Functional Analysis of MsepOR13 in the Oriental Armyworm Mythimna separata (Walker)

Olfaction in insects has a critical role in recognizing the host, finding food, and choosing mating partners, as well as avoiding predators. Odorant receptors (ORs), which are housed in the dendritic membrane of sensory neurons and extended into the lymph of sensilla on insect antennae, are participating in the detection of volatile compounds in insects. In the present study, we identified an OR gene, named MsepOR13, in the oriental armyworm Mythimna separata (Walker). Quantitative real-time polymerase chain reaction revealed that MsepOR13 was expressed mainly in the antennae of male and female moths. In in vitro heterologous expression experiments, MsepOR13 was widely tuned to 32 of the 67 different compounds tested. Furthermore, MsepOR13 responded to eugenol at a low concentration of 10-9 M, with an EC50 value of 3.91 × 10-6 M. The high sensitivity suggests an important role for the OR13 gene in the moth olfactory system.


INTRODUCTION
Chemoreception of odorants in the environment is critically important for the survival of insects. During evolution, insects have evolved a powerful sense of olfaction to locate hosts and mating partners, identify oviposition sites, discriminate toxic food, and escape predators (Schneider, 1969;Bruce et al., 2005;Bruyne and Baker, 2008;Hansson and Stensmyr, 2011;Gadenne et al., 2016), as they are surrounded by various chemical compounds emitted from conspecifics, predators, and host plants (Bentley and Day, 1989;Schneider, 1992;Hansson and Stensmyr, 2011). These odorants are diffused to the surface on olfactory appendages, which mainly consisting of antennae and maxillary palps (Steinbrecht, 1997), and enter the lymph through pores of the sensilla, which are hair-like structures. Odorant molecules interact with odorant-binding proteins (OBPs) in the sensilla lymph and are transferred toward the dendrites of olfactory sensory neurons (OSNs), where odorant receptors (ORs) are expressed. Activation of ORs leads to chemical information being transduced to electrical signals, which are conveyed to the antennal lobe and finally decoded by the insect brain (Vogt, 2003;Leal, 2013).
Owing to the availability of the Drosophila melanogaster genome sequence, the first insect OR was identified in D. melanogaster based on the homology of OR sequences in vertebrates and nematodes and the restricted expression of these genes in olfactory tissues (Clyne et al., 1999;Vosshall et al., 1999). Compared with G-protein coupled receptors (GPCRs), insect ORs have the opposite membrane topology, with their N-terminus inside and their C-terminus outside the cell; this is an inverse membrane topology to that found in vertebrate ORs (Buck and Axel, 1991;Benton et al., 2006;Fleischer et al., 2017;Butterwick et al., 2018). It is now generally accepted that insect ORs transduce chemical signals by forming heteromeric complexes with an OR co-receptor (Orco) that operate as non-selective cation channels (Koji et al., 2008;Wicher et al., 2008).
In recent decades, with progress in sequencing technology and bioinformatics tools, numerous ORs have been reported in many species from various insect orders, including Lepidoptera, Diptera, Hymenoptera, Coleoptera, Hemiptera, Orthoptera, and Phthiraptera. The number of OR genes varies considerably among insect species. For example, there are 65 ORs in Helicoverpa armigera (Liu et al., 2012;Zhang et al., 2015a) and 62 ORs in Mythimna separata (Du et al., 2018), based on antennal transcriptomic analysis, whereas 163 ORs have been obtained from the genome of Apis mellifera (Robertson and Wanner, 2006) and 256 ORs have been identified in the genome of Tribolium castaneum (Engsontia et al., 2008). The variation in number of ORs between insects is assumed to correlate with evolutionary adaption to certain ecological and physiological demands (Fleischer et al., 2017).
The oriental armyworm M. separata (Walker) (Lepidoptera: Noctuidae) is an economically important and common lepidopteran pest, which is widely distributed in eastern Asia and Australia, and attacks many crop plants including maize, sorghum, and rice. M. separata migrates long distances, resulting in widespread incidence, which can lead to complete crop loss (Sharma and Davies, 1983;Jiang et al., 2011). In recent years, M. separata has been observed in many regions of China and poses a severe threat to corn production. In order to control this pest, high doses of insecticides are often applied; however, this has some negative effects, including environmental pollution, insect resistance, and harm to non-target organisms (Lv et al., 2014;Duan et al., 2017). Outbreaks of M. separata represent a great challenge in crop protection worldwide .
Compared with the use of chemical pesticides, olfactorybaited trapping is an effective and environmentally friendly method to manage M. separata. The sex pheromone of M. separata has been used in this way (Wei, 1985;Zhu et al., 1987), but the effect was unsatisfactory for unknown reasons. Pterocarya stenoptera and Salix babylonica are also used to attract M. separata in the field (Lihuang et al., 2017), although the mechanism of attraction is unknown. In previous work, we identified the ORs in M. separata using transcriptomic analysis (Du et al., 2018), but no study on their function has been reported except for MsepOR1, responding to the major sex pheromone compound Z11-16:Ac (Mitsuno et al., 2010). In the present study, we cloned an OR, named MsepOR13, in M. separata and analyzed the expression patterns in different tissues of both sexes by quantitative real-time polymerase chain reaction (qRT-PCR). Functional analysis was completed using in vitro expression in a Xenopus oocyte system with two-electrode, voltage-clamp physiological recordings.

