Sub-Lethal Effects of Fenvalerate on the Development, Fecundity, and Juvenile Hormone Esterase Activity of Diamondback Moth, Plutella xylostella (L.)

The toxicities of fenvalerate （20% EC） to the 3rd instar larvae of diamondback moth （DBM）, Plutella xylostella （L.）, reared on three host plants viz., radish, oilseed rape, and cabbage were tested. The LC50 values of fenvalerate to the 3rd instar larvae of DBM varied with host plants, however, there wasn＇t any significant difference among them （P〉 0.05）. Similarly, DBM fed on three host plants had same pupal weight, pupal period, pupation rate, adult emergence rate, female ratio, and fecundity. The activity of juvenile hormone esterase （JHE, EC 3.1.1.1） in the 3rd instar larvae of DMB did not significantly vary with host plants, either. These suggested that DBM had similar fitness on the three host plant species. When fed on the host plants pretreated with fenvalerate at the concentrations equivalent to LC20, LC50 and LC50, the pupation rate, pupal weight, adult emergence rate, female ratio, fecundity, and JHE activity of the tested insects were declined as compared with insects in control treatments fed on the same host plant species. Furthermore, the pupal period of the tested insects was extended after fenvalerate treatment. The decrease in JHE activity after fenvalerate treatment in the tested insects could partly explain the changes in the mentioned growth parameters. Whether the role of fenvalerate in the inhibition of JHE activity could serve as a new way to control DBM needs further investigation.

In the monarch butterfly the juvenile hormone effect upon immune response depends on the immune marker and is sex dependent

In insects, juvenile hormone (JH) decreases or has any effect upon the phenoloxidase (PO) activity, and favors or decreases the Antimicrobial Peptides (AMPs) expression. Although there is no information about the differential effect of such hormone, two possibilities are that it depends on (a) the immune marker recorded and (b) sexual differences. Here, three commonly used immune markers, Phenoloxidase (PO), hydrogen peroxide (H2O2), and lytic activity, were measured 3, 6 and 24 hours after administration of methoprene (JHa, an analog of juvenile hormone) in male and female monarch butterflies (Danaus plexippus). At 3 and 6 h post-JHa administration, the PO activity increased in females but it only increased at 3 h in males, whereas H2O2 levels increased only in females at 3 h. For the remaining times the JHa had a null effect on POand H2O2. On the other hand, the JHa had a null effect for lytic activity in both sexes at 3, 6 and 24 h. To our knowledge, this is the first report of a positive effect of a JHa onPOand H2O2 and suggests that this effect is sex dependent.

Juvenile hormone induces reproduction via miR-1175-3p in the red imported fire ant, Solenopsis invicta

Juvenile hormone(JH)acts in the regulation of caste differentiation between queens and workers(i.e.,with or without reproductive capacity)during vitellin synthesis and oogenesis in social insects.However,the regulatory mechanisms have not yet been elucidated.Here,we identified a highly expressed microRNA(miRNA),miR-1175-3p,in the red imported fire ant,Solenopsis invicta.We found that miR-1175-3p is prominently present in the fat bodies and ovaries of workers.Furthermore,miR-1175-3p interacts with its target gene,broad-complex core(Br-C),in the fat bodies.By utilizing miR-1175-3p agomir,we successfully suppressed the expression of the Br-C protein in queens,resulting in reduced vitellogenin expression,fewer eggs,and poorly developed ovaries.Conversely,decreasing miR-1175-3p levels led to the increased expression of Br-C and vitellogenin in workers,triggering the“re-development”of the ovaries.Moreover,when queens were fed with JH,the expression of miR-1175-3p decreased,whereas the expression of vitellogenin-2 and vitellogenin-3 increased.Notably,the suppression of fertility in queens caused by treatment with agomir miR-1175-3p was completely rescued by the increased vitellogenin expression induced by being fed with JH.These results suggest the critical role of miR-1175-3p in JH-regulated reproduction,shedding light on the molecular mechanism underlying miRNA-mediated fecundity in social insects and providing a novel strategy for managing S.invicta.

