently expressed (e.g., delayed mortality) (Turnock and Bodnaryk, 1991; Leopold, 2000; Overgaard et al., 2005). We noted that mortality after acute cold stress increased gradually with time of observation in control and flies acclimated during development suggesting latent damages. In contrast, flies that have been subjected to adult acclimation, combined or not with developmental acclimation, showed high and constant survival rates. Temporal increase of chill injuries in control and flies acclimated during development may be correlated with the temporal metabolic disorders that presented the highest distortion amplitudes in these two phenotypes.

The first axis of PCA, which described the temporal deviation of metabolic profiles, was correlated with accumulation of several amino acids, including several essential amino acids (Ile, Leu, Phe, Thr, and Val, **Figure 6**). Furthermore, the most chillsusceptible flies (control and developmental acclimation) were characterized by a temporal augmentation of the global amino acids pool (**Figure 5**). Such accumulation of amino acids after a cold stress has been reported in several insect species and is assumed to relate to protein breakdown, especially for essential amino acids, which can't be synthetized de novo (Lalouette et al., 2007; Overgaard et al., 2007; Koštál et al., 2011b; Colinet et al., 2012). However, direct evidence of cold-induced protein degradation is scarce (Hochachka and Somero, 2002).

Chill-susceptible phenotypes (mainly control flies and to a lesser extent flies from developmental acclimation) were characterized by temporal accumulation of several polyols (e.g., erythritol, sorbitol, and inositol) after the acute cold stress (**Supplementary Figure S10**). As discussed previously, accumulation of polyols after acclimation or rapid cold hardening may have protective functions (Walters et al., 2009; Crosthwaite et al., 2011). Increase in polyol concentrations after a cold stress is also a general response in insects (Yoder et al., 2006; Lalouette et al., 2007; Michaud et al., 2008; Colinet et al., 2012). So far, it is not clear whether these accumulations have protective values or whether they represent biomarkers of complex metabolic deregulations due to cold stress (Colinet et al., 2012). Since only the two least cold-tolerant phenotypes (flies from control and developmental acclimation) displayed post-stress temporal increase in the level of some polyols, we can speculate that this most likely reflects a degenerative rather than a protective syndrome.

Concentration of amino acid pool didn't change in time after the cold stress in flies from combined acclimation (**Figure 5**), suggesting low magnitude of cold injuries to proteins structures. Conversely, global carbohydrates and TCA intermediates pools decreased with time in these flies (**Figure 5**). This may relate to rapid mobilization of energetic metabolism during recovery. Williams et al. (2016) reported that D. melanogaster lines selected for cold tolerance presented metabolic rate depression during cold stress, but that metabolic rate increased in a higher proportion than in chill-susceptible flies during recovery from a cold exposure at 0◦C. In the beetle Alphitobius diaperinus, recovery from cold stress during thermal fluctuation is also associated with an important metabolic rate augmentation (Lalouette et al., 2011). Repairing chilling injuries and reestablishing homeostasis likely induces energetic cost (MacMillan et al., 2012); therefore, reduction of TCA intermediates in flies from combined acclimation may be related to energy requirement for recovery.

#### CONCLUSION

This study revealed that combining both developmental and adult cold acclimation resulted in a particularly high expression of cold tolerance in D. suzukii. This fly is believed to overwinter on wooded areas under leaf litters (Kanzawa, 1936), and trappings in autumn and winter revealed that captured flies were bigger, darker and more cold tolerant than flies captured in summer (Shearer et al., 2016). During these periods, flies are maybe developing slowly in infected fruits (protected in the litter) and then cold-acclimate as adults. Emerging flies by the end of winter season may therefore express high level of cold survival (at temperatures as low as −5 ◦C) in the field. Our results indicate that cold tolerance plasticity of D. suzukii relies on physiological strategies similar to other drosophilids. Indeed, as frequently found in others drosophilids (e.g., Koštál et al., 2011a; Colinet and Hoffmann, 2012), we found that cold-acclimated D. suzukii accumulated low levels of cryoprotectants, such as sugars and amino acids, and were able to maintain metabolic homeostasis following cold stress. We highlighted that different acclimation treatments resulted in clearly distinct metabotypes, suggesting that physiological responses highly depend on thermal history. Collectively, these data contribute to the emerging understanding of the physiological strategies used by D. suzukii to acquire cold tolerance. The present metabolic analyses provided correlative but not causative effects of cold acclimation. Artificial variations of the candidate metabolites, for instance by diet supplementation or by disrupting their biosynthesis, could shed light on the function(s) of these metabolites in cold tolerance acquisition. In addition to metabolic adjustments and mobilization of cryoprotectants, cold tolerance plasticity relies on many other mechanisms and pathways, such as phospholipidic remodeling, maintenance of contractile function or altered thermal sensitivity of ion channel kinetics (Overgaard and MacMillan, 2016). Future studies should also explore these processes to gain better understanding of cold adaption of D. suzukii.

#### DATA AVAILABILITY

fphys-09-01506 October 30, 2018 Time: 15:22 # 14

The raw data supporting the conclusions of this manuscript will be made available by the authors on request, without undue reservation, to any qualified researcher.

#### AUTHOR CONTRIBUTIONS

TE and HC designed the experimental plan. TE conducted all experiments under the supervision of HC, DR, and MC. TE and HC analyzed the data and performed statistical analysis. TE and HC drafted the manuscript. All authors reviewed the manuscript.

#### REFERENCES


#### FUNDING

This study was funded by SUZUKILL project (The French National Research Agency): ANR-15-CE21-0017 and Austrian Science Fund (FWF): I 2604-B25.

#### ACKNOWLEDGMENTS

We would like to thank Maxime Dahirel for his advice on statistical analyses. The GC-MS analyses were performed at the analytical platform "PLAY" (University of Rennes 1, UMR CNRS EcoBio, Rennes, France).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01506/full#supplementary-material

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vary with gender, age, feeding, pregnancy or acclimation. J. Insect Physiol. 51, 861–870. doi: 10.1016/j.jinsphys.2005.03.017


metabolic plasticity during cold exposure in Drosophila melanogaster. Proc. R. Soc. B 283:20161317. doi: 10.1098/rspb.2016.1317


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Enriquez, Renault, Charrier and Colinet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Molecular Evidence for the Fitness of Cotton Aphid, Aphis gossypii in Response to Elevated CO<sup>2</sup> From the Perspective of Feeding Behavior Analysis

Shoulin Jiang1,2, Yang Dai<sup>1</sup> , Yongqing Lu<sup>1</sup> , Shuqin Fan<sup>3</sup> , Yanmin Liu<sup>1</sup> , Muhammad Adnan Bodlah<sup>1</sup> , Megha N. Parajulee<sup>4</sup> and Fajun Chen<sup>1</sup> \*

<sup>1</sup> Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China, <sup>2</sup> Personnel Department, Qingdao Agricultural University, Qingdao, China, <sup>3</sup> Qidong Agricultural Commission, Qidong, China, <sup>4</sup> Texas A&M University AgriLife Research and Extension Center, Lubbock, TX, United States

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Daniele Pereira Castro, Fundação Oswaldo Cruz (Fiocruz), Brazil Pin-Jun Wan, China National Rice Research Institute (CAAS), China

> \*Correspondence: Fajun Chen fajunchen@njau.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 04 July 2018 Accepted: 24 September 2018 Published: 12 November 2018

#### Citation:

Jiang S, Dai Y, Lu Y, Fan S, Liu Y, Bodlah MA, Parajulee MN and Chen F (2018) Molecular Evidence for the Fitness of Cotton Aphid, Aphis gossypii in Response to Elevated CO<sup>2</sup> From the Perspective of Feeding Behavior Analysis. Front. Physiol. 9:1444. doi: 10.3389/fphys.2018.01444 Rising atmospheric carbon dioxide (CO2) concentration is likely to influence insect– plant interactions. Aphid, as a typical phloem-feeding herbivorous insect, has shown consistently more positive responses in fitness to elevated CO<sup>2</sup> concentrations than those seen in leaf-chewing insects. But, little is known about the mechanism of this performance. In this study, the foliar soluble constituents of cotton and the life history of the cotton aphid Aphis gossypii and its mean relative growth rate (MRGR) and feeding behavior were measured, as well as the relative transcript levels of target genes related appetite, salivary proteins, molting hormone (MH), and juvenile hormone, to investigate the fitness of A. gossypii in response to elevated CO<sup>2</sup> (800 ppm vs. 400 ppm). The results indicated that elevated CO<sup>2</sup> significantly stimulated the increase in concentrations of soluble proteins in the leaf and sucrose in seedlings. Significant increases in adult longevity, lifespan, fecundity, and MRGR of A. gossypii were found under elevated CO<sup>2</sup> in contrast to ambient CO2. Furthermore, the feeding behavior of A. gossypii was significantly affected by elevated CO2, including significant shortening of the time of stylet penetration to phloem position and significant decrease in the mean frequency of xylem phase. It is presumed that the fitness of A. gossypii can be enhanced, resulting from the increases in nutrient sources and potential increase in the duration of phloem ingestion under elevated CO<sup>2</sup> in contrast to ambient CO2. In addition, the qPCR results also demonstrated that the genes related to appetite and salivary proteins were significantly upregulated, whereas, the genes related to MH were significantly downregulated under elevated CO<sup>2</sup> in contrast to ambient CO2, this is in accordance with the performance of A. gossypii in response to elevated CO2. In conclusion, rise in atmospheric CO<sup>2</sup> concentration can enhance the fitness of A. gossypii by increasing their ingestion of higher quantity and higher quality of host plant tissues and by simultaneously upregulating the transcript expression of the genes related to appetite and salivary proteins, and then this may increase the control risk of A. gossypii under conditions of climate change in the future.

Keywords: elevated CO2, Aphis gossypii, fitness, feeding behavior, molecular evidence

#### INTRODUCTION

fphys-09-01444 November 8, 2018 Time: 16:41 # 2

Global atmospheric carbon dioxide (i.e., CO2) concentration has continuously risen from about 280 ppm to 408 ppm as on May 2018 (Mauna Loa Observatory: NOAA-ESRL) and future estimations predict an increase up to 550 ppm within a few decades (Pachauri et al., 2014). Rising CO<sup>2</sup> has been an aspect of global climate change, being one great concern for the scientific community, owing not only to its "greenhouse effects" (Tubiello et al., 2000) but also its influences on the physiological and biochemical characters of the plant (Ainsworth and Rogers, 2007). The gaseous form of CO<sup>2</sup> is the direct substrate for photosynthesis in plants (Ziska, 2008), which shows typical increases of photosynthetic rate, biomass, leaf area, and carbon (C): nitrogen (N) ratio, especially in C<sup>3</sup> crops (During photosynthesis, "C" in CO<sup>2</sup> is fixed directly to "C3" in plants, such as, rice and cotton.) (Ainsworth and Long, 2005; Chen et al., 2005a; Ainsworth et al., 2007). Generally, elevated CO<sup>2</sup> alters plant chemistry by the assimilation and reassignment of C and N resources within plant tissues (Couture et al., 2010). Based on evidence provided by Chen et al. (2004) and Oehme et al. (2013), in spring wheat (Triticum aestivum), elevated CO<sup>2</sup> significantly increases the soluble components of plant tissues, such as free amino acids (FAAs), soluble proteins, and glucose. Similar results were also demonstrated by cotton (Gossypium hirsutum) plants, that is, elevated CO<sup>2</sup> significantly enhanced foliar soluble matters, including soluble sugars, FAAs, and fatty acids, which had further positive effects on the population growth of the cotton aphid, Aphis gossypii, in response to elevated CO<sup>2</sup> (Jiang et al., 2016). As noted in various studies, elevated CO<sup>2</sup> directly affects the primary and secondary metabolites of host plants, which, in turn, indirectly alter the performance of herbivorous insects (Couture et al., 2010; Guo et al., 2014a; Jiang et al., 2016).

Sap-sucking insects have shown consistently more positive responses in fitness to elevated CO<sup>2</sup> concentrations (Sun et al., 2015) than those shown by leaf-chewing insects (Bezemer and Jones, 1998). They feed exclusively on the phloem of their host plants (Douglas, 2003), and the phloem sap mainly contains sucrose (up to 80–85% of the organic components) and soluble proteins (SPs) (Avigad and Dey, 1997). Moreover, sucrose is also recognized as an important transportable sugar in most plant species and as the most effective phagostimulant for herbivorous insects (Hawker, 1985). Moreover, the concentration of SPs in the phloem sap is regarded as a key factor for identifying the nutritional quality of host plants by aphids (Nowak and Komor, 2010). Therefore, since elevated CO<sup>2</sup> inevitably alters plant metabolites, the performance of sap-sucking insects is affected by the bottom-up effects of the host plants in terms of nutritional status (Awmack and Leather, 2002). For example, rising atmospheric CO<sup>2</sup> increases the population growth of Acyrthosiphon pisum, owing to enhanced food ingestion and good food-quality plasticity; specifically, it increases amino acids' concentration and other nutrient components in leaves and phloem sap (Guo et al., 2013). Furthermore, the impact of elevated CO<sup>2</sup> on the growth, development, and fecundity of the cotton aphid A. gossypii was mainly indirect, which is affected by the nutritional status of the plant (Chen et al., 2005b; Jiang et al., 2016).

Electrical penetration graph (EPG) technique, which monitors the stylet penetration behavior via variation in electrical recording signals, is a well established and effective experimental method to quantify the sap-feeding behavior of aphids (McLean and Kinsey, 1964; Tjallingii, 1988, 1990; Jiang et al., 2015). Our previous study indicated that elevated CO<sup>2</sup> promoted the ingestion efficiency of the cotton aphid A. gossypii and simultaneously increased the leaf turgor and foliar soluble constituents of cotton plants (Jiang et al., 2015). Although the feeding behavior of aphids in response to elevated CO<sup>2</sup> has been well established, the underlying molecular mechanism of elevated CO2-induced changes in the ingestion in aphids remains largely unknown. It has been documented that the feeding behavior of insects is regulated by neuropeptide F (i.e., NPF) and angiotensin-converting enzymes (i.e., ACE) related to appetite (Nassel and Wegener, 2011; Wang et al., 2015). Wu et al. (2003) reported that the expression of NPF was high in the larvae of Drosophila melanogaster that were attracted to food, whereas its downregulation coincided with food aversion and hyperactivity of older larvae; the the over-expression of NPF in older larvae conversely promoted feeding and suppressed hypermobility and excessive behaviors. Numerous invertebrates, for example, Litopenaeus vannamei and Melicertus marginatus (Christie et al., 2011), Caenorhabditis elegans (de Bono and Bargmann, 1998), Periplaneta americana (Mikani et al., 2012), Latrodectus hesperus (Christie, 2015), and Schistocerca gregaria (Van Wielendaele et al., 2013), exhibit the fact that NPF has a function in the modulation of feeding behavior. Likewise, it was demonstrated that ACE modulates the aphid–plant interactions by affecting feeding behavior and survival of aphids, through evidence obtained from the knockdown of ACE genes (Wang et al., 2015). Previous studies showed that the salivary sheath protein and C002 play a critical role in the process of stylet penetration and food ingestion in aphids (Mutti et al., 2008; Bos et al., 2010; Abdellatef et al., 2015). Here, the question is what are the underlying molecular mechanisms that elicit the positive responses in the fitness of sap-sucking insects to elevated CO2.

The Cotton aphid, A. gossypii, as a typical phloem-feeding insect, is known as one of the most problematic insect pests

of cotton plants worldwide. In this study, an EPG experiment was carried out with cotton (G. hirsutum) plants and the cotton aphid A. gossypii under ambient and elevated CO<sup>2</sup> in opentop chambers; simultaneously, an assay to identify the foliar soluble constituents of cotton plants and the molecular biology analysis of the genes related to appetite and salivary proteins of cotton aphids were conducted. The purpose was to examine the effects of elevated CO<sup>2</sup> on stylet ingestion and fitness of phloem-feeding insects on host plants as well as elucidate the molecular mechanisms of feeding behavioral response of phloemfeeders when the host plant is exposed to rising atmospheric CO<sup>2</sup> concentrations.

# MATERIALS AND METHODS

# CO<sup>2</sup> Levels and Condition Setting

This study was conducted in six identical electronically controlled growth incubators (GDN-400D-4/CO2; Ningbo Southeast Instrument Co., Ltd., Ningbo, China) with a gas-tank system that maintained the desired CO<sup>2</sup> concentration. In these growth incubators, a periodic regime was maintained at 26◦C and 70% RH during the day, 25◦C and 70% RH at night, and L14: D10 photoperiod with light at 20000 Lux supplied by LED lamps. The CO<sup>2</sup> concentrations in the three growth incubators mentioned above were set at the current atmospheric CO<sup>2</sup> level (i.e., 400 ppm), and the rest of the three growth incubators were set at an elevated CO<sup>2</sup> level (i.e., 800 ppm), which was the predicted CO<sup>2</sup> level at the end of the 21st century (Mastrandrea et al., 2011). During the experiment, the six growth incubators were alternated by switching CO<sup>2</sup> concentration rates as well as swapping the entire content of each growth incubator every 5 days in order to equalize the possible bias on the cotton plants and aphids due to the incubator-specific growth conditions.

#### Host Plants and Cotton Aphids

Cotton (cv. C111) was planted in white plastic pots (12 cm diameter, 15 cm high) filled with nutritional soil (Xingnong Organic Fertilizer Co., Ltd., Zhenjiang, China). After the seedlings' emergence, cotton plants were thinned to one plant per pot and exposed to the above mentioned (about 400 ppm) and elevated (about 800 ppm) CO<sup>2</sup> conditions. The cotton plants were watered moderately every day; no additional chemical fertilizers or insecticides were used. At least 60 pots were randomly placed in each growth incubator (i.e., a total of 180 pots of cotton plants per CO<sup>2</sup> treatment) and re-randomized once a week to minimize position effects within each growth incubator.

The colony of the apterous cotton aphid A. gossypii used in this study was provided by Prof. Xiangdong Liu from the Department of Entomology, Nanjing Agricultural University. To obtain a standardized aphid colony for this experiment, only one clone in this colony was selected to establish an experimental population of A. gossypii. The colony was maintained on 35- to 60-day-old cotton seedlings planted in the same white plastic pots filled with the same nutritional soil in the same electronically controlled growth incubators mentioned above for the following experiments.

# Foliar Soluble Constituents of Cotton Seedlings

For the quantitative analysis of foliar soluble nutrition of cotton seedlings, 30 fully expanded leaves on the third to fourth main stem nodes were randomly selected and excised from the potted cotton seedlings in the above mentioned growth incubators of ambient and elevated CO<sup>2</sup> treatments, respectively. Cotton leaves were ground into a fine powder with a mortar and pestle in liquid nitrogen. For the determination of foliar FAAs, the leaf powder (accurately weighed 200–300 mg) was transferred to a 50 ml centrifuge tube, and then, it was diluted to a 10 ml solution by 0.02 mol/L HCl solution. The extraction buffer was sonicated for 15 min at 4◦C, and then centrifuged for 15 min at 4,000 rpm/min (RCF = 1503 g) at 4◦C to obtain the supernatant containing FAAs, 700 ml of the supernatant was transferred to a 1.5 ml microtube for deproteinization by an equal volume of 4% sulfosalicylic acid solution, then centrifuged for 15 min at 4,000 rpm/min (RCF = 1503 g) at 4◦C. The supernatants of the all the samples were individually filtered through 0.22 µm hydrophilic membranes, and, finally, the measurement of FAA concentrations was performed using an automatic FAA analyzer (L-8900; Hitachi High-Technologies Corporation, Tokyo, Japan). The values of FAAs were expressed as mg/g fresh weight.

The above obtained leaf powder was collected, approximately 30–40 mg fresh weight was transferred into a 1.5 ml microtube, and 0.9% saline was used as an extraction buffer at a ratio of 1:9 (tissue weight in g and buffer volume in ml) for the measurement of the foliar SP content. The supernatant of extraction buffer was used as a protein solution for the following test. The foliar SP content was determined by following the instructions of the corresponding diagnostic kit A045-2 (Jiancheng Bioengineering Institute, Nanjing, China). For sucrose determination, 30–40 mg of above obtained leaf power was collected in a 5 ml centrifuge tube with distilled water at a ratio of 1:10 (tissue weight in g and buffer volume in ml), and the mixture was boiled for 10 min and centrifuged at 4,000 rpm/min (RCF = 1503 g) for 10 min. The supernatants were used for assaying the foliar sucrose content according to the corresponding diagnostic kit for the determination of plant sucrose content (Jiancheng Bioengineering Institute, Nanjing, China). There were three replicates for assaying the foliar contents of soluble constituents (including FAA, SP, and sucrose) of cotton seedlings.

# Aphid Infestation

#### Life History Parameters of Cotton Aphids

A total of 45 newborn first instar nymphs were selected from the above mentioned aphid colony of A. gossypii and individually reared on fully expanded leaves, which were excised from the 35- to 60-day-old cotton seedlings grown under ambient and elevated CO2, respectively, in glass culture dishes (150 mm in diameter; one nymph per leaf × one leaf per dish × 15 dishes per growth incubator ×3 growth incubator per CO<sup>2</sup> treatment). Aphid nymphs were monitored twice a day to record molting until they developed into adults. The exuvia was removed, and the ecdysis time was recorded to quantify the nymphal duration of A. gossypii. Moreover, the number of offsprings laid per adult was recorded twice a day, and all the nymphs were removed until the adult aphid died to determine fecundity. The life history parameters of the reproductive period and adult longevity were also, finally, calculated and recorded. In this experiment, eight nymphs of the ambient CO<sup>2</sup> treatment and four nymphs of the elevated CO<sup>2</sup> treatment died in the rearing process, and actually, there were 37 and 41 individuals of A. gossypii in the treatments of ambient and elevated CO2, respectively. But, for assessing survival rate, we added data to 45 replicates in two CO<sup>2</sup> treatments.

#### Mean Relative Growth Rate (MRGR)

fphys-09-01444 November 8, 2018 Time: 16:41 # 4

A total of 30 newborn first instar aphids were randomly selected from the above mentioned aphid colony and weighed (i.e., W1) using a precision scale with an accuracy of ±1 µg (Mettler Toledo XP6, Switzerland) and then individually reared using the same protocol for the measurement of life history parameters of A. gossypii (i.e., one nymph per leaf × one leaf per dish × 10 dishes per growth incubator × 3 growth incubator per CO<sup>2</sup> treatment). These tested aphid nymphs were reweighed (i.e., W2) by using the same precision scale after 5 days of rearing, and the mean relative growth rate (i.e., MRGR) of A. gossypii nymphs was calculated based on the method described by Hodge et al. (2005): MRGR = (lnW2 − lnW1)/t, where W1 is the initial weight, W2 is the final weight, and t is the rearing time (here, 5 days) of A. gossypii nymphs. In this experiment, two nymphs of the ambient CO<sup>2</sup> treatment and five nymphs of the elevated CO<sup>2</sup> treatment died in the rearing process, and actually, there were 23 and 20 individuals of A. gossypii in the treatments of ambient and elevated CO2, respectively.

#### Electrical Penetration Graphs (EPG) to Monitor Aphid Feeding

To monitor the feeding behavior of the cotton aphid A. gossypii, 300 newborn first instar nymphs were randomly selected from the above mentioned aphid colony and reared on fully expanded leaves, which were excised from cotton seedlings grown under ambient and elevated CO<sup>2</sup> conditions, respectively, in 30 culture dishes (150 mm in diameter; 10 nymphs per leaf × one leaf per dish × 10 dishes per growth incubator × 3 growth incubator per CO<sup>2</sup> treatment). Once the newborn adult aphids emerged, they were randomly selected and used for the following EPG test.

The feeding activities of the cotton aphid A. gossypii were studied by using a Giga-8 DC-EPG amplifier system with 1 G input impedance, 50× amplification, and <1 pA input bias current (Wageningen University, Wageningen, Netherlands). The above mentioned newborn adult aphids were individually connected to a gold wire (0.5 mm diameter, 3 cm long) with conductive silver glue on their dorsum. After 1 h of starvation, the wired adult aphids were carefully placed on the abaxial surface of the fully expanded leaf in the same culture dishes mentioned above, and the other side of the gold wire was connected to the amplifier of the Giga-8 DC-EPG amplifier system. The experiment was conducted in a greenhouse at 26.5 ± 1 ◦C, 70 ± 10% RH, and L14:D10 photoperiod. Based on previous studies, probing behavior was continuously recorded for 5 h, and the 4-h effective records TABLE 1 | The electrical penetration graphs (EPG) of the cotton aphid Aphis gossypii and the respective correlated stylet penetration activities.


(which contained enough effective information for data analysis) from the beginning of the feeding test were analyzed using the EPG Stylet software (EPG Systems, Wageningen, Netherlands). All recorded signals were analyzed, including non-penetration period (i.e., the NP waveform indicating aphid walking and stylet not probing the host substrate), pathway phase (i.e., the C waveform indicating aphid stylet probing the host substrate to locate the feeding site), phloem phase (i.e., the E waveform, including two events: the E1 waveform showing salivation into phloem sieve elements; the E2 waveform showing ingestion of the phloem content), and xylem phase (i.e., the G waveform indicating ingestion of the xylem sap). In this study, there were eight types of EPG recordings, including the waveforms of NP, C, E1, E2, G, first E1, first E2, and E2 > 8 min (seen in **Table 1**). The waveform parameters of the first E1 waveform and the first E2 waveform indicated the duration of the first occurrence of E1 and E2, respectively; the waveform of E2 > 8 min indicated sustained phloem ingestion for more than 8 min (Kimmins and Tjallingii, 1985; Davis and Radcliffe, 2008).

#### RNA Preparation and Reverse Transcription

The newborn adults of A. gossypii sampled for the molecular test were randomly selected from the tested adult aphids used for the above mentioned EPG test. Once the newborn adults emerged, 20 of them were randomly collected from each growth incubator and mixed as one biological replicate, and there were three biological replicates for the treatments of ambient and elevated CO2, respectively. Total RNA was extracted from sampled newborn adult aphids by using the TRIzol <sup>R</sup> reagent (Invitrogen). The concentration and quality of samples were determined by using the NanoDropTM spectrophotometer (Thermo Scientific) and 1.5% agarose gel electrophoresis. The first-strand complementary cDNA templates were synthesized with 100 ng of total RNA by using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Reverse transcriptase reactions were performed in a 20 µl final volume reaction.

#### Real-Time PCR Analysis

Each cDNA product was diluted from 5× to 80× by diluting twice using RNase-free dH2O, in order to make the Ct value


TABLE 2 | The primers used for the qRT-PCR analysis of the related target genes of neuropeptide F (NPF), angiotensin converting enzyme (ACE), salivary proteins (C002a and C002b), salivary sheath protein (SHP), molting hormone (MH), and juvenile hormone (JHAMT and JHEH) of the cotton aphid A. gossypii.

to fall within the suitable range of 15–35 based on preliminary experiments. For fluorescence-based quantitative real-time PCR (qRT-PCR), 2 µl of cDNA dilution (100 ng/µl) and 0.2 µM of primers were used in 1× SYBR <sup>R</sup> Premix Ex Taq (TaKaRa) with the 7500 Real-Time PCR Detection System (Applied Biosystems), following the supplier's instructions. Reactions were performed in a 20 µl final volume. Specific primers for testing the genes were designed by Beacon DesignerTM 7.9 software, and the housekeeping gene RPL was used as the internal standard to analyze the expression levels of target genes, including appetite related genes [i.e., NPF and ACE], salivary protein genes [i.e., C002a, C002b and salivary sheath protein (SHP)], molting hormone (MH) gene (i.e., CYP314A1) and juvenile hormone genes (i.e., JHAMT and JHEH) of the cotton aphid A. gossypii, All the primers used for the qRT-PCR test are shown in **Table 2**. Quantification of the transcript levels of target genes was conducted by following the 2−11Ct normalization method. The expression levels of the internal control gene were examined in every PCR plate to eliminate systematic errors. Four biological replicates were made for each treatment in the qRT-PCR analysis, and each biological replicate contained three technical repeats.

#### Data Analysis

Statistical analysis of all data was performed by using the SPSS v.20.0 software (IBM Corporation, Armonk, NY, United States). One-way analyses of variance (ANOVAs) were used to analyze the effects of CO<sup>2</sup> levels on the foliar contents, on the insect life history parameters and feeding behavior, and on the relative transcript levels of the target genes. Also, the least significant difference (LSD) test was used to analyze the significant differences between the treatments of ambient and elevated CO<sup>2</sup> at P < 0.05. Survival data were calculated using the Kaplan–Meier survival curve and were compared using the log-rank test with a significance threshold of P < 0.05. Each experiment was compared with a control group, and all experiments were conducted independently at least three times.

# RESULTS

#### Effects of Elevated CO<sup>2</sup> on the Foliar Contents of Soluble Constituents of the Cotton Seedlings

The levels of CO<sup>2</sup> significantly affected the contents of foliar SPs (ie., SPs; F = 25.59, P ≤ 0.001; **Figure 1A**) and sucrose (ie., sucrose; F = 7.13, P ≤ 0.05; **Figure 1B**), whereas these levels failed to significantly affect the content of total FAAs (ie., FAA; F = 1.09, P ≥ 0.05; **Figure 1C**). The order of increase in SPs and sucrose was over 115% and 56% (**Figure 1**), respectively, in elevated CO<sup>2</sup> treatment when compared with ambient CO<sup>2</sup> treatment (P < 0.05; **Figures 1A,B**).

Moreover, CO<sup>2</sup> levels significantly affected the foliar serine (Ser) content (F = 13.54, P < 0.01), whereas these levels failed to significantly affect the contents of other FAAs (F ≤ 4.90, P ≥ 0.05; **Table 3**). Compared with ambient CO2, elevated CO<sup>2</sup> significantly decreased the foliar Ser content of cotton seedlings (−30.23%; **Figure 2**).

#### Effects of Elevated CO<sup>2</sup> on the Growth, Development, Fecundity, and Survival Rate of the Cotton Aphid A. gossypii

The levels of CO<sup>2</sup> failed to significantly affect the development duration of the first, second, and fourth instar nymphs, total nymphal stages (F ≤ 3.58, P ≥ 0.062), reproductive period (F = 0.07, P > 0.05), whereas these levels significantly affected

TABLE 3 | One-way analyses of variances (ANOVAs) for the effects of CO<sup>2</sup> levels (i.e., ambient vs. elevated) on the foliar contents of soluble constituents of cotton seedlings, and the transcript levels of the target genes related to growth, development, and fecundity of the cotton aphid A. gossypii fed on the fully expanded leaves excised from the 35- to 60-day-old cotton seedlings grown under ambient and elevated CO<sup>2</sup> conditions.


<sup>∗</sup>P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

the development duration of the third instar nymph (F = 4.07, P < 0.05), adult longevity (F = 4.95, P < 0.05), the whole life span (F = 5.02, P < 0.05), the fecundity (F = 4.23, P < 0.05), and the MRGR (F = 27.69, P < 0.001) of A. gossypii (**Table 4**).

Compared with ambient CO2, elevated CO<sup>2</sup> significantly shortened the development duration of the third instar nymph by 7.56% (P < 0.05) and significantly prolonged the adult longevity and whole life span of A. gossypii by 14.24% and

FIGURE 2 | Impacts of elevated CO<sup>2</sup> on the contents of different types of foliar free amino acids of cotton seedlings (Asterisks indicate a significant difference between the treatments of ambient and elevated CO<sup>2</sup> by the LSD test at P < 0.05).

TABLE 4 | Mean (±SE) values of the development indexes (including nymph duration, adult longevity, and whole life span), fecundity (including number of offsprings laid per adult and reproductive period), and mean relative growth rate (MRGR) of the cotton aphid A. gossypii fed on the fully expanded leaves excised from the 35- to 60-day-old cotton seedlings grown under ambient and elevated CO<sup>2</sup> conditions.


Asterisks indicate a significant difference between the treatments of ambient and elevated CO<sup>2</sup> by one-way ANOVAs at P < 0.05. Different lowercase letters indicate significant difference between the treatments of ambient and elevated CO<sup>2</sup> by the LSD test at P < 0.05.

11.26% (P < 0.05), respectively, and simultaneously enhanced the number of offsprings per adult and the MRGR of A. gossypii by 10.41% and 10.80%, respectively (P < 0.05; **Table 4**). The survival rate of A. gossypii from the newborn stage to the death of adult maintained under elevated CO<sup>2</sup> condition was significantly longer (P = 0.011), 24.42 ± 0.87 days, than that seen under ambient CO<sup>2</sup> condition 20.82 ± 0.98 days (**Figure 3**).

# Impacts of Elevated CO<sup>2</sup> on the Feeding Behavior of the Cotton Aphid A. gossypii

The EPG data were used to infer possible changes in feeding behavior of A. gossypii under ambient and elevated CO<sup>2</sup> conditions. The data analysis (**Table 5**) indicated that CO<sup>2</sup> levels significantly affected the frequency of G phase (F = 5.81, P < 0.05) and the mean time from the start of the EPG experiment to the first E1 waveform (F = 6.77, P < 0.05) and the first E2 waveform (F = 4.76, P < 0.05), whereas these levels failed to significantly affect the frequency of the other EPG waveforms (F ≤ 0.81, P ≥ 0.38) or the total duration of the EPG waveforms (F ≤ 2.89, P ≥ 0.10) of the cotton aphid A. gossypii.

In contrast to ambient CO2, elevated CO<sup>2</sup> significantly reduced the frequency of the G waveform by 75.99% (P < 0.05) and significantly shortened the time from the start of the EPG experiment to the E1 and E2 waveforms by 48.95% and 40.36%, respectively (P < 0.05; **Table 5**). Moreover, an increase in the total duration of the E2 (+33.12%) and E2 ≥ 8min (+29.58%) waveforms was found for the elevated CO<sup>2</sup> treatment in contrast to the ambient CO<sup>2</sup> treatment, respectively (P > 0.05; **Table 5**).

FIGURE 3 | Kaplan–Meier survival curves of Aphis gossypii fed on the cotton plant under different CO<sup>2</sup> levels (ambient vs. elevated) (The significant difference between the treatments of ambient and elevated CO<sup>2</sup> were obtained by log-rank test at P < 0.05).

TABLE 5 | Mean (±SE) values of the feeding behavior parameters of the cotton aphid A. gossypii fed on fully expanded leaves excised from the 35- to 60-day-old cotton seedlings grown under ambient and elevated CO<sup>2</sup> conditions.


NP, non-penetration period; C, stylet pathway activity; E1, saliva secretion to phloem tissues; E2, ingestion from phloem tissues; G, xylem ingestion; The first E, the first occurrence of E1; The first E2, the first occurrence of E2; E2 ≥ 8 min, sustained phloem ingestion for more than 8 min. Asterisks indicate a significant difference between the treatments of ambient and elevated CO<sup>2</sup> by one-way ANOVAs at P < 0.05.

### Impacts of Elevated CO<sup>2</sup> on the Expression of the Target Genes Related to Growth, Development, Reproduction, and Feeding of the Cotton Aphid A. gossypii

The levels of CO<sup>2</sup> significantly affected the expression levels of the appetite related genes of NPF (F = 10.65, P < 0.05) and ACE (F = 11.62, P < 0.05), the salivary protein genes of C002b (F = 7.89, P < 0.05) and SHP (F = 34.57, P < 0.01), and the MH gene of CYP314A1 (F = 9.32, P < 0.05), whereas these levels failed to significantly affect the expression levels of the salivary protein gene of C002a (F = 2.87, P > 0.05), the JH genes of JHAMT (F = 1.31, P > 0.05) and JHEH (F = 5.30, P > 0.05) in the cotton aphid A. gossypii fed on fully expanded leaves excised from the 35- to 60-day-old cotton

elevated CO<sup>2</sup> (Asterisks indicate a significant difference between the treatments of ambient and elevated CO<sup>2</sup> by the LSD test at P < 0.05).

seedlings grown under ambient and elevated CO<sup>2</sup> conditions (**Table 3**).

As compared with ambient CO2, elevated CO<sup>2</sup> significantly upregulated the relative transcript levels of the salivary protein genes of C002b and SHP by 20.80% and 111.85% and the appetite related genes of NPF and ACE by 34.27% and 22.66%, respectively (P < 0.05), simultaneously, downregulating the relative transcript level of the salivary protein gene of C002a by 31.80% (P > 0.05; **Figure 4**). Moreover, elevated CO<sup>2</sup> also upregulated the expression levels of the JH genes of JHAMT and JHEH by 11.20% and 39.64%, respectively (P > 0.05), simultaneously, significantly downregulating the expression levels of the MH gene of CYP314A1 by 13.60% (P < 0.05; **Figure 4**).

#### DISCUSSION

Currently, the global atmospheric CO<sup>2</sup> concentration continues to rise, standing now at 400 ppm and possibly reaching 800 ppm by the end of this century (Pachauri et al., 2014). As the main factor responsible for global warming, elevated CO<sup>2</sup> directly induces changes in plant growth, development, metabolism, and plant chemistry (Dader et al., 2016; Jiang et al., 2016); meanwhile, insects are sensitive to these environmental variations, which cause changes in their behavior, growth, development, fertility, and the occurrence of populations as a result of metabolic rate fluctuation (Sun et al., 2015, 2017; He et al., 2017). With the elevated CO<sup>2</sup> condition, Oehme et al. (2013), in their study, observed that the concentrations of fructose and glucose in spring wheat showed a significant increase, whereas the total amino acid concentration was not altered. These changes in plant chemistry positively affect the relative growth rate (RGR) of aphids. In this study, we found that elevated CO<sup>2</sup> had significant effects on the soluble nutrients of cotton, which, thereby, were beneficial to the performance of A. gossypii because of the bottom-up effects of the plant, which was in accordance with previous studies (Guo et al., 2013, 2014a). Moreover, the qPCR results also indicated that elevated CO<sup>2</sup> induced a certain degree of upregulation in JH transcription and a significant downregulation in MH transcription, whereas the transcription of genes related to appetite (NPF and ACE) and salivary proteins (C002b and SHP) was significantly upregulated under elevated CO2; all these molecular evidences determined here supported our findings well.

In general, elevated atmospheric CO<sup>2</sup> generally presents positive effects on foliar soluble nutrition of plants, especially in C<sup>3</sup> plants (Chen et al., 2005a; Wu et al., 2007; Guo et al., 2013). These alterations on the quality of plant host tissue can directly affect the performance of herbivorous insects. However, the response to elevated CO<sup>2</sup> varies between insects that have piercing and chewing mouthparts (Coll and Hughes, 2008; Sun et al., 2016). A recent meta-analysis examining the effects of elevated CO<sup>2</sup> on the life history traits of insects found that while the abundance of foliage feeders tends to decrease, phloem feeders on average tend to perform better under elevated CO<sup>2</sup> (Robinson et al., 2012). Generally, elevated CO<sup>2</sup> shows

Jiang et al. Aphid Ingestion Under Elevated CO<sup>2</sup>

negative effects on chewing insects with a decline in the foliar nitrogen content of host plants; as a recent study on the cotton bollworm, Helicoverpa armigera, showed that larval durations were significantly prolonged by elevated CO2, additionally, female pupal weight, fecundity, and total population size under elevated CO<sup>2</sup> were lower than ambient CO<sup>2</sup> (Liu et al., 2017). In contrast, aphids, as a kind of phloem feeders, are considered the only feeding guild that positively responds to elevated CO2. Our previous study has shown that, according to four successive generation data, elevated CO<sup>2</sup> significantly increases fresh body weight, fecundity, and population abundance of A. gossypii (Jiang et al., 2016). In regard to Rhopalosiphum padi reared on Hordeum vulgare, which was maintained under elevated CO2, there was a significant increase in aphid abundance and intrinsic rate of population increase; however, there were no statistically significant effects on fecundity and development time of the aphid, such beneficial performance of R. padi results from plant biochemical response; under elevated CO<sup>2</sup> (Ryan et al., 2015). However, piercing-sucking insect seems to have a species-specific response; in terms of studying the population responses of five aphid species to elevated CO2, one species showed an increase (Myzus persicae), one showed a decrease (A. pisum), and the other three remained unaffected (Aphis nerii, A. oenotherae, Aulacorthum solani) (Hughes and Bazzaz, 2001). Bemisia tabaci under elevated CO<sup>2</sup> treatment had a neutral response with no alterations in its life span, sex ratio, and fecundity (Sun et al., 2011). So, as CO<sup>2</sup> is the substrate for plant photosynthesis, elevated CO<sup>2</sup> may directly alter physiological and biochemical processes in plants. Furthermore, this indirectly affects insect physiological metabolism by changing plant nutrition and plant defense (Todgham and Stillman, 2013; Guo et al., 2014a,b).

According to our study on the expression of key genes of the JH and MH pathways, it indicated that elevated CO<sup>2</sup> slightly decreased MH transcription and mildly increased JH transcription. The JH and the main ecdysteroid (20E), known as highly versatile hormones, regulate many aspects of insect physiology, such as development, growth, reproduction, and aging (Riddiford, 1994; Flatt et al., 2008). Recent research suggested that JH was also involved in the regulation of final insect size and growth rates (Mirth et al., 2014). Studies on the tobacco hornworm Manduca sexta showed that a decline in circulating JH initiates the first step in the hormonal cascade that begins with the attainment of critical weight, and ends, after a terminal growth period (TGP), with the rise in circulating ecdysone that stops body growth (Fain and Riddiford, 1975; Cymborowski et al., 1982). Intriguingly, similar result was observed in D. melanogaster; additionally, it was demonstrated that the effect of JH on growth rate and final body size was mediated by ecdysone synthesis via the regulation of the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway by JH, without affecting the developmental timing (Colombani et al., 2005; Mirth et al., 2014). Our research speculated that JH might be cooperating with MH on regulating MRGR in A. gossypii through the key effector of the IIS pathway, like the results obtained of Mirth et al. (2014). These data were in line with previous studies that speculated a cross talk between JH and IIS in A. gossypii.

In this study, for the purpose of matching the higher growth rate observed under elevated CO2, the A. gossypii aphids needed to increase their food intake to obtain enough nutrition for growth. So, our EPG recordings showed that the A. gossypii aphids had higher efficiency of stylet penetration under elevated CO<sup>2</sup> compared with ambient CO2. In our previous study, one reason for this result might be that the increase in leaf turgor and soluble constituents of the leaf favored ingestion in A. gossypii (Jiang et al., 2016). Sun et al. (2015) also provided the evidence for increased ingestion under elevated CO2; that is, originally, A. pisum infestation triggered the abscisic acid (ABA) signaling pathway to decrease the stomatal apertures of Medicago truncatula, which consequently decreased leaf transpiration and helped to maintain the leaf water potential. Furthermore, elevated CO<sup>2</sup> upregulates an ABA-independent enzyme, carbonic anhydrase, which led to a further decrease in the stomatal aperture of aphid-infested plants. Thus, the effects of elevated CO<sup>2</sup> accentuated stomatal closure and synergistically increased leaf turgor in plants, resulting in enhanced aphid feeding. The second case might be that elevated CO<sup>2</sup> alters plant resistance. For piercing-sucking insects, the results obtained by Sun et al. (2013) indicated that the JA-regulated defense against M. persicae was more effective than the SA-regulated defense in Arabidopsis and that elevated CO<sup>2</sup> tends to enhance the ineffective SA signaling pathway and reduce the effective JA signaling pathway against aphids. Later, similar studies in regard to A. pisum reared on M. truncatula also demonstrated that elevated CO<sup>2</sup> enhances the SA-dependent defense pathway and suppresses the JA/ethylene-dependent defense pathway (Guo et al., 2014b, 2016). A recent research on elevated plant resistance in response to CO<sup>2</sup> indicated that the heat shock protein 90 plays a critical role in plant resistance against the aphid under elevated CO<sup>2</sup> (Sun et al., 2017). Taken together, elevated CO<sup>2</sup> increased host water potential and decreased plant resistance against piercing-sucking insects, which favored ingestion and growth of A. gossypii.

With respect to the appetite of A. gossypii, our results indicated that elevated CO<sup>2</sup> significantly increased aphid appetite and further regulated the feeding behavior. As known, SHP and C002 play important roles in the stylet probing phase and phloem feeding phase, respectively, in piercing-sucking insects. Insect stylet movement is accompanied by the secretion of gel saliva, which forms a salivary flange on the epidermis and an enveloping salivary sheath in the apoplast, both of which may provide stability, lubrication, and protection during feeding, while the latter also seals the plasma membrane at stylet penetration sites (Will and van Bel, 2006; Will et al., 2012). Abdellatef et al. (2015) showed that silencing the expression of SHP causes transgenerational feeding suppression in Sitobion avenae, additionally, reduced SHP expression correlates with a decline in growth, reproduction, and survival rates. The A. pisum aphids inject the protein C002 into the host plant during feeding to increase the acquisition of phloem sap. Knockdown of C002 in this aphid causes a decrease in the time that it spends in contact with the phloem sap (Mutti et al., 2008). In the current experiment, our results have revealed for the first time that the increased expression of the salivary protein genes was induced by the high expression of the insect appetite related genes and that this then leads to the higher efficiency of ingestion under elevated CO<sup>2</sup> condition. But, in the present study, still only little was known about how elevated CO<sup>2</sup> impacts the appetite of aphids; it needs further studies in the future. Overall, elevated CO<sup>2</sup> could increase plant phloem nutrition, which, in turn, favored the fitness of aphids via enhanced ingestion due to improved appetite. All the supporting evidences might point to the fact that the rising CO<sup>2</sup> concentration increases the risk of pest control under the conditions of climate change in the future.

#### AUTHOR CONTRIBUTIONS

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All the authors listed have made a substantial, direct, and intellectual contribution to the work and have approved its

#### REFERENCES


publication. SJ and FC designed the study. SJ, YD, YqL, and YmL performed the experiments. SJ and FC analyzed the data. SJ wrote the manuscript. SJ, FC, MP, SF, and MB reviewed and polished the manuscript.

# FUNDING

This research was supported by the National Key Research and Development Program of China (2017YFD0200400), the Fundamental Research Funds for the Central Universities (KYZ201818), the National Nature Science Foundation of China (31871963 and 31272051), the Basic Scientific Research Project in Colleges and Universities (2018), and the Qing-Lan Project of Jiangsu Province of China.

of neuropeptide F (NPF) in penaeid shrimp. J. Exp. Biol. 214, 1386–1396. doi: 10.1242/jeb.053173


bacteriocytes to favor aphid population growth under elevated CO2. Global Change Biol. 19, 3210–3223. doi: 10.1111/gcb.12260


biological variables. New Phytol. 194, 321–336. doi: 10.1111/j.1469-8137.2012. 04074.x


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Jiang, Dai, Lu, Fan, Liu, Bodlah, Parajulee and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fphys-09-01444 November 8, 2018 Time: 16:41 # 12

# Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctomaculata

Jing Lü<sup>1</sup>† , Shimin Chen<sup>1</sup>† , Mujuan Guo<sup>1</sup>† , Cuiyi Ye<sup>1</sup> , Baoli Qiu<sup>1</sup> , Jianhui Wu<sup>1</sup> , Chunxiao Yang<sup>2</sup> \* and Huipeng Pan<sup>1</sup> \*

<sup>1</sup> Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, Department of Entomology, South China Agricultural University, Guangzhou, China, <sup>2</sup> State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, China

#### Edited by:

Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China

#### Reviewed by:

Yingjun Cui, University of Missouri, United States Weihua Ma, Huazhong Agricultural University, China

#### \*Correspondence:

Chunxiao Yang yangchunxiao@scau.edu.cn Huipeng Pan panhuipeng@scau.edu.cn †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 06 September 2018 Accepted: 25 October 2018 Published: 14 November 2018

#### Citation:

Lü J, Chen S, Guo M, Ye C, Qiu B, Wu J, Yang C and Pan H (2018) Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctomaculata. Front. Physiol. 9:1614. doi: 10.3389/fphys.2018.01614 Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin, GAPDH, RPL13, RPL6, RPL32, RPS18, and ATPB, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the 1Ct method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata.

Keywords: Henosepilachna vigintioctopunctata, RT-qPCR analysis, reference gene, RefFinder, geNorm

# INTRODUCTION

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a frequently used technique for gene expression studies on account of its high specificity, high sensitivity, high throughput, and low cost (Hellemans and Vandesompele, 2014). However, many factors relevant to biological and technical variations, for instances, RNA isolation, integrity, purity; reverse transcription; PCR efficiency, can affect the precision of RT-qPCR analysis (Bustin et al., 2005, 2009). Generally, RT-qPCR involves standardization to the expression of a battery of appropriately stable reference genes concurrent. In spite of reference gene transcript levels should be stably

**192**

expressed in a serious of different biological or experimental conditions, previous studies have shown that many frequently used reference genes differ observably in different treatments (Li et al., 2013; Yang et al., 2014, 2015a,b,c, 2016, 2018; Pan et al., 2015a,b). Therefore, a systematic and customized study for each tested species is recommended for identifying appropriate reference genes.

Henosepilachna vigintioctopunctata (Fabricius) (Coleoptera: Coccinellidae) is one of the most serious insect pests in Asian countries (Ghosh and Senapati, 2001; Shinogi et al., 2005; Venkatesha, 2006; Zhou et al., 2015). H. vigintioctopunctata colonizes many different species of plants, for example, solanaceous plants such as eggplant, tomato, potato, and pepper; cucurbitaceous plants such as cucumber, white gourd, and loofah. In addition, it attacks many weeds, such as the black nightshade, winter cherry, thorn apple, and tobacco (Pang and Mao, 1979). The destructive potential of H. vigintioctopunctata is high at both the adult and larval stages, leading to up to 60% loss of fruit production (Sharma et al., 2012; Kawazu, 2014).

In China, H. vigintioctopunctata is widely distributed from Hainan Province in the south to Heilongjiang Province in the north and from Gansu Province in the west to Shanghai City in the east (Pang and Mao, 1979). Recently, because of climate warming, development of trade, and expansion of the cultivated area for protected vegetables, the food for H. vigintioctopunctata is constantly increasing throughout the year, and the occurrence and damage of H. vigintioctopunctata have become significant (Li et al., 2006; Zhou et al., 2015; Wang et al., 2017). Recently, we sequenced the transcriptomes of H. vigintioctopunctata at different developmental stages (unpublished data) and obtained a huge amount of genes involved in the development, energy metabolism, and reproduction. Recently, RNA interference (RNAi) has been used widely in the study of functional genomics, resulting in the development of new modes of action insecticides for insect pest management (Baum et al., 2007; Bellés, 2010; Burand and Hunter, 2013; Scott et al., 2013; Zhang et al., 2017). To identify novel target genes for controlling H. vigintioctopunctata, accurate gene expression


In this study, to discern the solidly expressed reference genes of H. vigintioctopunctata for RT-qPCR investigation under four experimental conditions (developmental stage, tissue, temperature, and host plant), 7 most commonly used reference genes, namely, actin (Actin), ribosomal protein L13 (RPL13), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L6 (RPL6), ribosomal protein L32 (RPL32), ribosomal protein S18 (RPS18), and vacuolar-type H+-ATPase subunit B (ATPB), were selected from the transcriptomes of H. vigintioctopunctata. The expression stability of each candidate reference gene was assessed under 2 biotic (developmental stage and tissue) and 2 abiotic (temperature and host plant) conditions by using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the 1Ct method, and BestKeeper). Finally, jim lovell (lov) and T-box transcription factor (TBX1) were used as the target genes to verify our findings. The results will be useful in improving the accuracy of RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata.

# MATERIALS AND METHODS

#### Insects

In April 2018, the adults of H. vigintioctopunctata were collected from Solanum nigrum (L.) in Guangzhou City, Guangdong Province, China, and reared in the incubator at 25 ± 0.5◦C temperature, 14L:10D photoperiod, and 80% relative humidity in petri dishes by using S. nigrum and S. melongena leaves.

#### Sample Treatment and Collection Biotic Factors

All stages of H. vigintioctopunctata were sampled: eggs, four larval instars, pupae, and female and male adults (collected on the first day of each stage). The number of sampled individuals for each replicate across the different developmental stage was


as follows: 20 eggs for the egg stage; 10 individuals for the first instar; 5 individuals for the second instar; 3 individuals for the third instar; 1 individual for the fourth instar; 1 pupa for the pupal stage; and 1 male or female individual for the adult male or female stage. Different body tissues, namely, the Malpighian tubule, fat body, midgut, and cuticle, were dissected from the fourth instar larvae, about 40 individuals were dissected for each replicate. The tissue samples were placed in RNAlater <sup>R</sup> (Thermo Fisher Scientific Inc., United Stats) and stored at 4◦C before total RNA isolation.

#### Abiotic Factors

For the temperature treatment, 5 s instars were exposed to 8, 25, and 35◦C for 3 h. For the host plant treatment, 2 third instars larvae were collected as one sample that reared on S. nigrum and eggplant.

fphys-09-01614 November 12, 2018 Time: 14:0 # 3

Each experiment was replicated 3 times. All the samples, except the ones used for the host plant treatment, were collected from the colony reared on S. nigrum. All the samples, except the tissue ones, were placed in 1.5 ml RNA free centrifuge tubes, rapidly frozen in liquid nitrogen, and stored at −80◦C before the total RNA extraction.

#### Total RNA Extraction and cDNA Synthesis

fphys-09-01614 November 12, 2018 Time: 14:0 # 4

The total RNAs of egg, Malpighian tubule, and fat body were isolated using TRIzol reagent (Invitrogen, United States), in line with our previously described methods (Pan et al., 2015a). Total RNAs from the other samples were isolated using a HiPure Total RNA Micro Kit (Magen, China), according to the manufacturer's instructions. A NanoDrop One<sup>C</sup> spectrophotometer (Thermo Fisher Scientific, Waltham, MA United States) was used to ascertain the RNA concentration. The total RNA was dissolved in 20–100 µl of ddH2O, and the concentrations were as follows: 494.3 ± 16.7 ng/µl [mean ± standard error of the mean (SEM)] for the eggs, 427.6 ± 28.6 ng/µl for the first instars, 683.9 ± 79.8 ng/µl for the second instars, 567.1 ± 38.7 ng/µl for the third instars, 619.8 ± 61.9 ng/µl for the fourth instars, 818.6 ± 59.1 ng/µl for the pupae, 760.2 ± 36.6 ng/µl for the male adults, 763.4 ± 78.9 ng/µl for the female adults, 677.5 ± 68.0 ng/µl for the cuticles, 565.3 ± 38.1 ng/µl for the fat body, 870.3 ± 22.6 ng/µl for the midguts, 483.8 ± 74.9 ng/µl for the Malpighian tubules, 468.4 ± 6.8 ng/µl for the secondinstar under 15◦C, 683.9 ± 79.8 ng/µl for the second-instar under 25◦C, 356.1 ± 7.9 ng/µl for the second-instar under 35◦C, and 422.1 ± 68.4 ng/µl for the third-instar under eggplant. The OD260/280 value of all samples was 1.9– 2.1. The PrimeScript RT kit (containing gDNA Eraser, Perfect Real Time, TaKaRa, China) was used for preparing the first-strand cDNA for gene expression investigation. The cDNA was diluted 10-fold for the pursuant RT-qPCR experiments.

#### Primer Design and Gene Cloning

In our study, 7 candidate reference genes that are most frequently used in RT-qPCR investigations were assessed (**Table 1**).

shown in terms of the Cq-value for each experimental condition. (A) Development stage, (B) Tissue, (C) Temperature, (D) Host plant.

The primers were designed based on the PrimerQuest Tool<sup>1</sup> , according to the sequences obtained from our recently sequenced transcriptomes for H. vigintioctopunctata (unpublished data).

The PCR reaction system and parameters were used according to our previous study (Yang et al., 2014). Amplicons of the expected lengths were purified using the TIANgel Midi Purification Kit (Tiangen, China), and subcloned into the pClone007 Blunt vector before transformation into Escherichia coli DH5α competent cells (Tsingke, China) for sequencing by Tsingke company. The reference genes were confirmed using sequence analysis.

#### RT-qPCR Analysis

fphys-09-01614 November 12, 2018 Time: 14:0 # 5

The RT-qPCR reactions and program were conducted, the melting curve and standard curve for each reference gene were generated according to our previous study (Yang et al., 2014, 2015a,b,c, 2016, 2018). The homologous RT-qPCR efficiencies (E) were calculated according to the equation: E = (10[−1/slope] −1) × 100.

<sup>1</sup>https://sg.idtdna.com/Primerquest/Home/Index

# Determination the Expression Stability of Reference Genes

The reference gene expression stability was assessed using RefFinder<sup>2</sup> , which integrates four different analytical tools, NormFinder (Andersen et al., 2004), geNorm (Vandesompele et al., 2002), the 1C<sup>t</sup> method (Silver et al., 2006), and BestKeeper (Pfaffl et al., 2004). The optimal number of reference genes for target gene expression normalization was decided by pairwise variation (Vn/Vn+1). A Vn/Vn+<sup>1</sup> cutoff value of 0.15 indicates the additional 1 more reference gene is not necessary, i.e., the starting n reference genes are enough for the target gene normalization; the V-values were calculated using geNorm (Vandesompele et al., 2002).

#### Determination of Gene Expression Levels on the Basis of Different Reference Genes

Lov is a nuclear protein has roles in various larval and adult behaviors (Bjorum et al., 2013). TBX1 plays a vital role in the upgrowth of Drosophila heart (Griffin et al., 2000). The stability

<sup>2</sup>http://150.216.56.64/referencegene.php

of these reference genes was investigated using lov and TBX1 as the target genes. The primer sequences of these two target genes were as follows: lov, forward (5<sup>0</sup> -CTCCCGCCCAACACTTTAT-3 0 ) and reverse (5<sup>0</sup> -TCGCTTTGCGGTAGTAGATG-3<sup>0</sup> ); TBX1, forward (5<sup>0</sup> -GAAACACCTCTGGGACGAAT-3<sup>0</sup> ) and reverse (5<sup>0</sup> - TCGGAGTGCAAGTCTAAACC-3<sup>0</sup> ). Lov and TBX1 expression levels in different tissues were computed on the basis of normalization to the 2 most- and 2 least-stable candidates. The relative gene expression of lov and TBX1was computed using the 2−11Ct method (Livak and Schmittgen, 2001). Oneway analysis of variance was used to detect significances in lov and TBX1 expression levels among different tissues (SPSS 17.0).

#### RESULTS

#### Reference Gene Expression Profiles

All of these candidate reference genes were expressed in H. vigintioctopunctata and intuitional with a single amplicon of the expected size for each gene (**Supplementary Figure S1**). Gene-specific amplification of all reference genes was verified by a single peak in the melting curve analysis (**Figure 1**). The PCR efficiency ranged between 92 and 100% (**Table 1**). The standard curve for each reference gene is also provided (**Supplementary Figure S2**).

The quantification cycle (Cq) values for all the reference genes under the 4 experimental conditions ranged from 20 to 29. RPS18 and RPL13 were the most abundant reference genes, whereas RPL32 and RPL6 were the least expressed ones (**Figure 2**).

#### Stability of the Reference Genes Under Different Experimental Conditions

According to RefFinder, across different development stages, the comprehensive reference gene rankings from the best to the least stable were as follows: RPL13, RPS18, ATPB, GAPDH, RPL6, RPL32, and Actin (**Figure 3A**). For the tissue treatment, the comprehensive reference gene rankings were as follows: RPL13, RPS18, RPL6, RPL32, ATPB, GAPDH, and Actin (**Figure 3B**). Under different temperature conditions, the integrated reference

TABLE 2 | Stability of 7 candidate reference gene expression in H. vigintioctomaculata under different experimental conditions calculated by the 4 different analytical tools geNorm, Normfinder, BestKeeper, and the 1Ct method, respectively.


<sup>∗</sup>Candidate reference gene.

gene rankings were as follows: RPL13, RPS18, RPL6, ATPB, Actin, RPL32, and GAPDH (**Figure 3C**). For the host plant treatment, the comprehensive reference gene rankings were as follows: RPL13, RPS18, ATPB, RPL32, RPL6, GAPDH, and Actin (**Figure 3D**). The expression stability value for each gene was also computed using Normfinder, geNorm, the 1Ct method, and BestKeeper under each experimental condition (**Table 2**).

#### Recommended Reference Genes Depended on geNorm

For each of four different experimental conditions, the initial V-value < 0.15 were all emerged at V2/3, respectively, suggesting 2 reference genes were enough for the target gene normalization. By coincidence, RPL13 and RPS18 were the best stable reference genes for each experimental condition, respectively (**Figure 4**).

#### Validation of the Selected Reference Genes

The relative expression levels of lov and TBX1 were used to validate the reference genes among the different tissues. The Lov expression patterns were similar, however, lov expression was about 15- and 99-fold higher in the fat body than in the Malpighian tubule when normalized to the 2 most- and leaststable reference genes, respectively (**Figure 5**). In contrast, TBX1 expression patterns were inconsistent among the different tissues when normalized to the 2 most- and least-stable reference genes. Different degrees of gene expression were obtained in each tissue under the 2 normalization conditions (**Figure 6**).

# DISCUSSION

Previous studies have demonstrated there is no "universal" reference gene applicable for various test conditions, even for the same insect species. For example, five papers have been published for the reference gene selection of the whitefly, Bemisia tabaci, a notorious and invasive insect species, in the past 5 years (Li et al., 2013; Su et al., 2013; Collins et al., 2014; Liang et al., 2014; Dai et al., 2017). It is pivotal to select and validate the reference genes expression stability under various experimental conditions precedence using them for normalizing gene expression.

We sequenced the transcriptomes for the developmental stages of H. vigintioctopunctata (unpublished data). In the future, we will use RNA interference to investigate the gene functions, and RT-qPCR will be widely applied for evaluating the gene expression changes. However, the reference genes have not been previously selected and validated in this insect pest. Therefore, in this study, seven frequently used reference genes were picked out and their stability was investigated using five software programs under four experimental conditions. The results displayed that the reference gene transcript levels vary with the experimental conditions. Thus, it is no doubt that the expression profiles of the target genes will show substantive variations relying on the reference gene and experimental treatments (Yang et al., 2016).

Our results certified that 5 computational methods yielded disparate stability rankings for the seven reference genes (**Table 2** and **Figure 3**). Coincidentally, the two most-suited references genes were the same under each experimental condition (**Figure 3**). Recently, researchers have been more receptive to the use of multiple reference genes to replace a single normalizer

in the RT-qPCR analysis (Vandesompele et al., 2002; Li et al., 2013; Yang et al., 2014, 2015a,b,c, 2016, 2018; Pan et al., 2015a,b), and the pairwise variations suggest that 2 reference genes were adequate for normalization under each experimental condition. Thus, RPL13 and RPS18 are suitable for use as the reference genes under each of the four experimental conditions.

In order to further validate the reference genes in H. vigintioctopunctata, the relative gene expression levels of lov and TBX1 were evaluated in different tissues. Our results showed that lov expression was sevenfold higher in the Malpighian tubule than in the fat body when normalized to the 2 least stable reference genes GAPDH and Actin than when normalized to the 2 most stable reference ones RPL13 and RPS18 (**Figure 5**). In addition, TBX1 expression patterns were inconsistent in the different tissues when normalized to the two best- and least-stable reference genes. The expression level was

different in each tissue under the two normalization conditions (**Figure 6**). These results indicated that the unreasonable use of reference genes may give rise to inaccurate results for target genes. Therefore, selection and validation of the best reference genes are crucial for determining the veracity of the expression results. Thus, our study provides a more strict way to normalize RT-qPCR data in H. vigintioctopunctata, which will boost our comprehension of the target gene functions in this serious pest.

# CONCLUSION

We identified stable reference genes for RT-qPCR analysis of H. vigintioctopunctata. RPL13 and RPS18 combinations are proposed as the reference genes for each experimental condition. This study on behalf of the initial step to establish the standardized RT-qPCR analyses of H. vigintioctopunctata and could contribute to the in-depth functional genomic dissection of H. vigintioctopunctata.

#### AUTHOR CONTRIBUTIONS

fphys-09-01614 November 12, 2018 Time: 14:0 # 10

HP and CY conceived and designed research. JL, SC, MG, and CY conducted experiments. BQ and JW contributed reagents. HP and JL analyzed data. HP and CY wrote the manuscript.

# FUNDING

This research was supported by the National Key R&D Program of China (2017YFD0200900), project supported by GDUPS

#### REFERENCES


(2017), and a start-up fund from the South China Agricultural University. The granting agencies have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01614/full#supplementary-material


L. and a wild host plant Solanum nigrum L. J. Ecol. Entomol. 110, 2084–2091. doi: 10.1093/jee/tox207


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Lü, Chen, Guo, Ye, Qiu, Wu, Yang and Pan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Corrigendum: Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctopunctata

#### Edited and reviewed by:

Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China

#### \*Correspondence:

Chunxiao Yang yangchunxiao@scau.edu.cn Huipeng Pan panhuipeng@scau.edu.cn

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 12 June 2019 Accepted: 15 July 2019 Published: 25 July 2019

#### Citation:

Lü J, Chen S, Guo M, Ye C, Qiu B, Wu J, Yang C and Pan H (2019) Corrigendum: Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctopunctata. Front. Physiol. 10:981. doi: 10.3389/fphys.2019.00981 Jing Lü1†, Shimin Chen1†, Mujuan Guo1†, Cuiyi Ye<sup>1</sup> , Baoli Qiu<sup>1</sup> , Jianhui Wu<sup>1</sup> , Chunxiao Yang<sup>2</sup> \* and Huipeng Pan<sup>1</sup> \*

<sup>1</sup> Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, Department of Entomology, South China Agricultural University, Guangzhou, China, <sup>2</sup> State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, China

Keywords: Henosepilachna vigintioctopunctata, RT-qPCR analysis, reference gene, RefFinder, geNorm

#### **A Corrigendum on**

#### **Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctopunctata**

by Lü, J., Chen, S., Guo, M., Ye, C., Qiu, B., Wu, J., et al. (2018). Front. Physiol. 9:1614. doi: 10.3389/fphys.2018.01614

In the original article, there was an error in the title. The title "Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctomaculata" should be "Selection and Validation of Reference Genes for RT-qPCR Analysis of the Ladybird Beetle Henosepilachna vigintioctopunctata."

The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.

Copyright © 2019 Lü, Chen, Guo, Ye, Qiu, Wu, Yang and Pan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Methoprene-Tolerant (Met) Is Indispensable for Larval Metamorphosis and Female Reproduction in the Cotton Bollworm Helicoverpa armigera

Long Ma<sup>1</sup> , Wanna Zhang<sup>2</sup> \*, Chen Liu<sup>3</sup> , Lin Chen<sup>3</sup> , Yang Xu<sup>2</sup> , Haijun Xiao<sup>2</sup> and Gemei Liang<sup>3</sup> \*

<sup>1</sup> Jiangxi Key Laboratory of Bioprocess Engineering, College of Life Sciences, Jiangxi Science and Technology Normal University, Nanchang, China, <sup>2</sup> Institute of Entomology, Jiangxi Agricultural University, Nanchang, China, <sup>3</sup> State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

#### Edited by:

Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China

#### Reviewed by:

Jalal Jalali Sendi, University of Guilan, Iran Cheolho Sim, Baylor University, United States Baiming Liu, Tianjin Institute of Plant Protection, China

> \*Correspondence: Gemei Liang gmliang@ippcaas.cn Wanna Zhang zhangwanna880210@yeah.net

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 22 January 2018 Accepted: 25 October 2018 Published: 15 November 2018

#### Citation:

Ma L, Zhang W, Liu C, Chen L, Xu Y, Xiao H and Liang G (2018) Methoprene-Tolerant (Met) Is Indispensable for Larval Metamorphosis and Female Reproduction in the Cotton Bollworm Helicoverpa armigera. Front. Physiol. 9:1601. doi: 10.3389/fphys.2018.01601 Juvenile hormone (JH) represses larval metamorphosis and induces adult reproduction in insects. Methoprene-tolerant (Met) is identified as an intranuclear receptor that mediates JH actions. In the present study, we characterized a Met from the severe agricultural pest, Helicoverpa armigera, namely HaMet. In the larval stage, HaMet is predominantly expressed in the epidermis and midgut, and is upregulated before each molting, whereas in adults HaMet is maximally expressed in the ovary, testis, and fat body. The immunofluorescence assay revealed that HaMet was distributed in the longitudinal and circular muscle layers of midgut in larvae, whereas in the ovary of female adults, HaMet was localized in the nucleus of the oolemma. Knockdown of HaMet in final-instar larvae shortened the time of pupation, induced abnormal pupation, and dampened pupation rate. In female adults, HaMet depletion severely suppressed the transcription of Vitellogenin (Vg) and Vitellogenin Receptor (VgR), disrupted the Vg accumulation in fat body and the yolk protein uptake in oocytes, and finally led to an impaired fecundity. Our findings therefore confirmed that HaMet acted as a nuclear receptor of JH and played an essential role in larval metamorphosis, vitellogenesis, and oocyte maturation.

Keywords: Helicoverpa armigera, methoprene-tolerant, juvenile hormone, vitellogenesis, reproduction

#### INTRODUCTION

Juvenile hormones (JHs) are sesquiterpenoid compounds that are synthesized and secreted by corpora allata. In insects, JHs play crucial roles in controlling metamorphosis, reproductive development, and some aspects of mating behavior in both male and female insects (Riddiford, 2008). In the larval stage, high titer of JH maintains larval molting and prevents metamorphosis, whereas a drop or absence of JH titer in the final instar allows insect metamorphosis with the rise of 20-hydroxyecdysone (20E) levels (Nijhout, 1994). Being an important hormone-regulating insect development, 20E has been well characterized by later studies (Siaussat et al., 2007; Jing et al., 2016). Conversely, the molecular mechanism addressing the action of JH has remained enigmatic until

**204**

recently, when the transcription factor methoprene-tolerant (Met) was identified as the potential JH receptor (Jindra et al., 2013).

Methoprene-tolerant protein has a typical basic helix-loophelix Per-Arnt-Sim (bHLH-PAS) domain. This protein is found to be capable of binding to both natural JH-III and a mimicker of JH (methoprene) with high affinity (Ashok et al., 1998; Miura et al., 2005; Charles et al., 2011). Since the first characterization of Met in D. melanogaster (Wilson and Fabian, 1986), homologs of Met have been identified from a broad range of insect species, including Aedes aegypti (Zhu et al., 2010), Tribolium castaneum (Konopova and Jindra, 2007), Bombyx mori (Kayukawa et al., 2012), Pyrrhocoris apterus (Konopova et al., 2011), and Nilaparvata lugens (Lin et al., 2015). Accumulating evidences suggest that the anti-metamorphic function of JH is correlated with Met. In the beetle T. castaneum, knocking down TcMet disrupted larval-pupal ecdysis and induced precocious adult development upon partial ecdysis, suggesting that Met was involved in anti-metamorphic JH signal transduction (Konopova and Jindra, 2007). In P. apterus, silencing Met at the penultimateinstar nymphs caused the development of adult features instead of molting to the final nymphal instar (Konopova et al., 2011). Moreover, studies in A. aegypti, T. castaneum, and B. mori have revealed that the heterodimerization of Met with a steroid receptor coactivator was required for the JH-induced transcription of JH target genes (Li et al., 2011; Zhang et al., 2011). Thus, Met was regarded as the JH intranuclear receptor (Jindra et al., 2013). Subsequently, Krüppel-homolog 1 (Kr-h1), a transcription factor with a DNA-binding domain consisting of eight zinc fingers, was reported to work on the downstream of Met in the JH signal pathway (Belles and Santos, 2014).

Vitellogenesis, a key process in female reproduction in insects (Sappington and Raikhel, 1998), is under the control of JH and/or ecdysone. These two hormones are the main inducers of vitellogenin (Vg) synthesis from the fat body and Vg uptake into the developing oocyte via vitellogenin receptor (VgR)-mediated endocytosis (Raikhel et al., 2005; Roy et al., 2018). Recent studies in mosquitos and locusts have revealed that JH regulated the expression of Vg based on its receptor Met (Zou et al., 2013; Guo et al., 2014). In these insects, Met is indispensable for egg production and Vg expression, and knockdown of Met resulted in the retardation of ovarian development (Zou et al., 2013) and the lower egg production (Li et al., 2011). Further study in the cockroach, Diploptera punctata, showed that knockdown of Met resulted in an arrest of oocyte development and Vg gene expression (Marchal et al., 2014). The similar function of Met in reproduction was detected in T. castaneum (Minakuchi et al., 2009), A. aegypti (Shin et al., 2012), and P. apterus (Konopova et al., 2011).

Methoprene-tolerant is undoubtedly an important transcription factor playing a crucial part in insect development and reproduction. However, the function and mechanism of Met regulation is not well understood owing to its complex roles in the JH pathway. In the present study, we obtained a full-length Met from the cotton bollworm, Helicoverpa armigera (HaMet). The expression patterns of HaMet were first investigated. An immunofluorescence assay was performed to determine the distribution of HaMet protein in the larval midgut and the adult ovary. Furthermore, the functions of HaMet in larvae-pupae transition and female reproduction were investigated by RNA interference (RNAi). Our study provides new insights into how Met functions in larval metamorphosis and female reproduction.

# MATERIALS AND METHODS

#### Insects and Tissues Sampling

The H. armigera used in this study were reared in the laboratory within a controlled environment of 27 ± 2 ◦C, 75 ± 10% RH, and a photoperiod of 14: 10 h (L:D). The H. armigera strain was divided into five instars according to their ecdysis times. The larvae were first reared on an artificial diet in the 24-well plate, and then transferred into 25 ml glass tubes containing an artificial diet at the 5th instar for pupation (one larva per tube). After emergence, the adults were placed in cages (30 cm × 60 cm × 30 cm) for oviposition and supplied with 10% sugar solution.

To examine the developmental expression profiles of HaMet, individuals were collected from egg, larvae (1st, 2nd, 3rd, 4th, and 5th instar), pupae, and adults. Samples were prepared at intervals of 1 day during larval stages, 2 days from pupation to adult emergence, and 2 days for adults. For tissue expression analysis, tissues (including head, epidermis, midgut, hemolymph, and fat body) were dissected from the 5th-instar larvae, and tissues from adults (including ovary, testis, head, epidermis, midgut, hemolymph, and fat body) were stripped in phosphatebuffered saline (PBS). All the samples were frozen immediately in liquid nitrogen and stored at −80◦C until RNA isolation. Four biological replicates containing three to 50 individuals were prepared for each experiment.

# RNA Isolation and cDNA Synthesis

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United States). The integrity of each RNA sample was checked with 1% agarose gel electrophoresis, and the RNA quantity was determined using a NanoVue spectrophotometer (GE-Healthcare, Germany). After digestion of residual genomic DNA with DNase I (Promega, Madison, United States), 2 µg total RNA was reverse transcribed in 20 µl reaction mixtures using the Fast Quant RT kit (TIANGEN, Beijing, China) according to the manufacturer's instruction. The synthesized first-strand cDNAs were stored at −20◦C.

# Molecular Cloning and Bioinformatics Analysis

A 768 bp cDNA fragment encoding the partial of HaMet was first amplified, and the rapid amplification of cDNA ends (RACE) was used to obtain the full-length cDNA. In brief, the 3<sup>0</sup> - and 5 0 -RACE cDNA templates were synthesized using the SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, United States). Gene-specific primers (GSP) were designed on the basis of the HaMet fragment. The PCR amplification was conducted by means of touchdown with the GSP and the

universal primer mixture (UPM). Then the 5<sup>0</sup> - and 3<sup>0</sup> -RACE products were purified and sequenced. After sequence splicing, the open reading frame (ORF) of HaMet was further confirmed by PCR amplification.

The BLASTx algorithm was employed to run the similarity searches. The tools available in the ExPASy proteomics server<sup>1</sup> were used to determine the putative molecular weight and isoelectric point. Moreover, the SMART program<sup>2</sup> was used to identify the conserved domains. The percent identity of the amino acid sequences was calculated from single pairwise alignments using Vector NTI. Finally, a phylogenetic tree was constructed with MEGA 7.1 using the neighbor-joining method with a p-distance model and a pairwise deletion of gaps. Bootstrap values of tree branches were assessed by resampling amino acid positions 1000 times.

#### Analysis of HaMet Expression by qRT-PCR

The qRT-PCR analysis was performed using SuperReal PreMix Plus (SYBR Green) (Tiangen Biotech, Beijing, China) on ABI 7500 Fast Real-Time system. Each reaction was performed in a 20 µl volume containing 1 µl of cDNA, 10 µl of SuperReal PreMix (2×), 0.6 µl of each primer (10 µM), 0.4 µl of ROX Reference Dye (50×), and 7.4 µl of ddH2O. The qRT-PCR program consisted of one cycle of 95◦C for 1 min, followed by 40 cycles of 95◦C for 5 sec, 60◦C for 15 sec, and a melt curve stage. Two house-keeping genes, Ef-a (accession no. XM\_021329970) and ß-actin (accession no. EU527017), were used as reference genes to normalize the target gene expression and to correct for sample-to-sample variation. The comparative 2−11C t method was used for the normalization of gene expression. To ensure reliability, each reaction for each sample was performed in triplicate with four biological replicates. Negative control without template was included in each reaction. The primer sequences of the genes were listed in **Supplementary Table S1**.

#### Immunofluorescence Staining of HaMet

The midgut from 5th-instar larvae and the ovary tubules from 2 day-old female adults were dissected, respectively, and prefixed in 4% paraformaldehyde for 30 min at 4◦C, followed by infusion overnight in a solution of 20% sucrose in 0.1 M PBS plus 0.1% Triton X-100 (PBST) at 4◦C. The tissues were then embedded in O.C.T. compound (optimum cutting temperature compound, Sakura, United States). Ultrathin sections of 12 µm thickness were cut, gathered on Super Frost Plus slides (Menzel-Gläser, Braunschweig, Germany), and dried at room temperature for 1 h. These sections were post-fixed in 4% paraformaldehyde for 30 min at 4◦C. The sections were then rinsed thrice in PBST, and blocked with 5% normal goat serum (NGS)-PBST at room temperature for 1h. After washing thrice with PBST, the slides were incubated at 4◦C overnight with HaMet antiserum (kindly provided by Pro. XiaoFan Zhao, Shandong University) diluted at 1:1000 in 5% NGS. The sides were then rinsed thrice in PBST, incubated for 2 h at room temperature with goat anti-rabbit

<sup>1</sup>http://www.expasy.org

<sup>2</sup>http://smart.emblheidelberg.de/

Alexa Fluor 488 dye (1:500 in TNB Buffer), and treated with DAPI (Beyotime Biotechnology, China) at room temperature for 10 min. Finally, the sections were observed with a confocal laser scanning microscope (Zeiss, Oberkochen, Germany) after washing with PBST thrice for 10 min each.

#### RNAi Experiment

Double-stranded RNA (dsRNA) was prepared using a HiScribeTM T7 Transcription Kit (New England BioLabs, Ipswich, MA, United States) from the PMD18 plasmid (Takara, Dalian, China) containing a 710 bp fragment of HaMet (from 872 to 1581), and the synthesized dsRNA was injected into larvae or female adults as described previously (Zhang et al., 2016b). A segment of GFP (green fluorescent protein) was used to produce dsRNA for GFP (dsGFP) as control.

To investigate the function of HaMet in larval development, 1 µl solution (3 µg/µl) of dsRNA targeting HaMet (dsHaMet) was injected into the abdomen of 5th-instar larva. The control individuals were treated with the same dose of dsGFP. Each treatment contained 72 individuals, then five individuals were randomly selected for qRT-PCR analysis at every 24 h after injection and the pupation time (from 5th instar 0 h to pupa) and the pupation rate of the remaining samples were recorded.

To examine the effect of HaMet depletion on fecundity, newly emerged female adults were injected in the abdomen with dsHaMet (5 µg), and the controls were injected with an equivalent dose of dsGFP. The injection point was sealed with geoline immediately. Subsequently, ten individuals were randomly selected at 24 h, 48 h, 72 h, and 96 h after injection, respectively; then the tissues of fat body and ovary were dissected to investigate the RNAi efficiency. In addition, in each treatment, the ovarian phenotypes of female moths were observed. In brief, the ovaries from dsRNA-treated individuals (3 days after treatment) were dissected in PBS and photographed with a stereomicroscope (Olympus SZX16, Tokyo, Japan), and the numbers of follicles at different developmental stages were recorded as described previously (Zhang et al., 2015). The observations were conducted for 15 females in each treatment. Meanwhile, 50 treated females were chosen for an oviposition bioassay. Each female was paired with two untreated males in one plastic cup (8 cm in diameter, 10 cm high). The plastic cups covered with one layer of 10 cm × 10 cm gauze were kept under the same condition as mentioned. The cotton wicks were placed on the gauze to supply 10% sugar solution, and both the gauze and the cotton wick were changed daily to count the number of eggs laid. All the experiments were performed thrice.

#### Hormone Treatments

For hormone mimic treatments, methoprene (Sigma-Aldrich, St. Louis, United States) and 20E (Sigma-Aldrich, St. Louis, United States) were dissolved in acetone. 20E or methoprene was topically applied to the dorsal abdomen of 5th-instar larvae (2 µg/larva), and controls were treated with the same dose of acetone.

When RNAi and JH mimic treatments were to be combined, newly emerged moths after dsRNA treatment were further treated with methoprene (5 µg per moth) 1 day after the dsRNA treatment. At 2 days after the methoprene application, the tissue of fat body was dissected and subjected to qRT-PCR and western blot analysis.

#### Western Blot Analysis

fphys-09-01601 November 14, 2018 Time: 13:26 # 4

The tissue of fat body was homogenized in lysis buffer (8 M urea, 4% chaps, 40 mM Tris–HCl, 5 mM EDTA, 1 mM PMSF and 10 mM DTT, pH 8.0) containing a mixture of protease inhibitors (Roche, Switzerland). The concentration of crude protein was determined by a Bio-Rad protein assay using bovine serum albumin (BSA) as the standard. The samples were then diluted with loading buffer to obtain an equal amount of total protein. After the proteins were separated by 12% (w/v) SDS–PAGE, the samples were transferred onto nitrocellulose (NC) membrane blotting filters at 100 V for 1 h at 4◦C. The membranes were then blocked with 5% (w/v) skimmed milk in PBST at 4◦C overnight. After washing thrice with PBST, the blocked membrane was incubated with H. armigera β-actin antibody (1:2000 dilution) and HaVg antibody (1: 4000 dilution) (Zhang et al., 2016b) for 1 h at room temperature.

No. EHJ75902.1), and Operophtera brumata (ObMet, Accession No. KOB7445.1). Domains: bHLH (Blue), PAS-A (Red), PAS-B (Green), and PAC (Black) are boxed. (B) Phylogenetic analysis of Met homologs from Diptera, Lepidoptera, Coleoptera, Dsphnia, Orthoptera, Blattaria, and Hemiptera species. Bootstrap values (%) were marked above the tree branches.

After three washes with PBST, the membrane was incubated for 1 h at room temperature with goat anti-rabbit IgG HRP-linked secondary antibody (Sigma, St. Louis, United States) at 1:10,000 dilution with PBST. The immunoreactivity was visualized using an enhanced electrochemiluminescence detection kit (TransGen, Beijing, China) and photographed by Image Quant LAS4000 mini (GE-Healthcare, Germany).

#### Data Analysis

fphys-09-01601 November 14, 2018 Time: 13:26 # 5

All the data in this study were presented as means ± SE. Significant differences were determined by Student'st-test or oneway analysis of variance (ANOVA) followed by a least significant difference test (LSD) for mean comparison. All statistical analyses were performed with SAS 9.20 software (SAS Institute, Cary, NC, United States).

# RESULTS

#### Cloning and Sequence Analysis of HaMet

The 5<sup>0</sup> - and 3<sup>0</sup> -end sequences of HaMet were obtained using the RACE technology. After assembling, a full-length transcript encoding HaMet was obtained (Accession no. KJ825895). This transcript is 2511 bp, including a 351 bp 5<sup>0</sup> -untranslated region (UTR), a 579 bp 3<sup>0</sup> -UTR, and 1581 bp ORF. The latter encodes

#### a 526-amino acid protein with a predicted molecular mass of 59.88 kDa and an estimated isoelectric point of 7.1.

The predicted protein sequence of HaMet was aligned with homologs from B. mori, Danaus plexippus, and Operophtera brumata. The result showed that the HaMet protein exhibited typical bHLH, PAS-A, PAS-B, and PAC (PAS C terminal motif) domains (**Figure 1A**). Phylogenetic analysis was constructed on the basis of the protein sequences of Met homologs from various insect species. As expected, HaMet is clustered with Mets from lepidopteran B. mori, D. plexippus, and O. brumata, forming an orthologous group of Met1. The result indicated that they originated from the same ancestors and shared conserved functions. However, HaMet appears to be more closely related to the Dipteran homologs than other lepidopteran Met homologs of the Met2 group (**Figure 1B**).

#### Tissue and Temporal Expression of HaMet During H. armigera Development

The expression pattern of HaMet in different stages was determined by qRT-PCR analysis (**Figure 2A**). The results showed that HaMet was detected throughout the entire life cycle, and the transcription level of HaMet fluctuated during developmental stages. HaMet was highly expressed in the embryonic stage (egg), sharply increased before each molting in the initial four larval instars, decreased at the end of the 5th instar,

difference between specimens (P < 0.05).

and maintained at a low level during the pupal stage. In the adult stage, HaMet had a notably higher expression in females than in males (**Figure 2A**).

The tissue expression profiles of HaMet in larvae and adults were also examined. In the larval stage, HaMet was highly expressed in epidermis and midgut, whereas low expression was observed in head, fat body, and hemolymph. In adults, the HaMet transcripts were maximally expressed in the ovary, testis, and fat body, and relatively low expression levels were found in other tissues (**Figure 2B**).

#### Immunostaining of HaMet Protein in Midgut and Ovary

In the preparation for HaMet localization, the specificity of HaMet antiserum was verified by western blot analysis. The result showed that the staining of crude ovarian extracts with HaMet antiserum showed a single band at approximately 60 kDa which is of the predicted size of HaMet protein (**Supplementary Figure S1**).

Immunofluorescence microscopy revealed that HaMet was localized in the longitudinal and circular muscle layers of the midgut in larvae (**Figure 3A**). In contrast in the ovary of female adults, the labeled cells were highly enriched in the oocyte membrane, and HaMet was localized in the cell nucleus (**Figure 3B** and **Supplementary Figure S2**).

#### Knockdown of HaMet Induced Larval Metamorphosis

The metamorphic action of larval-pupal transition was regulated by JH under the control of ecdysteroids. To examine whether

merge is the overlapped images of green and blue. Lu, midgut lumen; LM, larval midgut; OM, oolemma.

HaMet was regulated by JH or 20E, methoprene (JH analog) and 20E were applied to the 5th-instar larvae. The results showed that HaMet expression was upregulated by 3.78 times after methoprene treatment; however, HaMet expression was not induced by 20E or acetone (**Figure 4A**). These results indicated that JH analog (JHA) was the regulator of HaMet expression.

The function of HaMet in larval development was further investigated by RNAi of HaMet in the 5th-instar larvae. The result showed that 62% of the HaMet transcripts were silenced as compared with those in the dsGFP-treated group at 48 h postinjection. At 72 h and 96 h post-treatment, the transcription levels of HaMet were decreased by 70 and 55% in dsHaMet-treated individuals relative to the controls, respectively (**Figure 4B**).

Caste-differentiation bioassays were used to examine the effect of HaMet knockdown in terms of phenotype and development time. The pupation time in dsGFP-treated larvae was 48 h longer than that in dsHaMet-treated individuals (**Figure 4C**), indicating that knockdown of HaMet resulted in an early pupation. Besides, phenotypic analysis revealed that dsHaMet treatment caused the abnormal pupation in approximately 35% of the treated larvae, such as the persistence of larval-pupal intermediates (**Figure 4D**). The pupation rate in the dsHaMet-treated group declined to 26.68%, which indicated a 47.06% decrease compared

FIGURE 5 | Knockdown of HaMet in 1-day-old female adults of H. armigera. The relative expression levels of HaMet (A), HaVg (B), and HaVgR (C) after dsGFP or dsHaMet injection examined at 24, 48, 72, and 96 h after the treatment. Error bars indicate SE. <sup>∗</sup>Denotes P < 0.05, ∗∗denotes P < 0.01 compared with the respective dsGFP control (Student's t-test).

with that in control (P < 0.01). Taken together, these results demonstrated that HaMet functioned in suppressing larval metamorphosis.

# Met Is Required for Vg Synthesis and Uptake in Female Reproduction

In insects, vitellogenin (Vg) is primarily synthesized in fat body to meet the nutrient requirement for egg development. To explore the participation of HaMet in Vg synthesis, we knocked down the expression of HaMet in newly emerged female adults, and subsequently examined Vg transcription in fat body of treated females. The results showed that the expressions of HaMet in the fat body were reduced by 62.7, 87.6, 60.0, and 43.8% at 24, 48, 72, and 96 h post-treatment, respectively (**Figure 5A**). Meanwhile, knockdown of HaMet caused the significant reduction in Vg expression. As shown in **Figure 5B**, injection of dsHaMet reduced the HaVg mRNA levels to 40.5% (24 h post-treatment), 22% (48 h), 28% (72 h), and 30% (96 h) of its normal levels in fat body. Besides, the expressions of HaVgR in the ovary were also significantly reduced after HaMet knockdown, and an average of 50% decrease in HaVgR mRNA levels was observed (**Figure 5C**).

To evaluate the effect of HaMet silencing on oviposition and ovary development, the ovaries from both the treated and control female adults were observed 3 days after the treatment, and the number of eggs was recorded. The ovarian morphology showed that knockdown of HaMet resulted in an apparent decrease in yolk protein deposition, causing a small degree of yolk uptake in oocytes (**Figure 6A**). The number of follicles, particularly the mature follicles, was significantly fewer in HaMet-silenced moths than that in controls (**Figures 6A,B**) (P < 0.001). Moreover, dsHaMet-treated moths exhibited a 44% decline in

sum of daily egg number per female. Error bars indicate SE. <sup>∗</sup>Denotes P < 0.05, ∗∗denotes P < 0.01 (Student's t-test).

oviposition compared with those treated with dsGFP (P < 0.001) (**Figure 6C**).

To further explore the role of Met in JH-mediated vitellogenesis, JHA was applied to the HaMet-silenced moths (**Figure 7**). The results showed that the expression of HaVg and the content of Vg protein failed to recover in the HaMet-silenced moths, indicating that the capacity of methoprene to induce HaVg expression in the fat body was completely blocked by HaMet knockdown.

#### DISCUSSION

In insects, JH has primary roles in repressing metamorphosis and stimulating several aspects of reproduction (Riddiford, 2008). Multiple studies have confirmed that Met is an essential receptor in the JH signaling pathway (Konopova and Jindra, 2007). Here, we identified and characterized HaMet from H. armigera. Similar to other known Met genes, HaMet contains an HLH structure with two variably spaced PAS domains (A and B), and a hallmark of the bHLH-PAS protein family (Ashok et al., 1998). Phylogenetic analysis revealed that HaMet was clustered with three lepidopteran Mets into a separate clade (Met1 group) that was closely related to Dipteran Mets.

The temporal expression pattern showed that HaMet was sharply increased before each molting in the larval stage, and maintained low level during the pupal stage. This stage-specific expression profile is overall consistent with the changes of the in vivo JH titer in larvae (Zhang et al., 2017), which supports the previous findings showing that JH exerts its anti-metamorphic effect through its receptor Met (Jindra et al., 2013; Zhao et al., 2014). The similar expression pattern of Met was reported in T. castaneum (Parthasarathy et al., 2008). For tissue expression, HaMet is highly transcribed in larval midgut, and immunolocalization confirms the distribution of HaMet protein in different layers of longitudinal and circular muscle cells in larval midgut. Coincidentally, the previous study in H. armigera has documented that Met depletion could repress the suppressive effect of JH on midgut remodeling (Zhao et al., 2014). Similarly, in D. melanogaster, Met protein is present in the midgut imaginal cells during larval-pupal transition (Pursley et al., 2000), and the overexpression of Met leads to the precocious and enhanced programmed cell death (Liu et al., 2009). Interestingly, Rahman et al. (2017) described that JHs were also synthesized by the gut in adult Drosophila, and this gut-specific JH activity is synthesized by and acts on the intestinal stem cell and enteroblast populations, regulating their survival and cellular growth through the JH receptors Gce/Met. Consequently, we presume that the midgut-abundant HaMet acts to promote tissue remodeling during the larvalpupal transition in H. armigera. However, this function may not be applicable to all insects. In T. castaneum, the depletion of TcMet did not completely block the remodeling of midgut tissue (Parthasarathy et al., 2008). Therefore, how Met acts in midgut remodeling and whether Met alone is sufficient to promote the midgut metamorphosis needs further study.