Insect Rearing
The M. separata colony, maintained at the laboratory of Henan Agricultural University, Zhengzhou, China, was reared on an artificial diet at 28 ± 1 • C, 70% ± 5% relative humidity, and a 14 h:10 h light:dark (L:D) photoperiod. Adult male and female moths were fed with 10% sugar solution.

RNA Extraction and cDNA Synthesis
Male and female antennae, proboscises, labial palps, and legs (a mixture of female and male) of virgin male or female individuals were collected 3 days after eclosion, immediately frozen in liquid nitrogen, and stored at −70 • C until RNA extraction. Total RNA of 20 adult male or female moths was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer's instructions. Total RNA was dissolved in RNasefree water and gel electrophoresis was performed to assess its integrity. RNA concentration and purity were determined on a Nanodrop ND-2000 spectrophotometer (NanoDrop products, Wilmington, DE, United States).
First, total RNA was treated with DNase I (Fermentas, Glen Burnie, MD, United States) for 30 min at 37 • C to remove residual gDNA. Then, 1 µg total RNA was used to synthesize singlestranded cDNA as per the First Strand cDNA Synthesis Kit (Fermentas) manufacturer's instructions. The cDNA of antennal samples was used as a template to clone the MsepOR13 gene. The cDNA samples isolated from different female and male tissue types were used as templates for RT-qPCR.

Cloning of MsepOR13 Gene From M. separata
The sequence of MsepOR13 was identified in M. separata by transcriptomic analysis (Du et al., 2018). Specific primers were designed by Primer 5.0 (PREMIER Biosoft International, Palo Alto, CA, United States) to clone the full-length sequence of MsepOR13 (Table 1). Antennal cDNA from female and male moths was used to amplify the full-length sequence of MsepOR13 using primeSTAR HS (Premix) (TaKaRa, Dalian, China). PCR reactions of 50 µL contained 25 µL 2×primeSTAR HS (Premix), 1.5 µL sense and anti-sense primers (10 µM), 2 µL cDNA, and 20 µL double-distilled H 2 O. Reactions were carried out under the following conditions: 95 • C for 3 min; 35 cycles of 95 • C for 30 s, 57 • C for 30 s, and 72 • C for 1 min; and 72 • C for 10 min; before being held at 16 • C. PCR products were analyzed on a 1.5% agarose gel and the sequence was sub-cloned to the vector pEASY-Blunt (TransGene, Beijing, China). The sequencing was completed in Sangon Biotech, Shanghai, China.

Tissue Expression Profile of MsepOR13
Quantitative polymerase chain reaction was performed to determine the expression of MsepOR13. Male and female antennae, proboscises, labial palps, and legs (a mixture of female and male) were collected from 3-day-old M. separata adults after eclosion. RNA extraction and cDNA synthesis were 1 http://web.expasy.org/translate/

MsepOrco-A TCAgggcccGCCACCATGAT GACCAAAGTGAAGGC
MsepOrco-S TCAgcggccgcTTACTTGAG TTGCACCAACAC performed following the protocol described above. MsepRPS3 was chosen as the reference gene. The primers are listed in Table 1. GoTaq qPCR Master Mix (Promega, Madison, WI, United States) was used for qPCR, and the reactions were carried out on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI, Carlsbad, CA, United States). The reactions (20 µL) consisted of 10 µL GoTaq qPCR Master Mix, 0.8 µL gene primer (10 µM), 1 µL cDNA, and 7.4 µL RNase-free water. The reactions were carried out under the following conditions: 95 • C for 2 min; 40 cycles of 95 • C for 15 s, and 60 • C for 50 s. Each qPCR reaction was performed in triplicate with three independent biological samples to check reproducibility. The melting curves were inspected to check the specificity of the primers, and the amplification efficiencies were calculated by the standard curve method. The efficiency of the primers for MsepOR13 and MsepRPS3 were 97 and 105%, respectively. MsepOR13 relative expression levels were analyzed using the relative 2 − CT quantitation method, where C T = C T (MsepOR13) -C T (MsepRPS3), C T = C T (different samples) -C T (legs (female and male mixture)). Statistical comparison of expression of MsepOR13 was assessed using one-way nested analysis of variance (ANOVA), followed by least-significant difference (LSD) tests.

