Juvenile Hormone Biosynthesis Research Articles

Roles of the circadian clock and endocrine regulator in the photoperiodic response of the brown‐winged green bug Plautia stali

AbstractThe physiological mechanisms underlying photoperiodism in insects have been studied extensively, although the associated molecular machinery remains largely unknown. In the present study, we investigate the roles of the circadian clock gene cycle (cyc) and the endocrine regulator gene myoinhibitory peptide (Mip) in the photoperiodic response of the brown‐winged green bug Plautia stali Scott (Hemiptera, Pentatomidae). Typically, adult females of this species develop their ovaries under long‐day conditions, whereas they suppress its development under short‐day conditions. We find that RNA interference (RNAi) directed against cyc causes malfunction of the circadian clock governing the locomotor activity rhythm and yields abnormal activity profiles not only under constant darkness, but also under light/dark conditions. RNAi directed against cyc and Mip disrupts the photoperiodic response in ovarian development. cyc RNAi suppresses the ovarian development even under long‐day conditions, whereas Mip RNAi induces it even under short‐day conditions. We propose that the core circadian clock gene cyc regulates the photoperiodic response and that Mip is the causal regulator of juvenile hormone biosynthesis in the corpus allatum. Neither photoperiod, nor cyc RNAi affect Mip mRNA levels, and therefore it remains unknown how the photoperiodic information is processed and mediated by Mip.

Myoglianin triggers the premetamorphosis stage in hemimetabolan insects.

Insect metamorphosis is triggered by a decrease in juvenile hormone (JH) in the final juvenile instar. What induces this decrease is therefore a relevant question. Working with the cockroach Blattella germanica, we found that myoglianin (Myo), a ligand in the TGF-β signaling pathway, is highly expressed in the corpora allata (CA, the JH-producing glands) and the prothoracic gland [(PG), which produce ecdysone] during the penultimate (fifth) nymphal instar (N5). In the CA, high Myo levels during N5 repress the expression of juvenile hormone acid methyl transferase, a JH biosynthesis gene. In the PG, decreasing JH levels trigger gland degeneration, regulated by the factors Krüppel homolog 1, FTZ-F1, E93, and inhibitor of apoptosis-1. Also in the PG, a peak of myo expression in N5 indirectly stimulates the expression of ecdysone biosynthesis genes, such as neverland, enhancing the production of the metamorphic ecdysone pulse in N6. The Myo expression peak in N5 also represses cell proliferation, which can enhance ecdysone production. The data indicate that Myo triggers the premetamorphic nymphal instar in B. germanica and possibly in other hemimetabolan insects.-Kamsoi, O., Belles, X. Myoglianin triggers the premetamorphosis stage in hemimetabolan insects.

Double-Stranded RNAs High-Efficiently Protect Transgenic Potato from Leptinotarsa decemlineata by Disrupting Juvenile Hormone Biosynthesis.

RNA interference (RNAi) has been developed for plant pest control. In this study, hairpin-type double-stranded RNA (dsRNA) targeting the juvenile hormone (JH) acid methyltransferase ( JHAMT) gene ( dsJHAMT) was introduced in potato plants via Agrobacterium-mediated transformation. The results indicated that the transcriptional RNA of dsJHAMT accumulated in the transgenic plants. The transcripts and proteins of the L. decemlineata JHAMT gene were significantly reduced in larvae feeding on dsJHAMT transgenic foliage. The dsJHAMT had a significant negative effect on the growth and development of L. decemlineata, especially resulting in less oviposition. Importantly, in the field trials, transgenic plants are high-efficiently protected from insect damage mainly because surviving insects laid fewer or no eggs. Even full protection from beetle damage can be acquired by continuously lowering insect population size at large scale in the field over the years. Therefore, the transgenic plants expressing dsJHAMT successfully provided an additional option for plant pest control.

Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor β, farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35–56% mortality. Transgenic tobacco plants expressing hairpin dsRNA targeting Met gene were generated and tested. About 48% mortality was observed in TSSM raised on transgenic tobacco plants expressing dsMet. These studies not only broaden our knowledge on understanding hormone action in TSSM but also identified target genes that could be used in RNAi-mediated control of TSSM.

Juvenile hormone affects the splicing of Culex quinquefasciatus early trypsin messenger RNA.