Many studies have documented that JH inhibits the larvalpupal transition via Met and the downstream transcription factor Kr-h1 in both holometabolous and hemimetabolous species (Hiruma and Riddiford, 2010; Riddiford, 2012; Jindra et al., 2013; Zhao et al., 2014). Our present study showed that depletion of HaMet significantly reduced the pupation time, which supported its function of anti-metamorphosis. Similarly, disruption of Met by RNAi led to precocious metamorphosis in the linden bug Pyrrhocoris apterus (Konopova et al., 2011). Moreover, in T. castaneum, depletion of TcMet at the end of the last larval instar resulted in the premature upregulation of the adult-specifier factor TcE93, and led to a direct transformation from larva to the adult form, bypassing the pupal stage (Ureña et al., 2016). Our RNAi study showed that HaMet knockdown caused the malformation individuals with the complex characters of larvae and pupae, and the deficiency in pupation was largely attributed to the fact that the treated individuals were incapable of fully forming puparium. Other studies in T. castaneum and B. mori reported that Met depletion in the late larval stage induced a certain level of mortality and complications in ecdysis (Konopova and Jindra, 2007; Parthasarathy et al., 2008; Guo et al., 2012). We presume that the early pupation or the malformation

occurring during larval-pupal transition may be attributed to the metabolic deficiencies caused by the Insulin signaling pathway. Actually, the involvement of Met in the insulin signaling pathway has been reported in T. castaneum and Blattella germanica, where Met depletion impaired the expression of insulin-like-peptides (Sheng et al., 2011; Lozano and Belles, 2014).

In addition to its role during metamorphosis, JH also plays a primary role in regulating Vg expression in fat body, and is crucial for the maintenance of follicle patency, uptake of Vg, and choriogenesis (Ramaswamy et al., 1997). The previous study in H. armigera has reported the tight correlation between the JH titer and ovariole development (Zhang et al., 2016a). The expression of Vg, the major yolk protein in oocyte, is regulated by JH via its receptor Met, which has been documented in A. aegypti (Raikhel et al., 2005; Zou et al., 2013), P. apterus (Smykal et al., 2014), and T. castaneum (Parthasarathy et al., 2010). Other studies in Cimex lectularius (Gujar and Palli, 2016) and Nilaparvata lugens (Lin et al., 2015) also confirmed that Met was required for Vg expression and ovarian development. Our RNAi experiment revealed that knockdown of HaMet resulted in the significant decline of Vg expression in the fat body, the reduced expression of VgR in the ovary, and the atrophied ovaries with less yolk protein deposition. In other words, the declined transcription of Vg from the HaMet-depleted fat body blocked the Vg synthesis. Meanwhile, the depleted VgR expression in the ovary blocked the uptake of yolk proteins, both of which caused the atrophied ovaries and ultimately impaired female fecundity. Interestingly, the cellular immunolocalization revealed that HaMet was abundantly expressed in the nucleus of oocyte membrane, which corroborated the hypothesis that HaMet acted as the JH-nuclear receptor regulating the uptake of yolk protein during the oocyte formation. Indeed, oogenesis involves the internalization of proteins and lipids from the circulating hemolymph and this internalization process is thought to be mediated in large part by the membraneassociated receptors, and the immunoreactivity showed that most B. mori VgRs are localized in the inner oocyte membrane (Han et al., 2017). Taken together, we presume that the abundant expression of HaMet at oolemma is involved in Vg uptake by oocytes.

Juvenile hormone has been proved to play a major role in inducing vitellogenesis in many hemimetabolous insects (Glinka and Wyatt, 1996; Comas et al., 2001). In our study, JHA application induces Vg expression in the control group; however, JHA application failed to recover Vg expression in the HaMet-depleted moths. These results correspond to data from locusts and P. apterus, where methoprene treatment on Met-depleted individuals failed to restore the defective phenotypes (Smykal et al., 2014; Song et al., 2014). Taken together, these studies indicated that Met mediated the induction of Vg expression in a JH-dependent manner.

The cotton bollworm, H. armigera, is a major agricultural pest worldwide. Transgenic crops that produce Bacillus thuringiensis (Bt) Cry toxins have become an important tool against this pest. Currently, to counter the increasing pest resistance to transgenic cotton-expressing Bt toxin, plant-mediated RNAi provides a new strategy. Transgenic plants producing dsRNA targeting the crucial genes in insect reproduction and metamorphosis were designed to control agricultural pests (Xiong et al., 2013; Tian et al., 2015; Luo et al., 2017). Our results help to unveil the complex roles of HaMet in insect development and reproduction, and highlight Met as a target for suppression of lepidopteran pests.

# AUTHOR CONTRIBUTIONS

GL, LM, and WZ conceived and designed the experimental plan. LM, WZ, CL, LC, and YX performed the experiments. LM, WZ, and HX processed and analyzed the data. GL, LM, WZ, and HX wrote and edited the manuscript.

# FUNDING

This research was supported by the National Key R&D Program of China (2017YFD0201900), Key Project for Breeding Genetically Modified Organisms (Grant No. 2016ZX08011–002), Science and Technology Program of Department of Education of Jiangxi Province (GJJ170660 and GJJ160354), and Natural Science Foundation of Jiangxi Province (20171BAB214028 and 20171BAB214004).

# ACKNOWLEDGMENTS

We thank Professor Xiaofan Zhao for kindly providing HaMet antiserum.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01601/full#supplementary-material

FIGURE S1 | Western blot analysis of the specificity of HaMet antibody.

FIGURE S2 | Immunocytochemical localization of HaMet protein in oocyte section using colloidal gold labeling. Black spots represent the immunostained HaMet protein. (A) The negative control used the serum supernatant from an uninfected healthy rabbit as the secondary antibody in immunochemistry and (B) the enlarged image of oocytes section. (C) Cross section through an oocyte shows the strongly immunostained HaMet in oolemma. (D) Enlarged images of oocyte section reveal the heavy labeling of HaMet protein in the oolemma (arrow). The secondary antibody was anti-rabbit IgG conjugated with 10 nm colloidal gold granules at a dilution of 1:20.

TABLE S1 | Primers used in the experiment.

#### REFERENCES

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Ma, Zhang, Liu, Chen, Xu, Xiao and Liang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fphys-09-01601 November 14, 2018 Time: 13:26 # 12

# Identification and Characterization of Three New Cytochrome P450 Genes and the Use of RNA Interference to Evaluate Their Roles in Antioxidant Defense in Apis cerana cerana Fabricius

#### Edited by:

Sylvia Anton, Institut National de la Recherche Agronomique (INRA), France

#### Reviewed by:

Zhaojiang Guo, Chinese Academy of Agricultural Sciences, China Antonio Biondi, Università degli Studi di Catania, Italy

#### \*Correspondence:

Weixing Zhang 1308054313@qq.com Baohua Xu bhxu@sdau.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 07 June 2018 Accepted: 25 October 2018 Published: 15 November 2018

#### Citation:

Zhang W, Chen W, Li Z, Ma L, Yu J, Wang H, Liu Z and Xu B (2018) Identification and Characterization of Three New Cytochrome P450 Genes and the Use of RNA Interference to Evaluate Their Roles in Antioxidant Defense in Apis cerana cerana Fabricius. Front. Physiol. 9:1608. doi: 10.3389/fphys.2018.01608 Weixing Zhang\*, Wenfeng Chen, Zhenfang Li, Lanting Ma, Jing Yu, Hongfang Wang, Zhenguo Liu and Baohua Xu\*

College of Animal Science and Technology, Shandong Agricultural University, Tai'an, China

Cytochrome P450s play critical roles in maintaining redox homeostasis and protecting organisms from the accumulation of toxic reactive oxygen species (ROS). The biochemical functions of the P450 family have essentially been associated with the metabolism of xenobiotics. Here, we sequenced and characterized three P450 genes, AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5, from Apis cerana cerana Fabricius; these genes play a critical role in maintaining biodiversity. Quantitative PCR (qPCR) analysis indicated that the three genes were all predominantly expressed in the epidermis (EP), followed by the brain (BR) and midgut (MG). In addition, the highest expression levels were detected in the dark-eyed pupae and adult stages. The three genes were induced by temperature (4◦C and 44◦C), heavy metals (CdCl<sup>2</sup> and HgCl2), pesticides (DDV, deltamethrin, and paraquat) and UV treatments. Furthermore, Western blot analysis indicated that the protein expression levels could be induced by some abiotic stressors, a result that complements the qPCR results. We analyzed the silencing of these three genes and found that silencing these genes enhanced the enzymatic activities of peroxidase (POD) and catalase (CAT). Additionally, we investigated the expression of other antioxidant genes and found that some were upregulated, while others were downregulated, suggesting that the upregulated genes may be involved in compensating for the silencing of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5. Our findings suggest that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 may play very significant roles in the antioxidant defense against damage caused by ROS.

Keywords: Apis cerana cerana Fabricius, cytochrome P450s, RNA interference, abiotic stresses, antioxidant enzyme

# INTRODUCTION

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As social insects and pollinators of flowering plants, Chinese honeybees (Apis cerana cerana Fabricius, A. c. cerana) play an important role in the balance between agricultural economic development and regional ecology. Compared with Apis mellifera (A. mellifera), A. c. cerana has acquired some incomparable advantages through the history of evolution. A. c. cerana has a strong resistance to mites, an acute sense of smell, and can forage more widely of pollens, which are irreplaceable (Oldroyd and Wongsiri, 2006). Nevertheless, the population of A. c. cerana has been reported to have severely declined, which has been referred to as colony collapse disorder (CCD), likely due to various abiotic stresses that exist in the environment, such as extreme changes in temperature and widely used insecticides that may generate reactive oxygen species (ROS). Therefore, understanding the antioxidant system and its molecular mechanisms of defense against ROS damage has become a primary focus of research.

Excessive ROS can lead to lipid peroxidation and protein damage, which cause cell apoptosis and the loss of enzymatic activity, respectively (Green and Reed, 1998; Stadtman and Levine, 2003). Sperm storage was also affected by ROS in A. mellifera (Collins et al., 2004). Organisms protect themselves through a range of antioxidant enzymes, such as catalase (CAT), glutathione-S-transferases (GSTs), superoxide dismutase (SOD), peroxidases (POD), glutathione peroxidase (GPX), and cytochrome P450s (P450s) (Dubovskiy et al., 2008; Matteis et al., 2012).

P450s, a supergene family, are widely distributed in almost all living organisms and play crucial roles in insect biology and physiology (Feyereisen, 1999). P450s are classified into different clades on the basis of their evolutionary relationships (Claudianos et al., 2006; Moktali et al., 2012). In insects, P450s belong to four clades: the mitochondrial P450s (Mit P450s), CYP2 clades, CYP3 clades (including CYP6 and CYP9), and CYP4 clades. The Mit P450s and CYP2 clades have been implicated in essential roles in hormone biosynthesis (Gilbert, 2004; Semak et al., 2008; Sangar et al., 2010). Meanwhile, the CYP3 and CYP4 clades are largely responsible for abiotic stresses, such as pesticide metabolism, detoxifying functions, and chemical communication (Yu et al., 1984; Claudianos et al., 2006; Musasia et al., 2013).

Previous studies concerning the roles of CYP3 P450s have focused on their functions in the environmental response and detoxification in insects. In the pyrethroid-resistant Anopheles gambiae, CYP6Z1 (belonging to the CYP3 clade) is overexpressed (Nikou et al., 2003). Similarly, in Helicoverpa zea, CYP321A1, a member of the CYP3 clade, can metabolize cypermethrin (Sasabe et al., 2004). Meanwhile, CYP3A enzymes play a crucial role in preventing the bioaccumulation of xenobiotics substances (drugs, environmental pollutants, and fungal products) that enter the body and metabolize endogenous substrates, such as sex hormones, in steroidogenic tissues (Celander et al., 2000; Danielson, 2002; Hegelund and Celander, 2003).

In insects, the CYP4 clade, a highly diversified group of enzymes, are involved in both pesticide metabolism and chemical communication (Scott et al., 1994; Claudianos et al., 2006; Kirischian and Wilson, 2012). For example, some CYP4 clade genes are over expressed in several pesticide-resistant insects (Culex pipiens and Diabrotica virgifera virgifera), and CYP4G8 is over expressed in pyrethroid-resistant strains of Helicoverpa armigera (Pittendrigh et al., 1997; Scharf et al., 2001; Shen et al., 2003). Moreover, the expression of CYP4G11 was significantly induced by external factors such as temperature challenges, UV radiation, insecticide treatment, and the expression patterns under oxidative stress, suggesting that CYP4G11 may be involved in protecting honeybees from oxidative injury (Shi et al., 2013). These studies demonstrate that the CYP3 and CYP4 clades play key roles in protecting organisms from ROS damage.

Although the functions of the P450s have been investigated in other species, there is limited knowledge regarding the P450s in honeybees, particularly in A. c. cerana. The above discussion demonstrates that the P450s play key roles in the response to oxidative stress in most species, and we predict that the P450s may also be involved in antioxidant defense in A. c. cerana. To gain insight into the roles of the P450s, we isolated and characterized three genes (AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5) from A. c. cerana and analyzed the expression patterns of these genes in different tissues and different developmental stages. We also investigated the transcripts of these three genes when exposed to various oxidative stresses, including different temperatures (4 and 44◦C), heavy metals (HgCl<sup>2</sup> and CdCl2), pesticides (DDV, deltamethrin, and paraquat), and ultraviolet light (UV). Moreover, we used RNAi technology to knockdown AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5. We also investigated the activity of two antioxidant enzymes (POD and CAT) and examined the expression levels of other antioxidant genes. A very broad conclusion can be drawn from our results, with some considerable degree of certainty: AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 might play crucial roles in the response to oxidative stress in A. c. cerana.

# MATERIALS AND METHODS

#### Laboratory Feeding of Honeybees and Experimental Design

Animal housing facilities and handling protocols were approved by the Animal Welfare and Health Committee of Shandong Agricultural University. Honeybees were collected from the experimental apiary of Shandong Agricultural University (Tai'an, China). Honeybees of different development stages, including larvae (L1–L5), pupae [pre-pupal phase (Pp), white-eyed pupae (Pw), brown-eyed pupae (Pb), and dark-eyed, dark pigmented thorax pupae (Pbd)] (de F Michelette and Soares, 1993), and newly emerged worker bees (less than 12 h of age), were obtained from five healthy hives. Newly emerged workers were collected at 15 days after being labeled with paint. The brain (BR), midgut (MG), muscle (MS), and epidermis (EP) of 15-day postemergence adults (n = 90; 15/group) were dissected on ice, frozen immediately in liquid nitrogen, and stored at −80◦C until ready for use. Adult bees (post-emergence age: 15 days) were collected in wooden cages (dimensions of 10 cm × 7 cm × 8 cm), which were maintained in an incubator (33 ± 1 ◦C, 60 ± 10% relative humidity, darkness). Cages were divided into nine groups

(n = 60/group). Groups 1 and 2 were fed with a sucrose solution containing HgCl<sup>2</sup> and CdCl2, respectively. Group 3 was subjected to UV light. Groups 4 and 5 were treated with different temperatures. In addition, groups 6–8 were treated with one of three different pesticides (DDV, deltamethrin, and paraquat). Group 9 served as the negative control. The abiotic stress conditions for each experimental group are shown in **Supplementary Table S1**. The final effective concentrations of the pesticides were determined according to the manufacturer's instructions. All bee samples were immediately frozen in liquid nitrogen at the appropriate time points and were stored at −80◦C until ready for use. All experiments were performed with at least six biological replicates.

# Primers and PCR Amplification Conditions

All sequences of the PCR primers and the amplification conditions are listed in **Tables 1**, **2**, respectively. In this study, all primers used for quantitative PCR (qPCR) were designed based on the principle of quantitative primer design. The E value and R 2 value of each qPCR primer pair are listed in **Table 1**. All primer pairs were synthesized by the Sangon Biotechnological Company (Shanghai, China).

#### Total RNA Isolation, cDNA Synthesis, and DNA Isolation

Total RNA was extracted from samples using the E.Z.N.A. Total RNA Kit II (OMEGA, United States), according to the manufacturer's instructions. The purity and quality of the RNA were controlled by using a spectrophotometer and were estimated by electrophoresis. The PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan) was used to generate first-strand cDNA, according to the manufacturer's instructions.

#### Cloning the cDNA Sequences of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5

To obtain the open reading frame (ORF) sequences of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5, special primers (**Table 1**) were designed and synthesized based on the conserved regions of the genes from A. mellifera, Apis florea, and Apis dorsata. The cDNA of newly emerged workers was used to clone the ORFs of these three genes. The PCR reaction used 1 µL cDNA as a template, 12.5 µL 2× Es Taq MasterMix (Dye) (CWBIO, Beijing, China), 1 µL forward primer, 1 µL reverse primer, and 9.5 µL ddH2O in a 25-µl volume with the following cycling

TABLE 1 | Primers for ORF subcloning, qPCR analysis, and dsRNA primers sequence of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 genes.


Underlined sequences in primers for dsRNA production are added adaptors containing T7 polymerase promoters.

#### TABLE 2 | PCR amplification conditions.

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<sup>∗</sup>Primers for dsRNA productions.

conditions: initial denaturation program (94◦C for 10 min), followed by 35 cycles of 94◦C for 40 s, N◦C for 40 s, 72◦C for M s (N: the advisable annealing temperature of the primers; M: determined by the length of the gene sequences), and 72◦C for 10 min. All PCR products were separated by electrophoresis and purified using a Gel Extraction Kit (Solarbio, China), cloned into pEASY-T1 vectors (TransGen, China), and transformed into competent Escherichia coli DH5 α (E. coli DH5 α) cells for sequencing (Sangon, China).

#### Bioinformatics Analysis and Phylogenetic Tree Construction

DNAman version 5.2.2 (Lynnon Biosoft, Canada) was used to search for the ORFs of the three genes and to predict the theoretical isoelectric points (PIs) and molecular weights (MWs) of the protein products. Conserved domains in the three P450 genes were detected using bioinformatics tools available at the NCBI server.<sup>1</sup> Phylogenetic analyses were conducted using Molecular Evolutionary Genetics Analysis 7 software (MEGA version 7), using the neighbor-joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the analyzed taxa (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates are collapsed. The evolutionary distances were computed using the Poisson correction method (Felsenstein, 1985) and are expressed as the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There was a total of 507 positions in the final dataset.

#### Real-Time Relative Quantitative PCR

Real-time relative qPCR was used to analyze the mRNA levels of the three P450 genes using the 7500 Real-time System (ABI, United States) and ABI SDS 1.4 for the 7500 system (ABI, United States). The A. c. cerana β-actin gene (GenBank accession no. XM\_017065464) was used as a reference gene. The levels of the target genes were compared among the groups of interest. qPCR reactions were performed in a final volume of 20 µL:7 µL sterile deionized water, 1 µL each primer (10 µmol/L), 10 µL Taq DNA polymerase (5 U/µL, TaKaRa), and 1 µL DNA template (50 ng/µL). All qPCR amplifications were performed using the following conditions: (1) 30 s at 95◦C for pre-denaturation; (2) 40 cycles of amplification (5 s at 95◦C for denaturation, 35 s at 60◦C for annealing and extension). Three technical replicates were performed for each experiment.

#### Antibody Production

Custom-made polyclonal antibodies were used. The epitopes were predicted using the GenScript OptimumAntigen design tool, and the peptide antigen sequences for AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were PFGAGRRICPGK, EAHRNNKIDDEGIRE, and PNPDSFDPERFDQDAMAS, respectively. The peptide antigens were then synthesized (Sangon, China). After the coupling reaction and mixing with complete adjuvant, the coupled antigen was used for injection. Next, the coupled antigen was mixed with incomplete adjuvant (Sigma, United States) and injected into white mice (Taibang, China), which were 6 weeks old and specific pathogen-free, four times at 4-week intervals. Subsequently, blood was collected by eyeball puncture, incubated at 37◦C for 1 h, and centrifuged at 3,000 × g for 10 min. Finally, the anti-serum was prepared. The collected antibody was hybridized to a blot containing the overexpressed proteins to detect the specificity of the anti-serum.

#### Western Blotting

The honeybee whole bodies were lysed in RIPA buffer (pH 7.5). The lysate was centrifuged at 10,000 × g for 15 min at 4 ◦C. The supernatant was then collected, and the protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China). The extracted protein was separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, Bedford, MA, United States). The membrane was blocked with QuickBlockTM Western buffer (Beyotime, China) for 1.5 h to reduce non-specific binding. Next, the blot was incubated with the primary antibody overnight at 4◦C. After washing, the blot was incubated with AP-labeled goat antimouse IgG (H + L) secondary antibody (Beyotime, China) for 4 h at 4◦C. Finally, the signal was detected using an enhanced BeyECL Plus kit (Beyotime, China) and visualized in Fusion Fx by Vilber Lourmat. The optical density of each band was quantified using Fusion Capt Advance Fx7 software (Beijing Oriental Science, China), using tubulin as an internal control. The antibodies used included anti-AccCYP314A1 (1:100), anti-AccCYP4AZ1 (1:100), anti-AccCYP6AS5 (1:100), and antitubulin (1:1,000).

<sup>1</sup>http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml

#### RNA Interference

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To synthesize dsRNA for AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5, gene-specific primers (listed in **Table 1**), with a T7 polymerase promoter sequence at their 5<sup>0</sup> ends, were used to amplify the target sequences of the three genes by PCR. The PCR amplification conditions are shown in **Table 2**. The PCR products were purified using the Gel Extraction Kit (Solarbio, China). Next, the dsRNAs for the three P450 genes were synthesized using RiboMAX T7 large-scale RNA production systems (Promega, United States), according to the manufacturer's instructions. To remove the DNA template, the synthesized dsRNA was digested using DNase I, precipitated with absolute ethyl alcohol, and then redissolved in RNase-free water. dsRNA of the green fluorescent protein gene (GFP) (GFP control; GenBank accession no. U87974) was also synthesized.

Newly emerged workers, divided randomly into six groups (n = 50/group), were used for RNA interference (RNAi) experiments. Four groups were injected with 0.5 µL (9 µg) of either dsAccCYP314A1, dsAccCYP4AZ1, dsAccCYP6AS5 or dsGFP, performed as previously described by Amdam et al. (2003) (Hossain et al., 2008). The fifth group was injected with 0.5 µL of sterile water (H2O control), and the sixth group was the negative control group without treatment. Honeybees were maintained in an incubator (60% relative humidity at 34◦C) under a 24-h darkness regimen. Healthy workers were subsequently sampled each day and flash frozen in liquid nitrogen and stored at −80◦C until ready for use. qPCR was performed to detect AccsHsp22.6 (GenBank accession no. KF150016), AccGSTO1 (GenBank accession no. KF496073), and AccTrx1 (GenBank accession no. JX844652) expression profiles when AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were knocked down. Six independent biological replicates were performed in each experiment.

#### Enzymatic Activities of RNAi-Mediated Silencing Samples of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5

Total proteins were extracted from adults injected with dsAccCYP314A1, dsAccCYP4AZ1, dsAccCYP6AS5 or dsGFP at 2 and 3 days post injection. The total proteins were quantified using the BCA Protein Assay Kit (Beyotime, China). Next, a CAT test kit (Jiancheng, China) was used to assay CAT capacity. In addition, a POD assay kit (Jiancheng, China) was used to assay the POD capacity, according to the manufacturer's protocols.

#### Assessment of Survival Rate

Honeybees were randomly assigned to six experimental groups with three cages per group (n = 3; 1,080 bees in total). Groups I and II were injected with 0.5 µL (9 µg) dsRNA-AccCYP314A1; groups III and IV were injected with 0.5 µL (9 µg) dsRNA-AccCYP4AZ1; groups V and VI were injected with 0.5 µL (9 µg) dsRNA-AccCYP6AS5. Groups I, III, and V were fed with a 50% sucrose diet (control group, CK); groups

II, IV, and VI were fed diets containing DDV, deltamethrin, and paraquat, respectively. The pesticides conditions for each experimental group are shown in **Supplementary Table S1**. The number of dead bees in the treatment groups were carefully recorded, and the dead were discarded (Malone and Burgess, 2001).

#### Statistical Analysis

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The significant differences were determined by a Tukey's honestly significant difference test (Tukey's HSD) using the Statistical Analysis System version 9.1 software program (SAS, United States). Equivalence of variance among groups was evaluated using the Levene's Test for homogeneity of variance. One-way ANOVA was used for the analyses. The data are presented as the mean + SEM (n = 6). For the same column, values with different small letter superscripts indicate a significant difference (P < 0.05), while those with the same or no letter superscripts indicate no significant difference (P > 0.05).

#### RESULTS

#### Isolation of the Sequences for AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 and Analysis

The ORF sequence of AccCYP314A1 (GenBank accession no. MH748719) was 1,554 bp. The ORF of AccCYP314A1 encodes a polypeptide of 517 amino acids, with a calculated molecular mass of 58.6 kDa and a theoretical PI of 8.48. The length of the AccCYP4AZ1 ORF sequence (GenBank accession no. MH748718) was 1,548 bp, encoding a polypeptide of 515 amino acids, with a calculated molecular mass of 61 kDa and a theoretical PI of 9.07. The AccCYP6AS5 ORF (GenBank accession no. MH748720) was 1,500 bp long. The ORF encoded a polypeptide of 499 amino acids, with a predicted molecular mass of 58.57 kDa and an pI of 8.57.

As shown in **Supplementary Figures S1–S3**, multiple sequence alignments also revealed that the deduced amino acid sequences of each P450 (AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5) have a high similarity with those of other insect genes. The deduced amino acid sequence of AccCYP314A1 was closely related to those of A. mellifera 314A1 (GenBank accession no. XP\_006610075, 96.36% protein sequence identity), A. florea 314A1 (GenBank accession no. XP\_012348791, 94.88% protein sequence identity), and A. dorsata 314A1 (GenBank accession no. XP\_006619840, 94.5% protein sequence identity). The deduced amino acid sequence of AccCYP4AZ1 was closely related to those of A. mellifera 4AZ1 (GenBank accession no. XP\_006610075, 100% protein sequence identity), A. florea 4AZ1 (GenBank accession no. XP\_012348791, 82.14% protein sequence identity), and A. dorsata 4AZ1 (GenBank accession no. XP\_006619840, 93.98% protein sequence identity). The deduced amino acid sequence of AccCYP6AS5 had 99.4 and 74.75% similarity with A. mellifera 6AS5 (GenBank accession no. NP\_001035324), and Habropoda laboriosa 6a2-like (GenBank accession no. XP\_017796836), respectively. The characteristic active-site motifs Helix-K (EXXR) (Grahamlorence et al., 1995) and Helix-C (WXXXR) (Nelson, 1998) of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were highly conserved among P450 genes (**Supplementary Figures S1–S3**). In addition, the predicted AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 proteins share several characteristics with other members of the P450 supergene family, such as the amino acid residues of FXXGXRXCXG in the haem-binding domain (Kasai and Scott, 2000) (**Supplementary Figures S1–S3**). As shown in the above results, we named the three P450 genes AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5.

Multiple sequence alignment revealed the nucleotide identity (54.84%) among the three P450 genes (**Supplementary Figure S4**). A phylogenetic tree was also built to investigate the evolutionary relationships among AccCYP314A1, AccCYP4AZ1, AccCYP6AS5 and their homologs in insects. As shown in **Figure 1**, the phylogenetic tree revealed that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 belong to the mitochondrial P450 clade, the CYP4 clade and the CYP3 clade, respectively.

FIGURE 2 | AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 expression profiles in various tissues and different developmental stages. (A) The three genes expression levels at diverse developmental stages including 1- to 5-instar larvae (L1–L5), pre-pupal phase (Pp), white-eyed pupae (Pw), brown-eyed pupae (Pb), and dark-eyed, dark pigmented thorax pupae (Pbd), emergence worker (NE). (B) AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 transcript levels in the brain (BR), muscle (MS), midgut (MG), and epidermis (EP). The β-actin gene is shown for comparison. Vertical bars and letters above vertical bars represent the mean + SEM of six different samples and significant differences (P < 0.05), respectively.

#### Tissue-Specific Distribution and Developmental Expression of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5

fphys-09-01608 November 13, 2018 Time: 14:50 # 7

To investigate the expression patterns of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 in various tissues and in different developmental stages of A. c. cerana, qPCR was performed using total RNA extracted from various tissues from larvae, pupae, and adults. The three P450 genes were expressed differently among various tissues (**Figure 2B**). Specifically, Acc314A1 was expressed predominantly in the EP, followed by the BR, and little expression was detected in MS. The relative expression level of AccCYP4AZ1 showed the highest level in the

EP, followed by the MG. There were also low levels of expression of this gene in the BR, and very little expression in the MS. Acc6AS5 showed higher levels of expression in the EP than in any other tissues.

Developmental stage-specific expression patterns of the three P450 genes, including in first- to fifth-instar nymphs (L1–L5), pupae (Pp, Pw, Pb, and Pd), and adults, were also analyzed by qPCR. The expression pattern of the three genes were different among all the developmental stages of A. c. cerana (**Figure 2A**). Specifically, AccCYP314A1 was predominantly expressed in Pd, followed by the third day instar larvae and the fourth day instar larvae; in adults, very little was observed. The relative expression levels of AccCYP4AZ1 were the highest in Pd and showed the lowest expression levels in Pb. AccCYP6AS5 was expressed at a relatively high level in adults, followed by Pd and Pb. AccCYP4AZ1 and AccCYP6AS5 were expressed in larvae at lower levels than pupae and NE, while AccCYP314A1 showed the lowest in NE.

#### Expression Pattern of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 Under Different Oxidative Stresses

Fifteen-day post-emergence workers were exposed to 4◦C, 44◦C, HgCl2, CdCl2, UV light, and pesticides (DDV, paraquat, and deltamethyrin). The relative expression levels were normalized to those of the control workers (CK). As shown in **Figure 3A**, under 4 ◦C treatment, AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were all induced and reached their highest levels at 3, 4, and 1 h, respectively. Acc314A1 was obviously induced, while AccCYP4AZ1, and AccCYP6AS5 were hardly induced, when exposed to 44◦C (**Figure 3B**). Under CdCl<sup>2</sup> treatment, the transcript levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were all upregulated and reached the maximum levels at 1, 6, and 1 h, respectively (**Figure 3C**). Under HgCl<sup>2</sup> stress (**Figure 3D**), AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were all upregulated and reached their highest transcripts at 6, 1, and 3 h, respectively. As shown in **Figure 3E**, AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were upregulated when exposed to DDV and reached their peaks at 0.5 h. Under deltamethyrin and paraquat treatments, AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were all induced (**Figures 3F,G**). As shown in **Figure 3G**, AccCYP314A1, and AccCYP6AS5 reached their peaks at 2.0 h, whereas AccCYP4AZ1 reached its peak at 0.25 h. As shown in **Figure 3H**, the levels AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were upregulated after treatment with UV and reached maximums at 2, 0.5, and 0.5 h, respectively.

#### Western Blot Analysis

The above qPCR results showed that the AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 genes were induced in A. c. cerana in response to various types of oxidative stress. The entire Western blot using the three specific antibodies against the AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 proteins is shown in **Supplementary Figure S5**. Western blot was used to analyze the changes in AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 protein levels after 4 ◦C (A), CdCl<sup>2</sup> (B), paraquat (C), and deltamethrin (D) treatments (**Figure 4**). In our Western blot results, the protein expression levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 are basically consistent with the qPCR results (**Figure 4**), although there were small differences in the time points and degree of expression.

#### Knockdown of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 and the Expression Profiles of Other Antioxidant Genes

To investigate the functions of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 in the antioxidant defense of honeybee workers, RNAi experiments were performed to ascertain their functions. Newly emerged adult workers were injected with H2O, dsRNA-GFP, dsRNA-AccCYP314A1, dsRNA-AccCYP4AZ1, or dsRNA-AccCYP6AS5. The transcripts of the control groups, the H2O and dsRNA-GFP groups, were almost equal, showing that injection with dsRNA-GFP did not influence the expression of the above three genes in A. c. cerana (**Figure 5**). qPCR results showed that the AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 genes were successfully silenced compared with the control groups,

and the lowest transcript levels were discovered at 2 or 3 days post-injection. Additionally, the Western blot results showed the protein expression levels of the three P450 genes at 2 or 3 days post-injection (**Figure 6**). As shown in **Figures 6A,C**, 3 days after injection, the protein expression level of AccCYP314A1 and AccCYP6AS5 were obviously silenced; the protein expression level of AccCYP4AZ1 was downregulated 2 days after injection (**Figure 6B**). The protein expression levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 after injection are consistent with the qPCR results.

To evaluate the response of the other two P450 genes after one was silenced, qPCR was used. As shown in **Figure 7A**, when AccCYP314A1 was silenced, no differences in the transcription levels of AccCYP4AZ1 and AccCYP6AS5 were apparent among the treatment groups 3 days after injection. When AccCYP4AZ1 was silenced, the transcription level of AccCYP314A1 increased, but the transcription level of AccCYP6AS5 in the H2O-injected group, the GFP-injected group and the dsRNA-AccCYP4AZ1 injected group were all downregulated compared with the uninjected group after 2 days (**Figure 7B**). When AccCYP6AS5 was knockdown for 3 days, the expression level of AccCYP314A1 was upregulated, while the expression level of AccCYP4AZ was downregulated (**Figure 7C**).

Quantitative PCR was performed to further evaluate the responses of several genes that have been reported to be involved in oxidative stress responses after AccCYP314A1, AccCYP4AZ1, and AccCP6AS5 were silenced (Meng et al., 2014; Yao et al., 2014; Zhang et al., 2014). When AccCYP314A1 was knocked down, AccGSTO1 was induced (**Figure 8A**). As shown in **Figure 8B**, AccGSTO1 and AccsHsp22.6 were upregulated compared with the control groups. The qPCR results showed that AccGSTO1, AccsHsp22.6, and AccTrx1 were all suppressed when AccCYP6AS5 was silenced (**Figure 8C**). All of the above results suggest that the induced genes participate in the compensation for the knockdown of AccCYP314A1, AccCYP4AZ1, and AccCP6AS5 in A. c. cerana.

#### Determination of Enzymatic Activities After Knockdown of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5

When the AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were knock down, the enzymatic activities were all higher compared with control groups, respectively (**Figure 9**). As shown in **Figures 9A,B**, the CAT and POD capacities of A. c. cerana after the silencing of AccCYP314A1 were all higher than those of the control groups. Overall, for CAT, the activity increased at 3-day time point significantly higher than AccCYP4AZ1 was silenced after 2 days (**Figure 9C**). In A. c. cerana, silenced AccCYP4AZ1 had a significant effect on the activity of POD. The enzymatic activity of POD were significantly higher compared with control bees (**Figure 9D**). Both CAT and POD activity dose significantly enhanced compared with the control groups when AccCYP6AS5 was silenced after 2 or 3 days (**Figures 9E,F**).

#### Survival Rate of Artificially Reared Bees

The three different pesticides treatment groups were all survived shorter compared with control groups, respectively (**Figure 10**). For AccCYP314A1, there was no significant difference between control group and DDV treatment until day 1. By day 2, the control group had significantly higher survival rate compared with the DDV treatment (**Figure 10A**).

For AccCYP4AZ1, there was no significant difference between control group and deltamethrin treatment until day 1. By day 2, honeybees fed the control diet had significantly higher survival rate compared with honeybees fed deltamethrin (**Figure 10B**).

For AccCYP6AS5, honeybees fed the control diet had significantly higher survival rate compared with paraquat treatment starting on day 2, and the trend continued until day 4 (**Figure 10C**).

# DISCUSSION

In many insects, P450 genes are known to play crucial roles in the metabolism of chemicals from host plants and the degradation of diverse insecticides (pyrethroids, organophosphates, and carbamates), resulting in the bioactivation or detoxification of these compounds (Feyereisen, 1999; Scott, 1999; David et al., 2013). However, only a few P450 genes from A. c. cerana have been reported. Therefore, the identification and characterization of new P450 genes have become very attractive research areas. In this study, we obtained the ORF sequences of three new P450 genes (AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5). P450 genes are classified into four clades based on their amino acid sequence similarities. Within the same clades, P450s share ≥55% identities among amino acid (AA) sequences, whereas individual P450 enzymes can have up to 97% identity with any other P450 enzyme (Nebert et al., 1987). As shown in **Figure 1**, AccCYP314A1 and AccCYP4AZ1 were clustered in the mitochondrial P450 and CYP4 clades, respectively. Phylogenetic analysis showed that AccCYP6AS5 was clustered in the CYP3 clade (**Figure 1**). The CYP3 and CYP4 clades were once thought to be largely responsible for xenobiotic metabolism and insecticide resistance and are induced by phenobarbital, pesticides, and natural products (Berenbaum, 2002). Meanwhile, the CYP4 clade has been reported to be involved in chemical communication and pesticide metabolism in several insects (Claudianos et al., 2006). Additionally, previous research reported that the CYP4 clade is implicated in the response to oxidative stress and may play crucial roles in protecting A. c. cerana from oxidative damage (Shi et al., 2013).

Considering the conserved functional motifs of the P450s, which are present in their protein sequences, almost all of the identified P450 genes are likely to be functional. There are three conserved sequences and functionally important motifs, including WXXXR, EXXR, and FXXGXRXCXG (**Supplementary Figures S1–S3**). The three motifs are universal among almost all P450 enzymes and are often considered to be a "signature" of P450 enzymes (Feyereisen, 2012). Their sequences are highly diversified, which suggests that each of the identified P450 genes may play a different role or that they may have different substrate specificities (Guo et al., 2016).

Previous research reported that several tissues, including BR, MG, MS, and EP, were involved in pesticide detoxification in several insect species. For example, the predominant expression pattern in the EP suggests that the expression profiles of the three genes are tissue-specific. A plausible explanation for this may be that the EP is the most exposed to external attack and plays a key role in the resistance to abiotic stress (Marionnet et al., 2003). The BR of an insect has been found to detoxify drugs and xenobiotics to protect the insect nervous system (Holmes et al., 2008). In insects, the MG is known to be the most important organ of digestion and absorption and has a defensive function against xenobiotics. Thus, the diversification of expression profiles for P450 genes in the BR, MG, MS, and EP support their potential roles as antioxidants (**Figure 2B**), although these P450 genes may also play other physiological roles in these tissues.

The relative expression levels of different P450 genes were found to be diversified in different developmental stages of A. c. cerana. For example, CYP4G11 was expressed at higher levels in the 14-day post-emergence stage than in other developmental stages, a finding that has been previously reported (Shi et al., 2013). As shown in **Figure 2A**, AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were all observed to be expressed in all developmental stages. Additionally, AccCYP314A1, and AccCYP4AZ1 were expressed at higher levels of transcript accumulation in Pd, and AccCYP6AS5 showed significantly higher expression in NE than in the other developmental stages, for which stage AccCYP314A1 showed the lowest expression (**Figure 2A**). The above results suggest that the three genes could be involved in specific functions in the Pd and NE stages. Previous studies also found similar results for P450s in L. migratoria, where CYP408B1 was detected at a higher expression level in adults (Guo Y. et al., 2012). However, the expression profiles of these three P450 genes were different from those of AccCYP336A1 in A. c. cerana and CYP409A1 in L. migratoria (Guo Y.Q. et al., 2012; Zhu et al., 2016). In addition, the differences in the expression patterns of the P450 genes during different developmental stages and in different tissues imply their specific functions in insects (Chung et al., 2009).

Previous research findings have revealed that environmental conditions, such as temperature (cold or heat), heavy metals, insecticides, and UV exposure, can induce oxidative stress

(Lushchak, 2011; Kottuparambil et al., 2012). Deltamethrin and pyrethroid are still being widely used as major insecticides. In insects, a previous study reported that P450 genes were involved in the process of insecticide resistance. AccCYP336A1, a stressinducible gene, could be induced by various stresses, such as deltamethrin, heat, H2O2, and UV (Zhu et al., 2016). CYP409A1 and CYP408B1 from L. migratoria were upregulated after deltamethrin treatment, revealing that CYP409A1 and CYP408B1 play key roles in reducing harmful ROS (Guo Y.Q. et al., 2012). Previous research showed that AccCYP4G11 was upregulated by treatments with 4◦C temperatures, H2O2, several insecticides (cyhalothrin, acaricide, parquet, phoxime) and HgCl<sup>2</sup> (Shi et al., 2013). Previous results have revealed that some P450 genes (CYP6FD2, CYP6FF1, and CYP6FG2) showed higher expression levels when treated with the LD10 of deltamethrin but were not induced at the LD30 and LD50 levels of deltamethrin

FIGURE 8 | Expression profiles of other antioxidant genes performed using qPCR when (A) AccCYP314A1, (B) AccCYP4AZ1, and (C) AccCYP6AS5 were knocked down. The β-actin gene was used as an internal control. Each value is given as the mean + SEM. Different letters above the bars indicate significant differences (P < 0.05), according to SAS software 9.1.

(Guo et al., 2016). These differences could be due to deltamethrin affecting the expression profiles of different P450 genes differently and in a dose-dependent manner. As a previous report described, temperature (heat or cold stress) is one of the key mediators of ROS generation, which could cause physiological changes in organisms (An and Choi, 2010). For example, research has shown that heat stress can induce polyamine oxidation due to ROS damage; cold stress can result in the apoptosis of hepatocytes and liver endothelial cells and the generation of ROS (Rauen et al., 1999). As shown in **Figure 3**, after treatment with cold (4 ◦C) or heat (44 ◦C), the expression levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were increased in this study. These results suggest that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 may be involved in regulating body temperature and the heat ceiling, thus protecting A. c. cerana from ROS damage. It was also demonstrated that heavy metals can damage normal development and enhance the endogenous ROS levels in insects (Narendra et al., 2007). Mercury (Hg) and cadmium (Cd), the most poisonous heavy metals in nature, can directly bind to metal ion sites on enzymes, resulting in the inactivation of enzymes (Rashed, 2001). When foragers forage for pollen and nectar, foragers may come into greater contact with heavy metals in the environment. qPCR results also proved that the expression levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were enhanced by CdCl<sup>2</sup> (**Figure 3C**) and HgCl<sup>2</sup> (**Figure 3D**) treatment. These results suggest that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 are involved in avoiding injury under conditions of HgCl<sup>2</sup> and CdCl<sup>2</sup> stress. Insecticides, which are the primary threat to honeybees in the environment, could destroy physiological and biochemical functions due to lipid biomembrane oxidation damage (Narendra et al., 2007). In insects, P450 genes have been reported to be involved in insecticide resistance. Several insecticides, such as deltamethrin and pyrethroid, have been widely used and are still being used. In this study, we investigated the effect of three insecticides (deltamethrin, paraquat, and DDV) on AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 expression at the mRNA and protein levels, to determine whether the three P450 genes are involved in the process of insecticide metabolization. As shown in **Figure 3**, the expression levels of AccCYP314A1,

time intervals. (B) Effect of deltamethrin on AccCYP4AZ1 knockdown workers mortality at different time intervals. (C) Effect of paraquat on AccCYP6AS5 knockdown workers mortality at different time intervals. Means capped with different letters are significantly different (Tukey's HSD, P < 0.05).

AccCYP4AZ1, and AccCYP6AS5 were all induced compared with the control samples, although the time to reach peak expression were different for all three (**Figures 3E–G**). It is a complex manner in which an insecticide affects the expression pattern of different P450 genes differently and which is a time-dependent manner. The differences could be due to the more pronounced toxic effects of pesticides over the long term. UV radiation, a typical oxidant, causes oxidative damage (Schauen et al., 2007; Nguyen et al., 2009). Here, our data showed that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 can be induced by UV radiation treatment (**Figure 3H**). These findings support the hypothesis that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 play crucial roles in protecting A. c. cerana against ROS damage. The regulation of molecular mechanisms by different P450 genes in honeybees is not well understood. If the gene are involved in the process of insecticide resistance, the expression patterns of P450 genes may affect the susceptibility of insects to insecticides.

Western blot analysis was performed to explore the protein expression patterns of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 after A. c. cerana were treated with 4 ◦C, CdCl2, paraquat and deltamethrin. As a whole, the protein expression patterns of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were consistent with the qPCR data. Concerning the mRNA and protein levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5, there were certain differences in time points and degree of change (**Figure 4**). There are some explanations for these differences: first, the increased protein levels of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 could be the result of accumulation; second, the inconsistent mRNA and protein levels could be due to post-transcriptional regulation. Indeed, Hfq (RNA-binding protein) was shown to regulate the expression of invE through post-transcriptional regulation in S. sonnei; although the mRNA expression of invE was easy to detect, the protein of invE was barely detected (Mitobe et al., 2009). The above results support the hypothesis that AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 play important roles in protecting A. c. cerana against ROS damage.

To further understand gene roles, RNAi technology has been used in many insects. To examine the potential roles of these three genes, we first established gene-silencing procedures, in which H2O, dsRNA-GFP, dsRNA-AccCYP314A1, dsRNA-AccCYP4AZ1, or dsRNA-AccCYP6AS5 was microinjected into A. c. cerana. The controllable dose and minimal invasiveness make the microinjection method widely useful for diverse insect types. The results showed that the effect of dsGFP was less obvious; there were no off-target effects because there is no GFP target in A. c. cerana (Elias-Neto et al., 2010). As shown in **Figures 5**, 6, the gene silencing efficiency was different for the three genes. In this study, 40–70% gene silencing efficiency was achieved for the different genes. Above results showed that the three P450 genes of AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 might be involved in environmental response and detoxification in insects. The qPCR method was performed to elucidate the relationship among the three P450 genes. As shown in **Figure 7**, the transcription levels of the other two genes were significant changed compared with control groups when one of the three genes was silenced. These results should be useful for understanding the relationship and the functions of the three P450 genes.

The RNAi technology provides a valuable research tool for future research into the functions of P450s in A. c. cerana. Compared with the control groups, AccCYP314A1 silencing markedly down-regulated AccsHSP22.6 and AccTrx1 on the

second day post injection, but AccGSTO1 expression was upregulated on the third day post injection, suggesting that AccGSTO1 may be involved in the process of reducing the production of ROS after AccCYP314A1 is silenced (**Figure 8A**). The qPCR results also showed that, when AccCYP4AZ1 was silenced, the expression levels of AccGSTO1, AccTrx1, and AccsHSP22.6 were induced (**Figure 8B**). In addition, when AccCYP6AS5 was knocked down, the expression levels of AccGSTO1, AccTrx1, and AccsHSP22.6 were lower than those of the control groups (**Figure 8C**). Among these genes, AccGSTO1 and AccTrx1 were demonstrated to be involved in different abiotic stress responses (Meng et al., 2014; Yao et al., 2014). Previous research showed that AccsHSP22.6 could not only been involved in the abiotic stress response but also in development (Zhang et al., 2014). Thus, we speculate that AccCYP314A1 and AccCYP6AS5 might also be involved in the process of abiotic stress defense and development in A. c. cerana.

The antioxidant enzymes CAT and POD play crucial roles in scavenging ROS, which can cause oxidative damage to DNA, proteins, and lipids in an organism (Lee et al., 2005; Corona and Robinson, 2006). As shown in our results, the activities of CAT and POD were all enhanced when AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were knocked down, suggesting that A. c. cerana was exposed to a high level of oxidative stress when AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were silenced, and the two antioxidant enzymes may be involved in scavenging ROS (**Figures 9A–C**). When AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were silenced, the DDV, deltamethyrin, and paraquat treatments significantly decreased survival rate, respectively (**Figure 10**). This results further confirmed that enzymes encoded by the three P450 genes might contribute to the detoxification of the three pesticides in A. c. cerana (**Figure 10**).

In this study, we identified and characterized the expression patterns of three novel P450 genes from the major agricultural insect A. c. cerana. The expression patterns of the three novel genes showed developmental stage-specific and tissue-specific expression in A. c. cerana. Our findings on the three antioxidant genes in A. c. cerana reveal that there may be more extensive diversification within the P450 supergene family in insects (Sasabe et al., 2004). Our results also imply that the three genes were involved in antioxidant activities, based on the results of using RNAi knockdown for each of the three genes in A. c. cerana. The knowledge gained from this study may help us to better understand the roles of insect P450s and their interactions with pesticides at the mRNA and protein levels. Thus, further functional research on these P450s is essential for the assessment of their functions in abiotic stress and in agriculturally important insects.

#### REFERENCES


# AUTHOR CONTRIBUTIONS

WZ carried out the experimental work and wrote the paper. BX designed the experiments. WC participated in the SDS-PAGE and Western blot work. ZL designed the primers and analyzed the data of Western blot. LM analyzed the data of RT-qPCR. JY carried out the breeding of Apis cerana cerana. HW assisted with transcript expression assessments. ZL carried out RNA extraction and cDNA synthesis. All authors read and approved the final manuscript.

#### FUNDING

This work was financially supported by Funds of Shandong Province "Double Tops" Program (2016–2020), the National Natural Science Foundation of China (No. 31572470), and the earmarked fund for the China Agriculture Research System (No. CARS-44).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01608/full#supplementary-material

FIGURE S1 | The alignment of the deduced AccCYP314A1 amino acid sequence with other species known AccCYP314A1. Characteristic regions are underlined including Helix-C (WXXXR), Helix-K (EXXR), and the consensus sequence FXXGXRXCXG are marked with two triangle (N).

FIGURE S2 | The alignment of the deduced AccCYP4AZ1 amino acid sequence with other species known AccCYP4AZ1. Characteristic regions are underlined including Helix-C (WXXXR), Helix-K (EXXR), and the consensus sequence FXXGXRXCXG are marked with two triangle (N).

FIGURE S3 | The alignment of the deduced AccCYP6AS5 amino acid sequence with other species known AccCYP6AS55. Characteristic regions are underlined including Helix-C (WXXXR), Helix-K (EXXR), and the consensus sequence FXXGXRXCXG are marked with two triangle (N).

FIGURE S4 | The nucleotide sequences multiple alignment among the three target genes.

FIGURE S5 | The specific antibodies against the three target proteins. Protein marker molecular weights are given in kDa. (A) the specific antibodies anti-AccCYP314A1 against AccCYP314A1; M, protein marker; lines 1–4: the bands of AccCYP314A1. (B) The specific antibodies anti-AccCYP4AZ1 against AccCYP4AZ1; M, protein marker; lines 1–4: the bands of AccCYP4AZ1. (C) The specific antibodies anti-AccCYP6AS5 against AccCYP6AS5; M, protein marker; line 1; the negative sample (only SDS-PAGE loading buffer and RIPA buffer); lines 2–4: the bands of AccCYP6AS51.

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strains of Culex pipiens pallens. Pestic. Biochem. Physiol. 75, 19–26. doi: 10.1016/ S0048-3575(03)00014-2


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Chen, Li, Ma, Yu, Wang, Liu and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# FoxO Transcription Factor Regulate Hormone Mediated Signaling on Nymphal Diapause

Zhen-Juan Yin<sup>1</sup> , Xiao-Lin Dong<sup>2</sup> , Kui Kang<sup>3</sup> , Hao Chen<sup>1</sup> , Xiao-Yan Dai<sup>1</sup> , Guang-An Wu1,2 , Li Zheng<sup>1</sup> , Yi Yu<sup>1</sup> and Yi-Fan Zhai1,2,4 \*

1 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan, China, <sup>2</sup> College of Agriculture, Yangtze University, Jingzhou, China, <sup>3</sup> School of Life Sciences, Sun Yat-sen University, Guangzhou, China, <sup>4</sup> College of Life Sciences, Shandong Normal University, Jinan, China

Diapause is a complex physiological adaptation phenotype, and the transcription factor Forkhead-box O (FoxO) is a prime candidate for activating many of its diverse regulatory signaling pathways. Hormone signaling regulates nymphal diapause in Laodelphax striatellus. Here, the function of the FoxO gene isolated from L. striatellus was investigated. After knocking-down LsFoxO in diapausal nymphs using RNA interference, the titers of juvenile hormone III and some cold-tolerance substances decreased significantly, and the duration of the nymphal developmental period was severely shorted to 25.5 days at 20◦C under short day-length (10 L:14 D). To determine how LsFoxO affects nymphal diapause, analyses of RNA-sequencing transcriptome data after treatment with LsFoxO–RNA interference was performed. The differentially expressed genes affected carbohydrate, amino acid and fatty acid metabolism, and phosphatidylinositol 3-kinase/protein kinase B signaling pathway. Thus, LsFoxO acts on L. striatellus nymphal diapause and is, therefore, a potential target gene for pest control. This study may lead to new information on the regulation of nymphal diapause in this important pest.

Keywords: FoxO, RNAi, nymphal diapause, Laodelphax striatellus, RNA-sequencing (RNA-Seq)

# INTRODUCTION

Insects have evolved multiple strategies to adapt to environmental changes, such as diapause and migration. Diapause enables insects to decrease metabolism, arrest development and increase stress resistance under unfavorable conditions, and it is regulated by external environmental signals and internal genetic factors (Denlinger, 2002, 2008). Most insects rely on photoperiod and temperature signals to reach diapause, and there are several regulatory features of diapause, such as hormonal molecular regulation, the circadian clock and energy utilization (Eizaguirre et al., 1998; Anspaugh and Roe, 2005; Xu et al., 2012; Kauranen et al., 2016; Li et al., 2017). In addition, some cold tolerance substances accumulate and key enzymes catalyze, such as trehalase (TRE), sorbitol dehydrogenase (SDH), and pyruvate kinase (PK), etc., to improve the cold tolerance and overcome severe winter environments, (Rozsypal et al., 2013; Zhai et al., 2016). In addition, diapause typically occurs at a

Edited by: Guy Smagghe, Ghent University, Belgium

#### Reviewed by:

Takashi Koyama, University of Copenhagen, Denmark Christen Kerry Mirth, Monash University, Australia

> \*Correspondence: Yi-Fan Zhai zyifan@saas.ac.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 10 April 2018 Accepted: 02 November 2018 Published: 20 November 2018

#### Citation:

Yin Z-J, Dong X-L, Kang K, Chen H, Dai X-Y, Wu G-A, Zheng L, Yu Y and Zhai Y-F (2018) FoxO Transcription Factor Regulate Hormone Mediated Signaling on Nymphal Diapause. Front. Physiol. 9:1654. doi: 10.3389/fphys.2018.01654

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specific developmental stage for each species, such as the embryo, larvae/nymph, pupae or adult (Kostal, 2006; Williams et al., 2006; Zhang et al., 2013; Fan et al., 2017). Larval/nymphal diapause occurs because many insect species overwinter as larvae/nymph, in which the molting processes is usually arrested, and some related signaling pathways are suppressed, such as hormone and energy metabolism (Hahn and Denlinger, 2011; Jindra et al., 2013).

The Forkhead box (Fox) proteins form a family of transcription factors that has several subclasses. The member proteins are diverse but are characterized by a conserved DNA-binding domain. FoxO is the main transcriptional effector of the insulin signaling pathway and is normally suppressed in the presence of insulin (Martins et al., 2016). The insulin signaling pathway plays a critical role in regulating diapause in some invertebrates, such as Caenorhabditis elegans, Drosophila melanogaster, and Culex pipiens (Junger et al., 2003; Sim and Denlinger, 2008; Matsunaga et al., 2018). High insulin levels activate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway, which in turn phosphorylates FoxO, promoting FoxO inactivation. In C. pipiens, the shutdown of insulin signaling prompts the activation of downstream FoxO and leads to the adult diapause phenotype (Sim et al., 2015).

The small brown planthopper, Laodelphax striatellus Fallén (Hemiptera: Delphacidae), is a notorious pest in a variety of graminaceous crop systems, including rice, wheat, corn and barley (Kisimoto, 1989). It causes serious damage to plants due to the transmission of viruses associated with plant diseases, such as rice stripe virus and rice black-streaked dwarf virus (Otuka, 2013). L. striatellus exhibits more cold resistance than other rice planthopper species, Nilaparvata lugens (Stål) and Sogatella furcifera (Horváth), and the most L. striatellus northern populations showed the highest diapause incidence and a longer critical photoperiod (Hou et al., 2016). L. striatellus nymphal diapause has been studied under different laboratory conditions; however, little research has focused on the molecular regulatory mechanisms related to nymphal diapause (Kisimoto, 1989; Wang et al., 2014). In our previous study, we determined the 4thinstar nymph as the main diapause stage through investigation under field and laboratory conditions (Zhai et al., 2018). Here, we characterized the functions of the LsFoxO gene in nymphal diapause. The injection of dsLsFoxO significantly altered the levels of some cold-tolerance substances, metabolic enzymes activities and hormone titers, and the duration of diapause in nymphs was shortened.

# EXPERIMENTAL SECTION

#### Ethics Statement

The small brown planthopper, Laodelphax striatellus is an economically important pest insect in East Asia, which attacks a wide range of graminaceous crops. The field studies did not involve endangered or protected species, and no specific permissions were required for our research activities in these locations.

#### Insect Rearing

We obtained the original L. striatellus colony from the Shandong Rice Research Institute (SRRI; Shandong, China) in 2010. These insects were reared on fresh rice seedlings and maintained in the laboratory at 25 ± 1 ◦C under a 16 L: 8 D photo regime and 70–80% relative humidity. Newly hatched 1st instar nymphs were reared on fresh rice seedlings at 20◦C under long day-length (16 L: 8 D), which resulted in all 4th instar nymphs individuals continuing through direct development (non-diapause). On the contrary, newly hatched 1st instar nymphs were reared at 20◦C under short day-length (10 L:14 D), resulting in substantially all 4th instar nymphs individuals entering nymphal diapause, and developmental delay often characterized nymph population diapause (Wang et al., 2014; Zhai et al., 2018).

#### The Cloning and Sequence Analyses of LsFoxO

The Total RNA Kit II (Omega Bio-Tek, Norcross, GA, United States) was used to isolate the total RNA from larvae and pupae of L. striatellus, and first strand cDNA synthesis was performed using the 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan). Two pairs of intermediate fragments primers were designed from the L. striatellus transcriptome database by local blast (**Table 1**). The full-length complementary DNA (cDNA) of LsFoxO was cloned using the rapid amplification of cDNA ends RACE kit (Takara, Japan), and the evolutionary analyses were conducted using MEGA software, version 4.0.


after 1D4N. (C) The survival rates of diapause nymphs from 3rd instar to the initiation of adult stage. (D) The developmental duration of diapause nymphs. Three replicates were conducted, with the data presented as mean ± SEM. Significant differences between treatment and control are indicated with asterisks (∗p < 0.05; ∗∗p < 0.01), and the different letters means significant difference (p < 0.05, Tukey's post hoc test).

### Quantitative Real-Time PCR Analysis

The primers used for real-time PCR are listed in **Table 1**. The synthesized first-strand cDNA was amplified by PCR in 10 µL reaction mixtures using a Light Cycler 480 system (Roche, United States), and ADP-ribosylation factor (ARF) and elongation factor-1 (EF-1) genes were used as an internal standard (Wan et al., 2014). The quantitative variation was calculated using three independent biological samples by a relative quantitative method (2−11CT).

#### Western Blotting

The proteins were separated using 12% SDS-PAGE gel and transferred to PVDF membranes (0.4 µm; EMD Millipore, Hayward, CA, United States), and the membranes were immunoblotted with anti-LsFoxO serum (1:3000) prepared by our laboratory. IgG goat anti-mouse and goat anti-rabbit antibodies conjugated with HRP were used for secondary antibodies (1:5000, Abcam, Cambridge, United Kingdom), and the membranes were visualized by ECL.

# RNA Interference and Sampling

The dsRNA of LsFoxO was produced using the T7 RiboMAXTM Express RNAi System (Promega, Sunnyvale, CA, United States). After synthesis, dsLsFoxO (MF197906, 431 bp) and dsGFP (DQ389577, 495 bp) were quantified by an ultramicrospectrophotometers (NanoDrop 2000, Thermo Fisher, Scotts Valley, CA, United States) and were maintained at −80◦C until use (**Table 1**). The sequence was verified by sequencing (Sangon Biotech, Shanghai, China). Before injection, the dsRNA and phenol red solution were mixed for observations. Under carbon dioxide anesthesia, nymphs were immobilized on the agarose injection plate with the ventral side upward, under CO<sup>2</sup> anesthesia. The purified dsLsFoxO and dsGFP were slowly injected on one side of the metathorax using the Nanoject II (Drummond, Broomall, PA, United States). The injected individuals were

difference (p < 0.05, Tukey's post hoc test).

placed in a glass tube (length: 200 mm; diameter: 25 mm) on fresh rice seedlings for further observation. Data on developmental duration were recorded every day until the adult emerged.

# Assessment of Metabolic Enzyme Activities and Biochemical Substances

To clarify the physiological adaptation of first day fourthinstar diapause nymphs with dsLsFoxO or dsGFP treated. Glycogen (MAK016) and triglyceride (TR0100) were measured by commercial kits (Sigma-Aldrich Co., LLC., United States), and trehalose (K-TREH) was measured by commercial kits (Megazyme, Ireland). Some cold tolerance-related metabolic enzymes, such as TRE, SDH and PK were quantified. The activities of metabolic enzymes were measured by commercial kits (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China), and the absorbance of TRE, SDH and PK were measured at 340 nm.

# Quantitative Determination of Hormone

Laodelphax striatellus samples were separately ground in grinder and ultra sonicated with methanol and isooctane. After centrifugation at 12,000 g for 10 min, the upper layer was transferred into a test tube, the ultrasound-assisted extraction was repeated twice. The combined extracts were evaporated to dryness in a 40◦C water-bath under a stream of nitrogen. The residue was reconstituted in methanol, then transferred to injection vials and analyzed using HPLC-MS/MS, (Agilent 6420; Waldbronn, Germany). JH III was separated using gradient elution and the hormone titer was expressed as ng per mg body weight.

# Transcriptomic Analyses

The total RNA was extracted using the E.Z.N.A. <sup>R</sup> Total RNA Kit II (Omega Bio-Tek, Norcross, GA, United States) according to the manufacturer's instructions. To obtain ideal gene expression information after the RNAi, first day fourth-instar diapause

nymphs were injected with dsLsFoxO or dsGFP, and after 48 h, the samples were used for transcriptomic analyses. The genes differentially expressed between the two samples were identified using an algorithm as previously described (Schulze et al., 2012). Each cDNA library was sequenced using the Illumina sequencing platform (Hiseq 2500; Illumina, Hayward, CA, United States) according to the manufacturer's instructions.

#### Statistical Analyses

The statistical analyses were performed using SPSS 17.0 software, differences between treatments levels were examined using ANOVA, followed by Tukey's analysis.

# RESULTS

#### Isolation and Characterization of LsFoxO cDNA

The full-length LsFoxO sequence was 2,766 bp (GenBank accession No: MF197906) and had an open reading frame (ORF) of 1,275 bp, which encoded a protein of 424 amino acids with a predicted mass of approximately 46.41 kDa and an isoelectric point of 7.04, with a 5<sup>0</sup> -untranslated region (UTR) of 781 bp and a 3<sup>0</sup> -UTR of 710 bp (**Supplementary Figure 1**).

A phylogenetic tree was constructed based on the fulllength sequences of known FoxO genes from insects and other organisms (**Figure 1**). The BLAST results showed that the amino acid sequence of LsFoxO had the greatest similarity to FoxO from Nilaparvata lugens (Hemiptera) (88%) (XP\_022196038), Halyomorpha halys (Hemiptera) (69%) (XP\_014290452), Cimex lectularius (Hemiptera) (69%) (XP\_014254467), Helicoverpa armigera (Lepidoptera) (58%) (XP\_021186671), Pieris rapae (Lepidoptera) (56%) (XP\_022130764).

# The Spatiotemporal Expression of LsFoxO

To determine whether LsFoxO was present during developmental stages and in various tissues in the female adult L. striatellus, total RNA from each sample was isolated. We used qRT-PCR to characterize the LsFoxO gene's expression pattern in all of the developmental stages. The LsFoxO mRNA expression level was high in the 4th–5th instar nymphal period, but the expression level was higher in the female adult period (**Figure 2A**). The expression of LsFoxO mRNA was investigated in various tissues in adult females. LsFoxO was highly expressed in the fat body, hemolymph and ovary, with lower expression levels in the integument, Malpighian tube and midgut (**Figure 2B**).

# Effect of Knocking Down LsFoxO on Nymphal Performance

To evaluate the effects of an LsFoxO knockdown on nymphal diapause, we used a microinjection-based RNA interference (RNAi) method. Before analyzing the RNAi efficiency, we detected the LsFoxO mRNA expression level in diapausal nymphs (DNs) and non-diapausal nymphs (NNs), including 1st day 3rd instar nymph (1D3N), 1st day 4th instar nymph (1D4N) and 1st day 5th instar nymph (1D5N). The LsFoxO mRNA expression levels were significantly up-regulated in DNs, and the protein levels were also increased in DNs at 1D4N (**Figure 3A**). At 48 h after an injection of dsLsFoxO, the expression of LsFoxO decreased by 73.19, 57.90, and 65.40% in the three different periods, respectively, compared with

an injection of ddH2O. Western blot analyses showed that the LsFoxO protein levels also decreased after injections of dsLsFoxO (**Figure 3B**). These results indicated that the RNAi was effective.

The injection of dsLsFoxO significantly decreased the nymphal survival rate to 45.4%, and the nymphal period from 3rd to 5th instar was significantly shortened after injection. By contrast, over 82% of the nymphs injected with water or dsGFP survived (**Figure 3C**). The average duration to adult eclosion of nymphs injected with dsLsFoxO was 25.5 days, which was significantly shorter than the mean periods of other treatments at 20◦C under short day-length conditions (10 L:14 D) (**Figure 3D**).

#### Assessment of Physiological and Biochemical Changes

Diapause regulates several physiological and biochemical mechanisms, particularly modifying the activities of some cold-tolerance substances and metabolic enzymes. At 72 h after an injection of dsLsFoxO, the glycerol and trehalose levels significantly decreased (43.63 and 53.62%, respectively) (**Figure 4A**), the enzymatic activities of TRE and PK significantly increased (34.79 and 57.62%, respectively) (**Figure 4B**), and the juvenile hormone (JH) III titer indicated that the hormone levels were significantly decreased (**Figures 4C,D**).

# Global Changes at the Transcript Level After LsFoxO RNAi

A total of 38,547,651 clean pair-end reads were generated by Illumina sequencing and were de novo assembled into 42,273 unigenes, with an N<sup>50</sup> length of 1,357 bp (**Supplementary Table 1**). The saturated gene number achieved with the increase in sequenced reads indicates that sufficient and effective information was applied in this study (**Figure 5A**). L. striatellus lacks a reference genome; therefore, 31,254 unigenes were annotated from the Clusters of Orthologous Groups (COG, 10,671), GO (10,083), Kyoto Encyclopedia of Genes and Genomes (KEGG, 9,794), EuKaryotic Orthologous Groups (KOG, 18,483), Protein family (Pfam, 15,526), SwissProt (9,384) and NR (27,325) databases using a cut-off e-value of 10−<sup>5</sup> (**Supplementary Table 2**). The differentially expressed genes (DEGs) following RNAi treatment were analyzed according to the gene expression level (FPKM). Based on the DEG analysis, 384 genes had significantly different expression levels between the dsLsFoxO- and dsGFP-treated libraries, including 208 up- and

nymphal diapause.

176 down-regulated genes (**Figure 5B**). According to the KEGG analysis, most of the DEGs correlated with metabolic and environmental information processes, including carbohydrate metabolism, amino acid metabolism, fatty acid metabolism and the PI3K-Akt signaling pathway (**Figure 5C**).

#### DISCUSSION

fphys-09-01654 November 19, 2018 Time: 11:57 # 8

Diapause usually occurs during a specific stage, such as embryo, larvae/nymph, pupae or adult stages (Jiang et al., 2010; Liu et al., 2010; Kobayashi et al., 2014). Some research have found that diapause occurs in several nymphal stages of L. striatellus, and which varies with geographic location (Wang et al., 2014; Hou et al., 2016). In our previous study, we discovered that the overwintering diapause occurred in 3rd to 5th instars, with the 4th instar nymph being the predominant diapause stage (Zhai et al., 2018). Diapause is a complex physiological response process with many regulatory features, number of genes, proteins, and metabolites that are differentially expressed in diapause (Zhang et al., 2013). Diapause is regulated by the JH, and ecdysone in diverse species. A high JH titer inhibits ecdysone secretion during diapause maintenance, and the JH titer decreases significantly during late diapause. Hormone signaling regulates nymphal diapause in L. striatellus (Zhai et al., 2017). In D. melanogaster, JH and ecdysone synthesis are regulated by the insulin signaling pathway (Colombani et al., 2005; Tu et al., 2005), and insulin signaling is a regulator of diapause (Sim and Denlinger, 2013). FoxO is a well-known regulator of life span extension (Martins et al., 2016), insulin activates p-Akt levels repress FoxO activity, which activate other cross-talk genes that promote the insects' development. In contrast, a low level of insulin signaling represses PI3K/Akt and increases the FoxO activity, which regulates life-span extension and generates the diapause phenotype (Sim et al., 2015). High levels of p-Akt fail to phosphorylate FoxO through PRMT1-mediated methylation, blocking FoxO phosphorylation reduces FoxO protein degradation, thus promoting the accumulation of FoxO in brains, which leads to pupal diapause in Helicoverpa armigera (Zhang et al., 2017). The overexpression of FoxO during early larval stages inhibits development and extends life-spans. Our results, in which LsFoxO mRNA expression and protein levels were significantly up-regulated in diapause nymphs, corroborates previous findings (**Figure 3A**).

After evaluating the spatiotemporal expression of the LsFoxO gene in L. striatellus using qRT-PCR, we found that higher expression levels were detected in 4th–5th instar nymphs and the female adults. FoxO is expressed during all of the developmental stages in other insects (Hwangbo et al., 2004; Carter and Brunet, 2007). In the present study, the injection of dsLsFoxO significantly inhibited the gene's mRNA in the three different nymphal periods and the protein level in the 4th instar nymph (**Figure 3B**). Compared with these results, the developmental duration of 3rd to 5th instar nymphs may be a better indicator of the functional relationship between LsFoxO and nymphal diapause. Diapause is a form of dormancy used by insects to survive under adverse environmental conditions and is usually related to some cold-tolerance substances, such as glycerol, trehalose and glucose, which can improve the insects' cold tolerance and help overcome severe winter environments (Denlinger, 2002; Zhai et al., 2016). TRE is a key enzyme in trehalose hydrolysis and changes in the activity of this enzyme directly affect energy metabolism (Kamei et al., 2011). PK is the key enzymes in the glycolytic pathway, and PK mediates the conversion of phosphoenolpyruvic acid and ADP into pyruvic acid and ATP (Rider et al., 2011). Here, TRE and PK levels significantly increased after an injection of dsLsFoxO, and the glycerol and trehalose levels significantly decreased (**Figures 4A,B**). Thus, the diapausal nymphs had increased their cold tolerance through the accumulation of cold-tolerance substances.

Targeting a gene using RNAi may reveal its function, and this method has been most recently used for agricultural pest control (Baum et al., 2007; Mao et al., 2007). However, there have been limited studies on the global changes in the mRNA profile that occur after RNAi targeting of a specific gene (Wang et al., 2008). We determined previously that4th instar nymphs represent the main diapausal. Therefore, we selected 1D4N diapausal nymphs to inject with dsRNA and for transcriptomic analyses. We showed that in 384 annotated DEGs between LsFoxO-RNAi and control samples, 208 and 176 genes were up- and downregulated, respectively (**Figure 5B**). Among the down-regulated genes, we found the RNAi target gene LsFoxO (c11725.graph\_c0), indicating that the RNAi was effective. To review the global changes in the signaling pathway after RNAi, we used the KEGG analytical method. Most of the DEGs correlated with metabolic and environmental information processes, including carbohydrate metabolism, amino acid metabolism, fatty acid metabolism and the PI3K-Akt signaling pathway (**Figure 5C**). The latter is a signal transduction pathway that promotes survival and growth in response to extracellular signals and is a key component of the insulin signaling pathway. Insulin activates PI3K-Akt, and high p-Akt levels repress FoxO activity and activate other genes that inhibit diapause.

In summary, FoxO is a key downstream regulator that acts as a key developmental switch in insect diapause, which is regulated by the insulin signaling pathway. LsFoxO can regulate some cold-tolerance substances and JH III titers in the hemolymph to control the nymphal diapause status. This supports the conclusion that physiological levels of FoxO are beneficial for diapause. We propose a model to explain how different photoperiod signals interact with LsFoxO to regulate nymphal diapause in L. striatellus (**Figure 6**). There are still many issues to be studied in the future, such as how LsFoxO regulates JH expression and the accumulation of some cold-tolerance substances? The present results offer new insights into nymphal diapause and contribute to a comprehensive understanding of insect diapause.

#### AUTHOR CONTRIBUTIONS

Z-JY, X-LD, YY, and Y-FZ conceived and designed the experiments. Z-JY, KK, G-AW, HC, and XY-D preformed the

experiments. Z-JY, LZ, and Y-FZ analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This work was financially supported through a grant from the National Natural Science Foundation of China (31401803) and

#### REFERENCES


the Shandong Provincial Natural Science Foundation, China (ZR2014CQ014).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01654/full#supplementary-material


for management of planthopper Sogatella furcifera and Laodelphax striatellus. PLoS One 9:e86675. doi: 10.1371/journal.pone.0086675


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yin, Dong, Kang, Chen, Dai, Wu, Zheng, Yu and Zhai. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera

Jizhen Wei <sup>1</sup> , Shuo Yang<sup>1</sup> , Lin Chen<sup>2</sup> , Xiaoguang Liu<sup>1</sup> , Mengfang Du<sup>1</sup> , Shiheng An<sup>1</sup> \* and Gemei Liang<sup>2</sup> \*

*<sup>1</sup> State Key Laboratory of Wheat and Maize Crop Science, College of Plant Protection, Henan Agricultural University, Zhengzhou, China, <sup>2</sup> State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China*

*Helicoverpa armigera* can develop resistance to *Bacillus thuringiensis* (Bt), which threaten

#### Edited by:

*Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China*

#### Reviewed by:

*Gustavo Bueno Rivas, University of Florida, United States Dandan Wei, Southwest University, China*

> \*Correspondence: *Shiheng An anshiheng@aliyun.com Gemei Liang gmliang@ippcaas.cn*

#### Specialty section:

*This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology*

Received: *11 May 2018* Accepted: *02 November 2018* Published: *22 November 2018*

#### Citation:

*Wei J, Yang S, Chen L, Liu X, Du M, An S and Liang G (2018) Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera. Front. Physiol. 9:1653. doi: 10.3389/fphys.2018.01653* the long-term success of Bt crops. In the present study, RNAseq was employed to investigate the midgut genes response to strains with different levels of resistance (LF5, LF10, LF20, LF30, LF60, and LF120) in *H. armigera.* Results revealed that a series of differentially expressed unigenes (DEGs) were expressed significantly in resistant strains compared with the LF-susceptible strain. Nine trypsin genes, *ALP2*, were downregulated significantly in all the six resistant strains and further verified by qRT-PCR, indicating that these genes may be used as markers to monitor and manage pest resistance in transgenic crops. Most importantly, the differences in DEG functions in the different resistant strains revealed that different resistance mechanisms may develop during the evolution of resistance. The immune and detoxification processes appear to be associated with the low-level resistance (LF5 strain). Metabolic process-related macromolecules possibly lead to resistance to Cry1Ac in the LF10 and LF20 strains. The DEGs involved in the "proton-transporting V-type ATPase complex" and the "proton-transporting two-sector ATPase complex" were significantly expressed in the LF30 strain, probably causing resistance to Cry1Ac in the LF30 strain. The DEGs involved in binding and iron ion homeostasis appear to lead to high-level resistance in the LF60 and LF120 strains, respectively. The multiple genes and different pathways seem to be involved in Cry1Ac resistance depending on the levels of resistance. Although the mechanisms of resistance are very complex in *H. armigera*, a main pathway seemingly exists, which contributes to resistance in each level of resistant strain. Altogether, the findings in the current study provide a transcriptome-based foundation for identifying the functional genes involved in Cry1Ac resistance in *H. armigera*.

Keywords: Helicoverpa armigera, DGE, Cry1Ac, trypsin, receptors, mechanisms of resistance

# INTRODUCTION

The Cry1Ac toxin from Bacillus thuringiensis (Bt) is harmless to most organisms and considered as an environmentally friendly pesticide. Hence, Cry1Ac toxin is used commercially as a bioinsecticide and expressed in transgenic plants for controlling insect pests (Wu et al., 2008; Hutchison et al., 2010; Edgerton et al., 2012; Lu et al., 2012; Klümper and Qaim, 2014). The area

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of Bt transgenic crops planted worldwide has rapidly increased to 98.5 million hectares in 2016 and has accumulated more than 830 million hectares since 1996 (James, 2016). Although Bt crops have brought great economic and environmental benefits (Wu et al., 2008; Hutchison et al., 2010; Edgerton et al., 2012; Lu et al., 2012; Klümper and Qaim, 2014), the development of resistance to Bt toxins can reduce or even eliminate these benefits (Tabashnik et al., 2013; Van den Berg et al., 2013; Farias et al., 2014; Gassmann et al., 2014; Tabashnik and Carrière, 2017). Unfortunately, the cumulative number of cases of practical resistance to the Bt toxins in transgenic crops surged from 3 in 2005 to 16 in 2016 according to a report from Tabashnik and Carrière (2017) attracting researcher's attention.

Understanding the mode of Bt action and the mechanisms that confer resistance to Bt toxins can help to sustain and even enhance their efficacy to control pests. Models of Bt action agree that Bt protoxins are first converted to activated toxins by insect midgut proteases and then the activated toxins bind to the insect midgut receptors, finally leading to insect death (Gill et al., 1992; Pardo-López et al., 2013; Adang et al., 2014). Four main functional receptors of the Cry1Ac toxin have been identified and verified from the brush border epithelium, including alkaline phosphatase (ALP) (Flores-Escobar et al., 2013), cadherin (Chen et al., 2014), aminopeptidase (APN) (Zhang et al., 2009; Tiewsiri and Wang, 2011; Valaitis, 2011; Flores-Escobar et al., 2013; Wei et al., 2016b), and ATP-binding cassette transporter proteins (ABCs) (Tanaka et al., 2017). The identification of Cry1A receptors has broadened our understanding of Cry1Ac action. However, the mode of action of Cry1Ac is very complex and a change in any step of the toxicology process will inevitably lead to insect resistance. The most common Bt-resistant mechanisms had been reported in Lepidoptera, including a reduced binding capacity of Bt toxins to midgut receptors by a decrease in the activity and transcription of ALP or APN as well as mutations of APN, cadherin, and ABCC<sup>2</sup> (Xu et al., 2005; Zhang et al., 2009, 2012; Gahan et al., 2010; Baxter et al., 2011; Jurat-Fuentes et al., 2011; Atsumi et al., 2012; Xiao et al., 2014; Chen et al., 2015) and a reduced conversion of the protoxin to the toxin by downregulation of trypsin (Rajagopal et al., 2009; Cao et al., 2013; Liu et al., 2014; Wei et al., 2016a). These studies indicate that many genes are involved in resistance of insects to Bt toxins. In addition, it sounds like it is hard to identify the common marker to monitor and manage pest resistance in transgenic crops.

Most importantly, different genes may show different contributions to resistance at different development levels. The decreasing protoxin activation in Ostrinia nubilalis (HD-1 Bt kurstaki-resistant strain) caused a 47-fold resistance to Dipel, which contained Cry1Ab, Cry1Aa, Cry1Ac, and Cry2Aa (Li et al., 2004, 2005). In a 100-fold more Cry1Ab-resistant Diatraea saccharalis strain, the resistance to the Cry1Ab toxin is due to the lower gene expression level of cadherin (Yang et al., 2011). In the H. armigera GYBT-resistant strain, a deletion between exons 8 and 25 of the cadherin gene resulted in a 564-fold resistance to Cry1Ac-activated toxin (Xu et al., 2005). For the Cry1Ac-resistant BtR strain, it was identified that a deletion mutation of APN3 and the downregulation of cadherin lead to Cry1Ac resistance. A subsequent study confirmed that a deletion mutant in the APN1 gene caused a more than 2,971-fold resistance to Cry1Ac in the BtR strain (Wang et al., 2005; Zhang et al., 2009). Recently, the mutations in the ABCC2 transporter and the cadherin genes were reported to have a different affect on the binding of Cry toxins to the proteins on the brush border membrane vesicle (BBMV) from H. virescens (Gahan et al., 2010). The mutations of the ABCC2 transporter showed a higher level of resistance to Cry toxins. These studies demonstrate that different genes have different impacts on resistance levels.

Moreover, the resistant pathways were studied also to find the key factors that regulated the expression of resistant genes. Guo et al. (2015b) reported a novel transregulatory signaling mechanism in which the mitogen-activated protein kinase (MAPK) signaling pathway was confirmed to be responsible for regulating the expressions of ALP and ABCC genes in a fieldevolved resistant strain of P. xylostella. Also, the constitutively transcriptionally activated upstream gene, MAP4K4, in the MAPK signaling pathway is responsible for this transregulatory signaling mechanism (Guo et al., 2015b). Importantly, the key resistant factor, MAP4K4, may be used for molecular control of the Cry1Ac resistance.

However, the genes and pathways affected the Cry1Ac resistance depending on the Cry1Ac selection stresses in H. armigera have not been comprehensively assessed. The next-generation DNA sequencing provides a research technique to study the changes in gene expression in the midgut transcriptomes of Bt-resistant and -susceptible strains; this technology can detect differentially expressed genes in biochemical pathways involved in Bt resistance and provide new insights into resistance mechanisms (Lei et al., 2014; Nanoth Vellichirammal et al., 2015; Zhang et al., 2017). In this study, a susceptible LF (a laboratory strain collected from Langfang) strain and six resistant LF strains were selected for an analysis of related resistance genes, in particular, six substrains of LF came from the LF strain by selecting a series of gradually increasing resistant strains (Cao et al., 2013, 2014; Liu et al., 2014; Xiao et al., 2014; Chen et al., 2015; Wei et al., 2016a). First, RNA sequencing was employed to construct a complete and comprehensive reference transcriptome database from midgut samples of these seven strains. The differentially expressed genes were detected further among these seven strains by digital gene expression analysis (DGE). These data provide a foundation for understanding the systemic differences between Cry1Acresistant strains and Cry1Ac-susceptible strain and might aid in finding candidate resistance. Most importantly, the analysis of differently expressed genes among the seven strains will uncover the role of different genes in the different resistance phases and might explain how selection can cause fixed changes of the expression levels of numerous genes.

#### MATERIALS AND METHODS

#### Insect Strains

The LF-susceptible H. armigera strain was established from a field population by collecting from the Langfang County, Hebei Province of China in 1998. The LF strain was reared in a lab environment without exposure to any insecticides (Wu and Guo,

2004). The six LF substrains came from the LF-susceptible strain via a series of selections: LF5, LF10, LF20, LF30, LF60, and LF120 (Cao et al., 2013, 2014; Liu et al., 2014; Xiao et al., 2014; Chen et al., 2015; Wei et al., 2016a; **Table 1**). The strain name is in accordance to the selection concentration for each strain, where the number from 5 to 120 follows LF: such as LF5 was selected with a 5µg/ml Cry1Ac protoxin artificial diet (Liang et al., 2008). In this study, LF5, LF10, LF20, LF30, LF60, and LF120 had been selected for 60, 52, 42, 38, 21, and 17 generations with corresponding Cry1Ac diets, respectively (**Table 1**).

#### Bioassay of Resistance to Cry1Ac Toxin

Larval responses to Cry1Ac toxin were evaluated using the methods reported by Wei et al. (2015). The Cry1Ac protoxin crystals were obtained from the HD-73 strain of B. thuringiensis (kindly supplied by Biotechnology Research Group, Institute of Plant Protection, Chinese Academy of Agricultural Sciences). Totally, 72 neonates per concentration for each treatment were tested. About 7 days later, dead insects and those that were still first instars were scored as dead. Five or more toxin concentrations were used to calculate the LC<sup>50</sup> values of each strain (**Table 1**).

#### Dissection of Midgut and Extraction of RNA

Larvae from different colonies (LF, LF5, LF10, LF20, LF30, LF60, and LF120) were reared with a non-Bt toxin diet under standard rearing conditions. The midgut tissues of larvae in fifth instars (n = 30 per pool) were dissected from different strains. The lumen was then rapidly washed with a solution of 0.7% NaCl (w/v) to remove debris. Two biological replicates were employed. Total RNA from every replicate was extracted separately from each pool (LF, LF5, LF10, LF20, LF30, LF60, and LF120) using the Trizol reagent according to manufacturer's suggestions (Invitrogen, CA). In order to remove genomic DNA contamination, resulting RNA was treated with DNase I (Promega, Madison, WI, USA) following manufacturer's instructions. Quantity and quality of total RNA were assessed

TABLE 1 | Responses to Cry1Ac of the susceptible strain (LF) and six resistant strains (LF5, LF10, LF20, LF30, LF60, and LF120) of *H. armigera*.


*<sup>a</sup>Generation.*

*<sup>b</sup>Concentration killing 50% with 95% fiducial limits in parentheses, units are* µ*g toxin per cm<sup>2</sup> diet.*

*<sup>c</sup>Resistance ratio, the LC<sup>50</sup> for a strain divided by the LC<sup>50</sup> for LF.*

by denaturing gel electrophoresis and spectrophotometry on a Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA).

#### Preparation of Library for Analysis of Transcriptome

Sequencing libraries were generated from 3 µg total RNA per sample using NEBNext <sup>R</sup> UltraTM Directional RNA Library Prep Kit for Illumina <sup>R</sup> (NEB, USA) following manufacturer's recommendations. Briefly, mRNA was separated from total RNA using poly-T oligo-attached magnetic beads. First-strand cDNA was generated using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-) followed by secondstrand cDNA synthesis using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. The 3′ ends of DNA fragments were firstly adenylated and then NEBNext Adaptors with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). About 3 µL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37◦C for 15 min followed by 5 min at 95◦C before PCR. Then PCR was performed with Phusion high-fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. The PCR products were purified finally (AMPure XP system) and the quality of library was assessed on the Agilent Bioanalyzer 2100 system.

#### Analysis of Results of Illumina Sequencing

The clustering of the index-coded samples was carried out on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3 cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000 platform and paired-end reads were generated. Transcriptome assembly was accomplished by using Trinity r20121005 (Grabherr et al., 2011) with min\_kmer\_cov set to 2 by default and all other parameters set default. Raw data (raw reads) of fastq format were firstly processed through inhouse perl scripts. Clean data (clean reads) were then obtained by removing noise signals (reads containing adapter, reads containing ploy-N, and low-quality reads) from raw data. The following data analyses were performed based on the clean data. After eliminating redundancy using cd-hit and cap3 software, these data were mixed with our unpublic data of cotton bollworm transcriptome database. The resulting unigene database was used as a reference transcriptome database for subsequent analysis of DGE. The homology searches of all unigenes were performed based on BLASTx and BLASTn programs against the GenBank non-redundant protein (nr) and nucleotide sequence (nt) database at NCBI (v2.2.28). Matches of an Evalue < 1.0E-5 were considered to be significant (Altschul et al., 1997). Gene ontology term (GO, http://www.geneontology.org/) annotations were assigned by Blast2GO software (b2g4pipe\_v2.5) (Götz et al., 2008). The KOG (euKaryotic Ortholog Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations were performed using Blastall software against the KOG database (http://www.ncbi.nlm.nih.gov/COG/) and KEGG database (http://www.genome.jp/kegg/), respectively.

#### DGE Library Preparation and Sequencing

Library for DGE sequencing was prepared according to earliermentioned method (see "Library preparation for transcriptome analysis"). After cluster generation, the library sequencing was performed on an Illumina Hiseq 2000 platform and 100 bp single-end reads were generated.

#### Analysis and Mapping of DGE

After removing reads containing adapter, reads containing ploy-N, and low-quality reads from raw data, the clean data were then obtained. All analyses were performed according to the clean data. For unigene DEG, single-end clean reads were aligned to the unigene sequences by Bowtie v0.12.9. The HTSeq v0.5.4p3 was used to count unigene DEG numbers mapped to each unigene. Reads per kilobase of exon model per million mapped reads (RPKM) of each gene were calculated based on the length of the gene and reads count mapped to this gene (Mortazavi et al., 2008). The differentially expressed unigenes were used for mapping and annotation.

#### Evaluation of DGE Libraries

The frequency of each unigene in the different cDNA libraries was analyzed to compare gene expression in different strains. The DEGSeq R package (1.12.0) was used to analyze the differential expression of two conditions. The P-values were adjusted using the Benjamini & Hochberg method. Significant differential expression genes were obtained using set threshold values [corrected P-value of 0.005 and log<sup>2</sup> (Fold-change) of 1]. For pathway-enrichment analysis, we mapped all the differentially expressed genes to terms in the GO data database and KEGG database. The GO-enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. The GO terms with corrected P-value < 0.05 were considered significantly enriched by differentially expressed genes. We used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways.

# Validation of qRT-PCR

The first-strand cDNA of each strain was used as the template for real-time PCR analysis. Each strain of H. armigera included 90 larvae (30 larvae per biological replicate). The mRNA expression levels of ALP-like, ALP2, APN5, and APN1 in different strains were analyzed by a quantitative real-time PCR (qRT-PCR). The β-actin and GAPDH of H. armigera were used as internal reference genes (Liu et al., 2014). The primers of the said genes used for qRT-PCR analysis are listed in **Table S1**. Each qRT-PCR (TaqMan) (TIANGEN, FP206, China) reaction was performed individually in a 20-µL system containing 1 µL of the template cDNA, 10 µL of the 2 × SuperReal PreMix (Probe), 0.6 µL of the 10uM of each primer, 0.4 µL of the 10 uM of the probe, 0.2 µL of the 50 × ROX Reference Dye<sup>∗</sup> 3, and 7.2 µL of the RNase-Free ddH2O. The thermal cycler conditions used for realtime PCR were: 40 cycles of 3 s at 95◦C and 30 s at 60◦C. The mRNA expression levels of trypsin genes were tested by SYBR Green Supermix (TaKaRa). The primers of trypsin genes used for qRT-PCR analysis can be found in **Table S2**. The H. armigera 18S (Du et al., 2017) and EF1-α (Yuan et al., 2006) were used as internal reference genes. The qRT-PCR was performed at 95◦C for 3 min, followed by 40 cycles of 95◦C for 15 s and 60◦C for 20 s. Real-time PCR of trypsin and reference genes was done in a 20-µL reaction system containing 10 µL of 2 × SYBR Mix and 10µM forward primer and reverse primer (1.0 µL each), 1 µL template cDNA, and 7.0 µL nuclease-free water. All qRT-PCR reactions were performed in 96-well optical plates in an ABI 7500 Real-time PCR System (Applied Biosystems).

The expression levels of all the earlier-mentioned genes were calculated with their amplification efficiency (E) and mean Ct, and the expression levels of the candidate genes were normalized with the geometric mean of the expression of each of the two reference genes (GAPDH and EF-1α/18S and EF1-α; Livak and Schmittgen, 2001; Vandesompele et al., 2002; Liu et al., 2015). The results of each gene among different strains were determined with one-way analysis of variance (ANOVA), followed by Tukey's honestly significance difference (HSD) test for mean comparison. All statistical analysis was performed with SPSS v.18.0 (SPSS Inc., Chicago, IL, USA) at P < 0.05 level of significance.

# RESULTS

# Insect Resistance Levels

After 60 generations of Cry1Ac selection, the LF5 laboratory colony had an estimated resistance ratio of 540 compared with the susceptible LF strain (**Table 1**). The LF10 was divided from LF5; then, after selection for 52 generations using diets containing 10-µg/mL Cry1Ac toxin, the resistance ratio of the LF10 strain cotton bollworm reached 640 for Cry1Ac toxin (**Table 1**). The LF20 was divided from LF10 and after selection for 42 generations on diets containing 20µg/mL Cry1Ac toxin, the resistance ratio of LF20 strain cotton bollworm increased to 850. Similarly, LF30 was divided from LF20 and selected for 38 generations on diets containing 30-µg/mL Cry1Ac toxin; in addition, LF60 was divided from LF30 and selected for 21 generations on diets containing 60-µg/mL Cry1Ac toxin. The resistance ratio of these two strains was 1,000 when compared with the susceptible-LF strain (**Table 1**). The LF120 was divided from LF60 and selected for 17 generations on diets containing 120-µg/mL Cry1Ac toxin; the LF120 strain showed the highest resistance levels (2,000-fold) (**Table 1**). Generally, with the increase of the selection concentration of Cry1Ac toxin, the resistance levels were improved correspondingly (**Table 1**).

#### Illumina Sequencing and Transcriptome Assembly

In total, 77,422,352 clean reads were obtained from transcriptomics analysis of samples obtained from the midguts of the seven strains and were assembled into 66,502 transcripts. The mean length of the transcripts was 1,324 bp with lengths ranging from 201 to 49,954 bp. After mixing with our private cotton bollworm transcriptome database, a total of 139,012 unigenes were obtained. The size distribution of these unigenes is shown in **Figure S1**.

#### Annotation of Predicted Proteins

Annotation of gene function was performed by running Blast on the following databases: Nr (NCBI non-redundant protein sequences), Pfam (Protein family), Nt (NCBI non-redundant nucleotide sequences), Swiss-Prot (A manually annotated and reviewed protein sequence database), KOG (euKaryotic Ortholog Groups), GO (Gene Ontology), and KO (KEGG Ortholog database). The results demonstrated that 64.96% unigenes were annotated in NR, 35.14% were annotated in NT, 15.83% were annotated in KO, 43.77% were annotated in SwissPort, 41.29% were annotated in PFAM, 48.11% were annotated in GO, and 32.96% were annotated in KOG. In total, 6.12% unigenes were annotated in all Databases. Finally, most of the 139,012 unigenes (72.89%) were matched to known genes. This transcriptome database will be used as a reference database to analyze differences in gene expression among different strains of cotton bollworm.

