Unique peptidic agonists of a juvenile hormone receptor with species-specific effects on insect development and reproduction.

Juvenile hormones (JHs) control insect metamorphosis and reproduction. JHs act through a receptor complex consisting of methoprene-tolerant (Met) and taiman (Tai) proteins to induce transcription of specific genes. Among chemically diverse synthetic JH mimics (juvenoids), some of which serve as insecticides, unique peptidic juvenoids stand out as being highly potent yet exquisitely selective to a specific family of true bugs. Their mode of action is unknown. Here we demonstrate that, like established JH receptor agonists, peptidic juvenoids act upon the JHR Met to halt metamorphosis in larvae of the linden bug, Pyrrhocoris apterus. Peptidic juvenoids induced ligand-dependent dimerization between Met and Tai proteins from P. apterus but, consistent with their selectivity, not from other insects. A cell-based split-luciferase system revealed that the Met-Tai complex assembled within minutes of agonist presence. To explore the potential of juvenoid peptides, we synthesized 120 new derivatives and tested them in Met-Tai interaction assays. While many substituents led to loss of activity, improved derivatives active at sub-nanomolar range outperformed hitherto existing peptidic and classical juvenoids including fenoxycarb. Their potency in inducing Met-Tai interaction corresponded with the capacity to block metamorphosis in P. apterus larvae and to stimulate oogenesis in reproductively arrested adult females. Molecular modeling demonstrated that the high potency correlates with high affinity. This is a result of malleability of the ligand-binding pocket of P. apterus Met that allows larger peptidic ligands to maximize their contact surface. Our data establish peptidic juvenoids as highly potent and species-selective novel JHR agonists.

Sex- and caste-specific developmental responses to juvenile hormone in an ant with maternal caste determination.

Juvenile hormone is considered to be a master regulator of polyphenism in social insects. In the ant Cardiocondyla obscurior, whether a female egg develops into a queen or a worker is determined maternally and caste-specific differentiation occurs in embryos, so that queens and workers can be distinguished in a non-invasive manner from late embryogenesis onwards. This ant also exhibits two male morphs - winged and wingless males. Here, we used topical treatment with juvenile hormone III and its synthetic analogue methoprene, a method that influences caste determination and differentiation in some ant species, to investigate whether hormone manipulation affects the development and growth of male, queen- and worker-destined embryos and larvae. We found no effect of hormone treatment on female caste ratios or body sizes in any of the treated stages, even though individuals reacted to heightened hormone availability with increased expression of krüppel-homolog 1, a conserved JH first-response gene. In contrast, hormone treatment resulted in the emergence of significantly larger males, although male morph fate was not affected. These results show that in C. obscurior, maternal caste determination leads to irreversible and highly canalized caste-specific development and growth.

Insulin/IGF signaling and TORC1 promote vitellogenesis via inducing juvenile hormone biosynthesis in the American cockroach.

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient sensors insulin/IGF signaling (IIS) and target of rapamycin complex1 (TORC1), was investigated to elucidate the molecular mechanisms of vitellogenesis regulation in the American cockroach, Periplaneta americana Our data showed that a block of JH biosynthesis or JH action arrested vitellogenesis, in part by inhibiting the expression of doublesex (Dsx), a key transcription factor gene involved in the sex determination cascade. Depletion of IIS or TORC1 blocked both JH biosynthesis and vitellogenesis. Importantly, the JH analog methoprene, but not bovine insulin (to restore IIS) and amino acids (to restore TORC1 activity), restored vitellogenesis in the neck-ligated (IIS-, TORC1- and JH-deficient) and rapamycin-treated (TORC1- and JH-deficient) cockroaches. Combining classic physiology with modern molecular techniques, we have demonstrated that IIS and TORC1 promote vitellogenesis, mainly via inducing JH biosynthesis in the American cockroach.

The ecdysis triggering hormone system is essential for reproductive success in Mythimna separata (Walker).

Ecdysis triggering hormone (ETH) was originally discovered as a key hormone that regulates insect moulting via binding to its receptor, ETH receptor (ETHR). However, the precise role of ETH in moth reproduction remains to be explored in detail. ETH function was verified in vivo using Mythimna separata (Walker), an important cereal crop pest. RT-qPCR analysis revealed that transcriptional expression profiles of MsepETH showed evident sexual dimorphism in the adult stage. MsepETH expression increased in the females on day 3 and persisted thereafter till day 7, consistent with female ovarian maturation, and was merely detectable in males. Meanwhile, MsepETH expression levels were significantly higher in the trachea than in other tissues. MsepETHR-A and MsepETHR-B were expressed in both sexes and were significantly higher in the antennae than in other tissues. MsepETH and MsepETHR knockdown in females by RNA interference significantly reduced the expression of MsepETH, MsepETHR-A, MsepETHR-B, MsepJHAMT, and MsepVG, which delayed egg-laying and significantly reduced egg production. RNAi 20-hydroxyecdysone (20E) receptor (EcR) decreased MsepETH expression whereas injecting 20E restored egg production that had been disrupted by MsepETH interference. Meanwhile, RNAi juvenile hormone (JH) methoprene tolerant protein (Met) also decreased MsepETH expression and smearing JH analog methoprene (Meth) restored egg production. In conclusion, the reproduction roles of ETH, JH, and 20E were investigated in M. separata. These findings will lay the foundation for future research to develop an antagonist that reduces female reproduction and control strategies for pest insects.

CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.

Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.

Methoprene-tolerant and Krüppel homolog 1 are actors of juvenile hormone-signaling controlling the development of male sexual behavior in the moth Agrotis ipsilon.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-mediated vitellogenesis of female Liposcelis entomophila (End.) (Psocoptera: Liposcelididae).

Methoprene-tolerant (Met) as an intracellular receptor of juvenile hormone (JH) and the Krüppel-homolog 1 (Kr-h1) as a JH-inducible transcription factor had been proved to contribute to insect reproduction. Their functions vary in different insect orders, however, they are not clear in Psocoptera. In this study, LeMet and LeKr-h1 were identified and their roles in vitellogenesis and ovarian development were investigated in Liposcelis entomophila (Enderlein). Treatment with exogenous JH III significantly induced the expression of LeKr-h1, LeVg, and LeVgR. Furthermore, silencing LeMet and LeKr-h1 remarkably reduced the transcription of LeVg and LeVgR, disrupted the production of Vg in fat body and the uptake of Vg by oocytes, and ultimately led to a decline in fecundity. The results indicated that the JH signaling pathway was essential to the reproductive process of this species. Interestingly, knockdown of LeMet or LeKr-h1 also resulted in fluctuations in the expression of FoxO, indicating the complex regulatory interactions between different hormone factors. Besides, knockdown of both LeMet and LeKr-h1 significantly increased L. entomophila mortality. Our study provides initial insight into the roles of JH signaling in the female reproduction of psocids and provided evidence that RNAi-mediated knockdown of Met or Kr-h1 is a potential pest control strategy.

Methoprene treatment and its effect on male reproductive organ size and female remating in a fruit fly.

Methoprene, a juvenile hormone analog, is used to accelerate sexual maturation in males of species of economic importance in support to the sterile insect technique (SIT). In the SIT, mass-reared sterile males are released into the field and need to survive until they reach sexual maturation, find a wild female, mate with her and then induce female sexual refractoriness, so she will not remate with a wild counterpart. The use of methoprene shortens the time between release and copulation. However, in South American fruit flies, Anastrepha fraterculus, the ability of methoprene-treated males to inhibit female remating has been shown to be lower than wild males, when methoprene was applied by pupal immersion or topical application. Here we evaluated the possibility of incorporating methoprene into the male diet at different doses and the ability of those males to inhibit female remating, as well as the effect of methoprene on male reproductive organ size, due to the possible correlation between male accessory gland size and their content, and the role of male accessory gland proteins in female inhibition. We found that A. fraterculus males fed with methoprene in the adult protein diet at doses as high as 1% were less likely to inhibit female remating, however, at all other lower doses males had the same ability as untreated males to inhibit female remating. Males fed with methoprene had bigger male accessory glands and testes compared to methoprene-deprived males. We demonstrate that the incorporation of methoprene in adult male diets is possible in this species and potentially useful as a post-teneral, pre-release supplement at doses as low as 0.01%. Even at higher doses, the percentage of females remating after 48 h from the first copulation is sufficiently low in this species so as not compromise the efficiency of the SIT.

Juvenile hormone suppresses aggregation behavior through influencing antennal gene expression in locusts.

Animals often exhibit dramatically behavioral plasticity depending on their internal physiological state, yet little is known about the underlying molecular mechanisms. The migratory locust, Locusta migratoria, provides an excellent model for addressing these questions because of their famous phase polyphenism involving remarkably behavioral plasticity between gregarious and solitarious phases. Here, we report that a major insect hormone, juvenile hormone, is involved in the regulation of this behavioral plasticity related to phase change by influencing the expression levels of olfactory-related genes in the migratory locust. We found that the treatment of juvenile hormone analog, methoprene, can significantly shift the olfactory responses of gregarious nymphs from attraction to repulsion to the volatiles released by gregarious nymphs. In contrast, the repulsion behavior of solitarious nymphs significantly decreased when they were treated with precocene or injected with double-stranded RNA of JHAMT, a juvenile hormone acid O-methyltransferase. Further, JH receptor Met or JH-response gene Kr-h1 knockdown phenocopied the JH-deprivation effects on olfactory behavior. RNA-seq analysis identified 122 differentially expressed genes in antennae after methoprene application on gregarious nymphs. Interestingly, several olfactory-related genes were especially enriched, including takeout (TO) and chemosensory protein (CSP) which have key roles in behavioral phase change of locusts. Furthermore, methoprene application and Met or Kr-h1 knockdown resulted in simultaneous changes of both TO1 and CSP3 expression to reverse pattern, which mediated the transition between repulsion and attraction responses to gregarious volatiles. Our results suggest the regulatory roles of a pleiotropic hormone in locust behavioral plasticity through modulating gene expression in the peripheral olfactory system.

