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.

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.

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.

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.

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.

The juvenile hormone receptor as a target of juvenoid "insect growth regulators".

Synthetic compounds that mimic the action of juvenile hormones (JHs) are founding members of a class of insecticides called insect growth regulators (IGRs). Like JHs, these juvenoids block metamorphosis of insect larvae to reproductive adults. Many biologically active juvenoids deviate in their chemical structure considerably from the sesquiterpenoid JHs, raising questions about the mode of action of such JH mimics. Despite the early deployment of juvenoid IGRs in the mid-1970s, their molecular effect could not be understood until recent discoveries of JH signaling through an intracellular JH receptor, namely the ligand-binding transcription factor Methoprene-tolerant (Met). Here, we briefly overview evidence defining three widely employed and chemically distinct juvenoid IGRs (methoprene, pyriproxyfen, and fenoxycarb), as agonist ligands of the JH receptor. We stress that knowledge of the target molecule is critical for using these compounds both as insecticides and as research tools.

Molecular mechanism underlying juvenile hormone-mediated repression of precocious larval-adult metamorphosis.

Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval-pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysone-induced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93 Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis.

Does juvenile hormone prompt oxidative stress in male damselflies?

In invertebrates, it has recently been reported that secondary sexual characteristics (SSCs) reflect the antioxidant defense of their bearers, but it is not known what physiological link maintains the honesty of those signals. Here, we used the damselfly Hetaerina americana to test whether juvenile hormone plays such a role. First, we analyzed whether oxidative damage is a real threat in natural damselfly populations by examining the accumulation of oxidized guanines as a function of age in males. Then, we injected paraquat (a pro-oxidant agent) and added the juvenile hormone analog methoprene (JHa) to the experimental group and the JHa vehicle (acetone) to the control group, to determine whether JHa increases the levels of pro-oxidants and antioxidants. We found that DNA oxidation increased with age, and that levels of hydrogen peroxide and superoxide dismutase, but not catalase or glutathione, were elevated in the JHa group compared with the control group. We propose that juvenile hormone is a mediator of the relationship between SSCs and antioxidant capacity and, based on the literature, we know that JHa suppresses the immune response. We therefore suggest that juvenile hormone is a molecular mediator of the general health of males, which is reflected in their SSCs.

The FOXO transcription factor controls insect growth and development by regulating juvenile hormone degradation in the silkworm, Bombyx mori.

Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects. However, how FOXO interacts with hormone signaling to modulate insect growth and development is largely unknown. Here, using the transgene-based CRISPR/Cas9 system, we generated and characterized mutants of the silkworm Bombyx mori FOXO (BmFOXO) to elucidate its physiological functions during development of this lepidopteran insect. The BmFOXO mutant (FOXO-M) exhibited growth delays from the first larval stage and showed precocious metamorphosis, pupating at the end of the fourth instar (trimolter) rather than at the end of the fifth instar as in the wild-type (WT) animals. However, different from previous reports on precocious metamorphosis caused by juvenile hormone (JH) deficiency in silkworm mutants, the total developmental time of the larval period in the FOXO-M was comparable with that of the WT. Exogenous application of 20-hydroxyecdysone (20E) or of the JH analog rescued the trimolter phenotype. RNA-seq and gene expression analyses indicated that genes involved in JH degradation but not in JH biosynthesis were up-regulated in the FOXO-M compared with the WT animals. Moreover, we identified several FOXO-binding sites in the promoter of genes coding for JH-degradation enzymes. These results suggest that FOXO regulates JH degradation rather than its biosynthesis, which further modulates hormone homeostasis to control growth and development in B. mori In conclusion, we have uncovered a pivotal role for FOXO in regulating JH signaling to control insect development.

Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein.

Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JH-regulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membrane-initiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH.

Effects of juvenile hormone analogue (methoprene) and 20-hydroxyecdysone on reproduction in Polygonia c-aureum (Lepidoptera: Nymphalidae) in relation to adult diapause.

We investigated the effects of juvenile hormone analogue (methoprene) and 20-hydroxyecdysone on female and male reproduction in a nymphalid butterfly, Polygonia c-aureum. This butterfly has a facultative adult diapause controlled by the corpora allata and brain. Methoprene seems to terminate reproductive diapause, although transplantation experiments indicate that the activity of the corpora allata does not affect male mating behavior Endo (Dev Growth Differ 15:1-10, 1973a), suggesting that the brain may be involved in diapause. We found that exposure to methoprene promoted the development of ovaries and of the male accessory glands and simplex. On the other hand, exposure to 20-hydroxyecdysone did not promote the development of female and male reproductive organs and eupyrene sperm movement from the testis to the duplex in the adult stage. Ecdysteroid titer in both sexes was consistently low in adults. These results suggest that imaginal diapause is largely regulated by juvenile hormone in this butterfly.