Methoprene does not affect juvenile hormone titers in honey bee （Apis mellifera） workers

Methoprene, a juvenile hormone （JH） analog, is a widely used insecticide that also accelerates behavioral development in honey bees （Apis mellifera）. JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers ofmethoprene-treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene- treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal-aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers＇ JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone （JH） is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase （JHDK） is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA （cDNA） was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate-binding motifs were predicted in the protein. Modeling of the 3-D structure showed that the protein consisted of eight α-helixes linked with loops, with no β-sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae ofS. litura.

Juvenile hormone signaling-a mini review

Since it was first postulated by Wigglesworth in 1934, juvenile hormone (JH) is considered a status quo hormone in insects because it prevents metamorphosis that is initiated by the molting hormone 20-hydroxyecdysone (20E). During the last decade, significant advances have been made regarding JH signaling. First, the bHLH-PAS transcription factor Met/Gce was identified as the JH intracellular receptor. In the presence of JH, with the assistance of Hsp83, and through physical association with a bHLH?PAS transcriptional co-activator, Met/Gce enters the nucleus and binds to E-box-like motifs in promoter regions of JH primary?response genes for inducing gene expression. Second, the zinc finger transcription factor Kr-hl was identified as the anti-metamorphic factor which transduces JH signaling. Via Kr-hl binding sites, Kr-hl represses expression of 20E primary?response genes (i.e. Bi\ E93 and E5) to prevent 20E-induced metamorphosis. Third, through the intracellular signaling, JH promotes differ ent aspects of female reproduction. Nevertheless, this action varies greatly from species to species. Last, a hypothetical JH membrane receptor has been predicted to be either a GPCR or a tyrosine kinase receptor. In future, it will be a great challenge to understand how the JH intracellular receptor Met/Gce and the yet unidentified JH membrane receptor coordinate to regulate metamorphosis and reproduction in insects.

Dual roles of juvenile hormone signaling during early oogenesis in Drosophila

Juvenile hormone(JH)signaling plays crucial roles in insect metamorphosis and reproduction.Function of JH signaling in germline stem cells(GSCs)remains largely unknown.Here,we found that the number of GSCs significantly declined in the ovaries of Met,Gee and JHAMT mutants.Then we inhibited JH signaling in selected cell types of ovaries by expressing Met and Gee or Kr-hl double-stranded RNAs(dsRNAs)using different Gal4 drivers.Blocking of JH signaling in muscle cells has no effect on GSC numbers.Blocking of JH signaling in cap cells reduced GSCs cells.Inductive expression of Met and Gee dsRNA but not Kr-hl by Nos-Gal4 increased GSC cells.These results indicate that JH signaling plays an important role in GSC maintenance.

Juvenile hormone membrane signaling phosphorylates USP and thus potentiates 20-hydroxyecdysone action in Drosophila

Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases.Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C(PKC) which phosphorylated ultraspiracle(USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20 E through its nuclear receptor complex Ec RUSP. The usp;mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20 E signaling that delayed developmental timing. The usp;mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20 E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.

Expression of juvenile hormone acid O-methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone （JH）, a sesquiterpenoid synthetized by the insect corpora allata （CA）, plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O-methyltransferase （JHAMT）. Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica （L.） （BgJHAMT）. BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3＇-UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi-triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body viteUogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin （TOR）. Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH-specifie branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.

Juvenile hormone acts on male accessory gland function via regulating L-asparaginase expression and triacylglycerol mobilization in Aedes aegypti

Hormones control the reproductive development of Aedes aegypti mosquitoes.The adult male reproductive process and mating behavior require adequate nutrients and energy.Understanding the molecular mechanism linking hormones,energy metabolism,and reproduction in male mosquitoes is important.In this study,we found that the size of the male accessory gland,an essential part of the male reproductive system,gradually increased after eclosion.However,it was significantly reduced in male mosquitoes deficient in methoprene-tolerant(Met),the receptor of juvenile hormone.Likewise,egg hatchability of females that mated with Met-depleted males showed the same downward trend.The mRNA level of the gene encoding accessory gland protein,l-asparaginase(ASNase),was reduced in Met dsRNA-treated males.Electrophoretic mobility shift assay and quantitative reverse transcription-PCR results revealed that Met was capable of binding directly to the promoter of ASNase and activated its transcription.RNA interference of ASNase in males resulted in the reduction of egg hatchability of the females with which they mated.These results showed that Met influenced the fecundity of male mosquitoes by directly upregulating the expression of the ASNase gene.Moreover,the levels of triacylglycerol and the sizes of lipid droplets were decreased by 72-78 h after eclosion in the fat body cells,whereas both of them increased in Met-depleted male mosquitoes,indicating that Met knockdown reduced lipid catabolism.These data demonstrate that Met might influence the egg hatchability of females by regulating lipid metabolism and the development of the male accessory gland in male mosquitoes.

