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.

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.

Identification of species-specific juvenile hormone response elements in the fall armyworm, Spodoptera frugiperda.

Juvenile hormones (JH) regulate insect development and reproduction. The JH analogs (JHA) are used as insecticides. However, JHAs are rarely used in managing pests such as the fall armyworm, Spodoptera frugiperda that cause damage during larval stages. The insecticides that antagonize JH action and induce stoppage of feeding and precocious metamorphosis might work better to control these pests. Treating insects with JHA insecticides induces the expression of an early JH response gene, Krüppel homolog 1 (Kr-h1) by working through JH response elements (JHRE) present in its promoter. In this study, we identified JHREs present in the promoter of Kr-h1 gene of a global pest, S. frugiperda, and used them to develop a JHRE-reporter cell platform to screen for JH analogs. JHA, methoprene induced the expression of SfKr-h1 both in vitro and in vivo. JHRE present in the promoters of two SfKr-h1 isoforms, SfKr-h1α and SfKr-h1ß were identified. In Sf9 cells, the knockout of isoform-specific JHRE affected JH response in an isoform-specific manner. We also found that S. frugiperda JHRE (SfJHRE) did not function in the mosquito Aedes aegypti Aag2 cells and Tribolium castaneum TcA cells. Similarly, Ae. aegypti AaJHRE and T. castaneum TcJHRE were only functional in cells derived from these insects. The nucleotide sequence at the 3'end to the conserved core JHRE E-box sequence seems to be responsible for the species specificity observed. Two stable cell lines expressing the luciferase and enhanced green fluorescent protein genes under the control of SfJHRE were established. These cell lines responded well to JHA; these two JHRE-reporter cell lines could be used in screening assays to identify insecticides to manage S. frugiperda and other major pests.

CRISPR-Cas9 Genome Editing Uncovers the Mode of Action of Methoprene in the Yellow Fever Mosquito, Aedes aegypti.

Methoprene, a juvenile hormone (JH) analog, is widely used for insect control, but its mode of action is not known. To study methoprene action in the yellow fever mosquito, Aedes aegypti, the E93 (ecdysone-induced transcription factor) was knocked out using the CRISPR-Cas9 system. The E93 mutant pupae retained larval tissues similar to methoprene-treated insects. These insects completed pupal ecdysis and died as pupa. In addition, the expression of transcription factors, broad complex and Krüppel homolog 1 (Kr-h1), increased and that of programmed cell death (PCD) and autophagy genes decreased in E93 mutants. These data suggest that methoprene functions through JH receptor, methoprene-tolerant, and induces the expression of Kr-h1, which suppresses the expression of E93, resulting in a block in PCD and autophagy of larval tissues. Failure in the elimination of larval tissues and the formation of adult structures results in their death. These results answered long-standing questions on the mode of action of methoprene.

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.

Helicoverpa armigera miR-2055 regulates lipid metabolism via fatty acid synthase expression.

Insect hormones and microRNAs regulate lipid metabolism, but the mechanisms are not fully elucidated. Here, we found that cotton bollworm larvae feeding on Arabidopsis thaliana (AT) leaves had a lower triacylglycerol (TAG) level and more delayed development than individuals feeding on artificial diet (AD). Association analysis of small RNA and mRNA revealed that the level of miR-2055, a microRNA related to lipid metabolism, was significantly higher in larvae feeding on AT. Dual-luciferase reporter assays demonstrated miR-2055 binding to 3' UTR of fatty acid synthase (FAS) mRNA to suppress its expression. Elevating the level of miR-2055 in larvae by agomir injection decreased FAS mRNA and protein levels, which resulted in reduction of free fatty acid (FFA) and TAG in fat body. Interestingly, in vitro assays illustrated that juvenile hormone (JH) increased miR-2055 accumulation in a dosage-dependent manner, whereas knockdown of Methoprene tolerant (Met) or Kruppel homologue 1 (Kr-h1) decreased the miR-2055 level. This implied that JH induces the expression of miR-2055 via a Met-Kr-h1 signal. These findings demonstrate that JH and miRNA cooperate to modulate lipid synthesis, which provides new insights into the regulatory mechanisms of metabolism in insects.

