A male steroid controls female sexual behaviour in the malaria mosquito.

Insects, unlike vertebrates, are widely believed to lack male-biased sex steroid hormones1. In the malaria mosquito Anopheles gambiae, the ecdysteroid 20-hydroxyecdysone (20E) appears to have evolved to both control egg development when synthesized by females2 and to induce mating refractoriness when sexually transferred by males3. Because egg development and mating are essential reproductive traits, understanding how Anopheles females integrate these hormonal signals can spur the design of new malaria control programs. Here we reveal that these reproductive functions are regulated by distinct sex steroids through a sophisticated network of ecdysteroid-activating/inactivating enzymes. We identify a male-specific oxidized ecdysteroid, 3-dehydro-20E (3D20E), which safeguards paternity by turning off female sexual receptivity following its sexual transfer and activation by dephosphorylation. Notably, 3D20E transfer also induces expression of a reproductive gene that preserves egg development during Plasmodium infection, ensuring fitness of infected females. Female-derived 20E does not trigger sexual refractoriness but instead licenses oviposition in mated individuals once a 20E-inhibiting kinase is repressed. Identifying this male-specific insect steroid hormone and its roles in regulating female sexual receptivity, fertility and interactions with Plasmodium parasites suggests the possibility for reducing the reproductive success of malaria-transmitting mosquitoes.

Naturally Occurring Ecdysteroids in Triticum aestivum L. and Evaluation of Fenarimol as a Potential Inhibitor of Their Biosynthesis in Plants.

Ecdysteroids (ECs) are steroid hormones originally found in the animal kingdom where they function as insect molting hormones. Interestingly, a relatively high number of these substances can also be formed in plant cells. Moreover, ECs have certain regulatory effects on plant physiology, but their role in plants still requires further study. One of the main aims of the present study was to verify a hypothesis that fenarimol, an inhibitor of the biosynthesis of ECs in the animal kingdom, also affects the content of endogenous ECs in plants using winter wheat Triticum aestivum L. as a model plant. The levels of endogenous ECs in winter wheat, including the estimation of their changes during a course of different temperature treatments, have been determined using a sensitive analytical method based on UHPLC-MS/MS. Under our experimental conditions, four substances of EC character were detected in the tissue of interest in amounts ranging from less than 1 to over 200 pg·g-1 FW: 20-hydroxyecdysone, polypodine B, turkesterone, and isovitexirone. Among them, turkesterone was observed to be the most abundant EC and accumulated mainly in the crowns and leaves of wheat. Importantly, the level of ECs was observed to be dependent on the age of the plants, as well as on growth conditions (especially temperature). Fenarimol, an inhibitor of a cytochrome P450 monooxygenase, was shown to significantly decrease the level of naturally occurring ECs in experimental plants, which may indicate its potential use in studies related to the biosynthesis and physiological function of these substances in plants.

Control of the insect metamorphic transition by ecdysteroid production and secretion.

Ecdysteroids are a class of steroid hormones that controls molting and metamorphic transitions in Ecdysozoan species including insects, in which ecdysteroid biosynthesis and its regulation have been extensively studied. Insect ecdysteroids are produced from dietary sterols by a series of reduction-oxidation reactions in the prothoracic gland and in Drosophila they are released into the hemolymph via vesicle-mediated secretion at the time of metamorphosis. To initiate precisely controlled ecdysteroid pulses, the prothoracic gland functions as a central node integrating both intrinsic and extrinsic signals to control ecdysteroid biosynthesis and secretion. In this review, we outline recent progress in the characterization of ecdysone biosynthesis and steroid trafficking pathways and the discoveries of novel factors regulating prothoracic gland function.

Characterization of ecdysteroid biosynthesis in the pond wolf spider, Pardosa pseudoannulata.

