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.

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.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

Transcriptome-wide identification of development related genes and pathways in Tribolium castaneum.

The growth and development in Tribolium castaneum were poorly understood at the transcriptome level. Currently, we identified 15,756, 9941 and 10,080 differentially expressed transcripts between late eggs VS early larvae, late larvae VS early pupae, and late pupae VS early adults of T. castaneum by RNA-seq, which was confirmed by qRT-PCR analysis on nine genes expression. Functional enrichment analysis indicated that DNA replication, cell cycle and insect hormone biosynthesis significantly enriched differentially expressed genes. The transcription of DNA replication and cell cycle genes decreased after hatching but increased after pupation. The juvenile hormone (JH) and ecdysteroid biosynthesis genes decreased after hatching, and the JH degradation genes were stimulated after pupation and eclosion while the ecdysteroid degradation gene CYP18A1 decreased after pupation. Silencing CYP18A1 elevated the titer of ecdysteroids and caused developmental arrest at the late larval stage. This study promotes the understanding of insect growth and development.

Analyses of ecdysteroid transporters in the fat body of Tribolium castaneum.

The control of insect moulting and metamorphosis involves ecdysteroids that orchestrate the execution of developmental genetic programs by binding to dimeric hormone receptors consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). In insects, the main ecdysteroids comprise ecdysone (E), which is synthesized in the prothoracic gland and secreted into the haemolymph, and 20-hydroxyecdysone (20E), which is considered the active form by binding to the nuclear receptor of the target cell. While biosynthesis of ecdysteroids has been studied in detail in different insects, the transport systems involved in guiding these steroid hormones across cellular membranes have just recently begun to be studied. By analysing RNAi phenotypes in the red flour beetle, Tribolium castaneum, we have identified three transporter genes, TcABCG-8A, TcABCG-4D and TcOATP4-C1, whose silencing results in phenotypes similar to that observed when the ecdysone receptor gene TcEcRA is silenced, that is, abortive moulting and abnormal development of adult compound eyes during the larval stage. The genes of all three transporters are expressed at higher levels in the larval fat body of T. castaneum. We analysed potential functions of these transporters by combining RNAi and mass spectrometry. However, the analysis of gene functions is challenged by mutual RNAi effects indicating interdependent gene regulation. Based on our findings, we propose that TcABCG-8A, TcABCG-4D and TcOATP4-C1 participate in the ecdysteroid transport in fat body cells, which are involved in E → 20E conversion catalysed by the P450 enzyme TcShade.

An ecdysteroid-regulated 16-kDa protein homolog participates in the immune response of the crayfish Procambarus clarkii.

An ecdysteroid-regulated 16-kDa protein homolog (named Pc-E16), encoding 150 amino acid residues with a conserved MD-2-related lipid-recognition domain, was first identified in Procambarus clarkii. Phylogenetic analyses indicated similarity between Pc-E16 and 16-kDa proteins from Aplysia californica and insects. Recombinant Pc-E16 protein was successfully expressed in BL21 (DE3) Escherichia coli cells, and polyclonal antibodies against purified Pc-E16 proteins were prepared. In comparison with other tissues, Pc-E16 was highly expressed in the intestine; real-time PCR and Western blotting results indicated that Pc-E16 expression was significantly induced by lipopolysaccharides in hepatopancreas and hemocytes. Pc-E16-mediated signaling pathways were investigated by digital gene expression analysis following RNA interference targeting Pc-E16. A total of 6103 differentially expressed genes (DEGs) were identified, of which 3318 were up- and 2785 were downregulated. Many DEGs were involved in binding and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that DEGs were clustered into 225 pathways, and 15 significantly enriched pathways were identified at the immune system level. In addition, the expression level of Pc-E16 in hemocytes and hepatopancreas was obviously downregulated at 48 h after dsRNA injection, and Pc-E16-RNAi treatment affected the expression levels of immune-related genes. Altogether, our results suggest that Pc-E16 is involved in the innate immune response of P. clarkii.

The prothoracicotropic hormone (PTTH) of Rhodnius prolixus (Hemiptera) is noggin-like: Molecular characterisation, functional analysis and evolutionary implications.

