Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ).

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

Phylogenomics of the Ecdysteroid Kinase-like (EcKL) Gene Family in Insects Highlights Roles in Both Steroid Hormone Metabolism and Detoxification.

The evolutionary dynamics of large gene families can offer important insights into the functions of their individual members. While the ecdysteroid kinase-like (EcKL) gene family has previously been linked to the metabolism of both steroid molting hormones and xenobiotic toxins, the functions of nearly all EcKL genes are unknown, and there is little information on their evolution across all insects. Here, we perform comprehensive phylogenetic analyses on a manually annotated set of EcKL genes from 140 insect genomes, revealing the gene family is comprised of at least 13 subfamilies that differ in retention and stability. Our results show the only two genes known to encode ecdysteroid kinases belong to different subfamilies and therefore ecdysteroid metabolism functions must be spread throughout the EcKL family. We provide comparative phylogenomic evidence that EcKLs are involved in detoxification across insects, with positive associations between family size and dietary chemical complexity, and we also find similar evidence for the cytochrome P450 and glutathione S-transferase gene families. Unexpectedly, we find that the size of the clade containing a known ecdysteroid kinase is positively associated with host plant taxonomic diversity in Lepidoptera, possibly suggesting multiple functional shifts between hormone and xenobiotic metabolism. Our evolutionary analyses provide hypotheses of function and a robust framework for future experimental studies of the EcKL gene family. They also open promising new avenues for exploring the genomic basis of dietary adaptation in insects, including the classically studied coevolution of butterflies with their host plants.

Ecdysteroid Biosynthesis Halloween Gene Spook Plays an Important Role in the Oviposition Process of Spider Mite, Tetranychus urticae.

In insects, the ecdysteroid hormone regulates development and reproduction. However, its function in the reproduction process of spider mites is still unclear. In this study, we investigated the effect of the Halloween gene Spook on the oviposition of the reproduction process in a spider mite, Tetranychus urticae. The expression patterns of the ecdysteroid biosynthesis and signaling pathway genes, as analyzed by RT-qPCR, showed that the expression pattern of the Halloween genes was similar to the oviposition pattern of the female mite and the expression patterns of the vitellogenesis-related genes TuVg and TuVgR, suggesting that the Halloween genes are involved in the oviposition of spider mites. To investigate the function of the ecdysteroid hormone on the oviposition of the reproduction process, we carried out an RNAi assay against the Halloween gene Spook by injection in female mites. Effective silencing of TuSpo led to a significant reduction of oviposition. In summary, these results provide an initial study on the effect of Halloween genes on the reproduction in T. urticae and may be a foundation for a new strategy to control spider mites.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

Understanding molt control switches: Transcriptomic and expression analysis of the genes involved in ecdysteroidogenesis and cholesterol uptake pathways in the Y-organ of the blue crab, Callinectes sapidus.

The crustacean molting process is regulated by an interplay of hormones produced by the eyestalk ganglia and Y-organs (YO). Molt-inhibiting hormone and crustacean hyperglycemic hormone released by the sinus gland of the eyestalk ganglia (EG) inhibit the synthesis and secretion of ecdysteroid by the YO, hence regulating hemolymph levels during the molt cycle. The purpose of this study is to investigate the ecdysteroidogenesis pathway, specifically genes linked to changes in ecdysteroid levels occurring at early premolt (ePM). To this end, a reference transcriptome based on YO, EG, and hepatopancreas was de novo assembled. Two genes (cholesterol 7-desaturase Neverland and cytochrome p450 307a1-like Spook) involved in ecdysteroidogenesis were identified from the YO transcriptome using sequence comparisons and transcript abundance. Two other candidates, Hormone receptor 4 and probable cytochrome p450 49a1 potentially involved in ecdysteroidogenesis were also identified. Since cholesterol is the ecdysteroid precursor, a putative cholesterol carrier (Apolipoprotein D-like) was also examined to understand if cholesterol uptake coincided with the increase in the ecdysteroid levels at the ePM stage. The expression level changes of the five candidate genes in the YO were compared between intermolt (IM) and induced ePM (iePM) stages using transcriptomic analysis. Expression analysis using qPCR were carried out at IM, iePM, and normal ePM. The increase in Spook and Neverland expression in the YO at the ePM was accompanied by a concomitant rise in ecdysteroid levels. The data obtained from iePM stage were congruent with those obtained from the normal ePM stage of intact control animals. The present findings support the role of Halloween genes in the ecdysteroidogenesis and molt cycle in the blue crab, Callinectes sapidus.

