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.

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.

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.

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.

Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression.

The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box-like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.

Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling.

In temperate regions, the shortening day length informs many insect species to prepare for winter by inducing diapause. The adult diapause of the linden bug, Pyrrhocoris apterus, involves a reproductive arrest accompanied by energy storage, reduction of metabolic needs, and preparation to withstand low temperatures. By contrast, nondiapause animals direct nutrient energy to muscle activity and reproduction. The photoperiod-dependent switch from diapause to reproduction is systemically transmitted throughout the organism by juvenile hormone (JH). Here, we show that, at the organ-autonomous level of the insect gut, the decision between reproduction and diapause relies on an interaction between JH signaling and circadian clock genes acting independently of the daily cycle. The JH receptor Methoprene-tolerant and the circadian proteins Clock and Cycle are all required in the gut to activate the Par domain protein 1 gene during reproduction and to simultaneously suppress a mammalian-type cryptochrome 2 gene that promotes the diapause program. A nonperiodic, organ-autonomous feedback between Par domain protein 1 and Cryptochrome 2 then orchestrates expression of downstream genes that mark the diapause vs. reproductive states of the gut. These results show that hormonal signaling through Methoprene-tolerant and circadian proteins controls gut-specific gene activity that is independent of circadian oscillations but differs between reproductive and diapausing animals.

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.

Heat shock protein 83 (Hsp83) facilitates methoprene-tolerant (Met) nuclear import to modulate juvenile hormone signaling.

Juvenile hormone (JH) receptors, methoprene-tolerant (Met) and Germ-cell expressed (Gce), transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with PAS-B and basic helix-loop-helix domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of fat body cells of the larval when the JH titer is low and JH induces Met nuclear import. Hsp83 was accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuated JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR.

The hormone-dependent function of Hsp90 in the crosstalk between 20-hydroxyecdysone and juvenile hormone signaling pathways in insects is determined by differential phosphorylation and protein interactions.

BACKGROUND: Heat shock protein 90 (Hsp90) interacts with steroid hormone receptors, signaling kinases, and various transcription factors. However, the mechanism by which Hsp90 interacts with different proteins in various pathways remains unclear. METHODS: Western blot was used to study Hsp90 expression profile in Helicoverpa armigera (Lepidoptera). RNA interference was performed to investigate the function of Hsp90 in 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal pathways. The binding of Hsp90 to the transcription factor ultraspiracle protein (USP1) and JH candidate receptor methoprene-tolerant (Met1) was analyzed by co-immunoprecipitation. Phospho-(Ser) PKC substrate antibody was used to detect Hsp90 phosphorylation. RESULTS: Hsp90 participated in 20E- or JH-induced gene expression. 20E induced the interaction between Hsp90 and USP1, whereas JH III and methoprene induced the interaction between Hsp90 and Met1, respectively. 20E and JH counteracted each other for these protein interactions. Both JH III and methoprene induced protein kinase C (PKC) phosphorylation of Hsp90. This process could be inhibited by phospholipase C (PLC) and PKC inhibitors. 20E suppressed JH III- or methoprene-induced PKC phosphorylation of Hsp90. CONCLUSION: 20E maintained the non-PKC-phosphorylation status of Hsp90. Hsp90 interacted with USP1 to induce gene expression in the 20E pathway. JH regulated the PKC-phosphorylation status of Hsp90. Hsp90 also interacted with Met1 to induce gene expression in the JH pathway. GENERAL SIGNIFICANCE: Our study describes a novel mechanism of Hsp90 action by altering phosphorylation and protein interaction in various hormonal signaling pathways.

Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant.

Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met-Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met-Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors.

The juvenile hormone signaling pathway in insect development.

