Involvement of Two Paralogous Methoprene-Tolerant Genes in the Regulation of Vitellogenin and Vitellogenin Receptor Expression in the Rice Stem Borer, Chilo suppressalis

Besides the function of preventing metamorphosis in insects, the juvenile hormone (JH) plays a role in female reproduction; however, the underlying mechanism is largely unknown. The methoprene-tolerant (Met) protein belongs to a family of basic helix-loop-helix–Per-Arnt-Sim (bHLH-PAS) transcription factors and functions as the JH intracellular receptor. In this study, two full length cDNAs encoding Met (CsMet1 and CsMet2) were isolated from the rice stem borer, Chilo suppressalis. Structural analysis revealed that both CsMet1 and CsMet2 exhibited typical bHLH, PAS-A, PAS-B, and PAC (PAS C terminal motif) domains. Comparative analysis of transcript level using reverse transcription-quantitative PCR (RT-qPCR) revealed that CsMet1 was predominant in almost all examined developmental stages and tissues. Treatment with methoprene in vivo induces the transcription of both CsMet1 and CsMet2. Notably, injection of dsCsMet1 and dsCsMet2 suppressed the expression levels of vitellogenin (CsVg) and Vg receptor (CsVgR). These findings revealed the potential JH signaling mechanism regulating C. suppressalis reproduction, and provided evidence that RNAi-mediated knockdown of Met holds great potential as a control strategy of C. suppressalis.


INTRODUCTION
The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is one of the most serious rice pests in Asia, Middle East, and southern Europe, and causes large crop losses through feeding on the stems of rice. In China, the change of cultivation patterns has led to the frequent outbreaks of C. suppressalis in recent years (Mao et al., 2019). To date, spraying chemical insecticides remains the primary strategy for controlling C. suppressalis. However, intensive use of insecticides has driven C. suppressalis to develop resistance to a wide range of insecticides (Su et al., 2014;Lu et al., 2017;Yao et al., 2017). Hence, the development of RNA interference (RNAi)mediated disruption of reproduction represents an alternative control strategy of C. suppressalis (Miao et al., 2020).
The reproductive success of insects depends on vitellogenin (Vg) biosynthesis and uptake of Vg into developing oocytes mediated by vitellogenin receptor (VgR). Recently, we have characterized the C. suppressalis CsVg and CsVgR at the molecular levels (Huang et al., 2016;Miao et al., 2020). In this study, full-length cDNAs of two paralogous Met genes (named as CsMet1 and CsMet2) were isolated from C. suppressalis. We report the structural features and temporal-spatial expression patterns of CsMet1 and CsMet2. Furthermore, RNAi was employed to reveal the role of CsMet1 and CsMet2 in the regulation of CsVg and CsVgR.
To examine the developmental expression profiles of CsMet, individuals were collected from larvae (third, fourth, fifth, and sixth instar), pupae at intervals of 2 days from pupation, and adults at intervals of 12 h from eclosion. For tissue expression analysis, tissues (including head, epidermis, midgut, hemolymph, and fat body) were dissected from the fifth-instar larva. All the samples were frozen immediately in liquid nitrogen and stored at -80 • C until RNA isolation. Each experiment was performed with three biological replicates containing three to 10 individuals.

RNA Isolation, RT-PCR, and RACE
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United States). First-strand cDNA was synthesized from total RNA using the Primescript TM First-Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) according to the manufacturers' instruction.
The amino acid (aa) sequences of B. mori BmMet1 (GenBank: NP001108458) and BmMet2 (GenBank: BAJ05086) were searched against the transcriptome database of C. suppressalis in Insect Base 1 , and specific primer pairs were designed based on the sequences of putative transcripts ( Table 1). PCR reactions were performed with LA Taq TM DNA polymerase (TaKaRa, Dalian, China). To obtain full length cDNA sequences of CsMet1 and CsMet2, 5 -RACE and 3 -RACE were conducted using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, United States), following the manufacturers' instructions. Gene specific primers (GSPs) used for RACE are listed in Table 1.

Molecular Cloning and Sequence Analysis
RT-PCR and RACE products were subcloned into the pMD18−T vector (TaKaRa) and sequenced. The molecular mass and isoelectric point (pI) of the deduced protein sequences were 1 http://www.insect-genome.com/ TABLE 1 | Oligonucleotide primers used for RT-PCR, RACE, RT-qPCR, and RNAi.

Primer name
Sequence (5 to 3 ) Description predicted by using the online ExPASy proteomics server 2 . Conserved domains were predicted by NCBI conserved domain search tool 3 or by alignment to other published insect Mets. A phylogenetic tree was constructed with MEGA 7.0 using the neighbor-joining method with a p-distance model and a pairwise deletion of gaps (Kumar et al., 2016). The reliability of the NJ GSPs used are listed in Table 1. The stably expressed gene encoding EF1-α was used as a reference gene (Hui et al., 2011;Meng et al., 2019). The mRNA levels were normalized to reference gene with the 2 − CT method (Livak and Schmittgen, 2001). The means and standard errors for each time point were obtained from the average of three biologically independent samples.