Ecdysteroids, as the key growth hormones, regulate moulting, metamorphosis and reproduction in arthropods. Ecdysteroid biosynthesis is catalysed by a series of cytochrome P450 monooxygenases (CYP450s) encoded by Halloween genes, including spook (spo), phantom (phm), disembodied (dib), shadow (sad) and shade (shd). The ecdysteroid biosynthesis in insects is clear with 20-hydroxyecdysone (20E) as the main ecdysteroid. However, the information on the major ecdysteroids in arachnids is limited. In this study, Halloween genes spo, dib, sad and shd, but not phm, were identified in the pond wolf spider, Pardosa pseudoannulata. Phylogenetic analysis grouped arachnid and insect Halloween gene products into two CYP450 clades, the CYP2 clan (spo and phm) and the mitochondrial clan (dib, sad, and shd). In P. pseudoannulata, the temporal expression profile of the four Halloween genes in concurrence with spiderling moulting with steady increase in the course of the 2nd instar followed by a rapid dropdown once moulting was completed. Spatially, the four Halloween genes were highly expressed in spiderling abdomen and in the ovaries of female adults. In parallel, ponasterone A (PA), but not 20E, was detected by LC-MS/MS analysis in P. pseudoannulata, and it was demonstrated as a functional ecdysteroid in the spider by accelerating of moulting with PA addition. The present study revealed the different ecdysteroid biosynthesis pathways in spiders and insects.

Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.

The Corazonin-PTTH Neuronal Axis Controls Systemic Body Growth by Regulating Basal Ecdysteroid Biosynthesis in Drosophila melanogaster.

Steroid hormones play key roles in development, growth, and reproduction in various animal phyla [1]. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis [2]. In Drosophila melanogaster, the prothoracicotropic hormone (PTTH)-producing neurons stimulate peak levels of ecdysteroid biosynthesis for maturation [3]. Additionally, recent studies on PTTH signaling indicated that basal levels of ecdysteroid negatively affect systemic growth prior to maturation [4-8]. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Crz belongs to gonadotropin-releasing hormone (GnRH) superfamily, implying an analogous role in growth and maturation [9]. Inhibition of Crz neuronal activity increased pupal size, whereas it hardly affected pupariation timing. This phenotype resulted from enhanced growth rate and a delay in ecdysteroid elevation during the mid-third instar larval (L3) stage. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons during the mid-L3, but not the late-L3, stage. Furthermore, we found that octopamine neurons contact Crz neurons in the subesophageal zone (SEZ), transmitting signals for systemic growth. Together, our results suggest that the Crz-PTTH neuronal axis modulates ecdysteroid biosynthesis in response to octopamine, uncovering a regulatory neuroendocrine system in the developmental transition from growth to maturation.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo.

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

Structure and steroid isomerase activity of Drosophila glutathione transferase E14 essential for ecdysteroid biosynthesis.

Ecdysteroids are critically important for the formation of the insect exoskeleton. Cholesterol is a precursor of ecdysone and its active form 20-hydroxyecdysone, but some steps in the ecdysteroid biosynthesis pathway remain unknown. An essential requirement of glutathione (GSH) transferase GSTE14 in ecdysteroid biosynthesis has been established in Drosophila melanogaster, but its function is entirely unknown. Here, we have determined the crystal structure of GSTE14 in complex with GSH and investigated the kinetic properties of GSTE14 with alternative substrates. GSTE14 has high-ranking steroid double-bond isomerase activity, albeit 50-fold lower than the most efficient mammalian GSTs. Corresponding steroid isomerizations are unknown in insects, and their exact physiological role remains to be shown. Nonetheless, the essential enzyme GSTE14 is here demonstrated to be catalytically competent and have a steroid-binding site.

Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET.

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.

Effects of atrazine on life parameters, oxidative stress, and ecdysteroid biosynthetic pathway in the marine copepod Tigriopus japonicus.