Changes in expressions of ecdysteroidogenic enzyme and ecdysteroid signaling genes in relation to Bombyx embryonic development.

Although the role of ecdysteroids in regulating egg diapause process in Bombyx mori is well documented, temporal changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling are less well understood. In the present study, we studied changes in expression levels of genes involved in ecdysteroid biosynthesis and its downstream signaling during embryonic development of B. mori. Results showed that in diapause eggs, the expression of ecdysteroid-phosphate phosphatase (EPPase) gene and Halloween genes (Spook [Spo] and Shade [Shd]) remained at very low levels. However, in eggs whose diapause initiation was prevented by HCl, significant increases in the messenger RNA (mRNA) levels of EPPase, Spo, and Shd were detected during embryonic development. Other Halloween genes (Neverland [Nvd] and Phantom [Phm]) also showed different changes between diapause and HCl-treated eggs. However, genes of Disembodied (Dib) and Shadow (Sad) showed similar changes in both diapause and HCl-treated eggs. We further investigated changes in expression levels of ecdysone receptor genes (EcRA, EcRB1, and USP) and downstream signaling genes (E75A, E75B, E74A, E74B, Br-C, HR3, HR4, KR-H1, and FTZ-F1). Results showed that genes of EcRA and the other nuclear receptors (E75A, E75B, E74A, HR3, HR4, KR-H1, and FTZ-F1) exhibited significant differential patterns between diapause and HCl-treated eggs, with increased levels being detected during later stages of embryonic development in HCl-treated eggs. Differential temporal changes in expressions of genes involved ecdysteroid biosynthesis and its downstream signaling found between diapause and HCl-treated eggs were further confirmed using nondiapause eggs. Our results showed that nondiapause eggs exhibited the same changing patterns as those in HCl-treated eggs, thus clearly indicating potential correlations between expressions of these genes and embryonic development in B. mori. To our knowledge, this is the first comprehensive report to study the transcriptional regulation of ecdysteroidogenic and ecdysteroid signaling genes, thus providing useful information for a clearer understanding of insect egg diapause mechanisms.

RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages.

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world's poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

Roles of and cross-talk between ecdysteroid and sesquiterpenoid pathways in embryogenesis of branchiopod crustacean Daphnia magna.

The ecdysteroid and sesquiterpenoid pathways control growth, developmental transition, and embryogenesis in insects. However, the function of orthologous genes and the cross-talk between both pathways remain largely uncharacterized in non-insect arthropods. Spook (Spo) and Juvenile hormone acid o-methyltransferase (Jhamt) have been suggested to function as rate-limiting factors in ecdysteroid and sesquiterpenoid biosynthesis, respectively, in insects. In this study, we report on the functions of Spo and Jhamt and the cross-talk between them in embryos of the branchiopod crustacean Daphnia magna. Spo expression was activated at the onset of gastrulation, with the depletion of Spo transcript by RNAi resulting in developmental arrest at this stage. This phenotype could be partially rescued by supplementation with 20-hydroxyecdysone, indicating that Spo may play the same role in ecdysteroid biosynthesis in early embryos, as reported in insects. After hatching, Spo expression was repressed, while Jhamt expression was activated transiently, despite its silencing during other embryonic stages. Jhamt RNAi showed little effect on survival, but shortened the embryonic period. Exposure to the sesquiterpenoid analog Fenoxycarb extended the embryonic period and rescued the Jhamt RNAi phenotype, demonstrating a previously unidentified role of sesquiterpenoid in the repression of precocious embryogenesis. Interestingly, the knockdown of Jhamt resulted in the derepression of ecdysteroid biosynthesis genes, including Spo, similar to regulation during insect hormonal biosynthesis. Sesquiterpenoid signaling via the Methoprene-tolerant gene was found to be responsible for the repression of ecdysteroid biosynthesis genes. It upregulated an ortholog of CYP18a1 that degrades ecdysteroid in insects. These results illuminate the conserved and specific functions of the ecdysteroid and sesquiterpenoid pathways in Daphnia embryos. We also infer that the common ancestor of branchiopod crustaceans and insects exhibited antagonism between the two endocrine hormones before their divergence 400 million years ago.