#### Classification of Gene Ontology (GO)

The GO classification demonstrated that 66,886 sequences could be assigned into 48 functional groups (**Figure 1A**). In the three main categories of the GO classification, "metabolic process," "binding," and "cell and cell part" terms were dominant, respectively.

The GO analysis showed that the functions of the identified genes involved various biological processes. Totally, 40,256 unigenes were annotated in the "metabolic process" category, 38,541 unigenes were annotated in the "cellular process" category, and 36,898 unigenes were annotated in the "binding" category (**Figure 1A**).

#### KOG Classification

In total, 45,832 unigenes were categorized into 26 functional groups (**Figure 1B**). The main groups were "post-translational modification, protein turnover, chaperone" (5,184 unigenes), "general functional prediction only" (7,232 unigenes), and "signal transduction" (4,453 unigenes). These results demonstrated that based on high-throughput sequencing, novel genes that might play roles in CryAc resistance can be identified (**Figure 1B**).

# Functional Classification by KEGG

The KEGG classification revealed that 35,802 annotated unigenes were mapped to the reference canonical pathways in KEGG and categorized into 5 KEGG pathways. The unigenes were clustered into various classifications, including metabolism (25,537 members), organismal systems (13,554 members), genetic information processing (8,519 members), cellular processes (6,849 members), and environmental information processing (5,583 members). These annotations of unigenes provide a valuable resource for investigating functions, specific processes, and pathways in cotton bollworm Cry1Ac-resistance research (**Figure 1C**).

#### Estimates of Differential Expression Among the Midgut Transcripts

The DGE was used to analyze gene expression among the seven strains, including one susceptible and six Cry1Ac-resistant strains. Fourteen DGE libraries (containing two biological replicates): LF-1, LF-2, LF5-1, LF5-2, LF10-1, LF10-2, LF20-1, LF20-2, LF30-1, LF30-2, LF60-1, LF60-2, LF120-1, and LF120- 2 were sequenced and between 6.4 and 11.3 million clean reads were generated. The number of clean read entities with unique nucleotide sequences ranged from 5,935,643 to 10,511,449 (**Table 2**). Moreover, 94.62% (9,885,875) of the sequences in the transcriptome database were unequivocally identified by unique genes (**Table 2**).

#### Differentially Expressed Genes in Different Resistant Developmental Strains

The DEG numbers detected to confer resistance level in six resistant LF strains did not increase along with the increase of resistance level to Cry1Ac (**Table 1**; **Figure 2**); the changes in the trends in the numbers of DEGs in six resistant LF strains are similar to the letter "N" in **Figure 2A**. Commonly, more upregulated genes than downregulated genes were detected in each of the six resistant LF strains (**Figure 2A**). Compared with the susceptible LF strain, 3,688 unigenes were expressed differentially in the LF5 strain, which presents the lowest resistance level. The highest numbers of DGEs occurred in the LF10 strain (9,712) followed by the LF20 stain (8,558) (**Figure 2B**). The lowest numbers of DGEs occurred in the LF30 strain (2,085) (**Figure 2**). Although LF60 showed the same resistance level as LF30, more DEGs were found in the LF60 strain (4,990), possibly due to the greater exposure to Cry1Ac toxin for the LF60 strain (**Table 1**; **Figure 2**). In total, 6,859 unigenes were expressed differentially in the LF120 strain, although the larvae of this strain showed the highest resistance. Comparing two neighboring strains, more genes showed significant differences in expression levels between LF10 vs. LF5 and between LF20 vs. LF10, and fewer genes showed significant differences in expression levels between LF30 vs. LF20 and between LF60 vs. LF30 (**Figure 2A**). However, the declining trend did not continue between LF120 vs. LF60, possibly due at least in part to the greater number of mutations in more genes or alleles involved in conferring a higher resistance level in the LF120 strain (**Figure 2A**). To analyze the function of DEGs between the LF and LF-resistant strains, these genes were classified in GO terms. The 30 significantly enriched (according to the corrected pValue) GO terms are shown in **Figure S2**. The differentially expressed genes showed significant enrichment in "catalytic activity," "endopeptidase activity," "aminopeptidase activity," "serine-type endopeptidase activity," "proteolysis," "biological process," "metabolic process," "peptidase activity," "serine-type peptidase activity," "metallopeptidase activity," "exopeptidase activity," "hydrolase activity," "serine hydrolase activity," "protein metabolic process," "peptidase activity," "acting on L-amino acid peptides," and "organic substance metabolic process" terms in all resistant strains (**Table 3**). These DEGs may help the cotton bollworm to enhance their physiology to adapt to the Cry1Ac toxin. The LF10, LF20, LF30, and LF60 have moderate resistance level and some of the differentially expressed genes in these four resistant strains were significantly enriched in "ribosome," "translation," "non-membrane-bounded

organelle," "ribonucleoprotein complex," "structural constituent of ribosome," and "intracellular non-membrane-bounded organelle" (**Table 3**). The LF5 has the least resistance and some of the differentially expressed genes had the functions related to xenobiotics because they showed significant enrichment in "xenobiotic metabolic process," "response to xenobiotic stimulus," "antioxidant activity," "cis-stilbene-oxide hydrolase activity," "coenzyme binding," "cellular response to chemical stimulus," and "cellular response to xenobiotic stimulus" (**Table 3**; **Figure S2**). As the resistance level increased, the genes of the cotton bollworms showed some significant differences in macromolecule metabolic processes in the LF10 and LF20 strains (**Table 3**; **Figure S3**). Different from other strains, some genes involved in "proton-transporting V-type ATPase complex" and "proton-transporting two-sector ATPase complex" showed significant changes in the LF30 strain (**Table 3**; **Figure S2**). For the LF60 strain, some genes involved in "chitin binding," "sterol binding," and "alcohol binding" showed more significant expression differences than other Go terms, and this was specifically true in this strain (**Table 3**; **Figure S2**). As the highest resistance-level strain LF120, the differentially expressed genes were enriched more significantly in "cellular iron ion homeostasis," "ferric iron binding," "hexachlorocyclohexane metabolic process," "chlorinated hydrocarbon metabolic process," "halogenated hydrocarbon metabolic process," "cellular



transition metal ion homeostasis," "transition metal ion homeostasis," and "iron ion homeostasis" (**Table 3**; **Figure S2**). The enrichment of DEGs in the same or in different pathways provides information that can aid in understanding the development of resistance and the resistance mechanisms in different strains.

In organisms including cotton bollworm, different genes possess special biological functions and coordinate with each other. In another way, through KEGG pathway analysis, significant enrichment can identify DGEs that are involved in the main biochemical pathways and signal transduction pathways. The most significantly enriched (according to the corrected p-value) 20 KEGG pathways are shown in **Figure S3**. The results indicated that the DEGs were enriched more significantly in "propanoate metabolism," "two-component system," and "protein processing" in the endoplasmic reticulum in all resistant strains (**Table 4**). In the five least-resistant strains, the differentially expressed genes were enriched more significantly in "citrate cycle (TCA cycle)" and "carbon fixation pathways in prokaryotes." For the LF5 strain, some DEGs were enriched significantly in "starch and sucrose metabolism" and in "plant-pathogen interaction" pathways. For the LF10 strain, some differentially expressed genes were enriched significantly in "ABC transporters" pathways (**Table 4**) and other genes that differentially expressed between from LF5 and LF 10 were involved significantly in "glutathione metabolism," a pathway that may help to detoxify Cry1Ac toxins. Some DEGs from LF30 were found to be involved significantly in the "mTOR signaling pathway" (**Table 4**), which is a crucial signaling pathway that mediated cell growth and proliferation (Kazuyoshi Yonezawaa, 2004). These results indicated that these genes in LF30 may affect larval growth. Further details of differentially expressed genes that were enriched significantly in the KEGG pathway are shown in **Table 4** and **Figure S3**.

TABLE 3 | Gene ontology (GO) classification of differentially expressed genes in susceptible and resistant strains.


*The most significantly enriched pathways are shown between susceptible and resistant strains.*

# Expression Level of Trypsin Genes Involved in Development of Resistance

The trypsin family is present widespread in animals and plays a variety of roles, especially in the digestive system. Lower expression and activity of trypsin proteases result in decreased activation of the Cry1Ac protoxin and is a mechanism of resistance to Cry1Ac in H. armigera (Liu et al., 2014; Wei et al., 2016a). Fourteen unigenes (comp35781\_c0\_seq1, my\_s32485, comp41058\_c1\_seq5, Unigene12105, Unigene47996, Unigene20462, Unigene36451, Unigene15742, Unigene43312, Unigene8736, comp41058\_c4\_seq1, Unigene31995, my\_rep\_c15473, and my\_rep\_c25150) were matched


TABLE 4 | The KEGG ortholog classification of differentially expressed genes between the susceptible and the resistant strains.

*The most significantly enriched pathways are shown between susceptible and resistant strains.*

to nine corresponding candidate trypsin genes (Gene bank: XM\_021340600, XM\_021340599, XM\_021340597, XM\_021333008, XM\_021329499, XM\_021338512, XM\_021344340.1, XM\_021344341.1, and XM\_021344468.1), and the mRNA levels of these genes were found to be decreased significantly in resistant strains in comparison with the susceptible LF strain (**Table 5**), consistent with the qRT-PCR results obtained in all six resistant strains (**Figure 3**).

#### Expression Level of Cry1Ac-Receptors Genes Involved in Development of Resistance

Several known Bt receptors and Bt-resistance genes including ALP-like (XM\_004928089.1), ALP2 (EU729323.1), and APN5 (AY894814.1, EU325551.1, and EF417486.1) showed the same changed trend in all resistance strains (**Table 5**). The ALPlike genes encoded by fourteen unigenes (Unigene39578, Unigene40436, comp36273\_c0\_seq1, Unigene29103, Unigene5645, Unigene6346, Unigene6678, Unigene29103, Unigene33242, Unigene37010, Unigene40165, Unigene40714, Unigene40726, and Unigene49748) were found to be upregulated significantly based on DGE results (**Table 5**) and these results were further verified by qRT-PCR in all six resistant strains (**Figure 4A**). Another ALP gene, ALP2 (comp33523\_c0\_seq1), was significantly upregulated in the LF5, LF10, and LF20 strains, but there was no significant change in LF30, LF60, and LF120. However, qRT-PCR analysis demonstrated that this ALP2 (comp33523\_c0\_seq1) of H. armigera was ubiquitous and significantly reduced in all six resistant strains (**Figure 4B**). The APN5 (Contig1878, Unigene13560, Unigene13606, Unigene39423, and Unigene5729) was upregulated significantly in the LF-resistant strains according to DGE results (**Table 3**), However, qRT-PCR analysis indicated that this gene was upregulated significantly in the LF5, LF20, and LF30 strains, unchanged in the LF120 strain, and significantly downregulated in the LF10 and LF60 strains (**Figure 4C**). In contrast, APN1 (AF441377) was downregulated significantly in all these six resistance strains according to the qRT-PCR analysis, but not according to the DGE results (**Figure 4D**).


to

each

unigene.

TABLE

*differential expression.*

#### DISCUSSION

Resistance to Cry1Ac is controlled by multiple genes involved in fitness costs and in the selection of recessive or dominant receptors (and even alleles with different types of mutations on the same locus) and their interactions (Tabashnik et al., 2005; Xu et al., 2005; Zhang et al., 2009, 2012; Gahan et al., 2010; Baxter et al., 2011; Jurat-Fuentes et al., 2011; Atsumi et al., 2012; Xiao et al., 2014; Chen et al., 2015). Fitness costs contain longer period of development and reduction in survival, pupal weight, and fecundity (Sayyed et al., 2008). Fitness costs are expected to increase steadily with the development of increased resistance (Cao et al., 2014). According to Cao et al. (2014), who established multiple regressions to predict overall fitness cost and resistance level with fitness costs, these LF-resistant strains may use a second phase of resistance. In this stage, resistance gene-encoding enzymes, such as digestive enzymes, hydrolase, detoxification enzymes, and catalytic enzymes are considered the most important factor to produce fitness cost (Rajagopal et al., 2009; Zhu et al., 2011; Guo et al., 2012; Cao et al., 2013; Lei et al., 2014; Liu et al., 2014; Wei et al., 2016a; Zhang et al., 2017). As a significant and universal phenomenon, we found in this study that a significant portion of DEGs were enriched predominantly in "catalytic activity," "endopeptidase activity," "aminopeptidase activity," "serine-type endopeptidase activity," "proteolysis," "biological process," "metabolic process," "peptidase activity," "metallopeptidase activity," "serine-type peptidase activity," "exopeptidase activity," "serine hydrolase activity," "hydrolase activity," "protein metabolic process," "acting on L-amino acid peptides," "peptidase activity," and "organic substance metabolic process" for all the resistant strains (**Table 3**; **Figure S2**). High-level expression of the genes involved in the earlier-mentioned pathways will help resistant insects to avoid Cry1Ac damage. However, in compensation, these resistant insects develop a lower hatching rate, a lower copulation rate, a lower emergence rate, and even a lower survival rate (Cao et al., 2014). These findings suggest that the resistance to Cry1Ac in H. armigera might also be associated with increased catalytic activity, digestive activity, hydrolase activity, and detoxification activity.

The current understanding of Bt-toxin resistance in insects is associated generally with either conversion of Bt protoxins to activated toxins by insect midgut proteases (Rajagopal et al., 2009; Cao et al., 2013; Liu et al., 2014; Wei et al., 2016a) or by altered binding capacity of toxins to midgut proteins (Xu et al., 2005; Zhang et al., 2009, 2012; Gahan et al., 2010; Baxter et al., 2011; Jurat-Fuentes et al., 2011; Atsumi et al., 2012; Xiao et al., 2014; Chen et al., 2015). Decreased transcript levels of trypsins have been associated with reduced Cry1Ac protoxin activation (Rajagopal et al., 2009; Liu et al., 2014). This result is consistent with our present results, in which nine transcripts encoding trypsin serine protease were found to be downregulated in six LF-resistant strains (**Table 5**; **Figure 3**). Previous studies also found that reduced trypsinlike activity is correlated with reduced expression levels of trypsin gene transcripts in the LF120 strain (Wei et al., 2016a). Similar results were also reported in Ostrinia nubilalis, in which defense against Bt toxins was considered as main mechanism of resistance (Yao et al., 2012; Nanoth Vellichirammal et al., 2015). These results revealed that reduced protoxin activation is considered generally as a resistance mechanism against Bt proteins. More importantly, for the first time, we revealed that these nine trypsin serine proteases downregulated in Cry1Ac-resistant H. armigera was probably a common phenomenon. This result indicated that some trypsin activators may be used to improve the toxicity of Bt to a certain degree.

High levels of resistance are most commonly associated with mutations that disrupt the binding of Cry proteins to midgut receptors, decreased expression of these specific receptors in the midgut, and decreases of the toxin binding to its midgut proteins in the resistant strain (Ferré and Van Rie, 2002; Wu, 2014). While investigating the universal mechanism of resistance to Bt proteins, downregulation of ALP2 and APN1 and upregulation of ALP-like were found in these LF-resistant strains (**Table 5**; **Figure 4**). Reduced activity and transcription of ALP, which binds Cry1Ac, caused the resistance to Cry1Ac in the LF10, LF30, LF60, and LF120 strains when compared with a 96S-susceptible strain (Chen et al., 2015). The ALP1 and ALP2 all had been reported as the receptors of H. armigera (Ning et al., 2010; Chen et al., 2015); however, the total ALP activity was decreased and the transcription of the conserved region of HaALP2 (accession no. EU729323) and HaALP1 (accession no. EU729322) isoforms was also reduced in the resistance strains. It was difficult to know whether ALP1 or ALP2 caused the resistance to Cry1Ac. Here, we pinpoint accurately that the downregulation of ALP2 transcription leads to Cry1Ac resistance in LF resistance. Moreover, in LF Cry1Acresistant H. armigera larvae, a decrease in ALP activity may be correlated with reduced levels of ALP2 transcripts. However, ALP1 seems not to be a gene that importantly associated with resistance to Bt Cry1Ac since ALP1 was verified to be upregulated in all the LF-resistant strains (Zhang, 2013, PhD thesis).

In our study, ALP1 and ALP-like (**Table 5**; **Figure 4**) were found to be upregulated in all these LF-resistant strains. The increased expression of both genes in the LF-resistant strains was associated probably with the gut defensive response to Cry1Ac intoxication. In fact, ALP expression is considered as a marker for stem cell proliferation, which is crucial to gut defensive responses to Cry toxins (Singh et al., 2012). Moreover, midgut regeneration has been proposed as a mechanism of Cry1Ac resistance in H. virescens (Forcada et al., 1999). Further studies are needed to determine the molecular mechanisms responsible for the upregulation of ALP1 and ALP-like in these LF-resistant larvae.

Various studies with glycosylphophatidylinositol (GPI) anchored APN1 from lepidopteran insects consistently demonstrated that APN1 is one of the midgut receptors for Cry1Ac and related to the resistance to Bt toxins (Zhang et al., 2009; Tiewsiri and Wang, 2011; Valaitis, 2011; Flores-Escobar et al., 2013; Wei et al., 2016b). However, our DGE data analysis demonstrated that the expression level of APN1 transcript was not significantly different between the LF-susceptible and -resistant strains. This result contradicted our qRT-PCR analysis (**Figure 4**), which demonstrated that APN1 transcript was decreased significantly in the LF-resistant strains (**Figure 4**). The discrepancy between the DGE analysis and the qRT-PCR analysis may be due to the sensitivity of qRT-PCR, which is higher than that for DEG. The APN5 can also bind to Cry1Ac in H. armigera (Wang et al., 2005), but its involvement in Cry1Ac resistance has not been documented. In this study, we found that APN5 is widely upregulated in the LF-resistant strains using DGE analysis, and this result was confirmed further by qRT-PCR in the LF5, LF20, LF30, and LF120 strains. The upregulation of APN6 has been reported to act as a compensation of APN1 loss in order to minimize the fitness costs of resistance in Trichoplusia ni (Tiewsiri and Wang, 2011). Whether a similar function of APN5 exists in LF5, LF20, LF30, and LF120 H. armigera warrants further study. Other Cry1Ac receptors (cadherin and ABC transports) and related genes showed different expression levels in individual LF-resistant strains, but they did not show a universal mechanism of resistance to Cry1Ac in all LF-resistant strains. This finding suggests that changes in expression of one or more of the Cry1Ac receptors and related genes can influence Cry toxin resistance traits to different degrees. Indeed, additional study is required to decipher the individual roles of the interactions between Bt receptors within the framework of toxin modes of actions. Nevertheless, our results provide evidence that the downregulation of ALP2 and APN1 affected the resistance in both lower and higher levels of resistance. Therefore, these genes may be used as markers to monitor and manage pest resistance in transgenic crops.

Lei et al. (2014) identified unigenes that are differentially expressed between Cry1Ac-susceptible and two resistant Plutella xylostella strains by RNA-seq analysis, and further analysis found that the higher resistance strain showed the greater number of EDUs. However, the higher resistance to Cry1Ac in insects does not always involve the use of more DEGs to adapt to more toxins. Our results showed the numbers of DEGs increased from LF5 to LF10 and LF30, LF60 to LF120, consistent with the resistance ratios of these strains (**Figure 2B**). However, the DEG numbers in the LF30, LF60, and LF120 strains were all lower than those in the LF10 and LF20 and a negative correlation was found between DEG numbers and resistance ratios in the LF10, LF20, and LF30 strains (**Figure 2**). Obviously, our data indicate that this is in response to different selection pressures. Different genes are used to adapt to the new environment, including the evolution of resistance to Cry1Ac, finally developing different resistant mechanisms. This conclusion was confirmed further by previous studies in these LF-resistant strains. For example, the cismediated downregulation of HaTryR expression is considered as the main resistance mechanism in the LF5 strain (Liu et al., 2014). In the LF60 strain, an ABCC2 mutant (in which a 6bp deletion in genomic DNA introduces a premature stop codon) leads to the resistance to Cry1Ac (Xiao et al., 2014). Decreased ALP activity and transcription are considered to cause the Cry1Ac resistance in LF10, LF30, LF60, and LF120 (Chen et al., 2015). Although trypsin activity was decreased significantly in LF120, the high level of resistance to protoxin and activated toxin indicated that reduced activation of the protoxin was not a major Cry1Acresistance mechanism in LF120 (Wei et al., 2016a). Interestingly, the finding of the lowest number of DGEs indicated that a new domain mechanism of resistance has evolved in the LF30 strain (**Figure 2**). This result indicated that different genes and pathways were involved in Cry1Ac resistance in H. armigera. Also, these pathways seem to be differently affected depending on the level of resistance.

The GO and KEGG category analyses provided an important cue to uncover the different mechanisms involved in the development of resistance. First, the initial resistance appeared to increase immune and detoxification processes as shown by the series of DEGs in LF5 strains, predominantly DEGs involved in "xenobiotic metabolic process," "response to xenobiotic stimulus," "antioxidant activity," "cis-stilbene-oxide hydrolase activity," "coenzyme binding," "cellular response to xenobiotic stimulus," and "cellular response to chemical stimulus" (**Table 3**). These DEGs in the LF5 strain lead to increased immune and detoxification functions in the body, thus initiating defense to Bt invasion. Similar results were found in the KEGG pathway analysis, in which a series of DEGs were also enriched significantly in "plant-pathogen interaction," "glutathione metabolism," and "microbial metabolism in diverse environments" (**Table 4**). The changes of these DEGs in the LF5 stain indicated that low-level resistance is probably associated with insect immune and detoxification processes. With the increase of selection pressure involving Cry1Ac, the LF10 and LF20 strains showed different gene changes to that in the LF5 strain. These genes were found to participate in macromolecule metabolic process (**Table 3**) involved in the degradation, metabolism, transport, secretion, and absorption of macromolecular substances (**Table 4**; **Figure S2**). In particular, the 51 unigenes that encode ABC transporters were found to be expressed significantly in the LF10 strain. The ABC transporters, such as ABCG1, ABCC2, and ABCC<sup>3</sup> have been confirmed to be Bt receptors and are related to the resistance to Bt (Xiao et al., 2014; Guo et al., 2015a,b; Tanaka et al., 2017). These results indicate that these genes are associated probably with insect resistance to Bt in the LF10 and LF20 strains. Convincing evidence shows that ABC transporters have been verified to be involved in the mode of Cry1Ac action and the mechanism of resistance to Cry1Ac.

The DEGs involved in "proton-transporting V-type ATPase complex" and "proton-transporting two-sector ATPase complex" were found to be expressed significantly in the LF30 strain (**Table 3**). It is not surprising that V-ATPase takes part in the resistance to Bt because previous reports have demonstrated that a number of V-ATPase subunits can bind to different Bt proteins, including Cry1Ab, Cry1Ac, and Cry4Ba (Bayyareddy et al., 2009; Chen et al., 2010; Xu et al., 2013). Similar results were reported in a study of a Cry1F-resistant strain, in which seven transcripts encoding V-ATPase subunits were identified significantly in downregulation (Nanoth Vellichirammal et al., 2015). It has been reported that V-ATPase subunits are involved in maintaining the alkaline conditions of the midgut (Onken et al., 2008). Further work should be carried out to verify the effect of V-ATPase subunits on midgut pH in the LF30 strain, as the results may provide further insight into resistance mechanisms used by this resistant strain.

At high levels of resistance to Cry1Ac, receptor mutations may be the main reasons underlying the resistance. However, the resistance mechanisms in LF60 have been identified (Xiao et al., 2014). In addition, other factors must be involved in the resistance to Cry1Ac because our results identified 4,990 DEGs that were expressed significantly in the LF60 strain, compared with the LF-susceptible stain. Interestingly, some DGEs were found to act in "chitin binding," "sterol binding," and "alcohol binding" (**Table 3**). Other DGEs were found to function in "geraniol degradation" and "ether lipid metabolism" based on the KEGG ortholog classification. Future studies are needed to determine the roles of these DGEs in the development of resistance in the LF60 strain.

Our previous study confirmed that trypsin activity was decreased significantly in LF120 (Wei et al., 2016a). Correspondingly, in the present study, nine trypsins were found to be significantly downregulated in the LF120 strain (**Table 3**). However, LF120 has the highest levels of mRNA for different trypsins among all resistant strains. These results indicate that the reduced activation of protoxin seems not to be a main mechanism of resistance to Bt proteins in this strain. Meanwhile, DGEs in LF120 were enriched significantly in "cellular iron ion homeostasis," "ferric iron binding," "cellular transition metal ion homeostasis," "transition metal ion homeostasis," and "iron ion homeostasis" (**Table 3**). This result suggested to us that ion homeostasis in the insect's body may play a important role in affecting the resistance to Bt, because unbalanced ion homeostasis can impair the normal functions of proteins within cells, hinder pore formation, and lead to cell death. As reported, Cry toxins can induce the formation of non-selective channels and then lead to imbalance of cations, anions, neutral solutes, and water, finally causing cell swelling and lysis (Knowles and Ellar, 1987). The role of ions, especially iron, in the mechanisms of resistance in the LF120 strain will be explored in future studies.

From LF5 strain to LF120 strain, many pathways seemingly exist. However, a domain pathway contributes hugely to Cry1Ac resistance. The upregulated or downregulated genes may not be fully illustrated in the resistance mechanisms that occur in the LF-resistant strains. Importantly, key genes in different pathways regulating the expressions of resistance-associated genes should be identified further. Also, these key genes may be used to modify via gene edition (CRISPR) for molecular control of the resistance. For example, the interplay between ALP and ABCC is controlled by MAP4K4 in the MAPK signaling pathway in P. xylostella (Guo et al., 2015b). However, as the firstly discovered pathways, V-ATPase, ABC transporters, or ion homeostasis, which are involved in Cry1Ac resistance, are lesser known, and more functional experiments need to be carried out in the future.

### CONCLUSION

Based on our observations, several factors are associated with Cry1Ac resistance in the LF-resistant H. armigera strains. Changes in catalytic activity, digestive activity, hydrolase activity, and detoxification activity and in the downregulations of receptors and related genes, including ALP2, APN1, and trypsin, unavoidably result in resistance to Cry1Ac. The ALP2 and APN1 can, therefore, be considered as probes to monitor the resistance of H. armigera to first-generational Cry1Ac crops in the field. Most importantly, the results here revealed multiple genes and pathways that are probably involved in resistance. Also, these pathways seem to be differently affected depending on the level of resistance. For controlling the lower level Cry1Ac-resistance, some enzyme inhibitors or activators can be explored to improve the toxicity of Bt. As the resistance increases, the catalytic activity, digestive activity, hydrolase activity, and detoxification activity may not be the main role of resistance. Special pathways and genes may be involved in the resistance, such as V-ATPase, ABC transporters, or ion homeostasis, and they seem to differently contribute to resistance depending on the level of resistance. The identification of the key genes that regulate the main pathway contributing to resistance are underway. Also, these genes could be used via gene edition (CRISPR) for molecular control of resistance.

#### REFERENCES


#### DATA ACCESSIBILITY STATEMENT

Original sequencing reads are available from the GenBank Under accession numbers: PRJNA451313.

#### AUTHOR CONTRIBUTIONS

JW and GL conceived and designed the experiments. JW, LC, and SY performed the experiments. GL, SA, and JW analyzed the data. JW, XL, MD, SA, and GL wrote the manuscript. JW, SA, and GL shared the microscopic observations and writing responsibilities. All authors have read and approved the manuscript for publication.

# FUNDING

This research was supported by the Key Project for National Key R&D Program of China (2017YFD0201900), Breeding Genetically Modified Organisms (grant number 2016ZX08011– 002), the National Natural Science Funds of China (grant number 31621064), and the State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201708).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01653/full#supplementary-material


larval midgut. Arch. Insect Biochem. 42, 51–63. doi: 10.1002/(SICI) 1520-6327(199909)42:1<51::AID-ARCH6>3.0.CO;2-6


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responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae. PLoS ONE 7:e44090. doi: 10.1371/journal.pone.0044090


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Wei, Yang, Chen, Liu, Du, An and Liang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Adipokinetic Hormone Receptor Mediates Lipid Mobilization to Regulate Starvation Resistance in the Brown Planthopper, Nilaparvata lugens

Kai Lu1,2† , Xinyu Zhang<sup>1</sup>† , Xia Chen<sup>2</sup> , Yue Li<sup>2</sup> , Wenru Li<sup>2</sup> , Yibei Cheng<sup>2</sup> , Jinming Zhou<sup>1</sup> , Keke You<sup>1</sup> and Qiang Zhou<sup>1</sup> \*

<sup>1</sup> State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China, <sup>2</sup> College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Jae Park, The University of Tennessee, Knoxville, United States Pin-Jun Wan, China National Rice Research Institute (CAAS), China

\*Correspondence: Qiang Zhou lsszhou@mail.sysu.edu.cn †These authors share first authorship

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 18 June 2018 Accepted: 16 November 2018 Published: 29 November 2018

#### Citation:

Lu K, Zhang X, Chen X, Li Y, Li W, Cheng Y, Zhou J, You K and Zhou Q (2018) Adipokinetic Hormone Receptor Mediates Lipid Mobilization to Regulate Starvation Resistance in the Brown Planthopper, Nilaparvata lugens. Front. Physiol. 9:1730. doi: 10.3389/fphys.2018.01730 Lipid storage must be efficiently mobilized to sustain the energy demands during processes of exercise or starvation. In insects, adipokinetic hormone (AKH) and brummer lipase are well-known regulators of lipid mobilization. We recently demonstrated that brummer-dependent lipolysis regulates starvation resistance in the brown planthopper, Nilaparvata lugens, one of the most destructive rice pests. The present work investigated the roles of the AKH signaling system in lipid mobilization during the starvation process in N. lugens. NlAKHR is a typical G protein-coupled receptor (GPCR) and possesses high structure and sequence similarity to other insect AKHRs. Spatial and developmental expression profiles suggested that NlAKH is released from the corpora cardiaca to activate NlAKHR mainly expressed in the fat body. Starvation significantly induced the expression of NlAKH and NlAKHR, indicating a potential role of the AKH signaling system in starvation resistance. To reveal the functions of the AKH signaling system, a double-stranded RNA (dsRNA)-mediated knockdown of NlAKHR and NlAKH peptide injection was performed. The results show NlAKHR silencing decreased the levels of 1,2-diacylglycerol (DAG) in the hemolymph and increased triacylglycerol (TAG) levels in the fat body, whereas NlAKH injection led to a critical accumulation of DAG in the hemolymph and a severe reduction of TAG content in the fat body. Knockdown of NlAKHR resulted in prolonged lifespan and high levels of whole-body TAG, indicating an inability to mobilize TAG reserves during starvation. Conversely, the NlAKH injection reduced the survival and accelerated TAG mobilization during starvation, which further confirms the role of NlAKH in lipolysis. Moreover, NlAKHR silencing caused obesity in N. lugens, whereas NlAKH injection depleted organismal TAG reserves in vivo and produced a slim phenotype. These results indicate that lipid mobilization is regulated by the AKH signaling system, which is essential for adjusting body lipid homeostasis and ensuring energy supplement during starvation in N. lugens.

Keywords: adipokinetic hormone (AKH), adipokinetic hormone receptor (AKHR), lipid mobilization, starvation resistance, Nilaparvata lugens

#### INTRODUCTION

fphys-09-01730 November 28, 2018 Time: 11:3 # 2

The balance between lipid storage and mobilization is a critical characteristic of organismal energy homeostasis (Grönke et al., 2007). Most insects accumulate triacylglycerol (TAG), a strongly hydrophobic neutral lipid droplet with high energy content, as the primary lipid reserve for energy storage (Brown, 2001; Martin and Parton, 2006). TAG is mainly deposited in the fat body, an insect equivalent of adipose tissue, during periods of excessive food resources (Azeez et al., 2014). The ability to mobilize stored TAG reserves is important for the survival of insects under energy-demanding conditions. A tightly regulated balance between lipogenesis and lipolysis adjusts the TAG content in insects and matches acute energy needs in response to the fluctuation of environments (Grönke et al., 2007).

Two lipolytic systems, adipokinetic hormone (AKH) mediated lipolysis and brummer lipase-dependent lipolysis, were reported to be involved in the regulation of TAG mobilization in insects (Stone et al., 1976; Grönke et al., 2005). This contrasts with vertebrates, where only one lipolytic system that involves adipocyte triglyceride lipase (ATGL), a homolog of insect brummer, is known to metabolize TAG (Zimmermann and Zechner, 2004). In insects, the levels of hemolymph lipid and carbohydrates are regulated by AKH, a peptide hormone which is thought to be functionally analogous to the mammalian glucagon (Lee and Park, 2004; Bharucha et al., 2008). AKH was first identified as an insect neurohormone that stimulates lipolysis and locomotor activity in Locusta migratoria (Mayer and Candy, 1969; Stone et al., 1976). To date, more than 60 different kinds of AKHs have been identified or predicted from genome sequencing projects as highly conserved peptide hormones with similar structural characteristics in insect species (Gäde and Marco, 2013). Functionally, insect AKHs have been shown to possess pleiotropic actions. For example, AKHs stimulate lipolysis of TAG into diacylglycerols (DAG) and the fat body-based conversion of glycogen into trehalose in response to starvation (Grönke et al., 2007). In addition to the energy-mobilizing activity, some other functions of AKHs have also been revealed, such as inducting foraging activity in starved Drosophila melanogaster (Lee and Park, 2004), stimulating midgut proteolytic activity in the flesh fly Sarcophaga crassipalpis (Bil et al., 2014) and playing an important role in oxidative stress (Bednáˇrová et al., 2013). Recently, AKH signaling has also been shown to be strongly associated with insect reproduction (Lorenz, 2003; Lindemans et al., 2009; Attardo et al., 2012). AKH is synthesized and released from the corpora cardiaca (CC) into the hemolymph, then binds to a G protein-coupled receptor (GPCR) at the membrane of fat body cells, and eventually mobilizes lipid and carbohydrate reserves (Gäde and Auerswald, 2003; Caers et al., 2012; Gáliková et al., 2015).

The AKH receptor (AKHR) was first identified in the fruit fly D. melanogaster (Park et al., 2002) and the silkworm Bombyx mori (Staubli et al., 2002) as a rhodopsin-like GPCR with seven transmembrane-spanning alpha-helices, which is structurally and functionally analogous to the vertebrate gonadotropinreleasing hormone (GnRH) receptor (Lindemans et al., 2009). To date, AKHRs have been identified or predicted from genome sequencing projects in several other insect species (**Supplementary Table 1**). AKH signaling is achieved by binding peptide hormone with AKHR and then activating various cellular signaling pathways (Staubli et al., 2002). Insect AKH/AKHR signaling involves Ca2<sup>+</sup> and cyclic adenosine monophosphate (cAMP) as intracellular messengers, and the signaling cascades of AKH/AKHR-stimulated lipolysis also involve the activation of protein kinase A (PKA) (Arrese et al., 1999; Van der Horst et al., 2001; Gäde and Auerswald, 2003). AKHR is ubiquitously expressed, however, it is highly accumulated in fat body, where AKH possibly functionsin vivo. In the two-spotted cricket Gryllus bimaculatus, knockdown of AKHR led to the reduction of DAG and trehalose in the hemolymph whilst to elevated levels of TAG in the fat body (Konuma et al., 2012). Meanwhile, AKHR deficiency caused increased starvation resistance and decreased locomotory activity in the crickets (Konuma et al., 2012). Recently, it was shown that flies carrying AKHR mutations suffer from increased TAG and glycogen accumulation, supporting the idea that AKHR contributes to the lipolysis (Isabel et al., 2005; Bharucha et al., 2008). Along with these findings, AKHR mutant flies possess a phenotype of increased starvation resistance, which corresponds to the observation found in the AKH-deficient flies (Lee and Park, 2004; Grönke et al., 2007; Bharucha et al., 2008). Similar results were also obtained in Rhodnius prolixus (Alves-Bezerra et al., 2016) and Bactrocera dorsalis (Hou et al., 2017), wherein the silencing of AKHR reduced the levels of DAG and trehalose in the hemolymph and caused TAG accumulation in the fat body, which further underscores the central role of AKHR in insect lipolysis. The transcript expression of AKHR was also detected in the reproductive tissues, supporting the idea that energy metabolism mediated by the AKH signaling system might be regulated to meet the demands for female reproduction. In the tsetse fly Glossina morsitans, knockdown of AKHR resulted in an accumulation of stored lipids during pregnancy and caused a severe reduction of fecundity (Attardo et al., 2012). Recently, AKHR was reported to modulate female sexual traits, fecundity and flight duration in B. dorsalis (Hou et al., 2017). Therefore, lipolysis mediated by the AKH/AKHR signaling system is not only required for basic physiological functions, but also for survival during prolonged starvation and reproduction in insects.

The migratory brown planthopper Nilaparvata lugens is one of the most destructive rice pests in Asia, with a strong reproductive capacity. Moreover, many field populations have developed high levels of insecticide resistance. Since the resistance and fecundity both heavily rely on proper lipid metabolism, lipolytic systems related to lipid mobilization might significantly contribute to planthopper outbreaks (Lu et al., 2018; Zhou et al., 2018a). In previous studies, we identified brummer, a lipase which regulates lipid mobilization and starvation resistance in the planthopper (Zhou et al., 2018b). However, insect lipolysis is under control of multiple regulatory systems, and AKH-dependent mechanisms underlying the regulation of lipolysis have yet to be elucidated in this insect.

To reveal the role of AKH signaling pathway in lipid storage control, and to address the question of how these components orchestrate acute lipolysis in response to starvation, we identified the AKHR and analyzed its functions in vivo. We first investigated the evolutionary relationship of AKHR orthologs from other insect species, and then analyzed the expression patterns of AKH (Tanaka et al., 2014) and AKHR in different tissues and developmental stages. Then, the expressions of AKH/AKHR signaling system components under starvation conditions were characterized. Finally, the roles of this system in lipid mobilization and starvation resistance were tested by AKHR knockdown via RNA interference and AKH peptide injection. Our results suggest that the AKH/AKHR signaling system is indispensable for acute lipid mobilization and contributes to starvation resistance in the planthopper.

# MATERIALS AND METHODS

fphys-09-01730 November 28, 2018 Time: 11:3 # 3

#### Insects and Sample Preparation

Individuals of N. lugens were maintained at Sun Yat-sen University, which were originally sourced from a colony from the South China Agriculture University in September 2008 (Lu et al., 2015). Insects were reared on fresh rice seedlings (Taichung Native 1) and kept at 26 ± 1 ◦C with 65 ± 5% humidity under a 16/8 h (light/dark) photoperiod condition. Newly emerged females were collected and kept isolated until used for treatments.

Tissues, including head, midgut, ovary, fat body and epidermis, were dissected from 30 females that were 3 days old to investigate tissue-specific expression profiles. The females were anesthetized on ice and dissected in a precooled phosphate-buffered solution (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4; pH 7.4) under a stereomicroscope (SMZ745, Nikon, Tokyo, Japan). Dissected tissues were placed in a 1.5 mL RNase-free centrifuge tube and immediately frozen in liquid nitrogen and stored at −80◦C until use.

Hemolymph was collected using a centrifugation method reported previously with moderate modification (Xu et al., 2006). Briefly, the females were anesthetized with carbon dioxide and the body wall of the thorax was pulled open using a tungsten needle. Then the punctured females were placed in a 0.5 mL RNase-free centrifuge tube with several small holes at the tube bottom. Next, the 0.5 mL tube was placed into a 1.5 mL RNasefree centrifuge tube and centrifuged at 9000 × g at 4◦C for 5 min. Thirty females were combined as one biological sample for hemolymph collection with three independent replicates.

#### RNA Isolation, cDNA Synthesis and Cloning of NlAKHR

Total RNAs from whole bodies or different tissues were isolated using TRIzol reagent (Invitrogen, CA, United States) according to the manufacturer's instructions. RNA integrity was confirmed by 1.5% agarose gel electrophoresis and RNA concentration was determined using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, West Palm Beach, FL, United States). In order to avoid genomic DNA contamination, total RNA was treated with RNase-free DNase I (Promega, Madison, WI, United States). A GoScript Reverse System (Promega) was used to synthesize the first-strand cDNA with 5 µg of total RNA in a 20 µL reaction mixture volume.

A partial cDNA sequence of putative NlAKHR was identified from a transcriptome of N. lugens (SRX023419) by performing a tBLASTn search using D. melanogaster AKHR sequence (NP\_995639) as a query, and then verified by searching against a N. lugens genome database (PRJNA177647) (Xue et al., 2014). The full-length of NlAKHR was amplified using a SMARTTM RACE cDNA amplification kit (Clontech, Mountain View, CA, United States). Gene-specific primers were designed based on the cDNA partial sequence obtained as described above, and DNA polymerase GoTaq Master Mix (Promega) was used for RACE-PCR. Gene-specific outer primers and Universal Primer Mix (UPM) were used for the first round PCR (AKHR-F1 and UPM for 30RACE; AKHR-R1 and UPM for 50RACE) with the following amplification conditions: initial denaturation at 95◦C for 2 min, followed by 35 cycles at 95◦C for 30 s, 50◦C for 30 s, 72◦C for 2 min, and final extension at 72◦C for 5 min. Nested PCR was carried out with gene-specific inner primers and Nested Universal Primer (NUP) (AKHR-F2 and NUP for 3 <sup>0</sup>RACE; AKHR-R2 and NUP for 50RACE) with 35 cycles of amplification (95◦C for 30 s, 50◦C for 30 s, 72◦C for 1.5 min), and the diluted primary PCR amplification product was used as template. PCR products were separated by 1.5% agarose gel electrophoresis and purified (Tiangen, Beijing, China), subcloned into a pGEM-T easy vector (Promega) and transformed into E. coli DH5α competent cells (Tiangen). The inserted cDNA was sequenced by Life Technologies Company (Guangzhou, China).

# Sequence Characterization and Phylogenetic Analysis

The amino acid sequence of NlAKHR was deduced from the corresponding cDNA sequence using the ExPASy Proteomics translation tool, and the transmembrane domains were predicted using the TMHMM server 2.0 (Krogh et al., 2001). The potential signal peptide position was predicted using the SignalP 4.1 Server (Mccarthy et al., 2004). Protein sequences for AKHR were downloaded from GenBank and aligned by ClustalW algorithm and a phylogenetic tree was constructed by the MEGA 6 software using the Maximum Likelihood (ML) method with a bootstrap of 1000 replicates (Tamura et al., 2013).

#### Reverse Transcription PCR (RT-PCR) and Real-Time Quantitative PCR (qPCR)

RNA extraction and cDNA reverse transcription were performed as described above. Primers used for RT-PCR and qRT-PCR were designed by Primer 3 program (Untergasser et al., 2012) and are presented in **Table 1**. Partial cDNA fragments of NlAKHR, NlAKH, NlTUB (alpha 2-tubulin, FJ810204) and NlRPS11 (ribosomal protein S11, FJ810197) were amplified using GoTaq Master Mix (Promega) under the following conditions: initial denaturation at 95◦C for 2 min, followed by 30 cycles at 95◦C for 30 s, 60◦C for 30 s, 72◦C for 45 s, and final extension at 72◦C for 5 min. qRT-PCR was performed with a StepOnePlusTM Real-Time PCR system (Applied Biosystems) using the UltraSYBR Mixture (CWBIO, Beijing, China) under the following reaction conditions: one cycle for 10 min at 95◦C, followed by 40 cycles of 10 s at 95◦C, 15 s at 60◦C and 20 s at 72◦C. A melting curve analysis was performed at the end of each qPCR to check the amplification specificity and to rule out the possibility of primer-dimer formation. All PCR reactions were carried out in triplicate, and at least two technical replicates were performed for each sample. The relative expression levels were calculated using the 2−11CT method (Livak and Schmittgen, 2001) and the stable reference genes NlTUB and NlRPS11 were used for normalization (Yuan et al., 2014).

#### RNA Interference and Bioassay

fphys-09-01730 November 28, 2018 Time: 11:3 # 4

The RNA interference experiment was performed as described previously (Liu et al., 2010). Briefly, double-stranded RNA (dsRNA) was synthesized using T7 RiboMAXTM Express RNAi System (Promega) with the specific primers linked by the T7 promoter sequence at the 5<sup>0</sup> end. The integrity of dsRNA was confirmed by 1.5% agarose gel electrophoresis and dsRNA concentration was determined by a spectrophotometer NanoDrop 2000C (Thermo Fisher Scientific). The conjunctive between prothorax and mesothorax was selected as the dsRNA injection site. Newly emerged females (within 24 h) were anesthetized by carbon dioxide and injected with 23 nL dsRNA (about 100 ng) against the NlAKHR sequence or a


GFP-Ri 5<sup>0</sup> -ggatcctaatacgactcactatagggCAGCAGGACCATGTGATCGCGC-3<sup>0</sup>

F, forward primer; R, reverse primer. Lowercase letters indicate the T7 promoter sequences.

control dsRNA designed against a green fluorescent protein (GFP) gene (ACY56286) using a Nanoject II microinjection device (Drummond Scientific, Broomall, PA, United States). The knockdown efficiency of NlAKHR was measured at 24 and 48 h after dsRNA injection using RT-PCR and qRT-PCR as describe above. Females injected with dsRNA were starved and the number of dead females was counted every 8 h. One hundred individuals were used in each set of repetition and three independent biological replicates were performed in RNAi experiments.

#### NlAKH Treatment

NlAKH (pQVNFSPNW-NH2) was chemically synthesized (GenScript Biotech Inc., Nanjing, China) and dissolved in dimethyl sulfoxide (DMSO). In the present study, 20 pmol of NlAKH was injected twice daily into newly emerged females (within 24 h) using a Nanoject II microinjection device (Drummond Scientific). Females injected with the same volume of DMSO were used as experimental controls. The effects of NlAKH injection on the mobilization of lipid reserves were measured on the third day after treatment. Hemolymph and fat bodies were collected from thirty females in each replicate and three independent biological replicates were performed.

# Lipid Determination

Lipids were extracted as described previously, with moderate modification (Lorenz, 2003; Konuma et al., 2012). Briefly, hemolymph or fat bodies dissected from thirty females were mixed with 100 µL of 75% methanol, containing 10 mg of sodium sulfate and homogenized in a 300 µL mixture of chloroform/methanol (1:1), and then centrifuged at 12000 × g at 4◦C for 10 min. The supernatant was removed into a new tube and mixed with 150 µL of chloroform and 250 µL of 1 M NaCl, and the solvent was evaporated using a vacuum with centrifugation. The lipids from the organic layer were used for lipid quantification using a standard sulfo-phospho-vanillin method (Van, 1985; Xu et al., 2013).

Extracted lipids in chloroform/methanol (2 µL) mixed with 1 mL of sulfuric acid were heated at 100◦C for 10 min and then cooled to room temperature, followed by adding 1 mL of vanillin reagents (0.2% vanillin in 67% ortho-phosphoric acid). Samples were measured at 540 nm using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific), and lipid content was calculated against a lipid standard (cholesterol). Lipids derived from hemolymph and fat bodies were quantified as 1, 2-diacylglycerol (DAG) and TAG, respectively.

# Nile-Red Staining

Fat bodies were dissected in the precooled PBS buffer (pH 7.4) and the adherent tissues were carefully removed with forceps as thoroughly as possible under a stereomicroscope (SMZ745, Nikon). The dissected fat bodies were fixed with 4% paraformaldehyde on a glass slide for 2 h at room temperature and then washed with PBS for three times (3 min × 5 min). For lipid staining, fat bodies were submerged in Nile red solution [1 µL of Nile red (1 mg/mL) in 100 µL of PBS] and visualized using a Ti-S inverted fluorescence microscope (Nikon) within 2 h.

#### Statistical Analysis

fphys-09-01730 November 28, 2018 Time: 11:3 # 5

Results were presented as means ± SE (standard error) based on at least three independent biological replications. Differences between two groups were analyzed by Student's t-test. One-way ANOVA followed by Duncan's multiple comparison was used for the comparison among more than two different conditions. P-values less than 0.05 (<sup>∗</sup> ) or 0.01 (∗∗) were considered to be statistically significant. Graphical representations and all statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, United States).

# RESULTS

# Gene Identification and Phylogenetic Analysis

NlAKH (AB817235) was identified in a previous study (Tanaka et al., 2014) and NlAKHR (MH238458) was cloned in this work. The full-length sequence of NlAKHR is 1610 bp, including a 1212 bp open reading frame (ORF) that encodes a protein consisting of 403 amino acid residues. No signal peptide cleavage was found in NlAKHR. Seven transmembrane domains (TM) were identified in NlAKHR, which indicates that this protein is a member of the GPCR superfamily (**Figure 1**). Alignment of NlAKHR with other insect AKHRs showed that the transmembrane domain regions possessed particularly high conservation. The conserved sequence of DRY (positions 153– 155), which was suggested to participate in AKHR signaling transduction and G-protein coupling, was identified (Wess, 1997). Global alignment showed that NlAKHR possessed a highly conserved structure and sequence homologies with other known insect AKHRs, and the deduced amino acid sequence of NlAKHR was approximately 60% identical to those described previously. Phylogenetic analysis showed that NlAKHR and AKHRs from other insect species clustered in a group, and NlAKHR is most closely related to G. bimaculatus homologs (GbAKHR) (**Figure 2**).

# Spatial and Developmental Expression Profiles of NlAKHR and NlAKH

Expression patterns of NlAKHR and NlAKH in different tissues and developmental stages were determined by RT-PCR and

FIGURE 1 | Nucleotide and its deduced amino acid sequence of Nilaparvata lugens adipokinetic hormone receptor (NlAKHR). The numbering for each sequence is marked on the right. Seven predicted transmembrane (TM) regions 1–7 are shaded in gray. Red: amino acid residues predicted for AKHR signaling transduction and G-protein coupling. The stop codon is labeled by an asterisk.

qRT-PCR. For the tissue-specific expression patterns, different tissues were dissected from 3-day-old females. The highest transcript level of NlAKHR was detected in fat body, followed by head and epidermis, with lower levels in the midgut and ovary (**Figure 3A**). It should be noted that part of the NlAKHR expression found in the head and epidermis of female N. lugens is attributable to the existence of fat body cells in these two organs. Developmental expression profile results showed that NlAKHR was highly expressed in adult males and females, with relatively lower levels in nymphal stages (**Figure 3B**). In contrast, NlAKH was exclusively expressed in the head, but not in the other tissues (**Figure 4A**). The highest expression of NlAKH was observed in adult males, with the lower levels in other nymphal stages and females (**Figure 4B**).

# Effects of NlAKHR Knockdown and NlAKH Injection on Female Starvation Resistance, Lipid Content and Body Weight

Compared to normally fed females, the gene expression levels of NlAKHR increased significantly by 1.4-, 2.4-, 2.4-, and 3.1 fold at 6 (P = 0.035), 12 (P = 0.028), 24 (P = 0.001) and 48 h (P < 0.001) after starvation, respectively (**Figure 5A**). The expression levels of NlAKH were elevated significantly, by 1.8-fold at 48 h after starvation (P = 0.015) (**Figure 5B**). To further confirm the roles of the AKH signaling system in lipid metabolism and starvation resistance in N. lugens, the dsRNA-mediated knockdown of NlAKHR was performed. The dsAKHR treatment of females resulted in a reduction of NlAKHR transcripts by 68.1% (P = 0.005) and 81.9% (P < 0.001) compared to dsGFP-injected controls at 24 and 48 h after dsRNA injection, respectively (**Figure 6A**). Females under starvation conditions after NlAKHR knockdown lived 24 h longer than the dsGFPinjected controls (P = 0.0027) (**Figure 6B**). AKH injection significantly reduced the median lifespan of females by 40% after starvation compared to DMSO-injected controls (P = 0.0226) (**Figure 6C**). Knockdown of NlAKHR led to an excessive accumulation of TAG (1.6-fold) (P = 0.004) and glyceride (1.3 fold) (P = 0.047) compared to the dsGFP-injected controls at 48 h after dsRNA injection. Conversely, AKH injection significantly depleted the TAG and glyceride contents of starved females by 43.2% (P = 0.016) and 55.6% (P = 0.002), respectively (**Figures 6D,E**). Females lacking AKHR function showed signs of obesity, accumulating 12.7% more body weight compared to the dsGFP-injected controls (P = 0.036) (**Figure 6F**). On the contrary,

FIGURE 3 | Expression analyses of NlAKHR in different tissues and developmental stages. (A) qRT-PCR and RT-PCR analyses of NlAKHR expression levels in different tissues from 3-day-old adult females. (B) qRT-PCR and RT-PCR analyses of NlAKHR expression levels in fat bodies from the first instar nymph to adults. Results are represented as means ± SE of three independent samples, and samples are normalized to TUB and RPS11 expression levels. Different lowercase letters represent significant difference of NlAKHR levels among various tissues and developmental stages determined by one-way ANOVA followed by Duncan's multiple comparison test (P < 0.05).

FIGURE 4 | Expression analyses of NlAKH in different tissues and developmental stages. (A) qRT-PCR and RT-PCR analyses of NlAKH expression levels in different tissues from 3-day-old adult females. (B) qRT-PCR and RT-PCR analyses of NlAKH expression levels in heads from the first instar nymph to adults. TUB and RPS11 were used as normalization controls. Results are displayed as means ± SE of three independent replicates. Different lowercase letters represent significant difference of NlAKH levels among various tissues and developmental stages determined by one-way ANOVA followed by Duncan's multiple comparison test (P < 0.05).

AKH injection, which accelerated lipid mobilization, resulted in a 21.9% reduction of body weight and produced slim females (P = 0.001) (**Figure 6G**).

∗∗P < 0.01) by Student's t-test. n.s. represents no significant difference (P > 0.05).

# Effects of NlAKHR Knockdown and NlAKH Injection on Lipid Mobilization

Knockdown of NlAKHR resulted in reduced levels of DAG (34.6% decrease) (P < 0.001) in the hemolymph compared with the dsGFP-injected controls. Conversely, the increased lipid levels in the hemolymph were confirmed by the results that AKH injection led to a critical accumulation of DAG (1.45 fold increase) in the hemolymph (P = 0.002) (**Figure 7A**). TAG levels in the fat body of NlAKHR-silenced females significantly increased compared to that of dsGFP-treated controls (1.9-fold increase) (P < 0.001), whereas AKH injection resulted in a 39.1% reduction of TAG content in the fat body (P < 0.001) (**Figure 7B**). To measure the stored lipid reserves in the fat body, Nile-red staining was performed to visualize the lipid droplets. As shown in **Figure 7C**, the size and number of visualized lipid droplets critically increased after NlAKHR knockdown, whereas AKH injection resulted in a critical reduction of lipid storage droplets in the fat body compared with the DMSO-treated controls.

# DISCUSSION

Two AKHRs, which only differ at their C-terminus by containing phosphorylation sites for GPCR internalization, were identified in Aedes aegypti (Kaufmann et al., 2009) and G. morsitans (Attardo et al., 2012). However, in the present study, only a single copy gene that exhibits a high degree of homology to other AKHRs, was identified from N. lugens. Several lines of evidence support the idea that the putative NlAKHR reported here is indeed an insect AKHR. Firstly, seven transmembrane domains that are involved in GPCR ligand binding and receptor activation are functionally conserved in NlAKHR. In addition, it contains specific amino acid motifs typical for the GPCR family (Gonzalez et al., 2012). Secondly, the isolated NlAKHR was highly analogous to other receptors that have been functionally characterized as AKHR from various insect species. Furthermore, NlAKHR knockdown resulted in decreased levels of circulating DAG in the hemolymph and an accumulation of TAG in the fat body. Our results clearly demonstrate that AKHR is critical for the maintenance of energy homeostasis, possibly due to the structural and functional conservation of the AKH signaling system in the regulation of lipolysis.

Adipokinetic hormone neuropeptides are primarily synthesized in the corpora cardiaca and are responsible for lipid mobilization during energy-demanding processes in a wide diversity of insect species (Auerswald et al., 2005; Auerswald and Gäde, 2006). In D. melanogaster, only a single AKH gene was identified (Gäde, 2010), while two AKH precursors that possess a similar structure typical of the AKH family were identified from the tsetse fly G. morsitans (Attardo et al., 2012) and the mosquito A. aegypti (Kaufmann et al., 2009). Both tsetse AKH peptides are the cognate ligands of GmAKHR, indicating these two AKH genes derived from recent gene duplication (Caers et al., 2016). NlAKH is exclusively expressed in the head of N. lugens, as has been demonstrated to be present in several insect species (Siegert, 1999; Kaufmann and Brown, 2006;

FIGURE 6 | Effects of NlAKHR knockdown and AKH injection on the starvation resistance, whole body lipids and body weight. For RNAi, newly emerged females (within 24 h) were injected with 100 ng of dsRNA against NlAKHR (dsAKHR) or with a control dsRNA (dsGFP). For AKH treatment, newly emerged females were injected with 20 pmol of AKH or DMSO (control). (A) Fat bodies were dissected at 24 and 48 h after dsRNA injection. Differences between NlAKHR expression levels were determined by qRT-PCR and RT-PCR. TUB and RPS11 were used as internal reference controls. Survival rates for dsAKHR-injected females (B) and AKH-treated females (C) under starvation condition were analyzed with Kaplan-Meier plot with a log rank test. TAG (D) and glyceride (E) contents in the whole body of N. lugens were determined at 48 h after injection. (F) Body weight of females was measured on the sixth day after dsRNA injection (n = 23–28). (G) Body weight of females was measured on the third day after AKH treatment (n = 12). Results are represented as means ± SE of three independent replicates and asterisks indicate significant differences (∗P < 0.05 and ∗∗P < 0.01) by Student's t-test. Scale bar, 0.5 mm.

Kaufmann et al., 2009). We also observed the highest expression levels of NlAKH in adult males of N. lugens, which indicates an interesting role for AKH in the energy mobilization associated with flight and, in particular, the reproduction of males. These high expression levels suggest a conserved function of the AKH signaling system in the regulation of lipid mobilization and energy homeostasis. NlAKHR was mostly expressed in the fat body of adult females, as also observed in A. aegypti (Kaufmann et al., 2009), Anopheles gambiae (Kaufmann and Brown, 2006), D. melanogaster (Staubli et al., 2002), and Manduca sexta (Ziegler et al., 2011). This corresponds with the main function of AKH on lipid and carbohydrate mobilization from the fat body under conditions of high energy demand (Marco et al., 2013). In insects, the fat body is the main organ for lipid storage and energy utilization (Arrese and Soulages, 2010). The highest expression level in the fat body also suggests a conserved role of AKHR in the regulation of energy mobilization in N. lugens.

Many studies have demonstrated that the AKH signaling system plays an important role in lipid mobilization. Nutritional status, either feeding or starvation, can significantly affect the expression of AKH signaling system components. For example, AKHR transcript levels decreased after protein meals and it is likely that TAG synthesis may be stimulated and lipolysis may be inhibited in the blood-fed mosquito A. gambiae (Kaufmann and Brown, 2006) and liver-fed flesh fly S. crassipalpis (Bil et al., 2016). It also corresponds well with the decrease of AKH neuropeptides observed in the liver fed S. crassipalpis (Bil et al., 2014). Here, we found that starvation induces the expression of AKH and its receptor AKHR, indicating that the AKH signaling system is involved in the regulation of starvation resistance.

It seems likely that the higher abundance of AKH and AKHR stimulates the use of stored lipid reserves and meets energy demands when food is unavailable. In D. melanogaster, AKHR mutant flies possessed high levels of lipids at the time of death, and this is likely due to the inability of flies to mobilize stored lipids under starvation conditions (Grönke et al., 2007). Our results showed that knockdown of NlAKHR resulted in extended survival during starvation and accumulated lipid reserves, as previously observed in D. melanogaster (Grönke et al., 2007) and G. bimaculatus (Konuma et al., 2012). Conversely, the rate of lipolysis under starvation conditions accelerated and starvation resistance decreased after AKH exposure. Since the lipid reserves deposited in the fat body are the main energy resources under food-deprived conditions, it is likely that AKH signaling systemmediated lipolysis is essential for starvation resistance. NlAKHR knockdown females appear incapable of mobilizing lipid reserves, resulting in an obese phenotype. Similar results have been obtained in the cricket G. bimaculatus, wherein knockdown of AKHR resulted in an increase of TAG in the fat body and a decrease of DAG in the hemolymph (Konuma et al., 2012). AKHR mutants of D. melanogaster also accumulated high levels of TAG in the fat body (Grönke et al., 2007; Bharucha et al., 2008). These results support the idea that TAG mobilization is closely related to AKHR expression, and the AKH signaling system is critical for maintaining energy homeostasis. Here, we demonstrate that knockdown of NlAKHR increased the TAG levels in the fat body and decreased circulating DAG contents in the hemolymph. Conversely, NlAKH-treated females are slim with a severe reduction in stored lipid levels. This phenotype is a probable result of reduced fat body lipid reserves during starvation as AKH accelerates lipid mobilization. The inability to accumulate lipid reserves after AKH exposure was demonstrated in the locust Schistocerca gregaria, and the mosquito A. aegypti (Ziegler, 1997; Gokuldas et al., 2010). In addition, the exposure of NlAKH also affected the distribution of lipids, decreasing TAG levels in the fat body while increasing circulating DAG levels in the hemolymph. Based on these results, we speculate here that lipid mobilization in the fat body is tightly regulated by the AKH signaling system, which is essential in adjusting body lipid homeostasis and ensuring energy supplementation during starvation in N. lugens.

Two lipolytic systems, including the AKH/AKHR signaling system and brummer lipase system, are conserved in N. lugens, as has been shown in D. melanogaster (Grönke et al., 2005, 2007) and G. morsitans (Attardo et al., 2012). These two pathways are somewhat functionally redundant in regard to lipid mobilization; that is, when one pathway is suppressed the other may account for partial compensation. Our previous studies have demonstrated that brummer lipase plays an important role in maintaining hemolymph lipid levels during the starvation period (Zhou et al., 2018a). Given the scale of lipid mobilization required for survival during periods of nutritional stress, it appears that both the AKH/AKHR and brummer pathways are critical for lipid mobilization and starvation resistance (Grönke et al., 2005, 2007). The combination of these two lipolytic systems in N. lugens provides the flexibility to resist various kinds of stress by utilizing the strengths of one system to compensate for the weakness of the other. The molecular mechanisms that govern the cross-talk between these two lipolytic pathways in the regulation of lipid homeostasis and starvation resistance are the focus of research in our laboratory.

#### AUTHOR CONTRIBUTIONS

fphys-09-01730 November 28, 2018 Time: 11:3 # 11

KL and QZ designed the research and wrote the paper. XZ, XC, YL, WL, and YC performed the experiments and analyzed the

#### REFERENCES


data. JZ and KY revised the manuscript. All authors listed have approved the manuscript for publication.

#### FUNDING

This work was supported by the National Natural Science Foundation of China (31772159 and 31601634), Natural Science Foundation of Fujian Province (2017J01428), and Distinguished Youth Talent Program of Fujian Agriculture and Forestry University (xjq201722).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01730/full#supplementary-material


for two peptides and a putative receptor. Insect Biochem. Mol. Biol. 36, 466–481. doi: 10.1016/j.ibmb.2006.03.009


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Lu, Zhang, Chen, Li, Li, Cheng, Zhou, You and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

\*

# Comparative Transcriptome Analysis Between Resistant and Susceptible Rice Cultivars Responding to Striped Stem Borer (SSB), Chilo suppressalis (Walker) Infestation

\*, Dianrong Ma2,3,4 and Xiaoqi Wang<sup>1</sup>

#### Edited by:

Yue Wang<sup>1</sup>†

, Di Ju<sup>1</sup>†

, Xueqing Yang<sup>1</sup>

Su Wang, Beijing Agriculture and Forestry Academy of Sciences, China

#### Reviewed by:

Nianwan Yang, Chinese Academy of Agricultural Sciences, China Abid Ali, University of Agriculture Faisalabad, Pakistan David Mota-Sanchez, Michigan State University, United States

#### \*Correspondence:

Xueqing Yang sling233@hotmail.com Xiaoqi Wang wxq1120@sina.com †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 14 February 2018 Accepted: 15 November 2018 Published: 30 November 2018

#### Citation:

Wang Y, Ju D, Yang X, Ma D and Wang X (2018) Comparative Transcriptome Analysis Between Resistant and Susceptible Rice Cultivars Responding to Striped Stem Borer (SSB), Chilo suppressalis (Walker) Infestation. Front. Physiol. 9:1717. doi: 10.3389/fphys.2018.01717 <sup>1</sup> Key Laboratory of Economical and Applied Entomology of Liaoning Province, College of Plant Protection, Shenyang Agricultural University, Shenyang, China, <sup>2</sup> Rice Research Institute, Shenyang Agricultural University, Shenyang, China, <sup>3</sup> Key Laboratory of Northeast Rice Biology and Breeding, Ministry of Agriculture, Shenyang, China, <sup>4</sup> Key Laboratory of Northern Japonica Super Rice Breeding, Ministry of Education, Shenyang, China

The striped stem borer, Chilo suppressalis (Walker), is a notorious pest of rice that causes large losses in China. Breeding and screening of resistance rice cultivars are effective strategies for C. suppressalis management. In this study, insect-resistant traits of 47 rice cultivars were investigated by C. suppressalis artificial infestation (AI) both in field and greenhouse experiments, using the susceptible (S) cultivar 1665 as a control. Results suggest that two rice cultivars, namely 1688 and 1654, are resistant (R) and moderately resistant (MR) to C. suppressalis, respectively. Then, a comparative transcriptome (RNA-Seq) was de novo assembled and differentially expressed genes (DEGs) with altered expression levels were investigated among cultivars 1688, 1654, and 1665, with or without C. suppressalis infestation for 24 h. A total of 2569 and 1861 genes were up-regulated, and 3852 and 1861 genes were down-regulated in cultivars 1688 and 1654, respectively after artificial infestation with C. suppressalis compared to the non-infested control (CK). For the susceptible cultivar 1665, a total of 882 genes were up-regulated and 3863 genes were down-regulated after artificial infestation with C. suppressalis compared to the CK. Twenty four DEGs belong to proteinase inhibitor, lectin and chitinase gene families; plant hormone signal transduction and plant-pathogen interaction pathways were selected as candidate genes to test their possible role in C. suppressalis resistance. RT-qPCR results revealed that 13 genes were significantly up-regulated and 8 were significantly down-regulated in the resistant cultivar 1688 with C. suppressalis artificial infestation (1688AI) compared to the CK. Three genes, LTPL164, LTPL151, and LOC Os11g32100, showed more than a 10-fold higher expression in 1688AI than in 1688CK, suggesting their potential role in insect resistance. Overall, our results provide an important foundation for further understanding the insect resistance mechanisms of selected resistant varieties that will help us to breed C. suppressalis resistant rice varieties.

Keywords: Chilo suppressalis (Walker), rice, resistance identification, RNA-Seq, systemic induced defense

# INTRODUCTION

fphys-09-01717 November 28, 2018 Time: 20:56 # 2

Rice (Oryza sativa L.) is the most widely consumed food crop in the world (Chen et al., 2011), being the food staple for over 1 billion people in China and 2 billion people in other countries (Herdt, 1991). However, rice is frequently attacked by rice stem borers, a major group of lepidopteran pests of rice (Lu et al., 2017) causing annual losses of US\$1.69 billion (Sheng et al., 2003). Among these stem borers, Chilo suppressalis (Walker), commonly known as the striped stem borer, is a widely distributed destructive pest of rice that has greatly reduced rice production in China (Qu et al., 2003). Rice plant damage by C. suppressalis larvae includes boring into the stem and feeding inside, resulting in "dead hearts" (yellowing and withering of the stem) at the vegetative stage and "white heads" at the reproduction stage (Pathak, 1968). Once infested, such a rice plant will fail to produce an effective panicle.

Various control strategies including mechanical, biological, chemical, and cultural control have been used for striped stem borer management (Li, 1982). However, effective management of striped stem borer mainly depends on chemical insecticides (Su et al., 2014). Misuse of insecticides has resulted in severe insect outbreaks, environmental pollution, insecticide resistance, and food security problems (Chen et al., 2011). Thus, newer and safer pest management strategies are urgently needed for rice production in China. Agricultural biotechnology has been extensively explored to solve these problems in China, and genetic engineering approaches have raised the possibility of achieving high levels of resistance to stem borers in rice (Vila et al., 2005). However, such genetic engineering approaches are technically difficult, and their long term effects and safety have not yet been determined (Chow et al., 2016). Screening rice varieties for high levels of resistance to the striped stem borer is an effective strategy for pest management. However, the identification and screening for rice varieties resistant to the striped stem borer have not yet been well studied.

In this study, we screened and identified the resistance traits of 47 rice cultivars to the striped stem borer by artificial infestation in both field and greenhouse experiments. We then adopted the Illumina sequencing platform to construct a transcriptome database for comparative analysis to identify susceptible and resistant rice varieties with or without infestation by striped stem borer larvae. Differentially expressed genes (DEGs) with altered expression between susceptible and resistant varieties were identified and verified by quantitative real-time PCR (RTqPCR). This is the first data set to select resistant rice varieties and analyze resistance-related genes; this resource provides an important foundation for further understanding of the insect resistance mechanisms of selected resistant varieties and also can help us breed for rice resistance to C. suppressalis.

# MATERIALS AND METHODS

#### Insects

Larvae of susceptible C. suppressalis to host plant were supplied by the Test Center of Pesticides, Shenyang Chemical Industry Research Institute and were fed on japonica rice seedings in an artificial climate chamber (MLR-352H-PC, Panasonic). The insects had been maintained without insecticide exposure for over 30 generations in the laboratory at 25 ± 1 ◦C, 80 ± 1% RH and a photoperiod of 16:8 (L:D) hours.

#### Rice Cultivars

Tested rice cultivars (n = 47) including japonica, indica and weedy rice were provided by Rice Research Institute of Shenyang Agriculture University, Shenyang, Liaoning Province of China.

# Resistance Identification

#### Field Identification

The germinated rice seeds were sown on April 21, 2014 and transplanted 1 month later. Forty seven rice cultivars were designated as 47 treatment plots. The rice seedlings were transplanted in triplicate; thus a total of 141 experimental plots were conducted at the Rice Research Institute of Shenyang Agricultural University. Each experimental plot consisted of one row with 10 holes. Holes were spaced 16.7 cm apart and plants within each row were spaced 26.6 cm apart. The highly susceptible cultivar 1665 was used for the control because its tillering ability is stronger than the weedy rice and its growth period is close to the other cultivars (Wang et al., 2015; Wang, 2016). No pesticides were used during the whole growth period of plants.

During the tillering period, an artificial infestation was introduced from July 7 to 8, 2014. The density of infestation was 2 seedlings with one worm, which is according to the standard of the International Rice Research Institute (Pathak et al., 1971) and Zhou's methods (1985). The primary hatching larvae were inoculated into the ligule between the stem and the second or third leaf sheath. To minimize confounding factors, any natural enemies and other insects on the plants were removed before inoculation and the plants were covered with a 40 mesh cage. At this time, the total number of tillers of each plant was recorded. The number of "dead hearts" was surveyed 30 days later, and the "dead heart" rate was calculated according to methods in a previous study (Wang et al., 2015). The damage index was calculated by the following formula: Damage index = dead heart rate/dead heart rate of control. The resistance levels of rice cultivars are determined according to Pathak et al. (1971) and are shown in **Table 1**.

#### Indoor Identification

Based field identifications, 15 highly representative rice cultivars with similar growth periods were selected for further resistance identification in the greenhouse. The highly susceptible cultivar 1665 was used as the control.

This study was conducted in the Pesticide Creation Team of Shenyang Sinochem Agrochemicals R&D Co., Ltd. No other rice pests were reared and no pesticides were used in the greenhouse during the whole growth period of these plants. The rice was planted using the pot-culture method in the greenhouse at 28 ± 2 ◦C, 70 ∼ 80% RH, and a photoperiod of 16:8 (L:D) hours. On 12th January 2015, the seeds were


TABLE 1 | Grading standard for the strength of resistance<sup>∗</sup> .

fphys-09-01717 November 28, 2018 Time: 20:56 # 3

<sup>∗</sup>Grading standard of resistance refers to Pathak et al. (1971).

soaked in water with 6 days for germination, and were then planted in small plastic cups (10 cm in diameter and 12 cm high). Rice seedlings were then transplanted into square pots (18 cm<sup>∗</sup> 26 cm<sup>∗</sup> 12 cm) with soil after 16 days. Three replicates of each rice cultivar were planted in individual pot (with 8 holes). On March 10, as the rice was in the tillering stage, larvae were artificially introduced as described above. After infestation, each pot was placed 20 cm apart to prevent the larvae from an adjacent pot affecting the results of the experiment by heteroicous. The total number of tillers and "dead hearts" were surveyed, and the "dead heart" rate was calculated as described above.

#### Feeding Induction Treatment

Based on resistance identification results both in field and greenhouse, a series of high resistance cultivars (1688), moderate cultivars (1654) and susceptible cultivars (1665) were placed in the greenhouse as described above. The experiment contains 4 replicates. For each replicate, two pots were randomly divided into two groups, one was the control group (with infestation) and the other was the experimental group (with infestation). Two rows (one row was used for artificial infestation and the other one was regarded as a guarding row), each with 4 rice holes were planted in each pot. For the treatment group (CK, for short), a third instar larva of striped stem borer was placed onto the stem of rice when the rice seedlings had grown to the tillering stage. When half of the larva body had drilled into a stem of rice, a label that marked the time and tillering was tagged on the stem. After 24 h of this artificial infestation, the leaf sheath in which the tiller with larva was located was split gently and the larva was removed. The plant was then packaged in silver paper and was subsequently frozen in liquid nitrogen and stored at −80◦C for later use. The plant without any artificial infestation was similarly sampled and treated.

#### Effect of Diet Adding Rice Plant Powder on the Survival Rate and Larval Weight of C. suppressalis

Larval rearing diet was prepared according to Li et al. (2015) with or without adding rice plant powder (145 g) of different cultivars at the tillering stage. Diet was placed in a glass tube (2cm dia., 10 cm long). Newly hatched larvae were inoculated into different diets with a density of 30 larvae/tube. After 10 days, survival rate and larvae weight were determined. Three replicates were run.

#### RNA Extraction, cDNA Synthesis and Illumina Sequencing

Frozen samples were ground in liquid nitrogen using a mortar and pestle. Total RNA of 1688, 1654, and 1665 with or without artificial infestation was isolated using the Ultrapure RNA Kit (CWBIO, Beijing, China) according to the manufacture's protocol and treated with RNase-free DNase I. The quality and concentration of DNase I-treated RNA was determined using a NanoDrop 2000 UV–vis Spectrophotometer (Thermo Scientific, Waltham, MA, United States). mRNA was isolated from the total RNA using magnetic beads with oligo (dT) and sheared into short fragments using fragmentation buffer. The cDNA was then synthesized using the mRNA fragments as templates using Reverse Transcription Kit Prefect (TaKaRa) following the manufacture's protocol. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. The short fragments were then connected with sequencing adapters and analyzed by agarose gel electrophoresis. Suitable fragments were enriched by PCR amplification to construct the cDNA library. Six pooled cDNA libraries were constructed using an mRNA-Seq assay for paired-end transcriptome sequencing and sequenced on an Illumina HiSeq2000 system at Beijing Genomics Institute (BGI, Shenzhen, China). The raw data from Illumina deepsequencing are available in the NCBI Short Read Archive (SRA) (SRP142306).

# Functional Annotations and Deep Analysis of Gene Expression

The raw reads generated by Hiseq2000 were filtered to remove low-quality reads (reads containing adaptor, reads containing >10% unknown nt "N," and reads with >50% quality value ≤ 10). After filtering, the remaining reads, which were called clean reads, were used for downstream bioinformatics analysis. We used BWA to map clean reads to the entire genome reference<sup>1</sup> and used Bowtie to gene reference. Clean reads were mapped to the selected references, and the statistics of alignment results were presented for each reference. After clustering, the unigenes were divided into clusters and singletons. Assembled unigenes were subjected to blastx (BLAST, the basic local alignment search tool) alignment (E-value < 1e-5) and several protein databases, including blast nt, description, KEGG Orthology, Gene Ontology (GO) analysis (GO Component, GO Function and GO Process) and PCA analysis. GO function of all-unigenes were categorized by Blast2GO, according to molecular function, biological process and cellular component. The genes' complex biological behaviors were further examined by pathway annotation using KEGG identifiers<sup>2</sup> .

<sup>1</sup>http://rice.plantbiology.msu.edu/

<sup>2</sup>http://www.genome.jp/kegg/

# Differential Gene Expression in Resistant and Susceptible Rice Cultivars With or Without C. suppressalis Artificial Infestation

Differential expressed genes (DEGs) were determined based on their expression abundances in different treatment groups. The FPKM method was used to calculate expression levels of genes and to quantify transcript levels among the different samples, which eliminates the influence of different gene lengths. The GO database was used to annotate DEGs and the numbers of DEGs in each GO term were calculated. KEGG pathway analysis of the DEGs was also performed to identify the associated biochemical and signal transduction pathways.

#### Real-Time Quantitative PCR (RT-qPCR) Validation

The expression abundance of selected genes was determined by RT-qPCR to verify the transcriptome results. The 18s rRNA was chosen as a housekeeping gene (Kim et al., 2003). Using the Prime3 software tool (Rozen and Helen, 2000), primers were synthesized by Sangon Biotech (Shanghai, China). The sequences of primers used are shown in **Table 2**.

The expression patterns of candidate genes were analyzed by RT-qPCR using a Bio-Rad CFX96 (Bio-Rad, Hercules, CA, United States). Each reaction contained 12.5 µl of 2 × SYBR Premix Ex Taq II (TaKaRa), 2 µl of cDNA, and 1 µM of genespecific primers in a final volume of 25 µl. The RT-qPCR was biologically repeated three times with independently synthesized cDNA. Three technical replicates were used for each sample. A negative control using RNase-free water instead of cDNA and a no transcription control were also conducted. Relative expression levels of each gene were calculated using the 2−11Ct algorithm. In addition, a correlation analysis between RNA-Seq and RT-qPCR results was conducted.

# Data Analysis

Results are reported as mean ± standard deviation (SD). The data for resistance identification was analyzed by one way analysis of variance (ANOVA, P < 0.05). Fisher's least significant difference (LSD) test was used to separate means (P < 0.01). The software SPSS v17.0 (IBM Inc., Chicago) was used for data analyses.

# RESULTS

# Resistance Identification

#### Field Identification

After 30 days of artificial infestation, all rice cultivars demonstrated the symptoms of "dead heart" except the rice cultivar 1688. The dead heart rate of 47 rice cultivars ranged from 0 (1688) to 43.5% (1677) and the damage index ranged from 0 to 87.33% (**Table 3**). The variance analysis results showed that there was a significant difference between different cultivars (P < 0.001; F = 3.085; df = 46, 94). One highly resistant (HR) cultivar (1688), 3 resistant cultivars (Z46, 1611 and 1689), 13 moderately resistant (MR) cultivars, 15 endurance cultivars, 12 susceptible cultivars and 2 highly susceptible cultivars (Z52 and 1677) were determined according to their resistance grades (**Table 4**). The control cultivar 1665 exhibits the highest dead heart rate (49.81%). The 13 MR cultivars are 1610, Z48, 1654, 1663, 1683, 1681, 1676, Z43, 1660, Z47, 1682, 1687, and 1678. The high endurance cultivars are 1669, 1656, 1661, 1690, 1670, 1609, 1572, 1685, 1565, 1571, 1659, 1655, 1686, 1679, and Z50. The 12 susceptible cultivars are 1668, 1657, 1674, 1684, 1664, 1667, Z49, 1666, 1612, 1652, 1653, and Z51 (**Table 3**).

#### Indoor Identification

Based on the results of field identification, 15 highly representative rice cultivars (**Table 4**), including 1688, were selected for further resistance identification in the greenhouse. Results further showed that 1688 is a resistant cultivar, with the lowest dead heart rate of 5.56%, whereas the control cultivar 1665 exhibits a 85.83% dead heart rate (**Table 4**). The highly susceptible cultivar1677 determined by field identification also performed as a susceptible cultivar, and had the highest rate of dead hearts (112.63%) in 15 rice cultivars (**Table 4**). Combining the results of field identification with indoor identification, 1688 is a resistant cultivar and 1654 is a MR cultivar.

#### Effect of Diet Adding Rice Plant Powder on the Survival Rate and Larval Weight of C. suppressalis

Statistics suggest that the C. suppressalis larvar survival rate of rearing with basal diet + 1665 powder was significant higher (P < 0.05) than that rearing with basal diet, basal diet + 1688 powder, and basal diet + 1654 powder. The larvae weight of C. suppressalis was significant higher (P < 0.05) when rearing with basal diet than those rearing with basal diet adding rice plant powder; larvae weight of C. suppressalis rearing with basal diet + 1688 powder was significant higher (P < 0.05) than those rearing with basal diet adding 1665 or 1654 rice plant powder (**Supplementary Table S1**).

#### Results of RNA-Seq

After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) were used to isolate mRNA (for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA was fragmented into short fragments. Then cDNA was synthesized using the mRNA fragments as templates. Short fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments were connected with adapters. After agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. During the QC steps, the Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. Six cDNA libraries were prepared from three cultivars of 1688, 1654, and 1665, which included the control group (CK) and Artificial Infestation treatment (AI) for each cultivar. Then the six cDNA libraries were subjected to Illumina sequencing. Illumina paired-end sequencing generated

#### TABLE 2 | Sequences of primers.

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a total of 295.5 million raw reads. After cleaning and quality checks, 293.6 million clean reads (14.8 Gb) were obtained, with an average of 48.9 million reads (∼2.5 Gb) per sample. The 1654AI, 1654CK, 1665AI, 1665CK, 1688AI, and 1688CK cDNA libraries generated 49,054,738, 48,913,078, 49,011,744, 48,730,342, 48,937,446, and 48,997,360 clean reads, respectively



HR, highly resistant; R, resistant; MR, moderately resistant; E, endurance; S, susceptible; HS, highly susceptible.

(**Table 5**). The sample of 1654AI has the highest clean reads (49,054,738) and genome map rate (86.98%). The highest rate of genome map is 84.61% (1688AI) and the lowest rate is 83.09% (1654AI).

#### Number and Expression Levels of Genes Among Three Cultivars

The 1654AI, 1654CK, 1665AI, 1665CK, 1688AI, and 1688CK cDNA libraries generated 30,060, 29,289, 27,961, 29,569, 29,803,


R, resistant; MR, moderately resistant; E, endurance; S, susceptible; HS, highly susceptible.

TABLE 5 | Sequence and assembly summary of transcriptome.


AI, artificial infestation; CK, control treatment group without artificial infestation.

and 29,897 expressed genes, respectively (**Table 5**) and 37,608, 36,616, 34,915, 36,814, 36,880, and 37,134 expressed transcripts, respectively (**Table 5**). Genes with an adjusted p-value of <0.05 found by DESeq were assigned as differentially expressed. There were 1,144, 1,874, and 1,819 up-regulated genes and 1,426, 2,286, and 1,801 down-regulated genes in 1665-VS-1654, 1665-VS-1688, and 1654-VS-1688, respectively. After artificial infestation with C. suppressalis, the resistant (R) cultivar 1688 had 4,774 upregulated genes and 3,794 down-regulated genes compared with the susceptible (S) cultivar 1665. The MR cultivar 1654 had 5,126 up-regulated genes and 1,556 down-regulated genes when compared with 1665. Moreover, in the group of 1654AI-VS-1688AI, there were 2,757 up-regulated genes and 5,270 downregulated genesin 1654 compared with the 1665 (**Figure 1**).

#### Number and Expression Levels of Genes Between Different Treatments

Using DESeq software to analyze the expression level of differential genes, a total of 6,421 DEGs with 2,569 up-regulated genes and 3,852 down-regulated genes were identified in 1688 after artificial infestation with C. suppressalis, compared with the non-infested control. For 1654, only 1,861 genes were upregulated and 971 genes were down-regulated after artificial infestation with C. suppressalis compared to the non-infested control. For susceptile cultivar 1665, a total of 4,745 DEGs were identified, but only 882 genes were up-regulated and 3,863 genes were down-regulated after artificial infestation with C. suppressalis compared to the non-infested control (**Figure 1A**). As shown in **Figure 1**, the differentially-expressed genes in the group of 1688CK-VS-1688AI are more than in the groups 1665CK-VS-1665AI and 1654CK-VS-1654AI. When comparing 1654AI and 1688AI, there are 5,270 down-regulated genes. In other words, cultivar 1688 has 5,270 down-regulated genes with cultivar 1654 after artificial infestation which had 1,801 downregulated genes before that (**Figure 1**). Those changes among all the groups may be caused by feeding induction. The stress response of moderate cultivar 1654 to feeding induction is less significant than for the other two cultivars (1688 and 1665) (**Figure 1**).

#### Possible Genes Related to C. suppressalis Resistance

A deep analysis based on DEGs, including Gene Ontology (GO) enrichment analysis, pathway enrichment analysis and so on, was conducted. All the differentially-expressed genes of sequencing samples in GO enrichment analysis are divided into three main categories: biological processes, cellular components and molecular functions (**Figure 2**).

FIGURE 1 | Statistics of differentially-expressed genes in three rice cultivars after SSB artificial infestation compared with the non-infested control: (A) within; and (B) between cultivars. (AI) artificial infestation; (CK) non-infested control.

The DEGs are divided into 45 sub-categories, including 20, 13 and 12 sub-categories in biological processes, cellular components, and molecular functions, respectively in the cultivar 1688 after artificial infestation (**Figure 2A**). The DEGs in 1665 after artificial infestation are divided into 41 sub-categories belonging to three main GO categories, including 17 in biological

represents the name of the pathway, while the horizontal axis represents the rich factor. The greater the rich factor, the greater the degree of enrichment. The size of the point indicates the number of DEGs in this pathway, and the color of the points corresponds to different Q-value ranges. The values of Q were between 0 and 1. The closer the Q-value is to zero, the more significant the enrichment).

processes, 12 in cellular components and 12 in molecular function sub-categories, respectively (**Figure 2B**). The DEGs in 1654 after artificial infestation are divided into 37 subcategories belonging to three main GO categories, including 15 in biological processes, 11 in cellluar components and 11 in molecular function sub-categories, respectively (**Figure 2C**). **Figure 2** showed that those DEGs were mainly enriched in the biologicalprocess group. Those DEGs were mainly involved in functions of the metabolic process, biological regulation,cellular processes, localization and signaling. These results indicate that the majority of DEGs in response to C. suppressalisfeeding might be related to various metabolic processes, implying the resistance of rice involves changes of metabolites.

We also used KEGG to analyze the pathways of the DEGs. KEGG provides the integration of pathways, such as metabolic pathways, plant–pathogen interaction pathways and plant hormone signal transduction pathways, in which the DEGs are involved. In the pathway enrichment analysis, 66,360 differentially-expressed genes of samples were annotated. A total of 22,120 differentially-expressed genes of cultivar 1688 were annotated using pathway enrichment analysis, which consisted of 125 pathways. There were 121 pathways in cultivar 1654

which were the same as in cultivar 1665. Using a probability of false discovery rate (FDR) of ≤0.05, the top 20 statistics for pathways are shown in **Figure 3**. These were significantly enriched in each cultivar in response to C. suppressalis feeding. In addition, the plant hormone signal transduction pathway and plant–pathogen interaction pathway, which involve jasmonic acid (JA), salicylic acid (SA), ethylene (ET), mitogen-activated protein kinase (MAPK) and the WRKY family, all play an important role in rice resistance (Eulgem et al., 2000; Zheng et al., 2006; Pandey and Somssich, 2009; Wu and Baldwin, 2010; Arimura et al., 2011; Birkenbihl et al., 2012; Erb et al., 2012; Fu et al., 2012; Chujo et al., 2013; Wu et al., 2012; Wei et al., 2013). Therefore, it is interesting to investigate DEGs within these two pathways.

#### Validation the Expression of DEGs Using RT-qPCR

A total of 24 DEGs (P-values < 0.01) were screened as candidate genes to test theirpossible role in C. suppressalis resistance. DEGs that were significantly enriched for resistance and C. suppressalis feeding induction were investigated using RT-qPCR. These 24 candidate genes include 5 proteinase inhibitor genes (LOC\_Os07g11650, LOC\_Os03g02050, LOC\_Os01g12020, LOC\_Os03g59380, and LOC\_Os08g03690), 3 lectin genes (LOC\_Os07g03880, LOC\_Os06g10790, and LOC\_Os07g18230), 3 chitinase genes (LOC\_Os10g39700, LOC\_Os08g41100, and LOC\_Os05g33150), 9 genes in the plant hormone signal transduction pathway (LOC\_Os07g04220, LOC\_Os01g12160, LOC\_Os03g15880, LOC\_Os05g37690, LOC\_Os09g26780, LOC\_Os02g34320, LOC\_Os04g51070, LOC\_Os03g56950, and LOC\_Os11g32100) and 4 genes in plant-pathogen interaction pathway (LOC\_Os12g02420, LOC\_Os01g09080, LOC\_Os08g09900, and LOC\_Os03g58420).

Of the 24 candidates identified from RNA-Seq differential expression analysis, 21 were verified using RT-qPCR. Statistical analysis of the RT-qPCR data revealed that 13 genes were significantly up-regulated and 8 were significantly downregulated in the artificially infested group of resistant cultivar 1688 (1688AI) compared to the non-infested control group of 1688 (1688CK) (**Figure 4**). These results are in accord with those of RNA-Seq. Three genes showed a more than 10-fold higher expression in 1688AI than in 1688CK: LTPL164 (86-fold), LTPL151 (13-fold), and LOC Os11g32100 (17-fold) (**Figure 4**).

A correlation analysis between RNA-Seq and RT-qPCR results was conducted. The high correlation (r = 0.899, P < 0.0001) between RNA-Seq and RT-qPCR results (**Figure 5**) suggests that the results of RNA-Seq and RT-qRCR were consistent, and also suggests that the selected DEGs maybe involved in insect resistance.

#### DISCUSSION

#### Resistance Identification

In this study, the resistance traits of 47 rice cultivars from three different types (japonica rice, indica type rice, and weedy rice) to the striped stem borer were determined by field identification and

indoor identification. Using a resistance identification method proposed by Zhou (1985), Shu et al. (2003), cultivar 1688 was identified as a HR cultivar in the field identification trial, but indoor trial result suggests that it a resistant cultivar.

In this study, the dead heart rates for control cultivar 1665 are different between field identification (49.81%) and indoor identification (85.83) trails (**Figure 6**). The different results of resistance identification between field identification and indoor identification are similar with the result of Zhou's research (1985). The possible reason for such a large difference may be the fact that the method can be easily affected by many factors, such as the density of plants, spacing of experiments, climate conditions and so on (Luo et al., 2006). Compared with field conditions, the greenhouse has suitable temperatures and a mild climate, with no natural enemies and other insects. As a result, the survival rate and the boring rate of larvae were increased in the greenhouse, and the larvae transferred frequently to find suitable boring spaces after artificial infestation. In addition, the pot-culture method limited the thickness of the soil and therefore the absorption rates of water, nitrogen and phosphorus decreased, causings lower rice growth. Therefore, the dead heart

FIGURE 6 | Comparison of the growth among 1688, 1654, and 1665. (A) Growth status of 1688 after 30 days of artificial infestation. (B) Growth status of 1654 after 30 days of artificial infestation. (C) Growth status of 1665 after 30 days by artificial infestation. (Method: a third instar larva of striped stem borer was placed onto the stem of rice when the rice seedlings had grown to the tillering stage. When half of the larva body had drilled into a stem of rice, a label that marked the time and tillering was tagged on the stem. After 24 h of this artificial infestation, the leaf sheath in which the tiller with larva was located was split gently and the larva was removed. The plant was then packaged in silver paper and was subsequently frozen in liquid nitrogen and stored at −80◦C for later use. The plant without any artificial infestation was similarly sampled and treated).

rate and damage index are higher in the greenhouse than in the field.

In this study, we found that japonica rice is more resistance to striped rice borer than is indica rice. Eleven of 32 (34.4%) japonica rice units were found to be resistant (R), MR or HR to striped rice borer. But this rate was 28.6% (2 of 7) in indica rice. This result is in line with previous research (Zhou, 1985; Gu et al., 1989; Hao, 2011). Previous studies have found that the damage of SSB is closely related to the width of a leaf, the degree of compactness of the sheath, and the vascular bundle interval, etc. (Hao, 2011). Thin short stems and appressed sheaths are all adverse factors for boring or the growth of larvae, which in turn can cause growth and developmental delays and a high mortality rate (Gu et al., 1989). Compared with japonica rice, indica type rice is more sensitive because it has thick and strong stems and loose sheaths, etc. (Zhou, 1988). In addition, the HR cultivar 1688 and MR cultivar 1654 observed in this study have erect and lodging-resistant stems, more grains and a higher density of leaf-trichomes. This might be adverse to attack by striped rice borer. Actually, we found that some of the larvae died in the process of drilling. Our results suggest that 1688 and 1654 can be cultivated as striped rice borer resistant cultivars in the rice-planting regions of Northeast China.

