Juvenile Hormone Synthesis Research Articles

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.

Juvenile hormone enhances aversive learning performance in 2-day old worker honey bees while reducing their attraction to queen mandibular pheromone.

Previous studies have shown that exposing young worker bees (Apis mellifera) to queen mandibular pheromone (QMP) reduces their aversive learning performance, while enhancing their attraction to QMP. As QMP has been found to reduce the rate of juvenile hormone (JH) synthesis in worker bees, we examined whether aversive learning in 2-day old workers exposed to QMP from the time of adult emergence could be improved by injecting JH (10 µg in a 2 µl volume) into the haemolymph. We examined in addition, the effects of JH treatment on worker attraction to QMP, and on the levels of expression of amine receptor genes in the antennae, as well as in the mushroom bodies of the brain. We found that memory acquisition and 1-hour memory recall were enhanced by JH. In contrast, JH treatment reduced the bees’ attraction towards a synthetic strip impregnated with QMP (Bee Boost). Levels of expression of the dopamine receptor gene Amdop1 were significantly lower in the mushroom bodies of JH-treated bees than in bees treated with vehicle alone (acetone diluted with bee ringer). Expression of the octopamine receptor gene, Amoa1, in this brain region was also affected by JH treatment, and in the antennae, Amoa1 transcript levels were significantly lower in JH-treated bees compared to controls. The results of this study suggest that QMP’s effects on JH synthesis may contribute to reducing aversive learning performance and enhancing attraction to QMP in young worker bees.

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.

Crystal structure of juvenile hormone epoxide hydrolase from the silkworm Bombyx mori.

The juvenile hormone (JH) is a kind of epoxidecontaining sesquiterpene ester secreted by a pair of corpora allatum behind the brain of insects.1 It controls the metamorphosis development of insects together with the ecdysone.2,3 Thus the synthesis and degradation of JH are tightly regulated in different development stages.4 The degradation of JH is catalyzed by two hydrolases, juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone esterase. JHEH is responsible for opening the epoxide ring of JH to produce JH diol, whereas JHE catalyzes the removal of the methyl ester moiety of JH to form JH acid.5,6 JHEH belongs to the microsomal epoxide hydrolase (mEH) (EC 3.3.2.9) family, which is one of the most widely distributed families of epoxide hydrolases (EHs). EHs can transform epoxides to compounds with decreased chemical reactivity, increased water solubility, and altered biological activity.7,8 In addition to participating in the catabolism of JH in insects, mEHs also play important roles in cytoprotection, steroid metabolism, bile acid transport, and xenobiotic metabolism.9 To date, the only structure of the mEH from the fungus Aspergillus niger (termed AnEH, PDB 1QO7) revealed a typical a/b-hydrolase core composed of a twisted eightstranded b-sheet packing on both sides with several a-helices.10,11 Structural analyses suggested a bimolecular nucleophilic substitution (SN2) reaction mechanism involving a standard nucleophile–histidine–acid catalytic triad of Asp–His–Glu/Asp.11 However, the mechanism of substrate recognition and catalysis of mEHs remains unclear. Here we report the crystal structure of Bombyx mori JHEH (BmJHEH) at 2.30 A resolution. Structural analyses together with molecular simulation reveal insights into the specific binding of JH in the active-site pocket. These findings increase our understanding of the substrate recognition and catalysis of mEHs and might help the design of JH-derived pesticides.

Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata, and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.

The POU factor ventral veins lacking/Drifter directs the timing of metamorphosis through ecdysteroid and juvenile hormone signaling.