MicroRNA let-7-5p targets the juvenile hormone primary response gene Krüppel homolog 1 and regulates reproductive diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate various biological processes in insects. However, their roles in the regulation of insect diapause remain unknown. In this study, we address the biological function of a conserved miRNA, let-7-5p in the regulation of a juvenile hormone primary response gene, Krüppel homolog 1 (Kr-h1), which modulates reproductive diapause in Galeruca daurica. The dual luciferase reporter assay showed that let-7-5p depressed the expression of Kr-h1. The expression profiles of let-7-5p and Kr-h1 displayed opposite patterns in the adult developmental stage. Injection of let-7-5p agomir in pre-diapause adult females inhibited the expression of Kr-h1, which consequently led to delay ovarian development, increase lipid accumulation, expand fat body, and induce reproductive diapause just as depleting Kr-h1 did. Conversely, injection of let-7-5p antagomir resulted in opposite effects by reducing fat storage and stimulating reproduction. Moreover, JH receptor agonist methoprene reduced the expression of let-7-5p, and rescued the ovarian development defects associated with let-7-5p overexpression. These results indicate that let-7-5p plays an important role in the regulation of reproductive diapause and development of G. daurica adults through its target gene Kr-h1.

Detection of juvenile hormone agonists by a new reporter gene assay using yeast expressing Drosophila methoprene-tolerant.

Juvenile hormones (JHs) are sesquiterpenoids that play important roles in the regulation of growth, metamorphosis, and reproduction in insects. Synthetic JH agonists (JHAs) have been used as insecticides and are categorized as a class of insect growth regulators (IGRs). Natural JHs and synthetic JHAs bind to the JH receptor methoprene-tolerant (Met), which forms a functional JH-receptor complex with steroid receptor coactivators, such as Drosophila melanogaster Taiman (Tai). The ligand-bound Met-Tai complex induces the transcription of JH response genes by binding to specific DNA elements referred to as JH response elements (JHREs). In the present study, we established a reporter gene assay (RGA) for detecting natural JHs and synthetic JHAs in a yeast strain expressing D. melanogaster Met and Tai. The yeast RGA system detected various juvenoid ligands in a dose-dependent manner. The rank order of the ligand potencies of the juvenoids examined in the yeast RGA linearly correlated with those of RGAs for Met-Tai established in mammalian and insect cells. Our new yeast RGA is rapid, easy to handle, cost-effective, and valuable for screening novel JHAs.

Krüppel homolog 1 regulates photoperiodic reproductive plasticity in the cabbage beetle Colaphellus bowringi.

Many insects exhibit reproductive plasticity where the photoperiod determines whether the insect becomes reproductively active or enters diapause. Adult reproductive diapause is a strategy that allows insects to survive harsh environmental conditions. A deficiency in juvenile hormone (JH) leads to reproductive diapause. However, little is known about the molecular mechanisms by which JH signaling regulates reproductive diapause. In this study, we used the cabbage beetle Colaphellus bowringi, a serious pest, to investigate the role of Krüppel homolog 1 (Kr-h1) in controlling photoperiodic plasticity of female reproduction. We focused on Kr-h1, since it acts as a key mediator of JH signaling. We show here that JH-Methoprene-tolerant signaling upregulated the expression of Kr-h1 in reproductively active C. bowringi females when reared under short day conditions. In the long day-treated diapausing females, Kr-h1 transcripts decreased dramatically. Interfering with Kr-h1 function repressed reproductive development by blocking vitellogenesis and ovarian growth. Further, Kr-h1 depletion induced other diapause-like traits, including elevated lipid accumulation and high expression of diapause-related genes. RNA-Seq showed that Kr-h1 played both activating and repressive roles, depending on whether downstream genes were acting in reproduction- or diapause pathways, respectively. Finally, we identified the DNA replication gene mini-chromosome maintenance 4 and two triacylglycerol lipase genes as critical downstream factors of Kr-h1 that are critical for reproductive plasticity in C. bowringi. These results reveal that Kr-h1 is a key component of the regulatory pathway that coordinates reproduction and diapause in insects in response to photoperiodic input.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-signaling regulating the maturation of male accessory glands in the moth Agrotis ipsilon.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

Juvenile hormone induces methoprene-tolerant 1 phosphorylation to increase interaction with Taiman in Helicoverpa armigera.