Prothoracicotropic hormone (PTTH) is a central regulator of insect development that regulates the production of the steroid moulting hormones (ecdysteroids) from the prothoracic glands (PGs). Rhodnius PTTH was the first brain neurohormone discovered in any animal almost 100 years ago but has eluded identification and no homologue of Bombyx mori PTTH occurs in its genome. Here, we report Rhodnius PTTH is the first noggin-like PTTH found. It differs in important respects from known PTTHs and is the first PTTH from the Hemimetabola (Exopterygota) to be fully analysed. Recorded PTTHs are widespread in Holometabola but close to absent in hemimetabolous orders. We concluded Rhodnius PTTH likely differed substantially from the known ones. We identified one Rhodnius gene that coded a noggin-like protein (as defined by Molina et al., 2009) that had extensive similarities with known PTTHs but also had two additional cysteines. Sequence and structural analysis showed known PTTHs are closely related to noggin-like proteins, as both possess a growth factor cystine knot preceded by a potential cleavage site. The gene is significantly expressed only in the brain, in a few cells of the dorsal protocerebrum. We vector-expressed the sequence from the potential cleavage site to the C-terminus. This protein was strongly steroidogenic on PGs in vitro. An antiserum to the protein removed the steroidogenic protein released by the brain. RNAi performed on brains in vitro showed profound suppression of transcription of the gene and of production and release of PTTH and thus of ecdysteroid production by PGs. In vivo, the gene is expressed throughout development, in close synchrony with PTTH release, ecdysteroid production by PGs and the ecdysteroid titre. The Rhodnius PTTH monomer is 17kDa and immunoreactive to anti-PTTH of Bombyx mori (a holometabolan). Bombyx PTTH also mildly stimulated Rhodnius PGs. The two additional cysteines form a disulfide at the tip of finger 2, causing a loop of residues to protrude from the finger. A PTTH variant without this loop failed to stimulate PGs, showing the loop is essential for PTTH activity. It is considered that PTTHs of Holometabola evolved from a noggin-like protein in the ancestor of Holometabola and Hemiptera, c.400ma, explaining the absence of holometabolous-type PTTHs from hemimetabolous orders and the differences of Rhodnius PTTH from them. Noggin-like proteins studied from Hemiptera to Arachnida were homologous with Rhodnius PTTH and may be common as PTTHs or other hormones in lower insects.

Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.

Effects of molting on the expression of ecdysteroid biosynthesis genes in the Y-organ of the blackback land crab, Gecarcinus lateralis.

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.

The MicroRNA Ame-Bantam-3p Controls Larval Pupal Development by Targeting the Multiple Epidermal Growth Factor-like Domains 8 Gene (megf8) in the Honeybee, Apis mellifera.

20-Hydroxyecdysone (20E) plays an essential role in coordinating developmental transitions in insects through responsive protein-coding genes and microRNAs (miRNAs). However, the interplay between 20E and miRNAs during insect metamorphosis is unknown. In this study, using small RNA sequencing, a comparative miRNA transcriptomic analysis in different development stages, and 20E treatment, we identified ame-bantam-3p as a key candidate miRNA involved in honeybee metamorphosis. Target prediction and in vitro dual-luciferase assays confirmed that ame-bantam-3p interacts with the coding region of the megf8 gene and promotes its expression. Meanwhile, temporal expression analysis revealed that the expression of ame-bantam-3p is higher in the larval stage than in prepupal and pupal stages, and that this expression pattern is similar to that of megf8. In vivo, we found that the mRNA level of megf8 was significantly increased after the injection of ame-bantam-3p agomir. A 20E feeding assay showed that 20E downregulated the expression of both ame-bantam-3p and its target gene megf8 on larval days five, six, and seven. Meanwhile, the injection of ame-bantam-3p agomir also reduced the 20E titer, as well as the transcript levels of essential ecdysteroid synthesis genes, including Dib, Phm, Sad, and Nvd. The transcript levels of 20E cascade genes, including EcRA, ECRB1, USP, E75, E93, and Br-c, were also significantly decreased after ame-bantam-3p agomir injection. However, ame-bantam-3p antagomir injection and dsmegf8 injection showed the opposite effect to ame-bantam-3p agomir injection. Ame-bantam-3p agomir treatment ultimately led to mortality and the failure of larval pupation by inhibiting ecdysteroid synthesis and the 20E signaling pathway. However, the expression of 20E signaling-related genes was significantly increased after megf8 knockdown, and larvae injected with dsmegf8 showed early pupation. Combined, our results indicate that ame-bantam-3p is involved in the 20E signaling pathway through positively regulating its target gene megf8 and is indispensable for larval-pupal development in the honeybee. These findings may enhance our understanding of the relationship between 20E signaling and small RNAs during honeybee development.