Although endocrine changes are known to modulate the timing of major developmental transitions, the genetic mechanisms underlying these changes remain poorly understood. In insects, two developmental hormones, juvenile hormone (JH) and ecdysteroids, are coordinated with each other to induce developmental changes associated with metamorphosis. However, the regulation underlying the coordination of JH and ecdysteroid synthesis remains elusive. Here, we examined the function of a homolog of the vertebrate POU domain protein, Ventral veins lacking (Vvl)/Drifter, in regulating both of these hormonal pathways in the red flour beetle, Tribolium castaneum (Tenebrionidae). RNA interference-mediated silencing of vvl expression led to both precocious metamorphosis and inhibition of molting in the larva. Ectopic application of a JH analog on vvl knockdown larvae delayed the onset of metamorphosis and led to a prolonged larval stage, indicating that Vvl acts upstream of JH signaling. Accordingly, vvl knockdown also reduced the expression of a JH biosynthesis gene, JH acid methyltransferase 3 (jhamt3). In addition, ecdysone titer and the expression of the ecdysone response gene, hormone receptor 3 (HR3), were reduced in vvl knockdown larvae. The expression of the ecdysone biosynthesis gene phantom (phm) and spook (spo) were reduced in vvl knockdown larvae in the anterior and posterior halves, respectively, indicating that Vvl might influence ecdysone biosynthesis in both the prothoracic gland and additional endocrine sources. Injection of 20-hydroxyecdysone (20E) into vvl knockdown larvae could restore the expression of HR3 although molting was never restored. These findings suggest that Vvl coordinates both JH and ecdysteroid biosynthesis as well as molting behavior to influence molting and the timing of metamorphosis. Thus, in both vertebrates and insects, POU factors modulate the production of major neuroendocrine regulators during sexual maturation.

Neuropeptides affecting the transfer of juvenile hormones from males to females during mating in Spodoptera frugiperda

In the polyandric moth, Spodopterafrugiperda, juvenile hormone (JH) is transferred from the male accessory reproductive glands (AG) to the female bursa copulatrix (BC) during copulation (see Hassanien et al., 2014). Here we used the RNA interference technique to study the role of allatoregulating neuropeptides in controlling the synthesis and transfer of JH during mating.Knockdown of S. frugiperda allatostatin C (Spofr-AS type C) in freshly emerged males leads to an accumulation of JH in the AG beyond that in the control and mating results in a higher transport of JH I and JH II into the female BC. Knockdown of S. frugiperda allatotropin 2 (Spofr-AT2) significantly reduces the amount of JH in the AG as well as its transfer into the female BC during copulation. Knockdown of S. frugiperda allatostatin A (Spofr-AS type A) and S. frugiperda allatotropin (Spofr-AT; Hassanien et al., 2014) only slightly affects the accumulation of JH in the AG and its transfer from the male to the female.We conclude that Spofr-AS type C and Spofr-AT2 act as true allatostatin and true allatotropin, respectively, on the synthesis of JH I and JH II in the male AG. Moreover, both peptides seem to control the synthesis of JH III in the corpora allata of adult males and its release into the hemolymph.

Metabolic analysis reveals changes in the mevalonate and juvenile hormone synthesis pathways linked to the mosquito reproductive physiology

Juvenile hormone (JH) regulates reproductive maturation in insects; therefore interruption of JH biosynthesis has been considered as a strategy for the development of target-specific insecticides. The corpora allata (CA) from mosquitoes is highly specialized to supply variable levels of JH, which are linked to ovarian developmental stages and influenced by nutritional signals. However, very little is known about how changes in JH synthesis relate to reproductive physiology and how JH synthesis regulation is translated into changes in the CA machinery. With the advent of new methods that facilitate the analysis of transcripts, enzymes and metabolites in the minuscule CA, we were able to provide comprehensive descriptions of the mevalonic (MVA) and JH synthesis pathways by integrating information on changes in the basic components of those pathways. Our results revealed remarkable dynamic changes in JH synthesis and exposed part of a complex mechanism that regulates CA activity. Principal component (PC) analyses validated that both pathways (MVAP and JH-branch) are transcriptionally co-regulated as a single unit, and catalytic activities for the enzymes of the MVAP and JH-branch also changed in a coordinate fashion. Metabolite studies showed that global fluctuations in the intermediate pool sizes in the MVAP and JH-branch were often inversely related. PC analyses suggest that in female mosquitoes, there are at least 4 developmental switches that alter JH synthesis by modulating the flux at distinctive points in both pathways.