Juvenile hormone regulates photoperiod-mediated male reproductive diapause via the methoprene-tolerant gene in the ladybeetle Harmonia axyridis

Juvenile hormone (JH) absence induces photoperiod-mediated reproductive diapause, which is characterized by reproductive cessation. Although the role of methoprene-tolerant (Met)-mediated JH signaling in photoperiod-mediated female reproduction has been well documented, its role in male reproduction remains unclear. In this study, we investigated the role of JH in regulating photoperiod-mediated development of the male internal reproductive system (IRS) in the predatory ladybeetle Harmonia axyridis (Pallas). In a previous study, we found that adult male H. axyridis reared under either a short-day (SD) or long-day (LD) photoperiod had obvious differences in IRS development, but we were unable to identify the regulators of male reproductive diapause. In this study, we found that beetles reared under an SD photoperiod had significantly lower JH titer and a relatively undeveloped male IRS compared with those reared under an LD photoperiod. Additionally, application of the JH analog (JHA) methoprene promoted IRS development. Furthermore, Met knockdown strongly blocked JH signaling in males reared under the LD photoperiod, thereby slowing IRS development. Moreover, exogenous JHA did not reverse the suppressed development of the male IRS caused by Met knockdown. These results indicate that photoperiod regulates male IRS development in H. axyridis through a conserved Met-dependent JH signaling pathway.

The biosynthesis of alarm pheromone in the wheat aphid Rhopalosiphum padi is regulated by hormones via fatty acid metabolism

Aphids are major insect pests in agriculture and forestry worldwide.Following attacks by natural enemies,many aphids release an alarm pheromone to protect their population.In most aphids,the main component of the aphid alarm pheromone(AAP)is the sesquiterpene hydrocarbon(E)-β-farnesene(EβF).However,the mechanisms behind its biosynthesis and regulation remain poorly understood.In this study,we used the bird cherry–oat aphid Rhopalosiphum padi,which is an important wheat aphid,to investigate the regulatory mechanisms of EβF biosynthesis.Our results showed that EβF biosynthesis occurs during the mature embryo period and the molting period of the 1st-and 2nd-instar nymphs.Triglycerides provide the prerequisite material for EβF production and release.Based on transcriptome sequencing,RNAi analysis,hormone treatments,and quantitative measurements,we found that the biosynthesis of EβF utilizes acetyl coenzyme A produced from fatty acid degradation,which can be suppressed by juvenile hormone but it is promoted by 20-hydroxyecdysone through the modulation of fatty acid metabolism.This is the first systemic study on the modulation of EβF production in aphids.The results of our study provide insights into the molecular regulatory mechanisms of AAP biosynthesis,as well as valuable information for designing potential aphid control strategies.

Molecular Characterization and Functional Analysis of Krüppel-homolog 1(Kr-h1) in the Brown Planthopper, Nilaparvata lugens(Stl)

The brown planthopper, Nilaparvata lugens（Stl）, is the most serious insect pest of rice. It has developed high resistance to traditional insecticides because of their intensive use. Juvenile hormone（JH） analogs have been used successfully to control this species and other pest insects. However, the molecular mechanism of JH signaling is not well understood. Krüppel-homolog 1（Kr-h1） is a transcription factor involved in the JH pathway. In this study, the Kr-h1 cDNA was cloned and characterized from N. lugens by rapid amplification of cDNA ends（RACE） and reverse transcription PCR（RT-PCR）. Its spatial and temporal expression profiles were examined by real-time quantitative PCR, and its function was also studied by RNA interference（RNAi）. The open reading frame of NlKr-h1 is 1 833 bp encoding for 611 amino acids. The protein contains eight conserved zinc-finger motifs. NlKr-h1 was expressed at all life stages, with the highest mRNA level in the 4-day embryo. NlKr-h1 mRNA levels rose during each nymphal molt after the 2nd instar. In the adults, the mRNA level in males was significantly higher than that in females of either the macropterous or brachypterous type. The highest expression was observed in the female midgut. NlKr-h1 was activated by juvenile hormone III（JH III） in the 3rd-5th instar nymphs. Disruption of Nlkr-h1 expression by RNAi caused stunted wing development and malformations of both male and female external genitalia. Our findings suggest that Kr-h1 may be a useful target for pest insect management.