The retinoid metabolism of Gammarus fossarum is disrupted by exogenous all-trans retinoic acid, citral, and methoprene but not by the technical formulation of glyphosate.

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Investigating the role of the ROS/CncC signaling pathway in the response to xenobiotics in Spodoptera frugiperda using Sf9 cells.

Spodoptera frugiperda (fall armyworm, FAW) is an invasive polyphagous lepidopteran pest that has developed sophisticated resistance mechanisms involving detoxification enzymes to eliminate toxic compounds it encounters in its diet including insecticides. Although its inventory of detoxification enzymes is known, the mechanisms that enable an adapted response depending on the toxic compound remain largely unexplored. Sf9 cells were used to investigate the role of the transcription factors, Cap n' collar isoform C (CncC) and musculoaponeurotic fibrosarcoma (Maf) in the regulation of the detoxification response. We overexpressed CncC, Maf or both genes, and knocked out (KO) CncC or its repressor Kelch-like ECH associated protein 1 (Keap1). Joint overexpression of CncC and Maf is required to confer increased tolerance to indole 3-carbinol (I3C), a plant secondary metabolite, and to methoprene, an insecticide. Both molecules induce reactive oxygen species (ROS) pulses in the different cell lines. The use of an antioxidant reversed ROS pulses and restored the tolerance to I3C and methoprene. The activity of detoxification enzymes varied according to the cell line. Suppression of Keap1 significantly increased the activity of cytochrome P450s, carboxylesterases and glutathione S-transferases. RNAseq experiments showed that CncC mainly regulates the expression of detoxification genes but is also at the crossroads of several signaling pathways (reproduction and immunity) maintaining homeostasis. We present new data in Sf9 cell lines suggesting that the CncC:Maf pathway plays a central role in FAW response to natural and synthetic xenobiotics. This knowledge helps to better understand detoxification gene expression and may help to design next-generation pest insect control measures.

Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect's life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editing method to knockout the methoprene-tolerant (Met) gene coding for a JH receptor. The Met knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, Met knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that Krüppel homolog 1, a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 Met mutants. These data suggest that, during the L3 and L4 stages, Met mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in Ae. aegypti.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones.

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 µL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

Dynamic transcriptome analysis and Methoprene-tolerant gene knockdown reveal that juvenile hormone regulates oogenesis and vitellogenin synthesis in Propylea Japonica.

Propylea japonica has been regarded as one of the most remarkable natural enemies against aphid in China. However, the mechanism of juvenile hormone (JH) regulation of reproduction in P. japonica is still unclear. In this study, we investigated the JH titers of P. japonica and the development of the ovaries. We selected the six different developmental stages of ladybeetle females for transcriptome sequencing. We identified 583 genes involved in insect reproduction regulation, including 107 insect hormone synthesis signaling pathway-related genes and 476 nutrition-sensing signaling pathway-related genes. Transcriptome analysis indicated that a large number JH synthesis- and metabolism-related enzyme genes and some potential nutrient signal sensing- and transduction-related genes were significantly differentially expressed during P. japonica development. We investigated the effects of Met gene silencing on the reproduction of female adults and found that the ovarian maturation, vitellogenesis, and follicular epithelium development in the dsMet treatment group were significantly inhibited.

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica. RESULTS: Transcriptome analysis of resistant and susceptible strains of this pest species identified a set of differentially expressed genes related to cytochrome P450s, indicating their potential role in resistance to s-methoprene. Laboratory bioassays were performed with s-methoprene treated wheat grains in presence and absence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor. The results indicate that PBO, when applied alone, at least at the concentration tested here, had no effect on R. dominica adult emergence, but has a clear synergistic effect to s-methoprene. The number of produced progeny decreased in presence of the inhibitor, especially in the resistant strain. In addition, we also identified CYP complement (CYPome) of R. dominica, annotated and analysed phylogenetically, to understand the evolutionary relationships with other species. CONCLUSIONS: The information generated in current study suggest that PBO can effectively be used to break resistance to s-methoprene in R. dominica.

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Juvenile hormone affects age polyethism, ovarian status and cuticular hydrocarbon profile in workers of the wasp Polybia occidentalis.