Methoprene-tolerant 1 (Met1) is a basic-helix-loop-helix Per/Arnt/Sim (bHLH-PAS) protein identified as the intracellular receptor of juvenile hormone (JH). JH induces phosphorylation of Met1; however, the phosphorylation site and outcomes of phosphorylation are not well characterized. In the present study, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we showed that JH III induced threonine-phosphorylation of Met1 at threonine 393 (Thr393) in the Per-Arnt-Sim (PAS) B domain. Thr393-phosphorylation was necessary for Met1 binding to the JH response element (JHRE) to promote the transcription of Kr-h1 (encoding transcription factor Krüppel homolog 1) because Thr393-phosphorylated Met1 increased its interaction with Taiman (Tai) and prevented the Met1-Met1 association. However, JH III could not prevent Met1-Met1 association after Met1-Thr393 was mutated, suggesting that Thr393-phosphorylation is an essential mechanism by which JH prevents Met1-Met1 association. The results showed that JH induces Met1 phosphorylation on Thr393, which prevents Met1-Met1 association, enhances Met1 interaction with Tai, and promotes the binding of Met1-Tai transcription complex to the E-box in the JHRE to regulate Kr-h1 transcription.

Regulation of antimicrobial peptides by juvenile hormone and its receptor, Methoprene-tolerant, in the mosquito Aedes aegypti.

The trade-off between reproduction and immunity has been established for a number of insect species. However, the regulatory mechanisms governing this event is not well understood. In the mosquito Aedes aegypti, the vector of dangerous human arboviral diseases, juvenile hormone (JH) is required for the female post-eclosion development and reproductive maturation. In this study, we have revealed the JH negative effect on the expression of immunity-related genes, such as antimicrobial peptides (AMPs), during the post-eclosion phase of the female mosquito gonadotrophic reproductive cycle. Mosquitoes treated with JH became more sensitive to microbial infection. Mosquitoes subjected to the RNA interference knockdown (RNAi) of the JH receptor, Methoprene-tolerant (Met), showed increased expression of several AMP genes. Met binds to the E-box-like recognition motifs in the regulatory region of the diptericin (Dpt) gene, indicating that JH can suppress the Dpt gene expression through its receptor Met. Hence, JH is involved in the modulation of immune responses during the post-eclosion phase of reproduction. The RNAi knockdown of the peptidoglycan recognition protein (PGRP-LC) led to a significant reduction of the Dpt transcript level, indicating the PGRP-LC activating role on this AMP gene. Thus, Dpt appeared to be under the dual regulation of both the JH and the immune deficiency (IMD) signaling pathways. Our study provides a better understanding of how JH regulates insect immunity in adult mosquitoes.

Molecular action of pyriproxyfen: Role of the Methoprene-tolerant protein in the pyriproxyfen-induced sterilization of adult female mosquitoes.

Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action in Aedes aegypti mosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fed Ae. aegypti females but blocked the development of primary follicles at Christopher's Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Krüppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, including HR3 and E75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism in Ae. aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF.

Transcription-inducing activity of natural and synthetic juvenile hormone agonists through the Drosophila Methoprene-tolerant protein.

BACKGROUND: Juvenile hormones (JHs) are a class of sesquiterpenoids that play a pivotal role in insect growth and reproduction. Synthetic JH agonists (JHAs), including pyriproxyfen, have been widely used as insecticides to control agricultural pests and disease vectors. The antimetamorphic action of JHAs is mediated by their intracellular receptor, the heterodimer of Methoprene-tolerant (Met) and Taiman (Tai) proteins. Although a range of bioassay systems has been developed to detect the activity of JHAs, each of these systems has its own drawback(s), such as poor reproducibility, the use of radioactive ligands or the effect of endogenous JH-signaling factors. RESULTS: To address these issues, we constructed a new luciferase reporter assay for JHAs in mammalian HEK293T cells transiently transfected with the Drosophila Met and Tai genes. This reporter system gave highly reproducible results and showed nanomolar sensitivity to natural JHs. We then applied this reporter system to a structure-activity relationship (SAR) analysis of 14 natural and synthetic JHAs, leading to identification of the ligand structural factors important for the transcription-inducing activity. CONCLUSION: Because this reporter system is not affected by the signaling cascade downstream of the JH receptors, it is suitable for evaluating the intrinsic activity of JHAs. The SAR results obtained in this study therefore provide invaluable information on the rational design of novel JHA insecticides.

Methoprene-tolerant is essential for embryonic development of the red flour beetle Tribolium castaneum.