The molecular action of juvenile hormone (JH), a regulator of vital importance to insects, was until recently regarded as a mystery. The past few years have seen an explosion of studies of JH signaling, sparked by a finding that a JH-resistance gene, Methoprene-tolerant (Met), plays a critical role in insect metamorphosis. Here, we summarize the recently acquired knowledge on the capacity of Met to bind JH, which has been mapped to a particular ligand-binding domain, thus establishing this bHLH-PAS protein as a novel type of an intracellular hormone receptor. Next, we consider the significance of JH-dependent interactions of Met with other transcription factors and signaling pathways. We examine the regulation and biological roles of genes acting downstream of JH and Met in insect metamorphosis. Finally, we discuss the current gaps in our understanding of JH action and outline directions for future research.

Methoprene influences reproduction and flight capacity in adults of the rice leaf roller, Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae).

Juvenile hormone (JH) influences many aspects of insect biology, including oogenesis-flight syndrome tradeoffs between migration and reproduction. Drawing on studies of many migratory insects, we posed the hypothesis that JH influences migratory capacity and oogenesis in the rice leaf roller, Cnaphalocrocis medinalis. We treated adults moths (days 1, 2 and 3 postemergence) with the JH analog (JHA), methoprene, and then recorded the influences of JHA treatments on reproduction. JHA treatment on day 1 postemergence, but not on the other days, shortened the preoviposition period, although JHA did not influence total fecundity, oviposition period, or longevity. We infer day 1 postemergence is the JH-sensitive stage to influence reproduction. Therefore, we treated moths on day 1 postemergence with JHA and recorded flight capacity, flight muscle mass, and triacylglycerol (TAG) accumulation. JHA treatments did not influence flight speed, but led to reductions in flight durations and flight distances. At day 3 posttreatment (PT), JHA-treated females flew shorter times and less distance than the controls; JHA-treated males, however, only flew shorter times than the controls. JHA treatments led to reductions in flight muscle mass in females at days 2-3 PT and reductions in TAG content in females at day 3 PT, but, these parameters were not influenced by JHA in males. These findings strongly support our hypothesis, from which we infer that JH is a major driver in C. medinalis oogenesis-flight syndrome tradeoffs. Our data also reveal a JH-sensitive stage in adulthood during which JH influences the oocyte-flight syndrome in C. medinalis.

Effect of juvenile hormone analog, methoprene on H-fibroin regulation during the last instar larval development of Corcyra cephalonica.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E), co-ordinately orchestrate insect growth and development. The process of silk synthesis and secretion in lepidopteran insects is known to be under hormonal control. However, the role of JH in this process has not been demonstrated hitherto. The present study is aimed to elucidate the role of JH in H-fibroin regulation in Corcyra cephalonica, a serious lepidopteran pest. Reiterated amino acid stretches and the large molecular weight of H-fibroin render its cloning and characterization cumbersome. To address this, a commercially synthesized short amino acid peptide conjugated with a carrier protein was used to generate antibodies against the N-terminal region of H-fibroin. ELISA and immunoblot experiments demonstrated the sensitivity and specificity of antibody. Further, immunohistochemical analyses revealed the antibody's cross-reactivity with H-fibroins of C. cephalonica and Bombyx mori in the silk gland lumen. Quantitative RT-PCR and Western blot analysis demonstrated the tissue-specificity and developmental expression of H-fibroin. Hormonal studies revealed that JH alone does not alter the expression of H-fibroin. However, in the presence 20E, JH reverses the declined expression caused by 20E administration to normal levels. This study provides molecular evidence for the regulation of H-fibroin by the cumulative action of JH and 20E.

Ligand-dependent protein interactions of the juvenile hormone receptor captured in real time.