Genomic and transcriptomic analyses in Drosophila suggest that the ecdysteroid kinase-like (EcKL) gene family encodes the 'detoxification-by-phosphorylation' enzymes of insects.

Phosphorylation is a phase II detoxification reaction that, among animals, occurs near exclusively in insects, but the enzymes responsible have never been cloned or otherwise identified. We propose the hypothesis that members of the arthropod-specific ecdysteroid kinase-like (EcKL) gene family encode detoxicative kinases. To test this hypothesis, we annotated the EcKL gene family in 12 species of Drosophila and explored their evolution within the genus. Many ancestral EcKL clades are evolutionarily unstable and have experienced repeated gene gain and loss events, while others are conserved as single-copy orthologs. Leveraging multiple published gene expression datasets from D. melanogaster, and using the cytochrome P450s-a classical detoxification family-as a test case, we demonstrate relationships between xenobiotic induction, detoxification tissue-enriched expression and evolutionary instability in the EcKLs and the P450s. We devised a systematic method for identifying candidate detoxification genes in large gene families that is concordant with experimentally determined functions of P450 genes in D. melanogaster. Applying this method to the EcKLs suggested a significant proportion of these genes play roles in detoxification, and that the EcKLs may constitute a detoxification gene family in insects. Additionally, we estimate that between 11 and 16 uncharacterised D. melanogaster P450s are strong detoxification candidates. Lastly, we also found previously unreported genomic and transcriptomic variation in a number of EcKLs and P450s associated with toxic stress phenotypes using a targeted phenome-wide association study (PheWAS) approach in D. melanogaster, presenting multiple future avenues of research for detoxification genetics in this species.

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.

Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana).

Insect molting hormone (ecdysteroids) and juvenile hormone regulate molting and metamorphic events in a variety of insect species. Mealybugs undergo sexually dimorphic metamorphosis: males develop into winged adults through non-feeding, pupa-like stages called prepupa and pupa, while females emerge as neotenic wingless adults. We previously demonstrated, in the Japanese mealybug Planococcus kraunhiae (Kuwana), that the juvenile hormone titer is higher in males than in females at the end of the juvenile stage, which suggests that juvenile hormone may regulate male-specific adult morphogenesis. Here, we examined the involvement of ecdysteroids in sexually dimorphic metamorphosis. To estimate ecdysteroid titers, quantitative RT-PCR analyses of four Halloween genes encoding for cytochrome P450 monooxygenases in ecdysteroid biosynthesis, i.e., spook, disembodied, shadow and shade, were performed. Overall, their expression levels peaked before each nymphal molt. Transcript levels of spook, disembodied and shadow, genes that catalyze the steps in ecdysteroid biosynthesis in the prothoracic gland, were higher in males from the middle of the second nymphal instar to adult emergence. In contrast, the expression of shade, which was reported to be involved in the conversion of ecdysone into 20-hydroxyecdysone in peripheral tissues, was similar between males and females. These results suggest that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females, although the final conversion into 20-hydroxyecdysone occurs at similar levels in both sexes. Moreover, expression profiles of ecdysone response genes, ecdysone receptor and ecdysone-induced protein 75B, were also analyzed. Based on these expression profiles, we propose that the changes in ecdysteroid titer differ between males and females, and that high ecdysteroid titer is essential for directing male adult development.

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.

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.

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.

Roles of the insulin signaling pathway in insect development and organ growth.