MsepOR13 Expression in Xenopus Oocytes and Electrophysiological Recordings
The full-length MsepOR13 was first cloned into a pEASY-Blunt vector and then ligated into a pT7Ts expression vector using primers containing Apa I (GGGCCC) and Not I (GCGGCCGC) sites. The expression vector was linearized using Sma I (CCCGGG) (Fermentas, Glen Burnie, MD, United States) and the cRNA was synthesized using an mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX, United States). Mature healthy Xenopus oocytes (stages V-VII) were incubated with 2 mg/mL collagenase I in pH 7.6 washing buffer consisting of 96 mM NaCl, 2 mM KCl, 5 mM MgCl 2 , and 5 mM HEPES at room temperature for about 1 h until almost of them were separated a signal one. Then, 27.6 ng MsepOR13 cRNA and 27.6 ng MsepOrco cRNA were microinjected together into oocytes, and the oocytes were cultured in 1× Ringer's buffer (washing buffer supplemented with 0.8 mM CaCl 2 , 5% dialyzed horse serum, 50 mg/mL tetracycline, 100 mg/mL streptomycin, and 550 mg/mL sodium pyruvate) for 4-7 days. The whole-cell currents of injected oocytes were recorded with an OC-725C oocyte clamp at a holding potential of −80 mV (Warner Instruments, Hamden, CT, United States), following previously described experimental procedures (Cui et al., 2018;Liu W. et al., 2018;Liu Y. et al., 2018). Oocytes were exposed to different compounds at 10 −4 M for 15 s each, in a random order, with intervals between exposures that allowed the current to return to baseline. Dose-response curves were acquired from 10 −9 to 10 −4 M in ascending order of concentration. All data acquisition and analysis were carried out with Digidata 1440A and Pclamp10.0 (Axon Instruments, Inc., Union City, CA, United States), and dose-response data were analyzed using GraphPad Prism 5. Statistical comparison   of responses to different odors of MsepOR13 was assessed using ANOVA, followed by LSD tests.

Odorant Panel
Sixty-seven plant volatile compounds purchased from Sigma-Aldrich were used in this experiment ( Table 2) and were classified into six groups: terpenoid, aromatic, alcohol, ester, aldehyde, and ketone. All compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 M as stock solutions.
Before the experiments, the stock solutions were diluted in 1× Ringer's buffer to working concentrations, and 1× Ringer's buffer containing 0.1% DMSO was used as a negative control.

Tissue Expression Profiles of MsepOR13
Quantitative polymerase chain reaction was carried out to evaluate the expression profile of MsepOR13 in different tissues of both sexes in M. separata. The results showed that MsepOR13 was mainly expressed in antennae compared with other tissues and exhibited much higher relative expression level in female antennae than male antennae (Figure 3). MsepOR13 was less expressed in proboscis and labial palp in both sexes and there was no significant difference in the expression levels of MespOR13 between leg (mixture of female and male moths) and female proboscis.

Functional Characterization of MsepOR13 in the Xenopus Oocyte Expression System
The Xenopus oocyte expression system was used to identify candidate ligands for MespOR13. The cRNA of MsepOR13 and MespOrco were co-injected into Xenopus oocytes, and responses to 67 compounds were recorded using a two-electrode voltage clamp. MsepOR13 was tuned to 32 odorants from all six classes and was most sensitive to eugenol, with responses of about 3011 nA (Figures 4A,B,D). In addition, methyl eugenol and methyl phenylacetate elicited the second strongest responses, of about 1655 and 1150 nA, respectively (Figures 4A,D). Interestingly, these three main legends shared similar structure, a benzene ring ( Figure 4C). The other 29 odorants elicited the same response level. Acetophenone elicited a relatively higher response (523.3 nA) and 1-hexanol elicited the lowest response with an amplitude of 60 nA (Figures 4A,D). In the dose-response study, Xenopus oocyte co-expressing MsepOR13/MsepORco responded to 10 −9 M of eugenol and the peak amplitude occurred at the concentration of 10 −5 M ( Figure 4E). The EC50 value of eugenol was 3.91 × 10 −6 M ( Figure 4F).