The full length of Culex quiquefasciatus early trypsin has been cloned and sequenced and a three-dimensional (3D) model of the enzyme was built showing that the enzyme has the canonical trypsin's active pocket containing H78, D123, S129, and D128. The biosynthesis of juvenile hormone (JH) III by the corpora allata (CA) in female Cx. quiquefasciatus is sugar-dependent. Females that were maintained on water after emergence synthesize very little JH III, JH III bisepoxide, and methyl farnesoate (MF) (3.8, 1.1, and 0.8 fmol/4 hr/CA, respectively). One hour after sugar feeding, the synthesis of JH III and JH III bisepoxide reached a maximum (11.3 and 5.9 fmol/4 hr/CA, respectively) whereas MF biosynthesis reached a maximum at 24 hr (5.2 fmol/4 hr/CA). The early trypsin is transcribed with a short intron (51 nt) is spliced when JH III biosynthesis is high in sugar fed and at 1 hr after the blood meal (22 and 15 fmol/4 hr/CA, respectively). We investigated the transcriptional and posttranscriptional regulation of the early trypsin gene showing that JH III concentrations influence splicing. In the absence JH III the unspliced transcript is linked by a phosphoamide bond at the 5'-end to RNA ribonuleoprotein (RNP). The biosynthesis of the early trypsin was followed in ligated abdomens (without CA) of newly emerged females that fed blood by enema. Our results show that the early trypsin biosynthesis depends on sugar and blood feeding, whereas the late trypsin biosynthesis does not depend on sugar feeding, or JH III biosynthesis. Downregulating the early trypsin transcript does not affect the late trypsin.

Remodeling of the juvenile hormone pathway through caste-biased gene expression and positive selection along a gradient of termite eusociality.

The evolution of division of labor between sterile and fertile individuals represents one of the major transitions in biological complexity. A fascinating gradient in eusociality evolved among the ancient hemimetabolous insects, ranging from noneusocial cockroaches through the primitively social lower termites-where workers retain the ability to reproduce-to the higher termites, characterized by lifetime commitment to worker sterility. Juvenile hormone (JH) is a prime candidate for the regulation of reproductive division of labor in termites, as it plays a key role in insect postembryonic development and reproduction. We compared the expression of JH pathway genes between workers and queens in two lower termites (Zootermopsis nevadensis and Cryptotermes secundus) and a higher termite (Macrotermes natalensis) to that of analogous nymphs and adult females of the noneusocial cockroach Blattella germanica. JH biosynthesis and metabolism genes ranged from reproductive female-biased expression in the cockroach to predominantly worker-biased expression in the lower termites. Remarkably, the expression profile of JH pathway genes sets the higher termite apart from the two lower termites, as well as the cockroach, indicating that JH signaling has undergone major changes in this eusocial termite. These changes go beyond mere shifts in gene expression between the different castes, as we find evidence for positive selection in several termite JH pathway genes. Thus, remodeling of the JH pathway may have played a major role in termite social evolution, representing a striking case of convergent molecular evolution between the termites and the distantly related social hymenoptera.

Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model.

Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT) antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C-terminal amidated octapeptide Manse-AT (6–13). We identified three residues essential for bioactivity (Thr4, Arg6 and Phe8) by assaying alanine-replacement analogs of Manse-AT (6–13). Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR) was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10–13), we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10–13) validated our hypothesis. The IC50 value of antagonist Manse-AT (10–13) is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10–13) was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

A comparative analysis of corpora allata-corpora cardiaca microRNA repertoires revealed significant changes during mosquito metamorphosis

The corpora allata (CA) are a pair of endocrine glands with neural connections to the brain and close association with another neuroendocrine organ, the corpora cardiaca (CC). The CA from adult female Aedes aegypti mosquitoes synthesize fluctuating levels of juvenile hormone (JH), which have been linked to the ovarian development and are influenced by nutritional signals. In this study, we investigated the potential involvement of microRNAs (miRNAs), a type of small non-coding RNAs, in the regulation of gene expression in CA-CC complexes during mosquito reproductive development, at stages with distinct JH biosynthesis patterns. We analyzed the miRNA repertoires expressed in the CA-CC of pupae, sugar-fed and blood-fed female Ae. aegypti. In total, 156 mature miRNAs were detected in the CA-CC, with 84 displaying significant differences in expression among the three CA-CC developmental stages. There were more miRNAs that were expressed in pupae, and decreased or were absent after adult emergence, when compared with changes between CA-CC of sugar and blood-fed females. Analysis of the genes identified as potential targets for the CA-CC miRNA repertoires classified them into the broad categories of metabolism, information storage and processing, and cellular processes and signaling; with genes involved in cellular processes and signaling representing the largest portion. Among them, the signal-transduction mechanisms and intracellular trafficking, secretion and vesicular transport contained almost 55% of the genes’ targets. A substantial number of miRNAs were differentially abundant in the libraries of the three developmental stages, and those changes were much more notable when pupae and adult stages were compared. We detected putative binding sites for some of the most abundant miRNAs on genes encoding JH biosynthetic enzymes and CC neuropeptides. These studies should help us to gain a better understanding of the regulation of CA-CC activity mediated by miRNAs during major developmental stages in mosquitoes.

Structure-Based Discovery of Nonpeptide Allatostatin Analogues for Pest Control.

FGLamide allatostatins (ASTs) are regarded as possible insecticide candidates, although their lack of in vivo effects, rapid degradation, poor water solubility, and high production costs preclude their practical use in pest control. In contrast to previous research, the C-terminal tripeptide (FGLa) was selected as the lead compound in this study. Five nonpeptide AST analogues (2-amino-1-[3-oxo-3-(substituted-anilino)propyl]pyridinium nitrate derivatives) were designed on the basis of the structure-activity relationship and docking results of FGLa. All of the nonpeptide analogues (S1-S5) were more potent against juvenile-hormone (JH) biosynthesis than the lead compound. They significantly inhibited the biosynthesis of JH in vivo following injection. A pest-control application demonstrated that S1 and S3 have larvicidal effects following oral administration (the IC50 values were 0.020 and 0.0016 mg/g, respectively). The good oral toxicities and excellent water solubilities of S1 and S3 suggest that they have considerable potential as insecticides for pest management.

JH biosynthesis and hemolymph titers in adult male Aedes aegypti mosquitoes

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low in late pupae, significantly increased during the first 24 h after adult eclosion, and then remained relatively constant during the first six days after adult eclosion. Feeding high sugar diets resulted in an increase of JH synthesis and titers, and starvation significantly decreased JH synthesis, but this effect could be reversed by changing the males back to a high sugar diet. JH synthesis rates were similar in virgin and mated males, but hemolymph JH levels were different in well-nourished virgin and mated males. Starvation resulted in a significant reduction in insemination rates; with well-nourished males inseminating 2 times more females than water-fed. Giving a 20% sugar meal for 24 h to those mosquitoes that were previously starved for 6 days, caused a significant rise in insemination rates, restoring them to levels similar to those recorded for 20% fed males. These results suggest that nutrition plays a role on male fecundity, and this effect might be mediated by JH.

Transcriptional response of Methoprene-tolerant (Met) gene to three insect growth disruptors in Leptinotarsa decemlineata (Say)

Abstract Some insect growth disruptors (IGDs), such as pyriproxyfen and halofenozide, may be used to control Leptinotarsa decemlineata. However, their mechanism of action remains elusive. Methoprene-tolerant (Met) mediates juvenile hormone (JH) signal to control numerous essential physiological processes. In the present paper, we identified a Met gene (LdMet). LdMet protein was a typical basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) transcription factor with a bHLH domain, two PAS domains (PAS-A and PAS-B) and a region called PAS associated C terminal (PAC). Eight conserved amino acids critical for JH binding were located in PAS-B and PAC domains. The temporal expression pattern of LdMet was in accordance with the variation of circulating JH titers. Feeding of juvenoid methoprene or pyriproxyfen, or provide for JH dose-dependently stimulated the expression of LdMet. RNA interference-mediated knockdown of two JH degradation genes increased the transcription of LdMet, while silencing of a JH biosynthesis gene repressed the transcription. Conversely, ingestion of an ecdysteroid agonist halofenozide or 20-hydroxyecdysone (20E) reduced the mRNA levels of LdMet, in a dose-dependent manner, whereas knockdown of either ecdysteroidogenesis or 20E signaling genes increased the mRNA accumulation. Providing that the expression of LdMet can be disturbed by methoprene, pyriproxyfen and halofenozide, LdMet may be a potential target of these IGDs in L. decemlineata larvae.

A rapid quantitative assay for juvenile hormones and intermediates in the biosynthetic pathway using gas chromatography tandem mass spectrometry

A method for rapid quantitation of insect juvenile hormones (JH) and intermediates in the biosynthetic pathway, both in vitro and in vivo (hemolymph and whole body), has been developed using GC–MS/MS. This method is as simple as the radiochemical assay (RCA), the most commonly used method for measurement of JH biosynthesis in vitro, without need for further purification and derivatization, or radioactive precursors or ligands. It shows high sensitivity, accuracy and reproducibility. Linear responses were obtained the range of 1–800 ng/mL (approximately 4–3000 nM). Recovery efficiencies for farnesol, farnesal, methyl farnesoate and JH III were approximately 100% in vitro and over 90% in vivo, with excellent reproducibility at three different spike levels. Titer of JH III in the hemolymph was relatively low at day 0 (adult female emergence) (79.68 ± 5.03 ng/mL) but increased to a maximum of 1717 ng/mL five days later. In whole body, JH III quantity reached a maximum on day 4 (845.5 ± 87.9 ng/g) and day 5 (679.7 ± 164.6 ng/g) and declined rapidly thereafter. It is in agreement with the hemolymph titer changes and biosynthetic rate of JH in vitro. Comparison with the results of inhibition of JH biosynthesis by two known inhibitors (allatostatin (AST) mimic H17 and pitavastatin) using RCA and GC–MS/MS, showed that there was little difference between the two methods In contrast to other methods, the present method with GC–MS/MS can be used to elucidate the mechanism of inhibition by inhibitors of JH biosynthesis without any derivatization and purification. This method is applicable to screening of JH inhibitors and the study of inhibitory mechanisms with high sensitivity and accurate quantification. It may also be useful for the determination of JH titer in other Arthropods.

Sublethal Effects of Ethanol Extract of Ocimum sanctum on Laboratory Bred Population of Dengue Mosquito Aedes aegypti L. (Diptera: Culicidae)

Objectives: Aedes aegypti is a vector of several diseases which cause serious discomfort and fatality to human being. Current investigations were carried out to assess sublethal effects of ethanol extract of the leaves of Ocimum sanctum against A. aegypti in order to find ecofriendly alternatives for integrated vector management‟. Methods: Lethal and sublethal effects of ethanol extract of O. sanctum against fourth instar larvae were assessed by using WHO protocol. The studies were extended to evaluate the effects of the extract on growth and development of the treated larvae. The chemical components present in the extracts were analyzed by Gas chromatography and mass spectroscopy (GC-MS). Results: Exposure of fourth instar larvae of A. aegypti revealed moderate toxicity. However, the treated larvae exhibited mortality during subsequent days of exposure. The treatment with the extract also has adverse impact on growth and development of the larvae. Consequently there was increase in the larval duration in the treated larvae. Larva-pupa intermediate and pupa-adult intermediate were the developmental anomalies observed in the treated fourth instar larvae. This suggested presence of juvenile hormone mimic or juvenile analogue activity in the ethanol extract of O. sanctum leaves. Presence of farnasene, a derivates of the intermediates in the juvenile hormone biosynthesis was reported in the GCMS analysis of the extract. Conclusion: Our studied indicated moderate lethal and efficient sublethal effects of ethanol extract of O. sanctum against A.aegypti. The bioactive compounds present in the extract, individually or synergistically may be used in formulating a strategy for the mosquito management

Comparative Proteomic Analysis of the Effect of Periplocoside P from Periploca sepium on Brush Border Membrane Vesicles in Midgut Epithelium of Mythimna separata Larvae.

Periplocoside P (PSP), a novel compound isolated from Periploca sepium Bunge, possesses insecticidal activity against some lepidopterans, such as Mythimna separata. In M. separata, the brush border membrane vesicles of the midgut epithelium are the initial site of action of periplocosides. We conducted two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry analysis to analyze differentially expressed proteins (DEPs) from periplocoside P (PSP)-treated M. separata. We successfully isolated seven up-regulated and three down-regulated DEPs that have been previously identified, as well as a novel DEP. The DEPs are implicated in protein degradation, transporter, folding, and synthesis, and in juvenile hormone biosynthesis. DEPs involved in the oxidative phosphorylation energy metabolism pathway are enriched. Through real-time polymerase chain reaction assay, we confirmed that vma1 expression is significantly up-regulated expression levels in PSP-treated M. separata larvae. Enzymology validation further indicated that PSP can significantly inhibit V-type ATPase activity in a concentration-dependent manner. Given these results, we speculate that in M. separata, the V-type ATPase A subunit in the midgut epithelium is the putative target binding site of periplocosides. This finding provides preliminary evidence for the mode of action of periplocosides.

Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila

In both vertebrates and insects, developmental transition from the juvenile stage to adulthood is regulated by steroid hormones. In insects, the steroid hormone, 20-hydroxyecdysone (20E), elicits metamorphosis, thus promoting this transition, while the sesquiterpenoid juvenile hormone (JH) antagonizes 20E signaling to prevent precocious metamorphosis during the larval stages. However, not much is known about the mechanisms involved in cross-talk between these two hormones. In this study, we discovered that in the ring gland (RG) of Drosophila larvae, JH and 20E control each other's biosynthesis. JH induces expression of a Krüppel-like transcription factor gene Kr-h1 in the prothoracic gland (PG), a portion of the RG that produces the 20E precursor ecdysone. By reducing both steroidogenesis autoregulation and PG size, high levels of Kr-h1 in the PG inhibit ecdysteriod biosynthesis, thus maintaining juvenile status. JH biosynthesis is prevented by 20E in the corpus allatum, the other portion of the RG that produces JH, to ensure the occurrence of metamorphosis. Hence, antagonistic actions of JH and 20E within the RG determine developmental transitions in Drosophila Our study proposes a mechanism of cross-talk between the two major hormones in the regulation of insect metamorphosis.

Identification and characterization of genes involving the early step of Juvenile Hormone pathway in Helicoverpa armigera

Juvenile hormones (JHs) are crucial regulators for multiple physiological processes in insects. In the current study, 10 genes in mevalonate pathway involved in JH biosynthesis were identified from Helicoverpa armigera. Tissue-specific expression analysis showed that six genes were highly expressed in the head which contained the JH biosynthetic gland (corpora allata). Temporal expression pattern showed that 10 of 12 genes were highly transcribed in the late 2nd-instar when the in vivo JH titer reached the peak, indicating a tight correlation between JH titer and the transcription of JH synthetic pathway genes. Moreover, ingestion of methoprene, a JH analogue, significantly suppressed the transcription of nine JH biosynthetic genes and caused a feedback upregulation of the JH degradation enzyme. Particularly, the Acetoacetyl CoA thiolase (HaAce) and Farnesyl diphosphate synthase gene 4 (HaFpps4) showed high transcript abundance, and their temporal expressions keep pace with JH fluctuations. Further study by RNAi showed that knockdown of HaFpps4 caused the decrease of JH titer, led to a negative effect on the transcript levels of other genes in JH pathway, and resulted in molting disturbance in larvae. Altogether, these results contribute to our understanding of JH biosynthesis in H. armigera and provide target genes for pest control based on JH-dependent regulation.

Exploring the N-terminus region: Synthesis, bioactivity and 3D-QSAR of allatostatin analogs as novel insect growth regulators

Allatostatins (ASTs), a family of insect neuropeptide, can inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA) in Diploptera punctata, and therefore be regarded as potential leads for the discovery of new insect growth regulators (IGRs). But several shortcomings, such as their sensitivity to peptidases and high cost, impeded their practical application in pest management. In order to discover new IGRs, one AST analog B1 possessing non-peptide group was discovered with high ability to inhibit JH biosynthesis in vitro (IC50: 0.09μmol/L) in our previous studies. In the present work, two series of B1 analogs with different substituents on the N-terminus region were designed and synthesized. The result suggested that benzene showed better activity than other heterocycles, and the para-substitution on the benzene was beneficial for activity. Moreover, analogs with logP value over 2.0 exhibited good activity, which indicated the hydrophobicity is important to the bioactivity. Three dimension quantitative structure-activity relationship (3D-QSAR) studies were performed to highlight the structural requirements of AST analogs, which demonstrated introduction of bulkier substituents on the N-terminus would increase the activity. Analog II12 (IC50: 0.08μmol/L) exhibited similar inhibitory activity to the lead B1, but its synthetic route was simpler than B1. Therefore, II12 could be used as a new lead compound for the discovery eco-friendly IGRs.