Juvenile hormone (JH) signalling provides vital regulatory functions during insect development via transcriptional regulation of genes critical for the progression of metamorphosis and oogenesis. Despite the importance of JH signalling, the underlying molecular mechanisms remain largely unknown. Our current understanding of the pathway depends on static end-point information and suffers from the lack of time-resolved data. Here, we have addressed the dynamic aspect of JH signalling by monitoring in real time the interactions of insect JH receptor proteins. Use of two tags that reconstitute a functional luciferase when in proximity enabled us to follow the rapid assembly of a JH receptor heterodimer from basic helix-loop-helix/Per-Arnt-SIM (bHLH-PAS) proteins, methoprene-tolerant (Met) and taiman (Tai), upon specific JH binding to Met. On a similar timescale (minutes), the dissociation of Met-Met complexes occurred, again strictly dependent on Met interaction with specific agonist ligands. To resolve questions regarding the regulatory role of the chaperone Hsp90/83 in the JHR complex formation, we used the same technique to demonstrate that the Met-Hsp83 complex persisted in the agonist absence but readily dissociated upon specific binding of JH to Met. Preincubation with the Hsp90 inhibitor geldanamycin showed that the chaperone interaction protected Met from degradation and was critical for Met to produce the active signalling dimer with Tai. Thus, the JH receptor functions appear to be governed by principles similar to those regulating the aryl hydrocarbon receptor, the closest vertebrate homologue of the arthropod JH receptor.

Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant.

Metamorphosis in insects is regulated by juvenile hormone (JH) and ecdysteroids. The mechanism of 20-hydroxyecdysone (20E), but not of JH action, is well understood. A basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family member, methoprene tolerant (Met), plays an important role in JH action. Microarray analysis and RNA interference (RNAi) were used to identify 69 genes that require Met for their hydroprene-regulated expression in the red flour beetle, Tribolium castaneum. Quantitative real time PCR analysis confirmed microarray data for 13 of the 16 hydroprene-response genes tested. The members of the bHLH-PAS family often function as heterodimers to regulate gene expression and Met is a member of this family. To determine whether other members of the bHLH-PAS family are required for the expression of JH-response genes, we employed RNAi to knockdown the expression of all 11 members of the bHLH-PAS family and studied the expression of JH-response genes in RNAi insects. These studies showed that besides Met, another member of this family, steroid receptor co-activator (SRC) is required for the expression of 15 JH-response genes tested. Moreover, studies in JH responsive Aag-2 cells revealed that Aedes aegypti homologues of both Met and SRC are required for the expression of the JH-response gene, kr-h1, and SRC is required for expression of ecdysone-response genes. These data suggest the steroid receptor co-activator plays key roles in both JH and 20E action suggesting that this may be an important molecule that mediates cross-talk between JH and 20E to prevent metamorphosis.

Juvenile hormone mediates developmental integration between exaggerated traits and supportive traits in the horned flour beetle Gnatocerus cornutus.

Sexually selected exaggerated traits are often coupled with modifications in other nontarget traits. In insects with weapons, enlargements of nontarget characters that functionally support the weapon often occur (i.e. supportive traits). The support of sexual traits requires developmental coordination among functionally related multiple traits-an explicit example of morphological integration. The genetic theory predicts that developmental integration among different body modules, for which development is regulated via different sets of genes, is likely to be coordinated by pleiotropic factors. However, the developmental backgrounds of morphological integrations are largely unknown. We tested the hypothesis that the juvenile hormone (JH), as a pleiotropic factor, mediates the integration between exaggerated and supportive traits in an armed beetle Gnatocerus cornutus. During combat, males of this beetle use exaggerated mandibles to lift up their opponents with the supportive traits, that is, the head and prothoracic body parts. Application of methoprene, a JH analog (JHA), during the larval to prepupal period, induced the formation of large mandibles relative to the body sizes in males. Morphometric examination of nontarget traits elucidated an increase in the relative sizes of supportive traits, including the head and prothoracic body parts. In addition, reductions in the hind wing area and elytra length, which correspond to flight and reproductive abilities, were detected. Our findings are consistent with the genetic theory and support the idea that JH is a key pleiotropic factor that coordinates the developmental integration of exaggerated traits and supportive characters, as well as resource allocation trade-offs.

The knockdown of Ha-GRIM-19 by RNA interference induced programmed cell death.