Organismal development is a complex process as it requires coordination of many aspects to grow into fit individuals, such as the control of body size and organ growth. Therefore, the mechanisms of precise control of growth are essential for ensuring the growth of organisms at a correct body size and proper organ proportions during development. The control of the growth rate and the duration of growth (or the cessation of growth) are required in size control. The insulin signaling pathway and the elements involved are essential in the control of growth. On the other hand, the ecdysteroid molting hormone determines the duration of growth. The secretion of these hormones is controlled by environmental factors such as nutrition. Moreover, the target of rapamycin (TOR) pathway is considered as a nutrient sensing pathway. Important cross-talks have been shown to exist among these pathways. In this review, we outline the control of body and organ growth by the insulin/TOR signaling pathway, and also the interaction between nutrition via insulin/TOR signaling and ecdysteroids at the coordination of organismal development and organ growth in insects, mainly focusing on the well-studied fruit fly Drosophila melanogaster.

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas.

BACKGROUND: Ecdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis. RESULTS: Transcriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult. In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis. CONCLUSIONS: The data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.

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.

Functional male accessory glands and fertility in Drosophila require novel ecdysone receptor.

In many insects, the accessory gland, a secretory tissue of the male reproductive system, is essential for male fertility. Male accessory gland is the major source of proteinaceous secretions, collectively called as seminal proteins (or accessory gland proteins), which upon transfer, manipulate the physiology and behavior of mated females. Insect hormones such as ecdysteroids and juvenoids play a key role in accessory gland development and protein synthesis but little is known about underlying molecular players and their mechanism of action. Therefore, in the present study, we examined the roles of hormone-dependent transcription factors (Nuclear Receptors), in accessory gland development, function and male fertility of a genetically tractable insect model, Drosophila melanogaster. First, we carried out an RNAi screen involving 19 hormone receptors, individually and specifically, in a male reproductive tissue (accessory gland) for their requirement in Drosophila male fertility. Subsequently, by using independent RNAi/ dominant negative forms, we show that Ecdysone Receptor (EcR) is essential for male fertility due to its requirement in the normal development of accessory glands in Drosophila: EcR depleted glands fail to make seminal proteins and have dying cells. Further, our data point to a novel ecdysone receptor that does not include Ultraspiracle but is probably comprised of EcR isoforms in Drosophila male accessory glands. Our data suggest that this novel ecdysone receptor might act downstream of homeodomain transcription factor paired (prd) in the male accessory gland. Overall, the study suggests novel ecdysone receptor as an important player in the hormonal regulation of seminal protein production and insect male fertility.

Identification and expression patterns of Halloween genes encoding cytochrome P450s involved in ecdysteroid biosynthesis in the cotton bollworm Helicoverpa armigera.

20-Hydroxyecdysone (20E) is a key hormone which regulates growth, development and reproduction in insects. Although cytochrome P450 enzymes (P450s) participating in the ecdysteroid biosynthesis of 20E have been characterized in a few model insects, no work has been published on the molecular entity of their orthologs in the cotton bollworm Helicoverpa armigera, a major pest insect in agriculture worldwide. In this study, four cytochrome P450 homologs, namely HarmCYP302A1, HarmCYP306A1, HarmCYP314A1 and HarmCYP315A1 from H. armigera, were identified and evolutional conservation of these Halloween genes were revealed among lepidopteran. Expression analyses showed that HarmCYP302A1 and HarmCYP315A1 were predominantly expressed in larval prothoracic glands, whereas this predominance was not always observed for HarmCYP306A1 and CYP314A1. The expression patterns of Halloween genes indicate that the fat bodies may play an important role in the conversion of ecdysone into 20E in larval-larval molt and in larval-pupal metamorphosis, and raise the possibility that HarmCYP315A1 plays a role in tissue-specific regulation in the steroid biosynthesis in H. armigera. These findings represent the first identification and expression characterization of four steriodogenic P450 genes and provide the groundwork for future functional and evolutionary study of steroid biosynthesis in this agriculturally important pest.

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs ß bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs ß and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs ß and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.