Methoprene Treatment
To study effects of JH on expression of CsMet, newly emerged adult females were injected with 1 µL JH analog methoprene (3 µg/µL, Sigma-Aldrich, St. Louis, MO, United States) or same volume of acetone as control. Insects were collected for RT-qPCR analysis at 1, 6, 12, and 24 h after treatment, respectively, and experiments contained three biological replications.

RNA Interference
Double-strand RNAs (dsRNAs) against CsMet1 or CsMet2 were synthesized using TranscriptAid TM T7 High Yield Transcription Kit (Thermo Fisher Scientific, Waltham, MA, United States). GSPs for dsRNA synthesis are listed in Table 1.
Using a Nanoliter 2010 injector system (WPI, Sarasota, FL, United States), 1 µL solution of dsRNA (3 µg/µL) was injected into the abdomen of 6-day-old female pupae under a stereomicroscope. Equal dose of dsRNA for enhanced green fluorescent protein (EGFP) was injected as a control. A total of 40 pupae were injected per treatment, and each treatment was performed in triplicate. The treatment was repeated in newly emerged female adults (within 24 h), and insects were collected at 24 and 48 h after injection, respectively, for expression analysis of CsMet1, CsMet2, CsVg, and CsVgR by RT-qPCR.

Data Analysis
The statistical analysis was done using graphpad.prism.6 by oneway analysis of variance, followed by student's t-test. All data are presented as the mean ± SE.

Sequence and Structural Analysis of CsMet1 and CsMet2
The FIGURE 4 | Relative mRNA levels of CsMet1 (A) and CsMet2 (B) in the whole body insects after methoprene treatment in newly emerged female adults. The bars represent mean ± SE of three biological replicates. Asterisks indicate the significant differences between the groups (*P < 0.05, **P < 0.01, and ***P < 0.001).
Frontiers in Genetics | www.frontiersin.org FIGURE 5 | Knockdown of CsMet1 and CsMet2. dsRNAs were injected into 6-day-old female pupae followed by the second injection into the newly emerged female moths. The expression levels of CsMet1, CsMet2 (A), CsVg (B), and CsVgR (C) in whole body insects were examined at 24 h and 48 h after treatment. The bars represent mean ± SE of three biological replicates. Asterisks indicate the significant differences between the groups (*P < 0.05, **P < 0.01, and ***P < 0.001).

Temporal and Spatial Expression of CsMet1 and CsMet2
The temporal expression patterns of CsMet1 and CsMet2 were detected from third instar to 3-day-old female adults and determined by RT-qPCR analysis. The results showed that the transcription level of CsMet1 and CsMet2 was detectable during all selected developmental stages. Specifically, the expression of CsMet1 was relatively stable in the larval stage, sharply decreased in the prepupal stage, maintained at a low level during the pupal stage, and reached a peak in 12-h-old female adults ( Figure 3A). The expression of CsMet2 gradually increased from fourth instar to 5-day-old pupae. In the adult stage, the highest and lowest expression levels of CsMet2 were observed in 48-h-old and 72-h-old female adults, respectively ( Figure 3B). FIGURE 6 | Effects of RNAi-mediated knockdown of CsMet1 and CsMet2 on ovary development with dsEGFP as a control. The ovaries were dissected from female adults at 24 h after the second injection of dsEGFP (a1, a2), dsCsMet1 (b1, b2) and dsCsMet2 (c1, c2).
Frontiers in Genetics | www.frontiersin.org Analysis of the spatial expression profiles of CsMet1 and CsMet2 in fifth-instar larva showed that CsMet1 was highly expressed in head, hemocytes, and midgut, while relatively low expression was observed in epidermis, fat body, and malpighian tube ( Figure 3A). The highest expression level of CsMet2 was observed in hemocytes, followed by head and Malpighian tube, whereas low expression was observed in epidermis, fat body, and midgut ( Figure 3B).

Effect of JHA Treatment
To examine whether CsMet1 and CsMet2 were regulated by JH, JHA methoprene was injected into the newly emerged female adults. Compared with control, the results showed that CsMet1 expression was upregulated by 2.74, 2.41, and 1.63 times at 1, 6, and 12 h after injection, respectively ( Figure 4A). Similarly, transcription level of CsMet2 was increased by 1.95, 1.45, and 3.68 times at 1, 6, and 1 2 h post-treatment, respectively ( Figure 4B).

Effects of RNAi-Mediated Knockdown of CsMet1 and CsMet2
To explore the role of CsMet1 and CsMet2 in the regulation of CsVg and CsVgR, female adults were collected at 24 and 48 h after the second dsRNA injection, respectively. The transcript level of CsMet1 in dsCsMet1-injected female adults was suppressed by 78.74% (24 h) and 70.32% (48 h) compared with dsEGFP-injected insects, whereas the expression of CsMet2 was significantly reduced by 94.69% (24 h) and 92.79% (48 h) ( Figure 5A). Meanwhile, RNAimediated suppression of CsMet1 and CsMet2 decreased CsVg expression by 70.85 and 44.37% at 48 h post-treatment, respectively ( Figure 5B). CsVgR expression in the dsCsMet1treated and dsCsMet2-treated group decreased significantly by 52.91 and 62.98% at 24 h post-injection, respectively ( Figure 5C). Furthermore, phenotypic observation revealed that silencing of both CsMet1 and CsMet2 resulted in suppressed ovarian development with decreased number of mature follicles (Figure 6).

DISCUSSION
The important roles played by JH in insect development and reproduction prompted the study on JH signaling, and the breakthrough had been the identification of transcription factor Met as an intracellular receptor for JH (Charles et al., 2011). Binding of JH to Met triggers dimerization of Met with another bHLH-PAS protein Taiman (Tai) to form a functional complex, which interacts with JH response elements (JHREs) of target genes (Smykal and Raikhel, 2015). While the Met null allele was expected to result in a lethal phenotype, the paralogous Met gene in D. melanogaster, Gce, ensured survival of the met null mutants (Baumann et al., 2010a,b). Similarly, two Met genes were also recently identified in several Lepidopteran insects (Zhan et al., 2011;Guo et al., 2012;Suetsugu et al., 2013;Derks et al., 2015;Kayukawa and Shinoda, 2015). However, only a single Met gene was found in those from other insect orders including A. aegypti (Dipteran, Wang et al., 2007), T. castaneum (Coleopteran, Konopova and Jindra, 2007), Blattella germanica (Blattarian, Lozano and Belles, 2014), N. lugens (Hemipteran, Lin et al., 2015), Sitodiplosis mosellana (Dipteran, Cheng et al., 2018), and so on. In this study, we isolated two paralogous Met genes, CsMet1 and CsMet2, from C. suppressalis. Both proteins showed conserved domain organization of bHLH-PAS protein family (Ashok et al., 1998;Kayukawa and Shinoda, 2015;Ma et al., 2018). Phylogenetic analysis revealed that CsMet1 and CsMet2 were clustered into Lepidopteran group 1 and group 2 Met, respectively, providing further evidence of the occurrence of Met gene duplication in Lepidopteran insects.
The temporal and spatial expression patterns of a gene may provide hints as to its function. In this study, we found a marked reduction in expression levels of CsMet1 in the prepupae stage, supporting its involvement in larvalpupal metamorphosis. Similar results were also observed in HaMet1 of H. armigera (Ma et al., 2018) and SmMet of S. mosellana (Cheng et al., 2018), and was in agreement with the fact that JH exerts its anti-metamorphic effect through its receptor Met (Parthasarathy et al., 2008;Li et al., 2019). CsMet2 transcript was hardly detected during the larval stage, and its expression reached the peak in 48-h-old female adults, suggesting that CsMet2 was involved in JH signaling in adults, as had been reported for BmMet2 in B. mori (Kayukawa and Shinoda, 2015). For tissue expression, CsMet1 was highly expressed in larval head, midgut, and hemocytes, whereas CsMet2 showed the highest expression in larval hemocytes. Interestingly, the temporal and spatial expression analysis revealed that CsMet1 was dominant at almost all developmental stages and in all tissues examined. This result was somewhat different from the observation in B. mori that BmMet2 transcript was more abundant than BmMet1 transcript in the adult stage (Kayukawa and Shinoda, 2015). Whether CsMet1 played a more important role in C. suppressalis needed further study in the future.
Juvenile hormone plays a crucial role in insect reproduction, but its molecular mode of action only became clear recently. It has been reported in H. armigera, Diploptera punctata, N. lugens, and S. mosellana that Met is significantly upregulated by JH III or JHA methoprene (Marchal et al., 2014;Zhao et al., 2014;Lin et al., 2015;Cheng et al., 2018). Our hormone treatment results also indicated that CsMet1 and CsMet2 expression was upregulated after JHA methoprene treatment. Further RNAi analysis revealed that knockdown of CsMet1 and CsMet2 resulted in significant decline of both CsVg and CsVgR expression in female adults, leading to decreased number of mature follicles in ovaries. Similarly, expression of Vg is regulated by JH via Met in A. aegypti, H. armigera, D. punctata, N. lugens, and Bactrocera dorsalis (Zou et al., 2013;Marchal et al., 2014;Lin et al., 2015;Ma et al., 2018;Yue et al., 2018). Downregulation of VgR transcription as well as suppressed ovarian development were also observed in dsMet-treated H. armigera and Colaphellus bowringi (Ma et al., 2018;Liu et al., 2019). Whether expression of Vg and VgR were directly regulated by Met was not well known.
It had been recently reported that knockdown of Krüppelhomolog 1 (Kr-h1), a direct target of JH-Met signaling, also decreased VgR expression in C. bowringi . On the other hand, Met is also a repressor of 20E pathway gene expression. It is likely that Met regulated the relative genes of 20E pathway (Zhao et al., 2014), which contributes to vitellogenis to some extent. Further study needs to be carried out to confirm these presumptions.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article.

AUTHOR CONTRIBUTIONS
JW conceived and designed the study. LM, NZ, HJ, FD, XY, and XX performed the experiments. LM, JW, KQ, and XM wrote the manuscript. All authors reviewed and approved this submission.