Expression of FAMeT and JHE Influenced by Kruppel homolog-1 Gene from Brown Planthopper

The characteristics of zinc finger of Kruppel homolog-1 （N/kr-hl） gene from brown planthopper and its effect on expression of FAMET and JHE were studied in the paper, in order to understand the regulation role of Nlkr-hl. The evolutionary tree of each zinc finger in functional area of Nlkr-hl was analyzed via MEGAS. 0 software, and expression status of FAMeT and JHE after interference of N/kr-hl was determined using qPCR technology. The results showed that the functional area of N/kr-hl mainly included eight zinc fingers, and the conservativeness of Znl and Zn8 was relatively lower. In the brown planthopper with N/b＇-hl gene knockout, the expression levels of FAMeT and JHE increased. The paper provided the theoretical basis for understanding molecular mechanism of juvenile hormone signaling pathway and biological control of brown planthopper.

Diapause Regulation in the Moth Sesamia nonagrioides （Lepidoptera： Noctuidae）

Insects enter in diapause in response to diverse environmental cues. During diapause, insects arrest their development and many genes are down-regulated while a small number of genes uniquely expressed at this time. This review aims to present available data regarding the regulation of diapause in the moth Sesamia nonagrioides （Lepidoptera： Noctuidae）. Studying the transcriptional regulation of several genes （five heat shock proteins, two storage proteins and one juvenile hormone esterase） showed that these genes may play various roles in the diapause programming. The results show that SnoHsp19.5 gene was consistently expressed, while SnoHsp20.8 was down-regulated in deep diapause and was up-regulated at the termination of diapause. SnoHsc70 may play important roles in assisting protein conformation during specific stages of diapause. SnoHsp83 displays a similar pattern to SnoHsc70 under diapause conditions, when extra larval moults occur, indicating that could be involved in the developmental process that occurs between two moults. Expression of two SnoSP1 and SnoSP2 hexamerin genes was also observed throughout diapause. And the results lead us to the conclusion that larval diapause of S. nonagrioides is associated with continuous synthesis and accumulation of storage proteins. In addition, the transcript level of the carboxylesterase SnoJHER was higher in non-diapausing larvae than in diapausing ones. During the fifth instar of the non-diapausing and the ninth instar of the diapausing larvae, SnoJHER mRNAs reached higher expression levels on the days close to each larval molt.

Hexamerin and allergen are required for female reproduction in the American cockroach,Periplaneta americana

Reproduction is of great importance for the continuation of the species.In insects,the fat body is the major tissue for nutrient storage and involved in vitellogenesis,which is essential for female reproduction.Here,2 proteins,hexamerin and allergen,were separated from the fat bodies of adult female American cockroaches(Periplaneta americana)and identified as storage proteins,encoding for 733 amino acids with molecular weight of 87.88 kDa and 686 amino acids with molecular weight of 82.18 kDa,respectively.The encoding genes of these 2 storage proteins are mainly expressed in the fat body.RNA interference-mediated knockdown of Hexamerin and Allergen in the early stage of the first reproductive cycle in females suppressed vitellogenesis and ovarian maturation,indicating that these storage proteins are involved in controlling reproduction.Importantly,the expression of Hexamerin and Allergen was repressed by knockdown of the juvenile hormone(JH)receptor gene Met and the primary response gene Kr-h1,and was induced by methoprene,a JH analog,in both in vivo and in vitro experiments.Altogether,we have determined that hexamerin and allergen are identified as storage proteins and play an important role in promoting female reproduction in the American cockroach.The expression of their encoding genes is induced by JH signaling.Our data reveal a novel mechanism by which hexamerin and allergen are necessary for JH-stimulated female reproduction.