DISCUSSION
Detection of chemical odors in the environment is essential for the survival of insects. Accordingly, insects have evolved remarkable sensitive and discriminatory olfactory systems for locating hosts and food sources, identifying mating partners and oviposition sites, or escaping predators (Schneider, 1969;Hansson and Stensmyr, 2011;Gadenne et al., 2016). Previous studies have shown that ORs play an important part in the recognition of odorants and the process of chemo-electrical transduction (Leal, 2013;Wicher, 2014;Bohbot and Pitts, 2015).
In this study, we cloned an OR gene, MsepOR13, in M. separata. The sequence contained 1227 bp, encoding 408 amino acids. As showed in the qPCR experiment, MsepOR13 exhibited female antennae-biased expression, which suggested that it might play a vital role in regulating female-specific behaviors, such as oviposition sites selection (Liu et al., 2015). Meanwhile, we found MsepOR13 was also expressed in legs indicating that legs might assist insects to choose suitable oviposition sites. Previous studies found that female butterflies perceive oviposition stimulant by their foreleg tarsus and further determine the suitable feeding plant for larvae in Papilio polytes (Nakayama et al., 2003). Furtherly, 4 ORs were also identified by the legs transcriptome analysis in Ectropis obliqua (Ma et al., 2016), indicating that ORs expressed in legs was a ubiquitous phenomenon. During the past decade, the sex pheromone receptors have been well-deorphanized in many Lepidoptera species. However, the identification of ligands for the non-pheromone receptor ORs has significantly lagged behind, except for a few species such as D. melanogaster (Hallem et al., 2004;Kreher et al., 2005;Hallem et al., 2006), A. gambiae (Lu et al., 2007;Carey et al., 2010;Wang et al., 2010), and S. littoralis (Montagné et al., 2012;de Fouchier et al., 2017). In this study, MsepOR13 responded to 32 odorants and only three ligands elicited relative large response; this phenomenon was also found in studies of S. littoralis (de Fouchier et al., 2017) and H. armigera (Di et al., 2017). Narrowly tuned receptors are thought to be important in the detection of odors of high biological salience. In D. melanogaster, several ORs selectively responded to odors that are necessary and sufficient for vital behaviors such as avoiding toxic microbes and choosing oviposition sites (Stensmyr et al., 2012;Dweck et al., 2013Dweck et al., , 2015Ronderos et al., 2014). In mosquitoes, receptors that selectively respond to human emanations play a crucial part in host recognition and blood feeding (Hughes et al., 2010;Mcbride et al., 2014). Sex pheromone perception in moths also involves such specific pathways (Miura et al., 2010;Liu Y. et al., 2018). The homolog of MsepOR13 in S. littoralis, SlitOR31 shared 80% amino acid identity with MsepOR13. But SlitOR31 was narrowly tuned to eugenol, which is different from the function of MsepOR13. The difference of their function might relate with the different environment and the selective pressures they face. In M. separata, the three main ligands containing a benzene ring were structurally similar; a similar phenomenon has been found in functional studies of ORs in A. gambiae (Wang et al., 2010), S. littoralis (de Fouchier et al., 2017), and H. armigera (Di et al., 2017). Among all the ligands, eugenol activated the strongest response in MsepOR13/Orco Xenopus oocytes, and could response at a 10 −9 M concentration, with an EC50 value of 3.91 × 10 −6 M. Actually, MspeOR13 responding to eugenol showed the similar sensitivity with the reported pheromone receptors to sex pheromones Chang et al., 2016;Liu W. et al., 2018;Liu Y. et al., 2018), suggesting that eugenol might be important to M. separata. It has been reported that eugenol can repel the H. armigera moth (Xu, 2004), and also repel Populus yunnanensis oviposition (Ma et al., 2016). In Tribilium castaneum, eugenol has apparently repellent activity toward adults and toxic effects on both larvae and adults (Han and Huang, 2009). However, in Mamestra brassicae, eugenol was found to attract larvae and moths (Yan, 2015). The functions of eugenol with respect to M. separata require further study, especially behavioral experiments, in order to develop environmentally friendly approaches to control this economically significant insect. Based on the high sensitivity of MsepOR13 to eugenol, we predict that MsepOR13 may have an important role in the reception of eugenol in M. separata; thus, its function could be further explored using the CRISPR-Cas9 system.