Division of labor is one of the most striking features in the evolution of eusociality. Juvenile hormone (JH) mediates reproductive status and aggression among nestmates in primitively eusocial Hymenoptera (species without morphologically distinct castes). In highly social species it has apparently lost its gonadotropic role and primarily regulates the division of labor in the worker caste. Polybia occidentalis, a Neotropical swarm-founding wasp, is an ideal model to understand how JH levels mirror social context and reproductive opportunities because of the absence of a clear morphological caste dimorphism. In this study, we tested the hypothesis that JH influences division of labor, ovary activation and cuticular hydrocarbon profiles of workers. Our observations confirmed that a JH analog (methoprene) and an inhibitor of JH biosynthesis (precocene) affected the cuticular chemical profile associated with age polyethism. Also, methoprene and precocene-I treatment of females influenced ovarian activation differently (individuals treated with methoprene expressed more activated ovaries while precocene treatment did not have significant effect). These results suggest that different hormonal levels induce a differential expression of cuticular chemicals associated with workers' age polyethism, which may be essential for keeping the social cohesion among workers throughout their lives in the colony. Furthermore, JH is likely to play a gonadotropic role in P. occidentalis. JH has apparently undergone certain modifications in social Hymenoptera, presenting multifaceted functions in different species.

Behavioral flexibility in Wasmannia auropunctata (Hymenoptera: Formicidae).

Worker division of labor is a defining trait in social insects. Many species are characterized by having behavioral flexibility where workers perform non-typical tasks for their age depending on the colony's needs. Worker division of labor and behavioral flexibility were examined in the little fire ant Wasmannia auropunctata (Roger, 1863), for which age-related division of labor has been found. Young workers perform nursing duties which include tending of brood and queens, and colony defense, while older workers forage. When nurses were experimentally removed from the colony, foragers were observed carrying out nursing and colony defense duties, yet when foragers were removed nurses did not forage precociously. We also administered juvenile hormone analog, methoprene, to workers. When methoprene was applied, foragers increased their nursing and defense activities while nurses became mainly idle. The behavioral flexibility of foragers of the little fire ant may be evidence of an expansion of worker's repertoires as they age; older workers can perform tasks they have already done in their life while young individuals are not capable of performing tasks ahead of time. This may be an important adaptation associated with the success of this ant as an invasive species.

Juvenile hormone signaling in short germ-band hemimetabolan embryos.

The role of juvenile hormone (JH) in insect embryos is far from understood, especially in short germ-band hemimetabolan species. To shed light on this issue, we depleted the mRNA levels of Krüppel homolog 1, Methoprene-tolerant and JH acid O-methyltransferase, key elements of JH signaling, in embryos of the short germ-band hemimetabolan species Blattella germanica This precluded the formation of the germ-band anlage in a group of embryos. Hatchability was also reduced, which might have been caused by premature upregulation of laccase 2, a promoter of cuticle tanning. In other cases, development was interrupted in mid embryogenesis, involving defects related to dorsal closure and appendage formation. These phenotypes possibly result from the low levels of Broad-complex (BR-C) produced under JH-depleted conditions. This contrasts with holometabolan species, in which JH does not promote BR-C expression, which remains low during embryo development. Possibly, the stimulatory role of JH on BR-C expression and the morphogenetic functions of BR-C in hemimetabolan embryos were lost in holometabolan species. If so, this might have been a key driver for the evolution of holometabolan metamorphosis.

Molecular Insights into Structural and Ligand Binding Features of Methoprene-Tolerant in Daphnids.

Juvenile hormone (JH) is an important endocrine factor regulating many biological activities in arthropods. In daphnids, methoprene-tolerant (Met) belongs to a basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family protein which has recently been confirmed as a JH receptor and can bind and be activated by JHs and JH agonists. Although the activation of the JH signaling pathway causes many physiological effects, the molecular basis for the structural feature and ligand binding properties of Daphnia Met are not fully understood. To study the ligand preference in terms of structural features of Daphnia Met, we built in silico homology models of the PAS-B domain of Daphnia Mets from cladoceran crustaceans, Daphnia pulex and D. magna. Structural comparison of two Daphnia Met PAS-B domain models revealed that the volume in the main cavity of D. magna Met was larger than that of D. pulex Met. Compared with insect Met, Daphnia Met had a less hydrophobic cavity due to polar residues in the core-binding site. Molecular docking simulations of JH and its analogs with Daphnia Met indicated that the interaction energies were correlated with each of the experimental values of in vivo JH activities based on male induction and in vitro Met-mediated transactivation potencies. Furthermore, in silico site-directed mutagenesis supported experimental findings that Thr292 in D. pulex Met and Thr296 in D. magna Met substitution to valine contribute to JH selectivity and differential species response. This study demonstrates that in silico simulations of Daphnia Met and its ligands may be a tool for predicting the ligand profile and cross species sensitivity.