Atrazine is a widely used pesticide which acts as an endocrine disruptor in various organisms. The aim of this study was to investigate adverse effects of atrazine on life parameters, oxidative stress, and ecdysteroid biosynthetic pathway in the marine copepod Tigriopus japonicus. In T. japonicus, no mortality was shown in response to atrazine up to 20 mg/L in acute toxicity assessment. In nauplii, retardation in the growth and prolonged molting and metamorphosis resulted under chronic exposure of atrazine at 20 mg/L. In addition, body sizes of T. japonicus nauplii were significantly decreased (P < 0.01 in length and P < 0.001 in width) in response to 20 mg/L of atrazine. Furthermore, atrazine induced oxidative stress by the generation of reactive oxygen species at all concentrations compared to the control in the nauplii. Also, significant increase in glutathione-S transferase activity was observed in adult T. japonicus at low concentration of atrazine. To understand effects of atrazine on ecdysteroid biosynthetic pathway-involved genes (e.g., neverland, CYP307E1, CYP306A1, CYP302A1, CYP3022A1 [CYP315A1], CYP314A1, and CYP18D1) were examined with mRNA expressions of ecdysone receptor (EcR) and ultraspiracle (USP) in response to 20 mg/L atrazine in nauplii and adults. In the nauplii, these genes were significantly downregulated (P < 0.05) in response to atrazine, compared to the control but not in the adult T. japonicus. These results suggest that atrazine can interfere in vivo life parameters by oxidative stress-induced retrogression and ecdysteroid biosynthetic pathway in this species.

Ecdysteroidogenesis in Heliothis virescens (Lepidoptera: Noctuidae): Recombinant Prothoracicotropic Hormone and Brain Extract Show Comparable Effects.

Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.

Expression dynamics of key ecdysteroid and juvenile hormone biosynthesis genes imply a coordinated regulation pattern in the molting process of a spider mite, Tetranychus urticae.

In insects, the ecdysteroid 20-hydroxyecdysone coordinates with juvenile hormone (JH) to regulate the process of molting, development and metamorphosis; however, this interaction is still unclear in the mites. In this study, we investigated the gene related to ecdysteroid and JH biosynthesis pathways, including four ecdysteroid and 11 JH biosynthesis genes. We examined their expression patterns during molting of different developmental stages of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), an important agricultural pest that feeds on more than 1100 plant species. The expression of ecdysteroid biosynthesis Halloween genes exhibited a positive zigzag-like pattern, with a peak after 8 h of molting and a drop 8 h after entering each quiescent stage. In contrast, JH biosynthesis genes expression displayed a negative zigzag-like pattern, with a peak at 8 h after entering each quiescent stage and a drop after 8 h of each molting. These opposite patterns imply that ecdysteroid and JH expression is coordinated during the developmental transition. Our data provide an initial perspective on the co-expression of ecdysteroid and JH biosynthesis genes to regulate this important developmental process in the two-spotted spider mite.

Fat body lipolysis connects poor nutrition to hypopharyngeal gland degradation in Apis mellifera.

The hypopharyngeal glands (HGs) of honey bee nurse workers secrete the major protein fraction of jelly, a protein and lipid rich substance fed to developing larvae, other worker bees, and queens. A hallmark of poorly nourished nurses is their small HGs, which actively degrade due to hormone-induced autophagy. To better connect nutritional stress with HG degradation, we looked to honey bees and other insect systems, where nutrient stress is often accompanied by fat body degradation. The fat body contains stored lipids that are likely a substrate for ecdysteroid synthesis, so we tested whether starvation caused increased fat body lipolysis. Ecdysteroid signaling and response pathways and IIS/TOR are tied to nutrient-dependent autophagy in honey bees and other insects, and so we also tested whether and where genes in these pathways were differentially regulated in the head and fat body. Last, we injected nurse-aged bees with the honey bee ecdysteroid makisterone A to determine whether this hormone influenced HG size and autophagy. We find that starved nurse aged bees exhibited increased fat body lipolysis and increased expression of ecdysteroid production and response genes in the head. Genes in the IIS/TOR pathway were not impacted by starvation in either the head or fat body. Additionally, bees injected with makisterone A had smaller HGs and increased expression of autophagy genes. These data support the hypothesis that nutritional stress induces fat body lipolysis, which may liberate the sterols important for ecdysteroid production, and that increased ecdysteroid levels induce autophagic HG degradation.

Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain.

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of "Qiufeng", a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and ßFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi.

The prothoracic gland (PG) is an important endocrine organ of synthesis and secretion of ecdysteroids that play critical roles in insects. Here, we used a comparative transcriptomic approach to characterize some common features of PGs from two lepidopteran species Bombyx mori and Antheraea pernyi. Functional and pathway annotations revealed an overall similarity in gene profile between the two PG transcriptomes. As expected, almost all steroid hormone biosynthesis genes and the prothoracicitropic hormone receptor gene (Torso) were well represented in the two PGs. Impressively, two ecdysone receptor genes, eleven juvenile hormone related genes, more than 10 chemosensory protein genes, and a set of genes involved in circadian clock were also presented in the two PGs. Quantitative real time -PCR (qRT-PCR) validated the expression of 8 juvenile hormone and 12 clock related genes in B. mori PG, and revealed a different expression pattern during development in whole fifth larval instar. This contribution to insect PG transcriptome data will extend our understanding of the function and regulation of this important organ.

[Exploration of Insect Growth Regulators Targeting Noppera-bo, an Enzyme Involved in Ecdysteroid Biosynthesis].

Insect growth regulators (IGRs) are chemicals that adversely affect the physiological processes associated with insect development and cause abnormalities that impair insect survival. Ecdysone, an insect steroid hormone originally identified as a molting hormone, plays an essential role in developmental transition, such as during molting and metamorphosis. Recently, a member of the epsilon class of glutathione S-transferases (GST), GSTe14, also called Noppera-bo (Nobo), has been identified as essential for regulating the biosynthesis of ecdysone. Knockout or knockdown of the nobo gene causes ecdysone deficiency, leading to either death or arrested phenotype development at the larval stage. It is therefore considered that Nobo is potentially well suited as a target for novel IGRs. In this review, we focus on the development of a high-throughput screening strategy for Nobo inhibitors using a GST fluorogenic substrate.

Transcriptomic analysis of crustacean molting gland (Y-organ) regulation via the mTOR signaling pathway.

The intermolt crustacean Y-organ (YO) maintains a basal state mediated by pulsatile release of molt inhibiting hormone (MIH), a neuropeptide produced in the eyestalk ganglia, inhibiting YO ecdysteroidogenesis. Reduction of MIH results in YO activation and the animal enters premolt. In the crab, Gecarcinus lateralis, molting was induced by eyestalk ablation (ESA). ESA animals were injected with either rapamycin, an mTOR inhibitor, or DMSO vehicle at Day 0. YOs were harvested at 1, 3, and 7 days post-ESA and processed for high throughput RNA sequencing. ESA-induced increases in mRNA levels of mTOR signaling genes (e.g., mTOR, Rheb, TSC1/2, Raptor, Akt, and S6 kinase) declined following rapamycin treatment. In concert with mTOR inhibition, mRNA levels of ecdysteroid biosynthesis genes (e.g., Nvd, Spo, Sad, Dib, and Phm) were decreased and accompanied by a decrease in hemolymph ecdysteroid titer. By contrast, rapamycin increased the mRNA level of FKBP12, the rapamycin-binding protein, as well as the mRNA levels of genes associated with Wnt and insulin-like growth factor signaling pathways. Many MIH and transforming growth factor-ß signaling genes were down regulated in ESA animals. These results indicate that mTOR activity either directly or indirectly controls transcription of genes that drive activation of the YO.

Involvement of methoprene-tolerant (Met) in the determination of the final body size in Leptinotarsa decemlineata (Say) larvae.