Correction: Starvation Increases Insulin Sensitivity and Reduces Juvenile Hormone Synthesis in Mosquitoes

Correction: Starvation Increases Insulin Sensitivity and Reduces Juvenile Hormone Synthesis in Mosquitoes

Insulin receptor-mediated nutritional signalling regulates juvenile hormone biosynthesis and vitellogenin production in the German cockroach

Female reproductive processes, which comprise, amongst others, the synthesis of yolk proteins and the endocrine mechanisms which regulate this synthesis, need a considerable amount of energy and resources. The role of communicating that the required nutritional status has been attained is carried out by nutritional signalling pathways and, in particular, by the insulin receptor (InR) pathway. In the present study, using the German cockroach, Blattella germanica, as a model, we analysed the role of InR in different processes, but mainly those related to juvenile hormone (JH) synthesis and vitellogenin production. We first cloned the InR cDNA from B. germanica (BgInR) and then determined that its expression levels were constant in corpora allata and fat body during the first female gonadotrophic cycle. Results showed that the observed increase in BgInR mRNA in fat body from starved compared to fed females was abolished in those females treated with systemic RNAi in vivo against the transcription factor BgFoxO. RNAi-mediated BgInR knockdown during the final two nymphal stages produced significant delays in the moults, together with smaller adult females which could not spread the fore- and hindwings properly. In addition, BgInR knockdown led to a severe inhibition of juvenile hormone synthesis in adult female corpora allata, with a concomitant reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, HMG-CoA reductase and methyl farnesoate epoxidase. BgInR RNAi treatment also reduced fat body vitellogenin mRNA and oocyte growth. Our results show that BgInR knockdown produces similar phenotypes to those obtained in starved females in terms of corpora allata activity and vitellogenin synthesis, and indicate that the InR pathway mediates the activation of JH biosynthesis and vitellogenin production elicited by nutrition signalling.

Comparative Proteomics and Expression Analysis of Five Genes in Epicauta chinensis Larvae from the First to Fifth Instar

Blister beetle is an important insect model for both medicinal and pure research. Previous research has mainly focused on its biology and biochemistry, but very little data is yet available in the molecular biology. This study uses differential proteomics technology to analyze the soluble proteins extracted from each of the 5 instars larvae of Epicauta chinensis. 42 of the differentially-expressed proteins were identified successfully by MALDI-TOF/TOF-MS. Some of these proteins’ function and their expression profiles are analyzed. Our analysis revealed dynamics regulation of the following proteins: Axin-like protein pry-1 (APR-1), dihydrolipoyl dehydrogenase (DLD), vitellogenin (Vg) and lysozyme C (Lmz-S). APR-1 negatively regulates the Wnt signaling pathway. Its overexpression could result in embryo, leg, eye and ovary ectopica or malformation. DLD catalyzes the pyruvate into acetyl-CoA, the latter is the starting material of juvenile hormone (JH) and ipsdienol biosynthesis through the MVA pathway in insects. While Vg synthesis can be regulated by JH and stimulated by food factors. So DLD may affect the synthesis of JH, ipsdienol and Vg indirectly. The activity of lysozyme is an indicator of the immunity. Nutrition/food should be taken into account for its potential role during the development of larva in the future. Among the five genes and their corresponding proteins’ expression, only hsc70 gene showed a good correspondence with the protein level. This reflects the fluctuating relationship between mRNA and protein levels.