#### RNA-Seq

Although the experimental design was reasonable and its operation was normative, there are still some drawbacks in this work for reducing the error of research results. Firstly, no time gradients were set for feeding induction. Rice samples were artificially infested with striped rice borer for 24 h and then rice plants were sampled and sequenced. In other words, the results of RNA-Seq reflect the changes of gene expression during the 24-h period. If there were time gradients such as 0, 3, 6, 12, 24, and 48 h, results could be more comprehensive and differentiallyexpressed genes and pathways could be analyzed more accurately. Secondly, a mechanical damage study could be conducted. Daily cultivation management, investigation techniques, artificial infestation or sampling may cause mechanical damage, resulted in changes in gene function and metabolic pathways. Therefore, artificial mechanical damage should be carried out in the future research. Although no biological replicate was conducted for RNA-Seq, each treatment was sampled in a mixed pool and was RNA-sequenced. A correlation analysis between RNA-Seq and RT-qPCR was conducted; the high correlation suggests that the results of RNA-Seq and RT-qPCR were consistent.

# Screening of DEGs

#### Proteinase Inhibitor Genes

Proteinase inhibition plays an important role in plant defense systems that can inhibit the activity of substances such as trypsin and chymotrypsin,etc. In 1989, Johnson found that the transgenetic tobacco plants carrying the pi-I gene and pi-II gene all showed a good ability to resist insects (Johnson et al., 1989). In this category, the LTPL164, LTPL151, LTPL28, LTPL24, and LTPL18 gene expression levels increased. These five candidate genes belong to the cytoplasmic vesicle of the cellular component in the GO enrichment analysis. Meanwhile, LTPL151, LTPL28, and LTPL24 also belong to the binding of molecular function and localization of biological processes. LTPL 18 is involved in both of starch and sucrose metabolism pathways. LTPL164, LTPL24, and LTPL18 are the significant differentially-expressed genes of the HR cultivar 1688 (P < 0.01), but they are not significant in

the moderate cultivar 1654 and highly susceptible cultivar 1665 (P > 0.05). The gene expression of LTPL164 and LTPL24 was 0 FPKM in 1688CK; then the expression level increased after artificial infestation (6082 FPKM and 5 FPKM, respectively). The expression of LTPL151 were 1FPKM in 1688CK and 1850 FPKM in 1688AI which was not a significant differentiallyexpressed gene of 1665 (P > 0.05); the expression level of this gene was up-regulation from 1654CK to 1654AI. The expression level of the candidate gene of LTPL28 increased in cultivar 1688 and cultivar 1654 after artificial infestation, but decreased in cultivar 1665. These changes of data may be caused by induced feeding.

#### Lectin Genes

Galanthus Nivalis Agglutinin (GNA) is one of the lectin genes, which is most recognized now (Zhang et al., 2003, 2005, 2010; Feng et al., 2006; Wang and Fang, 2014). Research has shown that GNA can inhibit the growth of aphids and provides moderate resistance to Lepidoptera. GNA also has a significantly toxic effect on rice leafhopper (Nephotettix cinciteps), whitebacked planthopper (Sogatella furcifera), rice brown planthopper (Nilaparvata lugens) and so on (Xue et al., 2008). In this study, there are 3 significant differentially-expressed genes selected as candidate genes in the group of 1688CK-VS-1688AI (LOC\_Os07g03880, LOC\_Os06g10790, and LOC\_Os07g18230, respectively). They are not only classified as cytoplasmic vesicle of cellular component and protein kinase of molecular function, but also in the both plant hormone signal transduction pathway and the plant-pathogen interaction pathway. LOC\_Os06g10790 is the up-regulated significant differentially-expressed gene of cultivar 1688, but is not significant in cultivars 1654 and 1665. LOC\_Os07g03880 and LOC\_Os07g18230 are down-regulated genes of 1688 and 1654, which are up-regulated genes of 1665.

#### Chitinase Genes

Wheat α-amylase inhibitors and pea α-amylase are the focus of current research. The mortality rate increases by 30–40% after feeding lepidopterous larvae with the tobacco leaves of transgenic wheat α-amylase inhibitor gene (Chen et al., 2008). Shade found that transgenic pea seeds expressing the alphaamylase inhibitor of the common bean were resistant to bruchid beetles, and could significantly inhibit the growth of those insects (Shade et al., 1994). LOC\_Os10g39700, LOC\_Os08g41100, and LOC\_Os05g33150 were selected in this part. In the group of 1688CK-VS-1688AI, LOC\_Os10g39700, and LOC\_Os08g41100 are the significant differentially-expressed genes but are not the significant ones of cultivars 1665 and 1654. LOC\_Os05g33150 is the down-regulated differentially-expressed gene in both cultivars 1688 and 1665, but is the reverse in the MR cultivar 1654.

#### Plant Hormone Signal Transduction Pathway

Induced feeding can stimulate a plant to synthesize signaling molecules and pathways, such as JA, SA, ET, and MAPK etc. The transmission of signaling molecules can induce efficient transcription expression of defense-related genes and produce and release a large amount of volatile organic compounds, which can help parasitic or predatory natural enemies to locate insects and effectively control feeding and spawning of insects (Wu and Baldwin, 2010; Arimura et al., 2011; Erb et al., 2012). JA plays a significant role in inducing synthesis and signaling transmission. For this reason, a plant hormone signal transduction pathway that begins with α-linolenic acid, produces JA and eventually produces a stress response may participate in insect resistance. In this pathway, 9 candidate genes were selected in which the expression level in cultivar 1688 (up-regulated) was contrary to that in cultivars 1665 or 1654. 5 of 9 candidate genes were upregulated and another 4 candidate genes were down-regulated.

#### Plant–Pathogen Interaction Pathway

WRKY25 and WRKY29 of the WRKY family participate in and produce defense-related gene induction in two pathways of the plant–pathogen interaction pathway. In recent years, a few studies have reported that the WRKY family plays an important role in mechanisms of plant insect-resistant, in which the WRKY family has been studied and contributes much more in plant disease resistance (Van Eck et al., 2010; Atamian et al., 2012). As a result, four significant differentially-expressed genes (WRKY97, WRKY107, WRKY118, and WRKY6) in cultivar 1688 were selected as candidate genes in this pathway, all of which were up-related genes. These candidate genes were not the significant differentially-expressed genes of cultivar 1654. And they were not significant differentially-expressed genes except LOC\_Os12g02420 (down-regulated) in cultivar 1665.

In the process of screening candidate genes, the plantpathogen interaction pathway was selected. In this pathway, there are two biological pathways that the WRKY family is involved in and result in defense-related gene induction. Previously, studies found that the WRKY family transcription factor can regulate plants' biological and abiological stress to acquire resistance (Rushton et al., 2015). Currently, most of the WRKY family transcript factors that have been studied are associated with disease-resistant properties. For example, the WRKY33 transcription factor performs positive regulation to acquire resistance to Alternaria brassicicola in Arabidopsis; the WRKY family transcription factors can increase the resistance to A. brassicicola (Eulgem et al., 2000; Zheng et al., 2006; Pandey and Somssich, 2009; Birkenbihl et al., 2012; Fu et al., 2012; Wu et al., 2012; Chujo et al., 2013; Wei et al., 2013). In recent years, a few studies reported changes of expression levels in rice after feeding by herbivorous insects (Zhou et al., 2011). WRKY3 and WRKY6 have been found to produce significant resistance to tobacco hawkmoth (Manduca sexta) in tobacco (Nicotiana attenuata) (Skibbe et al., 2008). Thus, the significant differentially-expressed genes of WRKY97, WRKY107, WRKY118, and WRKY 6 in the HR cultivar 1688 were selected as candidate genes for further research.

# RT-qPCR Verification

In this part of study, the expression trend of 21 candidate genes were similar to the results of RNA-Seq on the HR cultivar 1688, the MR 1654 and highly susceptible cultivar 1665, indicating that the expression of the 21 candidate genes are reliable. However, we

did not acquire the RT-qPCR data for three genes LTPL 24, LTPL 28 (proteinase inhibitor genes) and CHIT15 (chitinase gene). The possible reason for this RT-qPCR failure may be the low expression level of these genes. RNA-Seq results indicate that the expression of LTPL24 is only 5 FPKM in 1688AI and 0 FPKM in 1688CK.

In this study, three genes OsLTPL164, OsLTPL151, and LOC Os11g32100 showed a more than 10-fold higher expression in 1688 under C. suppressalis infestation compared to that without C. suppressalis infestation. Our previous studies have demonstrated that both OsLTPL164 and OsLTPL151 genes are involved in resistance to C. suppressalis in rice based on the tissue-specific expression patterns and expression profiles in C. suppressalis infestation and mechanical damage treatment in three conventionally grown rice lines 1654, 1665, and 1688 (He et al., 2018). The expression level of OsLTPL164 and OsLTPL151 in stem and leaf was significantly higher than that in root after C. suppressalis infestation (He et al., 2018). This result was probably associated with the feeding behavior of C. suppressalis in host plant. Based on our observation, the newly-hatched larvae firstly wandered and fed for a period of time (about 30 min) on rice leaves, then they found a suitable borer site and bored into the stem of rice for feeding. Therefore, the higher expression level of OsLTPL164 and OsLTPL151 genes in leaf and stem may suggest a potential role of these genes in resistance to C. suppressalis.

# CONCLUSION

In summary, we identified the resistance traits of 47 rice cultivars to the striped stem borer by artificial infestation in both field and greenhouse experiments. We then used RNA-Seq to obtain comprehensive sequences from identified susceptible and resistant rice cultivars, and those cultivars with or without striped stem borer larvae infestation. From the comparison of transcriptomes, we identified several possible pathways associated with striped stem borer resistance and feeding induction and further verified the transcript level of genes belonging to those pathways. Our results suggest that induced expression of proteinase inhibitor, lectin and chitinase gene families, and plant hormone signal transduction and plant-pathogen interaction pathways are involved in striped

#### REFERENCES


stem borer resistance. Three genes LTPL164, LTPL151, and LOC Os11g32100 showed more than 10-fold higher expression in 1688AI than 1688CK, suggesting their potential role in insect resistance. Collectively, our results given here provide an important foundation for further understanding of the insect resistance mechanisms of selected resistant cultivars, which could help us breed for rice resistance to C. suppressalis.

# AUTHOR CONTRIBUTIONS

YW and DJ collected the samples, analyzed the data, and drafted the article. XY critically revised the article. DM selected the experimental rice cultivars. XY and XW conceived and designed the work, and approved the final version to be published.

# FUNDING

This research was funded by the National Key R&D Program of China (2018YFD0300300), the Special Fund for Agro-Scientific Research in the Public Interest of China (201203026-3), and the Young Elite Scientists Sponsorship Program (TESS20160085) by CAST.

#### ACKNOWLEDGMENTS

We would like to thank the Rice Research Institute of Shenyang Agricultural University and the Test Center of Pesticides, Shenyang Chemical Industry Research Institute for providing experimental field and greenhouse facilities and larvae of striped stem borers. We also thank Prof. John Richard Schrock from Emporia State University for proofreading the manuscript text in English.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01717/full#supplementary-material



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Wang, Ju, Yang, Ma and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Comparative Transcriptome and iTRAQ Proteome Analyses Reveal the Mechanisms of Diapause in Aphidius gifuensis Ashmead (Hymenoptera: Aphidiidae)

Hong-Zhi Zhang, Yu-Yan Li, Tao An, Feng-Xia Huang, Meng-Qing Wang, Chen-Xi Liu, Jian-Jun Mao and Li-Sheng Zhang\*

#### Edited by:

*Bin Tang, Hangzhou Normal University, China*

#### Reviewed by:

*Yifan Zhai, Shandong Academy of Agricultural Sciences, China Hamzeh Izadi, Vali-E-Asr University of Rafsanjan, Iran*

> \*Correspondence: *Li-Sheng Zhang zhangleesheng@163.com*

#### Specialty section:

*This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology*

Received: *28 July 2018* Accepted: *12 November 2018* Published: *30 November 2018*

#### Citation:

*Zhang H-Z, Li Y-Y, An T, Huang F-X, Wang M-Q, Liu C-X, Mao J-J and Zhang L-S (2018) Comparative Transcriptome and iTRAQ Proteome Analyses Reveal the Mechanisms of Diapause in Aphidius gifuensis Ashmead (Hymenoptera: Aphidiidae). Front. Physiol. 9:1697. doi: 10.3389/fphys.2018.01697* *Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Sino-American Biological Control Laboratory, USDA-ARS/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China*

*Aphidius gifuensis* Ashmead (Hymenoptera: Aphidiidae) is a solitary endoparasitoid used in the biological control of various aphids. Diapause plays an important role in the successful production and deployment of *A. gifuensis*. Diapause can effectively extend the shelf life of biological control agents and solve several practical production problems like long production cycles, short retention periods, and discontinuities between supply and demand. In recent years, studies have been conducted on the environmental regulation and physiological and biochemical mechanisms of diapause in *A. gifuensis.* Nevertheless, the molecular mechanism of diapause in this species remains unclear. In this study, we compared the transcriptomes and proteomes of diapause and non-diapause *A. gifuensis* to identify the genes and proteins associated with this process. A total of 557 transcripts and 568 proteins were differentially expressed between the two groups. Among them, (1) genes involved in trehalose synthesis such as glycogen synthase, glycogen phosphorylase, and trehalose 6-phosphate synthase were upregulated in diapause at mRNA or protein level while glycolysis and gluconeogenesis-related genes were downregulated, suggesting that *A. gifuensis* stores trehalose as an energy resource and cryoprotectant; (2) the expression of immune-related genes like C-type lectins, hemocyanin, and phenoloxidase was increased, which helps to maintain immunity during diapause; (3) a chitin synthase and several cuticular protein genes were upregulated to harden the cuticle of diapausing *A. gifuensis* larval. These findings improve our understanding of *A. gifuensis*. diapause and provide the foundation for further pertinent studies.

Keywords: aphidius gifuensis, diapause, molecular mechanism, transcriptome, proteome

**285**

# INTRODUCTION

Aphidius gifuensis Ashmead is widely distributed in East Asian countries (Tang and Chen, 1984; Nakata, 1995). It is a common parasitoid of many aphid species. This wasp has high parasitic capacity and adaptability and has been tested as a biological control agent against the aphids Myzus persicae (Sulzer), Sitobion avenae (Fabricius), and Aphis gossypii (Glover) (Pan and Liu, 2014; Khan et al., 2016). It is thought that the release of A. gifuensis in greenhouses or open fields might control aphid populations and prevent catastrophic crop damage. In practice, A. gifuensis has already been used to control M. persicae and has proven to be highly effective (Yang et al., 2009).

Diapause is a form of dormancy that occurs before environmental conditions become unfavorable for development. It ends when favorable environmental conditions return (Denlinger and Armbruster, 2014). The diapause process consists of six phases: induction, preparation, initiation, maintenance, termination, and post-diapause development (Kostál, 2006). When insects enter diapause, their metabolic rates are depressed and their nutrient reserves are increased. In this way, they can survive predictable adverse conditions and synchronize their life cycles with the seasons conducive to growth, development, and reproduction (Hahn and Denlinger, 2011). In general, insect diapause is induced by the low temperatures and short photoperiods of late autumn in preparation for overwintering. However, several insect species enter diapause in summertime to avoid heat and drought (Saulich and Musolin, 2017). Other insects can enter both summer and winter diapause, which results in differential voltinism (Takeda, 1998; Xue et al., 2002; Ding et al., 2003; Ren et al., 2018). Diapause is of great importance in pest management because it enables the prediction of pest emergence, facilitates pest control planning, and increases the shelf life and utility of biological control agents (Denlinger, 2008). In recent years, insect diapause research has elucidated its environmental regulation (Saunders, 2014), maternal effect (Voinovich et al., 2016), energetics (Hahn and Denlinger, 2011), hormonal control (Denlinger et al., 2012), and molecular mechanisms (Hand et al., 2016).

It is believed that the parasitoid wasp A. gifuensis does not enter diapause and can even develop in winter (Ohta and Ohtaishi, 2006). In China, however, several scientists found that A. gifuensis may enter diapause as mature larvae at low temperatures and short day lengths (Li et al., 1999; Xu et al., 2016). Our laboratory successfully induced diapause in A. gifuensis at 8◦Cand an 8 h light:16 h dark (L8:D16) photoperiod. We found that temperature played a more vital role than photoperiod in diapause induction (Li et al., 2013). When they enter diapause, the fourth instar larvae turn from white to mustard yellow. This body color change is an important identifying characteristic of diapause in A. gifuensis (**Figure 1**). Diapausing A. gifuensis have higher total sugar and lower protein levels than non-diapausing individuals. Therefore, A. gifuensis can accumulate nutrients and protective materials when they enter diapause (Li, 2011 ). Nevertheless, the molecular processes occurring in A. gifuensis diapause have not yet been determined.

In this study, we used high-throughput mRNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) technology to analyze the transcriptomes and proteomes, respectively, in A. gifuensis diapause (D) and non-diapause (ND) fourth instar larvae. We compared their relative gene and protein expression levels. We determined the functions of their differentially expressed genes (DEGs) and proteins (DEPs) using the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. We then compared transcriptome and proteome data between the D and ND groups and selected the DEGs with either the same or inverse change trends. We also ran quantitative real-time PCR (qRT-PCR) to verify the accuracy of the transcriptome analyses.

# METHODS

#### Insect Rearing and Treatment

The green peach aphid M. persicae was reared on tobacco (Nicotiana tabacum) seedlings and used as a host for the aphid parasitoid wasp A. gifuensis. The tobacco, aphids, and parasitoid wasps were obtained from Langfang Pilot Scale Base, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Hebei Province, China. The aphids were reared in homemade cages at 25◦C, 80% RH, and L16:D8. Adult A. gifuensis were released at a ratio of one parasitoid to 100 aphids. Daily observations were made and recorded. Mummies were dissected and fourth instar larvae were used as ND samples. Certain aphids were transferred to diapause-inducing conditions (8◦C, RH 80%, and L8:D16) 3 d after being parasitized. Once again, mummies were dissected and fourth instar larvae were used as D samples.

#### RNA Preparation, cDNA Library Construction, and Illumina Sequencing

Three replications were performed under each treatment and 50 larvae were used in each replication. Total RNA was extracted using RNAiso Plus Total RNA extraction reagent (TaKaRa Bio Inc., Kusatsu, Shiga, Japan) following the manufacturer's instructions. An Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and the RIN number were used to evaluate RNA integrity. Qualified total RNA was further purified with the RNeasy micro kit (Qiagen, Hilden, Germany) and the RNase-Free DNase Set (Qiagen, Hilden, Germany). Total RNA was extracted and oligo (dT) magnetic beads and fragmentation buffer were used to generate short fragments of the isolated poly(A) mRNA. First-strand cDNA was generated using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) with these short fragments and random hexamer primers. Second-strand cDNA synthesis was performed using the Second Strand cDNA Synthesis Kit (Beyotime, Shanghai, China). The adapter was ligated immediately after repair of the adenylated 3 ′ ends and purification with a QIAquick PCR purification kit (Qiagen, Hilden, Germany). A cDNA template enrichment was required for library QC. Concentration and library size were

**Abbreviations:** D, diapause; ND, non-diapause; PD, post-diapause; DEG, differentially expressed gene; DEP, differentially expressed protein; GO, Gene Ontology; iTRAQ, isobaric tags for relative and absolute quantification; KEGG, Kyoto Encyclopedia of Genes and Genomes.

assessed using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Three biological replicates of the non-diapause and diapause samples were sequenced with an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA). Paired-end sequencing was controlled with the data collection software provided by Illumina using real-time data analysis.

#### Transcript Identification and Quantification

The cDNA sequencing was performed using the Illumina HiSeq 2500 Sequencing System (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The clean reads were filtered from the raw reads using fastx v. 0.0.13 (http:// hannonlab.cshl.edu/fastx\_toolkit/index.html) to eliminate ribosomal RNA reads, adapter sequences, reads with more than 20% low quality bases, reads shorter than 50 bp, and reads with an N (unknown sequences) ratio>5% (Patel and Jain, 2012). De novo clean read assembly was performed using the scaffolding contig algorithm of CLC Genomics Workbench v. 6.0.4 according to the sequencing data of six samples (Bräutigam et al., 2011; Garg et al., 2011; Su et al., 2011). The primary unigenes obtained from the de novo assembly were then expressed sequence tag (EST)-assembled by CAP3. The final unigenes were then searched against the UniProt and Nr database with BLASTx. The best hits from the database were selected as unigene annotations. For quantitative gene expression analysis, reads from each sample were mapped to the final unigenes which acted as reference sequences. The read counts were normalized by trimmed mean M values (TMM; Robinson and Oshlack, 2010), and then a standard calculation was performed based on reads per kilobase of transcript per million mapped reads (RPKM; Mortazavi et al., 2008). The expressions of transcripts are summarized in **Table S1**.

#### Protein Extraction

Three replicates (50 larvae for each replication) were used for the D and ND treatments. Samples were quickly ground into a very fine powder in liquid nitrogen and added to centrifuge tubes containing 1 ml ice-cold BPP buffer [100 mm Tris, 100 mm ethylenediaminetetraacetic acid (EDTA), 50 mm borax, 50 mm vitamin C, 1% PVPP w/v, 1% Triton X-100 v/v, 2% β-mercaptoethanol v/v and 30% sucrose w/v, pH 8.0]. After the suspension was vortexed at room temperature for 10 min, 800 µl Trissaturated Phe were added and further vortexed for 10 min. The homogenates were then centrifuged at 15,000 × g and 4◦C for 15 min. The supernatants were then transferred to new centrifuge tubes (200 µl supernatants per tube). 1ml ammonium sulfate saturated-methanol was added to each tube and then the mixture was incubated over night at −20◦C. After being spun at 15,000 × g and 4◦C for 15 min, the precipitates were then re-suspended in 500 µl ice-cold methanol, and then centrifuged at 15,000 × g and 4◦C for 5 min. 500 µl ice-cold methanol acetone was added to the precipitates and centrifuged as described above. The protein precipitates were air-dried at room temperature and dissolved in 200 µl lysis buffer [50 mm Tris, 1 mm phenylmethanesulfonyl fluoride (PMSF), 2 mm ethylenediaminetetraacetic acid (EDTA), and 2 mm dithiothreitol (DTT), pH 7.4] at 22◦C for more than 2 h. The homogenates were then centrifuged at 17,000 × g and 20◦C for 30 min. The protein samples (supernatant) were packed into 1.5-ml microcentrifuge tubes and stored at −80◦C. Protein concentrations were determined by the Bradford method (Bradford, 1976). Each treatment included three biological and three technical repetitions.

# iTRAQ LABELING and UPLC-QTOF-MS/MS Analysis

200 µg protein from each sample solution was transferred to the centrifuge tube and diluted to 125 µl with 8 M UA buffer (8 M urea and 150 mm Tris-HCl, pH 8.0). 5 µl 1M DTT was added to the homogenates which were then incubated at 37◦C. After 1 h, 20 µl1 M iodoacetamide (IAA) were added to each centrifuge tube. These were incubated for 1 h in a dark room. The samples were then transferred to an ultrafiltration centrifuge tube and spun at 14,000 × g and room temperature for 20 min. The supernatants were then discarded. 100 µl 1M UA buffer was added to an ultrafiltration centrifuge tube and spun at 14,000 × g room temperature for 10 min. This step was repeated twice. One hundred microliters of dissolution buffer (AB SCIEX, Framingham, MA, USA) was added to an ultrafiltration centrifuge tube and spun at 14,000 × g room temperature for 20 min. This step was repeated three times. Samples were then transferred to new collection tubes. Protein samples were digested at 37◦C overnight with sequencing-grade trypsin (Worthington Biochemical, Lakewood, NJ, USA) at a protein:trypsin ratio of 50:1. After centrifugation at 14,000 × g room temperature for 20 min, the peptides were collected and the remaining homogenates were recentrifuged with 50 µl dissolution buffer. The filtrates were pooled with those from the previous step. The iTRAQ labeling was performed with an iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX, Framingham, MA, USA) according to the manufacturer's protocol. Six samples (three replicates each for ND and D) were labeled with iTRAQ reagent as follows: 113 (ND1), 114 (ND2), 115 (ND3), 116 Zhang et al. Diapause Mechanisms in *Aphidius gifuensis*

(D1), 117 (D2), and 118 (D3). After labeling, the peptides were fractionated by reversed phase liquid chromatography (RPLC) on a Gemini-NX C18 (4.6 mm × 150 mm, 5µm; Phenomenex, Aschaffenburg, Germany) to remove any remaining iTRAQ or other reagents. MS was performed using an AB SCIEX TripleTOFTM 5600(AB SCIEX, Framingham, MA, USA) coupled online with the LC-20AD Nano-HPLC system (Shimadzu Corp., Kyoto, Japan). The gas setting of the MS was as follows: curtain gas = 35 kPa; Gas1 = 4; Gas2 = 0. The ion spray floating voltage was 2,300 V and the collision energy voltage used for collision-induced dissociation (CID) fragmentation for MS/MS spectra acquisitions was 80 V. Each cycle consisted of a TOF-MS spectrum acquisition for 250 ms. The mass-tocharge ratio (m/z) scan range was 350-1,250 Da. There were 30 information-dependent acquisitions (IDA; m/z = 100-1,500 Da). The accumulation time of each IDA was 0.1 s. Masses were dynamically excluded for 25 s when MS/MS fragment spectra were acquired for them. MS was recalibrated at the start of each sample with a β-galactosidase digest standard.

#### Protein Identification and Quantification

Protein identification and quantification were performed with ProteinPilot 4.5 (AB SCIEX, Framingham, MA, USA). Raw MS/MS data were searched against the UniProt database. Peptides with unused scores ≥1.3 (corresponding to a confidence limit of 95%) were accepted. Predicted proteins with ≥1 unique peptide and a global false discovery rate (FDR) ≤1% were counted as identified. The quantitative result was outputted as ratios of proteins in every labeled sample to it in sample 113. A statistical comparison of the differences in protein expression between diapaused and non-diapaused A. gifuensis was conducted with one-way ANOVA in SPSS v. 13.0 (IBM Corp., Armonk, NY, USA). P-values were calibrated by the Benjamini method (Benjamini and Hochberg, 1995).

#### Bioinformatics Analysis

The identified transcripts and proteins were classified according to the Gene Ontology (GO; Ashburner et al., 2000) database using BLAST (E-value < 1e-5). KAAS (http://www.genome.jp/ tools/kaas/) was used to map gene and protein pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa and Goto, 2000) database. Enrichment analyses were performed on the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) based on the GO and KEGG annotations. DAVID v. 6.7 (https://david-d.ncifcrf.gov/) was used for these analyses.

# Quantitative Real-Time PCR

The qRT-PCR was carried out in a 7,900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the ABI Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for relative target gene quantification. The target genes included the upregulated fatty acid synthase (FAS), gamma-interferoninducible-lysosomal thiol reductase (GILT), cuticle protein 21 (CP21), uncharacterized protein LOC103571003, serine protease



homolog 21 precursor (SP21), trehalose phosphate synthase (TPS), troponin C (TnC), sodium/potassium-transporting ATPase (AT1A), and the downregulated venom carboxylesterase-6 (EST6). The primer sequences of GAPDH and the nine target genes are listed in **Table 1**. Nine replicates (three biological replicates × three technical replicates) were used per sample. The qRT-PCR conditions were as follows: incubation at 50◦C for 2 min and 95◦C for 10 min followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. To detect non-specific product amplification, the melting curve was analyzed from 60◦C to 95◦C. The amplification efficiency was automatically calculated using SDS v. 2.4 (Applied Biosystems, Foster City, CA, USA). Relative quantification of the target gene transcripts was determined by the comparative CT method 2−11Ct (Livak and Schmittgen, 2001).

# RESULTS

# Summary of Transcriptome Data

The RNA-seq experiment was performed on both non-diapaused and diapaused A. gifuensis according to the data obtained by sequencing with the Illumina HiSeq 2,500 platform. Using three biological replicates of two samples, the six libraries yielded 230,886,306 raw reads comprising 154,790,202 for the ND group and 145,249,536 for the D group. After quality filtering and fragment assembly, 47,077 unigenes were obtained. The assembled unigenes were mostly 200-1,000 bp long (70.49%). Another 17.47% were 1,001-2,000 bp long and 12.05% of them were >2,000 bp. Transcriptome data were deposited in the Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/ sra) of the National Center for Biotechnology Information

(NCBI) under the accession numbers SRR3717246 (ND) and SRR3717412 (D). Transcriptome datasets are also available at NCBI project PRJNA326701 under the accession number SRP077084.

We annotated the unigenes by aligning their sequences against the UniProt database with BLASTX (Altschul et al., 1990). There were 24,431 unigenes matched with a high homology (E-value < 1e-5; **Table S2**). Since the genome sequence of A. gifuensis was not yet determined, we performed a sequence alignment with known genomes of other species to establish whether the unigene belonged to A. gifuensis. BLAST results showed the greatest number of hits for Apis mellifera (19.03%) and Nasonia vitripennis (18.36%), followed by Cerapachys biroi (10.60%), Harpegnathos saltator (9.47%), Camponotus floridanus (8.37%), and Acromyrmex octospinosus (5.39%). Another 9.48% of the unigenes were found for species not belonging to the Hymenoptera (**Figure 2A**). These were treated as contaminants and filtered out.

In the differential expression analysis, transcripts with ≥ 1.25 fold change between ND and D and FDR (false discovery rate) ≤ 0.05 were considered DEGs. The expression levels of 557 transcripts were significantly changed; 389 were upregulated and 168 were downregulated.

# Protein Identification and Quantification

We identified 4,931 proteins from a total of 30,332 unique peptides using ProteinPilot v. 4.5. All of them had ≥1 unique peptide and 80.17% of them had ≥2 unique peptides. Of these, 2,852 proteins were quantified with iTRAQ ratios (**Table S3**). Species annotation showed that most of the matches belonged to Nasonia vitripennis (21.11%), Apis mellifera (11.68%), Harpegnathos saltator (10.83%), Camponotus floridanus (10.69%), and Cerapachys biroi (10.59%). Another 0.56% of the proteins were filtered out as contaminants (**Figure 2B**). The proteome datasets are available at Peptide Atlas (http://www. peptideatlas.org) under submission number PASS00902.

Correlations between biological replicates were evaluated before the differential expression analysis. Sample 113, which acted as the reference sample in protein quantification, was used as the denominator. The ratios from same treatment (ND or D) were log-transformed and plotted against each other. All correlations between the two biological replicates

were >0.6 (**Figure 3**). Therefore, reproducibility among the three replicates per treatment was satisfactory. Proteins with the same trend in all six comparisons and with average ≥1.25-fold change and FDR ≤0.05 were deemed DEPs. There were 253 upregulated and 315 downregulated DEPs.

# Correlation Analysis of Transcriptome and Proteome Data

There were 2,527 out of 2,852 proteins matching the corresponding genes by sequence alignment. The expression change ratios (D/ND) between transcriptome and proteome level were compared with correlation analysis. The result showed a low Pearson correlation coefficient between these two levels (r = 0.0046; **Figure 4**). All DEPs with ≥1.25-fold change matched their corresponding genes, while most of them did not significantly change. As shown in **Table 2**, 28 of the DEPs had the same change trend as their corresponding DEGs. Of these, 25 were upregulated and 3 were downregulated. Twelve DEPs had change trends that were the inverse of those for their corresponding DEGs.

# Functional Analysis of Differentially Expressed Transcripts and Proteins

To explore the biochemical reactions involved in A. gifuensis diapause, an enrichment analysis based on GO annotations was performed using hypergeometric testing to categorize the DEPs and DEGs. There were 168 (62 biological processes, 94 molecular functions, and 12 cellular components) and

167 (53 biological processes, 86 molecular functions, and 28 cellular components) enriched GO terms. These classified 152 DEGs and 181 DEPs, respectively. One gene or protein could have >1 GO annotation (**Tables S4**,**S5**). The same enrichment analysis was performed based on the KEGG annotation. At the mRNA level, 80 differentially expressed genes mapped to 143 pathways (**Table S6**). The greatest number of DEGs were linked to "metabolic pathways," (20) followed by "biosynthesis of secondary metabolites" (12) and "biosynthesis of antibiotics"

#### TABLE 2 | Correlated protein and mRNA pair expression comparisons.


(11). At the protein level, eight differentially expressed proteins (DEPs) mapped to 37 pathways (**Table S7**). The pathways with the most DEPs were "metabolic pathways," (7) "biosynthesis of secondary metabolites," (6) and "glycolysis/gluconeogenesis" (4). The aforementioned results suggest that glycometabolism, immunoreaction, and secondary metabolite accumulation play important roles in diapause.

#### Validation of Differentially Expressed Genes Using qRT-PCR

A qRT-PCR analysis was performed to investigate the transcriptional patterns of nine selected genes. As shown in **Figure 5**, the expression levels of all target genes were consistent between the qRT-PCR and RNA-seq data. This showed that the quality of transcriptome data was reliable.

FIGURE 5 | qRT-PCR validation of differentially expressed genes. The qRT-PCR results are shown in orange, and the values are shown on the left y-axis; the R result are shown with blue lines, and the value correspond to the right y-axis.

# DISCUSSION

# Metabolite Accumulation in Diapause

During diapause, an insect may feed little or not at all. For this reason, insects must store nutrients in preparation for diapause (Hahn and Denlinger, 2007). Certain metabolites like polyhydric alcohols and sugars can also serve as cryoprotectants that improve cold endurance in diapausing insects (Block, 1990). The insulin-signaling pathway may be a major player in diapause metabolite regulation (Hahn and Denlinger, 2011). The transcriptome analysis showed that the expression of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) gene (PDK1, contig\_31661) was upregulated 4.08-fold in the D group. PDK1 is an indispensable component of the PI3K-AKT signaling axis which, in turn, is a part of the insulin-signaling pathway. PDK1 phosphorylates and activates serine/threonine-protein kinase (AKT), which regulates glucose homeostasis (Sarbassov et al., 2005). The forkhead of the transcription factor Foxo1 is downstream of AKT and may regulate glucose homeostasis in insects (Sim and Denlinger, 2013). In the nucleus, FOXO1 binds the promoter of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK) and activates the expression of this enzyme (Puigserver et al., 2003). Activated AKT phosphorylates FOXO1. Thence, FOXO1 is exported from the nucleus and retained in the cytoplasm, and target gene expression is repressed (Biggs et al., 1999). PEPCK converts oxaloacetate to phosphoenolpyruvate. This reaction is generally considered to be the first committed step in gluconeogenesis (Rognstad, 1979). At the mRNA level, the expression of PEPCK (contig\_30482) was halved in the D group. Therefore, gluconeogenesis was probably inhibited in them. Another important gene downstream from AKT is glycogen synthase kinase 3 beta (GSK3β), which is involved in glycogen biosynthesis and metabolism. GSK3β inhibits glycogen synthase (GYS) by phosphorylating it. In this way, it inhibits glycogen biosynthesis (Cohen and Frame, 2001). By inhibiting GSK3β, AKT can overcome this repression and increase GYS activity (Beaulieu et al., 2009). In this study, GSK3β (K7JBD0\_NASVI) was downregulated at the protein level and GYS (contig\_31002) was upregulated at the mRNA level in the D group. Following this, glycogen synthesis was increased during A. gifuensis diapause.

It is generally accepted that glycogen is converted into polyhydric alcohols or trehalose in diapausing insects (Hayakawa and Chino, 1982). This transformation starts with phosphorolysis catalyzed by glycogen phosphorylase (GP). The product is D-glucose-1-phosphate (Steel, 1982). There are two alternative metabolic pathways for D-glucose-1-phosphate. In one scenario, it is converted into D-glucose-6-phosphate via phosphoglucomutase (PGM). In turn, D-glucose-6-phosphate may be transformed via glycolysis into pyruvate which would enter the TCA cycle. Glycerol and sorbitol may also be produced by glycolysis in diapausing insects (Chion, 1958). Contrarily, D-glucose-1-phosphate could be converted into trehalose via UTP-glucose-1-phosphate uridylyltransferase (UGP), trehalose 6-phosphate synthase (TPS), and trehalose 6-phosphate phosphatase (TPP). Several studies have shown that independent TPP genes were absent in insects whereas there were two conserved regions corresponding to TPS and TPP, respectively (Cui and Xia, 2009; Xu et al., 2009; Tang et al., 2010). In the D group, the expression levels of GP (contig\_3475) and TPS (contig\_3499) were 1.29-fold and 1.73-fold upregulated, respectively, while the protein pgm (K7IN84\_NASVI) was downregulated 0.80-fold. Therefore, we speculate that diapausing A. gifuensis decreases energy

relative to the ND group. Green boxes indicate genes/proteins downregulated in the D group compared with the ND group. Gray boxes indicate genes/proteins not differently expressed in the D group in comparison with the ND group. PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PIP3, Phosphatidylinositol-3,4,5-trisphosphate; PRKZC, atypical protein kinase C zeta type; PYK, pyruvate kinase.

expenditure and stores nutrients in the form of trehalose rather than glycerol or sorbitol.

Several insects like Colaphellus bowringi (Tan et al., 2017), Pieris napi (Lehmann et al., 2016), Arimania comaroffi (Bemani et al., 2012), and Ectomyelois ceratoniae (Heydari and Izadi, 2014) accumulate lipids in diapause whereas others like Cymbalophora pudica (Kostál et al., 1998) and Eurytoma amygdali (Khanmohamadi et al., 2016) do not. In group D, fatty acid synthase (FAS, K7IM26\_NASVI) and acetyl-CoA carboxylase (ACC, E2B9B3\_HARSA) were non-significantly downregulated at the protein level (0.88- and 0.89-fold change, respectively). The result suggested that A. gifuensis relies on sugars like trehalose rather than lipids as energy sources and cryoprotectants in diapause. The aforementioned signals and metabolic processes are summarized in **Figure 6**.

#### Immune Responses in Diapause

Comparatively little research has been conducted on immune responses in diapausing insects. Metabolic and developmental retardation in diapause are expected to weaken insect immune systems. However, diapause must not lower insect defenses against pathogens if it is to ensure insect survival under harsh environmental conditions. A report on Samia cynthia suggested that immunity continues to work well-during diapause (Nakamura et al., 2011). Insects may maintain immunity during diapause by regulating the expression of certain proteins. In the D group, pattern recognition proteins, C-type lectins (CTL, A0A088ACU2\_APIME), and the immune globulin hemocyanin (HC, F4X264\_ACREC) were all upregulated at the protein level and the humoral immune-related protein phenoloxidase (PO, contig\_33789) was upregulated at the mRNA level.

C-type lectins are a superfamily of proteins binding and agglutinating bacterial lipopolysaccharides and lipoteichoic acids (Dodd and Drickamer, 2001). Reports on endoparasitoid wasps indicated that CTL binds parasitoid eggs to mask their hemocytebinding sites. In this way, the wasp could evade detection by the host immune system (Glatz et al., 2003; Lee et al., 2008; Nalini et al., 2008). Since A. gifuensis enter diapause as fourth instar larvae, however the host is already dead by that time. Therefore, the role of CTL upregulation in A. gifuensis diapause is to improve pathogen defense.

Hemocyanin is the major protein component of invertebrate hemolymph. It participates in non-specific invertebrate immunity. A study on Scylla serrata suggested that HC agglutinates bacteria in preparation for host cell defensive measures (Yan et al., 2011). Zhang et al. (2004) reported that purified HC had non-specific antiviral properties. In response to microbial infection, HC can also be converted by proteolytic cleavage into antimicrobial peptides (Destoumieux-Garzón et al., 2001). Since hemocyanin is multifunctional, the 8.45-fold upregulation of HC expression in diapausing A. gifuensis may have been required for protein storage (Jaenicke et al., 1999), ecdysis regulation (Paul et al., 1994), and other processes besides immunity enhancement.

Phenoloxidase is an important enzyme in melanization. It oxidizes phenolic molecules to produce melanin around invading pathogens and wounds (Lu et al., 2014). Melanin and the melanization intermediates may be cytotoxic to microorganisms and impede pathogen invasion. They may also participate in insect wound healing (Eleftherianos and Revenis, 2011). In the D group, there was a 2.93-fold upregulation of phenoloxidase, which was manifested through increased melanin biosynthesis. These findings suggest that insects regulate immunity in diapause by accumulating immune-related proteins. Nevertheless, the precise nature of the changes in cellular immunity that occur during diapause remains unknown.

#### Melanism and Cuticle Sclerotization in Diapause

The cuticle is the protective barrier between the internal tissues and the external environment in holometabolous larval insects. It is composed of chitin fibers and cuticle proteins (Charles, 2010). Cross-links between the fibers and proteins result in hardening, dehydration, and close packing. This process and cuticle pigmentation are collectively referred to as tanning (Arakane et al., 2005). PO is present during the formation of the cuticulin layer. The quinones generated by PO cross-link the protein chains and stabilize the cuticle (Locke and Krishnan, 1971). A study on Bombyx mori reported that melanization occurred in the cuticle (Ashida and Bery, 1995). Therefore, melanin can form from quinones in the cuticle. Phenoloxidase upregulation, then, may explain the body color darkening observed in diapausing A. gifuensis. Several insect cuticular proteins were upregulated at the protein (A0A232F5H6\_9HYME) and mRNA (contig\_17805, contig\_2099, contig\_3789, contig\_9417) levels in the D group. Transcriptome analysis also identified a chitin synthase (contig\_135) that was upregulated during diapause. These modifications thickened and hardened the larval A. gifuensis cuticle to improve its resistance to mechanical damage.

#### Correlation Between Transcriptome and Proteome

In this study, the correlation between the transcriptome and proteome of the studied species was found to be very low. This phenomenon has been reported in several other studies as well (Tian et al., 2004; Huang et al., 2013). Transcription is an intermediate step in gene expression, and mRNA is the template for protein synthesis. In theory, protein and mRNA expression should be highly consistent. In practice, however, they may not align because of mRNA instability (Waggoner and Liebhaber, 2003), mRNA-ribosome binding (Zong et al., 1999), protein degradation (Beyer et al., 2004), and posttranslational protein modification (Futcher et al., 1999). Proteins directly regulate metabolism and physiology. Transcriptome analysis cannot completely reveal protein expression because of post-transcriptional regulation. Proteome studies may also overlook information for degraded or consumed proteins. Transcriptome and proteome analyses are both incomplete, but they could complement each other. For this reason, comprehensive transcriptome and proteome studies have been conducted in diverse research fields in recent years (Vogel and Marcotte, 2012; Kumar et al., 2016).

# CONCLUSION

In this study, we presented a comprehensive transcriptome and proteome analysis to explore possible molecular processes in A. gifuensis diapaus. As a result, we found three important molecular events (carbohydrate accumulation, immune enhancing and cuticle tanning) that may play roles in survival and stress resistance during diapause. Although the conclusions may need further metabology and immunological tests, and numerous other genes and pathways associated with diapause were not analyzed in this study, these findings still provide foundations and directions for the researche of diapause in Aphidius gifuensis and, more broadly, even all parasitic wasps.

#### AUTHOR CONTRIBUTIONS

H-ZZ, TA, F-XH, and L-SZ designed the research. H-ZZ, TA, and F-XH performed research. Y-YL, M-QW, C-XL, and J-JM provided assistance. H-ZZ, TA, and F-XH analyzed data. H-ZZ wrote the manuscript. Y-YL and L-SZ revised the manuscript.

#### ACKNOWLEDGMENTS

This research was supported by the National Key R&D Program of China (2017YFD0201000), National Natural

#### REFERENCES


Science Foundation of China (31572062), and the Major Science and Technology Project of China National Tobacco Corporation [110201601021(LS-01)]. We are grateful for the assistance of all staff and students in the Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Sino-American Biological Control Laboratory, USDA-ARS/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. Additionally, we would like to thank Editage (www.editage.com) for English language editing.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01697/full#supplementary-material

Figure S1 | Original file of Figure 1A.

Figure S2 | Original file of Figure 1B.


armyworm, Mamestra brassicae L. during long-term cold acclimation. J. Insect. Physiol. 49, 1153–1159. doi: 10.1016/j.jinsphys.2003.08.012


development in a pierid butterfly. J. Exp. Bio. 219(Pt 19), 3049–3060. doi: 10.1242/jeb.142687


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Li, An, Huang, Wang, Liu, Mao and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Proximal Remote Sensing to Non-destructively Detect and Diagnose Physiological Responses by Host Insect Larvae to Parasitism

Christian Nansen<sup>1</sup> \* and Michael R. Strand<sup>2</sup>

<sup>1</sup> Department of Entomology and Nematology, University of California, Davis, Davis, CA, United States, <sup>2</sup> Department of Entomology, University of Georgia, Athens, GA, United States

As part of identifying and characterizing physiological responses and adaptations by insects, it is paramount to develop non-destructive techniques to monitor individual insects over time. Such techniques can be used to optimize the timing of when indepth (i.e., destructive sampling of insect tissue) physiological or molecular analyses should be deployed. In this article, we present evidence that hyperspectral proximal remote sensing can be used effectively in studies of host responses to parasitism. We present time series body reflectance data acquired from individual soybean loopers (Chrysodeixis includens) without parasitism (control) or parasitized by one of two species of parasitic wasps with markedly different life histories: Microplitis demolitor, a solitary larval koinobiont endoparasitoid and Copidosoma floridanum, a polyembryonic (gregarious) egg-larval koinobiont endoparasitoid. Despite considerable temporal variation in reflectance data 1–9 days post-parasitism, the two parasitoids caused uniquely different host body reflectance responses. Based on reflectance data acquired 3–5 days post-parasitism, all three treatments (control larvae, and those parasitized by either M. demolitor or C. floridanum) could be classified with >85 accuracy. We suggest that hyperspectral proximal imaging technologies represent an important frontier in insect physiology, as they are non-invasive and can be used to account for important time scale factors, such as: minutes of exposure or acclimation to abiotic factors, circadian rhythms, and seasonal effects. Although this study is based on data from a host-parasitoid system, results may be of broad relevance to insect physiologists. Described approaches provide a non-invasive and rapid method that can provide insights into when to destructively sample tissue for more detailed mechanistic studies of physiological responses to stressors and environmental conditions.

Keywords: hyperspectral image classification, parasitization, reflectance, remote sensing, stress detecting, noninvasive method

# INTRODUCTION

In studies of basic insect physiology and evolutionary adaptations to environmental conditions and stressors, there is growing appreciation for the complex dynamics of physiological responses across different time scales. That is, a range of time scale factors can dramatically affect the magnitude and type of physiological responses by individual insects. Examples include: (1) minutes of exposure

#### Edited by:

Antonio Biondi, Università degli Studi di Catania, Italy

#### Reviewed by:

Gustavo Bueno Rivas, University of Florida, United States Marylène Poirié, Université Côte d'Azur, France

> \*Correspondence: Christian Nansen chrnansen@ucdavis.edu

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 25 July 2018 Accepted: 15 November 2018 Published: 04 December 2018

#### Citation:

Nansen C and Strand MR (2018) Proximal Remote Sensing to Non-destructively Detect and Diagnose Physiological Responses by Host Insect Larvae to Parasitism. Front. Physiol. 9:1716. doi: 10.3389/fphys.2018.01716

**298**

or acclimation to abiotic factors like temperature (Macdonald et al., 2004; Weidlich et al., 2012), UV irradiation (Meng et al., 2009), low oxygen levels (Deng et al., 2018); (2) when during circadian rhythms physiological processes are investigated (Danks, 2003, 2005; Weidlich et al., 2012; Bloch et al., 2013; Suszczynska et al., 2017; Nose et al., 2018); and 3) sampling time during field seasons (Danks, 2003, 2005; Matsuda et al., 2017). Time scale factors further vary among life stages and in response to biological stress factors such as infection or parasitism (Weidlich et al., 2012; Nose et al., 2018).

Parasitoids are free-living insects as adults whose offspring develop by feeding in or on the body of another arthropod. Most parasitoids complete their immature development by feeding on a single host and most hosts ultimately die as a consequence of being parasitized. For this reason, a number of parasitoids are used as biological control agents for management of important insect pest species. Parasitoids occur in several orders of insects but are most prevalent in the order Hymenoptera where estimates suggest more than 200,000 species exist (Godfray, 1994; Quicke, 1997). Most parasitoids are specialists that parasitize a restricted number of host species in a particular host life stage (eggs, larvae/nymphs, pupae, or adults) (Pennacchio and Strand, 2006). Parasitoids are often divided into idiobionts, whose hosts cease development after parasitism, and koinobionts, whose hosts continue to develop as the parasitoid's offspring grow (Haeselbarth, 1979; Askew and Shaw, 1986). Parasitoids also are commonly divided into ectoparasitic species whose offspring grow by feeding externally on hosts or endoparasitoids whose offspring grow by feeding internally. Most known idiobionts are either ectoparasitoids that paralyze and lay eggs on the surface of larval stage hosts or are endoparasitoids that lay their eggs inside sessile host stages like eggs or pupae. Microplitis demolitor Wilkinson (Hymenoptera: Braconidae) is a solitary larval koinobiont endoparasitoid that dramatically reduces host growth due to factors including venom, a polydnavirus and teratocytes this species introduces into its host (Strand, 2014; Strand and Burke, 2014). Due to parasitism-induced inhibition of growth, it is easy to differentiate soybean loopers parasitized by M. demolitor from non-parasitized larvae as long as the developmental stage of the host larva is known. In addition, a single M. demolitor offspring emerges from the host larva 7– 9 days post-parasitism to pupate, while non-parasitized larvae continue to increase in size to the final instar, which is followed by spinning of a silken cocoon and pupation (Strand, 1990; Strand and Noda, 1991). Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) is a polyembryonic (gregarious) egglarval koinobiont endoparasitoid that minimally alters host growth until late in the final instar, when thousands of wasp progeny complete their development (Ode and Strand, 1995; Giron et al., 2007). Distinguishing soybean loopers parasitized by C. floridanum from non-parasitized larvae based on morphology is very difficult until the ultimate (final) instar due to parasitized hosts exhibiting few differences in size or growth rates. However, C. floridanum parasitized larvae grow to a larger than nonparasitized larvae by the final instar (Ode and Strand, 1995). After the host spins a silken cocoon, C. floridanum larvae consume all internal organs and pupate inside the host's exoskeleton to form a "mummy," whereas non-parasitized larvae pupate inside of their silken cocoon to later emerge as an adult moth (Ode and Strand, 1995). Development of non-destructive methods for distinguishing parasitized from non-parasitized larval hosts, and also diagnosis of the parasitoid species, could be highly useful in both basic studies of parasitoid biology and pest management where estimates of parasitism rates can affect control decisions.

Proximal remote sensing is a fast and non-invasive imaging technique, in which target objects, such as insects, are placed within short distance under a camera lens for only a few seconds and reflectance or transmittance data are acquired with an imaging sensor. The imaging sensor can have three spectral bands (normally with the visible light divided into three regions: red, green, and blue), multi-spectral (typically with 5–12 spectral bands), or hyperspectral (with >50 spectral bands. Recent reviews describe the applications of proximal remote sensing in studies of insects (Nansen, 2016; Nansen and Elliott, 2016). Although most entomological applications of proximal remote sensing focus on identification of insects or plant responses to insect herbivory, there are several physiological studies, in which proximal remote sensing of insect body reflectance was used. These include: (1) age-grading of mosquitoes (Anopheles spp.) (Sikulu et al., 2010; Sikulu et al., 2014), biting midges (Culicoides sonorensis) (Reeves et al., 2010), and two species of fruit flies (Drosophila melanogaster and D. simulans) (Aw et al., 2012), (2) infection status of insects by intracellular bacteria in the genus Wolbachia (Aw et al., 2012), (3) mating status of honeybee queens (Webster et al., 2009); (4) ontogeny of puparia of two blowfly species (Voss et al., 2016), and (5) "terminal stress" responses caused exposure of maize weevils (Sitophilus zeamais) to insecticidal plant extracts and infection of darkling beetles (Cynaus angustus) by entomopathogenic nematodes (Nansen et al., 2015). Only two studies have previously used hyperspectral proximal remote sensing in parasitism of insects: (1) distinction of species of Trichogramma immatures developing inside parasitized host eggs (Nansen et al., 2014a) and (2) distinguishing non-parasitized fruit fly pupae (nine different species) from pupae parasitized by the wasp Pachycrepoideus vindemiae (Rondani) (Pteromalidae) (Nansen, 2016). Despite the steadily growing number of studies, in which proximal remote sensing technologies have been used in studies of insects, associations between body reflectance signals and underlying physiological processes are largely unknown. Although based on a study of plants, a recently published study characterized associations between leaf reflectance and phytocompounds (Ribeiro et al., 2018).

In this study, we used proximal remote sensing to acquire time series reflectance data from individual soybean looper [Chrysodeixis includens Walker (Lepidoptera: Noctuidae)] larvae, which were either non-parasitized (control) or parasitized by M. demolitor or C. floridanum. Due to the distinct differences in life histories of M. demolitor and C. floridanum, the key questions addressed in this study were: (1) when is the earliest time point post-parasitism that parasitized larvae can be accurately distinguished from non-parasitized larvae, and (2) do reflectance data acquired from the body of host larvae

enable accurate differentiation of parasitism by a solitary versus gregarious koinobiont endoparasitoid? We discuss the resultderived answers to these questions in the context of existing molecular and physiological knowledge about the host's immune responses.

#### MATERIALS AND METHODS

#### Insects

Soybean loopers were individually reared on an artificial diet at 27◦C as previously described (Strand, 1990) in plastic cups containing enough food for non-parasitized hosts to pupate or parasitized hosts to produce wasp offspring. Soybean loopers were parasitized by M. demolitor as day 1 s instars and as day 1 eggs by C. floridanum (Strand and Noda, 1991; Ode and Strand, 1995). Hosts were individually parasitized to assure that each host was only parasitized once by a single wasp female. Proximal remote sensing data were acquired from individual larvae in the following three treatments: (1) non-parasitized (referred to as "control") soybean loopers, (2) soybean loopers parasitized by a solitary koinobiont endoparasitoid, M. demolitor, and (3) soybean loopers parasitized by a gregarious koinobiont endoparasitoid, C. floridanum.

Initially, 28–30 replicated soybean loopers were included for each treatment, and proximal remote sensing time series data were acquired daily for 9 consecutive days post-parasitism. Some of the soybean loopers, especially those assigned to parasitism by C. floridanum, died during the 9 days of data acquisition. In addition, not all host larvae exposed to parasitism were actually parasitized. The latter was confirmed by rearing individual larvae until they pupated (control) or produced parasites (a single M. demolitor pupa or larvae forming a mummy containing thousands of C. floridanum). Consequently, the total number of observations was 605 (control = 376, M. demolitor = 123, and C. floridanum = 107). During the 9 days of proximal remote sensing data acquisition, host larvae were maintained under standard laboratory conditions (20–22oC and 30–40% RH).

#### Hyperspectral Proximal Remote Sensing

The acquisition of proximal remote sensing data was truly noninvasive as soybean loopers where not removed from their rearing cups (**Figure 1A**). The time to acquire proximal remote sensing data varied somewhat among soybean loopers (occasionally, the acquisition had to be repeated because of extensive movement by the larvae), but the vast majority of hyperspectral images from each larva was acquired within 30 s, which minimized exposure of test animals to stress from the lamp required for image acquisition.

Similar to previously published studies (Nansen, 2011, 2012; Nansen et al., 2013a, 2014b; Zhao et al., 2014; Zhang et al., 2015), we used a hyperspectral push broom spectral camera (PIKA II, Resonon Inc., Bozeman, MT, United States). The objective lens had a 35 mm focal length (maximum aperture of F1.4) and was optimized for the visible and NIR spectra. The main specifications of the spectral camera are as follows: interface, Firewire (IEEE 1394b); output, digital (14 bit); 240 spectral bands (from 383 to 1036 nm) by 1600 pixels (spatial); angular

field of view of 7<sup>0</sup> ; and spectral resolution of approximately 2.1 nm. All hyperspectral images were collected with artificial lighting from 15W, 12V LED light bulbs mounted in two angled rows, one on either side of the lens, with three bulbs in each row. A voltage stabilizer (Tripp-Lite, PR-7b<sup>1</sup> ) powered the lighting. Ambient climate conditions for data acquisition were between 19 and 22◦C and between 30 and 40% relative humidity. A piece of white Teflon (K-Mac Plastics, MI, United States) was used for white calibration, and "relative reflectance" refers to proportional reflectance compared with that obtained from Teflon. Consequently, relative reflectance values ranged from 0 to 1. Hyperspectral images were acquired at a spatial resolution of about 45 pixels mm<sup>2</sup> .

Prior to analysis, we excluded the first and last nine of the original 240 spectral bands, and the remaining 222 spectral bands (from 408 to 1011 nm) were spectrally 3X-binned (averaged) into 74 spectral bands. The spectral binning represents a loss of spectral resolution, but it was performed to ensure that the number of observations (average reflectance profiles from larvae) far exceeded the number of spectral bands. Moreover, model over-fitting due to the Hughes phenomenon or violation of the principle of parsimony (Hawkins, 2004) is a major concern when the number of explanatory variables is similar or exceeds the number of observations (Kemsley, 1996; Defernez and Kemsley, 1997; Nansen et al., 2013a; Nansen and Elliott, 2016).

#### Experimental Design and Data Analysis

"Robustness" or repeatability of proximal remote sensing data is a major technical challenge (Peleg et al., 2005; Nansen, 2011; Nansen and Elliott, 2016). Low robustness implies a high level of variability of proximal remote sensing data acquired from: (1) the same object at multiple time points, (2) different portions of the same object, (3) several objects in the same category or class. For instance, in this study, we acquired time series data to detect and diagnose the stress imposed by parasitoids, when at the same time the host larvae are growing over time. In other words, the body reflectance profiles of control (non-parasitized) larvae are changing over time in response to ontogeny, and this ontogeny-driven change in host body reflectance has to be separated from unique reflectance responses to parasitism.

Data processing and analyses were conducted in PC-SAS 9.4 (SAS Institute, NC, United States), and all statistical analyses were based on average host body reflectance profiles from individual larvae. Two data analyses were performed with average host body reflectance profiles as explanatory variables: (1) For each of the three treatments (three separate analyses), days of parasitism was used as response variable, and (2) the data was grouped into seven 3 days intervals: 1–3, 2–4, 3–5, 4–6, 5–7, 6–8, and 7–9 days, and treatment [1) non-parasitized (control) soybean loopers, (2) soybean loopers parasitized by M. demolitor, and (3) soybean loopers parasitized by C. floridanum was used as response variable. For both analyses, we performing a linear discriminant classifications (proc discrim) (Fisher, 1936). Initially, stepwise linear discriminant analysis (proc stepwise) was used to only select the spectral bands (out of the total of 74 spectral bands) with significant contribution to each of the linear discriminant classification models. The selected subset of explanatory variables was used to generate the linear discriminant classification models for each of the seven time intervals of parasitism, and the classification accuracy of each linear discriminant model was quantified on the basis of 80% of the data being randomly selected as training data set and the remaining 20% of the data used for independent validation. This validation procedure was repeated 10 times to calculate average accuracies for each linear discriminant classification model.

# RESULTS

**Figure 1A** shows four representative images (as acquired with the hyperspectral camera) of soybean loopers, when imaged noninvasively while moving around inside a plastic petri dish on top of food media. The hyperspectral camera used in this study is a line-scanning device, which means that each image is composed of a sequence of individual lines (frames) that are captured as the camera scans across the object (in this case a plastic petri dish). A hyperspectral image of a single soybean looper was acquired within about 10 s. As the larvae moved around, the hyperspectral images of individual larvae become slightly distorted, but it is still possible to identify pixels representing larval body and differentiate them from the background (food media).

# Temporal Trends in Average Body Reflectance Profiles

**Figures 1B–D** show the average host body reflectance profiles over 9 consecutive days for: (1) control soybean loopers (**Figure 1B**), (2) soybean loopers parasitized by M. demolitor (**Figure 1C**), and (3) soybean loopers parasitized by C. floridanum (**Figure 1D**). Each daily curve represents the average of 9–24 soybean loopers, and it is seen that, for all three treatments, there was considerable variation in body reflectance, especially in spectral bands within the range from 700 to 900 nm. In addition, in spectral bands from 408 to 600 nm, the average reflectance from non-parasitized soybean loopers remained fairly constant during the 9 days compared to average reflectance from parasitized soybean loopers.

In **Figures 1E,F**, the daily average host body reflectance profiles from larvae parasitized by M. demolitor or C. floridanum were divided with average host body reflectance profiles from control larvae on the same day. Consequently, these two figures illustrate the relative effect of parasitism for each of the 9 days, and: y-value < 1 implies that parasitism caused a decrease in larval body reflectance, y-value > 1 implies that parasitism caused an increase in larval body reflectance, and y-value close to 1 implies that parasitism had negligible effect on larval body reflectance. Parasitism by M. demolitor (**Figure 1E**) caused a decrease in reflectance in spectral bands from 410 to 450 nm 7–9 days post-parasitism, and it also caused an increase in reflectance in spectral bands near 680 nm 3–7 days postparasitism. Parasitism by C. floridanum (**Figure 1F**) caused an increase in reflectance in spectral bands from 408 to 450 nm 7–9

<sup>1</sup>www.radioreference.com

days post-parasitism, and it also caused a decrease in reflectance in spectral bands near 680 nm 3–7 days post-parasitism.

Average reflectance profiles in **Figure 1**, presented either as actual reflectance profiles or as relative to those from control larvae, were included to underscore an important challenge associated with analyses of time series of reflectance data acquired from living organisms (seeds, leaves, insects, etc.): that the data acquired from control organisms are non-constant, so the "baseline" is constantly changing. This obvious, but very important phenomenon, introduces considerable stochastic noise and it makes the identification of treatment effects more challenging. Despite the variation imposed by larval ontogeny, it appeared that solitary and gregarious koinobiont parasitoids caused somewhat opposite host body reflectance responses, and that these responses could be detected 3–7 days post-parasitism.

#### Linear Discriminant Analyses of Temporal Trends in Host Body Reflectance Data

To examine the temporal variability in daily proximal remote sensing data in detail, we conducted linear discriminant analysis of each of the three treatments separately, in which day postparasitism was used as explanatory variable. **Tables 1**–**3** show the classification results from validations of each classification model, and it was shown that: (1) the day of parasitism could be predicted with 39–100% accuracy, (2) early instars were only vary rarely misclassified as late instar larvae, (3) late instars were not misclassified as early instars, and (4) middle-aged early instars showed the widest spread of misclassification. All of these trends would be expected under the assumption of ontogeny markedly influencing the proximal remote sensing data. Based on the results presented in **Tables 1**–**3**, we grouped the data into 3 days time intervals (1–3, 2–4, 3–5, 4–6, 5–7, 6–8, and 7–9 days – as indicated by 3 by 3 cell rectangles). In **Tables 1**–**3**, it is seen that these day intervals capture most of the misclassification, so this was considered a reasonable grouping of the proximal remote sensing data.

TABLE 1 | Classification accuracy (%) of control (non-parasitized) soybean loopers (Chrysodeixis includens).


Linear discriminant analyses were performed for individual data sets acquired 1–9 days after parasitism. Bold rectangles illustrate 3 days time intervals.

TABLE 2 | Classification accuracy (%) of soybean loopers (Chrysodeixis includens) parasitized by Microplitis demolitor.


Linear discriminant analyses were performed for individual data sets acquired 1–9 days after parasitism. Bold rectangles illustrate 3 days time intervals.

TABLE 3 | Classification accuracy (%) of soybean loopers (Chrysodeixis includens) parasitized by Copidosoma floridanum.


Linear discriminant analyses were performed for individual data sets acquired 1–9 days after parasitism. Bold rectangles illustrate 3 days time intervals.

#### Linear Discriminant Analyses of Response to Parasitism

We used treatment (control, M. demolitor, or C. floridanum) as response variables, and replicated validations of the linear discriminant models showed that control larvae and those parasitized by C. floridanum could be classified with 65– 75% accuracy, when all data from 1 to 9 days postparasitism were included in the linear discriminant analysis (**Figure 2A**). This low level of classification accuracy was due to control larvae being misclassified as those parasitized by C. floridanum and vice versa. For comparison, when all data from 1 to 9 days post-parasitism were combined, host larvae parasitized by M. demolitor could be detected with >98% accuracy.

We suspected that the low classification accuracy of control larvae and those parasitized by C. floridanum was at least partially due to temporal variability in the body reflectance data, so the nine days post-parasitism were divided into seven 3 days intervals, and reflectance data from these were analyzed

presented for the three treatments (B). We performed an analysis of variance followed by a Tukey–Kramer post hoc test for all 74 spectral bands. Black circles indicate significant difference in average reflectance between control and M. demolitor parasitized larvae. White circles denote significant difference in average reflectance between control and C. floridanum parasitized larvae. Black squares denote spectral bands selected and included in the linear discriminant analysis of average reflectance profiles from the three treatments.

separately, and it was seen that the average classification accuracy of **Figure 2A**: (1) control larvae exceeded 80% when based on data from 1 to 6 days post-parasitism, (2) larvae parasitized by M. demolitor exceeded 99% for all seven 3 days intervals, and (3) larvae parasitized by C. floridanum varied considerably along the 9 days post-parasitism but exceeded 90% 3–5 days post-parasitism. As all three treatments were classified with >85% accuracy for reflectance data acquired 3–5 days postparasitism, this was considered the most suitable time interval for detection and diagnosis of parasitism. **Figure 2B** shows the average reflectance profiles from the three treatments 3–5 days post-parasitism, and it also shows the spectral bands that were selected and used in the linear discriminant function to accurately separate the three treatments. It is seen that the spectral bands included in the linear discriminant function were fairly evenly distributed within the examined spectrum.

#### DISCUSSION

Reflectance data acquired from the body of individual soybean loopers were used to address the following two questions: (1) when is the earliest time point post-parasitism that parasitized larvae can be accurately distinguished from non-parasitized larvae, and (2) do reflectance data acquired from the body of host larvae enable accurate differentiation of parasitism by a solitary or a gregarious koinobiont endoparasitoid? The results from this study demonstrated that a solitary (M. demolitor) and a gregarious koinobiont (C. floridanum) parasitoid have opposite effects on host body reflectance in spectral bands from 408 to 450 nm and in spectral bands near 680 nm. This result is both surprising and important, because it implies that "parasitism" per se may not be detectable, if a group of host larvae are parasitized by a complex of parasitoid species and afterward grouped as parasitized and non-parasitized. The reason being that unique reflectance responses to one parasitoid may be out-weighed by reflectance responses to another parasitoid species. This phenomenon was observed in this study, in which parasitism by a solitary and a gregarious koinobiont parasitoid caused somewhat opposite host body reflectance responses at 408–450 nm and near 680 nm 3–7 days post-parasitism. Consequently, our results suggest that hyperspectral imaging is potentially sensitive and reliable enough to differentiate physiological responses by host larvae to different parasitoid species and/or to parasitoids with different life histories. As noted in the Introduction, prior studies established that M. demolitor causes almost immediate physiological responses in host larvae. Consistent with these alterations, host body reflectance data distinguished hosts parasitized by M. demolitor with >98% accuracy when data from 1 to 9 days post-parasitism were combined. So regarding M. demolitor, reflectance-based detection of parasitism was possible within a few days of parasitism and with very high degree of accuracy. The physiological responses could reflect that hosts parasitized by M. demolitor exhibit very rapid changes in feeding behavior which correlate with reduced weight gain due to alterations in nutrient availability and delays in molting (Pruijssers et al., 2009). It also is not surprising that reflectance-based detection of parasitism by C. floridanum was associated with lower classification accuracy. These findings underscore that parasitism by C. floridanum cause negligible effects on growth and molting in the early phases of parasitism relative to non-parasitized control larvae (Strand et al., 1992; Baehrecke et al., 1993; Ode and Strand, 1995). However, the results presented here do demonstrate

that parasitism by C. floridanum could be detected with >85% accuracy if based on reflectance data acquired 3–5 days postparasitism.

Although reflectance data were acquired from the external surface of insect larvae (or other objects), it is well-described in the medical literature that radiometric energy in the range from 600 to 1300 nm penetrates deeply into soft human tissues (including brain, liver, lung, and skin) (Wilson and Jacques, 1990). Consequently, the absorption and scattering properties as well as penetration depth of radiometric energy in the range from 600 to 1300 nm is used in the medical field as part of therapeutic dosimetry and diagnostic spectroscopy (Jacques, 1989; Wilson and Jacques, 1990; Stolik et al., 2000; Bargo, 2003). In addition, it has been shown that the penetration depth of radiometric energy into fruits and vegetables is a valuable indicator of their quality and therefore physiological state (Lammertyn et al., 2000; Nicolaï et al., 2007). Proximal remote sensing was also used to non-invasively determine and characterize the ontogeny of fly pupae (Voss et al., 2016). Finally, a recent experimental study based on proximal remote sensing of different objects [pieces of candy (Skittles), sheets of paper, magnolia leaves, and mosquito eggs] clearly showed how the acquired reflectance signal is highly influenced by the physical structure and biochemical composition of both the given object's surface, the underlying tissues, and the thickness and size of objects (Nansen, 2018). The rather heterogeneous body of literature mentioned above is relevant to the current study, because of the important denominator that proximal remote sensing data acquired from the surface of objects provide partial insight into the composition and structure of below-surface tissues. Thus, it appears plausible to assume that internal physiological responses to parasitism can be detected and diagnosed on the basis of proximal remote sensing. In addition, it seems reasonable to speculate that general physiological variables, such as, larval content of water, carbohydrates, lips, protein, could partially explain why host body reflectance changes in response to parasitism. We are only aware of a few studies, in which reflectance data were directly associated with specific constituents of the imaged objects, and all of those studies were based on proximal remote sensing of plants (Nansen et al., 2013b; Lacoste et al., 2015; Ribeiro et al., 2018). Thus, future entomological research is needed, in which physiological, histological, and biochemical analyses are combined with acquisition of proximal remote sensing data from insects under different treatment regimes, including parasitism.

The growing appreciation for and adoption of proximal remote sensing is likely driven by the ability to, non-invasively and with only minor disturbance, acquire detailed spectral data over time from living animals assigned treatment groups. As an example with high relevance the to the current study, Nansen et al. (2015) acquired time series data to study insect responses to two terminal stressors: (1) adult weevils were maintained in petri dishes with maize kernels either untreated or treated with a plant-derived insecticide, and (2) adult darkling beetles were maintained in petri dishes with either control soil or soil inoculated with entomopathogenic nematodes. For both experiments, it was demonstrated that there was a significant change in adult insect body reflectance at time points, which coincided with published exposure times and known physiological responses by the insects to each of the two killing agents. An interesting result from this study was that the strongest insect body reflectance response was observed in spectral bands from 434 to 550 nm, and that: (1) exposure of adult weevils to a plant-derived insecticide caused a significant decrease in body reflectance, while (2) exposure of adult darkling beetles to a soil with entomopathogenic nematodes caused a significant increase in body reflectance. Moreover, the study highlighted a particular spectral region (434–550 nm) as a target for future and more in-depth studies of the relationship between host body reflectance and insect responses to terminal stressors. In addition, the study showed that two very different terminal stressors elicited either an increase or decrease in host body reflectance. The latter result suggests that proximal remote sensing data may be used to detect and diagnose insect responses to stressors. This particular study is highly relevant to the results presented in the current study, as we found that: (1) spectral bands in the same region (408–600 nm) responded strongly to a killing agent (in this case parasitism), and (2) the host larvae showed opposite reflectance responses to two parasitoids with markedly different life histories.

The main conclusions from the current study are: (1) larvae exposed to markedly different treatments (in this case, two parasitoids with different life histories) showed unique host body reflectance responses, and (2) despite considerable temporal variability, it was possible to detect clear trends and also identify a time interval (3–5 days of parasitism), in which the classification accuracy of proximal remote sensing data was higher than in other time intervals. In other words, time scale factors are of paramount importance, as the ability to classify objects (in this case parasitized larvae) vary over time. The latter conclusion implies that proximal remote sensing technologies can be used to monitor organismal responses over time and potentially identify time intervals with pronounced changes in host body reflectance. Such time intervals with pronounced changes in host body reflectance could be used to optimize the timing of when in-depth (i.e., destructive sampling of insect tissue) physiological or molecular analyses should be deployed. In other words, we argue that proximal remote sensing data analyses can be highly complementary to comprehensive physiological and molecular studies and be used to: (1) exclude outliers and reduce within-category variation among individuals, and (2) optimize when tissue should be sampled for costly and time-consuming physiological and molecular methods.

# AUTHOR CONTRIBUTIONS

CN and MS contributed equally to the writing. CN collected and analyzed the proximal remote sensing data.

#### REFERENCES

fphys-09-01716 December 1, 2018 Time: 14:5 # 8


armigera adults. J. Insect Physiol. 55, 588–592. doi: 10.1016/j.jinsphys.2009. 03.003



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Nansen and Strand. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effect of Sublethal Doses of Imidacloprid on the Biological Performance of Aphid Endoparasitoid *Aphidius gifuensis* (Hymenoptera: Aphidiidae) and Influence on Its Related Gene Expression

#### *Edited by:*

Bin Tang, Hangzhou Normal University, China

#### *Reviewed by:*

Abid Ali, University of Agriculture, Faisalabad, Pakistan Thorben Müller, Bielefeld University, Germany Rakesh Kumar Seth, University of Delhi, India

#### *\*Correspondence:*

Hong-Gang Tian tianhg@nwsuaf.edu.cn Tong-Xian Liu txliu@nwsuaf.edu.cn.

†These authors have contributed equally to this work

#### *Specialty section:*

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

*Received:* 06 July 2018 *Accepted:* 16 November 2018 *Published:* 11 December 2018

#### *Citation:*

Kang Z-W, Liu F-H, Pang R-P, Tian H-G and Liu T-X (2018) Effect of Sublethal Doses of Imidacloprid on the Biological Performance of Aphid Endoparasitoid Aphidius gifuensis (Hymenoptera: Aphidiidae) and Influence on Its Related Gene Expression. Front. Physiol. 9:1729. doi: 10.3389/fphys.2018.01729 Zhi-Wei Kang1,2†, Fang-Hua Liu3†, Rui-Ping Pang<sup>1</sup> , Hong-Gang Tian<sup>1</sup> \* and Tong-Xian Liu<sup>1</sup> \*

<sup>1</sup> State Key Laboratory of Crop Stress Biology for the Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, Northwest A&F University, Yangling, China, <sup>2</sup> Department of Entomology, University of Georgia, Athens, GA, United States, <sup>3</sup> State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

The integrated pest management (IPM) strategy was developed and used in combination with pesticides and beneficial biological control agents. To further develop IPM efficiency, it is important to evaluate the side effects of pesticides on biological control agents. Aphidius gifuensis is one of the most important aphid natural enemies and has been successfully used to control Myzys persicae and other aphid species. Imidacloprid (IMD) is a popular pesticide used worldwide and is highly toxic to non-target arthropods. Here, we investigated the short-term sublethal toxicity of IMD in Aphidius gifuensis and its impact on the biological performance and gene expression of this parasitoid. We found that sublethal IMD doses had a significant negative effect on the life history traits of female A. gifuensis, including shortening the lifespan and lowering parasitic capacity. Moreover, exposure to sublethal IMD also adversely affected the response of A. gifuensis to aphid-infested plant volatiles. Based on the transcriptome analysis, we found that the exposure to sublethal IMD doses significantly affected expression of genes involved in the central nervous system, energy metabolism, olfactory, and detoxification system of A. gifuensis. RT-qPCR also revealed that short term expose to sublethal IMD doses significantly induced the gene expression of genes related to the central nervous system (nAChRa7, nAChRa9, TbH, OAR1, NFR, TYR, and DAR1), olfactory system (OR28 and IR8a1), and detoxification system (CYP49p3, CYP6a2, and POD), while it suppressed the expression of genes involved in the central nervous system (nAChRa4 and nAChRb1), olfactory system (Orco1, IR8a2, and GR1), and detoxification system (GST2). Furthermore, exposure to sublethal doses of IMD also significantly increased the activities of CarEs and POD, whereas we observed no influence on the activities of CAT, GST, and SOD. Our results indicate that sublethal IMD doses might adversely

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affect the biological performance of A. gifuensis by altering gene expression related to the function of olfactory, nervous, energy metabolism, and detoxification systems. Thus, how the use of pesticides directly affect insect population should be considered when used in conjunction with natural pest parasitoids in IPM strategies.

Keywords: *Aphidius gifuensis*, imidacloprid, biological performance, transcriptome, integrated pest management

# INTRODUCTION

Over the past decade, numerous pesticides have been developed and introduced into agriculture, forestry, horticulture, grain storage, and public/personal health. Around the world, more than 2 million tons of pesticides are used annually (De et al., 2014). About 24.3, 18.2, and 9.7 kg/ha of pesticides were used in 12 villages in six counties in Guangdong, Jiangxi, and Hebei provinces, in China (Zhang et al., 2015)w. The global pesticide cost is estimated to be \$81.1 billion by 2021. However, the intensive use of pesticides has posed selective pressure on targeted pest species to develop pesticide resistance or pest resurgence (Desneux et al., 2007; Tabashnik et al., 2009). Over 500 species are resistant to at least one type of pesticide (De et al., 2014). For example, the diamondback moth (Plutella xylostella) has developed a resistance to over 91 pesticides, all within 3 years (2015–2017), Dysdercus koenigii has developed a very high resistance to acetamiprid (from 33 to 433-fold) and imidacloprid (from 21 to 173-fold) in Punjab, Pakistan (Zhang et al., 2016; Saeed et al., 2018). P. xylostella is also resistant to Bacillus thuringiensis and its derivatives. This higher resistance of pests lead to the development of novel pesticides and an increase in the quantity and frequency of pesticide application, which not only facilitates the resistance in the target pests but also results in environment contamination. In Thailand, the average pesticide residues found in surface water was 1.3757 ± 0.5014 mg/L (dicrotophos in summer) and 0.3629 ± 0.4338 mg/L (ethion in winter), and the average ethion residues in soil was 42.2893 ± 39.0711 mg/kg (summer), and 90 ± 24.16443 mg/kg (winter) (Harnpicharnchai et al., 2013). The persistent nature of pesticides has entered into various food chains and has bioaccumulated in higher trophic levels inlcuding bees, birds, and mammals (Bayen et al., 2005; Desneux et al., 2007; Kapoor et al., 2011; Dicks, 2013). Thus, to some extent, the adverse effects of pesticides have outweighed the benefits associated with their use.

To minimize chemical pesticides use, various candidate biological control agents have been evaluated, such as the application of trap crop systems, and entomopathogenic fungi, bacteria, predators, and parasitoids (Shah and Pell, 2003; Shelton and Badenes-Perez, 2006; Yang et al., 2009; Walker et al., 2017). For example, blue fluorescent light is widely used in rice paddy fields to control the rice stem borer, Chilo suppressalis Walker, and Tryporyza incertulas Walker moths (Ishikura, 1950). Alfalfa and mungbean are used as a trap crop in cotton fields for managing lygus bugs, Lygus Hesperus, and the mirid Apolygus lucorum, respectively (Godfrey and Leigh, 1994; Lu et al., 2009). Two parasitic wasps Trichogrammatoidea bactrae fumata Nagaraja and Trichogrammatoidea cojuangcoi Nagaraja are successfully applied to control the cocoa pod borer, Conopomorpha cramerella Snellen, in the field (Lim and Chong, 1987; Alias et al., 2005). However, these biological control agents such as trap crop systems and commercial inundated releases of parasitoids and predators may not be capable of reducing pest densities to levels that avoid economic losses in a timely manner (Yang et al., 2011). Thus, the proper amalgamation of various control techniques into a unified system may provide a powerful tool to keep pest population levels low and to avoid economic damage. However, amalgamation of various pest control techniques also poses a major challenge: how do we take advantage of each biological technique?

The pesticides that are used in pest management programs must be effective in controlling pests and have a low impact on non-target organisms, such as natural enemies (Desneux et al., 2007; Lu et al., 2009). To determine the residual period of control for an insecticide, is essential to plan insect management strategies, which will influence the spraying frequency and the release time of natural enemies, and in turn, affect the pest control cost. Thus, the residual and toxic effects of pesticides are the most serious bottlenecks in the successful use of pesticides and natural enemies.

Aphids are key insect pests that are responsible for major agricultural losses, particularly because they are vectors of various plant viruses (Van Emden and Harrington, 2017). In the Australian grain industry alone, aphid-related plant injuries, either through direct feeding or virus transfer, represent a potential economic cost of \$200–480 million/year (Murray et al., 2013; Valenzuela and Hoffmann, 2015). Current management strategies for broadacre aphids rely primarily on pesticides, either through seed dressings or foliar applications (Dedryver et al., 2010; Chollet et al., 2014). However, due to the strong adaptation and fecundity of aphids, they have developed strong resistance to various pesticides. For example, the green peach aphid (Myzus persicae) is resistant to more than 70 different types of synthetic insecticides (Silva et al., 2012).

Imidacloprid (IMD) is one of the most extensively used pesticides in the world (Li et al., 2018). It is sprayed directly onto plants or used as a seed or soil treatment on a number of agricultural products to control a variety of insect pests including plant- and leafhoppers, aphids, termites, whiteflies, and thrips (Li et al., 2018). However, IMD is highly persistent and toxic to non-target animals, including bees (Dicks, 2013). When a bumblebee (Bombus terrestris) colony was treated with IMD at a sublethal concentration, it significantly reduced the growth rate and production of queens and workers (Laycock et al., 2012; Whitehorn et al., 2012). In addition, there was a significant decrease in the fecundity of Orius insidiosus, Orius tristicolor, Hippodamia convergens, and Chrysoperla carnea, which are natural enemies of aphids, when treated with sublethal concentrations of IMD (Mizell and Sconyers, 1992; Sclar et al., 1998; Studebaker and Kring, 2003; Rogers et al., 2007; Funderburk et al., 2013).

Aphidius gifuensis Ashmead (Hymenoptera: Braconidae) is one of the most widely distributed and dominant endoparasitoid of pest aphids, including M. persicae and Sitobion avenae (Fabricius), and are successfully applied in greenhouses for controlling vegetable aphids and in fields for tobacco aphid (M. persicae, also known as green peach aphid) management in China (Yang et al., 2009, 2011; Ali et al., 2016; Kang et al., 2017a; Yang F. et al., 2017). Furthermore, Yang et al. (2009) reported that after augmentative releases of A. gifuensis, the frequency and quantity of pesticide application could be sustained at a low level for 8 years. However, A. gifuensis is sensitive to various agrochemicals (Ohta and Takeda, 2015). For example, after 14 days of exposure to residual permethrin and IMD, also showed high toxicities to A. gifuensis (Kobori and Amano, 2004). In this work, we not only evaluated the toxicity of IMD in A. gifuensis, but also investigated the biological performance of A. gifuensis exposed to sublethal doses of IMD. We hypothesized that sublethal doses of IMD would disrupt parasitoids performance through regulating some genes on the molecular level. Transcriptome technology was applied to explore which of the parasitoid genes could be modulated by IMD and how A. gifuensis adjusts its detoxification system to respond to the exposure of IMD.

# MATERIALS AND METHODS

#### Insect Species

Aphidius gifuensis used in this work were maintained on M. persicae, which was reared on chili pepper (Capsicum annuum L., var. "Lingxiudajiao F1") at 25 ± 1 ◦C with a 16 h light: 8 h dark photoperiod and a relative humidity of 60 ± 5% in an air-conditioned insectary.

# Performance of *A. gifuensis* Exposure to IMD

We used three different dilution magnifications to evaluate the toxicity of IMD on A. gifuensis, and distilled water was used as a control. Five plastic vials (length: 8 cm; diameter: 4 cm) were treated with 1 ml IMD or H2O. The IMD was swirled inside the vials for 30 s and allowed to air-dry in a hood to simulate the pesticide residues. At the time of exposure, twenty 2-day old A. gifuensis female adults were introduced into a vial. Twenty-four hours later, the mortality of A. gifuensis was counted and living parasitoids (at least 15) were individually collected to test the effects of IMD on the parasitism, longevity and sex ratio of offspring as described by Kang et al. (2017a) with little modification. Chili pepper plant with 200 second- or third- instar M. persicae were placed into a plastic cage (diameter: 13 cm; height: 30 cm) with screen mesh caps. Then, five females of A. gifuensis from different treatments (control (CK) or IMD), were introduced into each rearing cage for 8 h. After the parasitism, the aphids and Chili pepper complex was maintained in an incubator. Ten days later, the number of mummified aphids and the sex ratio of all wasps emerging from these mummified aphids were recorded. Five biological replicates were conducted in this work.

To analyze the effect of sublethal doses of IMD on the orientation behavior and gene expression of A. gifuensis, the LC<sup>20</sup> of IMD was used and 24 h later, surviving parasitoids were collected and separated into two groups: one group with thirty living parasitoids was flash-frozen in liquid nitrogen and stored at −80◦C for the gene expression analysis; the remaining parasitoids were placed into a PCR tube for orientation behavior. Y-tube olfactometers were used to assess the oriented responses of A. gifuensis toward healthy and aphid-infested plants. Y-tube was conducted as described by Kang et al. (2018a,b). In total, 100 living parasitoids were tested for the orientation behavior.

# RNA Sequencing

Total RNA was extracted from whole bodies of five female A. gifuensis using RNAiso Plus (Takara Bio, Tokyo, Japan), following the manufacturer's instructions. The high quality RNA was used for the further cDNA synthesis and Illumina library generation, which was completed at the Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).

# *De novo* Assembly and Gene Annotation

Transcriptome de novo assembly was conducted using a short read assembling program—Trinity with min\_kmer\_cov set to 2 by default and all other parameters set to default (Grabherr et al., 2011). In order to get comprehensive information about the genes, we aligned the unigenes larger than 150 bp to nr, Nt, KEGG, Swiss-Prot, and COG databases, with e-value <10−<sup>5</sup> . With nr annotation, we used the Blast2GO program to get GO annotation of unigenes (Conesa et al., 2005). The WEGO software was used next to perform GO functional classification for all unigenes (Ye et al., 2006). The unigene expression levels were calculated by fragments per kb per million reads (FPKM) method, using the formula, FPKM (A) = 10<sup>3</sup> (10<sup>6</sup> C)/NL (A: Unigene A; C: number of fragments that uniquely aligned to Unigene A; N: the total number of fragments that uniquely aligned to all Unigenes; L: the base number in the CDS of Unigene A). The FPKM method eliminates the influence of different gene lengths and sequencing levels on the calculation of gene expression; therefore, the calculated gene expression can be directly used for comparing differences in gene expression across samples.

#### Expression Analysis

Heat map analysis was performed by the R package of pheatmap (http://www.r-project.org/; R Foundation for Statistical Computing, Wien, Austria). Heatmap plots present the binary log of fold-change of IMD/CK for each gene with a three-color scale (navy, white and firebrick).

RT-qPCR was performed to validate the expression of several genes in A. gifuensis. Total RNA was extracted from five whole bodies of 2-day old female A. gifuensis, and cDNA was then synthesized from 1 µg total RNA using a PrimeScript <sup>R</sup> RT reagent Kit with gDNA Eraser (perfect Real Time) (Takara, Tokyo, Japan) according to the manufacturer's

protocol. Specific gene primers were designed by Primer Premier 6 (PREMIER Biosoft International, Palo Alto, CA, USA), which are presented in **Table S1**. In total, three biological replicates, with three technical replicates were conducted, and the qPCR was performed as previously described (Kang et al., 2017b). However, in this study, we used a 2−1Ct method to evaluate the expression of selected genes (Eakteiman et al., 2018).

#### Enzyme Activity Assay

The activities of CarE, SOD, CAT, POD, and GST were measured using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) as described previously (Kang et al., 2017a).

#### Data Analyses

The comparison of performance parameters was subjected to a one-way analysis of variance (ANOVA) followed by the separation of means by the Fisher's protected least significant difference (LSD) test at P = 0.05. The gene expression profiles were determined by a student's t-test at P < 0.05. The orientation behavior of A. gifuensis under the different treatments was separated by the Chi-square test (P < 0.05). A generalized linear mixed-effects model (GLMM) with a binomial family with the cbind function was then performed to analyze the response to the treatment: yes or no. Except for GLMM, SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used for the data analyses. GLMMs were performed in the R programming environment (version 3.5.1).

# RESULTS

#### Exposure of Sublethal Does of IMD Impaired the Performance of *A. gifuensis*

The influence of IMD on the mortality, parasitism, longevity and female proportion in offspring of A. gifuensis are shown in **Table 1**. Exposure to IMD significantly increased the mortality of female and male adults, and decreased the longevities of surviving female and male adults, as well as the parasitism of surviving female adults (Mortality: Female: F = 152.071, P < 0.001; Male: F = 62.448, P < 0.001; Longevity: Female: F = 27.952, P < 0.001; Male: F = 26.069, P < 0.001; Parasitism: F = 19.991, P < 0.001). However, exposure to IMD did not influence the female proportion of offspring produced by surviving female adults, compared to healthy female adults (F = 0.725, P < 0.504). Furthermore, IMD significantly reduced the sensitivity of A. gifuensis to the volatiles from aphid infested plants (Healthy wasps: χ <sup>2</sup> = 20.045, P < 0.001; IMD treated wasps: χ <sup>2</sup> = 0.636, P = 0.425, **Figure 1**). The GLMM analysis also revealed that IMD changed the response of A. gifuensis to these volatiles (P = 0.009).

#### An Overview of the Transcriptome

The transcriptome assembly was performed using the Trinity program, with an optimal K-mer length set to 25. A total of 48,033,980 and 53,409,010 raw reads were obtained from CK and IMD treatment groups, respectively. After removing adaptor sequences, low quality sequences and N-containing sequences, 46,760,944, and 51,668,492 clean reads were generated form the CK and IMD raw data, respectively. The assemblies produced 81,727 transcripts with a maximum sequence length of 19,224 bp and a N50 transcript length of 1,284 bp (**Table 2**). Furthermore, the GC content of the CK and IMD treatment groups were 31.47 and 30.76%, respectively. The quality of RNA samples and the expression file of genes were supplied as **Table S2** and **Datasheet 1**.

### Functional Gene Annotation and Classification

GO enrichment indicated that genes involved in the cellular process, metabolic process, single-organism process, biological regulation and the regulation of the biological process in the category of the biological process, cell, cell part, membrane and organelle in the category of cellular component, and binding and catalytic activity in the category of molecular function were dominant (**Figure 2**). The neuroactive ligand-receptor interaction, cAMP signaling pathway and MAPK signaling pathway were the major enrichment pathways in the IMD treatment group (**Figure 3**).

#### Genes Involved in the Central Nervous and Olfactory Systems Are Differentially Expressed in Response to Sublethal Doses of IMD

The first gene groups we examined focused on the central nervous and olfactory systems, which are the target of the IMD and influence target insect behavior. For the target of the IMD, we identified 15 acetylcholine receptors: 11 neuronal acetylcholine receptors and four muscarinic acetylcholine receptors (**Table 3**). Among these target genes, nAChRα4 and nAChRβ1 were significantly down-regulated in response to the IMD. Only nAChRα7 and nAChRα9 exhibited higher transcript abundances in the IMD treated A. gifuensis when compared to CK samples. Furthermore, no significant differences were detected in the rest of the nAChRs genes.

Apart from the potential target genes, we also analyzed the impact of IMD exposure on olfactory systems to explain the impaired orientation behavior we observed in A. gifuensis treated with IMD. We found that a very low proportion of olfactory related genes exhibited significant differences between the treatments and control samples (**Figure 4**). The decrease in the mean FPKM values for the odorant co-receptor (Cluster-8038.0), odorant receptors (Cluster-1578.0, Cluster-4221.1, and Cluster-3108.1), chemosensory protein (Cluster-8527.0), gustatory receptors (Cluster-7667.0 and Cluster-4878.0), and ionotropic receptors (Cluster-9767.24401 and Cluster-1662.0) was particularly striking. On the contrary, exposure to IMD significantly up-regulated the expression of the odorant-binding protein (Cluster-1704.0), odorant receptor (Cluster-3211.0), gustatory receptor (Cluster-9767.3034), and the ionotropic receptor (Cluster-6117.0), which were effected the most by IMD treatment in their gene group.

Furthermore, exposure to IMD also influenced the expression of genes involved in the central neurons. The dopamine receptor 1 (Cluster-9767.1884), tryptophan 5-hydroxylase (Cluster-1083.0), neuropeptide FF receptor (Cluster-9767.40897)


TABLE 1 | The side effects of IMD on the parasitism, longevity, and female proportion in offspring of A. gifuensis.

<sup>a</sup>Treatment: dilution magnification.

Means followed by different letters within a column indicate significant difference among the treatments (P < 0.05).

were significantly higher in IMD treatments compared to CK treatments.

#### Effects of Sublethal Doses of IMD on Detoxification Progress, Antioxidant System, and Biomolecule Damage Genes in *A. gifuensis*

We found that defense genes, such as cytochrome P450 (CYP4c1: Cluster-5030.0 and Cluster-9767.42126; CYP6a2: Cluster-9767.4090; CYP9p3: Cluster-9767.18925), cyt b5 (Cluster-6200.1), peroxidase (POD: Cluster-9767.17490), carboxylesterase (CarE, Cluster-9767.29708), glutathione Stransferaes (GSTs, Cluster-9767.30914), and heat shock proteins (HSPs, Cluster-9767.16364, and Cluster-9767.39176), were highly expressed in the IMD treated A. gifuensis (**Figure 5**), while three P450s (Cluster-9767.38298, Cluster-9767.30384, and Cluster-9767.36002), POD (Cluster-9767.24511), and HSP (Cluster-9767.32708) exhibited lower transcript abundances in the IMD treated A. gifuensis than in the CK group (**Figure 5**).