GRIM-19 (genes associated with retinoid-IFN-induced mortality-19) is a subunit of mitochondrial respiratory complex I in mammalian systems. However, its function in vivo is not really understood. We cloned GRIM-19 and explored its function and hormonal regulation in insect, the cotton bollworm, Helicoverpa armigera. The results showed that Ha-GRIM-19 was highly expressed during the larval stage. Its transcript levels could be upregulated by juvenile hormone (JH) analog methoprene or by methoprene plus 20E. The methoprene-upregulated transcription enhancement of Ha-GRIM-19 was mediated by the transcription factor Ha-Met1, the putative receptor of JH. Other transcription factors Ha-USP1 and Ha-Br-Z2 suppressed the action of methoprene in inducing Ha-GRIM-19 expression, but Ha-Br-Z2 introduced interaction between 20E and methoprene in upregulation of Ha-GRIM-19. The knockdown of Ha-GRIM-19 by RNA interference in larvae and in insect cell line induced programmed cell death. These data imply that Ha-GRIM-19 plays role in keeping the normal cellular growth and it is able to be upregulated by methoprene through putative JH receptor Met.

Methoprene and temperature effects on caste differentiation and protein composition in the Formosan Subterranean termite, Coptotermes formosanus.

The utilization of multiple castes is a shared feature of social insects. In termites, multiple extrinsic factors have been shown to impact caste differentiation; for example, increased temperature has been shown to increase soldier production. Also, application of exogenous methoprene has also been demonstrated to increase soldier production. The objective of this investigation was to examine and correlate the effects of temperature variation and methoprene treatments on termite caste differentiation, and identify the resulting changes in protein levels. Our results indicate that worker-to-soldier differentiation is modulated by temperature, where a greater number of soldiers developed at a higher rate at higher temperatures compared to lower temperatures. We analyzed total protein by sodium dodecyl sulfate Polyacrylamide gel electrophoresis and N-terminal sequencing and found several changes. Specifically, four proteins affected by temperature change were identified: Hexamerin-1, Hexamerin-2, Endo-beta 1,4 glucanase, and myosin. These proteins were further examined for their response to temperature, assay length (time), and exposure to the juvenile hormone analog methoprene. Hexamerin-1 protein showed a temperature-and assay length-dependent effect, while Hexamerin-2, Endo-beta 1, 4 glucanase, and myosin protein levels were all affected by temperature, assay length, and exposure to methoprene. Our analysis allows the correlation of temperature, assay length, and presence of methoprene with specific changes in protein levels that occur during caste differentiation. These results can be directly applied to better understand the complex developmental factors that control termite differentiation and guide the use of juvenile hormone analogs to maximize efficiency of termite eradication in the field.

Role of Methoprene-tolerant in the regulation of oogenesis in Dipetalogaster maxima.

Juvenile hormone (JH) signalling, via its receptor Methoprene-tolerant (Met), controls metamorphosis and reproduction in insects. Met belongs to a superfamily of transcription factors containing the basic Helix Loop Helix (bHLH) and Per Arnt Sim (PAS) domains. Since its discovery in 1986, Met has been characterized in several insect species. However, in spite of the importance as vectors of Chagas disease, our knowledge on the role of Met in JH signalling in Triatominae is limited. In this study, we cloned and sequenced the Dipetalogaster maxima Met transcript (DmaxMet). Molecular modelling was used to build the structure of Met and identify the JH binding site. To further understand the role of the JH receptor during oogenesis, transcript levels were evaluated in two main target organs of JH, fat body and ovary. Functional studies using Met RNAi revealed significant decreases of transcripts for vitellogenin (Vg) and lipophorin (Lp), as well as their receptors. Lp and Vg protein amounts in fat body, as well as Vg in hemolymph were also decreased, and ovarian development was impaired. Overall, these studies provide additional molecular insights on the roles of JH signalling in oogenesis in Triatominae; and therefore are relevant for the epidemiology of Chagas´ disease.