HORMONAL CONTROL OF THE VITELLOGENESIS IN THE JAPANESE OAK SILKWORM, ANTHERAEA YAMAMAI (LEPIDOPTERA : SATURNIIDAE)

Effects of ecdysteroid and juvenile hormone (JH) on vitellogenesis of the Japanese oak silkworm, Antheraea yamamai are reported in this article. After topical treatment with 20 hydroxyecdysone alone or JH analog (i.e. methoprene) alone and combined treatment with these two chemicals, vitellogenin (Vg) titers in the fat body and haemolymph at the pupal stage were mostly higher than those of the control, indicating that both ecdysteroid and JH exerted a promoting effect on the synthesis of Vg. In contrast, the Vg uptake was markedly inhibited by JH while stimulating effect of the ecdysteroid could be shown that vitellin (Vt) titer in the ovary was lower after methoprene treatments, but higher after 20 hydroxyecdyson treatments. Meanwhile, effects of these two hormones on Vg synthesis in the fat body were also tested with the incubation in vitro with Grace medium containing 3H leucine and the hormones. The results demonstrated that Vg synthesis was stimulated after treating with methoprene alone or 20 hydroxyecdysone alone and combined treating with these two chemicals, and particularly ecdysteroid had more marked positive effect. To comprehensively concluded our results, it could be regarded that ecdysteroid play the main role in the regulation of vitellogenesis for the Japanese oak silkworm.

A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori

Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells ofBombyx mori. BmSP36 contains an intact catalytic triad （H57, D102 and S195） and a conserved substrate-binding site （G189, H216 and G226）, suggesting that it is a serine protease with chymotrypsin- like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down- regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development.

Cloning and characterization of Methoprene-tolerant(Met)and Kruppel homolog 1(Kr-h1)genes in the wheat blossom midge,Sitodiplosis mosellana

Juvenile hormone(JH),a growth regulator,inhibits ecdysteroid-induced meta-morphosis and controls insect development and diapause.Methoprene-tolerant(Met)and Krippel homolog I(Kr-h1)are two proteins involved in JH action.To gain some insight into their function in development of Sitodiplosis mosellana,an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage,we cloned full-length complemen-tary DNAs of Met and Kr-h1 from this specics.SmMet encoded a putative protein,which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding.SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other specics,and contained all eight highly conserved Zn-finger motifs for DNA-binding.Expression patterns of SmMet and SmKr hl were de-velopmentally regulated and JH III responsive as well.Their mRNA abundance increased as larvae entered carly 3rd instar,pre-diapause and maintenance stages,and peaked during post-diapause quiescence,a pattern correlated with JH titers in this species.Different from reduced expression of SmMer,SmKr-h1 mRNA increased at mid-to-late period of post-diapause development.Topical application of JH II on diapausing larvac also induced the two genes in a dose-dependent manner.Expression of SmuMer and SmKr-h1 clearly declined in the pre-pupal phase,and was significantly higher in female adults than male adults.These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction,whereas metamorphosis from larvae to pupac might be correlated with their reduced expression.

Identification of G protein-coupled receptors required for vitellogenesis and egg development in an insect with panoistic ovary

G protein-coupled receptors(GPCRs),a superfamily of integral transmembrane proteins regulate a variety of physiological processes in insects.Juvenile hormone(JH)is known to stimulate Vitellogenin(Vg)synthesis in the fat body,secretion into the hemolymph and uptake by developing oocytes.However,the role of GPCRs in JH-dependent insect vitellogenesis and oocyte maturation remains elusive.In the present study,we performed transcriptomic analysis and RNA interference(RNAi)screening in vitellogenic females of the migratory locust Locusta migratoria.Of 22 GPCRs identified in ovarian transcriptome,LGR4,OR-A1,OR-A2,Mthl1,Mthl5 and Smo were most abundant in the ovary.By comparison,mAChR-C expressed at higher levels in the fat body,whereas Oct/TyrR,OARβ,AdoR and ADGRA3 were at higher expression levels in the brain.Our RNAi screening demonstrated that knockdown of six GPCRs resulted in defective phenotypes of Vg accumulation in developing oocytes,accompanied by blocked ovarian development and impaired oocyte maturation.While LGR4 and Oct/TyrR appeared to control Vg synthesis in the fat body,OR-A1,OR-A2,mAChR-C and CirlL regulated Vg transportation and uptake.The findings provide fundamental evidence for deciphering the regulatory mechanisms of GPCRs in JH-stimulated insect reproduction.