Insect juvenile hormone (JH) is well known to regulate post-embryonic development and reproduction in concert with ecdysteroids in a variety of insect species. In contrast, our knowledge on the role of JH in embryonic development is limited and inconsistent. Preceding studies indicate that JH biosynthesis or JH signaling genes are dispensable in holometabolous Drosophila melanogaster and Bombyx mori, while essential in hemimetabolous Blattella germanica. In the red flour beetle Tribolium castaneum, we performed functional analyses of key factors in JH signaling, i.e. the JH receptor Methoprene-tolerant (Met) and the early JH-response gene Krüppel homolog 1 (Kr-h1) using parental RNA interference. Knockdown of Met resulted in a significant reduction in hatching rates and survival rates in the first and second larval instars. Meanwhile, knockdown of Kr-h1 caused no significant effect on hatching or survival. The unhatched embryos under Met knockdown developed up to the late embryonic stage, but their body shape was flat and tubby compared with the controls. Attempts to suppress JH biosynthesis by parental RNA interference of JH biosynthetic enzymes were unsuccessful due to insufficient knockdown efficiency. These results suggested that Met but not Kr-h1 is essential for the embryonic development of T. castaneum, although involvement of JH still remains to be examined. Taken together, the function of Met in embryonic development seems to be diverse among insect species.

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.

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 receptor Met is essential for ovarian maturation in the Desert Locust, Schistocerca gregaria.

Juvenile hormones (JH) are key endocrine regulators produced by the corpora allata (CA) of insects. Together with ecdysteroids, as well as nutritional cues, JH coordinates different aspects of insect postembryonic development and reproduction. The function of the recently characterized JH receptor, Methoprene-tolerant (Met), appears to be conserved in different processes regulated by JH. However, its functional interactions with other hormonal signalling pathways seem highly dependent on the feeding habits and on the developmental and reproductive strategies employed by the insect species investigated. Here we report on the effects of RNA interference (RNAi) mediated SgMet knockdown during the first gonadotrophic cycle in female desert locusts (Schistocerca gregaria). This voracious, phytophagous pest species can form migrating swarms that devastate field crops and harvests in several of the world's poorest countries. A better knowledge of the JH signalling pathway may contribute to the development of novel, more target-specific insecticides to combat this very harmful swarming pest. Using RNAi, we show that the JH receptor Met is essential for ovarian maturation, vitellogenesis and associated ecdysteroid biosynthesis in adult female S. gregaria. Interestingly, knockdown of SgMet also resulted in a significant decrease of insulin-related peptide (SgIRP) and increase of neuroparsin (SgNP) 3 and 4 transcript levels in the fat body, illustrating the existence of an intricate regulatory interplay between different hormonal factors. In addition, SgMet knockdown in females resulted in delayed display of copulation behaviour with virgin males, when compared with dsGFP injected control animals. Moreover, we observed an incapacity of adult dsSgMet injected female locusts to oviposit during the time of the experimental setup. As such, SgMet is an essential gene playing crucial roles in the endocrine communication necessary for successful reproduction of the desert locust.

Juvenile hormone-independent function of Krüppel homolog 1 in early development of water flea Daphnia pulex.

Elaborate regulation of insect metamorphosis is the consequence of physiological cooperation among multiple endocrine factors such as juvenile hormones (JHs) and ecdysteroids. Hormone-induced transcription factors play important roles in substantive interactions between hormonal signaling pathways. In insects, zinc finger transcription factor Krüppel homolog 1 (Kr-h1) is a key gene of the endocrine signaling pathway in which it is directly upregulated by JH receptor Methoprene-tolerant (Met) in the presence of JH and then regulates multiple downstream factors, including components of the ecdysteroid signaling pathway. Although JH also plays a role in various biological phenomena in other arthropod species, little is known about the molecular basis of the JH signaling pathway. Here we cloned Kr-h1 from a branchiopod crustacean, Daphnia pulex, (DappuKr-h1) and analyzed its expression profile and developmental function together with consideration of its relationship to the JH signaling pathway. We suggest that DappuKr-h1 lacks JH responsiveness and regulatory relationship with the JH receptor. Moreover our loss-of-function analysis revealed that maternal mRNA of DappuKr-h1 plays a critical role in early development independent from the JH signaling pathway. These findings provide insights about whether and how the JH signaling pathway influenced evolution, leading to greater diversity in phylum Arthropoda.

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.