Transcription factor E93 specifies adult metamorphosis in hemimetabolous and holometabolous insects.

All immature animals undergo remarkable morphological and physiological changes to become mature adults. In winged insects, metamorphic changes either are limited to a few tissues (hemimetaboly) or involve a complete reorganization of most tissues and organs (holometaboly). Despite the differences, the genetic switch between immature and adult forms in both types of insects relies on the disappearance of the antimetamorphic juvenile hormone (JH) and the transcription factors Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) during the last juvenile instar. Here, we show that the transcription factor E93 is the key determinant that promotes adult metamorphosis in both hemimetabolous and holometabolous insects, thus acting as the universal adult specifier. In the hemimetabolous insect Blattella germanica, BgE93 is highly expressed in metamorphic tissues, and RNA interference (RNAi)-mediated knockdown of BgE93 in the nymphal stage prevented the nymphal-adult transition, inducing endless reiteration of nymphal development, even in the absence of JH. We also find that BgE93 down-regulated BgKr-h1 and BgBR-C expression during the last nymphal instar of B. germanica, a key step necessary for proper adult differentiation. This essential role of E93 is conserved in holometabolous insects as TcE93 RNAi in Tribolium castaneum prevented pupal-adult transition and produced a supernumerary second pupa. In this beetle, TcE93 also represses expression of TcKr-h1 and TcBR-C during the pupal stage. Similar results were obtained in the more derived holometabolous insect Drosophila melanogaster, suggesting that winged insects use the same regulatory mechanism to promote adult metamorphosis. This study provides an important insight into the understanding of the molecular basis of adult metamorphosis.

Behavioural effects of juvenile hormone and their influence on division of labour in leaf-cutting ant societies.

Division of labour in social insects represents a major evolutionary transition, but the physiological mechanisms that regulate this are still little understood. Experimental work with honey bees, and correlational analyses in other social insects, have implicated juvenile hormone (JH) as a regulatory factor, but direct experimental evidence of behavioural effects of JH in social insects is generally lacking. Here, we used experimental manipulation of JH to show that raised JH levels in leaf-cutting ants results in workers becoming more active, phototactic and threat responsive, and engaging in more extranidal activity - behavioural changes that we show are all characteristic of the transition from intranidal work to foraging. These behavioural effects on division of labour suggest that the JH mediation of behaviour occurs across multiple independent evolutions of eusociality, and may be a key endocrine regulator of the division of labour which has produced the remarkable ecological and evolutionary success of social insects.

Is Juvenile Hormone a potential mechanism that underlay the "branched Y-model"?

Trade-offs are a central tenet in the life-history evolution and the simplest model to understand it is the "Y" model: the investment of one arm will affect the investment of the other arm. However, this model is by far more complex, and a "branched Y-model" is proposed: trade-offs could exist within each arm of the Y, but the mechanistic link is unknown. Here we used Tenebrio molitor to test if Juvenile Hormone (JH) could be a mechanistic link behind the "branched Y-model". Larvae were assigned to one of the following experimental groups: (1) low, (2) medium and (3) high doses of methoprene (a Juvenile Hormone analogue, JHa), (4) acetone (methoprene diluents; control one) or (5) näive (handled in the same way as other groups; control two). The JHa lengthened the time of development from larvae to pupae and larvae to adults, resulting in adults with a larger size. Males with medium and long JHa treatment doses were favored with female choice, but had smaller testes and fewer viable sperm. There were no differences between groups in regard to the number of spermatozoa of males, or the number of ovarioles or eggs of females. This results suggest that JH: (i) is a mechanistic link of insects "branched Y model", (ii) is a double ended-sword because it may not only provide benefits on reproduction but could also impose costs, and (iii) has a differential effect on each sex, being males more affected than females.

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F1F0ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development.

Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti. Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methoprene-tolerant (Met) showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. Sequence motif analysis revealed the consensus for a 9-mer Met-binding motif, CACG(C)/TG(A)/G(T)/AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi-down-regulated (LPE) genes but not in Met RNAi-up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody supershift analyses confirmed the binding properties of the Met consensus motif variants. There was a striking temporal separation of expression profiles among major functional gene groups, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction.