Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP₃R-operated mobilization of endoplasmic reticulum Ca²⁺ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.     

Allatostatin-C reversibly blocks the transport of citrate out of the mitochondria and inhibits juvenile hormone synthesis in mosquitoes

Aedes aegypti allatostatin-C (AeaAST-C or PISCF-AST) is a strong and fast reversible inhibitor of juvenile hormone III (JH III) synthesis by the corpora allata (CA) of mosquitoes; however, its mechanism of action remains poorly understood. AeaAST-C showed no inhibitory activity in the presence of any of the intermediate precursors of JH III indicating that the AeaAST-C target is located before the entry of acetyl-CoA in the pathway. Stimulation experiments using different sources of carbon (glucose, pyruvate, acetate and citrate) suggest that AST-C acts after pyruvate is transformed to citrate in the mitochondria. In vitro inhibition of the citrate mitochondrial carrier (CIC) mimicked the effect of AeaAST-C, and was overridden by addition of citrate or acetate. Our results provide compelling evidence that AeaAST-C inhibits JH III synthesis by blocking the CIC carrier that transports citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of mosquitoes.     

Mosquito larvicidal activities of farnesol and farnesyl acetate via regulation of juvenile hormone receptor complex formation in Aedes mosquito

Due to their target specificity and low-toxicity, insect growth regulators (IGRs) are regarded as promising alternatives to chemical insecticides. In this study, farnesol and farnesyl acetate exhibited juvenile hormone (JH)-based IGR activities. While farnesyl acetate showed JH agonist (JHA) activity in concentration-dependent manner, farnesol was identified as JH antagonist (JHAN) by interfering pyriproxyfen-mediated binding JH receptor complex. Both compounds showed mosquito larvicidal activities and caused retardation of ovarian growth of female Aedes albopictus by modulating the formation of JH receptor complex, expression of JH-inducible genes, and thereby disrupting JH-based endocrine regulations. These results suggested that farnesol and farnesyl acetate could be applicable for investigating underlying mechanisms of JH-regulated insect physiologies as well as developing novel eco-friendly insecticides.     

The occurrence of a juvenile hormone binding protein and in vitro synthesis of juvenile hormone by the serosa of Locusta migratoria embryos

Summary At the end of blastokinesis, serosal epitheliae of 4- to 5-day-old embryos of Locusta migratoria contain an immunohistologically detectable cytosolic protein (M_r 240 kDa) which is related to the juvenile hormone carrier-protein in the haemolymph of the same species and which binds tritiated juvenile hormone 3 (JH3) (K_d10^–8 M). At this early stage of development the corpora allata of the embryo are not yet fully differentiated and do not synthesize JH3 in organ cultures. The earliest detectable JH3 production by corpora allata in isolated heads is on day 6. On the other hand, serosal epitheliae of 4- to 5-day-old embryos produce JH3 in organ cultures, as has been shown by methylation of (10-³H)-JH3-acid to (10-³H)-JH3, and by incorporation of tritiated CH₃ from l-(methyl-³H)-methionine into JH3. Isolated heads and abdomens of the embryos used as donors for the serosal preparations did not show methyl transferase activity responsible for JH3 biosynthesis. The serosal cells represent a hitherto unrecognized source of methyl transferase activity and of JH3 production. Degradation of JH3 to JH3-acid was also observed.Dedicated to Professor Herbert Röller on the occasion of his 60th birthday     

Characterization of an isopentenyl diphosphate isomerase involved in the juvenile hormone pathway in Aedes aegypti

Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterward IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg²⁺ or Mn²⁺ but not Zn²⁺ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect.     

20-hydroxyecdysone stimulation of juvenile hormone biosynthesis by the mosquito corpora allata

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. JH titer decreases in the last instar larvae allowing pupation and metamorphosis to progress. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which plays an essential role orchestrating reproductive maturation. 20-hydroxyecdysone (20E) prepares the pupae for ecdysis, and would be an ideal candidate to direct a developmental program in the CA of the pharate adult mosquito. In this study, we provide evidence that 20E acts as an age-linked hormonal signal, directing CA activation in the mosquito pupae. Stimulation of the inactive brain-corpora allata-corpora cardiaca complex (Br-CA-CC) of the early pupa (24 h before adult eclosion or −24 h) in vitro with 20E resulted in a remarkable increase in JH biosynthesis, as well as increase in the activity of juvenile hormone acid methyltransferase (JHAMT). Addition of methyl farnesoate but not farnesoic acid also stimulated JH synthesis by the Br-CA-CC of the −24 h pupae, proving that epoxidase activity is present, but not JHAMT activity. Separation of the CA-CC complex from the brain (denervation) in the −24 h pupae also activated JH synthesis. Our results suggest that an increase in 20E titer might override an inhibitory effect of the brain on JH synthesis, phenocopying denervation. All together these findings provide compelling evidence that 20E acts as a developmental signal that ensures proper reactivation of JH synthesis in the mosquito pupae.     

保幼激素合成的研究进展

保幼激素 (juvenile hormone,JH) 是通过甲羟戊酸途径合成的一类倍半萜化合物。以昆虫中普遍存在的JH Ⅲ为例,从分子水平上概述了JH合成途径中的各种酶,并对其中的两个关键酶：羟甲基戊二酰辅酶A还原酶和保幼激素酸甲基转移酶作了详细介绍。还从家蚕基因组数据库(http://silkworm.genomics.org.cn)中推测出了JH合成途径中大部分酶的编码基因,初探了JH合成的调节机制,讨论了JH合成的研究趋势。     

Ovary ecdysteroidogenic hormone functions independently of the insulin receptor in the yellow fever mosquito,Aedes aegypti

Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled.     

Identification of the juvenile hormone from the cricket, Teleogryllus commodus, and juvenile hormone titre changes

In the cricket, Teleogryllus commodus, eggs, haemolymph of 7th and 8th (last)-larval instars, and haemolymph of adults of both sexes contain only juvenile hormone III. While in the male the hormone titre is independent of previous mating experience, juvenile hormone concentration in haemolymph taken from females 36–38 hr after mating (an event which is followed by oviposition) is at a level 5 times higher than that of virgin females. Based on data gleaned from several research groups the identification of juvenile hormone III as the exclusive juvenile hormone in the Order Orthopteroidea is discussed.     

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.     

A mathematical model for the regulation of juvenile hormone titers

The titer of juvenile hormone (JH) is determined by three factors: its rate of synthesis, its rate of degradation, and the degree to which JH is protected from degradation by binding to a diversity of JH-binding proteins. All three of these factors vary throughout the life history of an insect and contribute to variation in the JH titer. The relative importance of each of these factors in determining variation in the JH titer is not known and can, presumably, differ in different life stages and different species. Here we develop a mathematical model for JH synthesis, degradation, and sequestration that allows us to describe quantitatively how each of these contribute to the titer of total JH and free JH in the hemolymph. Our model allows for a diversity of JH-binding proteins with different dissociation constants, and also for a number of different modes of degradation and inactivation. The model can be used to analyze whether data on synthesis and degradation are compatible with the observed titer data. We use the model to analyze two data sets, from Manduca and Gryllus, and show that in both cases, the known data on synthesis and degradation cannot account for the observed JH titers because the role of JH sequestration by binding proteins is greatly underestimated, and/or the in vivo rate of JH degradation is greatly overestimated. These analyses suggest that there is a critical need to develop a better understanding of the in vivo role of synthesis, sequestration and degradation in JH titer regulation.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

Network-level and population genetics analysis of the insulin/TOR signal transduction pathway across human populations

Genes and proteins rarely act in isolation, but they rather operate as components of complex networks of interacting molecules. Therefore, for understanding their evolution, it may be helpful to take into account the interaction networks in which they participate. It has been shown that selective constraints acting on genes depend on the position that they occupy in the network. Less understood is how the impact of local adaptation at the intraspecific level is affected by the network structure. Here, we analyzed the patterns of molecular evolution of 67 genes involved in the insulin/target of rapamycin (TOR) signal transduction pathway. This well-characterized pathway plays a key role in fundamental processes such as energetic metabolism, growth, reproduction, and aging and is involved in metabolic disorders such as obesity, insulin resistance, and diabetes. For that purpose, we combined genotype data from worldwide human populations with current knowledge of the structure and function of the pathway. We identified the footprint of recent positive selection in nine of the studied genomic regions. Most of the adaptation signals were observed among Middle East and North African, European, and Central South Asian populations. We found that positive selection preferentially targets the most central elements in the pathway, in contrast to previous observations in the whole human interactome. This observation indicates that the impact of positive selection on genes involved in the insulin/TOR pathway is affected by the pathway structure.     

Interaction of proteins involved in ecdysone and juvenile hormone signal transduction

Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.