#### Sublethal Doses of IMD Altered Expression of Genes Involved in Metabolic Signaling

To investigate the impact of IMD on energy metabolism, we analyzed the expression of genes involved in fatty acid, sugar, and amino acid metabolism (**Figure 6**). We found that almost all the genes involved in fatty acid metabolism were expressed at a TABLE 2 | Assembly summary of the A. gifuensis transcriptome.


higher level in the IMD treated A. gifuensis (**Figure 6A**), while only Cluster-9767.37118 and Cluster-6642.1 were expressed at a lower level of the IMD treatment. Consistent with fatty acid metabolism, the majority of genes that regulate sugar and amino acid metabolism, also exhibited higher mean FPKM values in the IMD treated A. gifuensis, whereas the expression of Cluster-9767.30238, Cluster-9767.39562, Cluster-8931.0, and Cluster-9767.35416 in sugar metabolism and Cluster-2788.0, Cluster-6624.0, Cluster-6867.0, Cluster-9767.27231, Cluster-9767.29443, Cluster-9767.39986, and Cluster-9767.5013 in amino acid metabolism, were down-regulated in response to the IMD treatment (**Figures 6B,C**).

#### Validation of Transcriptome Data by qPCR

To confirm the transcriptome data, we conducted the RT-qPCR of several genes identified in the transcriptome that were IMDsensitive. Exposure to IMD significantly increased the expression of CYP6a2, CYP9P3, POD, OR28, IR8a1, nAChRa7, nAChRa9, TbH, OAR1, NFR, TYR, and DAR1, whereas the expression of GST2, nAChRa4, nAChRb1, ORco, IR8a2, and GR1 decreased (**Figure 7**). Furthermore, exposure to IMD did not influence the expression of GST5, SOD1, and SOD2 (**Figure S1**).

#### Activities of CarEs, POD, and GSTs in *A. gifuensis* After IMD Exposure

Exposure to IMD significantly induced the activities of POD and CarEs, while it had no significant influence on SOD, CAT and GST activity (POD: t = −11.648, df = 4, P < 0.001; CarE: t = −10.552, df = 4, P = 0.003; SOD: t = 0.843, df = 4, P = 0.4467; CAT: t =0.6523, df = 4, P = 0.2298; GST: t = 1.886, df = 4, P = 0.1323; **Figure 8** and **Figure S2**).

# DISCUSSION

IPM program improvements requires an understanding of how pesticides influence natural enemies of the pests that are being targeted. Therefore, the effects of sublethal doses of pesticides are important for improving IPM programs. In this work, we found that oral ingestion of sublethal doses of IMD, adversely affected parasitoid performance, including the survival rate, parasitic capacity, and longevity of female adults, which was consistent with the performance of the Aphidius colemani, Microplitis mediator, O. insidiosus, C. flavipes, and Trichogramma species exposed to pesticides (D'Avila et al., 2018; Fontes et al., 2018). In the M. mediator, exposure to flonicamid, pymetrozine, spinosad, and thiacloprid reduced its parasitization activity, percentage of parasitism and female longevity. In addition, IMD impaired the longevity and parasitic capacity of Trichogramma species including Trichogramma achaeae, T. chilonis, T. platneri, and T. pretiosum (Khan and Ruberson, 2017; Fontes et al., 2018). The exposure to pesticides also adversely affected the biocontrol efficiency of pest predators (Moscardini et al., 2013; Nawaz et al., 2017). For example, IMD significantly repressed egg hatching, nymph survival and adult fecundity of the predatory bug, Orius albidipennis (Sabahi et al., 2010; Moscardini et al., 2013). Similarly, sublethal doses of diazinon, fenitrothion, and chlorpyrifos exhibited adverse effects on the biological performance of Andrallus spinidens, which is a predator of rice lepidopterous larvae (Gholamzadeh-Chitgar et al., 2015). Similar to chemical pesticides, other biological agents like entomopathogenic fungi and bacteria also adversely affect the biological performance of parasitoids and predators (Potrich et al., 2017). In addition, a high occurrence of wing deformities was observed when mummies of A. gifuensis were exposed to IMD (44.44%), acetamiprid (67.25%), and thiamethoxam (33.33%) (Sun et al., 2014). All of these results indicated that pesticide exposure adversely influenced the performance of natural enemies, which also means that the effectiveness of natural enemies can be reduced by the application of pesticide.

In addition to biological performance, we also investigated the side effects of IMD on the orientation behaviors of A. gifuensis after IMD treatment. We found that exposure of IMD significantly reduced the sensitivity of A. gifuensis to the volatiles produced by aphid infested plants. Consistent with this results, consuming IMD or aldicarb contaminated floral nectar, also reduced the response of Microplitis croceipes to the odors from its host Helicoverpa zea infested cotton (Stapel et al., 2000). In Anagrus nilaparvatae, survivors of IMD exposure had no response to volatiles from Nilaparvata lugens-infested rice (Liu et al., 2010). In addition, exposure to pyrethroids impaired the host-searching and oviposition behavior of the aphid parasitoids Aphidius ervi and Aphidius colemani, and Trissolcus basalis, which is an egg parasitic wasp of the southern green stinkbug, Nezara viridula (Ahmad and Hodgson, 1998; Salerno et al., 2002; Desneux et al., 2004a). Furthermore, Wang D. et al. (2017) found that exposure to beta-cypermethrin significantly decreased pheromone perception in male Trichogramma chilonis. All of these results indicate that IMD exposure impairs or reduces the sensitivity of the A. gifuensis olfactory system, thereby disrupting host searching and parasitizing.

To explore the potential mechanism of the negative effects of IMD on A. gifuensis, transcriptome technology was used to comprehensively analyze the gene expression of A. gifuensis in response to sublethal doses of IMD exposure. Our transcriptomic analysis pointed to a profound regulation of genes principally related to the olfactory and neuronal systems. The most

down-regulated genes were the odorant co-receptor (Cluster-8038.0), which is the most important odorant receptor in the detection of odorants; gustatory receptor 1 (Cluster-7667.0), a sugar receptor that is associated with host aphid discrimination; and the neuropeptides capa receptor (Cluster-9767.10302), a G protein-coupled receptor for the capa peptides and an important signaling molecule in the regulation of a wide range of physiological processes (Kang et al., 2017b; Schoofs et al., 2017). These results are generally consistent with recent studies of the interaction of neonicotinoid with OBPs and CSPs. For example, CSP3 and OBP21 were downregulated in honey bees exposed to thiamethoxam (Shi et al., 2017). Furthermore, a sublethal dose of IMD inhibited the binding affinity of OBP2 and CSP1 to a floral volatile β-ionone in Apis cerana and GOBP2 to a tea volatile E-2 hexenal in Agrotis ipsilon (Li et al., 2015, 2017a,b). Interestingly, in addition to these down-regulated genes, a considerable number of genes were up-regulated, such as the odorantbinding protein (Cluster-1704.0), gustatory receptor (Cluster-9767.3034), ionotropic receptor (Cluster-6117.0), and odorant receptor (Cluster-3211.0). Similarly, a single brief exposure to pesticides dramatically increased CSP expression in Bombyx mori (abamectin) and Bemisia tabaci (thiamethoxam) (Xuan et al., 2015; Liu G. et al., 2016). All of these results indicate that the impairment of olfactory systems from sublethal doses of some pesticides could be involved the disordered orientation behavior.

As a neurotoxin and agonist of nAChRs, IMD had high binding affinity for nAChRs, thereby disrupting neurotransmission (Ffrench-Constant et al., 2016). IMD was thought to impede information receiving and processing in N. vitripennis, which led to the disruption of sexual communication and foraging behavior (Cook et al., 2016; Tappert et al., 2017). Furthermore, in Solenopsis invicta, when treated with 0.25µg/ml IMD, there was a significant reduction in food consumption, digging and foraging behavior, while the neurotoxins flubendiamide and indoxacarb increased the walking time of Copidosoma truncatellum (Wang L. et al., 2015; Ramos et al., 2018). In the current work, we found that nAChRa4 and nAChRb1 were down-regulated, which was consistent with the expression of nAChRs in Rhopalosiphum padi (Wang K. et al., 2017). Similar to the findings of Desneux et al. (2004b) in A. ervi, we also found that some of the IMD exposed females bended their abdomen forward as they were attacking aphid, while no aphids were present. All of these results revealed that sublethal doses of IMD not only impaired the olfactory



\*Log2, Log2IMD/CK. Inf means this gene only expressed in IMD treated A. gifuensis.

system of A. gifuensis, but also disrupted the neurotransmission that influences their behavior. Furthermore, these results also indicate that the development of specific and environmentally safe pesticides, that present little or no harm to natural enemies of pest insects, are needed.

In addition to the effect IMD had on the olfactory and neuron systems, we also investigated the impact of IMD on the detoxification systems in A. gifuensis. Cytochrome P450 monooxygenases (P450s), carboxyl esterases (CarEs), and glutathione S-transferees (GSTs) are three major multiline enzyme families that are responsible for xenobiotic metabolism in most insect species (Li et al., 2007; Hsu et al., 2012; Chaimanee et al., 2016; Gong and Diao, 2017; Magesh et al., 2017; Traverso et al., 2017).

P450s are a group of important stress response-related genes that play significant roles in several physiological processes, including hormone metabolism, the adaptation to natural and synthetic toxins, and insecticide detoxification. As we know, overexpression of the gene coding of the P450 clades (CYP4, CYP6, and CYP9), contributes considerably to insecticide-resistance (Li et al., 2007; Bass et al., 2014). For example, in B. tabaci and M. persicae, over-expression of the cytochrome P450 genes CYP6CM1 and CYP6CY3, contribute to neonicotinoid insecticide resistance, as these enzymes can catalyze a more rapid conversion of imidacloprid to its less active form, 5-hydroxy-imidacloprid (Karunker et al., 2008; Puinean et al., 2010). Furthermore, CYP6AY1 and CYP6ER1 were highly overexpressed in the IMD resistant strain of N. lugens compared to the susceptible strain (Yang Y. X. et al., 2017). In A. mellifera, coumaphos and IMD treatment significantly decreased the expression of CYP306A1, CYP4G11, and CYP6AS14, whereas pyrethroid bifenthrin induced the expression of CYP9Q1 and CYP9Q2 but repressed the expression of CYP9Q3 (Mao et al., 2011; Chaimanee et al., 2016). In-vitro, CYP9Q1, CYP9Q2, and CYP9Q3 detoxify coumaphos independently and tau-fluvalinate with the cooperation of CarEs (Mao et al., 2011). Additionaly, CYP9Q1 and CYP9Q3 also contributed to the metabolism of quercetin (Mao et al., 2011). In this work, the most up-regulated genes were CYP4c1 and CYP6a2 (Cluster-9767.42126), which are associated with the IMD resistance in N. lugens; and cyt b5

gustatory receptors, and ionotropic receptors. (B) Genes identified as odorant receptors. (C) Genes involved in neuron functions.

(Cluster-6200.1), which is the electron transfer partners of P450 proteins and which modify the catalytic activity of P450 proteins (Paine et al., 2005; Ding et al., 2013).

CarEs are involved in the metabolic detoxification of dietary and environmental xenobiotics in insects (Xie et al., 2017; Wu et al., 2018). A higher expression or activity of CarEs have

been reported in the insecticide resistance strains of many insect species such as M. persicae, R. padi, Aphis gossypii, Pediculus humanus capitis, P. xylostella, and Bactrocera dorsalis (Hsu et al., 2012; Gong et al., 2013, 2014; Kwon et al., 2014; Wang L. et al., 2015; Wang L. L. et al., 2015; Xie et al., 2017). In A. mellifera, the induction of CarE activity by IMD, acetamiprid, pymetrozine, and pyridalyl was observed, while malathion and permethrin significantly inhibited CarE activity (Yu et al., 1984; Suh and Shim, 1988; Badawy et al., 2014; Li Z. et al., 2017). Furthermore, in P. xylostella, CarEs activity was positively correlated with resistance to spinosyn, beta-cypermethrin, chlorpyrifos, and abamectin (Gong et al., 2013). Moreover, RNA interferencemediated gene silencing (RNAi) tests revealed that the knockdown of CarE genes led to a decreased tolerance to some pesticides (Wang L. L. et al., 2015). In B. dorsalis, the knockdown of CarE4 and CarE6 significantly decreased the resistance to malathion, and the detoxification of malathion was observed when CarE4 and CarE6 were heterologously expressed (Wang L. L. et al., 2015). Furthermore, in Lygus lineolaris, IMD exposure significantly increased the expression of 13 esterase genes (Zhu and Luttrell, 2015). In this work, we found that the expression and enzyme activity of CarEs in IMD treated A. gifuensis were significantly higher than that in CK treatment, especially carboxylesterase (Cluster-9767.29708).

GSTs are part of another important detoxification enzyme family. GST activity in larvae, pupae, and nurse bees, but not in foragers, was induced by pyrethroid flumethrin (Nielsen et al., 2000). In the eastern honey bee Apis cerana cerana, the sigma-class AccGSTS1 was up-regulated by phoxim, cyhalothrin and acaricide and the theta-class GST gene GSTT1 and omega-class GST gene GSTO2 was induced by cyhalothrin, phoxim, pyridaben, and paraquat, indicating that they might be involved in the stress response to pesticides (Yan et al., 2013; Zhang et al., 2013; Liu S. et al., 2016). Furthermore, formetanate increased the activity of GST, whereas IMD and dimethoate had no influence on GST activity in A. mellifera (Li Z. et al., 2017; Staron et al., 2017). However, in this work, only one GST (Cluster-9767.30914) was found to be highly expressed in the IMD treated A. gifuensis compared to the CK treatment, while the rest of the GSTs did not show any response to sublethal doses of IMD treatment. Similarly, in Lygus lineolaris, only four of the 19 GSTs were significantly down-regulated after IMD exposure, while the rest of these genes did not show any detectable difference in expression (Zhu and Luttrell, 2015). All of these results indicate that GST might not be responsible for IMD resistance in A. gifuensis.

In addition to these three major detoxification pathways, other interrelated pathways might also contribute to the response of xenobiotics, such as superoxide dismutase (SOD), catalase, POD, and HSPs (Chaimanee et al., 2016). In honeybee queens, exposure to IMD and coumaphos significantly depressed the expression of SOD and thioredoxin peroxidase (Chaimanee et al., 2016). Conversely, the expression of catalase, SOD and thioredoxin peroxidase was significantly increased in worker bees (Chaimanee et al., 2016). In this work, POD (Cluster-9767.17490) and HSPs (Cluster-9767.16364 and Cluster-9767.39176) were up-regulated in IMD treated A. gifuensis, which is consistent with the HSP expression profiles in betacypermethrin treated R. padi (Li Y. T. et al., 2017). Further, exposure to IMD significantly decreased the expression of GSTs.

Together, these results indicate that detoxification and stress response systems are critical for protecting A. gifuensis from IMD damage.

To support or drive detoxification processes, the increased energy production through the up-regulation of enzymes involved in ATP synthesis, sugar metabolism, fatty acid metabolism, glycolysis, and the tricarboxylic acid (TCA) cycle were investigated. Our transcriptome data revealed that IMD treatment altered the expression of genes in energyproducing metabolic pathways such as fatty acid metabolism and sugar metabolism. Consistent with this finding, exposure to neonicotinoid also led to increased energy usage in honey bees (du Rand et al., 2017). Furthermore, exposure to a sublethal dose of beta-cypermethrin, led to an increase in respiratory quotient and respiratory rates in Harmonia axyridis, which is often coupled with the status of energy metabolism (Xiao et al., 2017). All of these results suggest that insects increase their energetic cost when undergoing detoxification after pesticide exposure. The increase in their energetic cost might result in the decrease of longevity and parasitism.

With the wide use of pesticides in agriculture and horticulture, understanding how pesticides impair, and influence biological efficiency of natural enemy insect species and how natural enemies adjust their detoxification mechanisms to metabolize pesticides is very important. In this work, we found that exposure to sublethal doses of IMD significantly affected the biological performance of A. gifuensis, potentially through changes in the expression of genes involved in the nervous, olfactory, detoxification systems and energy metabolism. Our results indicated that pesticides may block some physiological or biochemical processes that lead to the disruption of the survival, growth, development, reproduction, and behavior of the natural enemies of insect pests. Based on these results, we not only elucidated the sublethal effects of pesticides on the natural enemies, but also contributed to a better understanding of how residual pesticides influence the biological performance of natural enemies and how natural enemies respond to environmental xenobiotics. Our results provide an insight on how to improve experimental approaches, to investigate IPM.

# AUTHOR CONTRIBUTIONS

Z-WK and T-XL designed the study. Z-WK, R-PP, and F-HL performed research. Z-WK, F-HL, and H-GT analyzed data. Z-WK wrote the manuscript. H-GT and T-XL edited the manuscript. Z-WK revised the manuscript.

# FUNDING

This work was supported by the National Key Basic Research Program of China (973 Program) (No. 2013CB127600), China Agriculture Research System (CARS-23-D06), and the China Scholarship Council (award to Z-WK for two year's study abroad at the University of Georgia: 201706300121).

# ACKNOWLEDGMENTS

We are grateful for Dr. Shun-Hua Han (University of Georgia) for GLMM analysis and the assistance of all staff and students in the Key Laboratory of Applied Entomology, Northwest A&F University at Yangling, Shaanxi, China.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01729/full#supplementary-material

Figure S1 | qPCR results of SNF, GST5, SOD1 and SOD2. ns over the bars mean no significant difference, and the error bars is ± SE bars. N = 3.

Figure S2 | Activities of SOD, CAT, and GST. ns over the bars mean no significant difference, and the error bars is ± SE bars. N = 3.

Table S1 | Primers used for target genes and reference genes in qPCR.

Table S2 | RNA quality of transcriptomic samples.

Datasheet 1 | Gene expression differences between IMD and CK.

#### REFERENCES


Ishikura, S. (1950). Subsequent fluorescent light trap. J. Agric. Sci. 5, 15–19.


genes in an aphid endoparasitoid Aphidius gifuensis. Sci. Rep. 7:3939. doi: 10.1038/s41598-017-03988-z


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kang, Liu, Pang, Tian and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Insects With Survival Kits for Desiccation Tolerance Under Extreme Water Deficits

Leena Thorat\* and Bimalendu B. Nath\*

Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule Pune University, Pune, India

The year 2002 marked the tercentenary of Antonie van Leeuwenhoek's discovery of desiccation tolerance in animals. This remarkable phenomenon to sustain 'life' in the absence of water can be revived upon return of hydrating conditions. Today, coping with climate change-related factors, especially temperature-humidity imbalance, is a global challenge. Under such adverse circumstances, desiccation tolerance remains a prime mechanism of several plants and a few animals to escape the hostile consequences of fluctuating hydroperiodicity patterns in their habitats. Among small animals, insects have demonstrated impressive resilience to dehydration and thrive under physiological water deficits without compromising on revival and survival upon rehydration. The focus of this review is to compile research insights on insect desiccation tolerance, gathered over the past several decades from numerous laboratories worldwide working on different insect groups. We provide a comparative overview of species-specific behavioral changes, adjustments in physiological biochemistry and cellular and molecular mechanisms as few of the noteworthy desiccation-responsive survival kits in insects. Finally, we highlight the role of insects as potential mechanistic models in tracking global warming which will form the basis for translational research to mitigate periods of climatic uncertainty predicted for the future.

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Jose-Luis Martinez-Guitarte, Universidad Nacional de Educación a Distancia (UNED), Spain Kevin Hidalgo, INRA UR370 Qualité des Produits Animaux, France

#### \*Correspondence:

Leena Thorat leenathorat@gmail.com Bimalendu B. Nath bbnath@gmail.com

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 27 August 2018 Accepted: 06 December 2018 Published: 21 December 2018

#### Citation:

Thorat L and Nath BB (2018) Insects With Survival Kits for Desiccation Tolerance Under Extreme Water Deficits. Front. Physiol. 9:1843. doi: 10.3389/fphys.2018.01843 Keywords: insect ecology, humidity, temperature, climate change, stress, desiccation tolerance, anhydrobiosis, adaptation

# INTRODUCTION

Long-term drought conditions leading to physiological water deficits are a threat to the survival and distribution of all organisms. To this notion, what comes as a delightful surprise is the demonstration of water loss mediated resurrection of apparently 'dead' organisms (Keilin, 1959). Such organisms have a remarkable ability of desiccation tolerance whereby they sustain cellular integrity in the desiccated form by activating unique physiological mechanisms (Clegg, 2001). Interestingly, this phase is reversible upon rehydration causing the revival and resumption of active metabolism. At present, global concerns include the challenges associated in coping with climatic stressors, especially the fallout due to humidity-temperature imbalance (Bellard et al., 2012; Boggs, 2016). Under the global sustainable development agendas<sup>1</sup> , research priorities on "life on land" (item#15) and "climate action" (item#13) have warranted attention. Among small animals, insects have proved to be reliable biological systems to anticipate cause-and-effect relations of climate change stressors (Addo-Bediako et al., 2001; Hoffmann and Todgham, 2010).

<sup>1</sup>https://www.un.org/sustainabledevelopment/sustainable-development-goals/

This mini-review highlights the notable adaptive mechanisms employed by insects to evade dehydration bouts in their habitats. There have been a few reviews on similar topics (Watanabe, 2006; Cornette and Kikawada, 2011; Chown et al., 2011; Sogame and Kikawada, 2017); however, no recent competent review has emphasized on the profound diversity of hygropreference and associated strategies in insects. Most importantly, we discuss the desiccation tolerance profiles in insects irrespective of whether they possess a lower tolerance potential or are anhydrobiotic with a tolerance for severe water loss. These aspects have not been fully appreciated in the past, therefore, we aim to compile the diverse range of insect desiccation stress responses from a general perspective. Lastly, the present evaluation is by no means an exhaustive list of all desiccation tolerant insects; nonetheless, many case studies have been gathered within the ambit of insect water stress management.

#### DRY BUT NOT DEAD

The documented history of desiccation tolerance dates back to 370 BC when Theophrastus described conditions necessary to store 'dry seeds alive' (Leprince and Buitink, 2015). Later, Antonie van Leeuwenhoek described his amazement over the dry dust containing 'tiny dry animalcules' that came to life within a few hours after being rehydrated with water (Keilin, 1959). Little did Leeuwenhoek know that his meticulous observations would form the basis of the latent phases of life. To describe this phenomenon, Giard (1894) coined the term 'anhydrobiosis,' an extreme form of desiccation tolerance which in Greek implies 'life without water.' 'Desiccation avoidance' and 'desiccation tolerance' are distinguishable phenomena (Pallarés et al., 2016). The former refers to the maintenance of water uptake and/or minimization of body water loss (e.g., Folsomia candida, Collembola: Isotomidae) while the latter includes organisms that can afford loss of water and sustain a dry form without compromising on revival upon rehydration (e.g., all anhydrobiotes). The threshold for tolerance of water loss is highly species-specific and striking differences in desiccation tolerance strategies and traits in congeneric insect species have been linked with their geographic locations and the frequency and duration of drought exposure (Marron et al., 2003; Strachan et al., 2015). However, this is not true in all insects such as few heliconiine butterflies (Lepidoptera: Nymphalidae) (Mazer and Appe, 2001). Contrary to the rationale that desert insects can withstand higher water loss than mesic species, the aquatic beetle, Peltodytes muticus (Coleoptera: Haliplidae) is known for its highest tolerance in comparison to the desert spider beetle, Mezium affine (Coleoptera: Ptinidae) (Pallarés et al., 2016). Closely related Drosophila species (Diptera: Drosophilidae) have evolved different water balance mechanisms as demonstrated in D. nepalensis vs. D. takahashii and D. immigrans vs. D. nasuta (Parkash et al., 2012a,b).

Each organism may have its specific threshold longevity in the dry state; however, desiccation tolerance by no means confers 'immortality' or infinite survival but is rather influenced by the mode of desiccation, storage temperature, humidity and oxygen content (Tunnacliffe and Lapinski, 2003; Suemoto et al., 2004; Thorat and Nath, 2016). Depending on these factors, organisms display varying longevities in the desiccated form that may range from 1 day to several years (**Figure 1**). Notwithstanding these variations and by virtue of qualitative considerations, all such organisms have been considered as desiccation tolerant (Watanabe, 2006). To the best of our knowledge, a numerical method devised for grouping prokaryotes based on their degree of desiccation tolerance, was the first attempt made by Hernández et al. (2009). A recent study in animals proposed the 'desiccation tolerance index' (DTi) as a quantitative measure of endurance to desiccation stress (Thorat and Nath, 2016). This mathematical tool is based on the desiccation tolerance in nine oriental Chironomus species (Diptera: Chironomidae) which indicate varying degrees of the tolerance threshold based on their ecological habitats (**Figure 2**).

#### ANHYDROBIOSIS: AN EXTREME CASE OF DESICCATION TOLERANCE

Anhydrobiosis is characterized by extreme body water loss, generally over 95% (Benoit, 2010; Sogame and Kikawada, 2017). Thus, anhydrobiosis refers to complete desiccation, unlike desiccation tolerance, which refers to partial dehydration. In this context, we would like to introduce the term, 'euryhygrobiote' for such organisms that show a wide range of dehydration tolerance with a high anhydrobiotic potential. Conversely, we coin the term 'stenohygrobiote' for organisms that have a narrow dehydration tolerance range and can bear water loss only up to a certain limit. The extremophilic midges (Diptera: Chironomidae), Polypedilum vanderplanki (Hinton, 1951) and Belgica antarctica (Lopez-Martinez et al., 2009) are valuable models in understanding the gamut of molecular and biochemical signatures that render them anhydrobiotic. Anhydrobiotes can also be referred to as 'anhydrophiles' in comparison to 'anhydrophobes,' which lack desiccation tolerance. P. vanderplanki, the largest known anhydrobiotic eukaryote, endures water content as low as 3% through a gradual and optimized desiccation regime to sustain the dry state for 17 years until rehydration (Cornette and Kikawada, 2011). A new related species, Polypedilum pembai sp.n. also possesses anhydrobiotic potential and shares a few overlapping mechanisms with P. vanderplanki (Cornette et al., 2017). Recent work from our laboratory has demonstrated that the tropical midge, Chironomus ramosus and the fruit fly, Drosophila melanogaster possess a lower ability to tolerate water loss in comparison to the anhydrobiotic midges (Thorat et al., 2017) and are therefore stenohygrobiotic. Among invertebrates, other well-studied noninsect anhydrobiotes include brine shrimps, tardigrades, rotifers and nematodes (Tunnacliffe and Lapinski, 2003; Rebecchi, 2013). Interestingly, desiccation tolerance also confers cross tolerance to a variety of other stressors through multiple physiological defenses including physical and cellular protection via antioxidants, compatible solutes, proteins and DNA repair (Gusev et al., 2010b).

# DESICCATION TOLERANCE STRATEGIES IN INSECTS

Environmental cues cause dormancy in insects, a phenomenon triggered by climatic signals including humidity, photoperiod, temperature, etc. (Diniz et al., 2017). Dormancy is further classified into diapause and quiescence. While diapause is a pre-programmed predictive strategy, quiescence is an immediate response to adverse environmental conditions (Denlinger, 1986; Danks, 2002). Aestivation, a form of consequential dormancy is the reason behind the aridity survival strategies of several insect species (Colvin, 1996; Benoit and Denlinger, 2007; North and Godfray, 2018). Anhydrobiosis (ametabolism) is an adaptation against physiological water stress, whereas dormancy is characterized by interrupted or reduced metabolic and hormonal activities (hypometabolism) in response to environmental cues (Watanabe, 2006).

While external milieu trigger desiccation stress responses, interoception is central to tolerance, survival and propagation of species. Below, we discuss a few of the striking and widely established strategies that constitute part of the desiccation tolerance approach of insects (**Table 1**).

#### Behavior and Ecology

Hygrosensing abilities and behavioral responses suggest an evolutionary strategy for coping with water loss in insects (Chown et al., 2011). For instance, cockroaches show aggregation in order to control the water loss rate per individual (Dambach and Goehlen, 1999). Similar observations in Chironomus larvae indicate a 'clumping' behavior, forming a single bunch to reduce evaporative body water loss (Thorat and Nath, unpublished). Some beetles exhibit bimodal activity patterns in order to escape the hottest hours of the day whereas others display fog-basking for moisture absorption from the surroundings (Bedick et al., 2006; Chown et al., 2011). Other striking evidences for aridity protection, come from niche construction behaviors such as the housing nests of chironomid midges, termite nests, domiciles of some thrips and insect galls (Kikawada et al., 2005; Gilberta, 2014; Zukowski and Su, 2017; Thorat and Nath, 2018). The cuticle is the first portal of water loss in insects and the differential desiccation tolerance patterns in C. ramosus vs. D. melanogaster and P. vanderplanki vs. Paraborniella tonnoiri (Diptera: Chironomidae) have been attributed to striking differences in their cuticular thickness (Nakahara et al., 2008; Thorat et al., 2017). Furthermore, in some insects, restructuring of the cuticle and morphological changes in spiracular features are crucial to minimize water loss. Such restructuring mechanisms are important because water is mainly lost passively and/or actively throughout spiracular respiration and cuticular transpiration (Hadley, 1994; Benoit and Denlinger, 2007; Benoit, 2010; Bazinet et al., 2010; Wadaka et al., 2016; Hidalgo et al., 2018; Ferveur et al., 2018). Other behavioral traits for desiccation protection such as the arrangement of egg laying (layering and density) in the nymphalid butterfly, Chlosyne lacinia (Lepidoptera: Nymphalidae), increases desiccation survival chances of eggs (Clark and Faeth, 1998).

#### Development and Hormonal Regulation

Our current understanding on the desiccation-mediated developmental consequences in insects is rather fragmented. In the case of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae), desiccation does not exert significant effects on the average eclosion time (Xie and Zhang, 2007). In C. ramosus and D. melanogaster, modulations in 20-hydroxyecdysone affect recovery patterns and are linked with the desiccationmediated delay in metamorphosis (Thorat and Nath, 2015; Thorat et al., 2016b). Interestingly, in D. melanogaster, despite the developmental heterochrony, the overall duration of postembryonic development of the life cycle remains almost unaltered. This is reminiscent of Waddington's 'canalization' as an adaptive buffer to adjust their life histories around optimal seasonal conditions (Thorat et al., 2016b). Life cycle and aging in desiccation tolerant animals has been categorized into three hypothetical models, the first, known as the 'Sleeping Beauty' model, implies that organisms totally disregard the entire time spent in the dry state, the second model considers that organisms register partial discount of the time spent in the dry state and the third model, whereby organisms record the exact time spent in the dry state, exhibiting non-extended longevity. D. melanogaster follows the Sleeping Beauty model similar to the non-insect anhydrobiotic tardigrade, Milnesium tardigradum (Schill, 2010; Thorat et al., 2016b). Variations in insect hormonal titres are key players in synchronizing developmental changes in order to handle ecological ramifications of stressful environments such as hypoxia, high temperatures, starvation and sleep deprivation; however, investigations in the context of desiccation stress are warranted.

#### Physiological Biochemistry

A longstanding biochemical adjustment of survival under dry conditions, is the ability of desiccation-responsive synthesis and accumulation of biomolecules including trehalose, mannitol, glycerol, Heat-Shock (HS) and Late Embryonic Abundant (LEA) proteins, proline, glycine-betaine, gamma aminobutyric acid, alanine, and glucosamine (Crowe and Madin, 1974; Tunnacliffe and Lapinski, 2003; Yoder et al., 2006; Kikawada et al., 2008; Philip et al., 2008; Benoit et al., 2009; Mitsumasu et al., 2010; Thorat et al., 2012; Hidalgo et al., 2014; Shukla et al., 2015, 2016, 2018; Yoshida et al., 2016; Thorat et al., 2017; Mazin et al., 2018). These compatible solutes not only offer protection to the drying tissues but also trigger various signaling responses during recovery. Although trehalose was considered indispensable for desiccation tolerance, recent compelling evidences have affirmed that trehalose accumulation may be completely absent in some organisms in which the desiccation protective role is taken up by other biomolecules (Tunnacliffe et al., 2005; Thorat et al., 2017). Differential physiological mechanisms involving carbohydrates, lipids and proteins are known to contribute to the invasive potential of three related Ceratitis fly species (Diptera: Tephritidae) under episodic dehydration (Weldon et al., 2016). Osmoregulatory mechanisms in lepidopteran species have demonstrated the homeostatic control to readjust hemolymph osmolality triggered by body water loss (Willmer, 1980). Interestingly, eggs of Acanthoscelides obtectus (Coleoptera: Bruchidae) show water loss coping mechanisms that enhance egg tolerance and survival (Biemont et al., 1981). In the case of the flea beetle, Longitarsus bethae (Chrysomelidae: Alticinae), while low relative humidity has no influence on oviposition, aridity beyond a critical point is lethal for the eggs (Simelane, 2007). In contrast, egg desiccation did not affect embryo survival in xeric and mesic populations of the tobacco hawk moth, Manduca sexta (Lepidoptera: Sphingidae) (Potter and Woods, 2012).

#### Antioxidant Defense

Ionic imbalance and changes in osmolarity as a result of cellular water loss leads to the generation of reactive oxygen species (ROS) that are known to damage cellular macromolecules (Alpert, 2005; Benoit and Lopez-Martinez, 2012). Rebecchi (2013) has provided an excellent overview of the whole repertoire of antioxidant defenses under desiccation-responsive oxidative stress management in animals. P. vanderplanki shows the presence of both mitochondrial and cytosolic/extracellular superoxide dismutases (SODs) and abundant glutathione peroxidase and mitochondrial thioredoxin (Cornette et al., 2016; Nesmelov et al., 2016). Furthermore, genes that encode core components of enzymatic antioxidants in P. nubifer are similar to those in insects. However, in P. vanderplanki several groups of antioxidant genes have expanded (Gusev et al., 2014). In

#### TABLE 1 | List of representative desiccation tolerant insects from different orders.


(Continued)

#### TABLE 1 | Continued

fphys-09-01843 December 19, 2018 Time: 18:27 # 6


contrast, SOD serves as the major antioxidant in B. antarctica (Benoit and Lopez-Martinez, 2012). Recently, the role of unconventional antioxidant molecules such as trehalose, proline, polyamines and polyoils has gained attention (Goyal et al., 2004; Schill et al., 2009; Benoit and Lopez-Martinez, 2012). Trehalose, in particular, has been confirmed for its ROS-scavenging ability in SOD-deficient yeast cells and plants (Kranner and Birtic, 2005 ˇ ; França et al., 2007). Using the advantage of molecular genetic tools in Drosophila and a simple, non-invasive method of whole larval real-time imaging, Thorat et al. (2016a) have demonstrated for the first time that during desiccation, trehalose in collaboration with SOD is involved in the maintenance of redox homeostasis in insects.

#### Molecular and Evolutionary Biology

Cellular decline in water levels serves as a cue to elicit defensiveresponses of molecular indicators. Among the molecular responses mediated via proteins, Hsps, namely, smHsp, Hsp70 and Hsp90 have been linked with desiccation survival in insects (Tammariello et al., 1999; Sjursen et al., 2001; Hayward et al., 2004a; Benoit et al., 2009; Benoit, 2010). LEA proteins are another group of upregulated molecules that act as molecular shields to protect other proteins and bio-membranes against aggregation and denaturation resulting from drying (Goyal et al., 2005; Sogame and Kikawada, 2017). Interestingly, however, B. antarctica lacks genes encoding LEA proteins and Hsps are apparently not involved in conferring desiccation tolerance (Philip et al., 2008). Instead, metabolite synthesis and membrane phospholipids, distinct contractile and cytoskeletal protein patterns and aquaporins are among the key players essential for successful anhydrobiosis in the Antarctic midge (Benoit et al., 2007b; Michaud et al., 2008; Li et al., 2009; Teets et al., 2012; Kelley et al., 2014). In addition, desiccation response was shown to upregulate 'Frost,' 'Desi' and 'smp-30' genes whereas 'Desat2' was downregulated during postdesiccation recovery (Sinclair et al., 2007; Kawano et al., 2010). Metabolic fingerprint comparisons in mosquitoes have highlighted specific metabolic alterations, enabling them to survive seasonal aridity (Hidalgo et al., 2015). Diapause in Aedes albopictus (Diptera: Culicidae) promotes desiccation survival by overexpression of a transcript involved in lipid storage with a concomitant increase in hydrocarbon levels (Diniz et al., 2017). Seminal contributions from Davies et al. (2014) have deepened our understanding on the neuroendocrine regulation of salt and water balance in insects (Luan et al., 2015). Recently, the importance of capa neuropeptides as anti-diuretic hormones have been identified in D. melanogaster and is postulated to be a part of desiccation tolerance mechanisms in other insects as well (Davies et al., 2013; Terhzaz et al., 2015).

#### CONCLUSION

Adaptive mechanisms vary among organisms based on their ecological and evolutionary background. Thus, stress tolerance physiology is bound to vary even among closely related species and therefore cannot be generalized. In addition, variations in desiccation tolerance physiology is often a result of the desiccation protocols (acute/chronic) employed. It might therefore be possible to judge the desiccation tolerance or anhydrobiotic potential of organisms in the true sense, only when they are studied under a common denominator of reproducible protocols. Nature has a vast array of tactics

to safeguard its biodiversity and therefore, exploration of other aridity-induced mechanisms in known and unknown desiccation tolerant organisms will give way to our holistic understanding of the diversity in tolerance patterns from an evolutionary, ecological, physiological, cellular and molecular perspective. As reviewed here, although several molecular and biochemical underpinnings of desiccation tolerance in insects are thoroughly studied and well-established, an understanding of some other basic mechanisms remain elusive. For instance, there is a lack of information on the status of the immune responses elicited during desiccation survival. Another neglected area is the understanding of the neuronal basis governing recovery from desiccation that leads to the reactivation of coordinated sensory circuits. As an example, Pflüger and colleagues have determined the role of insect neurotransmitters in modulating multiple physiological and behavioral processes and have emphasized the involvement of biogenic amines under heat, mechanical stress, starvation and chemicals in insects (Verlinden et al., 2010). Similar studies on physiological water deficits in insects can hold great promise for translational research.

The role of insects as reliable mechanistic models presents endless research possibilities for the prediction of the consequences of climate change. The extreme desiccation tolerance of P. vanderplanki has been exploited as a prototype insect system for investigating the influence of spaceflight environments on life processes (Gusev et al., 2010a). Furthermore, the knowledge of insect desiccation biology offers ample ideas for exciting biomedical and pharmaceutical applications, e.g., anhydrobiotic engineering that targets at improving desiccation tolerance of desiccation-sensitive species, including humans (de Castro et al., 2000; Watanabe et al., 2016). These and many other applications that might have been previously viewed as science fiction, are now possible because of our knowledge of insect responses to water scarcity.

#### REFERENCES


Thus, research in desiccation stress response biology has come a long way from curiosity-driven explorations to present day technology-driven applications. Therefore, we hope that this review will trigger impetus for the development of methods and technology to mitigate the consequences of climate change in human and non-human biota.

#### AUTHOR CONTRIBUTIONS

LT and BN designed the review layout. LT prepared the manuscript draft, table, and figures. BN revised the manuscript with critical inputs. BN and LT approved the final version of the manuscript.

#### FUNDING

BN is thankful to partial funding received from UGC-ISF joint Indo-Israel Research Program [UGC-018 (191)] and UGC-CAS (Phase-III) grant. LT is grateful for logistic support received from the DBT Bio-CARe grant. The funders had no role in manuscript design, preparation or decision to publish.

#### ACKNOWLEDGMENTS

We are grateful to Drs. Takashi Okuda (NIAS, Japan), Tetsuo Suemoto (Oita University, Japan) and Koichiro Kawai (Hiroshima University, Japan) for their crucial advice, comments and suggestions on insect desiccation tolerance. We also thank Drs. S. C. Lakhotia (BHU, India) B. J. Rao (TIFR, India) and Amitabh Joshi (Evolutionary and Organismal Biology Unit, JNCSAR, Bengaluru) for their valuable insights.


larvae of the Antarctic midge Belgica antarctica. J. Insect Physiol. 53, 656–667. doi: 10.1016/j.jinsphys.2007.04.006




role for the brain in coordinating the response. J. Insect Physiol. 52, 202–214. doi: 10.1016/j.jinsphys.2005.10.005


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Thorat and Nath. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Protective and Detoxifying Enzyme Activity and ABCG Subfamily Gene Expression in Sogatella furcifera Under Insecticide Stress

Cao Zhou<sup>1</sup> , Hong Yang1,2 \*, Zhao Wang1,3, Gui-Yun Long<sup>1</sup> and Dao-Chao Jin<sup>1</sup>

<sup>1</sup> Provincial Key Laboratory for Agricultural Pest Management of Mountainous Regions, Institute of Entomology, Guizhou University, Guiyang, China, <sup>2</sup> College of Tobacco Science of Guizhou University, Guiyang, China, <sup>3</sup> College of Environment and Life Sciences, Kaili University, Kaili, China

Sogatella furcifera, an important migratory pest of rice, has substantial detrimental effects on rice production. To clarify the mechanism whereby S. furcifera responds to insecticide stress, we measured the activity of its protective [superoxide dismutase (SOD); peroxidase (POD); catalase (CAT)] and detoxifying [carboxylesterase (CarE); glutathione S-transferase (GST); mixed-function oxidase (MFO)] enzymes and the expression levels of its ATP-binding cassette subfamily G (ABCG) transporter genes in response to sublethal concentrations (LC<sup>10</sup> and LC25) of the insecticides thiamethoxam, buprofezin, and abamectin. On the bases of the transcriptome data and the ABCG genes of Laodelphax striatellus, we obtained 14 full-length ABCG sequences for S. furcifera. RT-qPCR results showed that 13, 12, and 9 sfABCG genes were upregulated in the presence of thiamethoxam, buprofezin, and abamectin, respectively, at LC10. Moreover, 13 and 7 sfABCG genes were upregulated following treatment with thiamethoxam and abamectin, respectively, at LC25. Enzyme activity assays showed that although thiamethoxam, buprofezin, and abamectin induced GST, CarE, CAT, POD, and SOD activity, they did so at different concentrations and exposure times. The activity of MFO was generally inhibited with prolonged exposure to the three insecticides, with the inhibitory effect being most significant at 72 h. These results indicate that S. furcifera differs in its response to different types or concentrations of insecticides. Taken together, our results lay the foundations for gaining a deeper understanding of the mechanisms underlying the adaptation of S. furcifera to different types of insecticides, which would be of considerable significance for the development of effective pest management strategies.

Keywords: white-backed planthopper, detoxifying enzyme, protective enzyme, ATP-binding cassette transporter, insecticide stress, response mechanism

# INTRODUCTION

Sogatella furcifera, an important pest of rice, causes serious problems in rice production by sucking phloem sap from the rice plant, inflicting damage through oviposition, and transmitting viral diseases (Zhou et al., 2008). Although the use of insecticides has traditionally been an important means of control for this rice pest (Endo and Tsurumachi, 2001; Nizamani et al., 2002), recent

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Jose Eduardo Serrão, Universidade Federal de Viçosa, Brazil Sengodan Karthi, Manonmaniam Sundaranar University, India

> \*Correspondence: Hong Yang axyridis@163.com

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 17 August 2018 Accepted: 13 December 2018 Published: 08 January 2019

#### Citation:

Zhou C, Yang H, Wang Z, Long G-Y and Jin D-C (2019) Protective and Detoxifying Enzyme Activity and ABCG Subfamily Gene Expression in Sogatella furcifera Under Insecticide Stress. Front. Physiol. 9:1890. doi: 10.3389/fphys.2018.01890

**333**

research has shown that sublethal concentrations of insecticides can affect the reproduction, development, and chemical susceptibility of insects in such a way that it could potentially result in the resurgence of pests (Zhou et al., 2017).

In general, the detoxification process in insects can be divided into three phases: phase I, phase II (involving metabolizing enzymes), and phase III (involving transporters) (Xu et al., 2005). The main enzymes involved in the phase I and phase II detoxification processes are P450 monooxygenase, glutathione S-transferase (GST), and carboxylesterase (CarE) (Xiao et al., 2018), whereas the ATP-binding cassette (ABC) transporters are the main components of phase III (Ferreira et al., 2014). In this regard, it has previously been observed that when the nymphs of Locusta migratoria were treated with chlorantraniliprole at LC50, only the activities of esterase (EST) and GST increased on the first day of treatment, whereas mixed-function oxidase (MFO) activity increased only at 3 days after treatment (Cao et al., 2017). In addition, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are three important protective enzymes in insects that play roles in immunity, preventing free-radical-associated damage, and protecting cells from adverse environmental effects (Dubovskiy et al., 2008; Bi et al., 2010). It has previously been reported that in response to treatment with abamectin at LC<sup>10</sup> and LC<sup>20</sup> for 12 h, the activities of SOD, POD, and CAT in Harmonia axyridis were higher relative to those in the untreated control group, although these activities gradually returned to normal levels as time progressed (Yang et al., 2015). Furthermore, in response to treatment with imidacloprid at LC<sup>10</sup> and LC20, the SOD and POD activities in Aphidius gifuensis initially appeared to be inhibited but were subsequently stimulated, with the highest activity occurring at 36 h. Moreover, it has been found that with an increase in insecticide concentration, SOD, POD, and CAT activities show a decreasing trend (Zhu et al., 2015).

The ABC transporters comprise a large family of proteins that mediate the transport of inorganic ions, sugars, amino acids, lipids, lipopolysaccharides, peptides, metals, xenobiotics, and chemotherapeutic drugs (Higgins, 1992). In insects, this family can be subdivided into eight major subfamilies (A– H) (Dean et al., 2001). Studies of the ABC transporters in eukaryotes have revealed that they are capable of transporting structurally unrelated compounds (Dassa and Bouige, 2001; Dean et al., 2001), and researchers are thus increasingly focusing on the roles of these proteins in the transport of exogenous substances and in insecticide resistance in insects. Recent studies have shown that the expression of ABC transporters is directly related to the development of insecticide resistance (Silva et al., 2012a; Dermauw and Van Leeuwen, 2014). After treatment of Bactrocera dorsalis with malathion, abamectin, and beta-cypermethrin at an LD<sup>50</sup> concentration, 4, 10, and 14 bdABC genes were significantly upregulated, respectively (Xiao et al., 2018). Quantitative polymerase chain reaction (qPCR) analysis has revealed that eight ABC transporters in the ABCB/C/D/G subfamilies were upregulated in strains of Laodelphax striatellus resistant to chlorpyrifos, deltamethrin, and imidacloprid, compared with those in a susceptible strain (Sun et al., 2017). In Plutella xylostella, RNA sequencing (RNA-seq) analysis showed that ABC transporters from the ABCA/C/G/H/F subfamilies were overexpressed in chlorpyrifos-resistant strains (You et al., 2013). Nevertheless, despite the insights gained from these studies, our current understanding of the role of ABC transporters in insect resistance to insecticides remains limited.

At present, little is known regarding the effects of insecticides on the activities of the detoxifying and protective enzymes and ABC transporters of S. furcifera. Accordingly, in this study, we sought to gain insights into the roles of these enzymes and the sfABCG subfamily genes in the response of S. furcifera to insecticide-induced stress. To this end, we exposed this insect to sublethal concentrations of three insecticides (thiamethoxam, abamectin, and buprofezin) and subsequently monitored the changes in enzyme activity and gene expression levels.

#### MATERIALS AND METHODS

#### Insects and Insecticides

In 2013, S. furcifera individuals were collected from a rice field in Huaxi, Guiyang, Guizhou, China (26◦ 31.302<sup>00</sup> N, 106◦ 62.294<sup>00</sup> E) and maintained on rice seedlings in the laboratory at 25 ± 1 ◦C and 70 ± 10% relative humidity under a 16:8 h (light:dark) photoperiod, without exposure to insecticides. For the purposes of this study, we used third-instar nymphs. Thiamethoxam (96%: technical formulation) was obtained from PFchem, Co., Ltd. (Nanjing, China); abamectin (96.4%: technical formulation) was obtained from Shandong Qilu King-Phar Pharmaceutical, Co., Ltd. (Shandong, China); and buprofezin (97%: technical formulation) was obtained from the Guangxi Pingle Pesticide Factory (Guangxi, China).

#### Insect Treatments and Sample Collection

For the insecticide treatments, we used the rice stem dipping method (Zhou et al., 2017). Three 100 third-instar nymphs were transferred to and reared separately in glass tubes (300 mm high × 30 mm diameter) that were open at both ends and contained rice seedlings dipped in a sublethal concentration (LC<sup>10</sup> or LC25) of thiamethoxam, abamectin, or buprofezin. Rice stems treated with distilled water were used as a control. The insects exposed to each treatment were maintained at 25 ± 1 ◦C and 70 ± 10% relative humidity under a 16:8 h (light:dark) photoperiod in an artificial climate box. After 48 h, 15 surviving insects from each treatment were randomly collected for extraction of RNA for a quantitative reverse-transcription PCR (RT-qPCR) assay. In addition, samples were taken at 6, 12, 24, 48, and 72 h after the treatment to determine the activity of the target enzymes. The LC<sup>10</sup> and LC<sup>25</sup> values (**Supplementary Table S1**) of thiamethoxam, abamectin, and buprofezin for S. furcifera were based on previously presented results (Liu et al., 2015).

#### Gene Identification

The RNA-seq transcriptome database of S. furcifera was sequenced and annotated as described previously (Zhou et al., 2018). With the reported ABCG gene of L. striatellus as a reference, Geneious R9 software (Kearse et al., 2012) was used to assemble the transcriptome data to obtain the corresponding sequences for S. furcifera. In addition, each of the putative ABCG sequences was used as a query to search the NCBI protein database<sup>1</sup> to further validate their identity.

#### Sequence Verification

fphys-09-01890 December 26, 2018 Time: 19:0 # 3

Specific primers were designed and used to amplify the internal cDNA fragments. PCRs were carried out using Sangon Biotech (Shanghai, China) Taq polymerase, under the following conditions: initial denaturation at 94◦C for 3 min; 30 cycles of denaturation at 94◦C for 30 s, annealing at 55–60◦C for 30 s, and elongation at 72◦C for 1–2 min; with a final elongation at 72◦C for 10 min. Specific primers for amplification of the 3<sup>0</sup> and 5<sup>0</sup> ends were designed using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, United States). Using a SMARTer <sup>R</sup> RACE 5<sup>0</sup> /3<sup>0</sup> Kit (Clontech, Mountain View, CA, United States), 3<sup>0</sup> and 5<sup>0</sup> rapid amplification of cDNA ends (RACE) were performed. Total RNA was extracted from 10 fifthinstar nymphs according to the instructions of an HP Total RNA Kit (Omega Bio-Tek, Norcross, GA, United States). Synthesis of the first-strand cDNA and PCR amplifications were carried out according to the instructions of a SMARTer <sup>R</sup> RACE 5<sup>0</sup> /30 Kit. SeqAmp DNA Polymerase (a SMARTer <sup>R</sup> RACE 5<sup>0</sup> /3<sup>0</sup> Kit component) was used for the RACE PCR, under the following conditions: 25 cycles of 94◦C for 30 s, 60–70◦C (depending on the primer) for 30 s, and 72◦C for 3 min. The overlapping PCR products were purified using an E.Z.N.A <sup>R</sup> Gel Extraction Kit, cloned into a linearized pRACE vector (a SMARTer <sup>R</sup> RACE 5<sup>0</sup> /30 Kit component), and sequenced by Sangon Biotech (Shanghai, China). The RACE sequences were assembled on the basis of the partial cDNA sequences corresponding to each fragment.

#### Sequence Alignment and Phylogenetic Analysis

Using ORF finder<sup>2</sup> , we identified the open reading frames (ORFs) of the sfABCG genes and determined the amino acid sequences of the encoded proteins. The Pfam program<sup>3</sup> and a search of the NCBI Conserved Domain Database<sup>4</sup> were used to identify the conserved domains (nucleotide-binding and transmembrane domains) of all putative ABCG genes. The ABCG gene sequences were then subjected to phylogenetic analysis, using the neighborjoining method and a bootstrap test with 1,000 replicates in the MEGA program package, v. 6.0 (Tamura et al., 2011).

#### Gene Expression Analysis

The mRNA levels of the ABC transporter genes under different insecticide treatments were measured by RT-qPCR using FastStart Essential DNA Green Master Mix (Roche, Indianapolis, IN, United States) in a CFX96TM real-time quantitative PCR system (BioRad, Hercules, CA, United States). Total RNA was extracted as described above and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer's protocols.

<sup>2</sup>http://www.ncbi.nlm.nih.gov/orffinder/

The RNA concentration was adjusted to 0.8 µg/µL with diethyl pyrocarbonate-treated H2O, and 0.8 µg of RNA was then reverse transcribed in a 20-µL reaction volume, using the PrimeScript RT Reagent Kit and gDNA Eraser (TaKaRa, Shiga, Japan), with ribosomal protein L9 (GenBank Accession No. KM885285) as an internal control. Specific primer pairs for each gene were designed using Primer Premier 6 (**Supplementary Table S2**). Each RT-qPCR was conducted in a 20-µL mixture containing 1 µL of sample cDNA, 1 µL of each primer (10 µM), 7 µL of diethyl pyrocarbonate-treated H2O, and 10 µL of FastStart Essential DNA Green Master Mix. The qPCR cycling parameters were as follows: 95◦C for 10 min, followed by 40 cycles of 95◦C for 30 s and 60◦C for 30 s. Melting curve generation was performed from 65 to 95◦C. To check the reproducibility of the assay results, the qPCR for each sample was performed using three technical replicates and three biological replicates. The comparative 2−11CT method (Livak and Schmittgen, 2001) was used to calculate the relative quantification.

#### Enzyme Activity Assay

In this study, we performed the following enzyme activity assays: the nitroblue tetrazolium reduction method for SOD; the guaiacol method for POD; a spectrophotometric method for CAT (based on the ultraviolet absorption of peroxide released from the activity of CAT on hydrogen peroxide); a colorimetric method for GST (based on the GST-catalyzed reaction between glutathione and 1-chloro-2,4-dinitrobenzene); and a colorimetric method for CarE (based on the CarE-catalyzed transformation of 1-naphthyl acetate to naphthyl ester, which then reacts with the Fast Blue RR salt to form an azo dye). These assays were conducted using respective commercial assay kits (Comin Biotechnology, Co., Ltd., Suzhou, China). MFO activity was measured according to the method reported by Qian et al. (2008). To check the reproducibility of the results, the enzyme activity assays for each insecticide treatment were performed using four biological replicates.

#### Statistical Analyses

All data were analyzed using Bonferroni corrections for multiple comparisons when the variance was homogeneous. When the variance was non-homogeneous, the Wilcoxon signed-rank test was used. In addition, the Kruskal–Wallis test was used to verify the temporal shifts within the effects of the same insecticide. All analyses were performed using SPSS version 22.0 (SPSS, Chicago, IL, United States) and the data are presented as the mean ± standard error (SE) of three or four biological replicates.

#### RESULTS

#### Identification and Characterization of ABC Subfamily G Transporter Genes

Using the reported ABCG gene of L. striatellus as a reference, Geneious R9 software was used to assemble the transcriptome data to obtain the corresponding sequences for S. furcifera.

<sup>1</sup>http://www.ncbi.nlm.nih.gov/

<sup>3</sup>http://pfam.xfam.org/

<sup>4</sup>https://www.ncbi.nlm.nih.gov/cdd

We verified 14 sfABCG genes by RT-qPCR and RACE (**Supplementary Table S3**). The designation, accession number, length, ORF size, theoretical isoelectric point, and molecular weight of all the sfABCG genes are summarized in **Table 1**. The ORFs of all gene sequences ranged from 603 to 967 bp. We initially identified the characteristic nucleotide-binding domains of ABC transporters using Pfam. The nucleotide-binding and transmembrane domains of all genes were similar to those of

TABLE 1 | Full-length ATP-binding cassette subfamily G (ABCG) transporter genes identified from Sogatella furcifera.


L. striatellus (**Supplementary Figure S2**). As determined from the neighbor-joining tree generated from phylogenetic analysis of the ABCG genes of S. furcifera, L. striatellus, Tribolium castaneum, and B. dorsalis, the corresponding genes of each subfamily are clustered together (**Supplementary Figure S1**).

# Effect of Insecticide Treatment on sfABCG Gene Expression

After exposing third-instar nymphs of S. furcifera to different concentrations of thiamethoxam for 48 h, we examined the relative expression levels of the 14 sfABCG genes. The results showed that the expression of only sfABCG7 was significantly downregulated (2.4-fold) after treatment with the insecticide at LC<sup>10</sup> (**Figure 1**), whereas the other 13 sfABCG genes were significantly upregulated. Among these 13 genes, sfABCG5 (766.6-fold) and sfABCG9 (5.8-fold) showed the highest and lowest upregulation, respectively. Responses to the LC<sup>25</sup> treatment were similar to those observed for the LC<sup>10</sup> treatment, with only sfABCG7 being significantly downregulated (3.5-fold) and the remaining 13 genes being significantly upregulated by 4.5- to 643.8-fold. However, we found that the expression levels of the upregulated genes showed a decreasing trend with increasing insecticide concentration, with sfABCG3 showing significantly different expression levels in response to the LC<sup>10</sup> and LC<sup>25</sup> treatments (Wilcoxon signed-rank test P < 0.05).

After treatment with buprofezin at LC10, the relative expression levels of the 14 sfABCG genes showed trends similar to those observed for thiamethoxam at LC10, with only the sfABCG7 gene being significantly downregulated (3.0-fold), and the other 13 genes all being upregulated (significantly in the case of 12),

from 6.0- to 924.0-fold. Although the expression of sfABCG9 was upregulated relative to that in the control, the difference was not significant (**Figure 1**). Buprofezin treatment at LC<sup>25</sup> resulted in a significant upregulation of sfABCG5 relative to the control, whereas sfABCG7 was significantly downregulated by 2.2-fold (Bonferroni-corrected P = 0.02) compared with the control level.

After treatment with abamectin at LC10, nine sfABCG genes (sfABCG1, sfABCG3, sfABCG4, sfABCG5, sfABCG6, sfABCG8, sfABCG10, sfABCG11, and sfABCG14) were significantly upregulated in the range of 3.2- to 97.4-fold (**Figure 1**). In contrast, compared with the control levels, the expression levels of sfABCG7, sfABCG9, sfABCG12, and sfABCG13 were significantly downregulated in response to abamectin treatment at LC<sup>10</sup> and LC<sup>25</sup> concentrations, with sfABCG13 being the most downregulated by 6.0-fold (LC10) and 13.3-fold (LC25). In response to abamectin exposure at the LC<sup>25</sup> concentration, sfABCG1, sfABCG4, sfABCG5, sfABCG6, sfABCG8, and sfABCG14 were significantly upregulated by 17.1-, 8.1-, 73.1-, 10.98-, 7.4-, and 2.7-fold, respectively, compared with the control levels. Interestingly, the expression levels of both sfABCG1 and sfABCG4 were upregulated with increasing abamectin concentration, with the difference being significant in the case of sfABCG4 (Bonferroni-corrected P = 0.04; **Figure 1**).

To gain a more intuitive understanding of the gene upregulation pattern in response to insecticide exposure, a Venn diagram was generated for the 13 significantly upregulated sfABCG genes after treatment with the three insecticides at LC<sup>10</sup> (**Figure 2**). Among these, sfABCG9 was upregulated by thiamethoxam only; sfABCG2, sfABCG12, and sfABCG13 were upregulated by thiamethoxam and buprofezin; and sfABCG1, sfABCG3, sfABCG4, sfABCG5, sfABCG6, sfABCG8, sfABCG10, sfABCG11, and sfABCG14 were upregulated by all

three insecticides. Among the latter group, the sfABCG5 gene showed the highest upregulation responses, with expression levels 766.6-, 924.0-, and 97.4-fold higher than those of the control in response to thiamethoxam, buprofezin, and abamectin treatments, respectively.

#### Activity of Detoxifying Enzymes

fphys-09-01890 December 26, 2018 Time: 19:0 # 6

Changes in the activity of the detoxifying enzymes in S. furcifera were examined after treatment with sublethal concentrations of the test insecticides for 6, 12, 24, 48, and 72 h (**Figures 3–5**). Compared with control levels, the activity of CarE was significantly increased after 6 and 12 h of treatment with thiamethoxam, buprofezin, and abamectin at the LC<sup>10</sup> and LC<sup>25</sup> levels, showing the same trend for all three insecticides and with the activity being highest at 6 h (**Figure 3**). It is worth noting that after treatment with the three insecticides at LC<sup>10</sup> and LC25, there was an initial increase in the overall activity of CarE with time, followed by a decrease, and then subsequently a further increase.

Glutathione S-transferase activity increased gradually and then decreased after treatment with thiamethoxam at LC10, peaking at 24 h (2.8-fold higher than that of the control). However, in response to treatment with thiamethoxam at LC25, there was no significant difference between the GST treatment and control groups after 24 h, and activity of the enzyme returned to normal levels at 72 h (**Figure 4A**). After treatment with buprofezin at LC<sup>10</sup> and LC25, GST activity showed an overall increasing trend, being highest at 6 h after the LC<sup>25</sup> treatment, and subsequently decreasing with the prolongation of treatment time, albeit at levels significantly higher than that of the control. In contrast, we observed a significant reduction in GST activity in response to treatment with buprofezin at LC<sup>10</sup> for 24 h (Bonferroni-corrected P = 0.04) compared with that of the control, although again the levels had returned to normal at 72 h (**Figure 4B**). In response to treatment with abamectin at LC<sup>10</sup> and LC25, the activity of GST increased significantly at 6 h, reached a maximum at 12 h, and then gradually decreased. Compared with control levels, the activity of this enzyme was significantly higher in response to the LC<sup>25</sup> treatment. However, similar to the response to buprofezin treatment at LC10, GST activity following abamectin treatment at LC<sup>10</sup> was not significantly different from that of the control at 24 and 72 h (**Figure 4C**).

Compared with the control, the activity of MFO showed a decreasing trend in response to treatment with thiamethoxam at LC10, with the difference being significant at 6, 24, and 72 h. In contrast, in response to treatment with thiamethoxam at LC25, although the activity of MFO had decreased at 6 and 72 h, we observed a significant increase at 12 h (Bonferroni-corrected P = 0.001) relative to the control level (**Figure 5A**). In response to buprofezin exposure at LC10, MFO activity was significantly increased at 6 and 12 h compared with that of the control, and reached a peak at 12 h (1.8-fold higher than that of the control). However, at 24, 48, and 72 h, the activity of MFO was significantly reduced. In addition, after treatment with buprofezin at LC25, we detected no significant difference between treatment and

control MFO activities at 6 and 12 h, whereas there was a significant increase in activity in response to treatment at 24 h, which thereafter gradually decreased (**Figure 5B**). In response to treatment with abamectin at LC<sup>10</sup> and LC25, MFO activity showed a decreasing trend compared with the control levels, with the difference being significant at 48 and 72 h. However, in response to abamectin treatment at LC10, MFO activity was significantly higher than that of the control after 12 and 24 h (**Figure 5C**).

#### Activity of Protective Enzymes

The activities of CAT, POD, and SOD were measured at 6, 12, 24, 48, and 72 h after exposure to sublethal concentrations of the test insecticides (**Figures 6–8**). Although at 6 h after treatment with thiamethoxam at LC<sup>10</sup> and LC25, we observed an inhibition of CAT activity, at 12 and 24 h the activity had increased significantly, respectively, but thereafter returned to normal levels (**Figure 6A**). Following treatment with buprofezin at LC<sup>10</sup> and LC25, CAT activity had increased significantly by 1.8- and 2.1-fold at 12 and 6 h, respectively, compared with the control, and in the LC<sup>25</sup> treatment group thereafter gradually returned to a normal level (**Figure 6B**). Similarly, after treatment with abamectin at LC<sup>10</sup> and LC25, CAT activity was 2.4- and 1.9-fold higher, respectively, than that of the control at 6 h, and in the LC<sup>25</sup> treatment group subsequently underwent a gradual return to normal levels. However, after 48 h of LC<sup>25</sup> treatment, the activity of this enzyme had increased significantly to a level 1.6-fold higher than that of the control (**Figure 6C**). Interestingly, in response to treatment with both buprofezin and abamectin at LC10, CAT activity initially increased, then decreased, and subsequently increased again with a prolongation of exposure time.

In response to treatment with thiamethoxam at both LC<sup>10</sup> and LC25, POD activity showed a tendency to initial increase and subsequently return to a normal level (**Figure 7A**). In the case of the LC<sup>10</sup> treatment, POD activity peaked at 24 h (53.7-fold higher than that of the control) and then gradually decreased, albeit at levels still significantly higher than those of the control. In the LC<sup>25</sup> treatment group, POD activity peaked at 12 h (45.9-fold higher than that of the control) and then decreased gradually until reaching the normal level at 72 h (**Figure 7A**). After treatment with buprofezin at LC10, POD activity began to increase significantly at 6 h (Bonferroni-corrected P = 0.004), peaked at 12 h (55.7-fold higher than that of the control), and then decreased gradually to a normal level after 72 h. Similarly, after treatment with buprofezin at LC25, POD activity showed a significant increase at 12 h (53.9-fold higher than that of the control) (Wilcoxon signed-rank test P < 0.05) and then gradually decreased to a normal level after 72 h (**Figure 7B**). The responses of POD activity following exposure to abamectin at LC<sup>10</sup> and LC<sup>25</sup> showed similar patterns to those following buprofezin treatment at LC<sup>10</sup> and LC25, whereby activity peaked at 12 h (70.3- and 97.7-fold higher than that of the control, respectively) and returned to a normal level after 72 h (**Figure 7C**).

Compared with the control level, the SOD activity levels following thiamethoxam treatment at LC<sup>10</sup> and LC<sup>25</sup> were significantly increased at 6 h (1.4- and 2.5-fold higher than that of the control, respectively) and returned to normal levels at 12 h. Subsequently, however, the SOD activity showed a secondary significant increase at 24 h, before eventually returning to a normal level thereafter (**Figure 8A**). In response to treatment with buprofezin at LC10, SOD activity increased significantly at 6 h (3.4-fold higher than that of the control), and then underwent a gradual decrease (**Figure 8B**), whereas following treatment at LC25, the activity of this enzyme increased significantly at 12 h to a level 1.6-fold higher than that of the control. After 48 h of exposure to buprofezin at LC<sup>10</sup> and LC25, SOD activity had decreased by 59.9 and 26.5%, respectively, compared with the control level, but had returned to a normal level at 72 h (**Figure 8B**). The responses of SOD activity to treatment with abamectin at LC<sup>10</sup> and LC<sup>25</sup> showed trends similar to those following buprofezin treatment at LC<sup>10</sup> and LC25; however, after 24 h of abamectin treatment at both sublethal concentrations, SOD activity showed a tendency to return to a normal level (**Figure 8C**).

#### DISCUSSION

Previous studies on insects have shown that the protective enzymes SOD, POD, and CAT are related to resistance and the response to insecticide-induced stress. In this regard, it has been reported that sublethal concentrations (LC<sup>10</sup> and LC25) of abamectin can promote upregulation of the SOD, POD, and CAT activities in Diadegma semiclausum adults, with activity increasing with increasing insecticide concentration (Jia et al., 2016). In third-instar H. axyridis nymphs exposed to LC<sup>10</sup> abamectin, the highest levels of SOD, POD, and CAT activity were recorded at 24, 12, and 24 h, respectively, and were significantly higher than those in the control group (Yang et al., 2015). In the present study, the overall levels of SOD, POD, and CAT activity in abamectin-treated (LC<sup>10</sup> and LC25) S. furcifer tended to undergo an initial increase and thereafter gradually return to normal levels, reaching their highest levels at 12, 12, and 6 h, respectively. Interestingly, at 12 and 24 h, POD and CAT activities showed an increase in response to increasing abamectin concentration, which is consistent with the observations on D. semiclausum previously reported by Jia et al. (2016). In contrast, the levels of SOD, POD, and CAT activity in thirdinstar H. axyridis nymphs were shown to decrease with an increase in abamectin concentration (Yang et al., 2015). In the present study, we found that exposure to thiamethoxam initially tended to promote upregulation of the overall activities of POD and SOD and then inhibit them with an increase in the insecticide concentration from LC<sup>10</sup> to LC25, which contrasts with the observations for buprofezin (LC<sup>10</sup> and LC25), which initially inhibited and then upregulated POD and SOD activities with increasing sublethal concentration. For CAT, buprofezin

initially upregulated and then inhibited enzyme activity with increase in concentration, whereas thiamethoxam tended to initially inhibit and then upregulate CAT activity with increase in concentration. Similar observations have previously be made in Aphidius gifuensis, in which the levels of SOD, POD, and CAT activity tended to decrease with an increase in imidacloprid concentration (LC10, LC20, LC30, and LC50) (Zhu et al., 2015). Such studies indicate that, in insects, SOD, POD, and CAT activities are related to insect resistance and the response to insecticide-induced stress, although the effects of these enzymes may be species, concentration, and time dependent.

The detoxifying enzymes CarE, GST, and MFO are also important components of insect resistance mechanisms, an increase in the activities of which is necessary during insecticide metabolism (Qi et al., 2016). Previously, it has been found that the levels of GST and MFO activity in two color morphs of the pea aphid Acyrthosiphon pisum increased in response to increasing sublethal concentrations of abamectin (LC5, LC10, and LC20) following exposure for over 24 h (Wang and Liu, 2014). Similarly, the activities of CarE, GST, and MFO in Tetranychus urticae were significantly upregulated at 12 h following exposure to abamectin (LC<sup>10</sup> and LC25) (Ru et al., 2017). In the present study, avermectin (LC<sup>10</sup> and LC25) resulted in a similar significant induction of CarE, GST, and MFO activities in S. furcifera, at 6, 12, and 24 h, respectively. These findings indicate that insects can adapt to the stress induced by avermectin by activating their detoxifying enzymes. In addition, after treatment with thiamethoxam and buprofezin (LC<sup>10</sup> and LC25), CarE activity showed an overall trend of initial upregulation and subsequent inhibition, with the activity being highest at 6 h. Thiamethoxam and buprofezin also significantly induced GST activity in S. furcifera, whereas these insecticides were found to have a generally inhibitory effect on the activity of MFO. Previously, it was found that GST and P450 activities in Aphis craccivora were significantly induced after treatment with cycloxaprid and imidacloprid (LC50) for 48 h, whereas in contrast, the activity of the CarE activity was inhibited, although the observed difference was not significant (Wu et al., 2016). In addition, after treating Cydia pomonella with imidacloprid (LC20), Shang et al. (2017) observed a significant induction of CarE and GST activity, whereas MFO activity was significantly inhibited. These findings suggest that MFO may not play a major role in the insect response to stress induced by neonicotinoid insecticides, and that the primary detoxifying enzymes are CarE and GST. The aforementioned findings indicate that detoxifying enzymes enable insects to respond to low levels of insecticide-induced stress; however, similar to protective enzymes, CarE, GST, and MFO are induced at different times in different insects. Moreover, the main enzymes involved in detoxification appear to be species dependent.

The ABC transporters are important participants in the third stage of detoxification and have been widely reported to be involved in insecticide resistance (Qi et al., 2016). In this regard, it has previously been found that the expression levels of an

ABCG gene and an ABCC gene were upregulated in S. furcifera treated with a high concentration (LC85) of cycloxaprid, whereas the expression levels of two ABCG genes were upregulated at a low concentration (LC15) of this insecticide (Yang et al., 2016). Transcriptome sequencing has revealed that the ABCB, ABCC, and ABCG subfamily genes are expressed at high levels in a pyrethroid-resistant strain of Aedes aegypti (Bariami et al., 2012). Similarly, results of microarray experiments have shown that genes of the ABCG and ABCH subfamilies are expressed at high levels in resistant strains of Myzus persicae (Silva et al., 2012b), and that the expression levels of ABCG subfamily genes are increased in DDT-resistant strains of Anopheles arabiensis (Jones et al., 2012). Given that ABCG subfamily genes play a role in insecticide resistance in many insects (You et al., 2013; Yang et al., 2016; Sun et al., 2017; Xiao et al., 2018), we decided to study the expression of 14 ABCG subfamily genes in S. furcifera in response to thiamethoxam, buprofezin, and abamectin. We accordingly found that 13 of these 14 sfABCG genes were significantly upregulated after treatment with at least one sublethal concentration of insecticide. On exposure to these insecticides at the LC<sup>10</sup> level, 13 sfABCG genes were significantly upregulated by thiamethoxam, 12 were significantly upregulated by thiamethoxam and buprofezin, and nine were upregulated by all three insecticides. Furthermore, 13 and seven sfABCG genes were significantly upregulated after treatment with LC<sup>25</sup> concentrations of thiamethoxam and abamectin, respectively. These findings provide further evidence that ABC transporters probably participate in the transport of various substrates related to the resistance to different types of insecticides. Moreover, it is conceivable that, in addition to enhancing the metabolism of S. furcifera, these highly expressed sfABCG genes are associated with cross-resistance in this insect. However, these inferences need to be verified with functional experiments.

The sublethal effects of insecticides on insects are multifaceted, including their effects on insect behavior, reproduction, development, and insecticide resistance. In addition, insect adaptation to insecticide stress is a complex metabolic detoxification process involving the activity of multiple enzymes. The results of our study show that S. furcifera can eliminate insecticides in the body by activating detoxifying enzymes and ABC transporters, and also activate the protective enzyme system to prevent injury to the body. Taken together, our research results lay the foundations for gaining a deeper understanding of the mechanisms contributing to the adaptation of S. furcifera to different types of insecticides, which is of considerable significance with regards to the development of effective pest management strategies.

#### DATA AVAILABILITY STATEMENT

The gene sequences obtained have been submitted to the NCBI database (Accession Nos. MH481837–MH481850). Other datasets for this study are included in the manuscript and the **Supplementary Files**.

#### AUTHOR CONTRIBUTIONS

fphys-09-01890 December 26, 2018 Time: 19:0 # 11

HY conceived and designed the experiments. ZW and G-YL measured the detoxifying and protective enzyme activities. CZ examined the ABCG gene expression levels and prepared the manuscript. CZ, HY, ZW, D-CJ, and G-YL finalized the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This research was supported by the National Natural Science Foundation of China (Grant No. 31560522), the Provincial Key

#### REFERENCES


Project for Agricultural Science and Technology of Guizhou (Grant Nos. NY20133006 and NY20103064), the International Cooperation Base for Insect Evolutionary Biology and Pest Control (Grant No. [2016]5802), and the Graduate Education Innovation Project of Guizhou Province (Qian Jiao He YJSCXJH, Grant No. [2018] 043).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01890/full#supplementary-material



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhou, Yang, Wang, Long and Jin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Inhibitory Effect of Protease Inhibitors on Larval Midgut Protease Activities and the Performance of Plutella xylostella (Lepidoptera: Plutellidae)

#### Aiping Zhao1,2, Yin Li1,2, Chunmeng Leng1,2, Ping Wang<sup>3</sup> and Yiping Li1,2 \*

<sup>1</sup> Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Northwest A&F University, Yangling, China, <sup>2</sup> State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, China, <sup>3</sup> Department of Entomology, Cornell University, Ithaca, NY, United States

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Fengliang Jin, South China Agricultural University, China Najmeh Sahebzadeh, Zabol University, Iran Xinzhi Ni, Agricultural Research Service (USDA), United States

\*Correspondence:

Yiping Li liyiping@nwsuaf.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 01 June 2018 Accepted: 31 December 2018 Published: 15 January 2019

#### Citation:

Zhao A, Li Y, Leng C, Wang P and Li Y (2019) Inhibitory Effect of Protease Inhibitors on Larval Midgut Protease Activities and the Performance of Plutella xylostella (Lepidoptera: Plutellidae). Front. Physiol. 9:1963. doi: 10.3389/fphys.2018.01963 Plutella xylostella L. (diamondback moth) is a pest of cruciferous plants. To understand the relationship among protease inhibitors, protease activities and the growth and development of this insect, the activities of midgut proteases of P. xylostella larvae were determined in this study. Protease samples were extracted from the midguts of P. xylostella larvae, and the protease activities were determined using enzyme specific substrates. The results showed that CaCl2, EDTA, and EGTA inhibited only the trypsin. Among the common protease inhibitors, phenylmethyl sulfonyl fluorine (PMSF), Nα-p-methyl sulfonyl-L-lysine chloromethylketone (TLCK), Nα-methyl sulfonyl-L- phenylalanine chloromethyl ketone (TPCK), soybean trypsin inhibitor (STI), and PMSF inhibited the total protease, high-alkaline trypsin (a trypsin subtype with highly alkaline pH optimum), low-alkaline trypsin (another trypsin subtype with slightly alkaline pH optimum), and chymotrypsin; TLCK inhibited the total protease and high-alkaline trypsin, whereas TPCK only activated the high-alkaline trypsin activities. STI had an inhibitory effect on all the proteases. These results showed that protease inhibitors had a certain extent inhibition to protease activities in the larval midgut of P. xylostella and that STI can potentially be used for effective pest control. The development of P. xylostella was delayed in the presence of different inhibitors. These effects were also related to the concentration of the inhibitor. A higher STI concentration showed a longer lasting effect but lower effect in this study compared to that of TLCK. The protease inhibitors had some inhibitory effect on the synthesis and secretion of proteases, and interfered with the protease activity, thereby inhibiting the absorption of nutrients and delaying the growth and development of P. xylostella and reducing their ability to reproduce. These findings should provide the baseline information about using for effective pest management in the future.

Keywords: Plutella xylostella, protease activity, protease inhibitors and activators, growth and development, midgut

#### INTRODUCTION

fphys-09-01963 January 14, 2019 Time: 16:23 # 2

The midgut of insects contains proteases that are involved in several physiological, biochemical processes and promote food digestion and nutrient absorption. Proteases can be divided into four groups, namely serine proteases, metalloproteinases (MMPs), cysteine proteases and aspartate proteases. The serine proteases are mainly involved in digestive processes. Serine proteases especially trypsin, chymotrypsin and elastase in particular have a digestive function, i. e., they can break peptide bonds in large proteins to generate smaller peptides (Jiang and Kanost, 2000; Li et al., 2004). Most serine proteases have an active serine residue at a conserved position (Ser-195). In lepidopteran species, the larval midgut was reported to contain serine proteases, particularly trypsin and chymotrypsin (Milne and Kaplan, 1993; Srinivasan et al., 2006). The midgut of lepidopteran larvae has an alkaline environment where serine proteases are reported to have a high level of activity (Berenbaum, 1980; Pritehett et al., 1981; Applebaum, 1985), however, the optimum pH for trypsin activity varies across insect species (Broadway, 1989; Kipgen and Aggarwal, 2014; Zhao et al., 2016).

Protease inhibitors can prevent the protease activity by binding to the active sites or allosteric sites of proteases or their zymogens (Xiao et al., 2004). For developing insectresistant plants using these inhibitors with the help of advanced technologies, such as genetic engineering, it is essential to have a thorough understanding about such inhibitors. Protease inhibitors can interfere with the synthesis and secretions of midgut proteases, and thereby, inhibit their trophic physiological function, growth and development (Hegedus et al., 2003; Chougule et al., 2005; Wu et al., 2013). The effects of some trypsin inhibitors on the growth of Heliothis zea (Boddie) have been examined (Broadway and Duffey, 1986). Few studies on the use of serine protease inhibitors for controlling herbivorous insects indicate that protease inhibitors can be employed in devising feasible and effective pest control strategies (Sagili et al., 2005; Tamhane et al., 2007).

Plutella xylostella L. (Lepidoptera: Plutellidae; diamondback moth) is a pest of cruciferous plants. There are several reports on the growth and development of P. xylostella, however, to date, only a few studies have examined the correlation between protease inhibitors and protease activities in the midgut of P. xylostella, and its effect on the growth and development of this insect. In the present study, we determined the midgut protease activity in the midgut of P. xylostella larvae fed a diet containing different protease inhibitors and examined the effect of protease inhibitors on its growth and development. The findings from this study could provide the foundation for establishing a new approach of effectively using protease inhibitors to control populations of this insect pest and other lepidopteran pests.

#### MATERIALS AND METHODS

#### Insects

For this study, P. xylostella samples were obtained from a laboratory colony maintained at the College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China. The larvae were reared on Brassica oleracea (cv. Qingan 70) at 24 ± 2 ◦C, 70 ± 10% relative humidity (RH) and under 15-h light:9-h dark photoperiod. The adults were provided with 10% honey solution for supplementary adult feeding to improve the female oviposition.

#### Extraction of P. xylostella Midgut Proteases

The larval midgut proteases were extracted as described by Wang and Qin (1996). Healthy late 3rd instar larvae of equal sizes were rapidly dissected on ice to collect the midgut, and the gut contents were flushed using 0.15 mol/L NaCl. Subsequently, the midgut was placed in a 1.5 mL centrifuge tube and rapidly homogenized over ice. The crude extract was centrifuged at 12,000 × g for 15 min at 4◦C, and the supernatant was collected and stored at −20◦C. Protein concentration in the extract was assayed using the method of Bradford (1976).

#### Determination of Protease Activity in the Midgut of P. xylostella

Total protease activity was determined as follows: A 20 mg/mL solution of azocasein, the substrate, was prepared in NaCl (0.15 mol/L) and stored at 4◦C. Subsequently, the following substances were added to a 1.5 mL centrifuge tube: 100 µL azocasein solution, 10 µL midgut protease extract and 40 µL glycine/NaOH reaction buffer (0.1 mol/L, pH 11.0). The mixture was incubated at 30◦C for 3 h, and then 150 µL of 20% (v/v) trichloroacetic acid (pre-cooled) was added to terminate the reaction. The mixture was centrifuged at 12,000 × g at 4◦C for 15 min to collect the supernatant, which was termed as the midgut protease extract in this study. Protease activity in the extract was determined by measuring absorbance at 415 nm using a plate reader (Wang and Qin, 1996).

The trypsin activity was determined using two specific substrates: BAρNA (Nα-Benzoyl-DL-arginine-p-nitroanilide) and TAME (Nα-P-Tosyl-L-arginine methyl ester hydrochloride). BAρNA was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL and stored at 4◦C. Then, 100 µL BAρNA, 10 µL midgut enzyme extract and 90 µL Tris-HCl reaction buffer (0.1 mol/L, pH 10.5) were added to a 1.5 mL centrifuge tube and incubated at 30◦C for 20 min. Subsequently, 100 µL of 20% (v/v) trichloroacetic acid (pre-cooled) was added to terminate the reaction. The reaction mix was centrifuged and 200 µL of the supernatant was used to determine the absorbance at 405 nm using a plate reader. TAME was dissolved in NaCl (0.15 mol/L) at a concentration of 2 nmol/L. Then, 100 µL TAME, 10 µL midgut enzyme extract and 90 µL Tris-HCl reaction buffer (0.1 mol/L, pH 8.5) were added to a 1.5 mL centrifuge tube and incubated at 30◦C for 20 min. The mixture was centrifuged, and the supernatant was used to measure the absorbance at 247 nm using a plate reader (Wirnt, 1974).

The chymotrypsin activity was determined using the substrate BTEE (substrate, N-Benzoyl-L-tyrosine ethyl ester) dissolved in NaCl (0.15 mol/L) at a concentration of 1 mmol/L and stored at 4 ◦C. Then, in a 1.5 mL centrifuge tube, 100 µL BTEE solution,

10 µL midgut enzyme extract and 90 µL glycine/NaOH reaction buffer (0.1 mol/L, pH 9.0) were added and incubated at 30◦C for 20 min and then centrifuged. An aliquot of the supernatant was used to measure absorbance at 256 nm using a plate reader (Wirnt, 1974).

# Effects of Protease Activators and Inhibitors on P. xylostella Larval Midgut Protease Activity

Four protease activators (MgCl2, CaCl2, EDTA and EGTA) and a total of six protease inhibitors IAA (In Alien Attitude), DTT (DL-Dithiothreitol), PMSF, TPCK, TLCK and STI (Soybean Trypsin Inhibitor) were used for these analyses. First, P. xylostella larval midgut protease extracts were obtained, and the effects of protease activators and inhibitors on the intestinal protease activity of P. xylostella larvae were determined. Each treatment had three replicates. The activity of the total protease was determined in a glycine/NaOH reaction buffer (0.1 mol/L, pH 11.0), that of high-alkaline trypsin enzyme (a trypsin subtype in the P. xylostella larval midgut protease extract having highly alkaline pH optimum) was determined in a Tris-HCl reaction buffer (0.1 mol/L, pH 10.5), low-alkaline trypsin (another trypsin subtype in the P. xylostella larval midgut protease extract having slightly alkaline pH optimum) was determined in a Tris-HCl reaction buffer (0.1 mol/L, pH 8.5), and that of chymotrypsin activity was measured in a glycine/NaOH reaction buffer (0.1 mol/L, pH 9.0; Zhao et al., 2017).

Each of the protease activators or inhibitors (10 µL) was mixed with 10 µL of the insect midgut enzyme extract and incubated at 30◦C for l5 min. The corresponding substrate was added to measure the protease activity. Protease activity was determined. Double-distilled water (ddH2O) served as the control and the reaction was performed in triplicates for each treatment.

# Effect of Protease Inhibitors on the Protease Activities of P. xylostella Larvae

The leaf immersion method (Guo et al., 2013) was used for introducing protease inhibitors: cabbage (cv. Qingan 70) leaves were immersed in solutions containing the protease inhibitors, TPCK (2 mmol/L), TLCK (2 mmol/L) and STI (100 µg/mL) for 10 s and were then dried. The 3rd instar P. xylostella larvae were starved for 4 h and then were allowed to feed on the inhibitor-soaked cabbage leaves. Double distilled water served as the control. Midgut samples were collected at 0 (starved for 4 h), 4, 8, 12, 24, 36, 48, 60, 72, and 84 h to extract the enzymes, which were then stored at −20◦C. For each treatment comprised 30 larvae were used in each replicate and there were three replicates per treatment. The protease activity was determined as described above.

# Effect of Different Protease Inhibitors on the Performance of P. xylostella

Cabbage (cv. Qingan 70) leaf sections (circular, 9 cm in diameter) were immersed for 10 s in solutions containing one of the three protease inhibitors: TPCK (2 mmol/L), TLCK (2 mmol/L) and STI (100 µg/mL, 50 µg/mL, 10 µg/mL)and were then dried. Double distilled water was used as the control. In each treatment, one hundred newly oviposited eggs were placed in Petri dishes (circular, plastic, 9 cm in diameter) containing the inhibitortreated leaves. The lids were closed and sealed. The leaves were changed every second day. The time was recorded when an egg hatched. Each neonate larva was transferred to another Petri dish (circular, plastic, 9 cm in diameter) and numbered. When a larva transformed into a pupa, the time was recorded to calculate the larval duration was recorded. For each treatment, 30 larvae were used in each replicate and there were three replicates per treatment. The duration of the growth and developmental stages (larval, pupal, adult and larval-adult), the time of larval survival, pupal weight, and time of emergence were recorded and the average of each treatment, for example, pupation rate, larval survival rate, emergence rate for each treatment were calculated. A newly emerged male and female moth were put together in paper cups and fed with 10% honey water. The number of eggs and the time of death were recorded at 0800 and 2000 h daily until the emergence ceased.

# Data Analyses

The statistical significance of the differences between the control and treatment groups were analyzed by ANOVA (analysis of variance) with alpha = 0.05, 0.01 and 0.001, and respectively. The means were separated using Tukey's HSD test, which are denoted with one (<sup>∗</sup> ), two (∗∗), or three asterisks (∗∗∗), corresponding to P-values are less than 0.05, 0.01 or 0.001. SPSS 20.0 was used for statistical analyses, and GraphPad Prism 5 was used to create the figures.

# RESULTS

# Effects of Protease Activators and Inhibitors on the Midgut Protease Activity of P. xylostella Larvae

Under optimal pH, we determined the effects of protease activators and inhibitors on the midgut protease activity of P. xylostella. Different effects were observed on the activities of total protease, high-alkaline trypsin, low-alkaline trypsin and chymotrypsin (**Figure 1**). Total protease activity remained similar for the ddH2O control and the groups treated with MgCl2, CaCl2, EGTA, EDTA, IAA and DTT. However, there were significant differences between the control and the remaining treatment groups: PMSF, TPCK, TLCK (1 mmol/L) and STI inhibited the activity of total proteases (F = 6.72, df = 20, 42, P < 0.05) (**Figure 1A**). Moreover, these inhibitory effects were concentration-dependent; higher concentrations resulted in greater inhibitory effects. On the other hand, the activity of highalkaline trypsin (F = 98.52, df = 20, 42, P < 0.05) was activated by IAA, DTT, TPCK and 5 mmol/L MgCl2, while TPCK had no effect on it, the remaining treatments had an inhibitory effect on the activity (**Figure 1B**). IAA and TLCK activated the activity of low-alkaline trypsin (F = 5.78, df = 20, 42, P < 0.05), whereas TPCK had no effect on low-alkaline trypsin, and the other

treatments had an inhibitory effect on the activity (**Figure 1C**). Lastly, the chymotrypsin (F = 4.96, df = 20, 42, P < 0.05) activity was inhibited by DTT, PMSF and STI, and the other treatments had an activating effect on the activity (**Figure 1D**). Overall, all the effects were concentration-dependent with higher concentrations of inhibitors resulting in maximum effects.

# Effects of Different Dietary Protease Inhibitors on the Midgut Protease Activity of P. xylostella Larvae

The ability of protease inhibitors to inhibit larval tryptic and chymotryptic activity was further examined using the protease inhibitors, TPCK (2 mmol/L), TLCK (2 mmol/L) and STI (100 µg/mL) at different time points after feeding these to P. xylostella larvae. Overall, the inhibitory effects of the different protease inhibitors significantly differed over time (**Figure 2**). The inhibitory effect of STI on all proteases was the lowest among all the inhibitors. In general, all the inhibitors reduced the activity of the total protease in comparison to the control group. The total protease (F = 14.02, df = 3, 36, P < 0.05) activity of P. xylostella larvae that ingested the inhibitors in the control and the TPCK-fed groups first increased and then decreased during the 0–8 h period. The STI treatment had fluctuating effects on total protease activity between 0 and 4 h after of the treatment (**Figure 2A**). TLCK and STI inhibited the activity of high-alkaline trypsin (F = 10.48, df = 3, 36, P < 0.05), while TPCK occasionally inhibited the activity of high-alkaline trypsin (**Figure 2B**). TPCK and STI also inhibited the activity of lowalkaline trypsin (F = 19.79, df = 3, 36, P < 0.05) (**Figure 2C**). Moreover, TPCK and STI inhibited the activity of chymotrypsin (F = 22.70, df = 3, 36, P < 0.05), whereas TLCK did not inhibit the activity of chymotrypsin except between 0 and 4 h (**Figure 2D**). In the control group, the activity of the total protease was not constant, there are fluctuations, the same goes for other groups, and the frequency of fluctuations were different, in other words, the inhibitors disrupted the enzyme system of P. xylostella.

#### Effects of Different Dietary Concentrations of STI on P. xylostella Larval Midgut Protease Activity

When P. xylostella larvae were fed with four different concentrations STI, i. e. 100, 50, 10, and 0 µg/mL, the activities of total protease, high-alkaline trypsin, low-alkaline trypsin and chymotrypsin were altered (**Figure 3**). Overall, after 4 h, STI increased the inhibitory activity of the total protease (F = 17.85, df = 3, 36, P < 0.05), with the highest STI concentration producing the greatest effect (**Figure 3A**). In the control group, two peaks appeared at 8 and 60 h, the larvae gone into the next instar, the activity intensified, and the fluctuation frequency of the total protease activity were less than that in other groups, whereas in the groups treated with the other inhibitors four peaks appeared at 4, 24, 48 and 84 h. The inhibitory effect was the

highest at 36 h. The interactions between the midgut protease and the inhibitors changed the activity of the midgut protease frequently. At 4 h, the inhibitory effect on the high-alkaline trypsin (F = 21.63, df = 3, 36, P < 0.05) activity was the highest with 100 µg/mL STI and lowest with 10 µg/mL STI (**Figure 3B**). The low-alkaline trypsin (F = 25.85, df = 3, 36, P < 0.05) activity in the control and 10 µg/mL STI groups increased, while there was no effect in the 50 µg/mL STI group, and the activity declined in the 100 µg/mL STI group (**Figure 3C**). In the treatment groups, there were three activity peaks at 4, 24 and 48 h with the lowest chymotrypsin (F = 31.80, df = 3, 36, P < 0.05) activity at 36 h. At 4 h the activities in the 100 µg/mL and 50 µg/mL STI groups were lower than those in the 100 µg/mL STI and the control groups (**Figure 3D**). At 36 h, the highalkaline trypsin activity was the lowest among the three treatment groups.