Starvation Increases Insulin Sensitivity and Reduces Juvenile Hormone Synthesis in Mosquitoes

BackgroundThe interactions between the insulin signaling pathway (ISP) and juvenile hormone (JH) controlling reproductive trade-offs are well documented in insects. JH and insulin regulate reproductive output in mosquitoes; both hormones are involved in a complex regulatory network, in which they influence each other and in which the mosquito's nutritional status is a crucial determinant of the network's output. Previous studies reported that the insulin-TOR (target of rapamacyn) signaling pathway is involved in the nutritional regulation of JH synthesis in female mosquitoes. The present studies further investigate the regulatory circuitry that controls both JH synthesis and reproductive output in response to nutrient availability.MethodsWe used a combination of diet restriction, RNA interference (RNAi) and insulin treatments to modify insulin signaling and study the cross-talk between insulin and JH in response to starvation. JH synthesis was analyzed using a newly developed assay utilizing fluorescent tags.ConclusionsOur results reveal that starvation decreased JH synthesis via a decrease in insulin signaling in the corpora allata (CA). Paradoxically, starvation-induced up regulation of insulin receptor transcripts and therefore “primed” the gland to respond rapidly to increases in insulin levels. During this response to starvation the synthetic potential of the CA remained unaffected, and the gland rapidly and efficiently responded to insulin stimulation by increasing JH synthesis to rates similar to those of CA from non-starved females.

Juvenile Hormone Biosynthesis Gene Expression in the corpora allata of Honey Bee (Apis mellifera L.) Female Castes

Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution.

Genome-Wide Transcriptional Response of Silkworm (Bombyx mori) to Infection by the Microsporidian Nosema bombycis

Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by N. bombycis was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, B. mori genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, N. bombycis can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. N. bombycis also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of N. bombycis-specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between B. mori and N. bombycis and may provide a foundation for further work on host-parasite interaction between insects and microsporidia.

Allatotropin: An Ancestral Myotropic Neuropeptide Involved in Feeding

BackgroundCell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms.MethodsA combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies.ConclusionsAT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion.SignificanceImmunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion.

Early manipulation of juvenile hormone has sexually dimorphic effects on mature adult behavior in Drosophila melanogaster

Hormones are critical for the development, maturation, and maintenance of physiological systems; therefore, understanding their involvement during maturation of the brain is important for the elucidation of mechanisms by which adults become behaviorally competent. Changes in exogenous and endogenous factors encountered during sexual maturation can have long lasting effects in mature adults. In this study, we investigated the role of the gonadotropic hormone, juvenile hormone (JH), in the modulation of adult behaviors in Drosophila. Here we utilized methoprene (a synthetic JH analog) and precocene (a JH synthesis inhibitor) to manipulate levels of JH in sexually immature male and female Drosophila with or without decreased synthesis of neuronal dopamine (DA). Locomotion and courtship behavior were assayed once the animals had grown to sexual maturity. The results demonstrate a sexually dimorphic role for JH in the modulation of these centrally controlled behaviors in mature animals that is dependent on the age of the animals assayed, and present DA as a candidate neuronal factor that differentially interacts with JH depending on the sex of the animal. The data also suggest that JH modulates these behaviors through an indirect mechanism. Since gonadotropic hormones and DA interact in mammals to affect brain development and later function, our results suggest that this mechanism for the development of adult behavioral competence may be evolutionarily conserved.

Aspergillus nidulans Synthesize Insect Juvenile Hormones upon Expression of a Heterologous Regulatory Protein and in Response to Grazing by Drosophila melanogaster Larvae

Secondary metabolites are known to serve a wide range of specialized functions including communication, developmental control and defense. Genome sequencing of several fungal model species revealed that the majority of predicted secondary metabolite related genes are silent in laboratory strains, indicating that fungal secondary metabolites remain an underexplored resource of bioactive molecules. In this study, we combine heterologous expression of regulatory proteins in Aspergillus nidulans with systematic variation of growth conditions and observe induced synthesis of insect juvenile hormone-III and methyl farnesoate. Both compounds are sesquiterpenes belonging to the juvenile hormone class. Juvenile hormones regulate developmental and metabolic processes in insects and crustaceans, but have not previously been reported as fungal metabolites. We found that feeding by Drosophila melanogaster larvae induced synthesis of juvenile hormone in A. nidulans indicating a possible role of juvenile hormone biosynthesis in affecting fungal-insect antagonisms.