In the tobacco hornworm Manduca sexta, juvenile hormone (JH) is critical for the control of species-specific size. However, whether the basic helix-loop-helix/Per-Arnt-Sim domain receptor methoprene-tolerant (Met) is involved remains unconfirmed. In the present paper, we found that RNA interference (RNAi)-aided knockdown of Met gene (LdMet) lowered the larval and pupal fresh weights and shortened the larval development period in the Colorado potato beetle Leptinotarsa decemlineata. Dietary introduction of JH into the LdMet RNAi larvae rescued neither the decreased weights nor the reduced development phase, even though JH ingestion by control larvae extended developmental time and caused large pupae. Moreover, the transcript levels of five genes involved in prothoracicotropic hormone and cap 'n' collar isoform C/Kelch-like ECH associated protein 1 pathways were upregulated in the LdMet silenced larvae. Ecdysteroidogenesis was thereby activated; 20-hydroxyecdysone (20E) titer was increased; and 20E signaling pathway was elicited in the LdMet RNAi larvae. Therefore, JH, acting through its receptor Met, inhibits PTTH production and release before the attainment of critical weight. Once the critical weight is reached, JH production and release are averted; and the hemolymph JH is removed. The elimination of JH allows the brain to release PTTH. PTTH subsequently stimulates ecdysteroid biosynthesis and release to start larval-pupal transition in L. decemlineata.

Cholesterol internalization and metabolism in insect prothoracic gland, a steroidogenic organ, via lipoproteins.

Dietary sterols including cholesterol and phytosterols are essential substrates for insect steroid hormone (ecdysteroid) synthesis in the prothoracic glands (PGs). In the silkworm Bombyx mori, one of the model species of insects, the steroidogenesis has been well demonstrated that cholesterol biotransformation into ecdysone in the PG cells. Because insects lack the ability to synthesize cellular sterol de novo, lipoprotein, lipophorin (Lp), has been thought to be the major cholesterol supply source; however, details of cholesterol behavior from Lp to the PG cells has not been analyzed till date. In this report, we developed Lp incorporation method using labeled cholesterols such as 22-NBD-cholesterol and cholesterol-25,26,26,26,27,27,27-d7 (cholesterol-d7), and analyzed the internalization and metabolism of cholesterol in PGs in vitro using the silkworm Bombyx mori. The internalization of cholesterol was visualized using 22-NBD-cholesterol. PGs showed an enriched cellular 22-NBD-cholesterol signal, which dissociated from the Lp localizing at the close area of cell membrane. The distribution pattern observed in the PGs was different from other tissues such as the brain, fat body, and Malpighian tubules, suggesting that the internalization of cholesterol in the PGs was distinct from other tissues. The metabolism of cholesterol was traced using LC-MS/MS methods to detect cholesterol-d7, 7-dehydrocholesterol-d7 (an expected intermediate metabolite), and the final product ecdysone-d6. 7-Dehydrocholesterol-d7 and ecdysone-d6 were detected in the PG culture incubated with labeled Lp, showing that the cholesterol of Lp was utilized for ecdysone synthesis in the PGs. Our results reveal the distinct behavior of cholesterol in the PGs, with the first direct evidence of biochemical fate of lipoprotein cholesterol in insect steroidogenic organ. This will aid in the understanding of the involvement of lipoprotein cholesterol in steroid hormone synthesis in insects.

Ecdysteroid and juvenile hormone biosynthesis, receptors and their signaling in the freshwater microcrustacean Daphnia.

The two essential insect hormones, ecdysteroids and juvenile hormones, are possessed not only by insects, but also widely by arthropods, and regulate various developmental and physiological processes. In contrast to the abundant information about molecular endocrine mechanisms in insects, the knowledge of non-insect arthropod endocrinology is still limited. In this review, we summarize recent reports about the molecular basis of these two major insect hormones in the freshwater microcrustacean Daphnia, a keystone taxon in limnetic ecology and a bioindicator in environmental studies. Comprehensive comparisons of endocrine signaling pathways between insects and daphnids may shed light on the regulatory mechanisms of various biological phenomena and, moreover, evolutionary processes of arthropod species.