#### Effect of Dietary Protease Inhibitors on the Performance of P. xylostella

Plutella xylostella larvae were fed the protease inhibitors TPCK, TLCK and STI, and their growth and development were observed (**Table 1**). The larval duration (F = 3.62, df = 5, 12, P < 0.05), pupal duration (F = 2.77, df = 5, 12, P < 0.05), adult duration (F = 2.32, df = 5, 12, P < 0.05), larva-adult duration (F = 6.71, df = 5, 12, P < 0.05) of P. xylostella fed different protease inhibitors were significantly different. The larval-adult treatment duration were all longer than that of the control treatments and were affected by the concentration of the inhibitor, with higher STI concentrations having the longer duration.

The larval survival rate (F = 2.53, df = 5, 12, P < 0.05), pupation rate (F = 4.39, df = 5, 12, P < 0.05), pupal weight (F = 1.81, df = 5, 12, P < 0.05) and number of eggs per female (F = 5.51, df = 5, 12, P < 0.05) of P. xylostella were significantly different among treatments (**Table 2**). Overall, the pupation rate, pupal weight, and the number of eggs per female for all the treatment groups were lower than those for the control group. The concentration of the inhibitor influenced the emergence rate with higher concentrations resulting in a higher inhibitory effect. The larval survival rate and number of eggs per female are two important parameters affecting the population dynamics of P. xylostella fed 100 µg/mL. The mean larval survival rate of P. xylostella ingested TLCK and STI (100 µg/mL) were 89.17 and 86.71%, respectively, and the number of eggs per female were 112.07 and 120.17, respectively, which were lower than the mean larval survival and the number of eggs per female for the control group.

#### DISCUSSION

The diverse protease inhibitors and activators had different effects on the activities of total protease, high-alkaline trypsin, lowalkaline trypsin and chymotrypsin in the midgut of P. xylostella larvae midgut. For example, EDTA inhibited all the four proteases, and EGTA activated only the high-alkaline trypsin.


Data in the table are means ± SE. Different lowercase letters in the same column indicate significant difference at P < 0.05 level as assessed by Tukey's HSD test.

TABLE 2 | Effects of ingesting different protease inhibitors on growth and development of Plutella xylostella.


Data in the table are means ± SE. Different lowercase letters in the same column indicate significant difference at P < 0.05 level as assessed by Tukey's HSD test.

Some divalent metal ions (Ca2+, Mg2+) are activators of a variety of proteases (Wang and Qin, 1996). Although Ahmad et al. (1980) reported that Mg2<sup>+</sup> and Ca2<sup>+</sup> had no effect on the activity of these four enzymes, the current study demonstrates that CaCl<sup>2</sup> inhibited the trypsin activity. Among the common protease inhibitors, such as PMSF, TLCK, TPCK and STI, PMSF had no effect on chymotrypsin but inhibited the total protease, highalkaline trypsin, and low-alkaline trypsin; TLCK inhibited the total protease and high-alkaline trypsin, whereas, TPCK activated high-alkaline trypsin. The inhibitory effect of STI on all the proteases was the greatest among the four inhibitors examined. This suggests that ability of STI to form a bonds with the insect

enzymes is strong, whice results in the protease being susceptible to STI.

In general, the inhibitory effects of the different protease inhibitors on the protease activities, and consequently, on the development of P. xylostella were varied greatly. During larval feeding period, the levels of inhibition for all the inhibitors varied, reflecting the complexity of the P. xylostella midgut protease profile. During the experiment, the protease activities of all the inhibitor treatment groups changed. The inhibition level of STI on the four proteases (total protease, high-alkaline trypsin, low-alkaline trypsin and chymotrypsin) was greater than that of the other three inhibitors. STI is a natural trypsin inhibitor, which has been reported to decrease the weight of Spodoptera litura Fabricius (Lepidoptera: Noctuidae) larvae and pupae, delay their growth, and prolong their generation time, with these inhibitory effects being more significant in the early developmental stages (Wu et al., 2013). We found that STI had obvious inhibitory effects on P. xylostella larvae, which is consistent with those reported by Wang and Qin (1996) and Upadhyay and Chandrashekar (2012), showing that the greater the inhibitor concentration, the more obvious is the inhibitory effect. On feeding protease inhibitors to larvae, the insects often change their intestinal proteases to adapt to the inhibitory effects, causing a change in protease activity. When these insect larvae ingested low concentrations of protease inhibitors for long period, the activity of chymotrypsin increased, that of high-alkaline trypsin activity decreased, whereas that of total protease activity remained the same. If larvae ingested high concentrations of the protease inhibitors for long period, then the total protease and high-alkaline trypsin activities decreased (Wang et al., 1995). Protease inhibitors can result in antinutritional effects and break the balance among the proteases, causing a disorder in the digestive system, which would affect the growth and reproduction of the insects (Chougule et al., 2005). Oppert (1999) found that for the coleopteran pest, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), the control was more effective when both serine and cysteine protease inhibitors were used simultaneously. In the current experiment, changes were observed in the midgut protease activity of P. xylostella after it ingested the protease inhibitors for only a short period. In future experiments, the feeding time should be further extended to explore the changes in protease activity due to long-term ingestion of protease inhibitors.

Protease inhibitors have an inhibitory or activating function on serine protease in the midgut of insects, destroying coordination among the proteases which disrupt the digestion process of the insect. This affects their growth, development and reproduction (Lawrence and Koundal, 2002; Giri et al., 2005). It is also reported that in cowpea bruchid, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae), protease inhibitors degrade the proteases (Zhu-Salzman et al., 2003). The growth and development period of P. xylostella that ingested different protease inhibitors were significantly different. The larva-adult treatment durations in the protease inhibitor treatments were longer than those in the control treatments. The inhibitors delayed the growth of P. xylostella. The results of this treatment were the same as those reported by Ortego et al. (1998), who reported that different inhibitors had different inhibition levels for the growth of Aubeonymus mariaefranciscae Roudier (Coleoptera: Curculionidae). The growth and development periods were affected by the concentration of the inhibitor, wherein treatment with higher STI concentrations had a longer duration and lower survival rate than that with the other inhibitors used in this study. At STI concentrations of 0, 10, 50, and 100 µg/mL, the pupal duration, pupal weight, and emergence rate showed no significant differences, suggesting that the concentration discrepancies in concentration were minimal. However, insects had certain ability for adaptation, when STI was fed to Prodenia litura Fabricius (Lepidoptera Noctuidae) through multiple generations, the inhibitory effect of STI on larval weight and pupal weight was reduced and the generation period was shortened (Ogunlabi and Agboola, 2007; Opitz and Mvller, 2009). The adaptive capacity of the insect to STI was improved by regulating the growth and development process, and thus, its inhibitory effect was gradually decreased (Chougule et al., 2005; Wu et al., 2013). Protease inhibitors can suppress the activities of the larval insect midgut protease, then disrupt the digestive system and delay the growth of insects. The same result was observed in previous studies (Tamhane et al., 2005; Bhattacharyya et al., 2007; Zhu et al., 2007). Several studies have focused on efficient inhibitors that can be used for pest control, and on their usage-pattern (Macedo et al., 2002; Oliveria et al., 2007). When bean flour containing a protease inhibitor was mixed with several insecticides, the insecticidal effect was doubled, thus protease inhibitors have significant potential as synergists for new pesticides (Hou et al., 2004).

#### CONCLUSION AND FUTURE PERSPECTIVES

In this study, we examined the effect of protease inhibitors on protease activities and on the growth and development of P. xylostella larvae, both in vitro and in vivo when larvae ingested protease inhibitors for a given period of time. We observed that protease inhibitors had varying degrees of inhibitory effect on protease activities and consequently, extended the growth and development periods of P. xylostella. Next possible step could be to use protease inhibitors as a new biological pesticides for controlling P. xylostella or to transfer the gene(s) of protease inhibitors to host plant which would reduce the feeding of P. xylostella on the plants, suppress its development, and minimize damage to the host plant.

#### AUTHOR CONTRIBUTIONS

AZ participated in the design of the study, performed the experiments and data analysis, generated the figures and tables, and helped to draft the manuscript. YinL and CL helped AZ to performed the experiments. YipL and PW conceived the study, designed the study, coordinated the study,

and drafted the manuscript. All the authors approved the final manuscript.

#### FUNDING

This study was supported by the National Natural Science Foundation of China (Grant Nos. 31871971 and 31772503), Agricultural Science and Technology Innovation Projection in Shaanxi Province (Grant No. 2016NY-058), and National Key R&D Program of China (Grant No. 2017YFD0200900).

#### REFERENCES


#### ACKNOWLEDGMENTS

We thank the reviewers for valuable comments on the manuscript. We thank Prof. T-XL from the Key Laboratory of Applied Entomology, Northwest A&F University at Yangling, Shaanxi, China for his advice and for editing of the manuscript. We thank Editors, BT and Su Wang, from Frontiers in Physiology for providing constructive comments aimed at strengthening the manuscript. We also thank Vikas Narang, Vice President of Author Services from Editage, a brand of Cactus Communications for editing this manuscript.



development and midgut proteinase activities of the bollworm, Helicoverpa zea. Pestic. Biochem. Physiol. 87, 39–46. doi: 10.1016/j.pestbp.2006. 05.004

Zhu-Salzman, K., Koiwa, H., Salzman, R. A., Shade, R. E., and Ahn, J. E. (2003). Cowpea bruchid Callosobruchus maculatus uses a threecomponent strategy to overcome a plant defensive cysteine protease inhibitor. Insect Mol. Biol. 12, 135–145. doi: 10.1046/j.1365-2583.2003. 00395.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhao, Li, Leng, Wang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# RNA-Seq Analyses of Midgut and Fat Body Tissues Reveal the Molecular Mechanism Underlying Spodoptera litura Resistance to Tomatine

Qilin Li<sup>1</sup>† , Zhongxiang Sun1,2† , Qi Shi<sup>3</sup> , Rumeng Wang<sup>1</sup> , Cuicui Xu<sup>3</sup> , Huanhuan Wang<sup>3</sup> , Yuanyuan Song<sup>1</sup> \* and Rensen Zeng<sup>1</sup> \*

#### Edited by:

Antonio Biondi, Università degli Studi di Catania, Italy

#### Reviewed by:

Muthugounder S. Shivakumar, Periyar University, India Jalal Jalali Sendi, University of Guilan, Iran Frederique Hilliou, UMR7254 Institut Sophia Agrobiotech (ISA), France Michael J. Stout, Louisiana State University, United States

#### \*Correspondence:

Yuanyuan Song yyuansong@163.com Rensen Zeng rszeng@fafu.edu.cn †These authors have contributed

equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 09 July 2018 Accepted: 07 January 2019 Published: 22 January 2019

#### Citation:

Li Q, Sun Z, Shi Q, Wang R, Xu C, Wang H, Song Y and Zeng R (2019) RNA-Seq Analyses of Midgut and Fat Body Tissues Reveal the Molecular Mechanism Underlying Spodoptera litura Resistance to Tomatine. Front. Physiol. 10:8. doi: 10.3389/fphys.2019.00008 <sup>1</sup> College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou, China, <sup>2</sup> State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fuzhou, China, <sup>3</sup> College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, China

Plants produce secondary metabolites to provide chemical defense against herbivorous insects, whereas insects can induce the expression of detoxification metabolism-related unigenes in counter defense to plant xenobiotics. Tomatine is an important secondary metabolite in tomato (Lycopersicon esculentum L.) that can protect the plant from bacteria and insects. However, the mechanism underlying the adaptation of Spodoptera litura, a major tomato pest, to tomatine in tomato is largely unclear. In this study, we first found that the levels of tomatine in tomatoes subjected to S. litura treatment were significantly increased. Second, we confirmed the inhibitory effect of tomatine on S. litura by adding moderate amounts of commercial tomatine to an artificial diet. Then, we utilized RNA-Seq to compare the differentially expressed genes (DEGs) in the midgut and fat body tissues of S. litura exposed to an artificial diet supplemented with tomatine. In total, upon exposure to tomatine, 134 and 666 genes were upregulated in the S. litura midgut and fat body, respectively. These DEGs comprise a significant number of detoxification-related genes, including 7 P450 family genes, 8 glutathione S-transferases (GSTs) genes, 6 ABC transport enzyme genes, 9 UDP-glucosyltransferases genes and 3 carboxylesterases genes. Moreover, KEGG analysis demonstrated that the upregulated genes were enriched in xenobiotic metabolism by cytochrome P450s, ABC transporters and drug metabolism by other enzymes. Furthermore, as numerous GSTs were induced by tomatine in S. litura, we chose one gene, namely GSTS1, to confirm the detoxification function on tomatine. Expression profiling revealed that GSTS1 transcripts were mainly expressed in larvae, and the levels were the highest in the midgut. Finally, when larvae were injected with double-stranded RNA specific to GSTS1, the transcript levels in the midgut and fat body decreased, and the negative effect of the plant xenobiotic tomatine on larval growth was magnified. These results preliminarily clarified the molecular mechanism underlying the resistance of S. litura to tomatine, establishing a foundation for subsequent pest control.

Keywords: tomatine, Spodoptera litura, RNA-sequencing, GSTS1, RNAi

# INTRODUCTION

fphys-10-00008 January 19, 2019 Time: 16:45 # 2

Plants produce toxic secondary metabolites that provide chemical defense against herbivorous insects and pathogens. For instance, plants can biosynthesize a variety of toxins and secondary metabolites, such as isoflavones, furanocoumarins, terpenoids, alkaloids and cyanogenic glycoside (Mithöfer and Boland, 2012; Medina et al., 2015). Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. In soybean (Glycine max: Fabaceae), multiple isoflavones are induced and accumulate in leaves after Spodoptera litura larvae attack (Nakata et al., 2016). Tomato (Lycopersicon esculentum L.), an economically important vegetable worldwide and a commonly used model plant for studying plant-insect interactions (Wei et al., 2011), can be attacked by many herbivorous insects, such as Trialeurodes vaporariorum (Westwood) and Helicoverpa armigera (Hubner), but tomato can biosynthesize chemical substances to defend against herbivorous insects. For example, tomatine (TOM), an important secondary metabolite found in high amounts in tomato, serves as a growth inhibitor of Trypanosoma cruzi, strain EP, in Liver Infusion Tryptose medium (Chataing et al., 1998). TOM can inhibit bacteria and herbivorous insects (such as S. litura) and is expected to be exploited as a biological insecticide.

The co-evolution of plants and their insect predators has resulted in remarkable development in insects, including the abilities to deter and detoxify host plant phytochemicals (Mithöfer and Boland, 2012). Insect detoxification enzymes typically include three main superfamilies: cytochrome P450 monooxygenases (P450s or CYPs for genes), glutathione S-transferases (GSTs) and carboxylesterases (CarEs) (Després et al., 2007). S. litura feeds on more than 290 species of plants belonging to 99 families and is one of the most destructive agricultural pests in tropical and subtropical regions worldwide (Zou et al., 2016). Numerous insect cytochrome P450 genes, which involved in detoxification of allelochemicals, have been identified by newly developed high-throughput sequencing technologies. For example, CYP6AB14 in S. litura has been suggested to play a key role in detoxifying plant allelochemicals, such as xanthotoxin, coumarin and flavone (Wang et al., 2015). CYP6B8 and CYP321A1 in the generalist Helicoverpa zea have been shown to metabolize a variety of allelochemicals, such as flavone, α-naphthoflavone, chlorogenic acid, and indole-3 carbinol (Li et al., 2004; Rupasinghe et al., 2007). Insect GSTs also play important roles in detoxifying toxic compounds; for example, GSTE1 in the midgut of S. litura may play an important role in the detoxification of chlorpyrifos, xanthotoxin and the heavy metal cadmium (Xu et al., 2015). In addition, GSTE2 in Anopheles gambiae showed enzymatic activity to detoxify dichlorodiphenyltrichloroethane (DDT) (Ding et al., 2003). Meanwhile, CarEs are implicated in the metabolic resistance of many different classes of insecticides, and two CarE genes, Pxae22 and Pxae31 in Plutella xylostella, have been shown to be involved in fipronil resistance (Ren et al., 2015). Plant secondary metabolites have a detrimental and toxic effect on the growth and development of herbivorous insects. However, tomatoes remain vulnerable to many pests, such as S. litura, indicating that S. litura may have adapted to the tomato defense mechanism. Studies have reported that tomatoes with TOM are highly toxic to insect attack. However, the specific effects of TOM on S. litura and how S. litura adapts and metabolizes TOM has not yet been reported.

In insects, immune and detoxification systems respond quickly to chemical and biological stresses (Lemaitre and Hoffmann, 2007) and are well expressed in the midgut (Hao et al., 2003; Pauchet et al., 2009), suggesting that this organ is the site of exposure to many stressors. However, detoxification also takes place in the fat body and hemolymph (Enayati et al., 2005; Dubovskiy et al., 2011). Transcriptome sequencing can systematically recognize the transcriptional regulation of all genes in an organism. Prior to this study, RNA sequencing was used to investigate the honeybee response to biotic and abiotic environmental stressors by measuring the midgut transcriptional changes induced by the parasite Nosema ceranae and one neurotoxic insecticide (fipronil or imidacloprid) alone or in combination (Aufauvre et al., 2014). In addition, the transcriptomic profiles of midgut genes and Cry1Ac gene networks resulting from challenging P. xylostella with the Cry toxin have also been studied using RNA-Seq (Lei et al., 2014). In addition, RNA-Seq and molecular docking reveal that CYP397A1V2 likely contributes to P450-mediated insecticide resistance in Cimex lectularius (Mamidala et al., 2012). Understanding the effects of plant secondary metabolites on the feeding behavior, growth and development of insects and clarifying the mechanisms by which insects metabolize and adapt to plant secondary metabolites is very significant for pest management practices. However, the transcriptional levels of S. litura midgut and fat body genes induced by TOM have not yet been reported.

In this study, we used RNA-Seq analyses of the S. litura midgut and fat body to reveal the molecular mechanism underlying S. litura resistance to TOM. First, we observed that the TOM content in tomatoes subjected to S. litura attack was significantly increased. Second, to verify the inhibitory effect of TOM on S. litura, we fed S. litura artificial diets supplemented with moderate commercial TOM. Then, we used RNA-Seq to analyze the TOM-induced detoxification enzyme genes in the midgut and fat body tissues of S. litura. The differentially expressed genes (DEGs) and their associated pathways identified provide insight into the genes adapted to metabolize TOM. Finally, the GSTS1 gene was silenced with RNA interference (RNAi) to further determine the likely contribution of GSTS1-mediated TOM resistance in S. litura.

#### MATERIALS AND METHODS

#### Insect Culture, Plants and Antibodies

Spodoptera litura was provided by the Institute of Crop Resistance & Chemical Ecology of Fujian Agriculture and Forestry University and maintained in an insectary without exposure to any insecticide. Larvae were reared on artificial diets composed of soybean powder (100 g), brewer's yeast (40 g), wheat bran (60 g), ascorbic acid (4 g), methyl p-hydroxybenzoate (2 g), sorbic acid (2 g), agar (16 g), cholesterol (0.8 g) and water (1 L)

(Qi et al., 2000) at 25 ± 2 ◦C and 70 ± 5% relative humidity with a photoperiod of 16:8 h (L:D). Adults were provided supplemented with 10% honey solution under the same conditions (Zhou et al., 2012).

Tomato (L. esculentum L.) cv Castlemart (CM) was used as the wild-type species for all experiments (Yan et al., 2013), and all tomato seeds were provided by Prof. Chuan-you Li (Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China). Tomato seedlings were grown in growth chambers and maintained under 16 h of light (Yan et al., 2013) at 22◦C and 8 h of darkness at 18◦C and 60% relative humidity. Five-week-old plants with five to seven leaves were used in the experiment.

Tomatine (TOM) (90% purity, T0329, Tokyo Chemical Industry CO. LTD, Japan) was dissolved in dimethyl sulfoxide (DMSO, Q/STXH234-2013, XiLong Chemical Industry, Guangdong, China) and mixed with an artificial diet. The control diets were supplemented with the same amount of DMSO.

#### Determination of Tomatine in CM Tomato Using HPLC

To examine the effects of S. litura damage on the changes of TOM content in wild-type tomatoes, we placed three fourth instar S. litura larvae on the fully expanded leaves of each 5-week-old tomato plant for 24 h. Control plants were not infested by S. litura larvae. After 24 h inoculation, tomato leaves from S. litura-infested plants and uninfested plants were weighed for TOM extraction. The TOM extraction method was referenced previously with some modifications (Kozukue et al., 2004). Briefly, 1. 200 mg tomato leaves were extract with 100 mL Chloroform/Methanol (2:1, v/v); 2. Add 2 mL of 0.2 N Hydrochloric acid; 3. Add 3 mL of 2% Ammonia, centrifuge at 18,100 × g/min for 1 min at 1◦C, discard the supernatant and repeat the previous step; 4. Dissolve the precipitate with 2 mL of Tetrahydrofuran/Acetonitrile/0.02 M Monobasic potassium phosphate (50:30:20, v/v/v), centrifuge at 18,100 × g/min for 1 min at 1◦C; 6. Pipette 1 mL into the sample vial for HPLC analysis.

To determine the sample TOM contents, HPLC analysis was carried out using a Waters liquid chromatography (model e2695), and clear supernatant extracts were injected into a stainless steel HPLC column (250 mm × 4.0 mm) filled with Inertsil ODS-2 (5 µM particles) and eluted with a mobile phase comprising acetonitrile/20 mM KH2PO<sup>4</sup> (24:76) at a flow rate of 1 mL/min−<sup>1</sup> . The UV detector (model 2998 PDA) was set at 208 nm. The standard substance of tomatine (≥97%, HPLC, Chengdu Purechem-Standard CO. LTD, China) were used to confirm and quantify the peaks from TOM extraction.

#### Insect Treatment, Sample Collection and RNA Extraction

Newly molted fourth instar S. litura larvae were used for all treatments to monitor weight growth rate. Synchronous larvae (80–100 mg) were first weighed (labeled as WT1) and fed artificial diets supplemented with TOM at 0.1 and 0.3 mg/g for 48 h. The control larvae were fed on artificial diet supplemented with the same amount of DMSO (labeled as WC1). The weight of larvae were measured again simultaneously after inoculation (for TOM treatment group, WT2; for control group, WC2). The relative weight growth rate of larvae from treatment group were calculated by normalized with control larvae, that is, (WT2−WT1)/(WC2−WC1) × 100%. Thirty synchronous individuals were used for each treatment, and three independent replicates were performed for all treatments.

After 48 h inoculation, midgut and fat body tissues from three S. litura larvae were, respectively, dissected prior to RNA extraction. Each treatment had four replicates. Total RNA was isolated from flash-frozen tissues using the Eastep Super Total RNA Extraction Kit (Promega Corporation, Madison, WI, United States) and quantified by measuring the absorbance at 280 and 260 nm. Then the equal RNA from four replicates were pooled together as a mix sample, including midgut and fat body tissues from control or TOM-treated samples. The pooled samples were subjected to RNA-Seq, and the four replicates samples were used for qRT-PCR analysis to verify the results of RNA-Seq and the induced effect of TOM stress.

#### Library Preparation and Sequencing

Total RNA was quantified by the NanoPhotometer <sup>R</sup> spectrophotometer (IMPLEN, United States) and RNA quality was assessed using the RNA Nano 6000 Assay Kit in the Bioanalyzer 2100 system (Agilent Technologies, United States). The transcriptome libraries were generated using Illumina TruSeqTM RNA Sample Preparation Kit (Illumina, San Diego, CA, United States) following the manufacturer's recommendations. RNA transcript was sequenced on an Illumina Hiseq 2000 in Novogene Bioinformatics Institute (Beijing, China).

#### Quantification and Differential Expression Analysis of Transcripts

Raw data (raw reads) in FASTQ format were processed through in-house Perl scripts to remove reads containing adapters, reads containing ploy-N, and low quality reads. Q20, Q30 and GC-content of the cleaned data were used to assess the sequencing quality. All the downstream analyses were based on clean data with high quality. A global de novo assembly of the resultant reads was performed using the Trinity method with min\_kmer\_cov set to 2 by default and all other parameters set default. To annotate the obtained unigenes, the databases of Nr (e-value ≤ 1e−5), Nt (e-value ≤ 1e−5), Pfam (e-value ≤ 1e−2), KOG/COG (**Supplementary Figures 2**, **3**) (e-value ≤ 1e−3), Swiss-protc (e-value ≤ 1e−5), KEGG (e-value ≤ 1e−10), and GO (e-value ≤ 1e−6) were searched.

For reads mapping, the transcriptome obtained by Trinity splicing was used as reference sequence, and all clean reads were mapped to the reference sequence using RSEM with bowtie 2 set to mismatch 0 by default (Li and Dewey, 2011). Splicing length and frequency distribution of transcripts and unigenes were listed in **Supplementary Table 4** and success rate of gene annotation were listed in **Supplementary Table 5**. For quantification of gene expression level, the number of expressed reads mapped to each gene was calculated and normalized to the number of

FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) (Trapnell et al., 2010).

The read counts were normalized using the edgeR Bioconductor (Robinson et al., 2010) with the TMM method (Storey, 2003), and the DESeq R package provided statistical routines for determining differential expression using a model based on the negative binomial distribution, and was used to identify DEGs between the control and TOM-treated samples. The p-values in multiple tests were adjusted as q-values using the Benjamini and Hochberg's approach for controlling the false discovery rate (FDR) (Dillies et al., 2013). We used "fold changes ≥ 1 and q < 0.005" as the threshold to assess DEGs between the TOM treatment and control groups.

#### Quantitative Real-Time PCR (qRT-PCR) Analysis

To validate the DEGs analysis results, quantitative realtime reverse transcriptase PCR (qRT-PCR) experiments were performed on an Applied Biosystems StepOne Plus Real-Time PCR System in a 10 µL reaction volume consisting of 5 µL of 2× SYBR GoTaq <sup>R</sup> qPCR Master Mix (Promega Corporation, Madison, WI, United States), 0.4 µL of each gene-specific primers (10 µM), 1 µL cDNA equivalent to 50 ng total RNA and sterilized water to reach the final volume. PCR conditions were set as: 1 cycle of 95◦C for 10 min; 40 cycles of 95◦C for 15 s, 55◦C for 30 s and 72◦C for 30 s. The reference gene elongation factor 1 alpha (EF-1α) was used as internal controls (Shu et al., 2018). A dissociation curve analysis program was performed to check the homogeneity of the PCR product. Relative standard curves of EF-1α and target genes were generated by using 10 fold serial dilutions cDNA to calculate the amplification efficiency of primers. The relative mRNA levels were normalized against EF-1α using the 2−M M Ct method (Livak and Schmittgen, 2001). Three independent biological repeats were performed, each sample had two technical replicates, and a calibrator sample was used to make comparisons between different plates. All the primers were listed on **Supplementary Table S1**. All designed primers were synthesized at BioSune Biotechnology Co., Ltd. (Shanghai, China).

#### Clone of the GSTS1 Gene

To develop full-length GSTS1, we performed 3<sup>0</sup> RACE and 5<sup>0</sup> RACE (rapid amplification of cDNA ends) using an oligo dT primer (Invitrogen) and gene-specific primers. The following gene-specific primers were utilized: 5<sup>0</sup> RACE primer 1, 5<sup>0</sup> -GCCATCACCAAGTATG TGGCAAGAGGA-3<sup>0</sup> ; 5<sup>0</sup> RACE primer 2, 5<sup>0</sup> -TGGGGTGA TTGAAGCCAGCGACAT-3<sup>0</sup> ; 3<sup>0</sup> RACE primer 1, 5<sup>0</sup> - GATAATCCTTGCTCAATTCGATGCCCAG-3<sup>0</sup> and 3<sup>0</sup> RACE primer 2, 5<sup>0</sup> -CACCCCAGGAAAGCTTGCCATTCA-3<sup>0</sup> . By merging the 3<sup>0</sup> and 5<sup>0</sup> cDNA ends with internal fragment sequences, full-length cDNAs of GSTS1 were generated and then deposited into the GenBank database (accession number: KY304480.1<sup>1</sup> ).

<sup>1</sup>http://www.ncbi.nlm.nih.gov/

# Preparation and Injection of dsRNA

Templates for in vitro transcription reactions were prepared by PCR amplification using cloned GSTS1 sequences as the template and the primers (T7-GSTS1dsRNA-F/GSTS1dsRNA-R and GSTS1dsRNA-F/T7-GSTS1dsRNA-R). The amplification conditions comprised 30 cycles at 98◦C for 10 s, 53◦C for 30 s and 72◦C for 45 s, with a final extension step at 72◦C for 5 min. PCR products were purified using the TIANGEN Universal DNA purification kit (Tiangen Biotech, Co., Ltd., Beijing, China), and DNA concentrations were determined using a microplate reader. Double-stranded RNA (dsRNA) corresponding to GSTS1 (dsGSTS1) was synthesized using the T7 RiboMAXTM Express RNAi System (Promega, United States) according to the manufacturer's instructions. Additionally, 688 bp dsRNA corresponding to the control green fluorescent protein (GFP) gene (ACY56286), used as a negative control, was synthesized by the same method using the following primer (T7-GSTS1dsRNA-F/GSTS1dsRNA-R and GSTS1dsRNA-F/T7- GSTS1dsRNA-R) pairs: T7-GFPdsRNA-F/GFPdsRNA-R and GFPdsRNA-F/T7-GFPdsRNA-R (Dong et al., 2011). The resulting dsRNA were analyzed by agarose gel electrophoresis and stored at −80◦C prior to use. All the primers were listed on **Supplementary Table S3**.

The dsRNA were adjusted to a final concentration of 1.5 µg/µL with ddH2O prior to use. For all dsRNA injection experiments, fourth instar larvae were used; 2 µL (3.0 µg) of dsRNA was injected into the side of each S. litura thorax using a manual microliter syringe (Shanghai High Pigeon Industry and Trade Co., Ltd., China), and the injection points were imprinted immediately with Vaseline. Following injection, S. litura were maintained on artificial diets supplemented with or without 0.1 mg/g TOM. The treatment larvae were injected with 3.0 µg dsGSTS1, while the control larvae were injected with equal dsGFP. At 24 h post injection, insect midguts and fat bodies were harvested, and total RNA were extracted as described above.

#### Statistical Analysis

All data are presented as the mean ± SE unless otherwise noted. Statistically significant differences (p < 0.05) were determined by one-way ANOVA followed by Duncan's multiple range test using the SPSS 10.0 software package (IBM Corp., Armonk, NY, United States).

# RESULTS

#### Tomatine Contributes to Tomato Chemoresistance Against S. litura

We hypothesized that tomatoes might produce tomatine (TOM), an important toxic compound, to improve resistance to insect damage. To test this hypothesis, we used HPLC to determine the TOM content in S. litura damaged wild-type tomatoes (cv Castlemart, CM) and S. litura undamaged CM. Compared with standard substance of TOM (**Figure 1A**), the peaks of TOM extracted from CM-undamaged (**Figure 1B**) and CMdamaged (**Figure 1C**) were confirmed. The physiological levels of

(n = 5, <sup>∗</sup>p < 0.05, t-test).

FIGURE 2 | The effect of exposing to tomatine on growth of S. litura and morphological of midgut tissue. (A) Effects of the presence and absence of tomatine in artificial diets (TOM, 0.1 mg/g, 0.3 mg/g) on weight growth rate (n = 10, p < 0.05, Dunnett's multiple range test). (B) Morphological changes in the phenotype and midguts of larvae fed a diet supplemented with tomatine; Control: larvae fed a diet supplemented with DMSO; tomatine: larvae fed a diet supplemented with 0.1 mg/g tomatine.

TOM in undamaged tomatoes were 0.61 ± 0.15 mg/g, however, when damaged by S. litura, the TOM content were significantly increased to 1.05 ± 0.01 mg/g (**Figure 1D**). The results verified our hypothesis that TOM contributes to tomato's defense against S. litura attack.

#### The Effect of Exposing to Tomatine on Growth of S. litura and Morphological of Midgut Tissue

Next, biological experiments were performed on fourth instar S. litura larvae maintained on artificial diets supplemented with pure TOM. In comparison with control larvae reared in the absence of TOM, the average weight gains observed for fourth instars fed diets containing 0.1 mg/g TOM were decreased by 54% following 48 h of treatment (**Figure 2A**). And S. litura fed artificial diets supplemented with 0.3 mg/g pure TOM were decreased by 80% (**Figure 2A**). Then we confirmed the growth inhibition phenotype of S. litura induced by TOM, revealing that larvae treated with TOM were significantly smaller (**Figure 2B**). To determine the effect of TOM on insect histomorphology, midgut samples of larvae were dissected and measured, revealing that the larval midgut was significantly smaller after TOM exposure (**Figure 2B**).

#### Analysis of DEGs in the Midgut and Fat Body Tissues of S. litura After TOM Treatment

To study the molecular mechanism underlying the S. litura counterdefense against TOM, we utilize RNA-Seq to explore differences in gene expression in midgut and fat body tissues after TOM treatment. S. litura larvae were maintained on artificial diets supplemented with TOM for 24 h. We then dissected the midgut and fat body tissues of S. litura and analyzed the DEGs by RNA-Seq. In total, 134 upregulated genes and 177 downregulated genes were observed in the midgut, whereas 666 upregulated genes and 302 downregulated genes were observed in fat body tissue (**Figure 3**, fold changes ≥ 1, q < 0.005). We mainly focused on screening detoxification genes; gene family enrichment analysis was performed on DEGs identified in each group, including P450 family genes, ABC transport enzymes, UDP-glucosyltransferases, CarEs, and glutathione transferases (**Table 1**). We focused mostly on upregulated genes, including 2 P450 family genes, 3 ABC transport enzymes, 5 UDPglucosyltransferases and 2 CarEs in the midgut and 5 P450 family genes, 1 CarE, 8 glutathione transferases, 3 ABC transport enzymes and 4 UDP-glucosyltransferases in fat body tissues (**Table 1**).

KEGG pathway enrichment analysis was done using KOBAS 2.0 with a hypergeometric test and the Benjamini-Hochberg FDR correction (Xie et al., 2011). KEGG analysis demonstrated that the upregulated genes were enriched in xenobiotic metabolism by cytochrome P450, ABC transporters, and drug metabolism by other enzymes (**Table 2**). These results showed that numerous detoxification enzymes were induced in S. litura subjected to TOM treatment.

#### Verification of RNA-Seq Results by qRT-PCR

To validate the gene expression data obtained using RNA-Seq, we combined 30 genes which were commonly upregulated in midgut and fat body tissues (**Figure 3A**) with the reported genes and KEGG analysis, and 34 DEGs were selected for quantitative real-time PCR (qRT-PCR) analysis. The heat map showed that almost all the upregulated genes of in midgut and fat body from RNA-seq data were also upregulated in the qRT-PCR analysis in biological replicates samples, except for the UGT40Q1 gene in midgut (**Figure 4**), which was upregulated in TOM treatment from RNA-seq, but was downregulated in the qRT-PCR analysis (**Supplementary Table S2**). The detailed fold change and p value were shown in **Supplementary Table S2**. The 97.06% (33/34) consistency of gene expression indicated that the RNA-Seq approach provided reliable differential gene expression data for this system.

#### Sequence Analysis and Spatial and Temporal Expression of GSTS1

Transcriptome sequencing data revealed that multiple GST family genes were induced by TOM in S. litura, suggesting

TABLE 1 | Up-regulated differentially expressed genes in Midgut and Fat body.


M, midgut; F, fat body.

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TABLE 2 | KEGG pathways containing genes differentially expressed in Midgut and Fat body.


M, midgut; F, fat body.

that GST family genes may play an important role in the counterdefense of S. litura to TOM. In addition, we identified a new GST family gene, whose function has not yet been reported, GSTS1, which we utilized for further functional studies. Using RACE, the full-length 748-bp GSTS1 cDNA sequence was determined to contain a 36-bp 5'-untranslated region (5'-UTR), a 639-bp open reading frame (ORF), and a 78-bp 3' UTR. The sequence was deposited into the GenBank database (accession number: KY304480.1). The ORF encodes a predicted protein of 213 amino acids. GSTS1 has a theoretical pI value of 6.62 and a predicted molecular mass of 24.57 kDa.

To study the spatial expression of GSTS1 in S. litura, we use RT-qPCR to test the relative expression patterns of GSTS1 mRNA at different ages. GSTS1 transcripts were mainly present in larvae, especially in first instar larvae, and slightly lower transcript levels were observed in female moths (**Figure 5A**). To study the temporal expression of GSTS1 in S. litura, RT-qPCR was used to test the relative expression patterns of GSTS1 mRNA at different developmental stages. Among the stages analyzed, Slitu-GSTS1 was almost exclusively expressed in the midgut (**Figure 5B**). The expression of GSTS1 was negligible in brain, fat body and hemolymph tissue. Thus, we speculated that the high GSTS1 expression in the midgut is likely related to metabolizing TOM in this region.

#### Effect of Silencing GSTS1

We validated the GSTS1 gene expression data by qPCR, revealing that GSTS1 was transcribed in both the midgut and fat body (**Figure 6A**). From the above results, we hypothesized that GSTS1 mediated the resistance of S. litura to TOM. To further confirm this function, the GSTS1 gene was knocked down by injecting dsRNA into fourth instar larvae. Approximately 24 h after the injection, RT-qPCR showed that GSTS1 expression was significantly reduced in the midguts and fat bodies of fourth instar S. litura larvae subjected to the dsRNA injection compared to that in control larvae (received a double-stranded GFP, dsGFP) injection (**Figure 6B**), showing that the RNAi procedure was successful.

RNAi experiments were next performed on fourth instar larvae maintained on artificial diets supplemented with TOM. Importantly, larvae injected with dsGSTS1 and fed TOM exhibited significantly lower weight gains than dsGFP-injected controls fed TOM (**Figure 6C**), showing that GSTS1 may plays an important role in metabolizing TOM.

FIGURE 4 | Biological (independent) qRT-PCR validation of the RNA-Seq data. The first 11 genes are highly expressed in the midgut when exposure to tomatine (TOM), and the latter 23 genes are highly expressed in fat bodies when exposure to TOM. The bar represents the scale of the expression levels of TOM treatment/Control. The red, black, and green bar indicates mRNA expression levels of TOM treatment are higher, similar or lower than the control groups, respectively. The detailed fold change and p value were shown in Supplementary Table S2.

# DISCUSSION

Insect organs, especially the midgut and fat body, are important for defense against xenobiotic compound toxicity. A previous study reported the transcription and expression of genes in the midgut tissue of Bombyx mori strain Daizo larvae subjected to persistent pathogenic infection with cytoplasmic polyhedrosis virus (BmCPV) (Kolliopoulou et al., 2015). Another study determined that heat shock proteins (HSPs) and their expression levels may play important roles in the resistance of various silkworms to high temperature stress by analyzing gene expression in their midguts (Li et al., 2014). In addition, another study reported the physiological shift of pre-blood-fed fat bodies from a resting state to vitellogenic gene expression after conducting transcriptome analysis of fat bodies of the yellow fever mosquito Aedes aegypti (Price et al., 2011). Prior research has suggested that the S. litura midgut plays a crucial role in growth physiology by influencing digestion and metabolism (Franzetti et al., 2015). In our study, we performed RNA-Seq analysis on midgut and fat body tissues to investigate the mechanisms underlying tomatine (TOM) resistance. In total, 134 and 666 upregulated genes were identified in S. litura midgut and fat body tissues, respectively, among which 30 genes were commonly differentially expressed. These results suggested that these genes may play a substantial role in TOM detoxification.

FIGURE 5 | The qRT-PCR analysis of GSTS1 expression in various S. litura tissues and life stages. (A) Relative spatial expression of GSTS1; the different life stages include 2-day-old 1st, 2nd, 3rd, 4th, 5th, and 6th (last) instar larvae, pupae, and adults. (B) Relative temporal expression of GSTS1; the different tissues include the cuticle, brain, midgut, fat body, and hemolymph. The different letters on the error bars indicate significant differences (p < 0.05, Duncan's multiple range test) among the developmental stages and tissues.

We classified DEGs according to their gene family and compared the differences in gene expression between midgut and fat body tissues. The results suggested that GSTs were significantly differentially expressed between control S. litura and S. litura treated with TOM. RNAi was performed on GSTS1 to further reveal the molecular mechanism underlying the

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resistance of S. litura to TOM. Previous studies on metabolic resistance have focused on the roles of P450s, CarEs, and GSTs in xenobiotic metabolism in the Lepidoptera midgut (Pauchet et al., 2010). Current research on GSTs has focused mainly on the relationship between GSTs and insecticide resistance in insects. For example, the organophosphorus insecticide chlorpyrifos increased the amounts of both GSTs and malonaldehyde in S. litura (Huang et al., 2011). GSTe2 and GSTe7 of A. aegypti are involved in resistance to pyrethroid deltamethrin (Lumjuan et al., 2011). In S. litura, both SlGSTe2 and SlGSTe3 responded to six insecticides, but SlGSTE2 showed a much higher detoxification activity than SlGSTE3 (Deng et al., 2009). Moreover, different GSTs in a species have different detoxification activities against various toxic compounds. Eight GSTs have been identified in S. litura, and the mRNA levels of SlGSTe1, SlGSTe3, SlGSTs1, SlGSTs3 and SlGSTo1 were shown to be increased after xanthotoxin ingestion (Huang et al., 2011). SlGSTE1 was shown to play a potentially critical role in S. litura host adaptation (Zou et al., 2016). However, plant allelochemicals can also present their toxicity via the oxidative stress pathway. For example, xanthotoxin, a plant allelochemical from Apiaceae, can generate superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, causing deleterious lipid peroxidation and increasing the antioxidative activity of glutathione peroxidase in several insects (Ahmad and Pardini, 1990). TOM is a tetrasaccharide linked to the 3-OH group of the aglycone tomatidine (**Supplementary Figure S1**) and could induces permeabilisation of the cell membrane and a loss of the cytosolic enzyme pyruvate kinase (Medina et al., 2015). Intriguingly, as some environmental compounds induce excessive GST expression, certain GSTs have been utilized as biomarkers of environmental pollution (Pérez-López et al., 2002; Ayoola et al., 2011), and GSTs have many other functions that remain to be explored. In this study, we further studied the contribution of the GSTS1 gene to mediating TOM resistance in S. litura, as this relationship has not yet been reported.

In our study, we suggest that GSTS1 expression is increased to metabolize TOM, thus revealing the molecular mechanism by which S. litura mediates TOM resistance. Furthermore, in addition to GSTS1, many other genes, such as P450s, CarEs, glutathione transferases, ABC transport enzymes and UDP-glucosyltransferases, may also mediate TOM resistance in S. litura. For example, a member of the CYP6 family, CYP6A8, catalyzes the hydroxylation of lauric acid and increase the resistance of Drosophila melanogaster against aldrin and heptachlor (Restifo, 2004). Many important molecules, including pheromones and other semiochemicals, are types of esters that are hydrolysed by esterases in insects (Montella et al., 2012). Further research should establish whether these genes function individually or in combination to mediate resistance, and their functions still need to be verified.

RNAi has been developed as an effective tool in plants and animals (Plasterk et al., 2000; Aravin et al., 2001; Wesley et al., 2001). Insect genes expression can be downregulated by dsRNA injection (Bettencourt et al., 2002; Eleftherianos et al., 2006; Ohnishi et al., 2006) or with artificial diets containing high concentrations of dsRNA (Turner et al., 2006), but an efficient method of delivering dsRNA to control pests in the field remains to be developed. Some plant-mediated herbivorous insect RNAis have been reported to suppress critical insect genes by feeding insects plant tissues engineered to produce a specific dsRNA (Mao et al., 2007). For example, when larvae are fed plant material expressing dsRNAs specific to CYP6AE14, the levels of these transcripts in the midgut are decreased, and larval growth is retarded. These results suggest that feeding insects plant material expressing dsRNA may be a general RNAi strategy and be applicable in entomological and insect pest field control research, which provides us inspiration. According to our study, dsRNA specific to GSTS1 can be expressed in plants to specifically control S. litura damage, but how these successes observed in the laboratory translate into effective pest control in the field remains unknown. However, researchers and farmer can believe that silencing insect-detoxifying genes via plant delivery could be a powerful strategy for controlling insect pests.

In conclusion, while insect-plant interactions have been studied for several years, the mechanisms underlying resistance in insects remain poorly understood, and many key genes and proteins involved in these interactions have not been elucidated. In the present study, we utilized an RNA-Seq approach to investigate control S. litura and S. litura treated with TOM. In total, 134 and 666 upregulated genes were identified in the S. litura midgut and fat body tissues, respectively, among which 30 genes were commonly differentially expressed. In addition, GSTS1 gene expression was induced by TOM treatment. Our study initially clarified the molecular mechanism underlying the adaptation of S. litura to TOM, laying the foundation for subsequent pest control by plant-mediated herbivorous insect RNAi.

# DATA AVAILABILITY STATEMENT

The raw data of the RNA-Seq have been submitted to NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA509528.

# AUTHOR CONTRIBUTIONS

ZS conceived and designed the experiments. QL, ZS, CX, QS, HW, and RW performed the experiments. QL and ZS performed analysis of the data. QL and ZS wrote the manuscript. ZS, YS, and RZ edited the manuscript.

# FUNDING

This work was supported by the National Natural Science Foundation of China (31601899, 31670414, and 31870361), Natural Science Foundation of Fujian Province (2016J01104), China Postdoctoral Science Foundation (2017M612111, 2018T110635), State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, National College Student Innovation Program, China (201810389039) and Fujian Provincial Excellent Youth Science Foundation (2017J06010).

#### SUPPLEMENTARY MATERIAL

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The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2019.00008/full#supplementary-material

#### REFERENCES


by the alkaloids tomatine and tomatidine. Memórias Do Instituto Oswaldo Cruz 110, 48–55. doi: 10.1590/0074-02760140097


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Li, Sun, Shi, Wang, Xu, Wang, Song and Zeng. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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**365**

# Effects of Fungicide Propiconazole on the Yeast-Like Symbiotes in Brown Planthopper (BPH, Nilaparvata lugens Stål) and Its Role in Controlling BPH Infestation

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Pin-Jun Wan, China National Rice Research Institute (CAAS), China Keiichiro Matsukura, NARO Kyushu Okinawa Agricultural Research Center, Japan Rui Pang, Guangdong Institute of Microbiology (CAS), China

#### \*Correspondence:

Xiaoping Yu yxp@cjlu.edu.cn †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 28 July 2018 Accepted: 25 January 2019 Published: 11 February 2019

#### Citation:

Shentu X, Wang X, Xiao Y and Yu X (2019) Effects of Fungicide Propiconazole on the Yeast-Like Symbiotes in Brown Planthopper (BPH, Nilaparvata lugens Stål) and Its Role in Controlling BPH Infestation. Front. Physiol. 10:89. doi: 10.3389/fphys.2019.00089 Xuping Shentu† , Xiaolong Wang† , Yin Xiao and Xiaoping Yu\*

Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, China

Yeast-like symbiotes (YLS), harbored in the abdomen fat-body cells of the rice brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), are vital to the growth and reproduction of their host. It is feasible to manipulate BPH infestation on rice by inhibiting YLS using fungicide. In this study, the fungicide propiconazole was injected into the hemolymph of BPH thorax via microinjection to investigate its effect on YLS, especially the dominant species, Hypomyces chrysospermus, and their host BPH. Propiconazole markedly reduced the total number of YLS and H. chrysospermus in BPH hemolymph and fat body, thereby leading to an obvious higher mortality and lower fecundity of BPH than the negative control (PBS, phosphate buffer solution). After microinjecting propiconazole, the survival rate of BPH nymphs at the 5th instar was significantly lower than that obtained after PBS treatment. Eight days after propiconazole microinjection, the BPH survival rate dropped to 40%, only half of BPH survival rate treated with PBS microinjection. For female adults (1-day-old), there were significant differences in the survival rates between BPHs treated with propiconazole and those treated with PBS at days 5–8. The fecundity of BPH decreased significantly by microinjecting propiconazole and averaged only 229 eggs per female, which was 20% less than that of the negative control. Furthermore, we reared BPH on the susceptible variety TN1 sprayed with propiconazole to prove the feasibility manipulating field occurrence of BPH by inhibiting YLS using fungicides. The number of YLS and H. chrysospermus in BPH obviously declined. Subsequently, the survival rate and fecundity of BPH significantly decreased after feeding on rice treated with propiconazole. Meanwhile, the propiconazole residue was detected in the hemolymph and gut of BPH by HPLC analysis within 1 day of feeding. Inhibiting YLS using fungicides was a novel and effective way to control BPH infestation.

Keywords: yeast-like symbiotes, Nilaparvata lugens, Hypomyces chrysospermus, microinjection, propiconazole

# INTRODUCTION

fphys-10-00089 February 7, 2019 Time: 19:37 # 2

The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is one of the most destructive monophagous insect pests of rice in Asia (Park et al., 2008). The insects suck nutrients from the phloem of rice plants and transmit plant viruses such as ragged stunt and grassy stunt viruses (Ge et al., 2011; Piyaphongkul et al., 2012; Wan et al., 2013). High BPH populations can destroy rice plants and cause hopper burn in a short period of time (Yang et al., 2002). Up to now, the control of BPH has predominantly relied on the use of synthetic chemicals (Puinean et al., 2010; Wan et al., 2013). However, due to the injudicious use of chemical insecticides, BPH has evolved a high level of resistance to major varieties of insecticides, including organophosphates, carbamates, pyrethroids, neonicotinoids, insect growth regulators, and phenylpyrazoles (Wu et al., 2018). In recent years, BPH outbreaks have occurred more frequently in China and other Asian countries, thereby causing a serious yield loss of rice (Wang et al., 2008; Wu et al., 2018). Thus, the global importance of rice, which supplies approximately 20% of the world's calorific intake, drives research on the development of BPH control methods.

Yeast-like symbiotes (YLS), harbored in the fat body cells of BPH abdomen, are dominant obligatory symbionts, although several bacterial symbionts have been reported (Tang et al., 2010). BPH establishes an intimate symbiotic relationship with YLS and provides a small habitat for YLS (Chaves et al., 2009). In turn, YLS has vital physiological and trophic functions in the growth and reproduction of their host BPH and provides complementary functions to their host such as essential amino acid synthesis, nitrogen storage and recycling, steroid synthesis, and vitamin supply (Xue et al., 2014).

In order to clearly elucidate the close relationship between YLS and its host BPH, many experiments have been conducted on the taxonomy and diversity of YLS populations. Kagayama et al. (1993) isolated seven morphologically different YLS strains from the eggs of BPH. In our previous studies, we used 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences analysis, Cryp-Like and Pichia-Like YLS symbiotes were identified (Dong et al., 2011). Furthermore, several fungal species, namely, Hypomyces chrysospermus (usually called Noda), Pichia guilliermondii, Candida sp., Saccharomycetales sp., and Debaryomyces hansenii, were detected by nested PCR-denaturing Gradient Gel Electrophoresis (DGGE) technology (Hou et al., 2013). Therefore, many species of YLS exists in BPH, according to these reports. Up to now, the species and amounts of YLS in the fat body of BPH keep unknown. H. chrysospermus is the dominant species of YLS in BPH (Noda et al., 1995; Cheng and Hou, 1996).

The YLS in the fat body of BPH is transmitted to the next generation by transovarial infection (Cheng and Hou, 1996, 2001). The transmission process is as follows: the symbiotes in mycetocytes move out of the syncytium, which is formed from a layer of fat body cells, by exocytosis and release into BPH's hemocoel in BPH females. Then, the free YLS in hemolymph approach the ovarioles near the pedicel and are enclosed by follicle cells. They enter the follicle cells around the primary oocyte by endocytosis at epithelial plug of the ovariole. The YLS aggregate at the posterior end of the mature egg after entering and finally form a symbiote ball (Cheng and Hou, 2001). Furthermore, the entry of YLS into BPH oocyte was triggered by oocyte vitellogenesis, as shown in our previous study (Nan et al., 2016). Whether or not the growth and fecundity of BPH are affected by the number of total YLS and H. chrysospermus in the transovarial process is unclear. In this paper, the fungicide propiconazole was injected into the hemolymph of BPH thorax using microinjection technology to observe its effects on the YLS and their host BPH. Furthermore, we reared BPH on the susceptible variety TN1 sprayed with propiconazole to prove the feasibility of manipulating BPH infestation by inhibiting YLS using fungicides. Inhibiting YLS using fungicides was a novel and effective way to control BPH infestation.

#### MATERIALS AND METHODS

#### Insect Mass Rearing and Rice Culture

Brown planthopper population used in the experiments were originally collected from rice fields in Hangzhou (E120◦ 12, N30◦ 16), China. Successive generations were maintained on the susceptible rice variety TN1 in a climatic chamber under constant conditions of 26 ± 1 ◦C, 70–80% relative humidity and a 16 h light/8 h dark photoperiod. The TN1 seedlings were cultured in 14 cm diameter plastic pots and used for BPH mass rearing at the tillering stage (height: 14–16 cm).

#### Microinjection of Fungicide Into Hemolymph of BPH Thorax

The fungicide 50% propiconazole ME was provided from Qingdao Hengyuanxiang Chemical Co., Ltd., Propiconazole was diluted with 0.01 mol/L phosphate buffer solution (PBS) to 0.17 ng/nL. The final concentration for microinjection was determined by the preliminary gradient experiments. Thirty BPH nymphs in the 5th instar or 1-day-old female adults were used for microinjection for each treatment with five replications. The BPH individuals were anesthetized using CO<sup>2</sup> for 30 s and fixed on 4% agarose plate with their abdomen facing up. Propiconazole at 17 ng (100 nL) was injected into each BPH thorax using the FemtoJet 4i microinjection device (Eppendorf, Germany), and the injection sites were located on the conjunction between prothorax and mesothorax (Lu et al., 2015). The treatments with 0.01 mol/L PBS and without microinjection were used as the negative and the blank controls, respectively. BPH samples were collected at day 1, day 2, and day 4 after injection to investigate the number of YLS and H. chrysospermus. The mortality and fecundity of BPH were investigated and calculated correspondingly.

# Effects of Propiconazole With Foliar Spraying on Rice Plants

Foliar spraying with propiconazole was carried out at the rice tillering stage using a mini-sprayer. Propiconazole was used

at the recommended concentration (0.5 mg/mL) in the field trial. Three stems of sprayed rice plants were placed in a test tube (2.5 cm in diameter and 30 cm in height) filled with 20 ml rice nutrient solution (Wilkinson and Ishikawa, 2001; Shentu et al., 2016). Thirty fifth-instar nymphs or 1-dayold female adults were released into the test tube, which was covered with two pieces of gauze. These were then kept in a constant temperature room at 26 ± 1 ◦C with 16 h light/8 h dark photoperiod. Five replications were performed in each treatment. The treatment sprayed with water only was used as the negative control. The mortality and fecundity of BPH were calculated. The number of YLS and H. chrysospermus in the BPH body was investigated at day 1, day 2, and day 4 after BPH release.

#### Quantification of Total YLS

Brown planthopper samples were sterilized by immersion in 75% ethanol for 3 min and then washed quickly with 0.01 mol/L PBS for 90 s. The fat bodies in the BPH abdomen and the hemolymph in the BPH thorax were collected by dissection and homogenized in 0.01 mol/L ice-cold PBS at pH 7.4 Percoll (Pharmacia, Sweden), respectively (Shentu et al., 2016). The total number of YLS in the fat body and the hemolymph were counted on a hemocytometer under a binocular microscope (400 ×). Each sample was observed in triplicate.

# Quantification of H. chrysospermus by Quantitative Real-Time PCR (qPCR)

Total DNA of YLS in the hemolymph and fat body was extracted using a Yeast DNA Mini Kit (Tiangen Biotech Co., Ltd., Beijing, China). The DNA concentration was measured using a spectrophotometer (Nanodrop). To estimate the abundance of H. chrysospermus, the copy number of the 18S rDNA (Genbank: AF267233.1) fragment was measured by qPCR (Applied Biosystems) using a FastStart Universal SYBR Green Master(ROX) (Roche Biotechnology Co., Ltd.), and the two primers used for qPCR amplification were F: 5<sup>0</sup> -CGTAGGAGAGCAGCAAAC-3<sup>0</sup> and R: 5<sup>0</sup> - CGATGCCAGAGCCAAGA G-3<sup>0</sup> . The actin (Genbank: KU365929.1) was used as the reference gene. The primers were F:5<sup>0</sup> -GATGAGGCGCAGTCAAAGAG-3<sup>0</sup> and R:5<sup>0</sup> - GTCATCTTCTCACGGTTGG C-3<sup>0</sup> . The primers were designed by Primer Premier 5.0 with DNA sequence and cDNA sequence. The resulting PCR products were cloned into a pMD18-T vector (TaKaRa Biotechnology (Dalian) Co., Ltd.). The inserted gene fragments were sequenced and were proven to correspond to a part of the target gene. The qPCR was performed in a 20 µL total reaction volume containing 10 µL of FastStart Universal SYBR Green Master(ROX), 2 µL of template DNA, 0.8 µL of forward primer (10 µM), 0.8 µL of reverse primer (10 µM), and 6.4 µL of ddH2O. The qPCR reactions were pre-denatured at 95◦C for 10 min, followed by 40 cycles of 95◦C for 15 s and 60◦C for 1 min. Each DNA template was analyzed in triplicate. The acceptable qPCR standard curve (0.9 ≤ E ≤ 1.0, R <sup>2</sup> ≥ 0.99) for each gene was optimized by altering the annealing temperature and time. The normalized fold changes of the target gene DNA copy number were expressed as 2−11CT (Schmittgen and Livak, 2008).

# Analysis of Propiconazole Residue in BPH

Propiconazole standard sample was purchased from Aladdin– Holdings Group. Fifteen BPH individuals in group were dissected, and their hemolymph and gut were collected and homogenized in 0.01 mol/L PBS at pH 7.4 Percoll (Pharmacia, Sweden), respectively (Shentu et al., 2016). Then, the PBS extraction solution was subjected to centrifugation (12,000 r/min, 5 min) to obtain the supernatant. The 500 µL supernatant was filtrated with 0.22 µm filter membrane. The residue of propiconazole was analyzed using a Waters e2695 HPLC (2998 PDA Detector, Waters Workstation, United States) (Mu et al., 2005). The mobile phase was methanol and water (85:15, v/v). The detection wavelength was 225 nm, and the flow speed was 1.0 mL/min. The column (RP-C18 column) temperature was maintained at 30◦C (250 mm × 4.6 mm, 5 µm, XBridgeTM, Waters, United States).

# Data Analysis

Values were expressed as mean ± SE. All data were analyzed with SPSS, version 24.0. Comparisons of the means were conducted based on Least Significant Difference (LSD) test following a oneway analysis of variance (ANOVA). Differences between means was deemed significant and highly significant when P < 0.05 and P < 0.01, respectively.

# RESULTS

# Effect of Propiconazole on the Number of YLS in BPH

Effect of propiconazole on the YLS in hemolymph and fat body of BPH was indicated in **Figures 1A–D**. The total number of all YLS in BPH generally increased with the growth of host BPH (**Figures 1A,B**). One day after propiconazole microinjection, the total number of YLS in the hemolymph of nymph decreased as low as 33.6% of that in the negative control (PBS treatment). At days 2 and 4 after microinjection, the numbers of YLS in the treatment with propiconazole significantly declined to 48.7 and 50.6% as compared with that in the negative control, respectively (P < 0.05). Similar results were also obtained from the treatment of BPH female adults. The total number of YLS in the hemolymph of female adults obviously declined when treated with propiconazole. Especially at day 4 after microinjection, the number of the YLS dropped to 18.9 % of that in the negative control (PBS treatment) (**Figure 1A**).

The total number of YLS in the BPH fat body after propiconazole microinjection was significantly lower than that of untreated or PBS-treated controls (P < 0.05). For the 5th instar nymph and 1-day-old female adults, the growth and reproduction of YLS was effectively suppressed at day 1 (a drop of 43.3 and 21.2%), day 2 (a drop of

with one another. Significant differences were indicated by different letters at P < 0.05. The quantification of H. chrysospermus 18S rDNA copy number in the

different treatments was analyzed by 2−11Ct method. 1 day after untreated BPH nymphs in the 5th instar were used as control.

32.7 and 53.8%), and day 4 (a drop of 35.2 and 42.6%) after propiconazole microinjection as compared with PBS microinjection, respectively (**Figure 1B**).

The number of H. chrysospermus was analyzed after microinjection (**Figures 1C,D**). In the 5th instar BPH nymph, the number of H. chrysospermus increased with BPH growth. However, the number of H. chrysospermus in the BPH hemolymph treated with propiconazole was significantly decreased compared with that of the PBS-control (P < 0.05). On 1-day-old female adults, the number of H. chrysospermus had rapid declined at day 1 (a decline of 90%), day 2 (a decline of 65%), and day 4 (a decline of 76%) after propiconazole microinjection as compared with PBS microinjection (**Figure 1C**). Meanwhile, a big decline in the number of H. chrysospermus was observed after propiconazole microinjection in BPH fat body (**Figure 1D**).

#### Effect of Propiconazole on the Survival Rate of BPH

The survival rates of BPH by hemolymph microinjection were indicated in **Figures 2A,B**. During days 1 and 3 after microinjection, there was a significant difference in the survival rates of the 5th instar nymph between BPH treated with propiconazole and PBS (P < 0.05) (**Figure 2A**). Furthermore, from days 4 to 8 after propiconazole microinjection, the survival rate of 5th instar nymph was highly significantly lower than that of the control treated with PBS (P < 0.01). The BPH survival rate was 40% after microinjection at day 8, which was

lower by half of the BPH survival rate after treatment with PBS microinjection (**Figure 2A**). During days 5 and 8, the survival rates of 1-day-old female adults treated with propiconazole were significantly different from those treated with PBS (P < 0.05) (**Figure 2B**).

#### Effect of Propiconazole on the Fecundity of BPH

The fecundity of BPH females significantly decreased after propiconazole microinjection (P < 0.05) (**Figure 3**).

Significant differences were indicated by different letters at P < 0.05.

Female adults (1-day-old) laid 229 eggs per female after propiconazole microinjection, which was 20% lower than the control treated with PBS. For BPH treated in the 5th instar nymph, the oviposition of BPH treated with propiconazole was 195 eggs per female, which was only 75% of the PBS treatment.

#### Effect of Propiconazole on the Number of YLS in BPH by Foliar Spraying

The effect of fungicide on YLS after foliar spraying was shown in **Figures 4A,B**. From day 1 to day 3 after spraying, the number of YLS in the BPH hemolymph in 5th instar nymph was similar to that of water-treated BPH. However, the numbers of YLS in 1-day-old female adults significantly declined to 32, 29, and 22% of the negative control at day 1, day 2, and day 4 after foliar spraying, respectively (**Figure 4A**).

In the fat body of the 5th instar nymph, the number of YLS reduced significantly at day 1 and day 4 after foliar spraying with propiconazole compared with the water-treated control (P < 0.05). Meanwhile the growth and reproduction of YLS in 1-day-old female adults was also significantly inhibited at day 2 and day 4 after foliar spraying with propiconazole (P < 0.05) (**Figure 4B**).

The number of the dominant YLS species, H. chrysospermus, decreased after foliar spraying with propiconazole in the 5th instar nymphs and 1-day-old female adults (**Figures 4C,D**). Particularly, the number of H. chrysospermus in 1-day-old female adults significantly decreased at day 1 (a drop of 86%), day 2 (a drop of 81%), and day 4 (a drop of 81%) after foliar spraying with propiconazole compared with the negative control (P < 0.05) (**Figure 4C**). In the BPH fat body, an obvious reduction in the number of H. chrysospermus at day 2 and day 4 was observed after foliar spraying with propiconazole (**Figure 4D**).

(B) Effect of foliar sprayed propiconazole on the total number of YLS in the fat body. (C) Effect of foliar sprayed propiconazole on H. chrysospermus in the hemolymph. (D) Effect of foliar sprayed propiconazole on H. chrysospermus in the fat body. CK, water control; SF, foliar spraying with propiconazole; N, BPH nymphs in the 5th instar; A, 1-day-old female adults; 1, 2, and 4, 1 day, 2 day, and 4 day after foliar spraying with propiconazole. One-way ANOVA with LSD test was used to compare all individual treatments with one another. Significant differences were indicated by "<sup>∗</sup> " (P < 0.05). The quantification of H. chrysospermus 18S rDNA copy number in the different treatments was analyzed by 2-11Ct method. 1 day after water treatment of BPH nymphs in the 5th instar was used as control.

# Effects of Propiconazole on the Survival and Fecundity of BPH by Foliar Spraying

From day 1 to day 5 after foliar spraying with propiconazole, there was no significant difference between the treatment and negative control in both the 5th instar nymph and 1-dayold female adults (P < 0.05) (**Figures 5A,B**). Furthermore, from day 6 to day 8 after foliar spraying, the survival rates of the 5th nymph and 1-day-old female adults of BPH showed significant differences between propiconazole and water treatments (P < 0.05).

The fecundity of BPH significantly decreased after foliar spraying with propiconazole (**Figure 6**). The BPH treated with fungicide at the 5th nymph and 1-dayold female adults laid 199 and 214 eggs per female, respectively, which were 27 and 31% drops compared with the water-treated BPH.

#### Propiconazole Residue in the Hemolymph and Gut of BPH After Treatments

In the foliar spraying test, the residue of propiconazole in the hemolymph and gut was detected at day 1 after BPH release and the result was shown in **Figure 7**. According to the retention time and ultraviolet absorption spectrum of propiconazole by HPLC, there were propiconazole residues in the hemolymph and gut of BPH, respectively. The concentration of propiconazole residue in the gut was higher than in the hemolymph.

5th instar. (B) Effect of foliar sprayed propiconazole on the survival of 1-old-day female adults. CK, water control; SF, foliar spraying with propiconazole; One-way ANOVA with LSD test was used to compare all individual treatments to each other. Significant differences were indicated by "<sup>∗</sup> " (P < 0.05).

#### DISCUSSION

Yeast-like symbiotes harbored in the fat body of BPH abdomen plays a vital role in the growth and reproduction to their host BPH. If the YLS in BPH significantly decreased, then BPH would not survive (Chang et al., 2011; Lee and Hou, 1987). Based on the intimate relationship between BPH and YLS, we tried to manipulate BPH population by inhibiting YLS using fungicides as additive of imidacloprid. Results showed the satisfactory effect of some fungicides on the abundance of YLS and the mortality of BPH in our previous study (Shentu et al., 2016). In order to clearly clarify the importance of YLS on their host BPH, the fungicide propiconazole was injected into the hemolymph of BPH to investigate its role on YLS and their host BPH. Propiconazole could cause a marked reduction of the total number of YLS in BPH's hemolymph and fat body, thereby resulting in a significantly higher mortality and lower fecundity as compared with the PBS treatment. Hence, the decrease of YLS number in BPH not only caused BPH mortality but also reduced fecundity of BPH.

In our previous study, there was no significant difference in the survival rate of nymphs at 1 day after treatment with water spray and fungicide spray (Shentu et al., 2016). It may take a certain time for the fungicide to enter the hemolymph of BPH. However, at 1 day after propiconazole microinjection, the survival rate of the 5th instar BPH nymphs was significantly lower than that of the negative control (PBS treatment). Undoubtedly the inhibitory effect of propiconazole microinjection into the hemolymph of BPH on YLS was stronger than that of foliar fungicide sprays on rice plants. Furthermore, this study directly showed that fungicide treatment led to the decrease of YLS, thereby resulting in the high mortality and low fecundity of BPH. For newly emerged females, there was significant difference in the BPH survival rates between propiconazole treatment and PBS-treated control until day 5 to day 8 after microinjection. BPH females may have better tolerance to YLS loss because they have complete physiological function, including more nutritional reserves and numerous mycetocytes in the fat body. Thus, the same dose of fungicide had less inhibition effect on the YLS in BPH adults than that on BPH nymphs.

Yeast-like symbiotes in the fat body of BPH is transmitted into the next generation by transovarial infection (Cheng and Hou, 1996, 2001). By microinjecting propiconazole into the hemolymph of BPH, the transovarial process of YLS was obviously affected. Thus, the fecundity-related pathways were possibly affected by YLS decrease. As a result, the BPH fecundity was significantly affected. The fungicide could influence the growth of BPH and the reproduction as well.

Using microinjection of fungicide into the hemolymph of BPH is not practical in BPH control. Effects of propiconazole on the YLS and BPH by foliar spraying showed that the number of YLS and the survival rate of BPH obviously decreased in the rice field. Furthermore, the propiconazole residue was detected in the hemolymph and gut of BPH using HPLC analysis. Thus, the fungicide was assimilated by BPH feeding and then entered the hemolymph. The fungicide led to the decrease of

YLS in BPH's hemolymph and resulted in the death of BPH and lower fecundity. However, the fungicide may inhibit the microorganisms in BPH intestines. The interaction between BPH and microorganisms in BPH's intestines needs further study.

According to previous reports, H. chrysospermus is the dominant species of YLS in BPH (Cheng and Hou, 1996, 2001). Hence, effects of propiconazole on H. chrysospermus in BPH using microinjection or foliar spraying were also studied. Similar results were found, the number of H. chrysospermus was significantly reduced in both nymphs and adults after treatments. Meanwhile we tried to detect the number variation of other YLS species, such as Pichia guilliermondii, Candida sp., Saccharomycetales sp., and D. hansenii, by qPCR technology, but the results were less well developed. One of the possible reasons was the number of these symbiotes was much less than that of the dominant species H. chrysospermus. Thus, it is difficult to quantify the number of these symbiotes by qPCR. DNA extraction efficiency and PCR reaction condition are required in future research to test the effect of other YLS species on the BPH.

We tested the direct influence of propiconazole on BPH. However, the elimination of intracellular symbiotes was difficult and rarely successful. BPH died if YLS was completely inhibited. We did not obtain the YLS-free BPH as the control in hemocoel microinjection and foliar spraying tests using fungicide. Thus, it is difficult to confirm the direct influence of propiconazole on BPH per se. In our previous study, the number of YLS in the BPH treated with imidacloprid was not reduced significantly (Shentu et al., 2016). In contrast, the mixture of fungicides with imidacloprid substantially reduced the number of YLS and subsequently caused high mortality of BPH. This finding

#### REFERENCES


indicated that some fungicides could significantly enhance the suppressive effect of the insecticide against the BPH population after the inhibition of YLS in BPH. Our research presents a new way to manipulate BPH occurrence through inhibition of YLS.

#### CONCLUSION

Our study demonstrated that the fungicide caused the decrease of YLS, thereby resulting in the higher mortality and lower fecundity of BPH. Hence, inhibiting YLS using fungicide is a novel and effective way to control BPH infestation. To our knowledge, this is first report on the effect of fungicide microinjection into the hemolymph on YLS and BPH.

#### AUTHOR CONTRIBUTIONS

XS and XY conceived and designed the experiments, and wrote the manuscript. XW and YX performed the experiments. XS and XW analyzed the data.

#### FUNDING

This work was supported by National Natural Science Foundation of China (31401793, 31640018) and Zhejiang Provincial Programs for Science and Technology Development (2017C32006, 2018C02030, 2019C02015).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Shentu, Wang, Xiao and Yu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Insect Behavior and Physiological Adaptation Mechanisms Under Starvation Stress

Dao-Wei Zhang\*, Zhong-Jiu Xiao, Bo-Ping Zeng, Kun Li and Yan-Long Tang

School of Biological and Agricultural Science and Technology, Zunyi Normal University, Zunyi, China

Intermittent food shortages are commonly encountered in the wild. During winter or starvation stress, mammals often choose to hibernate while insects—in the form of eggs, mature larvae, pupae, or adults opt to enter diapause. In response to food shortages, insects may try to find sufficient food to maintain normal growth and metabolism through distribution of populations or even migration. In the face of hunger or starvation, insect responses can include changes in behavior and/or maintenance of a low metabolic rate through physiological adaptations or regulation. For instance, in order to maintain homeostasis of the blood sugar, trehalose under starvation stress, other sugars can be transformed to sustain basic energy metabolism. Furthermore, as the severity of starvation increases, lipids (especially triglycerides) are broken down to improve hunger resistance. Starvation stress simultaneously initiates a series of neural signals and hormone regulation processes in insects. These processes involve neurons or neuropeptides, immunity-related genes, levels of autophagy, heat shock proteins and juvenile hormone levels which maintain lower levels of physiological metabolic activity. This work focuses on hunger stress in insects and reviews its effects on behavior, energy reserve utilization, and physiological regulation. In summary, we highlight the diversity in adaptive strategies of insects to hunger stress and provides potential ideas to improve hunger resistance and cold storage development of natural enemy insects. This gist of literature on insects also broadens our understanding of the factors that dictate phenotypic plasticity in adjusting development and life histories around nutritionally optimal environmental conditions.

Keywords: insect, starvation stress, behavior, trehalose, physiological adaptation, ecological regulation

#### INTRODUCTION

Food is a critical source of nutrients and an important external factor in insect survival. Lack of food over long periods of time affects the growth and reproduction of insects and may even result in death (Chang, 2015; Yang et al., 2016). However, insects can enter an anti-stress state to adapt to adverse conditions. Diapause, a decrease in metabolism, and increased lipid deposition help insects to adapt to food shortages and maintain homeostasis and recover once favorable conditions return (Sánchez-Paz et al., 2006; Buckemüller et al., 2017; McCue et al., 2017). Insects can enter diapause in the form of eggs, mature larvae, pupae or adults to survive the long and resourcescarce winter (Zhang X. Y. et al., 2015). In addition to natural seasonal changes, damage to the

#### Edited by:

Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China

#### Reviewed by:

Leena Thorat, Savitribai Phule Pune University, India Shoaib Freed, Bahauddin Zakariya University, Pakistan

> \*Correspondence: Dao-Wei Zhang zhangdw1000@163.com

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 23 September 2018 Accepted: 11 February 2019 Published: 05 March 2019

#### Citation:

Zhang D-W, Xiao Z-J, Zeng B-P, Li K and Tang Y-L (2019) Insect Behavior and Physiological Adaptation Mechanisms Under Starvation Stress. Front. Physiol. 10:163. doi: 10.3389/fphys.2019.00163

**376**

ecological environment can often lead to food shortages and is one of the most common and severe stresses in nature (Panizzi and Hirose, 1995; Rotkopf et al., 2013). Previous studies have found that insect responses to food deficiencies or hunger stress generally fall into one of the two categories: (1) behavioral activities, i.e., ecological adaptation strategies whereby insects find food-rich places through behaviors such as migration or other activities such as entry into diapause, etc. or (2) physiological countermeasures that regulate the metabolism of related physiological and biochemical substances in the body to improve the insect's ability to endure hunger (Yang et al., 2016). Since food availability is crucial for insect survival, studying the consequences associated with starvation stress helps reveal adaptive strategies. Understanding these strategies is useful for the storage and release of natural insect enemies for biological pest control (Fujikawa et al., 2009). Previous reviews have summarized insect tolerance to starvation and hunger regulation mechanisms such as physiological adaptations to sugar intake (Chng et al., 2017), neurohormone adaptation (Peric-Mataruga, ´ 2006) and diapause (Hahn and Denlinger, 2007). Here we review studies of insect behavioral and physiological adaption mechanisms under starvation, especially those involving energy changes between carbohydrates, lipids, and proteins, as well as nerve signals and hormone regulation.

#### REGULATION OF INSECT BEHAVIOR UNDER STARVATION

Under the stress of starvation, insects tend to change their behavior that includes migration, cannibalism, early entry into diapause or the pupal stage, and reduced numbers of eggs (**Table 1**). For example, when larvae of Drosophila melanogaster (Diptera: Drosophilidae) and Harmonia axyridis (Coleoptera: Coccinellidae) are nutritionally challenged, they exhibit cannibalistic behavior (Ahmad et al., 2015). The main behavioral responses toward seasonal food shortages are migration and diapause. The rate at which larvae secrete ecdysteroids is significantly increased because hunger affects the level and production of ecdysone (Chen and Gu, 2006). Thus, larvae enter the pupal stage earlier under starvation conditions and the duration of pupation is prolonged with increasing days of starvation (Ballard et al., 2008). Moreover, pupae are very resistant so many insects endure adverse environmental conditions in the pupal form (Zhang X. Y. et al., 2015). For example, the larvae of Psacothea hilaris (Coleoptera: Cerambycidae) reach the pupal stage early after fasting (Munyiri and Ishikawa, 2005; Helm et al., 2017). When food is scarce, some beetles begin metamorphosis earlier (Zauner et al., 2000) while Bactrocera dorsalis (Diptera: Tephritidae) larvae enter the pupal stage after 12 h of starvation (Cong et al., 2015).

Insufficient food also limits insect reproduction (Zhang H. H. et al., 2015; Ojima et al., 2018). The most typical survival mechanism is to reduce reproductive investment under starvation conditions to increase somatic cell maintenance (Elkin and Reid, 2005; García-Roger et al., 2006; Billings et al., 2018). Studies of Bactrocera minax (Diptera: Tephritidae) indicated that hunger was not conducive to ovary development and led to a significant reduction in the number of matings and egg production and a prolonged spawning period (Huang, 2015). Similarly, under starvation conditions, Arma chinensis (Hemiptera: Pentatomidae) reduced the number of eggs laid and also exhibited a decline in the hatching rate of eggs (Zhang et al., 2017). Compared with bees kept under normal environmental conditions, the ovaries of hungry Apis mellifera (Hymenoptera: Apidae) worker bees showed shrinkage and decrease in egg plaques (Wang et al., 2016a,b). These behavioral adjustments are aimed at limiting population size to ensure availability of sufficient food for the existing population. However, there are exceptions. In the case of Eriosoma lanigerum (Hemiptera: Aphididae), the early stages of starvation produced a large number of breeding individuals followed by a sharp decline with progressive duration of starvation. In this way, the production of future generations was prioritized and then their own life processes were maintained (Chen, 2013).

In nature, most insects enter diapause during winter food shortages. However, a small number of insects migrate to places more suitable for growth and reproduction. After the low temperatures and food shortage conditions are over, they return to their original habitats to continue normal survival and growth (Feng et al., 2014). For example, in the fall in China, Mythimna separata (Lepidoptera: Noctuidae) moves from high to low altitudes and latitudes for the winter (Li, 1993). Incompletely metamorphosed insects that do not experience the pupal stage seek food by moving. It must be noted that hunger stress can also affect the ability of insects to take off and fly. Hunger before flight can have a negative impact on flight endurance (Kehl and Fischer, 2012), for instance, the speed and angular velocity of beetles decrease with increasing hunger (Nguyen, 2008). Despite these challenges, migratory movement is still one of the most important ways in which insects respond to hunger stress.

# PHYSIOLOGICAL REGULATION OF INSECT ENERGY UNDER STARVATION STRESS

#### Carbohydrates

Carbohydrates are a key source of energy for insects. Carbohydrates, mainly in the form of glycogen, trehalose and glucose, play an important role in energy metabolism and metabolite synthesis (Gaxiola et al., 2005; Kehl and Fischer, 2012). They not only enhance insect hunger resistance, but also play a vital role in other physiological adaptations (Tang et al., 2012b; Chng et al., 2017). Studies have found that starved larvae of A. mellifera can still maintain a stable blood sugar trehalose concentration (Wang et al., 2016a).

When the blood sugar content of insects is low, glycogen can be broken down, especially during starvation (Marron et al., 2003; Arrese and Soulages, 2010; Parkash et al., 2012; Tang et al., 2012a; Rovenko et al., 2015). Glucose is an equally important carbohydrate and all sugars are eventually converted to glucose to provide ATP, an energy source and metabolic


TABLE 1 | Some insect behaviors and physiological adaptions under starvation conditions.

intermediate in living cells (Jensen et al., 2015). Trehalose, an important component of insect blood is also known as the "sugar of life" (Shukla et al., 2015). In harsh environments, such as severe cold, high temperature and drought, trehalose accumulates and is used to maintain normal life processes and enhanced survival (Yu et al., 2008). Glycogen, glucose and trehalose can be converted into other forms for energy storage and release in insects (Tang et al., 2010). When insects are starved, trehalose is used first as a source of energy (Tang et al., 2014), leading to a rapid decrease in glucose and trehalose in their hemolymph (Satake et al., 2000). Under starvation stress, the trehalose content of Trogoderma granarium larvae was significantly reduced (Mohammadzadeh and Izadi, 2018). In starved P. hilaris larvae, glucose levels significantly decreased and the level of trehalose showed an initial decrease, followed by an increase (Munyiri and Ishikawa, 2005). The hunger-tolerance mechanisms of insects differ. For example, the hemolymph glucose concentration of A. mellifera was significantly reduced under starvation stress (Buckemüller et al., 2017) whereas the phosphorylase activity of Bombyx mori and Manduca sexta larvae increased and the glycogen content gradually decreased (Meyer-Fernandes et al., 2000; Satake et al., 2000). When the intensity of starvation stress increases, glycogen is converted to trehalose and released into the blood to maintain energy metabolism (Bede et al., 2007). Therefore, as the intensity of starvation increases, the levels of trehalose and glycogen in insects decrease, but trehalose remains at a relatively low and stable concentration (Shi et al., 2017). After relief from hunger stress, the hemolymph glucose levels of insects increase (Sánchez-Paz et al., 2007) and are gradually converted into trehalose and glycogen for storage.

The most important substances that mediate the decomposition of carbohydrates are trehalose synthase (TPS), trehalase (TREH), glycogen synthase (GS), and insulin-like pathway-related genes (Tang et al., 2012b). In H. axyridis exposed to starvation stress, the trehalose level and trehalase activity were significantly lower 8 h after starvation treatment, but the relative expression of TRE1-1 increased. After 8–24 h of starvation, trehalose was maintained at a high level, but glycogen levels decreased. These results indicate that trehalose plays a key role in the starvation process through molecular and biochemical regulation of trehalose and glycogen metabolism (Tang et al., 2014; Shi et al., 2017). When human blood sugar levels are very low, the body increases the blood sugar concentration by regulating the insulin signaling pathway. For insects, insulin-like signaling pathways in vivo play a similar role in controlling the balance of blood glucose concentrations (Kuhn et al., 2015; Zhai et al., 2015). Similarly, in D. melanogaster and B. mori, a decrease in insulin-like signaling levels regulate the expression of related antimicrobial peptide genes (Becker et al., 2010; Yang et al., 2016; Lebreton et al., 2017). These genes participate in immune regulation and repair of damage and thereby enhance the resistance of insects to starvation (Riddell and Mallon, 2006).

#### Lipids

Fat bodies are the main units of lipid storage in insects and their storage function is key for normal life processes (Ballard et al., 2008; Kehl and Fischer, 2012; Park et al., 2013). To combat hunger, stored lipid resources are often used through reduced glucose oxidation and increased fatty acid mobilization and lipid oxidation (Gergs and Jager, 2014; McCue et al., 2015; Wang et al., 2016a). Lipid metabolism in insects is

mainly regulated by Adipokinetic hormone (AKH), the fat stimulating hormone, which is secreted under low nutrient conditions, thereby leading to lipolysis, glycogenolysis, and sugar and lipid nutrients moving from the fat body into the hemolymph (Kim and Rulifson, 2004; Lee and Park, 2004). AKH, a key regulator of energy—also mobilizes sugar and lipids from insect fat bodies during high-energy activities such as flight and exercise and contributes to the balance of hemolymph sugars, lipids, and carbohydrates (Staubli et al., 2002; Hou et al., 2017). Intensive research has found that the ability of insects to survive starvation is largely dependent on the ratio of triglycerides to lipids in the body (Renault et al., 2002; Ballard et al., 2008; Laparie et al., 2012). When insects fly under starvation conditions, lipids are the main source of energy for the flight muscles (Ryan and van der Horst, 2000). Lipids are, therefore, closely linked to insect movement during starvation. Of course, the use of lipids under starvation stress varies among different insects. Under laboratory starvation conditions, the concentration of lipids in the hemolymph of B. mori increased (Satake et al., 2000) whereas in Pachnoda sinuata (Coleoptera: Scarabaeidae), the lipids in the flight muscles and fat bodies significantly decreased (Auerswald and Gäde, 2000). The beetle Merizodus soledadinus (Coleoptera: Carabidae) significantly increased the hydrolysis of triglycerides during food deprivation which returned to normal levels after feeding (Laparie et al., 2012). This suggests that triglycerides are immediately mobilized to enhance hunger resistance in the event of food shortage.

It is worth noting that moderate hunger leads to the accumulation of fat, which is dependent on the developmental stage of the insect and the state of feeding (Lorenz, 2001). Increased fat mass in insects can increase resistance to hunger, indicating a significant correlation between lipid levels and hunger resistance (Parkash et al., 2014). Pure lipid-based triglycerides are used during starvation (Sinclair et al., 2011) and proteins that are starved by lipid synthesis or degradation are affected by the opposite form. For example, a fatty acid synthase and a glycocholine transfer protein were down-regulated four fold after 4 h of starvation, while triacylglycerol lipase levels increased 10 fold (Muhlia-Almazán et al., 2005).

#### Proteins

A modest decrease in protein content during insect starvation indicates that insects can cope with hunger by using different endogenous reserves (Gäde and Auerswald, 2002; Helland et al., 2003). After starvation, the concentrations of alanine in the flight muscles, lipid bodies, and hemolymph of insects rapidly decline whereas those of proline remain high (Kehl and Fischer, 2012). At the same time, the heat shock proteins 70 (HSP70s) are important stress protectants in H. axyridis and Rhodnius prolixus (Hemiptera, Reduviidae) and play a role in adaptation to food deprivation (Shen et al., 2015; Paim et al., 2016). In addition, studies have shown that the positive effects of adult amino acids are limited to females, presumably because their high protein demand significantly changes the catabolic rate (Mevi-Schütz and Erhardt, 2005; Bauerfeind and Fischer, 2009). There is ample evidence that amino acids extracted from protein breakdown are degraded during starvation (Haubert et al., 2005).

# Nerve Signals and Hormones

When insects are stressed by hunger, they initiate a series of anti-starvation mechanisms. These mechanisms include regulating the expression of related genes, synthesizing anti-stress substances and regulating the catabolism of energy substances in the body to maintain normal growth and development (Buckemüller et al., 2017). A study in D. melanogaster found that dG9a (histone methyltransferase) is a key factor in tolerance to hunger stress and is also a key regulator of behavioral strategies under starvation conditions (An P.N.T. et al., 2017; Shimaji et al., 2017). Hunger is a powerful driver of food intake and some neurons, neuropeptides and neurohormones play key roles in behavioral and physiological regulation (Peric-Mataruga, 2006 ´ ; Jourjine et al., 2016; Mena et al., 2016). At the same time, starvation increases the expression level of SLC5A11 neurons and enhances their excitability by inhibiting the dKCNQ channel, thereby conferring hunger status and promoting feeding and starvation-driven behaviors (Park et al., 2016).

Hunger itself affects stress in insects and the expression of immunity-related genes. Many immunity-related genes, such as interleukin 1-β, are significantly down-regulated during starvation (Riddell and Mallon, 2006; Buckemüller et al., 2017) leading to lower metabolism followed by a concomitant decline in immunity (Guo et al., 2014). Starvation stress can also significantly up-regulate the expression of two octopamine (OA) receptor genes (Li, 2017). OA has significant biological effects on the growth and behavior of various arthropods and poor living conditions pose differential consequences on the distribution and content of OA. Hunger not only has an effect on the transcription of the brain and surrounding tissues in Drosophila (Bos et al., 2016; Singh et al., 2018), but also induces autophagy in Drosophila larvae during metamorphosis by inhibiting the PI3KI/Akt-Tor pathway (Riddiford et al., 2000; Wang et al., 2012). Similarly, starvation can also induce autophagy in Spodoptera frugiperda (Lepidoptera: Noctuidae) Sf9 cells (Xie K. et al., 2017). Increased levels of autophagy are usually induced by signals such as starvation, while excessive levels of autophagy can cause autophagic programmed cell death. During starvation, the level of autophagy helps to reduce the level of apoptosis in fat cells, thus playing a protective role in the survival of adipocytes (Otomo et al., 2013; Li et al., 2015).

Dopaminergic signaling pathways and juvenile hormones (JH) are also important stress-resistance substances in insects and play a role in adaptation to hunger stress (Neckameyer and Weinstein, 2005; Lee and Horodyski, 2006). While the former directly affects the metamorphosis and development of insects (Yang, 2014), the latter confers damage protecting (Shi et al., 2016). After starvation treatment, the growth and development of Plutella xylostella (Lepidoptera: Plutellidae) were found to be delayed because the expression of the juvenile hormone acid methyl transferase (JHAMT) candidate gene Px009591 increased and the expression of the juvenile hormone esterase (JHE) gene Px004817 and JHE activity decreased. These changes led to an

increase in JH levels in insects, which in turn delayed growth and development (Duan, 2016). Similarly, the rate of biosynthesis of M. sexta gradually increased after starvation (Lee and Horodyski, 2006). Telang et al. (2010) also found that JHE mRNA levels in the 4th instar larvae of Aedes aegypti (Diptera: Culicidae) were close to zero after 36 h of starvation. In addition, when the expression of the hsp18.3 gene of Tribolium castaneum (Coleoptera: Tenebrionidae) was silenced by RNAi technology, the resistance of the starved group was significantly lower than that of the control group (Xie J. et al., 2017). Similarly, a study of three HSP70 genes in H. axyridis showed that their relative expression not only increased with increasing temperature, but also at the peak of starvation at 8 h (Shen et al., 2015). Thus, juvenile hormones and heat shock proteins have a significant effect on insect emergency stress responses.

#### FUTURE PROSPECTS

Nutrition plays an important role in the life history of insects, especially as reproduction is influenced by the quality and quantity of food (Fischer et al., 2004). While dietary restrictions can reduce reproductive yield, it can also extend an insect's lifespan (Partridge et al., 2005; Carey et al., 2008). At the same time, insects exposed to starvation or low temperatures may enter a state of diapause. In previous studies, 46 diapauserelated genes were found in Aphidius gifuensis (Hymenoptera: Aphididae) (Huang et al., 2015; An T. et al., 2017) and 443 in Coccinella septempunctata (Coleoptera: Coccinellidae) (Liu et al., 2014; Ren et al., 2015; Qi et al., 2016). These genes are associated with energy demand during diapause and inhibition of metabolism. During diapause and wintering in insects, nutrient levels, amino acid accumulation and transformation and regulatory mechanisms including insulin signaling pathways are similar to those under starvation stress (Hahn and Denlinger, 2007; Huang et al., 2015; Ren et al., 2016; Sinclair and Marshall,

#### REFERENCES


2018). Moderate hunger can also have a positive impact on insects and on pest control. Thus, a systematic examination of behavioral and physiological strategies under starvation stress can provide significant theoretical basis for the development of natural breeding in insects and storage of natural enemy insects for their use in pest control.

Nutritionally challenging situations in environments have evolved insects with diverse adaptive responses to withstand periods of food shortage. In this review we have attempted to showcase the starvation stress physiology of insects with respect to behavioral attributes and physiological regulation. Given their broad range of ecological habitats, insects are suitable and convenient models to evaluate the strategies they employ to cope with starvation. Moreover, starvation stress research in insects offers interesting cues from ecological and evolutionary perspectives that not only govern reproduction, survivorship and cross tolerance to other environmental stressors but also dictate phenotypic plasticity in adjusting development and life histories around nutritionally optimal environmental conditions. Such studies are critical to our understanding of starvation-induced physiological responses that precede death and in extrapolating them to vertebrates.

#### AUTHOR CONTRIBUTIONS

B-PZ, Z-JX, KL, and D-WZ conceived and manuscript structure design. B-PZ, Y-LT, and D-WZ wrote the manuscript.

#### FUNDING

This work was supported by the National Natural Science Foundation of China (Grant No. 31560511) and Science and Technology Foundation of Guizhou Province [Grant No. QKH.JZ (2014)2014].



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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhang, Xiao, Zeng, Li and Tang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Starvation Stress Causes Body Color Change and Pigment Degradation in Acyrthosiphon pisum

Xing-Xing Wang1,2, Zhan-Sheng Chen<sup>1</sup> , Zhu-Jun Feng<sup>1</sup> , Jing-Yun Zhu<sup>1</sup> , Yi Zhang<sup>1</sup> and Tong-Xian Liu<sup>1</sup> \*

<sup>1</sup> Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau, Ministry of Agriculture, College of Plant Protection, Northwest A&F University, Yangling, China, <sup>2</sup> College of Horiculture, Northwest A&F University, Yangling, China

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Jérôme Casas, Université de Tours, France Simon Leather, Harper Adams University, United Kingdom Kai Lu, Fujian Agriculture and Forestry University, China Angelique Christine Paulk, Massachusetts General Hospital, Harvard Medical School, United States

> \*Correspondence: Tong-Xian Liu txliu@nwsuaf.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 13 November 2018 Accepted: 15 February 2019 Published: 05 March 2019

#### Citation:

Wang X-X, Chen Z-S, Feng Z-J, Zhu J-Y, Zhang Y and Liu T-X (2019) Starvation Stress Causes Body Color Change and Pigment Degradation in Acyrthosiphon pisum. Front. Physiol. 10:197. doi: 10.3389/fphys.2019.00197 The pea aphid, Acyrthosiphon pisum (Harris), shows body color shifting from red to pale under starvation in laboratory conditions. These body color changes reflect aphid's adaptation to environmental stress. To understand the color-shifting patterns, the underlying mechanism and its biological or ecological functions, we measured the process of A. pisum's body color shifting patterns using a digital imagery and analysis system; we conducted a series of biochemical experiments to determine the mechanism that causes color change and performed biochemical and molecular analyses of the energy reserves during the color shifting process. We found that the red morph of A. pisum could shift their body color to pale red, when starved; this change occurred rapidly at a certain stress threshold. Once A. pisum initiated the process, the shifting could not be stopped or reversed even after food was re-introduced. We also discovered that the orange-red pigments may be responsible for the color shift and that the shift might be caused by the degradation of these pigments. The carbohydrate and lipid content correlated to the fading of color in red A. pisum. A comparative analysis revealed that these reddish pigments might be used as backup energy. The fading of color reflects a reorganization of the energy reserves under nutritional stress in A. pisum; surprisingly, aphids with different body colors exhibit diverse strategies for storage and consumption of energy reserves.