A virus carries a gene encoding juvenile hormone acid methyltransferase, a key regulatory enzyme in insect metamorphosis

Microbial parasitism, infection, and symbiosis in animals often modulate host endocrine systems, resulting in alterations of phenotypic traits of the host that can have profound effects on the ecology and evolution of both the microorganisms and their hosts. Information about the mechanisms and genetic bases of such modulations by animal parasites is available from studies of steroid hormones. However, reports involving other hormones are scarce. We found that an insect virus, a betaentomopoxvirus, encodes a juvenile hormone acid methyltransferase that can synthesize an important insect hormone, the sesquiterpenoid juvenile hormone. Phylogenetic analysis suggested that this gene is of bacterial origin. Our study challenges the conventional view that functional enzymes in the late phase of the juvenile hormone biosynthesis pathway are almost exclusive to insects or arthropods, and shed light on juvenoid hormone synthesis beyond Eukaryota. This striking example demonstrates that even animal parasites having no metabolic pathways for molecules resembling host hormones can nevertheless influence the synthesis of such hormones, and provides a new context for studying animal parasite strategies in diverse systems such as host-parasite, host-symbiont or host-vector-parasite.

Identification and Characterization of Novel Genes Expressed Preferentially in the Corpora Allata or Corpora Cardiaca During the Juvenile Hormone Synthetic Period in the Silkworm, Bombyx mori.

Juvenile hormone (JH) plays important roles in insect development and physiology. JH titer is tightly regulated to coordinately adjust systemic physiology and development. Although control of JH titer is explained by the expression of JH biosynthetic enzymes in the corpora allata (CA), molecular mechanisms that regulate the expression of these genes remain elusive. In the present study, to identify novel regulators of JH biosynthetic genes, we conducted a gene expression screen using the CA and corpora cardiaca (CC) of the silkworm, Bombyx mori, in the JH synthesis period. We identified seven candidate genes and characterized their properties through extensive expression analyses. Of these candidates, we found that a novel gene, which encodes type II phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] 4-phosphatase, shows highly correlated expression with JH titer. In addition, expression of this gene was strongly upregulated by starvation, when JH biosynthetic enzyme genes are concurrently upregulated. These results, for the first time, imply possible involvement of phosphoinositol signal in regulation of JH biosynthesis, providing novel insights into molecular mechanisms of nutrition-dependent regulation of JH biosynthesis.

Methyl farnesoate epoxidase (mfe) gene expression and juvenile hormone titers in the life cycle of a highly eusocial stingless bee, Melipona scutellaris

In social insects, juvenile hormone (JH) has acquired novel functions related to caste determination and division of labor among workers, and this is best evidenced in the honey bee. In contrast to honey bees, stingless bees are a much more diverse group of highly eusocial bees, and the genus Melipona has long called special attention due to a proposed genetic mechanism of caste determination. Here, we examined methyl farnesoate epoxidase (mfe) gene expression, encoding an enzyme relevant for the final step in JH biosynthesis, and measured the hemolymph JH titers for all life cycle stages of Melipona scutellaris queens and workers. We confirmed that mfe is exclusively expressed in the corpora allata. The JH titer is high in the second larval instar, drops in the third, and rises again as the larvae enter metamorphosis. During the pupal stage, mfe expression is initialy elevated, but then gradually drops to low levels before adult emergence. No variation was, however, seen in the JH titer. In adult virgin queens, mfe expression and the JH titer are significantly elevated, possibly associated with their reproductive potential. For workers we found that JH titers are lower in foragers than in nurse bees, while mfe expression did not differ. Stingless bees are, thus, distinct from honey bee workers, suggesting that they have maintained the ancestral gonadotropic function for JH. Hence, the physiological circuitries underlying a highly eusocial life style may be variable, even within a monophyletic clade such as the corbiculate bees.