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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

MicroRNA miR-2765-3p regulates reproductive diapause by targeting FoxO in Galeruca daurica

The forkhead box O(FoxO),as a conserved transcription factor,plays an indispensable role in regulating insect diapause.However,how FoxO is regulated to control diapause in insects remains unknown.In this study,we discovered functional binding sites for miR-2765-3p in the 3′untranslated region of FoxO in Galeruca daurica.The luciferase reporter assay showed that miR-2765-3p targeted FoxO and suppressed its expression.The expression profiles of miR-2765-3p and FoxO displayed opposite patterns during the female developmental process.Overexpression of miR-2765-3p by the injection of the miR-2765-3p agomir into adult females reduced FoxO expression,leading to the suppression of lipid accumulation,promotion of ovarian development,and inhibition of reproductive diapause.This is similar to the phenotype that results from the depletion of FoxO by injecting dsFoxO into adult females.In addition,the repression of miR-2765-3p by injecting the miR-2765-3p antagomir increased the FoxO transcript level,leading to the stimulation of lipid accumulation,depression of ovarian development,and induction of reproductive diapause.A hormone injection assay showed that the juvenile hormone(JH)agonist(methoprene)upregulated miR-2765-3p and downregulated FoxO.Notably,injecting methoprene rescued ovarian development defects associated with miR-2765-3p inhibition.These findings indicate that the JH/miR-2765-3p/FoxO axis plays a vital role in the regulation of reproductive diapause in G.daurica.

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition

Endoparasitoid wasps introduce venom into their host insects during the egglaying stage.Venom proteins play various roles in the host physiology,development,immunity,and behavior manipulation and regulation.In this study,we identified a venom protein,MmRhol,a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone,egg production,and cocoons formation in the female wasps.We demonstrated that MmRho1 entered the cotton bollworm's(host)hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining.Furthermore,wasp MmRhol interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down.In conclusion,this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.

Multiple endocrine factors regulate nutrient mobilization and storage in Aedes aegypti during a gonadotrophic cycle

Anautogenous mosquitoes must blood feed on a vertebrate host to produce eggs.Each gonadotrophic cycle is subdivided into a sugar-feeding previtellogenic phase that produces primary follicles and a blood meal-activated vitellogenic phase in which large numbers of eggs synchronously mature and are laid.Multiple endocrine factors including juvenile hormone(JH),insulin-like peptides(ILPs),ovary ecdysteroidogenic hormone(OEH),and 20-hydroxyecdysone(20E)coordinate each gonadotrophic cycle.Egg formation also requires nutrients from feeding that are stored in the fat body.Regulation of egg formation is best understood in Aedes aegypti but the role different endocrine factors play in regulating nutrient mobilization and storage remains unclear.In this study,we report that adult female Ae.aegypti maintained triacylglycerol(TAG)stores during the previtellogenic phase of the first gonadotrophic cycle while glycogen stores declined.In contrast,TAG and glycogen stores were rapidly mobilized during the vitellogenic phase and then replenishment.Several genes encoding enzymes with functions in TAG and glycogen metabolism were differentially expressed in the fat body,which suggested regulation was mediated in part at the transcriptional level.Gain of function assays indicated that stored nutrients were primarily mobilized by adipokinetic hormone(AKH)while juvenoids and OEH regulated replenishment.ILP3 further showed evidence of negatively regulating certain lipolytic enzymes.Loss of function assays indicated AKH depends on the AKH receptor(AKHR)for function.Altogether,our results indicate that the opposing activities of different hormones regulate nutrient stores during a gonadotrophic cycle in Ae.aegypti.

Comparison of the signaling pathways of wing dimorphism regulated by biotic and abiotic stress in the brown planthopper

Wing polymorphism is an evolutionary trait that is widely present in various insects and provides a model system for studying the evolutionary significance of insect dispersal.The brown planthopper(BPH,Nilaparvata lugens)can alter its wing morphs un-der biotic and abiotic stress.However,whether differential signaling pathways are induced by the 2 types of stress remain largely unknown.Here,we screened a number of candidate genes through weighted gene co-expression network analysis(WGCNA)and found that ornithine decarboxylase(NIODC),a key enzyme in the synthesis of polyamines,was as-sociated with wing differentiation in BPH and mainly responded to abiotic stress stimuli.We analyzed the Kyoto Encyclopedia of Genes and Genomes enrichment pathways of dif-ferentially expressed genes under the 2 stresses by transcriptomic comparison,and found that biotic stress mainly influenced insulin-related signaling pathways while abiotic stress mainly influenced hormone-related pathways.Moreover,we found that insulin receptor 1(NllnRI)may regulate wing differentiation of BPH by responding to both biotic and abiotic stress,but NllnR2 only responded to biotic stress.Similarly,the juvenile hormone epoxide hydrolase associated with juvenile hormone degradation and NIODC may regu-late wing differentiation mainly through abiotic stress.A model based on the genes and stresses to modulate the wing dimorphism of BPH was proposed.These findings present a comprehensive molecular mechanism for wing polymorphism in BPH induced by biotic and abiotic stress.

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.