#### Keywords: pea aphid, color fading, starvation, adaptation, pigment degradation

#### INTRODUCTION

Many animals, including insects, show variations in their body colors. This coloration could be used in making mating choices, sending warning signals, receiving external radiant energy for body temperature regulation, and many other functions (Huey and Kingsolver, 1989; Merilaita and Tullberg, 2005; Forsman et al., 2008). Aphids that show polyphenism in their sexual generation, wing formation, and body colors are flexible under varied environmental conditions. The pea aphid, Acyrthosiphon pisum (Harris), generally contains two strains based on body color, red and green. Ecological studies showed that red aphids on green plants are easily detected by predators, while green aphids can be easily parasitized by parasitoids (Losey et al., 1997; Libbrecht et al., 2007). The green peach aphid, Myzus persicae (Sulzer), is known to display a connection between body

color and esterase differences (Takada, 1979); the red strain of A. pisum might use its pigments (carotenoid pigments) for light-induced electron transfer and ATP synthesis (Valmalette et al., 2012).

Body color in aphid species is affected by many physical conditions. Studies on the English grain aphid, Sitobion avenae (Fabricius), showed that the aphid changes its body color from green to pink or brown with changes in light intensity (Alkhedir et al., 2010). Also, the red A. pisum can shift color to green in low temperatures within a few generations (Valmalette et al., 2012). Other studies showed that the body colors of M. persicae and S. avenae can also be affected by temperature (Matsumoto and Tsuji, 1979; Takada, 1981).

Biological conditions can similarly modify aphid body color. For instance, different color-esterase morphs in M. persicae might be determined by their hosts (Takada, 1979). In addition, an endosymbiont Rickettsiella sp. was reported to be responsible for the change of A. pisum body color from red to green over one generation (Tsuchida et al., 2010).

The insect's body color is basically determined by several kinds of chemical pigments. Aphid pigments that make them colorful comprise of two categories. The first category includes aphins, which specifically exist in the hemolymph of aphids (Bowie et al., 1966) and have multiple molecular structures with different colors, such as red or green, and constitute the base color for aphids (Bowie et al., 1966; Horikawa et al., 2004; Horikawa et al., 2006; Alkhedir et al., 2010). The second category includes carotenoid pigments, such as β-carotene, lycopene, and torulene, which are present in lower quantities than aphins, and are synthesized within aphids, and thus, can strongly affect body color (Andrewes et al., 1971; Jenkins et al., 1999; Moran and Jarvik, 2010). The pea aphids show a relatively stable red-green color polymorphism. They can make their own carotenoids based on the genes laterally transferred from fungi. Studies have shown red individuals to have a single carotenoid desaturase enzyme that is absent from green individuals (Moran and Jarvik, 2010). Laboratory crosses between the two strains and a Mendelian genetic analysis revealed that color polymorphism in the pea aphids is determined by a single bi-allelic locus named colorama (Caillaud and Losey, 2010). Previous studies have shown that the host plant might also affect the aphid's color. For instance, Losey and Eubanks (2000) found that the color morphs of pea aphids were not significantly different from those collected from forage crops and various vegetables. However, the frequency of occurrence of these color morphs was different on the three hosts: Pisum sativum (pea), Medicago sativa (alfalfa), and Trifolium pratense (red clover; Simon et al., 2003). Although the two morphs are stable, the red individuals can turn into green at low temperatures (Valmalette et al., 2012) or following an infection of endosymbionts (Tsuchida et al., 2010).

Another color-changing phenomenon exists in the red A. pisum, which is the shifting of body color from red (pink) to pale red; this change is reversible, even though the change is directly associated with the change in carotenoids (Valmalette et al., 2012; Tabadkani et al., 2013). The color shifting in red A. pisum could be triggered by low-quality diets and can be reversed, when nutrition habits change from a low-quality to a high-quality diet. The pea aphids with pale body color have significantly lower wet and dry weights, which may be attributable to a loss of lipids and soluble carbohydrates, as well as faster movements; in addition, this color reversion enables the aphids to quickly respond to deprived host plants and restore their original status, when they find appropriate host plants (Tabadkani et al., 2013). The shift in color in this process may be a type of stress symptom or may possibly play a role in response mechanisms to environmental threats.

In red A. pisum, body color represents the aphid's response to nutritional stress; scant information is available on the mechanism behind the shift from red to pale red. Previous studies simply divided the aphids into red (pink) or pale red categories. Furthermore, the ecological and biological functions of these changeable pigments are also unclear. In our study, we monitored the color shift process in A. pisum under starvation and revealed the details of this shift. In addition, we extracted the red pigments from aphids at every color-shifting stage for further analysis. Considering the connection between color changes and nutritional decline, the content changes of energy reserves (soluble carbohydrate, glycogen, lipid, and protein) and its correlation with body color at different color-shifting stages were also measured. We also conducted an analysis of the energy reserves to evaluate the potential function of reddish pigments by comparing the red and green strains of A. pisum under starvation.

# MATERIALS AND METHODS

#### Experimental Insects and Plants

A red strain of the pea aphid, A. pisum, was collected from Lanzhou, Gansu Province, northwestern China, while a green strain was obtained from our laboratory colony in Yangling, Shaanxi, China. Both strains were cultured on broad bean (Vicia faba L., var. "Jinnong") as a substrate under long-day conditions (16L: 8D; 20 ± 1 ◦C) for more than 30 generations at the Key Laboratory of Applied Entomology, Northwest A&F University, Yangling, Shaanxi, China. All aphids were reared at a low density (less than 30 individuals per plant) for more than three generations before they were subjected to the experiments below. As compared to nymphs, A. pisum adults show additional behaviors, such as nymph-production or dispersal (winged), which make their energy consumption more complicated; therefore, wingless fourth-instar nymphs (12 h after molting) were picked for experiments.

# Aphid Color Collection

#### Color Collection From Color-Shifted Aphids

In order to get color data, we used digital camera observing images for further analysis (Joiner, 2004; Murakami et al., 2005; Lebourgeois et al., 2008). Nutrition stress is one important stimulation for shifting of body color in our experience, but it is difficult to sustain a stable nutrition stress under laboratory conditions for analysis. We picked starvation as a stable and controllable nutrition stress in our treatments, which is also common in wild conditions. Starvation treatment is stable enough for laboratory experimentation and is easy to control.

Fourth-instar nymphs (12 h after molting) of the wingless red A. pisum were placed in a transparent plastic dish (90 mm in diameter) and a 24-well tissue culture plate (transparent, plastic, one individual per well) depending on the experiments. The aphids were starved for 24 h to stimulate shift in the body color. All devices were placed under 24-h light conditions (20 ± 1 ◦C). A cold light source (KL 1500 LCD, Zeiss, Germany, temperature of 3200 K) was used for lighting. Images were taken using a digital camera (Canon <sup>R</sup> EOS 5D Mark III, Canon, Tokyo, Japan; Lens: Canon <sup>R</sup> Macro lens EF 100 mm 1:2.8 L IS USM, Canon, Japan). The camera parameters were set as instructed in the manufacturer's manual (shutter speed: 1/250; aperture: F4.0; ISO: 320; picture style: faithful 0,0,0,0; white balance: color temp, 3200 K, AF mode: manual focus; metering mode: center-weighted average), and the images were recorded in RAW (.CR2); and the 24-Patch ColorChecker chart (Mennon, China) with standard colors on was used to correct images before experiments (**Supplementary Figure S1A**). The images were read and modified by Adobe Camera Raw and corrected by the 24-Patch ColorChecker chart in Adobe DNG Profile Editor, and the values of RGB channels (RGB for red, green and blue, respectively, as below) were read and recorded by Adobe Photoshop CS6 and Microsoft Excel 2013 (Schwarz et al., 1987; Zhang et al., 2003).

#### **Color collection during aphid color shifting**

To analyze the body color shifting pattern, 50 12-h-old wingless fourth instar nymphs of the red A. pisum (well-cultured and in low density) were used in each color shifting test. The newmolting nymphs were selected and reared in host plants for 12 h, and then collected and placed in a transparent plastic dish (90 mm in diameter; 10 individuals per dish) for starvation treatment. The color-shifting images of the aphids were captured at hourly intervals for 24 h. The values of RGB channels were read by Adobe Photoshop CS6 software.

#### **Body color reversibility test**

To understand if the color shifting could be reversed or not, 58 12-h-old wingless fourth instar nymphs of the red A. pisum (well-cultured and in low density) were individually picked into two 24-well tissue culture plates (transparent plastic; 18 mm in diameter of each well; one individual per well) for each starvation treatment. Aphid images were captured at an early stage (12 h after treatment began) and late stage (24 h after treatment began). To determine if the color of the aphids can be reversed after they are re-introduced to food, images of the aphids were collected in a condition of starvation, and then they were re-transferred onto V. faba leaf disks (with 1% agar) in two 24-well tissue culture plates and reared on the leaf within them. Images of the aphids were then captured after 24 h of being reared on the leaves to analyze changes in body color.

#### Pigment Extraction and Analysis

For a deeper understanding of the composition of body pigments and the changes in the shifting process, a series of studies were performed.

#### Extraction of Pigments for Spectral Analysis

The protocol of extraction has been presented before in Tsuchida et al. (2010) and Valmalette et al. (2012) and it was modified before use. The 12-h-old wingless fourth-instar nymphs of the red A. pisum strain were starved for 16 h to stimulate red to pale red color shift. The pale individuals (whose body color had changed) were collected for extraction of pigments; the red individuals were collected from prepared aphids without starvation. Collected samples were quick-frozen by liquid nitrogen and freeze-dried for 36 h in a lyophilizer (MSA3.6P, Sartorius, Germany). The pigments were then extracted with chloroform and ethyl alcohol. Following this, 20 mg of each sample was transferred into a 1.5 mL microtube and homogenized by a micropestle in 1 mL of chloroform. All samples were kept in the dark for 24 h. The supernatant was filtered into a new 1.5-mL microtube using a 1-mL injector and a nylon syringe filter (this mixture of pigments was marked as "pigment mix"). The solution was then condensed in a speed vacuum concentrator (ScanSpeed 40, Scanvac, Denmark; 2000 × g 1 h, 40◦C) until all red pigments separated out; 200 µL of ethyl alcohol was added to dissolve the solute by mixing for 2 h; the contents were then centrifuged at 20,000 × g for 15 min at 4◦C, and the supernatant was transferred into a new tube. The ethyl alcohol-insoluble deposits were washed twice with 1 mL of ethyl alcohol, followed by addition of 50 µL of chloroform to dissolve the remainder (this pigment mixture recognized as "tomato red" was marked Pigment 1); the supernatant which was transferred to a new tube was condensed in a speed vacuum concentrator till all solutes separated out and then resolved using 200 µL of chloroform (this pigment mixture that was recognized as "orange-red" was marked Pigment 2; **Supplementary Figure S1B**).

#### Pigment Absorbance Patterns

The 12-h-old wingless fourth-instar nymphs were starved for 16 h to stimulate the red to pale red color shift. The aphids were then individually placed into transparent plastic dishes (90 mm in diameter; 20 individuals per dish). The aphids were collected at 1 h intervals for 3 h. The collected samples were used for pigment extraction. UV–Vis absorbance spectra of the pigment solution were then collected using Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., United States).

#### Correlation of Pigment Absorbance and Three RGB Color Channels

The images of the wingless fourth-instar nymphs in different body colors were captured using a digital camera, and the aphids were then used for pigment extraction. The values for the RGB color channels and the corresponding absorbance at λmax (483 nm) were used for correlation analysis for the pigment 2 mixture.

#### Thin-Layer Chromatography of Aphid Pigments

The crude mixture of pigment extracts was used for thin-layer chromatography (TLC). The protocol of TLC was modified from Tsuchida et al. (2010). Based on the values for the G channel (as recorded using a digital camera), the aphids were categorized to have two colors: the red (G value: around 160) and the pale red (G value: around 190). The aphids were quick-frozen in liquid nitrogen and freeze-dried for 36 h in a lyophilizer (MSA3.6P, Sartorius, Germany). They were extracted using a chloroformethyl alcohol (1:2) mixture. The total solution was kept in the dark for 24 h and centrifuged at 20,000 × g for 15 min at 4 ◦C to remove deposition from the mixture. The clarified liquid was used for TLC.

#### Red Pigment Extraction

fphys-10-00197 March 4, 2019 Time: 10:55 # 4

The aphids were categorized according to the two body colors as described above; they were quick-frozen in liquid nitrogen and freeze-dried for 36 h in a lyophilizer (MSA3.6P, Sartorius, Germany). The samples were then used for extraction of the two red pigments (mixture of the pigments 1 and 2; **Supplementary Figure S1B**). Pigment mixtures from aphids with different body colors were then used for TLC.

#### Thin-Layer Chromatography

The extraction solutions were subjected to phase TLC (pTLC) on pre-coated silica gel plates using chloroform-methanol (1:1) mixture as a solvent for all pigments and ethyl alcohol-waterammonia-isopropyl alcohol-ethyl acetate (6:1:1:6:1) mixture for red pigments. The images of TLC were captured by a digital camera under visible and ultraviolet (295–365 nm) lights, which were then analyzed using the Adobe Photoshop CS6 software (Version 13.0 x64, Adobe, CA, United States).

#### Energy Reserves Assay

Considering that nutrition stress is a key trigger for the fading of color, pigment degradation and conversion are probably related to nutritional physiology. Studies of energy reserves were performed.

#### Energy Reserves Assay

Twelve-hour-old fourth instar nymphs of the wingless red and green A. pisum were transferred into transparent plastic dishes (90 mm in diameter; 20 aphids per dish) for starvation treatment. The aphids were collected at hourly intervals for 24 h. The collected samples were then immediately used in energy reserve assays. The aphids were freeze-dried for 36 h in a lyophilizer (Heto PowerDry LL3000 Freeze Dryer, Thermo Fisher Scientific, United States); 2 mg of dried samples was weighed out using a high-precision electronic balance (MSA3.6P, Sartorius, Germany) at ambient temperature. The aphids were transferred into a 1.5 mL microtube, homogenized by a micropestle, and dissolved in 800 µL of solution buffer (100 mM KH2PO4, 1 mM DTT, 1 mM EDTA, pH 7.4) for further analysis. This experimental step had three replicates.

#### Glycogen and Soluble Carbohydrate Assays

After all samples (2 mg of dried samples) were dissolved in the solution buffer as mentioned above, each tube with the aphid samples was centrifuged at 20,000 × g for 15 min at 4◦C to remove deposition from the mixture. The supernatant was then transferred into a new tube for soluble carbohydrate assay; the deposits were washed twice using methanol for glycogen assay. Both glycogen and soluble carbohydrates were measured using the colorimetric method based on the enthrone reagent with glucose as the standard as described in Foray et al. (2012).

#### Protein Assay

After all samples (2 mg of dried samples) were dissolved in the solution buffer, each tube with the samples was centrifuged at 1800 × g for 15 min at 4◦C. The protein content in these mixtures was measured using a modified Bradford protein assay kit (C503041-1000, Sangon Biotech, Shanghai, China). After preparation of the mixture, 2.5 µL of each supernatant was transferred into a 96-well microplate, together with 250 µL of Bradford reagent. Protein concentration was determined by a microplate reader (infinite M200, TECAN, Switzerland) at 595 nm.

#### Lipid Assay

After all samples (2 mg of dried samples) were dissolved in the solution buffer, 160 µL of NaOH (6 N) was added to hydrolyze the fat content in each tube; the tubes were waterbathed (75◦C) for 3 h. The hydrolytic fatty acid content in the mixtures was measured using a non-esterified free fatty acids (NEFA) assay kit (A042, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The raw sample volume of each test was 0.2 mL. First, the prepared mixture was transferred into glass tubes and buffer B (0.5 mL) was added, followed by reagent C (1 mL with Cu2+; in blue color); after 4 mL of reagent A was added, samples were centrifuged at 3500 × g for 10 min (all these reagents are named by Nanjing Jiancheng Bioengineering Institute). Afterward, the supernatant was discarded, while 2 mL of clear liquid from the lower layer was transferred to a quartz cuvette for further analysis. Concentration of fatty acids was determined by NanoDrop <sup>R</sup> 2000c (Thermo Fisher Scientific, Middletown, VA, United States) at 440 nm.

#### Color-Dependent Energy Reserves Assay (Red Strain)

New molt wingless aphids at the fourth-instar stage were collected and prepared for energy reserves assay. After 12 h of rearing, samples were transferred into a tissue culture plate (transparent, plastic, one individual per well) and starved for 12, 16, and 24 h; images of all aphids were then taken for color assay. Individuals from each treatment (12, 16, and 24 h) were divided into two groups (red and pale red) based on the values for the G channel (around 160 as red and 190 as pale red) observed in the images. Collected samples with three replications were then marked and used for energy reserve assays immediately. The protocols for this assay have been detailed in the previous section.

#### Transcriptional Analyses

Glycogen was considered to play an important role in this process, and we have conducted a preliminary study of its downstream metabolism. The two aphid strains were starved as described above. Aphid samples were collected at hourly intervals for 24 h. Samples were quick-frozen using liquid nitrogen immediately after collection. Samples of RNA were extracted with RNAiso Plus (Takara, Japan), and cDNA was synthesized from

them using a PrimeScriptTM RT reagent kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was performed with SYBR <sup>R</sup> Premix Ex TaqTM II (Takara, Japan) in an IQ-5 system (Bio-Rad, United States). Glycogen phosphorylase (EC 2.4.1.1) catalyzes the rate-limiting step in glycogenolysis in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond and breaks up glycogen into glucose subunits<sup>1</sup> . It is the key enzyme for glycogen metabolism. We selected it for evaluation of the Glycogen metabolism. Ribosomal protein L7 (Rpl7) was selected as a reference gene from Nakabachi et al. (2005) and Guo et al. (2016). The primers were designed by Primer-BLAST available online at NCBI<sup>2</sup> .

Primer (glycogen phosphorylase, GP, ACYPI001125; ribosomal protein L7, Rpl7, ACYPI010200) sequences were as follows:

GP-f: GCTCAGAAAATAACCAACGG; GP-r: GTGTGTCT ACTACTTTGCCA Rpl7-f: GCGCGCCGAGGCTTAT; Rpl7-r: CCGGATTTCTT TGCATTTCTTG

#### Data Analysis

Results from color shift analysis, energy reserves assay, and transcriptional analysis data were analyzed by Student's t-test and the Duncan's test; data on body color reversibility were analyzed by Chi-square test; correlation analysis of energy reserves was performed using ANCOVA. All data were subjected to statistical analysis using the SPSS software (version 22; SPSS Inc., Chicago, IL, United States). The RAW images with 24-patch ColorChecker chart were analyzed using Adobe DNG Profile Editor (Version 1.0.0.46 beta, Adobe, CA, United States). All captured images were read and modified using Adobe Camera Raw (Version 7.1 beta, Adobe, CA, United States), and analyzed using Adobe Photoshop CS6 (Version 13.0 x64, Adobe, CA, United States).

#### RESULTS

#### Values for the Color Channels in Color-Shifting Aphids

After the starvation treatment, the aphids showed variations in body color (**Figure 1A**). Although the values for the R channel varied greatly during the 24-h period (F = 2.105, df = 23, 1014, P = 0.002) (red), the changes were irregular (**Figure 1B**). The color channels moved to higher values during the red to pale red color shift as expressed by the values for both the G (F = 17.208, df = 23, 1014, P < 0.0001) (**Figure 1C**) and B channels (F = 6.758, df = 23, 1014, P = 0.002) (green and blue) (**Figure 1D**). The values of the G channel showed a marked increase, and the point in time at which the rise occurred was around 10 to 14 h after the starvation treatment, which plateaued at 19 h. The increasing trend of the B channel values was too weak to be identified (**Figure 1D**).

#### Body Color Reversibility After Starvation

Based on our pre-test, we selected the value of 170 as the shift point for the G channel. The proportion of body color shifted slightly (in pale red aphids, G > 170) and remained unchanged (in red aphids, G < 170) after 12- and 24-h starvation periods, which were separately but continuously monitored for color change. The 24-h starvation treatment stimulated a high proportion of aphids to shift color (χ <sup>2</sup> = 4.773, df = 1, P = 0.029, Chi-square test); most of these color-shifted or slightly color-shifted aphids then turned to pale red (G > 170) in another 24 h after being reared on leaf disks in both treatments (χ <sup>2</sup> = 0.918, df = 1, P = 0.338); about 1/3 of the aphids that had a red (G < 170) body color turned to pale red (G > 170) within another 24 h on leaf disks after the two treatments. The proportion of aphids that shifted to pale red was similar between the two treatments (χ <sup>2</sup> = 0.006, df = 1, P = 0.940; **Figure 2A**).

The distributions of R, G, and B values of all the starved aphids before and after being reared on leaf disks for 24 h are shown in **Supplementary Figure S3**. The values for the G channel in both treatments were categorized into two groups with a cutting point of 170 (G value) after 24 h of rearing (values distribution, 12 h: before treatment, P = 0.036; after treatment, P < 0.0001; 24 h: before treatment, P < 0.0001; and after treatment, P = 0.012; **Supplementary Figure S3**). The values of the R channel maintained a normal distribution (12 h: K.S. test, before treatment, P = 0.200; after treatment, P = 0.184; 24 h: before treatment, P = 0.200; after treatment, P = 0.200). The values of the B channel showed similar trends (12 h: before treatment, P = 0.200; after treatment, P = 0.200; 24 h: before treatment, P = 0.200; and after treatment, P = 0.200; **Supplementary Figure S3**).

#### Absorbance Patterns of Differently Colored A. pisum Pigments

Preliminary studies on the nature of pigments show some differences. The UV–Vis absorption spectral readings of the two crude extracts of the pigment are shown in **Supplementary Figure S4**. The pigment 1 solution in dark-red color showed two absorption bands: one at 240–300 nm (ultraviolet region) and another one at 340–590 nm (visible region, λmax = 520 nm, **Supplementary Figures S4A,B**); the pigment 2 solution in orange-red color also had two absorption bands: one at 200– 340 nm (ultraviolet region) and another one at 400–550 nm (visible region, λmax = 483 nm, **Supplementary Figures S4C,D**). Both the absorption bans and the absorbance peak of pigment 1 solution were located in the visible region, close to the infrared region, which formed the yellow-green absorption band (550–600 nm); this gave this pigment mixture a yellowgreen reflectance spectrum, and thus, the pigment 1 mixture appeared tomato red (**Supplementary Figure S4B**). The pigment 2 mixture, with its absorption band and absorbance peak located close to the ultraviolet region with little contribution toward the yellow-green absorption band, had a yellow-green reflectance spectrum; therefore, this mixture appeared as orange-red (**Supplementary Figure S4D**). Both pigment mixtures appeared

<sup>1</sup>https://enzyme.expasy.org/EC/2.4.1.1

<sup>2</sup>http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK\_LOC= BlastHome

to be oil-like in nature after they were concentrated; they were dried by vacuuming for 24 h (**Supplementary Figure S4F**).

# Detection of Pigments of the Two Different Colors in A. pisum

A comparative analysis of the two pigment solutions extracted from A. pisum revealed the amounts of red (G channel value around 160) and pale red (G channel value around 190) pigments to be similar in pigment 1 mixture (t = 0.056, df = 5, P = 0.957, **Figure 2B**), but significantly different in the pigment 2 mixture (t = −13.630, df = 3, P = 0.001, **Figure 2C**).

# Absorbance Potential of the Pigments

The contents of the crude extracts of the pigments, the pigment 1 and pigment 2, changed with the shifting of color in the aphid's body (**Figures 2D,E**). The absorbance pattern of the pigment 1 mixture was irregular at λmax = 520 nm and did not show any significant difference during the whole period of color shifting (F = 0.362, df = 12, 26, P = 0.966; **Figure 3A**); on the other hand, the absorbance pattern of the pigment 2 mixture showed a decreasing trend 12 h after starvation at λmax = 483 nm (F = 1.737, df = 12, 26, P = 0.116; **Figure 3B**), which revealed that pigment 2 mixture might be responsible for the color shift.

# Correlation of Pigment Absorbance and the RGB Color Channels

The results of absorption (based on λmax of pigment 2 solutions) and values for the RGB channels showed that there was no correlation between the pigment absorbance and values for the R channel (r = −0.170, P = 0.211, Kolmogorov–Smirnov test, **Figure 3C**). Values for both the G and B channels were negatively correlated to pigment absorbance; the values for the G channel showed a stronger correlation with absorbance than the values for the B channel (G channel, r = −0.826, P < 0.0001, Kolmogorov– Smirnov test, **Figure 3D**; B channel, r = −0.502, P < 0.0001, Kolmogorov–Smirnov test, **Figure 3E**).

# Thin-Layer Chromatography of Aphid Pigments

Thin-layer chromatography bands of extracts from A. pisum strains with the two different body colors (pale red and red, classified based on the value of G channel) were mostly similar in both their positions and quantities except for the red bands which were different; the extracts from red A. pisum had more red pigments (**Figure 2F**). Furthermore, TLC that was observed under ultraviolet light exhibited more bands than those observed in visible light; red pigments showed different contents between the two lanes (**Figure 2G**), but the two red pigments (pigment mixtures 1 and 2) could not be separated with this experimental protocol.

# Soluble Carbohydrate and Glycogen Assay

The change in carbohydrates is highly correlated to the degree of starvation treatment. The glycogen reserves in the earlier points in time were significantly different between the two strains of aphids (P < 0.001, tests of between-subjects effects, ANCOVA), while there were no significant differences in the later points in time. The green strain contained about sevenfold more glycogen than the red strain during the first 6 h, and this amount gradually decreased (**Figure 3A**). Although both curves displayed a decreasing trend, glycogen reserves in the green strain

declined more rapidly than in the red strain. The differences in values recorded at each point in time were also analyzed by Student's t-test and are marked in the diagrams (P <sup>∗</sup> < 0.05, P ∗∗ < 0.001, **Figure 4A**).

The soluble carbohydrates displayed fluctuations in both the aphid strains. The tests of between-subjects effects (ANCOVA) showed strong correlations between time and experimental strains (P < 0.001); comparative experiments showed differences at many points in time between the two strains (**Figure 4B**). The dynamic changes in soluble carbohydrates in both strains went up and down during the first 12 h and then declined during the last 12 h (**Figure 4B**).

The differences in values at each time point were analyzed using Student's t-test and are marked in corresponding diagrams (P <sup>∗</sup> < 0.05, P ∗∗ < 0.001, **Figure 4B**).

#### Glycogen Phosphorylase Transcriptional Expression

The metabolism of glycogen is not much different between the two aphid strains. Tests for the between-subjects' effects (ANCOVA) on the transcription levels of glycogen phosphorylase showed no correlation between time and strains (P = 0.113). Expression levels of glycogen phosphorylase in both aphid strains showed a decreasing trend with no significant differences between the two strains (P = 0.113; **Figure 4C**). The differences of expression levels at each point in time were also analyzed by Student's t-test and marked in the corresponding diagrams (P <sup>∗</sup> < 0.05, P ∗∗ < 0.001, **Figure 4C**).

#### Energy Reserves Assay Between the Red and Pale Red A. pisum (Red Strain)

There are differences in energy reserves contents between aphids before and after color fading. The red aphids contained significantly higher soluble carbohydrates (t = 9.592, df = 6, P < 0.0001, **Figure 5A**) and lipids (t = 2.711, df = 6, P = 0.035, **Figure 5D**) than the pale aphids. The contents of glycogen and proteins were not significantly different between the two strains of aphids (glycogen: t = 2.013, df = 10, P < 0.072; and protein: t = −1.284, df = 6, P = 0.228; **Figures 5B,C**).

# DISCUSSION

Understanding shifts in the body color of A. pisum can help reveal the adaptation mechanisms to environmental threats in these aphids. We believe that in the red-colored strain of A. pisum, the change in body color is a response to nutritional stress, and thus, represents the aphid's health status under such conditions. The reddish pigments are possibly used as backup energy reserves. The change in color was observed to occur in certain patterns and could be triggered under experimental conditions (starvation). Based on our results, we have concluded that the red A. pisum strain can remain red for about 10 h without food, after which it turns to a pale red color rapidly. Once an A. pisum initiates the color-shifting process, it cannot be stopped, regulated, or reversed even if the aphids are offered food. Between the two main pigment mixtures that were extracted in this study, the orange-red pigment mixture may be responsible for the color-shifting process and pigment degradation in A. pisum. All results from pigment analysis correlated with the colorshifting process. Carbohydrate and lipid contents correlated with color fading in the red A. pisum strain. The consumption patterns of energy reserves under starvation treatments between the two A. pisum strains were observed to be different; from this, we speculate that the reddish pigments may possibly be metabolized into carbohydrates for energy generation under nutrition stress, which may then lead to fading of color in the red A. pisum as well.

Based on the digital imagery analysis, we found out that the red A. pisum did not start color shifting during the first 10 h of starvation. Although it has been reported in previous studies that starvation stress can trigger color shift from red to pale red (Valmalette et al., 2012; Tabadkani et al., 2013), no detailed information has been provided. By using the digital imaging system, we were able to monitor the color change without interfering with them. The changes of aphids in color channel reflected the changes in the reflectance spectrum (Mancuso and Battiato, 2001; Vrhel et al., 2005). By analyzing the values for the RGB channels in the captured images, we found that the values for the R channel were always higher than those for the G and B channels. This might explain why the red A. pisum strain displays the red color in its body because there were no pigments that absorbed incoming red light in their body. Measurement values for the G and B channels showed that the aphids could reflect more green light than blue light (values for the G channel were higher than those for the B channel); the color of both bands of the reflected light (especially for G) was higher during the shift from red to pale red. We observed that the reflected red light during the shift from red to pale red did not show any change, while the green and blue lights increased, which made the reflected light whitish (Vrhel et al., 2005). Therefore, we assumed that some greenblue light absorbing pigments might have declined in their amount during the shift in body color in A. pisum, due to which they reflected more green-blue light and appeared to be pale red.

The red strain of A. pisum could remain red for about 10 h under no-diet conditions (**Figure 1** and **Supplementary Figure S2**). We believe that A. pisum could not initiate the colorshifting process until the stimulus for it was sufficiently strong. The aphids that were still red (color shifting was not initiated) after treatment stayed reddish after they were re-transferred into better diet conditions (**Supplementary Figures S2**, **S3**), but the individuals whose body color had changed (even slightly) would eventually turn to the pale red color even after being re-transferred (**Figure 2A** and **Supplementary Figure S3**). It has been concluded that once the shift from red to pale red color in A. pisum is initiated, it cannot be stopped, until the process finishes.

The shift in body color is actually caused by a dynamic change in pigment composition and their contents. Pigments extraction and analysis showed that the ethyl alcohol-soluble pigment mixture with orange-red color might be responsible for the shift in color. The content of these pigments declined during

the color-changing process. Earlier studies have reported that the content of some carotenoid pigments might be correlated to changes from red to pale red (Valmalette et al., 2012), but we could not identify each pigment in the pigment mixture so far. Based on earlier studies on the aphid pigments, these ethyl alcohol soluble pigments might be a type of aphins (Bowie et al., 1966; Horikawa et al., 2004, 2006). This pigments mixture showed a strong absorbance in the green band, a weak absorbance in the blue band, and almost no absorbance in the red band. Our absorbance analyses showed that the change in absorbance correlated with the fluctuation in the values for the G and B channels as well, indicating that the green-blue-absorption pigments degraded during the process of color shift. The TLC experiments showed similar results, where the red pigments in the pale red A. pisum strain were present in a relatively small amount. From this observation, we suggest that these reddish pigments are most likely degraded and metabolized for some other use. The details of such a process need to be further investigated.

Considering that nutrition stress is a key trigger for the fading of color, pigment degradation and conversion are probably related to nutritional physiology. Previous studies have shown

that energy reserves (soluble carbohydrate and lipid contents) are different in the red and pale red A. pisum strains (Tabadkani et al., 2013). After monitoring the changes in the energy reserves during color shift from red to pale red, we found that there is a connection between shift in body color and energy reserves. The results showed that changes in both lipid and carbohydrate contents have similar trends as those in color shifts. As soluble carbohydrates could be formed from glycogen and lipids, or even from some other precursors, the soluble carbohydrate content did not drop dramatically and was maintained at a certain level (**Figure 4** and **Supplementary Figure S5**). The energy reserves in red and pale red A. pisum strains were similar to those reported in literature (Tabadkani et al., 2013). The functions of these specific pigments in the red strain of A. pisum are unclear, so we conducted a comparative analysis of energy reserves with the green strain of A. pisum, which is genomically quite identical but has a different body color. Based on these comparative experiments, a significantly lower glycogen reserve was detected in the red strain than in the green strain; on the contrary, the carbohydrate level was slightly higher in the red strain. Based on the carbohydrate contents and absorbance patterns under starvation treatment, we assumed that there might be a way of carbohydrate refueling besides metabolization of glycogen in the red strain of A. pisum; these reddish pigments are the most likely candidates for being a precursor of such a process. The conversion of reddish pigments to carbohydrates may lead to the fading of the body color. It is also possible that the reddish pigments provide energy in some other pathway; more studies are needed to better understand this energy support system (**Figure 6**).

#### CONCLUSION

The shift in body color from red to pale red in the red A. pisum strain depicts an adaptation mechanism to environmental changes; this color shift also reveals that A. pisum strains

of different body colors exhibit diverse strategies of storing and using energy reserves. The connection between body color and A. pisum health status is a representation of the interactions between aphids and their environmental conditions from a new perspective. The shift in body color is actually a result of pigment degradation and even their consumption as energy reserves. Future studies should include the extraction and identification of key pigments, understanding of their molecular structures, related downstream metabolic pathways, especially the downstream metabolism, and conversion to reddish pigments.

#### AUTHOR CONTRIBUTIONS

X-XW, YZ, and T-XL designed research and wrote the manuscript. X-XW performed the research. J-YZ, Z-SC, and Z-JF provided assistance. YZ and X-XW analyzed the data.

#### REFERENCES


# FUNDING

Funding of this research was partially supported by the National Basic Research Program of China (Grant No. 2013CB127600) and Chinese Universities Scientific Fund (Grant No. Z109021718).

#### ACKNOWLEDGMENTS

We are grateful for the assistance of all staff and students in the Key Laboratory of Applied Entomology, Northwest A&F University at Yangling, Shaanxi, China.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2019.00197/full#supplementary-material



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wang, Chen, Feng, Zhu, Zhang and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Role of Modified Atmosphere in Pest Control and Mechanism of Its Effect on Insects

#### *Yu Cao, Kangkang Xu, Xiaoye Zhu, Yu Bai, Wenjia Yang and Can Li\**

*Guizhou Provincial Key Laboratory for Rare Animal and Economic Insect of the Mountainous Region, Department of Biology and Engineering of Environment, Guiyang University, Guiyang, China*

Pests not only attack field crops during the growing season, but also damage grains and

other food products stored in granaries. Modified or controlled atmospheres (MAs or CAs) with higher or lower concentrations of atmospheric gases, mainly oxygen (O2), carbon dioxide (CO2), ozone (O3), and nitric oxide (NO), provide a cost-effective method to kill target pests and protect stored products. In this review, the most recent discoveries in the field of MAs are discussed, with a focus on pest control as well as current MA technologies. Although MAs have been used for more than 30 years in pest control and play a role in storage pest management, the specific mechanisms by which insects are affected by and adapt to low O2 (hypoxia) and high carbon CO2 (hypercapnia) are not completely understood. Insect tolerance to hypoxia/anoxia and hypercapnia involves a decrease in aerobic metabolism, including decreased NADPH enzyme activity, and subsequently, decreases in glutathione production and catalase, superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase activities, as well as increases in carboxyl esterase and phosphatase activities. In addition, hypoxia induces energy and nutrient production, and in adapted insects, glycolysis and pyruvate carboxylase fluxes are downregulated, accompanied with O2 consumption and acetate production. Consequently, genes encoding various signal transduction pathway components, including epidermal growth factor, insulin, Notch, and Toll/Imd signaling, are downregulated. We review the changes in insect energy and nutrient sources, metabolic enzymes, and molecular pathways in response to modified O2, CO2, NO, and O3 concentrations, as well as the role of MAs in pest control. This knowledge will be useful for applying MAs in combination with temperature control for pest control in stored food products.

Keywords: modified atmosphere, physiological adaptation, pest control, hypoxia, molecular mechanisms

#### INTRODUCTION

Herbivorous insects not only attack field crops during the growing season, but also damage grains stored in granaries (Weaver and Petroff, 2005; Sadeghi et al., 2011). Losses of 5–10%, and up to 40% in developing countries, caused by insects in stored products have been reported worldwide (Shaaya et al., 1997; Weaver and Petroff, 2005). Fumigation is an optimal management practice to control all stages and kinds of pests in grain bins, warehouses, and

#### *Edited by:*

*Bin Tang, Hangzhou Normal University, China*

#### *Reviewed by:*

*Yujie Lu, Henan University of Technology, China Hongbo Jiang, Southwest University, China Mureed Husain, King Saud University, Saudi Arabia*

> *\*Correspondence: Can Li lican790108@163.com*

#### *Specialty section:*

*This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology*

*Received: 27 September 2018 Accepted: 18 February 2019 Published: 12 March 2019*

#### *Citation:*

*Cao Y, Xu K, Zhu X, Bai Y, Yang W and Li C (2019) Role of Modified Atmosphere in Pest Control and Mechanism of Its Effect on Insects. Front. Physiol. 10:206. doi: 10.3389/fphys.2019.00206*

**397**

other mass grain-storage structures. Therefore, fumigation with chemical insecticides presently is the most effective and widely used method to control storage pest infestations. However, the excessive use of chemical can lead to pesticide residues in treated grain or grain products that cause human health and environmental problems as well as potential resistance development in insects (Cheng et al., 2012). Modified or controlled atmospheres (MAs or CAs) depleted in oxygen (O2) and/or with elevated levels of carbon dioxide (CO2) or other gases provide an environmentally friendly and cost-effective approach to protecting grains and other stored food products (Cheng et al., 2012).

The concentrations of atmospheric carbon dioxide (CO2) are rising at an accelerated rate, which greatly affects the behavior and adaptation of herbivorous insects (Guo et al., 2014). In northern latitudes, natural insect enemies might benefit from the increasing temperature for their development, which in turn might facilitate integrated pest management (Castex et al., 2018). Our focus is not on the effects of global changes in CO2 concentrations on insect pests, as, importantly, insects can relatively easily adapt to CO2, O2, nitric oxide (NO), or ozone (O3) stress by changing their physiology and thus increase their survival rate under CA or other stresses. Therefore, we focus on the potential role of CAs in pest control.

Farmers and warehouse managers are interested in using hermetic storage for pest control in stored products (Njoroge et al., 2016, 2017). Several studies have evaluated the timing of insect die-off under CAs with reduced O2 or increased CO2 (Soderstrom et al., 1990; Ofuya and Reichmuth, 2002; Gunasekaran and Rajendran, 2005). For example, exposure for 17 days to a mixture of 40% CO2 and 2% O2 resulted in 100% mortality of grain weevils, *Calandra granaria* Linnaeus (Bailey, 1955). Under gradual reduction of O2 to 0% in 6–9 days in hermetic conditions, maize weevils (*Sitophilus zeamais* Linnaeus) produced a significantly lower number of offspring than weevils in non-hermetic conditions (Moreno-Martinez et al., 2000). *Cadra cautella* Walker and *Tribolium castaneum* Herbst showed significantly different susceptibilities to a high CO2 concentration of 99.9% at different developmental stages (Husain et al., 2017).

Insects have an effective respiratory system that allows direct air inflow from the atmosphere through muscular valves called spiracles. Insects accomplish respiratory gas exchange by controlling the opening and closing of these spiracles, and ventilate their tracheal system through muscular contractions (Matthews and White, 2011). As gaseous fumigants are mainly absorbed through the respiratory system, factors that influence respiration in insects also affect fumigant uptake (Lu et al., 2009). Changes in the concentrations of O2, CO2, and other gases can potentially affect the respiration rate and hence, the rate and biochemistry of metabolization and incorporation, and ultimately, the toxicity of a fumigant (Lu et al., 2009).

An MA with depleted O2 (hypoxia) and/or elevated CO2 (hypercapnia) is an environmentally friendly alternative to fumigants, which are currently widely used for stored-grain pest control (Cheng et al., 2012; Li et al., 2012; Mehmood et al., 2018). Although MAs have been used as a safe alternative to conventional fumigants for more than 30 years, the specific mechanisms by which insects are affected by and adapt to hypoxia and hypercapnia remain poorly understood (Boyer et al., 2012; Ingabire et al., 2013). Certain gas compositions, e.g., 100% CO2, 75% CO2, and 25% N2, and 22 ppm O3, can be used together with temperature control to effectively control pests in stored grains (Husain et al., 2015). MA treatments using CO2, O2, N2, and/or O3 together with other measures, e.g., controlled temperature or humidity, provide important means to reduce insect survival or postharvest disinfestation (Boardman et al., 2011). MA treatments usually involve either low O2 (0–11.5 kPa) or high CO2 (18–90 kPa) and are applied with augmented-temperature sterilization to combat pests in stored products.

#### MODIFIED ATMOSPHERE GASES COMMONLY USED FOR PEST CONTROL AND THEIR TOXICITIES

Ambient atmosphere consists of approximately 79% N2, 20–21% O2, and 0.04% CO2. MAs with hypoxia and/or hypercapnia in airtight storage, with O2 maintained at a level sufficient for insect development, have been used for preventing insect damage in stored grains (Banks and Annis, 1990; Fleurat-Lessard, 1990; Riudavets et al., 2009; Sanon et al., 2011; Navarro et al., 2012; Rasool et al., 2017).

MAs generally involve O2, CO2, NO, and O3. Insect tolerance to hypoxia and hypercapnia critically affects insect control (Cui et al., 2017). O2 is critical for the survival of aerobic life. However, oxidative injury can be induced by a too low or too high (hyperoxia) O2 level in organisms, which will induce morbidity and mortality (Zhao and Haddad, 2011). For example, egg laying in insects decreases with increasing CO2 concentration (Azzam et al., 2010). CO2 toxicity increases in a concentrationdependent manner, as reported for *Stegobium paniceum* Linnaeus and *Oryzaephilus surinamensis* Linnaeus (Cao et al., 2015a,b). Adult insects and larvae show different susceptibilities to CO2 stress. For example, at 90% CO2, the LT50 and LT99 of adult insects reportedly are 6.89 and 15.83 h, and those of larvae 18.76 and 60.58 h, respectively (Cao et al., 2015a). A 12-hour exposure to 80% CO2 and 20% N2 at 32.2°C resulted in 100% mortality of pupae of *Plodia interpunctella* Hübener (Sauer and Shelton, 2002). Larval mortality in *Ephestia cautella* Walker (Husain et al., 2015) and mosquito (Garcia et al., 2014) was higher after 48-h than after 24-h exposure to 100% CO2 or 75% CO2 at 25°C. An MA with 8% O2, 60% CO2, and 32% N2 at 30°C killed 100% of 4th instar larvae of *E. cautella* within 72 h, and resulted in 95% mortality in *Amyelois transitella* Walker after 60-h exposure at 27°C (Brandle et al., 1983). Under the same MA, the mortality of *E. cautella* significantly increased when the temperature was increased from 25 to 35°C (Husain et al., 2015). These results indicate that an MA combined with higher temperature is an effective method for pest control in stored products in future.

NO is a potent fumigant that shows excellent control effect on all insects, regardless of their life stage (Liu, 2013, 2015, 2016;

Liu and Yang, 2016; Yang and Liu, 2018). However, the application of NO MAs should follow a logical order (Li et al., 2009; Riudavets et al., 2009; Navarro, 2012). For example, when NO is used with nitrogen (N2) in an airtight fumigation chamber to protect fresh fruit and vegetables against pests infection, N2 should be flushed into the chamber first, to create an ultralow oxygen (ULO) environment, followed by injection of NO (Liu et al., 2016, 2017). Because nitrogen dioxide (NO2) will be produced when NO reacts with O2, NO fumigation must be applied under ULO conditions and under low temperature (Liu, 2013).

As a natural atmosphere component, O3 can rapidly decompose to molecular oxygen, without leaving residues (Lu et al., 2009). Gaseous O3 is used in food processing (Palou et al., 2002; Forney et al., 2007; Wei et al., 2007), and as a fumigant against stored-product pests (Kells et al., 2001; Sousa et al., 2008; Lu et al., 2009; Hansen et al., 2012; Pandiselvam et al., 2017). O3 treatment caused 100% larval mortality of *E. cautella* after 24-h exposure at two temperature regimes (Husain et al., 2015). O3 at 2.0 ppm induced 83 and 27% mortality of *E. cautella* adults and larvae, respectively, after 12-h exposure (Abo-El-Saad et al., 2011). Three-day exposure to 5–45 ppm O3 led to 92–100% mortality of larvae of *Tribolium castaneum* Herbst, *S. zeamais* adults, and *P. interpunctella* in stored maize (Kells et al., 2001) and other stored products (Abo-El-Saad et al., 2011; Husain et al., 2015). *Tribolium confusum* du Val and *Ephestia kuehniella* Zeller showed different susceptibilities to O3 reflush treatment at 30-min intervals for 5 h at different developmental stages, and *T. confusum* was more tolerant than *E. kuehniella* at all developmental stages (Isikber and Oztekin, 2009). Together, these findings indicate that various MA combinations are available to create hypoxia and/or hypercapnia, and different MA combinations can be used for different pests in stored products.

#### CHANGES IN ENERGY/NUTRIENT SOURCES UNDER MODIFIED ATMOSPHERE

High-CO2 stress suppresses the production of NADPH and subsequently, glutathione, which are involved in the protection against the toxic effects of reactive oxygen species (Boardman et al., 2011). Further, NADPH contributes to nucleotide synthesis, cholesterol synthesis, and fatty-acid synthesis (Feron, 2009). Trehalose is the primary carbohydrate in insects, and plays an important role in insect development and all physiological activities by serving as an instant energy source as well as by mitigating abiotic stressors (Shukla et al., 2015). Trehalose protects cells against various environmental stresses, such as heat, cold, desiccation, dehydration, and oxidation. Chen and Haddad (2004) reported that trehalose can protect *Drosophila* and mammalian cells from hypoxic and anoxic injury. The mechanism underlying this protective action might be related to the decrease in protein denaturation through protein-trehalose interactions (Chen et al., 2003). In the presence of trehalose, cells can be maintained in the dry state for up to 5 days. Moreover, trehalose reportedly protects cultured human corneal epithelial cells from death by desiccation (Chen et al., 2003). Trehalose-6-phosphate synthase (TPS), which produces trehalose, is vital to insect growth and development (Chen et al., 2018). Overexpression of TPS increased trehalose levels and tolerance to anoxia (Chen et al., 2003). Trehalose plays an important role in protecting flies against anoxia injury, and induction of TPS increased tolerance to anoxia by reducing anoxia-induced protein aggregation (Tang et al., 2018).

Several studies have demonstrated that stored-product insect pests have the genetic potential to develop resistance to MA. In *Liposcelis bostrychophila* Badonnel, this resistance is related to enhanced levels of triacylglycerol and polysaccharides (Wang et al., 2000; Wang and Zhao, 2003). However, contents of energy substances, including polysaccharides, soluble proteins, and lipids, decreased in a dose- and time-dependent manner in response to CO2 in larvae of *S. paniceum* and *L. serricorne* (Cao et al., 2016a) and adult *S. paniceum* (Cao et al., 2016b) and *O. surinamensis* (Cao et al., 2015b). In bean weevil (*Callosobruchus chinensis* Linnaeus), Cui et al. (2017) reported that the levels of carbohydrates, amino acids, and organic acids increased, whereas those of free fatty acids decreased in response to hypoxia. When hypercapnia was added, these changes were further enhanced, except for the decrease in free fatty acids (Cui et al., 2017).

Hypoxia-adapted flies tend to have decreased glycolysis and pyruvate carboxylase fluxes relative to the amount of O2 consumed, and tend to produce acetate rather than oxaloacetate (Feala et al., 2009). In addition, in hypoxia-adapted flies, fewer protons are generated and more ATP per glucose is produced, pyruvate carboxylase flux is lower, and complex I rather than complex II was used in the electron transport chain. Based on simulations, it has been suggested that ATP-per-O2 efficiency is greater in hypoxia-adapted metabolism in insects (Harrison and Haddad, 2011). During metabolic processes, the production of cytochrome oxidase and mitochondrial ATP is significantly affected by O2 (Hochachka et al., 1996). Under very low atmospheric O2 partial pressure or temperature, ATP production is directly limited, which results in reduced rates of feeding, digestion, absorption, and protein synthesis (Harrison and Haddad, 2011). Under hypoxia, besides the direct effects on ATP levels, the AMP-to-ATP ratio increases because AMP kinases are activated upon AMP accumulation. Accordingly, multiple cellular effects related to the control of energy metabolism and growth have been observed (Tao et al., 2010). In *D. melanogaster* Meigen flies adapted to severe hypoxia, Feala et al. (2009) suggested a network-level hypothesis of metabolic regulation, in which lower baseline rates of biosynthesis resulted in lower anaplerotic flux and consequently, lower rates of glycolysis, less acidosis, and more efficient substrate use.

#### CHANGES IN METABOLIC ENZYMES IN RESPONSE TO MODIFIED ATMOSPHERE GASES

In insects, CO2 is thought to inhibit respiratory enzymes at concentrations higher than 20%; however, the effect varies strongly among species (Zhou et al., 2001). In *S. paniceum* and *Lasioderma serricorne* Fabricius, carboxyl esterase activity increased compared to that in the normal condition after exposure to a CO2-enriched atmosphere (Li et al., 2007, 2009). Acid phosphatase activity also increased under CO2 stress with the extension of exposure time, whereas alkaline phosphatase was hardly affected (Li et al., 2008). In *Araecerus fasciculatus* Degeer, the activities of carboxyl esterase and acid phosphatase increased significantly under CO2-enriched MA (75% CO2, 5% O2 and 20% N2) for 3 h (Li et al., 2012), and glutathione-S-transferase (GST) activity also increased significantly in *S. paniceum*, *L. serricorne*, and *A. fasciculatus* under the same condition (Li and Li, 2009). In *L. serricorne*, *LsGSTd1* (encoding GST) did not change significantly following exposure to CO2 stress, whereas the expression levels of *LsGSTt1* and *LsGSTs1* were significantly increased (Xu et al., 2017).

The expression of antioxidant enzymes, including catalase, superoxide dismutase (SOD), GST, and glutathione peroxidase (GPx), was reportedly increased in *Achaea janata* Linnaeus subjected to different oxidative stress stimuli, which also slowed down its development and resulted in weight reduction (Pavani et al., 2015). In pupae of *Anastrepha suspensa* Loew, the total antioxidant capacity was increased by more than twofold after 1 h of anoxic exposure (López-Martínez and Hahn, 2012). The increase was maintained for 24 h and was associated with increases in mitochondrial SOD (MnSOD) and GPx, but not catalase. Further, after 2-h anoxic exposure, cytoplasmic SOD (Cu-ZnSOD) activity was significantly increased when compared to normoxia (López-Martínez and Hahn, 2012).

#### MOLECULAR MECHANISMS UNDERLYING ADAPTION OF INSECTS TO MODIFIED ATMOSPHERES

Hypoxia is generally defined as <21% O2 and hyperoxia as >21% O2. In addition to gases such as CO2, O3, and NO, CA can be used as a pest control practice. Insights into the molecular mechanisms underlying the responses in insects to hypoxic/ hypercapnic conditions are required to efficiently use MAs for pest control. Local hypoxia causes a rise in NO production in certain tissues of *Drosophila* larvae, and overexpression of NO synthase causes a greater hypoxia response, whereas knockout of protein kinase G or inhibition of NO synthase reduces such responses (Harrison and Haddad, 2011). Through microarray and bioinformatics analyses, Zhou et al. (2009) identified genes (*e.g.*, Notch pathway genes) that play important roles in the development of hypoxia resistance. Genes related to metabolism (*e.g*., carbon metabolism) were largely downregulated, whereas upregulated genes mainly encoded multiple components of epidermal growth factor (EGF), insulin, Notch, Toll, and immune deficiency (IMD) signal transduction (Zhou and Haddad, 2013). In addition, genes involved in protein digestion and tricarboxylic acid cycle as well as genes encoding stress-responsive heat shock proteins were increased in insects challenged by O2 deprivation (Cheng et al., 2012). Identification of the molecules that mediate the adaptation to hypoxia might lead to new therapeutic targets to protect or reverse hypoxia-induced pathologies (Zhou et al., 2009).

Under hypoxia, cells and tissues are challenged by O2 deprivation to the extent that energy production is inefficient. Trehalose reportedly protects *Drosophila* and mammalian cells from hypoxic and anoxic injury (Chen and Haddad, 2004). In mammalian cells transfected with the *Drosophila tps1* gene, the exogenous trehalose could protect the cells from hypoxic injury (Chen et al., 2003). Hypoxia-inducible factor, which is a key molecule produced in response to O2 deprivation, is mainly regulated by prolyl hydroxylase domain-containing enzymes (Hochachaka and Rupert, 2003; Wang et al., 2015). Organisms show different responses to constant hypoxia (CH) and intermittent hypoxia (IH), and the effect of hypoxia depends on the severity and duration of hypoxia (Farahani et al., 2008). In *D. melanogaster*, hypoxia resistance has been well studied. Severe short-term CH (2.5 h, 1% O2) and IH (cycles of 1–21% O2) triggered the expression of genes involved in immunity and unfolded protein, carboxylic acid, amino acid, and lipid metabolism (Azad et al., 2009; Zhou and Haddad, 2013). More importantly, gene families activated in response to CH include those involved in the metabolism of chitin, lipid, and carboxylic acid; the immune response; and the response to protein unfolding (Harrison and Haddad, 2011). Gene expression under CH and IH varies in both the number of responsive genes and the gene families affected. In a study by Zhou and Haddad (2013), gene families overrepresented in CH-treated flies included those involved in the response to unfolded proteins, lipids, carboxylic acid, amino acid metabolic processes, and immunity, whereas gene families overrepresented in IH were related to drug resistance. During CH exposure, strong upregulation of the chaperones heat shock protein *HSP70* and *HSP23* was observed in *D. melanogaster* (Harrison and Haddad, 2011). Overexpression of *HSP70*, which regulates CH tolerance, had no effect on IH tolerance, and overexpression of *Mdr49* enhanced adult survival under IH, but not CH (Zhou and Haddad, 2013). In *Sarcophaga crassipalpis* Macquart, *HSP* genes play a key role in the response to severe hypoxia (3% O2), with different HSPs having different functions (Michaud et al., 2011). Cryoprotective low-molecular-weight sugars and polyols can stabilize biological membranes and protect them from ice damage (Kostál et al., 2007; Overgaard et al., 2007), as do HPSs (*e.g*., *HSP70*) (Yi and Lee, 2003; Kostál and Tollarová-Borovanská, 2009).

Genes related to RNA editing are also involved in anoxia tolerance. For example, pre-mRNA adenosine deaminase plays an important role in IH tolerance through altering protein structure and function (Harrison and Haddad, 2011). Recent evidence suggests that atypical guanyl cyclases, which are heme-containing heterodimeric enzymes that are activated by hypoxia, but not NO (Morton, 2004) may mediate at least some of the rapid neuronal responses to O2 as conventional guanyl cyclases (Vermehren et al., 2006). NO-sensitive guanyl cyclases may also play a role in hypoxic responses (Wingrove and O'Farrell, 1999). Soluble guanylyl cyclases (sGCs) play a role in the synthesis of the intracellular messenger cyclic guanosine monophosphate (cGMP), and conventional sGCs are the main receptor for and mediate the majority of physiological actions of NO (Garthwaite, 2010). Atypical sGC subunits bind O2 to their heme group in a manner Cao et al. Insect Adaption Under Modified Atmosphere

analogous to NO binding to the conventional sGCs under normal atmospheric conditions. Atypical sGCs have a relatively low affinity for O2, a property that is necessary for a molecular O2 detector that can sensitively detect a reduction in O2 concentration from the atmospheric level (Vermehren et al., 2006). The physiological effects of cGMP are typically mediated by activation of a cGMPdependent protein kinase, a cyclic nucleotide-gated ion channel, or a cGMP-regulated phosphodiesterase (Lucas et al., 2000).

#### POTENTIAL ROLE OF MODIFIED ATMOSPHERES IN STORED-PRODUCT AND FRUIT PEST CONTROL

A study by Cui et al. (2017) showed that insect tolerance to hypoxia or hypoxia/hypercapnia is on the rise. The authors provided direct evidence of insect adaption to hypoxia, and reported free fatty acid regulation by hypercapnia in storedproduct pests (Cui et al., 2017). Combined hypoxia exposure and low temperature or high CO2/NO has been used to sterilize commodities in postharvest pest management programs, and the current knowledge on the mechanisms involved in insect cross-tolerance can be used to develop more targeted control measures (Follett et al., 2018). However, one important problem is that many insects develop stronger resistance or cold crosstolerance through physiological adaptions (Nilson et al., 2006; Cui et al., 2014). Therefore, more in-depth research is needed for the development and application of control measures in future.

At low temperature, MAs can increase pest mortality induced by low-temperature sterilization, and sometimes, the treatment duration can be shortened. Therefore, combined low temperature and hypoxia exposure have been used to sterilize commodities for pest control (Boardman et al., 2015; Saha et al., 2015). The control efficacy for storage pests can be enhanced by reducing O2 levels and increasing treatment time or temperature (Liu and Haynes, 2016). For example, Neven et al. (2014) indicated that heat treatment in combination with high CO2 and low O2 may be effective for the control of diapausing codling moth, *Cydia pomonella* Linnaeus, in walnut; especially, temperatures higher than 44°C rapidly killed the moths (Neven et al., 2014). Thus, high-temperature forced-air treatment combined with an O2-depleted and CO2-enriched atmosphere is an environmentally friendly postharvest mitigation approach to control quarantine

#### REFERENCES


pests (Johnson and Neven, 2010). In future, high or low temperature combined with low O2 and high CO2 or NO might have the potential to control or kill not only storage pests, but also fruit pests. Specialized machinery or technology for MA/temperature treatment can be developed for postharvest pest management (Villers et al., 2008; Mditshwa et al., 2018).

#### CONCLUSION

MAs provide a highly effective non-chemical control measure for stored-product pests. The control effect of MAs can be reasonably improved through combination with temperature stress, or by using suitable facilities and techniques or other measures. Although some combination approaches (*e.g.*, combination with natural enemies) and related underlying mechanisms (*e.g.*, cross-tolerance of pests) remain to be resolved, MA control systems should be further developed, improved, and applied in stored-product protection for their unique advantages.

#### AUTHOR CONTRIBUTIONS

YC, WY, and CL conceived and designed manuscript structure. YC, KX, XZ, YB, and CL wrote the paper.

#### FUNDING

We thank the National Natural Science Foundation of China (31460476), the Regional First-class Discipline Construction of Guizhou Province (No. [2017]85), Discipline and Master's Site Construction Project of Guiyang University financed by Guiyang City (SH-2019), Training Project for High-Level Innovative Talents in Guizhou Province (No. 2016 [4020]), The Program for Academician workstation in Guiyang University (20195605), and Special Funding of Guiyang Science and Technology Bureau and Guiyang University [GYU-KYZ(2019)02-06] for financial support.

#### ACKNOWLEDGMENTS

We greatly appreciate the useful suggestions to an earlier draft of this manuscript from Prof. Zhongshi Zhou (Chinese Academy of Agricultural Sciences).


review of biochemical mechanisms. *Front. Physiol.* 2:92. doi: 10.3389/ fphys.2011.00092


in *Drosophila suzukii* (Diptera: *Drosophilidae*) in sweet cherries. *J. Econ. Entomol.* 111, 141–145. doi: 10.1093/jee/tox337


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Cao, Xu, Zhu, Bai, Yang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Plant Defense Responses Induced by Two Herbivores and Consequences for Whitefly Bemisia tabaci

Dan Lin<sup>1</sup> , Yonghua Xu<sup>2</sup> , Huiming Wu<sup>1</sup> , Xunyue Liu<sup>1</sup> , Li Zhang<sup>1</sup> , Jirui Wang<sup>1</sup> and Qiong Rao<sup>1</sup> \*

<sup>1</sup> School of Agriculture and Food Science, Zhejiang A&F University, Hangzhou, China, <sup>2</sup> Zhejiang Branch of National Pesticide R&D South Center, Zhejiang Chemical Industry Research Institute, Hangzhou, China

Diverse herbivores are known to induce various plant defenses. The plant defenses may detrimentally affect the performance and preference to subsequent herbivores on the same plant, such as affecting another insect's feeding, settling, growth or oviposition. Here, we report two herbivores (mealybug Phenacoccus solenopsis and carmine spider mite Tetranychus cinnabarinus) which were used to pre-infest the cucumber to explore the impact on the plants and the later-colonizing species, whitefly Bemisia tabaci. The results showed that the whiteflies tended to select the treatments pre-infested by the mites, rather than the uninfected treatments. However, the result of treatments preinfested by the mealybugs was opposite. Total number of eggs laid of whiteflies was related to their feeding preference. The results also showed that T. cinnabarinus were more likely to activate plant jasmonic acid (JA) regulated genes, while mealybugs were more likely to activate key genes regulated by salicylic acid (SA). The different plant defense activities on cucumbers may be one of the essential factors that affects the preference of B. tabaci. Moreover, the digestive enzymes and protective enzymes of the whitefly might play a substantial regulatory role in its settling and oviposition ability.

Keywords: Bemisia tabaci, Phenacoccus solenopsis, Tetranychus cinnabarinus, plant defense, jasmonic acid pathway, defensive enzyme, salicylic acid pathway

# INTRODUCTION

In nature, plants possess a considerable diversity of resistance strategies and produce complex chemical reactions after experiencing mechanical damage or attacks by herbivores (Green and Ryan, 1972). The signal transduction pathways related to plant defense includes the ethylene (ET) pathway, jasmonic acid (JA) pathway, and salicylic acid (SA) pathway. The JA and ET pathway are induced against necrotrophic pathogens, chewing herbivores and cell-content feeders (Thaler et al., 2012; Godinho et al., 2016). However, the SA signaling pathway is primarily induced by bio-trophic pathogens and piercing- sucking herbivores, resulting in minimal tissue damage (Arena et al., 2016). Simultaneously, insects adapt to plant defense strategies by evolving their feeding patterns and feeding behavior (Hogenhout and Bos, 2011). In some cases, the herbivores may induce the accumulation of SA by attacking host plants and utilize this accumulation to inhibit JA-mediated defenses. That is, interference between the plant defense pathways may occur (Zhang et al., 2011;

#### Edited by:

Su Wang, Beijing Academy of Agricultural and Forestry Sciences, China

#### Reviewed by:

Jalal Jalali Sendi, University of Guilan, Iran Jing Zhao, Weifang University of Science and Technology, China

> \*Correspondence: Qiong Rao qiong.rao@zafu.edu.cn

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 19 July 2018 Accepted: 14 March 2019 Published: 04 April 2019

#### Citation:

Lin D, Xu Y, Wu H, Liu X, Zhang L, Wang J and Rao Q (2019) Plant Defense Responses Induced by Two Herbivores and Consequences for Whitefly Bemisia tabaci. Front. Physiol. 10:346. doi: 10.3389/fphys.2019.00346

**405**

Zhang X. et al., 2015). The interaction between separate signaling pathways in host plants carries out an irreplaceable role in regulating plant defense and preventing further invasions of pests. Between them, the JA pathway and SA pathway consistently showed mutual inhibition (Pieterse et al., 2012; Thaler et al., 2012; Glas et al., 2014). For example, several genes (like LOX and OPR3) regulated by the JA pathway can be restrained by the SA signaling pathway in Arabidopsis, suggesting that SA intensely antagonizes the JA signaling pathway (Guleria et al., 2005). Furthermore, ET is a critical modulator of SA-JA cross-talk, which equally participates in the regulation of a key gene (NPR1) in the SA pathway. Thereby, it regulates the inhibition of the JA pathway. As follows, the presence of the ET pathway is equally involved in the complex interaction between the SA pathway and the JA pathway. This intervention is mediated through the regulation of the NPR1 protein (Guleria et al., 2005; Thaler et al., 2012). Collectively, the cross-talk between JA, ET and SA signaling is ultimately to balance plant-defense strategies in response to multiple attackers (Guleria et al., 2005).

The complicated induced plant defenses may affect the performance and preference of subsequent herbivores, by affecting the feeding, settling, growth, or oviposition ability of another insect (Karban and Carey, 1984; Leitner et al., 2005). For instance, the plant may become more sensitive to another herbivore after previous attacks by other herbivores. Meanwhile, the phloem-feeding insects were susceptible to the change of nutrient substance and resistant material in the phloem sap (Denno and Roderick, 1992). A substantial number of secondary metabolites in plants induced by pests are extremely detrimental to the growth performance of insects, like terpenoids, phenolic compounds, and alkaloids (Chen et al., 2015). The resistance of insects to the secondary metabolites is due to various biochemical and physiological characteristics of the midgut. The midgut of insects can secrete various digestive enzymes (e.g., proteases, lipases, amylases, sucrases, trehalases) and defense enzymes (superoxide dismutase, catalase, and peroxidase), which occupy a crucial role for insects to combat plant defenses.

The whitefly Bemisia tabaci (Gennadius) is a phloemfeeding pest that causes extensive damage (De Barro et al., 2011; Liu et al., 2012). Due to the wide host range, intense competition exists between the invasive species B. tabaci and other local pests (De Barro et al., 2011; Rao et al., 2011). The carmine spider mite Tetranychus cinnabarinus (Boisduval) is a polyphagous pest that attacks crops, vegetables, and flowers. In particular, the economic yield of cucumbers decreases significantly after heavy spider mite infestations (Sertkaya et al., 2010; Park and Lee, 2016). The spider mite remains a cell-content feeder, which can puncture the host plant leaf epidermis cells and consume the contents of the mesophyll cells. Typically, the mite induces defenses of both the SA and JA pathways (Ament et al., 2004). Moreover, T. evansi down-regulates tomato defensive compounds, and this is correlated with a significantly more excellent performance of herbivores on plants pre-infested by mites of this species (Kant et al., 2008; Alba et al., 2015; Godinho et al., 2016). The mealybug Phenacoccus solenopsis (Tinsley) represent an aggressively invasive species on agricultural and ornamental plants in China. It is an obligate phloem-feeding pest that possesses a specialized stylet and causes minimal damage as it obtains nutrition from plants through the vascular tissue (Will et al., 2013; Huang and Zhang, 2016). All three pests have broad host distributions, and can damage the same crops in the field. Previous studies have shown diverse pests that eat plants often activate different signaling pathways (Zhang et al., 2011). Here we pre-infested cucumbers with T. cinnabarinus and P. solenopsis, insects with different foraging patterns, to explore the impact on B. tabaci.

# MATERIALS AND METHODS

#### Plant and Insects Plant

Cucumber, Cucumis sativus L. (Jinongjiejiegua, Tianjin, China), was the host plant for this experiment. The plants used in the tests were approximately 30 cm in height and had 4–6 true leaves.

#### Insects

The spider mites, T. cinnabarinus, were maintained on broad bean plants (Vicia faba). The mealybugs, P. solenopsis, were cultured on potato plants (Solanum tuberosum). The whiteflies, B. tabaci (MED), were cultured on cucumber plants (Cucumis sativus).

The experiments were conducted at 25 ± 2 ◦C, 65 ± 10% RH and a photoperiod of 16 h:8 h (L:D) with artificial lighting in a greenhouse.

# The Settling and Oviposition of Whitefly

The leaf disks were pre-infested by five 3rd-instar female mealybugs or five red mite females for 12 h. Then, five couples of new emerged whiteflies were placed at the middle of the plastic pipe and allowed to fly to either side. The positions of the two leaf disks were alternated among replicates. Each experiment was replicated 15–30 times. In the experiment, the location of B. tabaci was counted every 12 h. After 72 h post inoculation (hpi), the number of laid eggs deposited on each leaf disk was recorded. In total, there were 15–30 replicates in each treatment. The ratios of the whitefly number of landing on the plant to the total number of releasing in the test were subjected to a t-test (SPSS version 18.0).

# Enzymatic Assay of Defensive Enzymes in Host Plants

The activity of defensive enzymes in plants was tested as follows: (1) T(+), each plant pre-infested by 25 T. cinnabarinus females for 12 h and continue feed for 3 days; (2) P(+), each plant preinfested by 25 P. solenopsis for 12 h and continue feed for 3 days; (3) T(+)+B, after pre-infested for 12 h, the T. cinnabarinus were kept and B. tabaci was infested for 3 days; (4) P(+)+B, after preinfested for 12 h, the P. solenopsis were kept and B. tabaci was infested for 3 days; (5) CK, the plants were uninfected but were kept for the same time and were used as controls.


TABLE 1 | The specific primers for qRT-PCR.

fphys-10-00346 April 2, 2019 Time: 17:28 # 3

Enzymatic assay of PAL was determined according to the procedure of Burrell and Rees (1974). The enzymatic assay of LOX is slightly modified according to the method described by Surrey (1964). This experiment contains 5–8 biological experiments with three technical repetitions.

#### Quantitative Real-Time PCR

We quantified the transcript levels of several genes in the leaves treated. Leaves of the treated and untreated plants were collected, and total RNA was extracted consuming the RNAiso Plus reagent (TaKaRa, Dalian, China). The cDNA was synthesized using the PrimeScript <sup>R</sup> RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). For the relative quantification of gene expression, the comparative CT method (Livak and Schmittgen, 2012) was used with BIO-RAD CFX96 real-time PCR system (Bio-Rad, United States). The amount of the target was normalized to the endogenous reference gene TUA. The specific primers of all aimed genes for qRT-PCR were showed in **Table 1**. For qRT-PCR, three biological replicates were carried out, and triplicate quantitative assays for each replicate were performed.

#### The Digestive Enzymes, Detoxification Enzymes, and Protective Enzymes in Whiteflies

Here, the detoxification, digestive and protective enzymes in whiteflies were also tested to reveal the effect of induced plant response. The different treatments were as follows: (1) T(+)+B (described above); (2) P(+)+B (described above); (3) Whitefly (the plants that were uninfected but were kept for the same time, and then 25 couples of whiteflies were allowed to feed on the plants as a control). The whiteflies were collected at the end of the experiment and each treatment was repeated 8–10 times.

For the determination of trehalose and sucrase activity, the dinitrosalicylic acid method was adopted (Zhang et al., 2014). For the determination of superoxide dismutase (SOD) activity, the pyrogallol auto-oxidation method was used (Xu et al., 2006). The molybdate colorimetric method was used for determination of catalase (CAT) activity (Cheng and Meng, 1994). The glutathione S-transferase (GST) activity was measured according to Habig and Jakoby (1981). The carboxylesterase (CarE) activities were performed with slight modifications according to Stumpf and Nauen (2002).

#### RESULTS

#### Effects of Mealybug or Spider Mite Infestation on B. tabaci Response

The percentages of B. tabaci that settled on the treatments with T. cinnabarinus were significantly more than those on the control treatments without T. cinnabarinus (12 h, P = 0.008; 24 h, P = 0.002; 36 h, P = 0.003; 48 h, P = 0.009; 60 h, P = 0.007; 72 h, P = 0.007) (**Figure 1**). In contrast, less whiteflies settled on the plants infested with P. solenopsis (12 h, P = 0; 24 h, P = 0.001; 36 h, P = 0; 48 h, P = 0.001; 60 h, P = 0.005; 72 h, P = 0.014) (**Figure 1**). In all, B. tabaci were more likely to choose the leaf disk with T. cinnabarinus treatment than with P. solenopsis treatment. After 3 days, the total laid eggs were significantly higher in the plants with T. cinnabarinus than those on the leaf disk with no treatment (**Figure 2**). In contrast, the total eggs were significantly lower in the plants with P. solenopsis than control (**Figure 2**).

#### The Digestive Enzymes, Detoxification Enzymes, and Protective Enzymes in Whiteflies

According to **Table 2**, we found the trehalose, SOD and CAT activities in B. tabaci from cucumbers infected by mites were higher than those of whiteflies from the uninfected cucumber (P < 0.05). However, the sucrase and GST activities were significantly lower than those of whiteflies reared in cucumbers infected by mites (P < 0.05). The CarE activity of B. tabaci from cucumber fed on by mealybugs was higher than that of whiteflies from cucumbers infected by mites and the control. Similarly, the sucrase activity in whiteflies was significantly lower from cucumber infected by mealybugs than that of whiteflies in the control (P < 0.05).

#### Estimation of Phenylalanine Ammonialyase and Lipoxygenase Activity in Plants

The activity of PAL but not LOX was significantly increased through the induced response in cucumbers fed on by spider mites after 3 days. However, the activities of LOX and PAL in cucumbers were significantly increased by feeding mealybugs. The activities of LOX and PAL in cucumbers were both significantly increased after the whiteflies coexisted with mites for 3 days. Nevertheless, the activities of LOX and PAL in cucumbers were not significantly increased after the whiteflies coexisted with mites for 3 days (**Figure 3**).

#### Changes in Gene Expression

To assess cucumber response to five different combinations of the insect feeding treatments, time course experiments were conducted in which transcript levels of one ETdependent gene (ETR), two JA-dependent genes (LOX2 and

OPR3) and SA-dependent genes (PAL, PR-1, and PR-5) were monitored (**Figure 4**).

Ethylene receptor gene (ETR) is a marker gene of ET signal pathways (Shoresh et al., 2005). The relative expression levels of ETR did not significantly change in the cucumber leaves infested with T. cinnabarinus only or P. solenopsis only after 3 days. However, the transcript levels of ETR in the cucumber leaves induced by T. cinnabarinus were repressed in leaves infested with both T. cinnabarinus and B. tabaci compared to undamaged leaves (P < 0.05) (**Figure 4**).

Lipoxygenase (LOX) is typically a key enzyme in the octadecanoid pathway of the JA biosynthesis (Hu et al., 2009). All treatments apparently upregulated the transcript levels of LOX compared to undamaged leaves. However, the level of LOX induction in leaves infested with T. cinnabarinus was significantly lower than other treatments (**Figure 4**). The oxophytodienoate reductase (OPR3) gene is one marker of the JA upstream genes (Ramírez et al., 2012). The level of the OPR3 gene infested with either P. solenopsis or B. tabaci was significantly higher than other treatments (P < 0.05) (**Figure 4**).

PR-1 and PR-5 are known as two pathogenesis-related genes, which are mainly regulated by the SA signaling pathway (Zhang P.J. et al., 2015). Phenylalanine ammonia lyase (PAL), a ratelimiting enzyme of the phenylpropanoid pathway, is the key enzyme in the biosynthetic pathway of SA and plays an important role in plant development and defense (Hu et al., 2009; Wang et al., 2013; Zhang et al., 2016). The abundance of PAL, PR1, and PR5 transcripts was significantly increased in the cucumber leaves infested with P. solenopsis only after 3 days, but not in the leaves infested with T. cinnabarinus only (P < 0.05; **Figure 4**). In addition, the leaves infested with P. solenopsis and B. tabaci showed a marginally significant increase of the expression of PAL and PR5 in comparison with undamaged leaves. However, the leaves infested with P. solenopsis and B. tabaci witnessed a significant reduction of the expression of PR1 compared to undamaged leaves (**Figure 4**).

# DISCUSSION

From the perspective of temporal and spatial interactions, intraspecific insect interactions affect the population performance, especially because herbivores may affect the

TABLE 2 | The digestive enzymes, detoxification enzymes and protective enzymes in whiteflies.


Paired means ( ± SE) with different letters above bars indicate significant differences in the quantities between control and treatment (ANOVA, P < 0.05).

FIGURE 3 | The activity of two defense enzymes in each treatment on cucumber. Different letters above bars indicate significant differences in the quantities between control and treatment (ANOVA, P < 0.05).

performance of subsequent herbivores on the same plant (Green and Ryan, 1972; Hunter and Price, 1992; Messina et al., 2002; Beale et al., 2006; Xue et al., 2010; Tan and Liu, 2014). Our result showed whiteflies were extremely sensitive to pre-infestation with mealybugs, though there were few previous reports of mealybugs. In contrast, the plants pre-infested with T. cinnabarinus attracted more whiteflies than the plants with no mites. This may depend on the combination of the activation of defensive proteins and volatile organic compounds (Smith and Boyko, 2007; Howe and Jander, 2008), as well as the species of pests and feeding sequences of herbivore insects (Inbar and Gerling, 2008; Erb et al., 2011). Plants known to be impacted by herbivores are capable of producing a series of volatile substances that trigger an indirect plant defense. How plants respond to infestation by multiple herbivores, particularly if these belong to different feeding guilds, is important. The pattern of the plant defense is crucially important for regulating the behavior of herbivorous pests (Walling, 2000). Transcriptomic evidence has demonstrated that chewing insects activate the JA pathway, whereas sucking pests usually elicit the SA pathway.

Next, a cell-content feeder may induce defenses of both the SA and JA pathways (Zhang et al., 2009). In fact, inconsistencies in induced plant defense depend on the species of the pest or the host plant species (Walling, 2000; Diezel et al., 2009). The induced defense pathway is always considered to promote the production of secondary metabolites and is very unfavorable to insects (e.g., affecting pest feeding and digestion) (Walling, 2000; Jormalainen et al., 2001). In addition, the contents of the SA, total phenolic, and tannins were negatively correlated to the fecundity of B. tabaci. Simultaneously, the increase of plant secondary metabolites and the lack of nutrients may affect the feeding preference of B. tabaci.

Numerous investigations indicate that sucking insects activate the SA signaling defenses and inhibit the JA pathway more easily (Kempema et al., 2007; Poelman et al., 2012). For example, B. tabaci activate SA signaling and inhibit JA-dependent responses (Kempema et al., 2007; Zarate et al., 2007; Zhang et al., 2013a). P. solenopsis can limit the expression of JAdepended genes and defensive substances to enhance its nymph adaptive ability on cotton. Specifically, mealybugs could induce the expression of JA-related genes but reduce the production of JA-regulated defensive substances, like gossypol on cotton (Zhang et al., 2011). Due to the particular interaction between JA-SA, mealybugs can enhance the host adaptability of its nymphs (Zhang X. et al., 2015), but not for the performance of B. tabaci. Here, the result showed expression level of LOX was significantly higher in P. solenopsis- infested plants compared to undamaged plants. However, T. cinnabarinus had less ability to induce LOX genes in comparison to P. solenopsis. T. cinnabarinus remain a typical cell-content feeder that may activate JA and SA pathways. Previous research confirmed that insects and mites could suppress the JA pathway to enhance their performance on the same plant, but maybe not for another species (Sarmento et al., 2011; Glas et al., 2014). Here, P. solenopsis significantly upregulated the expression level of the PR genes and PAL gene, a downstream gene regulated by SA. However, T. cinnabarinus appeared to be following the reverse strategy. Although it has previously been reported, that mealybugs and aphids can upregulate PR genes after feeding on hosts (Zhang X. et al., 2015). The direct and substantial roles of PR proteins in defense against various threats (including insects) show diversified mechanisms (Ebrahim et al., 2011), which are not very clear against the phloem-feeding insects. In contrast, JA-regulated genes may perform a crucial role in preventing pest invasions (Rahbé and Febvay, 1993). In our study, a significantly higher level of the LOX gene was observed on leaves pre-infested with mites or mealybug after 3 days. The results suggested the effectiveness of plant defenses elicited by upregulating the level of LOX gene against P. solenopsis' invasion. Taken together, it is important to emphasize that JA/ET-induced defenses carry out an important role in the successful defense against the phloem-feeding pest, like B. tabaci. The P. solenopsis maintains a 'stealthy feeding strategy' that avoids massive cell damage, which may produce insufficient JA levels for gene induction (De Vos et al., 2006). There is antagonism between SA and JA, and the SA signaling pathway may inhibit the JA signaling pathway (Doares et al., 1995).

Previous studies have revealed pests always accumulate SA at the site of oviposition, which may interfere with the accumulation of JA (Bruessow et al., 2010). Therefore, the high content of PR protein not only does not adversely affect B. tabaci, but rather is conducive to reproduction and development (McKenzie et al., 2002). In addition, the leaves infested with P. solenopsis and B. tabaci showed a marginally significant increase of the expression of PAL and PR5 by comparison with the undamaged leaves. However, the leaves infested with T. cinnabarinus and B. tabaci witnessed a significant reduction in the expression of PR1 compared to undamaged leaves. Thus, the high expression of SA regulatory genes did not favor the selection and oviposition of B. tabaci. Another study confirmed that whitefly pre-infestation of the tomato reduced the fecundity of subsequent whiteflies (Cui et al., 2012). Moreover, JA-mediated defenses may in addition cause an effect on egg development (Bruinsma et al., 2007). This suggests that inhibition of plant defense is of importance to phloem feeders, allowing them to effectively utilize the host.

The cross-talk between JA-SA has been demonstrated adequately in the past. Studies have speculated insects can take advantage of this cross-talk to address plant defenses to enhance host adaptability (Zhang et al., 2013b). Like JA, it is equally common for ET signals in plants to be activated by insects. However, ET is not so much a direct stimulus as an indirect regulator, that rarely directly adjust the corresponding plant defense (Dahl and Baldwin, 2007; Diezel et al., 2009). In addition, the ET pathway may equally affect genes regulated by JA in numerous plants (Zhu-Salzman et al., 1998; Royo et al., 1999; Stotz et al., 2000; Winz and Baldwin, 2001). Moreover, the interference between plant hormones allows plants to minimize their own cost of capacity, thereby developing a flexible signaling pathway that is more effective against intruders (Zhang et al., 2009). The ability of plants to regulate signaling pathways may, in turn, provide benefits to other invasive pests (Sarmento et al., 2011; Glas et al., 2014). Comparing to the leaves infested with T. cinnabarinus only, B. tabaci caused a marginally significant reduction of the level of ETR and PR1. However, it facilitated the expression level of LOX, a JA-regulated gene.

Insects equally possess a range of defense strategies to adapt to harsh environments, including the adaptations of detoxifying enzymes, digestive enzymes and protective enzymes (Ahmad et al., 1986; Jongsma et al., 1995; Pearse et al., 2013). B. tabaci, as a sucking pest, will inevitably exchange material with the plant phloem. It is properly known that plants fed by pests can induce the production of secondary metabolites. These substances can stimulate the protective enzymes and detoxification enzymes of B. tabaci, affect the midgut digestive enzymes, and thus their feeding and digestion and absorption capacity (Green and Ryan, 1972; Ahmad et al., 1986). The difference in detoxifying enzymes frequently affects the interspecific competition between B. tabaci and other populations (Julian et al., 2003; Liang et al., 2007; Liu et al., 2008). In this study, the activity of GST in whiteflies was significantly inhibited after feeding by mites, and the activity of CarE increased significantly after co-existence of whiteflies and mealybugs. Previous studies have shown CarE is important for insect resistance and can detoxify a variety of environmental toxicants. Therefore, this study demonstrated that the increase in

CarE activity of B. tabaci may be one of the adaptation measures for it against adverse environmental conditions.

Insects advocate broader tolerance to secondary metabolites, and this tolerance is determined by a variety of biochemical and physiological characteristics of the midgut. As one of the most key components of plant phloem, sucrose can supply energy for insects. Not only that, it can maintain osmotic pressure balance for sucking insects to ensure insects survivein a high osmotic pressure environment (Becker et al., 1996). Trehalose is known as the sugar of life. Its vital function is to synthesize chitinase and participate in the metabolism of insects, therefore providing energy for insect host positioning, courtship, mating, resisting adversity, spawning and other activities (Tang et al., 2012; Qin et al., 2015). Therefore, host adaptability may be reflected by the activity of trehalase and invertase in insects. B. tabaci may respond to the unfavorable situation by increasing trehalase activity. However, the activity of trehalase in whiteflies was inhibited on the plants infected by mealybugs, which may affect the behaviors of B. tabaci, like the oviposition behavior. It was only significantly increased after the pre-infestation of mites. Therefore, it was adequately demonstrated that the presence of mealybugs may cause an undesirable effect on the performance of B. tabaci, like the abilities of digestion and feeding. In addition, trehalase is also involved in feeding regulation and provides energy for insect flight behavior, which may be directly related to the tendency of B. tabaci in the field to select a host (Shi et al., 2016).

SOD can scavenge superoxide anion radicals and prevent damage to the body, but the generated hydrogen peroxide is still harmful to the insect midgut, however, CAT and POD in the body will immediately dispose of it (Tang et al., 2016). The protective enzyme activities of B. tabaci increased significantly after coexisting with T. cinnabarinus. However, this phenomenon does not occur when coexisting with mealybugs.

Because of the broad distribution and overlapping of hosts, all three pests in this study are extremely likely to erupt simultaneously on the consenting host plants in the field. Therefore, exploring the differences in plant defenses induced by different pests helps to reveal an understanding of the rules

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and mechanisms of pest invasion in the field. Despite previous reports of competition between mealybugs and whiteflies or spider mites and whiteflies, there has been increasing interest in the interspecies relationships between sap-feeding insects and the interspecific effects between mites and sucking insects (Godinho et al., 2016). In this study, T. cinnabarinus was more likely to activate plant genes regulated by JA, while mealybugs were more likely to activate key genes regulated by SA. Either may be one of the chief factors that affect the resistance of host plants to B. tabaci. Moreover, B. tabaci was more adaptable to plants coexisting with T. cinnabarinus, which may be attributed to the increase in digestive enzyme activity and protective enzyme activity. This result is of great significance to the layout of field crops and pest control or prevention. It additionally provides a firm basis for exploring the functions of the detoxification enzymes GST, trehalase and the protective enzyme systems in the anti-defense of B. tabaci. Through the further study of plant defense mechanisms, it is of great significance to understand the direct interaction of pests as well as the monitoring and control of field pests. These will support us to take effect early warning measures as early as possible and use natural laws to prevent and control natural disasters.

#### AUTHOR CONTRIBUTIONS

QR and HW conceived and designed the experiments. DL, YX, XL, LZ, and JW performed the experiments. DL and QR analyzed the data. DL and QR drafted the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by Natural Science Foundation of Zhejiang Province, China (LY14C140003) and Agriculture, Rural Areas, and Farmers Six-Party Science and Technology Cooperation Projects of Zhejiang Province, China (CTZB-F180706LWZ-SNY1).


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**Conflict of Interest Statement:** YX was employed by company Zhejiang Chemical Industry Research Institute.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lin, Xu, Wu, Liu, Zhang, Wang and Rao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fphys-10-00346 April 2, 2019 Time: 17:28 # 9

# Microorganism-Based Larval Diets Affect Mosquito Development, Size and Nutritional Reserves in the Yellow Fever Mosquito Aedes aegypti (Diptera: Culicidae)

Raquel Santos Souza<sup>1</sup> , Flavia Virginio<sup>2</sup> , Thaís Irene Souza Riback<sup>3</sup> , Lincoln Suesdek4,5 , José Bonomi Barufi<sup>6</sup> and Fernando Ariel Genta1,7 \*

<sup>1</sup> Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil, <sup>2</sup> Laboratório Especial de Coleções Zoológicas, Instituto Butantan, São Paulo, Brazil, <sup>3</sup> World Mosquito Program, Rio de Janeiro, Brazil, <sup>4</sup> Laboratório de Parasitologia, Instituto Butantan, São Paulo, Brazil, <sup>5</sup> Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, Brazil, <sup>6</sup> Laboratório de Ficologia, Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brazil, <sup>7</sup> Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Raman Chandrasekar, Kansas State University, United States Qian Han, Hainan University, China Zhen Zou, Institute of Zoology (CAS), China

#### \*Correspondence:

Fernando Ariel Genta genta@ioc.fiocruz.br; gentafernando@gmail.com

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 12 September 2018 Accepted: 08 February 2019 Published: 09 April 2019

#### Citation:

Souza RS, Virginio F, Riback TIS, Suesdek L, Barufi JB and Genta FA (2019) Microorganism-Based Larval Diets Affect Mosquito Development, Size and Nutritional Reserves in the Yellow Fever Mosquito Aedes aegypti (Diptera: Culicidae). Front. Physiol. 10:152. doi: 10.3389/fphys.2019.00152 Background: Mosquito larvae feed on organic detritus from the environment, particularly microorganisms comprising bacteria, protozoa, and algae as well as crustaceans, plant debris, and insect exuviae. Little attention has been paid to nutritional studies in Aedes aegypti larvae.

Objectives: We investigated the effects of yeast, bacteria and microalgae diets on larval development, pupation time, adult size, emergence, survivorship, lifespan, and wing morphology.

Materials and Methods: Microorganisms (or Tetramin <sup>R</sup> as control) were offered as the only source of food to recently hatched first instar larvae and their development was followed until the adult stage. Protein, carbohydrate, glycogen, and lipid were analyzed in single larvae to correlate energetic reserve accumulation by larva with the developmental rates and nutritional content observed. FITC-labeled microorganisms were offered to fourth instar larvae, and its ingestion was recorded by fluorescence microscopy and quantitation.

Results and Discussion: Immature stages developed in all diets, however, larvae fed with bacteria and microalgae showed a severe delay in development rates, pupation time, adult emergence and low survivorship. Adult males emerged earlier as expected and had longer survival than females. Diets with better nutritional quality resulted in adults with bigger wings. Asaia sp. and Escherichia coli resulted in better nutrition and developmental parameters and seemed to be the best bacterial candidates to future studies using symbiont-based control. The diet quality was measured and presented different protein and carbohydrate amounts. Bacteria had the lowest protein and carbohydrate rates, yeasts had the highest carbohydrate amount and microalgae

showed the highest protein content. Larvae fed with microalgae seem not to be able to process and store these diets properly. Larvae were shown to be able to process yeast cells and store their energetic components efficiently.

Conclusion: Together, our results point that Ae. aegypti larvae show high plasticity to feed, being able to develop under different microorganism-based diets. The important role of Ae. aegypti in the spread of infectious diseases requires further biological studies in order to understand the vector physiology and thus to manage the larval natural breeding sites aiming a better mosquito control.

Keywords: Aedes aegypti, microorganism, development, nutritional reserves, digestion, yeast, bacteria, algae

#### INTRODUCTION

Mosquitoes are medically the most significant group of insects due to their important role in the widespread of several human infectious diseases including malaria, dengue fever, encephalitis, yellow fever and filariasis (Weaver and Reisen, 2010). The global magnitude of morbidity and mortality caused by arthropod-borne diseases has been a public health emergency of international concern. Early stages of mosquito development are related to aquatic environments, thus understanding the ecological factors involved in the aquatic habitats is essential in order to develop and improve effective strategies of mosquito control.

The biotic and abiotic environmental conditions experienced during the immature stage are determinant for the growth and development of mosquitoes. A considerable number of studies in the early 20th century devoted attention to investigating the food requirements of larvae in order to reduce or eliminate the nutritional supply of these insects in nature (Hinman, 1930). Studies on holometabolous insects suggest that well-nourished larvae become healthier adults (Zeller and Koella, 2016). The biomass accumulation of mosquitoes can be attributed to the efficiency of foraging by larvae and withstanding of starvation (Barrera, 1996). Quantitative and qualitative aspects of larval nutrition exert immediate effects on immature survivorship and development rate, which can alter population dynamics of mosquitoes and determine adults life traits (Subra and Mouchet, 1984; Gimnig et al., 2002; Barrera et al., 2006; Araújo et al., 2012; Radchuk et al., 2013; Kivuyo et al., 2014; Li et al., 2014).

Mosquito populations that develop in containers can be regulated by the availability and amount of food resources in the aquatic habitat (Washburn, 1995). Food deprivation can have several carry-over effects on mosquito life. A longer development time under conditions of food insufficiency has been observed before (Tun-Lin et al., 2000; Arrivillaga and Barrera, 2004; Dominic et al., 2005; Vantaux et al., 2016; Aznar et al., 2018), with mosquito larva that take longer time to reach pupa stage (Telang et al., 2007; Levi et al., 2014; Banerjee et al., 2015). An extended larval stage is generally associated with an increased risk of mortality as a consequence of predation, breeding site instability and human interference (Padmanabha et al., 2011). Beyond development time, the amount of food influences characteristics such as: nutritional reserves (Van Handel and Day, 1989; Briegel, 1990b; Gullan and Cranston, 1999; Arrivillaga and Barrera, 2004), adult emergence (Okech et al., 2007), body size (Grimstad and Walker, 1991; Strickman and Kittayapong, 2003; Jirakanjanakit et al., 2007; Foster et al., 2012; Aznar et al., 2018), response to repellents and insecticides (Xue et al., 1995; Xue and Bernard, 1996), survival (Landry et al., 1988; Dominic and Das, 1996; Sumanochitrapon et al., 1998; Aznar et al., 2018), sexual maturity, fecundity, egg production and longevity of the adult female (Briegel, 1990b; Nasci and Michell, 1994; Naksathit and Scott, 1998; Sumanochitrapon et al., 1998; Reiskind and Lounibos, 2009; Alto et al., 2012; Foster et al., 2012; Takken et al., 2013). The vector competence also could be influenced by the available food resource. Adults that emerge from larvae with low nutritional reserve are smaller (Lehmann et al., 2006) and require more blood feeds to produce eggs (Briegel, 1990b), which may lead to an increase in their vectorial capacity (Muturi et al., 2011). Restricted larval food can extend the time for mosquitoes to become infectious (Shapiro et al., 2016; Vantaux et al., 2016), modulate microbiota (Linenberg et al., 2016) and permissiveness to parasites (Takken et al., 2013; Linenberg et al., 2016), affecting immune traits (Suwanchaichinda and Paskewitz, 1998; Telang et al., 2012).