RNA interference of a putative S-adenosyl-L-homocysteine hydrolase gene affects larval performance in Leptinotarsa decemlineata (Say)

In Leptinotarsa decemlineata, juvenile hormones (JHs) play primary roles in the regulation of metamorphosis, reproduction and diapause. In JH biosynthetic pathway in insect corpora allata, methylation of farnesoic acid or JH acid using S-adenosyl-L-methionine generates a potent feedback inhibitor S-adenosyl-L-homocysteine (AdoHcy). Rapid removal of AdoHcy is hypothesized to be essential for JH synthesis. AdoHcy hydrolase (SAHase) is the only eukaryotic enzyme catalyzing the removal. In the present paper, we firstly cloned a putative LdSAHase gene from L. decemlineata. The cDNA consists of 1806bp and encodes a 525 amino acid protein. LdSAHase was expressed in all developmental stages. The gene had the highest and the lowest level of transcription respectively in the 3rd- and 4th-instars’ heads that contain corpora allata, which was positively correlated with JH titer in the haemolymph and the mRNA level of a JH early-inducible gene, the Krüppel homolog 1 gene (Kr-h1). Secondly, dietary ingestion of bacterially-expressed LdSAHase-dsRNA significantly decreased LdSAHase and LdKr-h1 mRNA levels, reduced JH titer, and caused the death of the larvae, and the failure of pupation and adult emergence. After continuous exposure for 12days, 42% of the larvae died, 65% of the prepupae failed to pupate and 100% of the pupae failed to emerge. Moreover, RNAi-mediated LdSAHase knockdown also reduced larval developing time, and decreased larval weight. Lastly, application of JH analogue pyriproxyfen to LdSAHase-dsRNA-exposed larvae did not greatly increase LdSAHase expression level and JH content, but up-regulated LdKr-h1 mRNA level. Expectedly, pyriproxyfen application could partially rescue the negative effects on the survival and the development. Thus, our results support the hypothesis that SAHase plays a critical role in JH biosynthesis in insects.

Characterisation of a functional allatotropin receptor in the bumblebee, Bombus terrestris (Hymenoptera, Apidae)

Allatotropins (ATs) are multifunctional neuropeptides initially isolated from the tobacco hornworm, Manduca sexta, where they were found to stimulate juvenile hormone synthesis and release from the corpora allata. ATs have been found in a wide range of insects, but appear to be absent in Drosophila. The first AT receptor (ATR) was characterised in 2008 in the lepidopteran Bombyx mori. Since then ATRs have been characterised in Coleoptera and Diptera and in 2012, an AT precursor gene was identified in hymenopteran species. ATRs show large sequence and structural similarity to vertebrate orexin receptors (OXR). Also, AT in insects and orexin in vertebrates show some overlap in functions, including modulation of feeding behaviour and reproduction.The goal of this study was to identify a functional ATR in a hymenopteran species. We used ATRs (insect sequences) and OXRs (vertebrate sequences) to search the genome of the bumblebee, Bombus terrestris. Two receptors (XP_003402490 and XP_003394933) with resemblance to ATRs and OXRs were found. Phylogenetic analysis provided the first indication that XP_003402490 was more closely related to ATRs than XP_003394933. We investigated the transcript level distribution of both receptors and the AT precursor gene by means of quantitative real-time reverse transcriptase PCR. XP_003402490 displayed a tissue distribution comparable with ATRs in other species, with high transcript levels in the male accessory glands. After pharmacological characterisation, it appeared that XP_003402490 is indeed a functional ATR. Activation of the receptor causes an increase in intracellular calcium and cyclic AMP levels with an EC50 value in the low nanomolar to picomolar range. XP_003394933 remains an orphan receptor.

Aldehyde dehydrogenase 3 converts farnesal into farnesoic acid in the corpora allata of mosquitoes

The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD+-dependent class 3 ALDH showing tissue- and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren–Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a “trade-off” system to balance the key role of farnesal as a JH precursor with its potential toxicity.