Previous studies reported that Aedes aegypti size is vulnerable to food amount and population density in immature stages (Jirakanjanakit et al., 2007). Direct measurement of the mosquito body is not satisfactory estimation of size, due to the variation of three-dimensional structures, besides the variation in the dryness of the abdomen. Weight is also an unreliable estimator of body size as it can be influenced by the blood feeding, egg production, among other factors. The mosquito body size may be adequately estimated using the wing length, or even better, using wing centroid size, an isometric and comprehensive estimator of body size (Bookstein, 1992; Siegel et al., 1992; Lounibos, 1994; Carron, 2007; Jirakanjanakit et al., 2007; Strickman and Kittayapong, 2003; Lehmann et al., 2006).

Immature stages of culicids are generally undemanding and have a pliant food behavior, ingesting through different feeding modes (e.g., filtering, suspension feeding, browsing, and interfacial feeding) organic particles in the water and almost everything available in the natural or artificial environments (Walker et al., 1988; Merritt et al., 1992; Clements, 2000). Particulate microorganisms and organic debris are commonly

the main nutritional source of mosquito larvae. Bacteria, viruses, protozoa, fungi (Timmermann and Briegel, 1996; Forattini, 2002) and algae (Merritt et al., 1992; Kivuyo et al., 2014) are some of the organisms that actively contribute to foraging and development during the larval stage. Bacteria seems the most abundant microorganisms present in the larval diet, and may even be the only nutritional source for insect growth and development (Merritt et al., 1992). Pollen particles dispersed in the aquatic environment can also be used as food sources by immature forms (Ye-Ebiyo et al., 2003; Kivuyo et al., 2014; Asmare et al., 2017).

The evolutionary success and extensive dispersal of mosquitoes may have been widely motivated by symbiotic relationships with microorganisms (Ricci et al., 2011a; Coon et al., 2014). Insects harbor numerous symbiont microbial communities, which possibly supplant the number of the cells of the invertebrate (Gusmão et al., 2010). Intracellular symbionts can occur in up to 70% of all insect species, and the intestinal compartment concentrates most of these microorganisms (Gusmão et al., 2010). The contribution of the intestinal microbiota of insects in nutritional ecology is quite relevant due to their impressive biosynthetic and degradative capacity (Douglas, 2009; Kukutla et al., 2014). The insect microbiota plays an important role in the synthesis of vitamins and essential amino acids, steroids and carbohydrates metabolism and promoting the growth and development using the insulin pathway (Shin et al., 2011; Storelli et al., 2011; Douglas, 2014). Besides nutrition, symbionts aid in nitrogen fixation, behavior, reproduction, development and enhance or suppress infections by pathogens (Dillon and Dillon, 2004; Hegde et al., 2015).

Aspects such as digestion, processing, absorption and detoxification of such generalist diets are the result of refined interactions with symbionts and digestive enzymes (Fisk and Shambaugh, 1952; Geering and Freyvogel, 1975; Marinotti and James, 1990; Ho et al., 1992; Souza et al., 2016). It is still unclear as the several microbial nutritional sources may influence the physiology of larval mosquito and which are the main enzymes involved in the digestion of these nutrients.

In this study, we investigated Ae. aegypti larval feeding using a range of microorganisms as a nutritional source. Life parameters including development rates, survival, sex ratio, body size, ingestion rates, quantity and quality of food and nutritional reserve accumulation were reported in this paper. The results suggest that microorganism-based diets can be supported by these insects in laboratory conditions and aim to provide information to laboratory breeding or studies for potential biological larvicides.

#### MATERIALS AND METHODS

#### Mosquito Rearing

The Ae. aegypti specimens eggs used for this study, were originated from eggs of Rockefeller strain gently ceded by Dr. José Bento Pereira Lima - from colonies of the Laboratory of Physiology and Control of Arthropod Vectors (LAFICAVE, - IOC/-FIOCRUZ; Dr. José Bento Pereira Lima). Insects were reared until the adult stage in the Laboratory of Insect Biochemistry and Physiology (LABFISI, IOC/FIOCRUZ) under standard conditions (temperature 26 ± 1 ◦C, relative humidity 80 ± 5% and photoperiod 12:12 h [L: D]). Newly hatched larvae derived from the same egg batch within 2 h of eclosion were fed Tetramin <sup>R</sup> sprinkled on the distilled water surface until the nutritional trials being performed.

#### Screening of Microorganisms

The nutritional physiology experiments were performed based on the follow microorganisms: Serratia marcescens (SM365), Escherichia coli (D31) and Staphylococcus aureus isolated and cryopreserved in the LABFISI, Saccharomyces cerevisiae (S14) kindly donated by Dr. Pedro Soares de Araújo (Chemistry Institute, University of São Paulo), Asaia sp. (A1), Ochrobactrum intermedium (Om17), Bacillus sp. and Pseudozyma sp. (Pa1) by Dr. Rod J. Dillon (Faculty of Health and Medicine, Lancaster University, United Kingdom), Arthrospira platensis (Spirulina) and Chlorella sp. by Dr. José Bonomi Barufi (Laboratory of Phycology, Federal University of Santa Catarina, Brazil).

#### Preparation of Microorganisms Diets

Aliquots of S. marcescens, E. coli, Bacillus sp, O. intermedium, and S. aureus were inoculated in Luria-Bertani agar plates (LB) and incubated overnight for 24 h at 30◦C. S. cerevisiae and Pseudozyma sp. were inoculated in YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose/dextrose, 2% agar). Growth conditions: S. cerevisiae overnight for 24 h at 30◦C and Pseudozyma sp. 48 h at 30◦C. Asaia sp. were inoculated on GCA agar plates (2% glucose, 0.8% yeast extract, 0.7% CaCO3, 2% agar) and incubated overnight for 72 h at 26◦C (Sant'Anna et al., 2014). Bacteria single colonies were transferred to LB medium, yeast-like fungus to YPD medium and Asaia sp. to GLY medium (glycerol 25 g/l, yeast extract 10 g/l, pH 5.0) in 50 mL polypropylene tubes. All strains were grown according to the incubation temperatures of each strain in a shaking incubator (150 rpm). The microbial suspensions were centrifuged (20 min, 21,000 g, 4◦C) and the supernatant was discarded to fresh mass (FM) measurements. Cells harvested by brief spin were washed with sterile PBS three times, and finally, the bacterial and yeast pellet was resuspended in sterile water and adjusted in a concentration of 800 mg/80 mL (w/v) per strain. Chlorella sp. were inoculated in Bold's Basal Medium (BBM), and A. platensis were inoculated in Spirulina Medium Modified (Andersen, 2005). Chlorella sp. and A. platensis were incubated at 21◦C with a photoperiod of 12:12 h [L: D] in a shaking incubator (100 rpm). They were centrifuged gently (5 min, 5,000 g), before the measure of their biomass. Cells harvested by brief centrifugation were resuspended in their respective medium and were adjusted in a concentration of 150 mg/15 mL (w/v).

#### Microorganisms Viability Trials

To evaluate the capacity of microorganisms used in this study remains alive in the aquatic environment, we observed the viability of these strains on water. Bacteria and yeast were inoculated in 50 mL polypropylene tubes in specific liquid media and growth conditions were described previously (see details in Preparation of Microorganisms Diets). Cells harvested by

centrifugation (20 min, 21,000 g, 4◦C) were resuspended in liquid media or sterile water. Twenty microliter aliquots of each suspension were placed on agar plates and the number of colonies forming units, CFU were recorded after 0, 24, 48, and 120 h. Five biological experiments were performed for statistical analysis.

#### Experimental Nutrition Protocol

Ten diets were compared. Groups of 150 first-instar larvae (L1) were manually counted and transferred to each of three sterile borosilicate glass recipients (22.5 cm × 12.8 cm × 3.59 cm). Under sterile conditions, each container was filled with QSP 250 mL of sterile water or distilled water (density = 0.22 larvae/cm<sup>2</sup> of surface area; depth of 39 mm). The same larval density was used in all experiments. The dietary supply was administered only at the L1, 80 mL (corresponding to 800 mg [w/v] and 16 mg/larva) of yeasts and bacterial suspensions and 15 mL (corresponding to 150 mg [w/v] and 3 mg/larva) of microalgae cultures were added to glass recipients. A slurry by mixing the components of Tetramin <sup>R</sup> in distilled water was prepared to fed standard group. We used 800 mg of Tetramin <sup>R</sup> resuspended in distilled water QSP to a final volume of 250 mL. Evaporated water was replaced as needed to maintain the initial volume. Three replicates were performed for each dietary experiment.

#### Effects of Diets on Development and Survivorship of Ae. aegypti

For comparison of diet effects, developmental rate and survivorship from eclosion to adult emergence were measured. Larvae were observed daily until pupation, and dead larvae and exuviae were removed. Pupae were collected daily, counted and transferred individually into 15 mL polypropylene tubes covered with mosquito netting and filled with 4 mL of breeding water until adult emergence. The number and the sex of adults emerged were determined. Adults received only cotton wool moistened with distilled water ad libitum. The median time in days for pupation, the emergence of adults (males and females) and adult survivorship were calculated using the number of individuals that reached pupae or the adult stage. We also recorded the proportion of larvae that survived from L1 to the pupal stage, time to metamorphosis (development duration in days, between pupa and adult stage), time to emergence (development duration in days, between L1 and adult stage), adult survival (time from adult emergence to dead), and survivorship full span (using larvae that survived from L1 to dead adult stage). Sex ratio was estimated as the number of males relative to total emerged adults.

#### Wing Length Measurements

To evaluate possible morphological variation in body size of adults reared with microorganism diets, we measured the size of males and females wings separately using standard methods of geometric morphometrics. In this study, we used the wings of adults emerged from larvae fed with standard diet Tetramin <sup>R</sup> , the yeasts S. cerevisiae and Pseudozyma sp., and the Gramnegative bacteria E. coli and Asaia sp. The wings (both sides) were removed from the thorax of individuals, mounted on Canadian balsam microscope glass slides and processed as reported by Lorenz et al. (2012). Images of the slides were digitized using a digital camera Leica DFC320 coupled to a Leica S6 (40×) stereoscope. To each image were registered coordinates x and y of 18 landmarks (Virginio et al., 2015) using TpsDig software V.2.05 (Rohlf, 2006). The wing size variations were assessed using measurements of the centroid size (CS) (Bookstein, 1992).

# Microorganism Staining and Larval Feeding Behavior

We decided to monitor the ingestion of live microorganisms labeled with fluorescein isothiocyanate (FITC) by Ae. aegypti larvae. The protocol of microorganism staining was performed according to Moraes et al. (2012). FITC-labeled microorganisms were resuspended in 3 mL of sterile water and added to 50 mL polypropylene tubes containing 7 mL of sterile water and 50 fourth instar larvae raised on Tetramin <sup>R</sup> . After 2 h incubation at 26◦C, 10 larvae were dissected, and single guts were placed in microtubes with 100 µL of sterile NaCl 0.9% (w/v). Samples were homogenized by shaking the tube for 30 s at 25 Hz (MiniBeadBeater; Biospec Products, Bartlesville, OK, United States). The gut fluorescence detection was performed in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, United States) on λEx = 495 nm and λEm = 520 nm. Aliquots (10 µL) of microorganisms FITClabeled were mounted on microscope glass slides for fluorescence observation in a Nikon Eclipse E200 (40×), fitted with a B-2A filter (Excitation Filter Wavelengths: 450–490; Dichromatic Mirror Cut-on Wavelength: 500; Barrier Filter Wavelengths: 515). Images were taken with a regular digital camera. Five experiments were performed.

#### Protein and Carbohydrate Contents of Microorganism-Based Diets

Culture samples of 20 mL were centrifuged (7,500 × g, 20 min, 4 ◦C). The supernatant was discarded, and cells were resuspended in 1 mL of water. Aliquots of 10 and 40 µL were withdrawn for protein and sugar measurements, respectively. We assessed total protein content using the bicinchoninic acid method procedure (Smith et al., 1985) and total carbohydrates were measured with the phenol-sulfuric method (Dubois et al., 1956). Eight experiments were performed for each diet.

#### Measurement of the Energy Reserves in Single Individuals

Fourth instar newly molted larvae were individually weighed and immediately frozen for analysis of nutritional reserves. We quantified protein, total carbohydrate, glycogen, and lipids in single individual fourth instar entire larvae, gut and rest of the body for comparison. Larvae were reared as reported in item "Experimental Nutritional Protocol" and dissected as described in "Microorganism Staining and Larval Feeding Behavior." The biochemical analysis was performed accordingly the Van Handel (1985a) method adapted by Foray et al. (2012). Protein content was measured as Bradford (1976) method using ovalbumin as a standard. Carbohydrates and glycogen were detected by an anthrone procedure using glucose as a standard (Van Handel, 1985a). Total lipid was determined in chloroform-methanol solvent solution by vanillin–phosphoric acid reaction (Van Handel, 1985b, 1988) using Glyceryl trioleate as standard (Van Handel and Lum, 1961). The assays were performed in 96-well microplates. Ten experiments were performed for each diet.

#### Statistical Analysis

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For all experiments, measurements were described using mean ± SEM. Developmental parameters (larval development, pupation, emergence, and survivorship) were analyzed using the GraphPad InStat v.3.01 (San Diego, CA, United States) and Excel <sup>R</sup> . The correlation among non-parametric variables was performed using the Log-rank (Mantel-Cox), Wilcoxon and Fisher tests. Tests for normality of the sample distribution were assessed by D'Agostino-Pearson omnibus test. Microorganism's viability significance and measurement of protein and carbohydrate content on diets were examined with a T-test. Analysis of variance (ANOVA 1) was used in ingestion of live microorganisms labeled with FITC and measurement of the energy reserves in the whole larva. The wings morphometric statistical analyses were managed with the software MorphoJ (Klingenberg, 2011), and GraphPad InStat v.3.01 (San Diego, CA, United States). The normality and homoscedasticity of samples distribution were assessed by Shapiro–Wilks with the software Past3. In populations that had a Gaussian distribution, the parametric T-test was used based on means. In populations that did not, the non-parametric Mann–Whitney test was used based on medians.

#### RESULTS

To verify if all the strains used in our experiments were viable on aquatic conditions we tested the viability of the microorganism cells in water. CFU counts revealed that re-suspension in water does not affect the viability and number of cells (p > 0.05, paired T-test, n = 15, **Figure 1**). S. cerevisiae, S. marcescens, Bacillus sp., and O. intermedium remained viable after being incubated in water for 48 h, and Pseudozyma sp., E. coli, Asaia sp., and S. aureus were viable until 24 h (**Figure 1**). These data demonstrate that it is possible to expose larvae to live cells and that these microbial cells might be used as a nutritional source.

#### Estimates of Development and Survivorship for Ae. aegypti Reared Using Exclusive Microorganism-Based Diets

To assess the development and survivorship until the adult stage of Ae. aegypti reared exclusively with microorganism-based nourishment at immature stages, we tested four strains of Gramnegative bacteria were used: S. marcescens (SM365), E. coli (D31), Asaia sp. (A1), and O. intermedium; two strains of Gram-positive bacteria, Bacillus sp. and S. aureus; the yeasts: S. cerevisiae (S14) and Pseudozyma sp. (Pa1), a genus of microalga Chlorella sp., and a species of cyanobacteria (blue–green algae), A. platensis (Spirulina). The biological life attributes measured for the Ae. aegypti Rockefeller strain under controlled laboratory conditions are presented from **Supplementary Tables S1–S6** and **Tables 1**–**5**, and summarized as follows.

The developmental time from L1 to pupae differed significantly (p < 0.0001) between the diets. Larvae of the standard group fed with Tetramin <sup>R</sup> developed in 5.3 ± 0.04 days. Pseudozyma sp. and S. cerevisiae (mean 6.5 ± 0.12; 8.1 ± 0.05 days) developed faster than the larvae on other microorganism-based diets. Larvae fed with the Chlorella sp. take longer to develop until pupation with a mean time of 61.5 ± 2.09 days until pupa (**Supplementary Table S1**). S. marcescens (42.0 ± 3.0 days) and Bacillus sp. (34.0 ± 0.0 days) showed the longest time to achieve pupal stage compared with the other bacterial diets.

The next biological parameter evaluated was the duration of the metamorphosis period of larvae in adult mosquitoes. No significant differences (p > 0,05) were detected in diets that used Asaia sp. (2.0 ± 0.04 days; p = 0.6804), O. intermedium (2.0 ± 0.0 days; p = 0.3652), Chlorella sp. (1.9 ± 0.07 days; p = 0.0978) and A. platensis (1.9 ± 0.09; p = 0.1042) compared to the standard group fed with Tetramin <sup>R</sup> (2.09 ± 0.02 days) (**Supplementary Table S2**). Time from L1 to adult emergence differed significantly (p < 0.0001) among each diet. Time until the adult stage was higher to Chlorella sp. (**Supplementary Table S3**).

The survival of adults maintained only with water was evaluated once a day until confirmation of the death of all insects. The diets containing E. coli (6.2 ± 0.3 days) and Asaia sp. (5.5 ± 7.52 days) showed the closest survival rates compared to the standard diet (8.9 ± 0.1 days). Bacillus sp. (2.0 ± 0.0 days), S. aureus (2.3 ± 6.1 days), and O. intermedium (2.5 ± 2.1 days) revealed the lowest survival rates (**Supplementary Table S4**). The full lifespan from L1 to adult death is significantly (p < 0.0001) different between diets (**Supplementary Table S5**).

Biological parameters were analyzed separately by gender to disclose possible sex-specific effects in development rates and survivorship. The development time of L1 to pupae differed significantly in females and males (p < 0.001; **Table 1**) from different diets. Larvae fed with yeast diet developed faster in both genders (mean 7.2 ± 0.2; 8.4 ± 0.06 days for females; 6.1 ± 0.1; 7.9 ± 0.07 days for males) than larvae on bacteria and microalgae diets. Male development time until pupa is shortest than female larvae in all diets used (**Table 1**). No significant differences (p = 0.7867) were detected for female adult metamorphosis on diets containing Asaia sp. However, a significant (p < 0.0001) effect was observed among all the other diets compared to the standard group fed with Tetramin <sup>R</sup> (**Table 2**).

Male development time until adult metamorphosis differed significantly (p < 0.0001) solely on yeasts diets, and no significant differences were observed among the other diets (**Table 3**). Female average development time until adult emergence was longest than males. In both genders a significant difference (p < 0.0001) was detected when compared microorganismbased diets with the group fed with the standard diet Tetramin (**Table 3**). The survival of adults (males or females) differs significantly (p < 0.0001) between diets. The average survival time of each female adult varied from 2.6 ± 0.93 to


NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

5.1 ± 0.73 days across the different dietary supply. Females fed with A. platensis (5.1 ± 0.73 days), Asaia sp. (4.8 ± 0.39 days) and E. coli (4.6 ± 0.40 days) exhibited an elongated average survival (**Table 4**).

The adult survival pattern observed in males differed partially from females. The average observed in the survival span for males varied from 2.1 ± 0.39 to 7.0 ± 0.44 days. Males fed with E. coli (7.0 ± 0.44) and Asaia sp. (6.1 ± 0.24 days) displayed the same longest survival observed for females. Additionally, males fed with Pseudozyma sp. (5.6 ± 0.19 days) and S. cerevisiae (5.5 ± 0.28 days) also presented a long survival (**Table 4**). The full lifespan of males and females from L1 larvae to pupation differ significantly among diets (p < 0.0001). The average life span varied from 13.0 ± 0.23 to 73.5 ± 4.74 days for females and 14.0 ± 0.17 to 65.0 ± 2.32 days for males (**Table 5**). The overall adult sex ratio was more male-biased (**Supplementary Table S6**). The development time of L1 to pupae, adult emergence, survivorship, and lifespan of all dietary studied have been depicted graphically in **Figures 2**–**5**.

Yeast diets revealed a similar development time to standard diet Tetramin <sup>R</sup> . The diets of bacteria and microalgae, in the opposite, presented a lethargic larval development (**Figures 2**, **3**). Regarding adult mortality, E. coli and Asaia sp. presented the highest survival mean in days, surpassing even the yeast diets (**Figure 4**). The full lifespan was extended in immature stages fed with bacteria and microalgae diets. These results suggest a possible badly nourishment which could breed smaller larvae with difficulty to attain the critical mass that is necessary

#### TABLE 2 | Average metamorphosis time for females and males in days.


NA: Not available. Differences were analyzed with Log-rank and Wilcoxon.

TABLE 3 | Average emergence time for females and males in days.


NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

#### TABLE 4 | Adult average survival time in females and males.


NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

#### TABLE 5 | The full lifespan of females and males in days.

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NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

for metamorphosis (**Figure 5**; Telang et al., 2007). All the compiled biological development data were detailed in the **Supplementary Spreadsheet S1**.

Concerning the wing centroid size analysis, which is herein used as a predictor of body size, the majority of samples showed a normal distribution (p > 0.05, **Supplementary Tables S7, S8**). In general terms, the comparisons between microorganismbased diets and the standard group (with normal and nonnormal distribution, respectively), showed a significant difference of sizes in both genders and wing sides (p < 0.05). The isometric size of males and females reared in Tetramin <sup>R</sup> showed larger adults than all the other diets (**Figure 6**). On average, among the experimental diets (excluding the standard group), the largest females were reared in S. cerevisiae, followed by Pseudozyma sp., Asaia sp., and E. coli diet. The males were largest in S. cerevisiae, Asaia sp., Pseudozyma sp., and E. coli diet, respectively (**Figure 6**).

#### Nutritional Evaluation of Diets, Energetic Reserve Accumulation Measurement and Feeding Behavior of Ae. aegypti Larvae Reared Exclusively With Microorganisms

Nutritional quality influences the physiology of Ae. aegypti immature stages directly. To evaluate the impact of quality of two important macronutrients on larval breeding, we quantified the protein and carbohydrate contents present in the dietary supplies used. Differences in the nutritional composition of the

FIGURE 3 | The impact of microorganism-based diets on adult emergence. A representative emergence curve is comparing larvae fed with Tetramin (Dark blue line, left y-axis) and larvae fed with microbial cells (red line, right y-axis). The dietary supply was administered only at L1. Dead individuals were removed daily.

microorganism diets were detected and may have influenced the immature stages development time (**Table 6**). S. cerevisiae and Pseudozyma sp. diets have higher amounts of protein (116 ± 6.1 mg; 75 ± 6.9 mg) and carbohydrate (180 ± 9.2 mg; 98 ± 3.8 mg) than the bacterial diets (7.0 ± 0.6 mg to 15.4 ± 1.4 mg and 0.3 ± 0.1 mg to 1.6 ± 0.02 mg). Yeast diets, on the other hand, have less protein than A. platensis (341 ± 34 mg) and Chlorella sp. (300 ± 17.5 mg) diets. Conversely, the content of carbohydrates was higher on diets with yeasts (180 ± 9.2 mg; 98 ± 3.8 mg) compared to the diets based in A. platensis (35 ± 4 mg) and Chlorella sp. (26 ± 1.5 mg).

Four major energetic components (protein, carbohydrate, glycogen, and lipids) and the body weight were measured in individual fourth instar entire larvae and compared with the contents recovered from their guts and rest of body tissues. The results are summarized in **Table 7**. The absolute values of the

nutritional reserves differed significantly (p < 0.0001) among individuals compared with the standard group Tetramin <sup>R</sup> . Larvae fed with S. cerevisiae, Pseudozyma sp, E. coli, and Asaia sp. accumulate the highest amounts of protein (194 ± 18 mg to 89 ± 3 mg), carbohydrates (103 ± 1.3 mg to 16 ± 0.3 mg), glycogen (12 ± 0.8 to 3 ± 0.4 mg) and lipids (71 ± 4.9 to 27 ± 0.8 mg). The nutritional reserve accumulation in A. platensis, Chlorella sp., S. aureus, S. marcescens, Bacillus sp., and O. intermedium showed the lowest amounts of energetic components (protein, carbohydrate, glycogen, and lipid) and these results are in agreement with the longer developmental rates observed in the experiments above (**Supplementary Tables S1–S5** and **Tables 1**–**5**). Mean body weight differed significantly with larval diet (**Table 7**). Only S. cerevisiae and Pseudozyma sp. did not differ significantly compared with the standard diet.

To further investigate the rates of ingestion of the different microorganisms by larvae, the consumption rate of each diet was measured by fluorescence in the gut of individual larvae after 2 h of incubation with FITC-labeled cells. Larvae fed with S. cerevisiae, Pseudozyma sp., and E. coli consumed more food than larvae fed with the other diets after 2 h (**Figure 7**). Our results showed that Ae. aegypti larvae actively consumed all the microorganisms used, but with some preference for the microorganisms above (**Supplementary Figures S1, S2**).

#### DISCUSSION

The nutrition environment experienced by larvae strongly influences the physiology and behavior of mosquitoes. The current work evaluated the impact of nourishment from live microbes in the development and survival of Ae. aegypti. The organisms selected for feeding were strains of Gram-negative bacteria S. marcescens (SM365), E. coli (D31), Asaia sp. (A1), and O. intermedium (Om17), Gram-positive bacteria Bacillus sp. and S. aureus, yeast-like fungi S. cerevisiae (S14), and Pseudozyma sp. (Pa1), the cyanobacteria (blue–green algae) A. platensis (also known as Spirulina), and the marine microalga Chlorella sp. All microorganisms used in this paper showed stability in water until 120 h. Therefore, we decided to perform the feeding experiments by dispersing the strains directly in rearing water under sterile conditions.

#### General Developmental Parameters in A. aegypti Raised on Microorganisms-Based Diets

All diets showed important differences in developmental and survival rates when compared individually to the standard group Tetramin <sup>R</sup> . Larva fed with yeast take less time to achieve pupation than all the other microorganism-based diets tested (**Figure 2**). Parameters as adult emergence (**Figure 3**), survival (**Figure 4**) and full lifespan (**Figure 5**) showed a slight delay in developmental rates when compared to the standard group Tetramin <sup>R</sup> . Due to the satisfactory developmental rates in all life parameters tested, and the rapid growth speed of cultures in lowcost media, S. cerevisiae, and Pseudozyma sp. seem to be suitable candidates for diets in the mass rearing of mosquitoes or the regular laboratory breeding (Imam et al., 2014). Trager (1935a,b) studies pioneered larval nutrition, demonstrating that Ae. aegypti larvae can reach adulthood being fed only with yeast powder. Confirming the results obtained by Trager, 1935a showed that autoclaved yeasts suspended in CaCl<sup>2</sup> 0.01% is sufficient for the

sp. and S. cerevisiae). Vertical lines: individuals. Asterisks: Non-normal distribution.

between control diet (Tetramin <sup>R</sup>

) and Gram-negative bacteria (Asaia sp. and E. coli). (E–H) Comparison between control diet [Tetramin (R)] and Yeasts (Pseudozyma

TABLE 6 | Protein and carbohydrate contents in microorganism-based diets.


(ANOVA 1; ∗∗∗∗p < 0.001).

full larval lifespan of Ae. aegypti. Souza et al. (2016) conducted a study based on feeding larvae with a specific diet containing live or dead S. cerevisiae cells only. The data obtained in this paper corroborated the works described previously, that highlighted the ability of Ae. aegypti larvae to feed and digest living yeast cells through the enzyme beta-1,3-glucanase. Yeast and fungi can also be suitability as a paratransgenic vehicles or integrated pest management (IPM) tools.

Interestingly, the symbiont Wickerhamomyces anomalus (Saccharomycetales) can be found in the gut and reproductive organs of some mosquito vector species. This symbiont can be easily cultured in cell-free media and seem to be a good candidate for the expression of effector molecules in the gut of mosquito vectors (Ricci et al., 2011b). Murphy et al. (2016) demonstrated that genetically modified S. cerevisiae could be used as biopesticide through oral delivery of species-specific dsRNA. This application as biopesticide decreases larval survivorship, reduces locomotor activity and reproductive fitness in the insect pest Drosophila suzukii. This yeast biopesticide approach could be adapted to a large number of species once many interactions have been observed between sylvatic yeasts and insect species as Diptera, Coleoptera, and Hymenoptera (Gonzalez, 2014; Abrieux and Chiu, 2016). The authors also postulate that biopesticide design may be favored in the managing of an insect pest that both consumes yeast as food and has systemic RNAi.

Studies of biological control agents based on entomopathogenic fungi have been reported in several vector mosquitoes. The potential of Metarhizium anisopliae fungus was tested in Anopheles gambiae, Ae. aegypti, Aedes albopictus, Culex quinquefasciatus (Alves et al., 2002; Scholte et al., 2007; de Paula et al., 2008; Pereira et al., 2009; Fang et al., 2011). Geneticengineered M. anisopliae inhibited Plasmodium sp. development within the mosquito and prevented malaria infection in Anopheles (Fang et al., 2011). In Ae. aegypti, A. albopictus, and C. quinquefasciatus, the full lifespan of M. anisopliaecontaminated mosquitoes was significantly reduced and showed high mortality rates compared to uninfected mosquitoes (Alves et al., 2002; Scholte et al., 2007; de Paula et al., 2008; Pereira et al., 2009). Recently, two strains of M. anisopliae were tested against Ae. aegypti and besides the increase in mortality, the fungus also reduced egg laying (Jemberie et al., 2018). The fungi: Lagenidium giganteum and Leptolegnia chapmanii were also tested as promising biological control agents for use against Ae. aegypti adults (McCray et al., 1973; McInnis and Zattau, 1982). The development of innovative strategies using yeast has, therefore, potential as an eco-productive alternative for the management of mosquito-borne diseases.

Bacteria are considered the most common microorganism present in the nourishment of mosquito larvae (Laird, 1956, 1988; Christophers, 1960). Previous studies reported that bacteria could be used as a unique food requisite to mosquito growth (Hinman, 1932; Rozeboom, 1935). Larvae fed with S. marcescens, Bacillus sp, S. aureus, O. intermedium, S. aureus, E. coli, and Asaia sp. showed a severe delay to achieve pupal stage when compared to the standard group Tetramin <sup>R</sup> (**Supplementary Table S1**). Other biological parameters as metamorphosis (**Supplementary Table S2**), emergence (**Figure 3**), survival (**Figure 4**) and full lifespan also strongly affected (**Figure 5**). Studies obtained by Dickson et al. (2017) provide the concept that larval exposure to different bacterial communities during larval development can drive variation in Ae. aegypti adult traits. Therefore, the results observed here are similar to other studies reported. Noteworthy, E. coli and Asaia sp. take result in shorter developmental times into pupa than the other bacterial diets (**Figure 2**). The survival span for larvae fed with E. coli and Asaia sp. were superior to those observed in diets using S. cerevisiae and Pseudozyma sp. E. coli and Asaia sp. seem to be the best bacterial models for laboratory-reared larvae up until now.

Food stress was expected to reduce survival, however, larva fed with Asaia sp. and E. coli had higher adult survival (**Supplementary Table S4**). There are different ways to interpret these results. Nutritional stress can increase the life-span by a hormetic model (Mattson, 2008). Larvae fed with E. coli and Asaia sp. might be presenting a stress-induced response, hormesis, that can be an overcompensation to environmental, nutritional stress (Calabrese, 2001). Hormesis induces cellular protective mechanisms through an increased in gene expression, working as a key regulator of many cellular defenses that allow survival in response to stress (Lin et al., 2000; Motta et al., 2004; Heilbronn et al., 2005). Enhanced levels of heat shock proteins (HSPs) and antioxidants to cellular maintenance are also considered as part of the hormetic mechanism (Gems and Partridge, 2008; Mattson, 2008). The beneficial effects of hormesis on survival and longevity have been described for years, and our results might thus exemplify a beneficial carry-over effect of the hormetic stress on larval development, reinforcing the Ae. aegypti phenotypic plasticity in limiting environments (Aznar et al., 2018). Previous studies had already shown that larvae reared in restricted diets might be associated with prolonged life in Ae. aegypti (Joy et al., 2010; Zeller and Koella, 2016) and Anopheles sp. (Vantaux et al., 2016).

The positive correlations between the presence of microbiota and larval development might be another way to explain the results observed in larvae fed with Asaia sp. and E. coli. The acetic acid bacterium genus Asaia have been shown to be stably associated with larvae and adults of anophelines and Ae. aegypti (Favia et al., 2007; Damiani et al., 2010; Ricci et al.,


NutritionalamountsofsolubleandtotalinA.larvaeraisedindifferentdiets,and

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2012a; Rossi et al., 2015). Mitraka et al. (2013) showed that a diet supplemented with Asaia in the Anopheles gambiae larval environment had a significant boost in developmental rate and Chouaia et al. (2012) observed a delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts. Besides the effects on development rates, a possible mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes (Rossi et al., 2015). These results drive us to believe that Asaia may play a significant role in mosquito larval development. Although the molecular nature of the developmental improvement caused by the Asaia symbiont needs to be identified, these bacteria can be considerate as candidate paratransgenic vehicle for the control of mosquito-borne diseases (Favia et al., 2008; Ricci et al., 2012b; Mitraka et al., 2013).

Coon et al. (2017) showed that each mosquito species including Ae. aegypti contains a simple bacterial community and that the composition of bacterial gut communities can also be strongly influenced by diet. Their results also showed that axenic larvae could not develop, but several community members and E. coli can rescue the larval development. Using E. coli K-12 as a model for studies of molecular interactions that underlie bacteria-dependent growth of larvae into adults, Coon et al. (2017) unveiled one of the molecular mechanisms involved in mosquito development. They showed that bacteria through the cytochrome b oxidase gene mediate a reduction of oxygen levels in the digestive tract of larvae, working as a signal for ecdysone-induced molting. Thereby, E. coli plays an essential role in mosquito development and may have important implications to be used in symbiont-based control techniques for disabling the growth of larvae into mosquito adults.

S. marcescens, Bacillus sp., O. intermedium, and S. aureus had a lower adult rate survival contrary to results described in diets with Asaia sp. and E. coli (**Supplementary Table S4**). The effect of a restricted diet in mosquito larva is a rather speculative issue. Previous studies reported that dietary restrictions can lead to longer development rates (Olivo et al., 1979; Tun-Lin et al., 2000; Arrivillaga and Barrera, 2004; Couret et al., 2014; Levi et al., 2014; Aznar et al., 2018), with larvae extending time to achieve the pupal stage (Chambers and Klowden, 1990; Telang et al., 2007; Foster et al., 2012; Banerjee et al., 2015). Some individuals with slower growth rates try to counterbalance this deficit through compensatory growth (Wilson and Osbourn, 1960). This ecological factor allows that once the same nutritionally deprived individuals have the opportunity to acquire more food, speed up or slow down the growth rates to employ the food resources in the maintenance of important biologicals traits as reproduction and survival (Dmitriew, 2011; Dmitriew and Rowe, 2011; Zeller and Koella, 2016).

Bacillus sp. and S. marcescens presented only two adults (**Supplementary Table S1**). S. marcescens is recognized by its entomopathogenic properties in some conditions (Flyg et al., 1980; Flyg and Xanthopoulos, 1983). However, in this study, it does not appear to be associated with infections in Ae. aegypti larvae. In the present study, larvae fed with bacterial diets, including S. marcescens, remained in the third instar for several weeks. Other studies have been shown that withstand to starvation could be measured by the time spent in the third larval instar without adequate nourishment (Wigglesworth,

1942). The duration of development from the L1 larval stage to adult mosquito is faster when food is abundant (Tun-Lin et al., 2000); thus, the prolongation in third instar larvae observed in our results may indicate a state of malnutrition. We did not observe the presence of melanized-killed larvae or any specific phenotype more related to pathogenicity. However, previous studies have already reported that food restriction alters immunological traits in Ae. aegypti (Grimstad and Walker, 1991; Alto et al., 2005, 2008; Muturi et al., 2011; Telang et al., 2012). More studies are necessary to evaluate the influence that the diets used in this study exert on the immune system of Ae. aegypti larvae.

Lowering of growth rates may be a response to dietary stress and adaptive behavior in calorie-depleted environments (Arendt, 1997; Badyaev, 2005). It is possible that Ae. aegypti larvae raised under bacterial diets suffer nutritional depletion, resulting in developmental delays. In natural environments, mosquitoes are commonly found with reduced sizes and low energy reserves. This remarkable capacity of withstanding starvation situations and recovery later in more favorable conditions can justify the great success for the establishment of these insects in the environment (Barrera, 1996; Barrera and Medialdea, 1996; Zeller and Koella, 2016). Therefore, our data show that Ae. aegypti larva can develop in all bacterial diets tested, even at a higher ecological cost.

In natural conditions, the biomass of algae is the major content of mosquito larvae guts (Hinman, 1930; Garros et al., 2008). Thus, algae seem to play an important role in the development and survival of larvae. Ae. aegypti larvae were not able to develop in microalgae diets with the standard concentration established for the other diets (16 mg/larva), so we adjusted the values offered to lower concentrations (3 mg/larvae). The development failure observed in the standard concentration (data not shown) might have been caused by several factors among them the junction between a controlled photoperiod environment as well as a low larval density. Together these factors might have accelerated the exponential microalgae growth triggering a toxicological response by larvae which impaired their development.

Larvae fed with the microalgae Chlorella sp or with A. platensis (3 mg/larvae) showed a delay in development similar to the observed in bacterial diets, but with shorter adult survivals (**Supplementary Table S4**). Kivuyo et al. (2014) evaluated larval survival and the development rate of the aquatic stage using green filamentous algae and dry powdered filamentous algae as diet. The survivorship and pupation rates in algae food diet had the worst performance between all foods assessed. Some species of algae seem to be resistant to digestion and are discarded entirely after passage through the larval gut (Laird, 1988). However, there is no evidence that Chlorella sp. and A. platensis belong to this specific group.

Interestingly, a genus of Chlorella was used as a larvicide against Ae. aegypti by Borovsky et al. (2016). Chlorella desiccata was engineered to express an insect peptide hormone, the trypsin modulating oostatic factor (TMOF), that is recognized by Ae. aegypti ovaries and controls the translation of the gut's trypsin mRNA. Feeding mosquito larvae with transformed C. desiccata cells kill by starvation 60% of Ae. aegypti larvae in 4 days (Borovsky et al., 2016). A. platensis (Spirulina) have never been used in nourishment experiments with mosquito larvae before. Our results showed that Chlorella and A. platensis could be used as a food source by mosquito larvae, but with slower developmental rates. Their rapid grow under heterotrophic culture, with a relatively low cost, make these microalgae interesting candidates for future methods of vector control through genetic engineering (Dawson et al., 1997; Madkour et al., 2012).

#### Sex-Related Parameters in A. aegypti Raised on Microorganism-Based Diets

The biological parameters evaluated in Ae. aegypti were analyzed separately for males and females in all diets (**Tables 1**–**5** and **Supplementary Table S6**). The results showed that, as already described in the literature, males take less time to reach adulthood and survive longer. When mosquitoes are fed only water as adults, the only nutritional resources available lie in reserve acquired during immature stages. The transference of reserves from the larval nutrition to the mosquito adult stage using only microbial cells as nourishment has never been investigated before. Thus, it is possible that males show more withstand to starvation during the larval stage and a lower nutritional threshold for pupation than females. These traits might have influenced the better developmental rates observed in males and increased their survival. The measured lifespan of adult males and females fed with nectar is approximately 9 weeks and around 12–17 weeks for females that were also blood fed (Putnam and Shannon, 1934). We were not able to find available data in the literature to compare the mortality rates of males and females under laboratory conditions without any nutritional stimulus. Aznar et al. (2018) reported that treatments with scarcity or excess of food might preferentially influence the proportions and survival of females over males. They have shown that females exhibit a larger extension of development time in response to food deprivation than males, and relate this result to a male fitness advantage, ability of an individual to survive, reproduce and spread genes, this advantage is obtained when males emerge early and can copulate with non-mated females. The longer development time on females is similar to the observed under a competitive environment (Bedhomme et al., 2003). Under variated conditions, it is expected that females take more time to develop and spend more time to enhance their fitness abilities, achieving larger body sizes and thus boost their fecundity (Bedhomme et al., 2003; Wormington and Juliano, 2014). Previous field studies already reported a faster development in a male over female mosquito larvae, that showed a slower development and a higher mortality rate (Yates, 1979).

Imbalances in sex ratio (males: females) were observed in all diets tested (**Supplementary Table S6**). This male-biased sex ratio may result from underfeeding. Some studies support a sex-related difference in larval nutrient metabolism, possibly due to the earlier ecdysteroid peak in Ae. aegypti male during pupal-adult development (Brust, 1967; Whisenston et al., 1989;

Chambers and Klowden, 1990; Puggioli et al., 2013; Balestrino et al., 2014). In field studies, distortion in sex ratio is frequent and associated to the slow development of female larvae and differential response of the sexes to egg hatching stimuli (Yates, 1979; Shroyer and Craig, 1981; Sims and Munstermann, 1983; Frank et al., 1985; Lounibos and Escher, 2008). Other studies observed a density-dependent alteration and a sexspecific response to a critical day period time, trough feedback mechanisms that are dependent on density or mortality by selective sexual predation (Chambers, 1985; Frank et al., 1985; Alto et al., 2012). The highest proportion of males observed in this study seem favorable to future studies using the Sterile Insect Technique (SIT). A greater male pupae production is important to SIT mass rearing and determine the number of males that can be selected for release in the natural environment (Puggioli et al., 2016).

In this study, it was also possible to realize that microbial diets (yeast and Gram-negative bacteria) influenced the wing size of Ae. aegypti adults, and presumably the whole body size, of both genders. The correlation of wing size with food concentration is interesting, mainly because the "centroid size" (Bookstein, 1992) is considered a more informative estimator of body size than the traditional size measurements (Jirakanjanakit and Dujardin, 2005; Jirakanjanakit et al., 2007). As previously mentioned, we quantified the wing centroid size as a readout of the adult size. In general, the wings of the mosquitoes fed with Tetramin <sup>R</sup> in their immature stages were larger than those that were fed with microorganism-based diets. Also, in all comparisons, females showed larger wings than males. Sexual dimorphism is present in wing shape and size of Ae. aegypti (Virginio et al., 2015; Sánchez and Liria, 2017). However, independently of the inherent wing sexual dimorphism, and some asymmetry, we recorded some patterns of differentiation. In males, the group fed with E. coli showed lower sizes than the Asaia sp. group, which in turn was more similar to Tetramin <sup>R</sup> . In females, Asaia sp. and E. coli were lower than Tetramin <sup>R</sup> , although Asaia sp. has been closer to the Tetramin <sup>R</sup> diet. On the yeast diets, males of Pseudozyma sp. and S. cerevisiae showed similar scores, and females of the Pseudozyma sp. were lower than S. cerevisiae, both being lower than the Tetramin <sup>R</sup> score. Among all the microorganism-based diets tested, S. cerevisiae showed the wing sizes that are closest to the Tetramin <sup>R</sup> diet. Jong et al. (2017), showed significant shorter wings in A. albopictus under sub-optimal food availability. Jirakanjanakit et al. (2007) and Aznar et al. (2018) reported that low food concentration in Ae. aegypti immature stages could alter mosquito size. Those results support this study, suggesting a strong influence of microbial food composition on mosquito body size.

# Nutritional Analysis of Microorganism-Based Diets and Nutrient Acquisition by Larvae

The differences reported in the developmental time and adult size with the microbial diets are consistent with other studies on environment stress (Badyaev, 2005). Response to stress conditions such as nutritional limitation (Bubliy et al., 2000), extreme temperatures (Sisodia and Singh, 2009) and larval crowding (Imasheva and Bubliy, 2003) in Drosophila have been studied and related to genetic modifications. Schneider et al. (2011) demonstrated a genotype variation in Ae. aegypti adult size in response to larval food depletion, reporting wide phenotypic plasticity and adaptive behavior to changing environments. Aznar et al. (2018), suggested that the variety in food stress conditions in the natural habitat can increase genetic modifications in Ae. aegypti. This genetic variety, as mutation and recombination rates, is usually hidden under regular food conditions and facilitates the development of novel adaptations to adverse environments (Badyaev, 2005).

The next step was to analyze the nutritional quality through the measurement of the main energetic components of diets. Our goal was to observe if these developmental and size variations are a result of malnourishment. The nutritional requirements of mosquitoes are divided into two majority classes: macronutrients (energetic nutrients) and micronutrients (nonenergetic nutrients) (Singh and Brown, 1957; Foster, 1995; Arrese and Soulages, 2010; Canavoso et al., 2011; Rivera-Pérez et al., 2017). Aquatic environments with abundant nutritional richness supply all the energy necessary to mosquito larvae metamorphosis. Carbohydrates and proteins are among the main nutritional requirements of Ae. aegypti (Singh and Brown, 1957).

The nutritional values of the diets were evaluated through protein and total sugar quantification (**Table 6**). Chlorella sp and A. platensis showed higher protein amounts than the other diets, including the standard diet Tetramin <sup>R</sup> . These results were already expected once both species are rich in proteins and can be employed even for human consumption (Mühling, 2000; Guccione et al., 2014; Bleakley and Hayes, 2017). Yeast diets also presented robust protein values. S. cerevisiae has more protein than Pseudozyma sp. Souza et al. (2016) showed that S. cerevisiae contains higher protein amounts than the standard diet Cat food. However, Tetramin <sup>R</sup> seems to be more nutritive and present a superior protein amount. Bacillussp., S. marcescens, S. aureus, and O. intermedium showed lower protein values than E. coli and Asaia sp. These results coincide with the developmental pattern observed (**Supplementary Tables S1–S5**). As previously mentioned, protein and amino acids consumption are directly related to growth and development in mosquitoes (Golberg and De Meillon, 1948; Merritt et al., 1992). Scarce consumption of proteins during the immature stages might interfere in life cycle duration, adult emergence, body size and fecundity (Singh and Brown, 1957; Dadd, 1977). Thus, our results are in line with other studies, showing that badly nourishment leads to delayed larval development and adults with low energetic reserves (Timmermann and Briegel, 1999; Arrivillaga and Barrera, 2004; Telang et al., 2007; Banerjee et al., 2015; Zeller and Koella, 2016).

The sugar amounts in S. cerevisiae and Pseudozyma sp. were higher than in the other diets. S. cerevisiae did not show significant differences (p < 0,005) when compared to the standard diet (**Table 6**). All the bacterial diets had low sugar contents and E. coli and Asaia sp. presented the

best values, reinforcing our hypothesis that both bacteria are better sources for larval feeding. Chlorella sp. and A. platensis presented high sugar values. However, in contrast to the protein amounts, their sugar content is lower than the yeast or the standard diet. Carbohydrate is directly associated with pupation (Chambers and Klowden, 1990; Telang et al., 2007). Carbohydrate storage is associated to pupal commitment and larval growth (Sneller and Dadd, 1977; Van Handel, 1988; Telang et al., 2007), and the low levels observed in our results suggest that larvae have not sufficient sugar to achieve the pupal stage in these diets, resulting in developmental delay. Although the microalgae species had a significant amount of protein and sugar, the development time in these diets was prolonged, leading us to believe that Ae. aegypti larvae may not assimilate nutrients adequately from these organisms.

Besides the nutritional quality measurements, we analyzed the nutrient reserves of Ae. aegypti larvae. We have undertaken a comparative study in the larval development rates and their storage of protein, free carbohydrates, glycogen and lipids concerning different dietary conditions. There is a positive correlation between body mass and caloric reserves stored during the larval stage, triggering endocrine responses that lead to insect molting (Chambers and Klowden, 1990). Insects must achieve a minimum weight in the immature stage in order for continuing their development (Nijhout and Williams, 1974; Nijhout, 1975; Lounibos, 1979; Safranek and Williams, 1984; Chambers and Klowden, 1990; Davidowitz et al., 2003; Lan and Grier, 2004; Mirth et al., 2005). In Ae. aegypti larvae, the metamorphic capacity depends on nutritional reserves and needs a minimum critical mass that is estimated between 2.7 and 3.2 mg (Telang et al., 2007). Critical mass is defined as the mass that results in 50% of starved larvae achieving the pupal stage. That usually occurs 24 h after the transformation into the final fourth instar, in optimal conditions (Chambers and Klowden, 1990; Davidowitz et al., 2003; Lan and Grier, 2004; Telang et al., 2007). In this minimum period these larvae require to acquire food so that at least 50% of them pupate and emerge as adults, and at this age, the ecdysteroid production begins to rise (Telang et al., 2007). Nutrient intake and energetic accumulation are important factors to metamorphosis. Most larvae that are starved after reaching their critical weight will molt, but if larvae are starved before they have achieved the critical weight, metamorphosis is delayed, or they eventually die without initiating this process (Chambers and Klowden, 1990; Davidowitz et al., 2003; Telang and Wells, 2004).

Insects fed exclusively with bacteria and microalgae seem unable to accumulate sufficient nutrients during the larval stage to reach the minimum critical mass required for pupation in regular time (**Table 7**). Restrictive food amount in Ae. aegypti larvae delay pupation, but larvae remain able to pupate even in underfeeding conditions if sufficient energetic accumulation has been done in previous larval stages (Telang et al., 2007). Thus, the delay observed in our results in the metamorphosis rates might afford to larvae an additional period to feed, grow, and meet the critical mass to achieve the pupal stage. In all diets tested, the energetic larval contents were lower than in larvae fed with the standard diet Tetramin <sup>R</sup> . These results were expected, once the insect physiological parameters were strongly affected (**Supplementary Tables S1–S5**). Additional evidence for developmental delays comes from studies in Manduca sexta. The underfeeding in M. sexta during the last larval stage resulted in death or a delay in metamorphosis (Nijhout and Williams, 1974).

The protein storage in larvae fed with bacteria or algae showed the lowest proteins levels and Asaia sp. and E. coli showed better levels than the others including Chlorella sp. (54 ± 19.1 mg) and A. platensis. Although microalgae have a high protein content, Ae. aegypti seems not able to store this protein satisfactorily. The energetic reserves in gut and rest of body of all diets tested were proportional to values obtained in whole larvae, and the rest of the body showed higher contents than the gut (**Table 7**). Yeast-fed larvae showed the highest levels of protein, suggesting that Ae. aegypti larvae can process and store this protein efficiently. In immature stages, proteins play an important role in metabolic processes as well for adults of several insect species (Hagen et al., 1984; Zucoloto, 1988). In immature stages ingestion of proteinaceous food is essential for growth, survival, nutritional reserve for the pupation and utilization in the adult stage, principally for egg production (Chan et al., 1990). Our results are in line with the decline in body size observed in Ae. aegypti, Culex pipiens, Anopheles albimanus, and Anopheles gambiae caused by different feeding regimes (Timmermann and Briegel, 1999).

Carbohydrates content in bacterial and microalgae diets showed significant storage differences (p < 0.0001). Larvae fed with S. cerevisiae and Pseudozyma sp. presented carbohydrates reserves similar to the standard diet, showing no significant differences (p > 0.05). As expected, E. coli and Asaia sp. had better carbohydrates storage amounts than other bacteria and microalgae, reinforcing the potential of these diets. Carbohydrate is necessary for optimal growth and developmental rates in larvae, and nutritional environment with low levels of sugar leads to a substantially delayed growth (Sneller and Dadd, 1977). Previous studies with M. sexta suggested the dependence of sugar ingestion to reaching the pupa stage and the requirement to both dietary sugar and protein to growth until the adults (MacWhinnie et al., 2005). Carbohydrate role in Ae. aegypti larvae development is associated with pupal commitment and an inverse relation between hemolymph trehalose levels, and juvenile hormone titers have been described (Jones et al., 1981; Chambers and Klowden, 1990; Telang et al., 2007). Therefore, our data confirm that sugar and other digestible carbohydrates are required for adult stage maintenance and larval development.

The larvae glycogen storage showed significant differences (p = 0.0001) in all diets tested. Bacterial diets resulted in low glycogen contents, and Asaia sp. and E. coli showed higher rates than other bacteria diets. Larvae fed with S. cerevisiae and Pseudozyma sp. showed better glycogen levels, confirming the excellent storage capacity of Ae. aegypti in yeast diets. Past studies reported a critical threshold of larval glycogen as a stimulant to metamorphosis through a drastic drop in juvenile hormone titer and a concomitant

increase in ecdysone that trigger molting (Chambers and Klowden, 1990; Timmermann and Briegel, 1999). Telang et al. (2007) reported that the timing of ecdysteroid release is not critical to start the larval-pupal molt for Ae. aegypti larvae, however, both the ecdysteroid titer and the nutritional condition of fourth instars are determinant factors in initiating the metamorphic molt. Earlier studies indicated that larval nutrient reserves (protein, lipid, and glycogen) are important for egg production and the endocrine regulation of egg development in Ae. aegypti and Ochlerotatus atropalpus (Telang et al., 2006). High levels of glycogen and protein overtake a threshold set in the insect nervous system that activates ovarian ecdysteroid production and inhibits juvenile hormone biosynthesis by the corpora allata, which together enable vitellogenesis and egg production. Without sufficient threshold levels, the corpora allata increase the juvenile hormone levels secretion, decreasing ovarian ecdysteroid production and as consequence egg maturation is delayed (Telang et al., 2006).

The low glycogen reserve in Ae. aegypti larvae fed with bacteria and microalgae might have affected the insect neuroendocrine system, resulting in the delaying of pupation time, due to a lack of the minimum glycogen threshold required for a successful molt (Nijhout and Wheeler, 1982; Chambers and Klowden, 1990; Lan and Grier, 2004; Noriega, 2004; Telang and Wells, 2004; Margam et al., 2006; Telang et al., 2006, 2007). Glycogen storage seems to be a larval strategy valid to withstand starvation when the larva is waiting for additional nutrition during the last larval instar, under badly nourishment conditions (Timmermann and Briegel, 1999). This strategy might be used by larvae fed with E. coli and Asaia sp., which showed higher glycogen levels and prolonged their survival.

Levels of lipids were lowest in larvae fed with S. marcescens and Bacillus sp. These diets showed the worst developmental rates (**Supplementary Table S1**) with a severe delay in all biological parameters evaluated (**Supplementary Tables S1– S5**). Although, the other experimental diets have also shown significant and substantial differences (p < 0.05) in the lipid contents. Lipid reserve influence pupal commitment, and the endocrine regulation of egg development in autogenous and anautogenous female mosquitoes (Briegel, 1990b; Foster, 1995; Ziegler and Ibrahim, 2001; Briegel et al., 2002; Zhou et al., 2004; Telang et al., 2006). Under favorable nutritional conditions, lipids start accumulating after glycogen has reached a plateau (Van Handel, 1984). Environments with nutritional stress lead to drastic reductions in whole-body lipid, protein and carbohydrate contents (Briegel, 1990b). Due to the diets poor in nutrients (**Table 6**), Ae. aegypti larvae storage lesser amounts of energetic components (**Table 7**), affecting the developmental rates directly. The metamorphic capacity clearly depends on all four nutrient reserves in different ways.

Nutrients accumulated by larvae are correlated with adult emergence and body size (Telang et al., 2007; Zeller and Koella, 2016). Nutrition, temperature and larval density also influence growth, development, energy reserves, egg production, longevity of adult females, immunity, vector capacity and insecticide-resistance (Stearns and Koella, 1986; Briegel, 1990a,b; Kitthawee et al., 1992; Lyimo et al., 1992; Juliano and Stoffregen, 1994; Ameneshewa and Service, 1996; Telang et al., 2006, 2007; Murrell and Juliano, 2008; Reiskind and Lounibos, 2009; Arrese and Soulages, 2010; Muturi et al., 2011; Kulma et al., 2013; Murray et al., 2013). Larvae fed with S. cerevisiae and Pseudozyma sp. were not affected in body weight, what is expected as these diets showed good developmental rates, high nutritional quality, and efficient energetic accumulation. The fresh mass of larva fed with bacteria and microalgae were significantly lower (p < 0.05) when compared to controls. In natural environments, it is common to find mosquitoes with reduced sizes and low energy reserves (Zeller and Koella, 2016). Telang et al. (2007) showed that nutritional stress leads to smaller adults with reduced hemocyte numbers. Muturi et al. (2011) reported that nutritional stress during larval development cause changes in phenotype and immunity of mosquitoes and increased susceptibility of these adults to pathogens. Therefore, the nutritional quality of larvae in diets with microorganisms may affect not only the entire life history of Ae. aegypti, but also their size, immunity, and vector competence.

#### Larval Preference for Ingestion of Microorganisms

Our results showed that Ae. aegypti larvae consumed all microorganisms used at different rates (**Figure 7**). The guts of larvae fed with S. cerevisiae, Pseudozyma sp., and E. coli presented high mean fluorescent intensity after 2 h of feeding. The lower consumption rates in other diets might suggest a possible food preference. Despite that, more studies must be performed, increasing the feeding exposure time. We have not found studies involving food preference in Ae. aegypti larvae, hence exploring this subject is necessary for a better understanding of larval physiology.

# CONCLUSION

Our results provide new knowledge into the effect of microorganism-based diets in different larval biological parameters as developmental rates, pupation time, emergence, survival, lifespan, and wing size. Larvae fed with bacteria and microalgae shown lethargic development and low survival, due to bad nourishment and low energetic reserve accumulation. Asaia sp. and E. coli seem the best bacterial models for future studies aiming the development of symbiont-based methods for mosquito control. Larvae fed with yeasts showed developmental rates that are similar to the standard diet Tetramin <sup>R</sup> , being nutritional rich and providing high energetic storage. S. cerevisiae and Pseudozyma sp. seem suitable candidates to improve mosquito laboratory breeding and a low-cost diet to mosquito mass rearing. Ae. aegypti larvae ingested all the tested microorganisms 2 h after addition of them to the water. Therefore, Ae. aegypti larvae showed very high plasticity about feeding, being able to develop under different microbial diets.

#### AUTHOR CONTRIBUTIONS

fphys-10-00152 April 8, 2019 Time: 17:47 # 19

RS and FV performed the experiments and analyzed the data. RS, FV, TR, LS, JB, and FG conceived the study. All authors wrote and revised the manuscript.

#### FUNDING

JB, LS, and FG are fellows of CNPq. LS, FV, JB, and FG are members of their respective departments. RS is a Ph.D. student of Oswaldo Cruz Institute and received a grant from CAPES.

#### REFERENCES


# ACKNOWLEDGMENTS

We thank Fernanda Almeida for technical support in microscopy and Dr. José Bento Pereira Lima for supplying Aedes aegypti eggs.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2019.00152/full#supplementary-material


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Zeller, M., and Koella, J. C. (2016). Effects of food variability on growth and reproduction of Aedes aegypti. Ecol. Evol. 6, 552–559. doi: 10.1002/ece3.1888

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Souza, Virginio, Riback, Suesdek, Barufi and Genta. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dietary Stress From Plant Secondary Metabolites Contributes to Grasshopper (Oedaleus asiaticus) Migration or Plague by Regulating Insect Insulin-Like Signaling Pathway

Shuang Li<sup>1</sup>† , Xunbing Huang<sup>2</sup> \* † , Mark Richard McNeill<sup>3</sup> , Wen Liu<sup>2</sup> , Xiongbing Tu1,4 , Jingchuan Ma1,4, Shenjin Lv<sup>2</sup> and Zehua Zhang1,4 \*

#### Edited by:

Su Wang, Beijing Academy of Agriculture and Forestry Sciences, China

#### Reviewed by:

Yifan Zhai, Shandong Academy of Agricultural Sciences, China Qiong Rao, Zhejiang Agriculture and Forestry University, China

#### \*Correspondence:

Xunbing Huang xunbingh@163.com Zehua Zhang lgbcc@263.net †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 22 July 2018 Accepted: 15 April 2019 Published: 03 May 2019

#### Citation:

Li S, Huang X, McNeill MR, Liu W, Tu X, Ma J, Lv S and Zhang Z (2019) Dietary Stress From Plant Secondary Metabolites Contributes to Grasshopper (Oedaleus asiaticus) Migration or Plague by Regulating Insect Insulin-Like Signaling Pathway. Front. Physiol. 10:531. doi: 10.3389/fphys.2019.00531 <sup>1</sup> State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, <sup>2</sup> College of Agriculture and Forestry Science, Linyi University, Linyi, China, <sup>3</sup> Canterbury Agriculture and Science Centre, AgResearch, Christchurch, New Zealand, <sup>4</sup> Scientific Observation and Experimental Station of Pests in Xilin Gol Rangeland, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Xilinhot, China

Diets essentially affect the ecological distribution of insects, and may contribute to or even accelerate pest plague outbreaks. The grasshopper, Oedaleus asiaticus B-Bienko (OA), is a persistent pest occurring in northern Asian grasslands. Migration and plague of this grasshopper is tightly related to two specific food plants, Stipa krylovii Roshev and Leymus chinensis (Trin.) Tzvel. However, how these diets regulate and contribute to plague is not clearly understood. Ecological studies have shown that L. chinensis is detrimental to OA growth due to the presence of high secondary metabolites, and that S. krylovii is beneficial because of the low levels of secondary metabolites. Moreover, in field habitats consisting mainly of these two grasses, OA density has negative correlation to high secondary metabolites and a positive correlation to nutrition content for high energy demand. These two grasses act as a 'push-pull,' thus enabling the grasshopper plague. Molecular analysis showed that gene expression and protein phosphorylation level of the IGF → FOXO cascade in the insulin-like signaling pathway (ILP) of OA negatively correlated to dietary secondary metabolites. High secondary metabolites in L. chinensis down-regulates the ILP pathway that generally is detrimental to insect survival and growth, and benefits insect detoxification with high energy cost. The changed ILP could explain the poor growth of grasshoppers and fewer distributions in the presence of L. chinensis. Plants can substantially affect grasshopper gene expression, protein function, growth, and ecological distribution. Down-regulation of grasshopper ILP due to diet stress caused by high secondary metabolites containing plants, such as L. chinensis, results in poor grasshopper growth and consequently drives grasshopper migration to preferable diet, such as S. krylovii, thus contributing to grasshopper plague outbreaks.

Keywords: grasshopper, diet stress, gene, plague, plant secondary metabolites

# INTRODUCTION

fphys-10-00531 May 2, 2019 Time: 17:43 # 2

Nearly half of all insect species are herbivores (Gatehouse, 2002; Wu and Baldwin, 2010), that have co-evolved with plants for 350 million years (Kessler and Baldwin, 2002). Phytophagous insects have specific adaptability to various host plants (Howe and Jander, 2008; Rominger et al., 2009; Unsicker et al., 2010), which also determine their ecological distribution and population dynamics (Whitman, 1990; Franzke et al., 2010). Such phytophagous insect-host plant relationships are examples of co-adaptation, co-evolution, and co-speciation (Scriber, 2002; Powell et al., 2006; Zhang and Fielding, 2011).

Plants have evolved various defense mechanisms against phytophagous insect (Padul et al., 2012). Such defenses can be broadly classified into two categories: constitutive defenses, including physiological barrier and nutritional hurdle; and inducible defenses, including secondary metabolites and protein inhibitors (PIs) (Zavala et al., 2004; Wu and Baldwin, 2010; Padul et al., 2012). Both types are achieved through similar means but differ in that constitutive defenses are present before herbivore attacks, while induced defenses are activated only when attacks occur (Wu and Baldwin, 2010; Padul et al., 2012). The regulatory elements in networks that modulate herbivory induced responses in plants mainly include jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) (Baldwin, 1998; Wu and Baldwin, 2010; Kessler and Baldwin, 2002). Plant defense mechanisms are also categorized into direct and indirect responses according to their role and function (Wu and Baldwin, 2010). Indirect defenses include volatile organic compounds produced when the plant is subject to herbivory that attract predators and parasitoids of the insect (Dicke and Loon, 2000; Despland and Simpson, 2005; Despres et al., 2007). Plant chemistry, especially those associated with secondary metabolites, is an important component of the phenotype that mediates plant-insect interactions (Mendelsohn and Balick, 1995; Despres et al., 2007). In general, insect dietary stress predominantly originates from high levels of plant secondary metabolites (Behmer, 2009; Despres et al., 2007; Wetzel et al., 2016).

Conversely, insect herbivores also evolved various detoxification mechanisms, mainly including avoidance, excretion, sequestration, metabolic resistance, and target mutation, which allow them to consume and develop on toxic plants producing high levels of secondary metabolites (Despres et al., 2007). Such insect feeding continues the selective pressure on plants to develop increased or novel chemical defenses (Musser et al., 2002; Becerra, 2003; Dussourd, 2003; Helmus and Dussourd, 2005). Insect herbivores' response to diet stress are well-documented in many insects, mainly focusing on insect behavioral, physiological, chemical, genetic, ecological, and evolutionary mechanisms (Raubenheimer and Simpson, 2004; Giri et al., 2006; Dicke and Baldwin, 2010; Ibanez et al., 2013). Some herbivorous species are strongly attractive or indispensable for some specific plant species (Schutz et al., 1997). Existence of those plants may contribute to, or even accelerate, insect population outbreaks (Powell et al., 2006). For example, Phragmites australis (Cav.) Trin. provides an optimal food source (Zhu, 2004; Ji et al., 2007), which could significantly benefit Locusta migratoria manilensis (Meyen) population growth.

Many genes and related pathways of insects, such as the insect insulin-like signaling pathway (ILP), play important roles in specific insect-diet relationship, and contribute to the variation of insect performance (Bishop and Guarente, 2007; Taguchi and White, 2008; Ragland et al., 2015). The insect ILP is considered to act as a sensor of the dietary status and to stimulate the progression of anabolic events when the status is positive (Taguchi and White, 2008; Badisco et al., 2013). It plays a crucial role in a number of fundamental and interrelated physiological processes, including insect growth, energy metabolism, and detoxification (Claeys et al., 2002; Wu and Brown, 2006; Kawada et al., 2010; Fujisawa and Hayakawa, 2012). Many studies in different metazoan species have indeed demonstrated that not only the insulin-related peptides are evolutionarily conserved, but also the components of their signaling pathway, such as IGF/INSR/IRS/PI3K/PDK/AKT/FOXO (**Figure 1**), which play an important role in insulin resistance, are also conserved (Sim and Denlinger, 2008; Badisco et al., 2013). Insulin signaling can be delivered by phosphorylation or dephosphorization of proteins, such as INSR/IRS/AKT/FOXO (Kramer et al., 2008; Hedrick, 2009). ILP changes associated with diet stress can influence insect growth (Kawada et al., 2010; Fujisawa and Hayakawa, 2012; Badisco et al., 2013), which can potentially affect pest distribution or even plague outbreaks. However, the role of the ILP signaling pathway in regulating pest plague outbreaks is poorly understood.

FIGURE 1 | IGF-PI3K-AKT-FOXO pathway of insulin signaling. The insulin-like signaling system includes different well-defined ligands, such as insulin-like growth factor (IGF), which regulate the activity of the homologous insulin receptor INSR. Insulin receptor substrate (IRS) proteins act as messenger molecule-activated receptors to signaling, and which is an important step in insulin's action. Phosphoinositide 3-kinase (PI3K), 3-phosphoinositide-dependent protein kinase (PDK), and protein kinase B (AKT), three major nodes downstream of IRS, and have been implicated in many of the metabolic actions of insulin. The forkhead transcription factor (FOXO) regulates transcription of genes involved in stress resistance, xenobiotic detoxification and DNA repair. FOXO is negatively regulated by insulin-like signaling when the PI3K → AKT cascade stimulates phosphorylation of FOXO and promotes its secretion from the nucleus and inactivation in the cytosol.

Oedaleus asiaticus Bey-Bienko is a specialist grass-feeder, with preference for Poaceae species, particularly Stipa krylovii Roshev (Poaceae) (Zhang et al., 2013; Qin et al., 2017). It is a member of the subfamily Oedipodinae (Orthoptera: Acrididae: Oedipodinae), and a dominant grasshopper of northern Asian grasslands, generally distributed in Inner Mongolia of north China (Cease et al., 2012; Zhang et al., 2014). Outbreaks of O. asiaticus have often lead to significant loss in grasses and economic disruption (Liu et al., 2013). From routine surveys of plant and grasshoppers composition in Stipa (S. krylovii) and Leymus (Leymus chinensis) (Trin.) Tzvelev (Poales: Poaceae) grasslands (Han et al., 2008), we found that O. asiaticus was mainly confined to the former (Huang et al., 2016, 2017a). In addition, we also found that the presence of plant secondary metabolites in L. chinensis can have a negative impact on O. asiaticus growth parameters (Huang et al., 2017a), while acting as a catalyst to drive grasshopper migration and plague outbreak. In the present study, we investigated how diet stress influences insect growth, distribution, and the ILP, to decipher the relationship between diet stress and pest outbreaks. We also discuss the role of diet stress in driving pest plague outbreaks, and how this information provides new insights into pest management.

# MATERIALS AND METHODS

#### Ethics Statement

Insects (O. asiaticus) were collected from the Xilin Gol grassland from 2011 to 2017. It is a common agricultural pest and not in the "List of Protected Animals in China." No permits were required for the described field studies.

#### Study Area

The research grassland (E115◦ 130–117◦ 060 , N43◦ 020–44◦ 520 ) is located in the Xilin Gol League, Inner Mongolia, northeast China. This grassland is a region representative of the Eurasian steppe grassland and characterized by L. chinensis- and S. krylovii dominated plant communities (Han et al., 2008). The aboveground biomass of these two plants accounts for more than 80% of the total community production (Huang et al., 2016). Plants in this grassland cover only 30 to 40% of the ground area with the remainder being bare ground for rapid steppe degradation in part driven by livestock over-grazing (Chen and Wang, 2000; Han et al., 2008). O. asiaticus is the dominante grasshopper species (Guo et al., 2006), and generally hatches between late-May and late-June, reaching adulthood between early to late July (Huang et al., 2016). This grasshopper mainly feeds on Poaceae species, particularly S. krylovii (Wu et al., 2012; Zhang et al., 2013).

#### Large-Scale Survey of Plant Biomass and O. asiaticus Density on Grazed Grassland

We examined the relationship between above-ground plant biomass composition and O. asiaticus density in this grazed grassland area, in mid-July for each year from 2011 to 2017. Those areas were mainly dominated by S. krylovii and L. chinensis, with rare distribution of other plant species. Each year, we selected eight 1 km<sup>2</sup> sample plots (∼10 km apart). In each plot, we selected five 1-m<sup>2</sup> quadrats (∼50 m apart) randomly. Grass S. krylovii and L. chinensis plants within each quadrat were cut to ground level and placed separately into envelopes. Those collected grass were dried at 90◦C for 24 h, and weighed to provide the relative mean above ground biomass (g DM/m<sup>2</sup> ) of the two plant species for each plot.

We estimated the relative density of the grasshopper O. asiaticus in each plot in mid-July for each year 2011 to 2017, using the same method described in our previous published paper (Huang et al., 2016). We averaged the four samples in each plot, to derive a relative O. asiaticus density (number of individuals per 100 sweep-nets) for each of the eight plots in each year.

Sampling produced eight means (one for each of the eight 1 km<sup>2</sup> plots) for both grass species and O. asiaticus relative densities for each year from 2011 to 2017.

#### Cage Study of O. asiaticus Growth in Grassland

To study O. asiaticus growth for different host plant species, a field cage study was carried out on S. krylovii and L. chinensis grasslands during late June of both 2016 and 2017. In each of those two grasslands, all of the other plants were removed to assure that only one host plant remained. We constructed 10 screen cages (1 m × 1 m × 1 m) using iron rod frames covered with 1 mm<sup>2</sup> cloth mesh. In each plant species, five cages were used as the biological replicates. All of the visible spiders and other natural enemies in the field cages were removed carefully before adding female 4th instar O. asiaticus.

We collected 4th instar O. asiaticus nymphs by sweep net from the grassland mainly containing these two grasses on 21 June, 2016 and 2017. Those collected individuals were then temporarily maintained in metal-frame cages and starved for 24 h. Then, female 4th instar O. asiaticus nymphs (total 160 individuals) were assigned to the 10 field cages (16 individuals per cage) randomly. Those experimental individuals were selected to be as uniform in size as possible, with fresh body mass weighed and verified by ANOVA to confirm there were no significant differences in the weight of O. asiaticus nymphs amongst the four treatments. Besides, we killed another cohort of 30 O. asiaticus 4th instar females by chloroform and dried them at 90◦C for 24 h. Those dried grasshoppers were individually weighed (mg), and a mean dry mass determined to serve as the baseline data. Once grasshoppers were assigned to every cage, we checked field cages to monitor survival every day. In each cage, grasshoppers were able to feed ad libitum on grass. The plant biomass in the cages could provide sufficient food to allow development through to adults. When all of the surviving individuals became female adults, they were also killed and dried using the same method above to get adult dry mass (mg). Adult body dry mass was used to calculate grasshopper increased body mass (mg) by subtracting the 4th instar body dry mass. Grasshopper survival rate (%) was calculated by the number of adult individuals / number of initial individuals (n = 16). Grasshopper development time (days) was calculated by the following formula: DT = P<sup>n</sup> i=1 i ∗Ni Nt , where i is the number of days from 4th instar to adult; N<sup>i</sup> is the number of individuals with the development time corresponding to the value of "i"; and N<sup>t</sup> is the number of adult individuals (Huang et al., 2016). Grasshopper growth rate (mg/day) was calculated by increased body dry mass / development time, and overall performance calculated from growth rate × survival rate (Cease et al., 2012).

#### Grass Chemical Traits

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In 2017, we cut the remaining plants from each cage at ground level after removing the adults. Each species were placed in a separate plastic container and used to detect the starch, nitrogen, and lipid content by Iodine-starch colorimetric method, Kjeldahl method, and Soxhlet extraction method, respectively. We then estimated crude protein content of each plant sample by 6.25 × nitrogen content (Bawa and Yadav, 1986). We also detected plant secondary compounds (tannins, phenols, alkaloids, terpenoids, and flavonoids) content in each sample by high performance liquid chromatography (HPLC), using the same method described in previous published papers (Ossipov et al., 1995; Griffin et al., 1999; Naczk, 2004; Friedman et al., 2006; Guo, 2007).

#### Gene Expression of ILP

We investigated seven genes of O. asiaticus ILP signaling pathway (IGF, INSR, IRS, PI3K, PDK, AKT, and FOXO) to compare their relative expression when exposed to different plant species. Unigene sequences were acquired from our previous transcriptome profiles (RSA accession number SRP072969) to design gene-specific primers (**Supplementary Table S1**). We randomly collected one adult sample from each replicate of the two treatments (10 samples). The relative expression of these genes was analyzed by qRT-PCR, using the same method described in our previous published paper (Huang et al., 2017b). Then, relative gene expression was normalized by the internal standard (actin), and calculated using the 2−11CT Method. Expression values were adjusted by setting the expression of O. asiaticus feeding on S. krylovii to be one for each gene. All qRT-PCRs for each gene with 10 samples (five biological replicates for each treatment) used 3 technical replicates per experiment.

#### Protein Phosphorylation Analysis

We used the rapid ELISA-Based Measurement to detect the protein phosphorylation of IRS, INSR, AKT, and FOXO in the insect ILP. Grasshopper samples were homogenized in 1 ml PBS, and the resulting suspension subjected to ultrasonication to further break the cell membranes. After that, we centrifuged the homogenates for 15 min at 5000 rpm, collected the supernatants and stored at −20◦C until required for further analysis.

We prepared all of the reagents (**Supplementary Table S2**) and brought all of the reagents and samples to room temperature (18◦C−25◦C). After 30 min at room temperature, we added 50 µl standard to each standard well, 50 µl sample to each sample well and 50 µl sample diluent to each blank/control well. Then, 100 µl of HRP-conjugate reagent was added to each well, and covered with an adhesive strip and incubated for 60 min at 37◦C. The Microtiter Plate was washed 4 times using Wash Buffer (undiluted), and then 50 µl Chromogen Solution A and 50 µl Chromogen Solution B was sequentially added to each well. This was then gently mixed and then incubated for 15 min at 37◦C in dark, after which 50 µl Stop Solution was added to each well. A change in the color of the solution from blue to yellow was expected. If the color in the wells was green or the color change did not appear uniform, the plate was gently taped to ensure thorough mixing. The Optical Density (O.D.) at 450 nm was read using a Microelisa Stripplate reader within 15 min of adding the Stop Solution. Then, we constructed the standard curve of each protein, and calculated the amount of phosphorylated protein in each sample.

#### Data Analysis

Student's t test was used to compare grasshopper growth variables (body size, survival rate, development time, growth rate, and overall performance), protein phosphorylation level, and relative gene expression. Linear regression was used to analyze the growth, distribution, ILP gene expression, and phosphorylation to grass chemical content in O. asiaticus. We used SAS version 8.0 for these analyses.

# RESULTS

#### Chemical Traits of Stipa krylovii and Leymus chinensis Grasses

The main nutrition and secondary metabolites were different in the two plant species (**Figures 2**, **3**). The highest starch content (t = 3.805, df = 8, P = 0.005) was present in S. krylovii, and the highest crude protein content (t = 4.085, df = 8, P = 0.004) was present in L. chinensis (**Figure 2**). The sum of the three nutritive substances (crude protein, lipid, and starch) was not significantly different between the two species.

For the five secondary metabolites, L. chinensis had higher levels of alkaloids (t = 9.440, df = 8, P < 0.001), tannins (t = 8.534, df = 8, P < 0.001), and terpenoids (t = 8.149, df = 8, P < 0.001) than S. krylovii. The total amount of all five secondary

metabolites was highest in L. chinensis (t = 11.099, df = 8, P < 0.001) (**Figure 3**).

#### Relationship Between O. asiaticus Performance and Grass Chemical Composition

The mean survival rate (**Figure 4A**) (2016: t = 7.732, df = 8, P < 0.001; 2017: t = 6.641, df = 8, P < 0.001), developmental time (t = 4.647, df = 8, P = 0.002; t = 5.077, df = 8, P = 0.001) (**Figure 4B**), adult fresh mass (t = 10.521, df = 8, P < 0.001; t = 13.311, df = 8, P < 0.001) (**Figure 4C**), growth rate (t = 7.838, df = 8, P < 0.001; t = 13.311, df = 8, P < 0.001) (**Figure 4D**), and overall performance (t = 7.732; df = 8; P < 0.001; t = 5.949; df = 8; P < 0.001) (**Figure 4E**) of O. asiaticus were significantly lower in insects that fed on L. chinensis, compared to those that

fed on S. krylovi. Feeding on L. chinensis provided less benefit for O. asiaticus growth and development. There was a significant negative linear relationship between grasshoppers and total plant secondary metabolites (**Figure 5**; R <sup>2</sup> = 0.893, F = 76.095, P < 0.001). Based on these results, we concluded that S. krylovi with less secondary metabolites resulted in better grasshopper growth. Conversely, high levels of secondary metabolites in L. chinensis, created a higher level of dietary stress, which lowered growth of O. asiaticus.

#### Relationship Between O. asiaticus Density and Grass Chemical Traits in Field Habitat

Survey results of 7 years showed that the relative density of O. asiaticus exhibited a significant positive relationship to S. krylovii above-ground biomass (**Figure 6**, linear correlation: y = 0.1886x + 0.7506, R <sup>2</sup> = 0.365, N = 56, F = 31.006, and

FIGURE 5 | Relationship between grasshopper overall performance and plant chemical composition. Red circles indicate total nutrition, and blue triangles indicate total secondary metabolites.

FIGURE 6 | Relationship between the relative density of O. asiaticus (mean number of individuals per 100 sweep-nets) and mean above-ground biomass (g/m<sup>2</sup> ) of S. krylovii (red circles) and L. chinensis (blue circles). Data combined from measurements recorded from 2011 – 2017 (N = 8 means per year), with 2011–2014 values from our published data.

P < 0.001). In contrast, the relative density of O. asiaticus was significant negative related to L. chinensis above-ground biomass (**Figure 6**, power correlation: y = 13.393x−0.<sup>523</sup> , R <sup>2</sup> = 0.283, N = 56, F = 21.298, and P < 0.001).

We also used above plant chemical data to evaluate total chemical traits of surveyed grassland. There was significant multiple linear relationship between grasshopper density and variable nutrition (x1) and secondary metabolites (x2) (**Figure 7**; y = 1.11x<sup>1</sup> − 49.435x<sup>2</sup> + 6.687, R <sup>2</sup> = 0.366, F = 15.285, and P < 0.001). From the standardization regression coefficient (x<sup>1</sup> = 0.638, x<sup>2</sup> = 0.624), we concluded that the content of secondary metabolites had the largest effect (negative) on grasshopper density.

# Change in O. asiaticus ILP Due to Diet Stress

#### Gene Expression

qRT-PCR to determine the relative expression of seven genes in ILP signaling pathway indicated that the genes IGF (t = 8.472, df = 8, P < 0.001), INSR (t = 17.851, df = 8, P < 0.001), IRS (t = 14.951, df = 8, P < 0.001), PI3K (t = 14.951, df = 8, P < 0.001), PDK (t = 8.944, df = 8, P < 0.001), AKT (t = 10.633, df = 8, P < 0.001), and FOXO (t = 6.529, df = 8, P < 0.001) were significantly down-regulated in O. asiaticus that fed on L. chinensis (**Figure 8**). Grass secondary metabolites also exhibited a significant negative relationship (**Figure 9**) to the gene expression of IGF (R <sup>2</sup> = 0.832, F = 39.626, P < 0.001), INSR (R <sup>2</sup> = 0.890, F = 65.039, P < 0.001), IRS (R <sup>2</sup> = 0.893, F = 76.095, P < 0.001), PI3K (R <sup>2</sup> = 0.810, F = 34.016, P < 0.001), PDK (R <sup>2</sup> = 0.884, F = 60.782, P < 0.001), AKT (R <sup>2</sup> = 0.866, F = 60.123, P < 0.001), and FOXO (R <sup>2</sup> = 0.720, F = 20.565, P = 0.002). Based on this evidence, we concluded that S. krylovi with less secondary metabolites determined the high gene expression involved in grasshopper ILP. But, L. chinensis

with high secondary metabolites, induced dietary stress and down-regulated gene expression in the grasshopper.

#### Protein P- Level of ILP

ELISA to determine the phosphorylation level of four proteins in ILP signaling pathway indicated that INSR (t = 3.0269, df = 8, P = 0.016), IRS (t = 3.247, df = 8, P = 0.012), AKT (t = 7.237, df = 8, P < 0.001), and FOXO (t = 7.498, df = 8, P < 0.001) were phosphorylated at the lowest levels in O. asiaticus fed on L. chinensis (**Figure 10**). Grass secondary metabolites exhibited a significant negative relationship to phosphorylation levels of INSR (R <sup>2</sup> = 0.462, F = 6.811, P = 0.03), IRS (R <sup>2</sup> = 0.509, F = 8.212, P = 0.021), AKT (R <sup>2</sup> = 0.925, F = 98.120, P < 0.001), and FOXO (R <sup>2</sup> = 0.901, F = 72.656, P < 0.001) (**Figure 11**). Based on these results, we concluded that low levels of secondary metabolites produced by S. krylovi determined the high protein phosphorylation level of proteins involved in ILP. But high levels of secondary metabolites found in L. chinensis, acted as a dietary stress, and down-regulated protein phosphorylation level.

# DISCUSSION

Migration or plague of grasshoppers generally can cause massive agricultural damage, and lead to tremendous economic losses (Stige et al., 2007; Liu et al., 2013). To achieve

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the control of pest species, it is essential to understand the factors that lead to migration and plague outbreaks. Plant species in the grassland could significantly influence population dynamics and spatial distribution of grasshoppers (Unsicker et al., 2010; Masloski et al., 2014). Some plant species are strong attractive or indispensable to some specific herbivore species, and may contribute to or even accelerate pest plague outbreaks or migration (Schutz et al., 1997; Scriber, 2002; Powell et al., 2006). Understanding the relationship of grasshoppers with their host plant species has great significance for improving management strategies (Cease et al., 2012; Huang et al., 2016).

Interactions between plants and insects are among the closest and most dynamic ecological relationships in nature, with both

taxa exerting mutual effects on one another. Such relationships can vary from beneficial to detrimental, as observed in both S. krylovii and L. chinensis in our present and previous studies (Huang et al., 2016, 2017a). Both S. krylovii and L. chinensis are dominant and widely distributed grasses across Inner Mongolia grasslands, with their above-ground biomass accounting for the most of the total community production (Huang et al., 2016). Interestingly, these two grasses had opposite roles in grasshopper migration and plague. Grasshoppers that fed on L. chinensis had reduced growth variables (size, growth rate, development, and survival) compared to those fed on S. krylovii, which indicated that L. chinensis was unsuitable for O. asiaticus compared to S. krylovii. These results are consistent with previous studies (Wu et al., 2012; Zhang et al., 2013), which also indicated that S. krylovii was the best food resource and a preferred plant host for O. asiaticus grasshopper. In the field, we also found that dry matter consumption and loss was highest for S. krylovii and that grasshoppers generally avoided L. chinensis.

In the present study, 7 years of extensive monitoring also showed that O. asiaticus density positively correlated with the above-ground biomass of S. krylovii, and negatively correlated with L. chinensis above-ground biomass. Grasshopper O. asiaticus mainly distributed in S. krylovii dominated grassland, with a lower distribution in Leymus-dominated grassland, a relationship also reported by other researchers, who found that grasshopper plague outbreaks usually occurred in Stipa-dominated grasslands (Cease et al., 2012). Wu et al. (2012) used redundancy analysis and Huang et al. (2015) used the projection pursuit model and showed that the existence of S. krylovii positively affected

O.asiaticus density in Inner Mongolia grassland, while L. chinensis had a negative effect on O. asiaticus density. Based on the perspective of grasshopper biology, S. krylovii is the favorable host plant while L. chinensis is a detrimental host plant.

Based on those researches that focused on plant associations and grasshopper performance or distribution, we hypothesized that S. krylovii, acts as the 'pull,' and the L. chinensis, as a 'push,' which contributes to grasshopper migration and consequently aggravates plague outbreaks in S. krylovii-dominated grassland (**Figure 12**). Particularly under the background of climate change (Stige et al., 2007), those areas of Stipa-dominated grasslands are the potential regions of O. asiaticus expansion or plague, such as the northern China, Siberia, Mongolia, Kazakhstan (Chen and Wang, 2000; Han et al., 2008). So, the monitoring and control of grasshopper O. asiaticus should be strengthened in those areas. Besides, the opposite function and role of these two grasses implied that the 'push-pull' strategy (Cook et al., 2007) would be a potential management tool to control future grasshopper outbreak.

The reasons underpinning the buildup of insect populations can be related to chemical traits of plants, such as the presence of important nutrients and plant secondary metabolites (Mendelsohn and Balick, 1995; Raymond et al., 2004). Available protein, carbohydrate and lipid content are important for insect herbivores growth (Simpson et al., 2004). They have well-defined nutritional requirements (Behmer, 2009), and generally prefer plants with suitable nutritional qualities, as the optimal food (Bernays et al., 1994; Powell et al., 2006). The availability of such plants may increase the probability of pest population outbreaks. Plant secondary metabolites, such as tannins, phenols, flavonoids, alkaloids, terpenoids, and glucosinolates, generally function as toxins or repellents (Despres et al., 2007). Those metabolites are detrimental to insect growth (Bernays and Chapman, 1994; Pérez et al., 2003; Unsicker et al., 2008). So, reducing access to key nutrients or increased levels of secondary compounds may decrease the probability of pest population outbreaks (Simpson et al., 2004; Cease et al., 2012). In the present study, we found that grasshopper density was positively related to nutrition content, but negatively related to secondary metabolites, which suggested that grasshopper plague events were confined to habitats providing high nutrition and low toxin levels whereby growth and development is optimal. These results are also supported by the general hypothesis that nutritious habitats benefit insect growth, but plant secondary compounds have detrimental effects on growth (Behmer, 2009; Despres et al., 2007; Wetzel et al., 2016). Dietary stress resulting from feeding on plants containing high levels of secondary metabolites could well explain the poor growth performance and low distribution of grasshopper, O. asiaticus, when confronted with L. chinensis, consequently resulting in the push role of L. chinensis. In contrast, S. krylovii, a preferred host species containing few secondary metabolites, benefited grasshopper growth and plague, and consequently acting as the pull.

Gene expression and related enzyme function were the underpinning mechanism of insect performance variations (Bishop and Guarente, 2007; Taguchi and White, 2008;

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kinase; AKT, protein kinase B; and FOXO, forkhead transcription factor.

Roy et al., 2016). Diet stress from plant chemical exposure can also result in different gene expression of insect herbivores (Badisco et al., 2013; Roy et al., 2016; Huang et al., 2017b). For example, the gene expression patterns of digestive and detoxifying enzymes, immunity, transporters, and peritrophic membrane associated transcripts varied significantly in Spodoptera spp when confronted with different diet stress (Janz and Nylin, 2008; Ragland et al., 2015; Roy et al., 2016). Those changed genes were the basis of genetic adaptation, and allowed the rapid induction of arrays of broader or more robustly active digestive or detoxifying enzymes in herbivores after the consumption of toxic plants (Bishop and Guarente, 2007; Despres et al., 2007). Such as, the gene expression and activity of CYP450s, glutathione-S-transferase, and carboxylesterase were generally positively correlated to levels of secondary plant metabolites (Despres et al., 2007; Roy et al., 2016). These rapid biochemical responses to diet stress from changing plant chemical traits are vital for insect survival and growth. From our previous study (Huang et al., 2017b), we also found that grasshoppers feeding on L. chinensis had high gene expression and enzyme activity associated with detoxification, which implies that grasshopper survival requires greater consumption to detoxify these compounds and consequently resulting in reduced phenotypic parameters (Karban and Agrawal, 2002; Castañeda et al., 2010), such as size and growth rate compared to grasshoppers feeding on a suitable host species. For example, S. krylovii has lower levels of secondary metabolites, grasshoppers feeding on this plant produce fewer detoxifying enzymes, and was required to expend less excess energy to survive. Consequently, the higher survival and growth rates contributed to plague outbreaks in S. krylovii-dominated grassland.

Gene expression of detoxification was mainly regulated by the ILP signaling pathway IGF → FOXO (Wolkow et al., 2002; Wu and Brown, 2006; Badisco et al., 2013). The high phosphorylation level of FOXO generally down-regulated the expression of detoxification-related genes, with dephosphorylated FOXO (low phosphorylation level) having the opposite effect (Bishop and Guarente, 2007; Kramer et al., 2008; Taguchi and White, 2008; Hedrick, 2009; Ragland et al., 2015). We found that high levels of secondary metabolites in L. chinensis significantly down-regulated gene expression and phosphorylation of the IGF → FOXO cascade (**Figure 13**), which could promote gene expression of detoxification enzymes in O. asiaticus that fed on L. chinensis. In addition, the significantly downregulated genes, IGF/INSR/IRS/PI3K/PDK/AKT, could also down-regulate growth-related gene expression, which is also generally detrimental to insect growth. The down-regulated ILP indicated poor adaptation of grasshopper to L. chinensis. These

#### REFERENCES


important gene variations revealed why grasshoppers prefer S. krylovii, and avoid L. chinensis.

# CONCLUSION

In conclusion, the grass L. chinensis contains high levels of secondary metabolites that down-regulated the ILP signaling pathway, resulting in the poor growth of O. asiaticus grasshopper, consequently driving migration to S. krylovii-dominated grassland. Therefore, we conclude that grasshoppers have an intelligent compromise to energy demand and detoxification cost, and propose a hypothesis that dietary stress from secondary metabolites contributes to grasshopper, O. asiaticus, migration, and plague outbreaks by regulating insect ILP.

# ETHICS STATEMENT

Insects (O. asiaticus) were collected from the Xilin Gol grassland from 2011 to 2017. It is a common agricultural pest and not in the "List of Protected Animals in China." No permits were required for the described field studies.

# AUTHOR CONTRIBUTIONS

ZZ and XH designed the experiments. XH, SuL, and JM performed the experiments. XH, WL, SeL, and XT analyzed the data. XH, SuL, and MM wrote the manuscript. All authors reviewed and considered the manuscript.

# FUNDING

This study was supported by the National Natural Science Foundation of China, 31672485, the Earmarked Fund for China Agriculture Research System, CARS-34-07, the Innovation Project of Chinese Academy of Agricultural Sciences, and the Forage Industrial Innovation Team, Shandong Modern Agricultural Industrial and Technical System, China (SDAIT-23-10).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2019.00531/full#supplementary-material


defense in pigeonpea). Plant Physiol. Biochem. 52, 77–82. doi: 10.1016/j.plap hy.2011.10.018 doi: 10.1016/j.plaphy.2011.10.018


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Li, Huang, McNeill, Liu, Tu, Ma, Lv and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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