Control of the ecdysteroid level plays a crucial role in density-dependent metamorphosis in the giant mealworm beetle Zophobas atratus.

Metamorphic transition in some tenebrionid beetles is dependent on population density. This phenomenon is useful for pupae that are vulnerable to cannibalism. The physiological mechanism of this adaptive developmental phenomenon remains unclear. In Zophobas atratus, which show density-dependent metamorphosis, larval isolation can induce metamorphosis. We herein demonstrated that the return of isolated larvae to a crowded condition (re-crowding) inhibited their metamorphosis. The timing of metamorphic initiation was slightly extended according to the duration of re-crowding experienced by the isolated larvae. Therefore, the re-crowding induced physiological changes needed for metamorphic inhibition. We investigated whether hormone-related genes involved in signaling of metamorphic inhibitor (juvenile hormone, JH) and molting hormone (ecdysteroid) responded to the re-crowding. An expression analysis showed that gene expression of ecdysteroid signaling was maintained at low levels under the re-crowded condition. Actually, ecdysteroid levels decreased responding to re-crowding. Ecdysteroid injections induced metamorphosis in re-crowded larvae. In contrast, the JH signaling gene showed little fluctuation in both isolated and re-crowded conditions, and knockdown of JH signaling factors did not affect inhibition of metamorphosis under the re-crowded condition. The present study suggests that regulation of ecdysteroid level rather than JH is more crucial in the density dependent metamorphosis in Z. atratus.

Protein kinase C signalling involved in prothoracicotropic hormone-stimulated prothoracic glands in the silkworm, Bombyx mori.

In the present study, the participation of protein kinase C (PKC) signalling in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx prothoracic glands (PGs) is demonstrated and characterized. PTTH stimulated phosphorylation of a 37-kDa protein in Bombyx PGs both in vitro and in vivo, as recognized by a PKC substrate antibody. Treatment with either A23187 or thapsigargin also stimulated this 37-kDa protein phosphorylation. PTTH-stimulated phosphorylation of the 37-kDa protein was markedly attenuated in the absence of Ca2+ . The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated phosphorylation of this protein, indicating the involvement of Ca2+ and PLC. A mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) and a chemical activator of adenosine 5'-monophosphate-activated protein kinase (AMPK) (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside) did not affect PTTH-stimulated phosphorylation of the 37-kDa protein, implying that ERK and PI3K/AMPK are not the upstream signalling pathways for PKC-dependent protein phosphorylation. The mitochondrial oxidative phosphorylation inhibitors (the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone and diphenylene iodonium) inhibited PTTH-stimulated phosphorylation of the 37-kDa protein, indicating its redox regulation. Treatment with PKC inhibitors (either calphostin C, chelerythrine C or rottlerin) reduced PTTH-stimulated phosphorylation of the 37-kDa protein. PTTH-stimulated ecdysteroidogenesis was also inhibited by treatment with rottlerin, thus further confirming participation of PKC-dependent phosphorylation in PTTH signalling. From these results, we demonstrated that redox-regulated PTTH-stimulated PKC signalling is involved in ecdysteroid secretion in Bombyx PGs.

The role of ecdysteroid in the regulation of ovarian growth and oocyte maturation in Rhodnius prolixus, a vector of Chagas disease.

Rhodnius prolixus is a blood-gorging insect that is medically important since it transmits Chagas disease via feces and urine that contain the parasite Trypanosoma cruzi. In adult females, ecdysteroid hormone (20-hydroxyecdysone, 20E) is involved in the growth of the ovary and development of eggs post-blood meal (PBM). Halloween genes are essential for ecdysteroid synthesis since they code for cytochrome P450 enzymes in the ecdysteroidogenic pathway. The ecdysone receptor (EcR/USP) binds 20E, resulting in activation of ecdysone-responsive genes. We have identified and characterized the Halloween genes, and the non-Halloween gene, neverland, in the R. prolixus ovary using transcriptomic data. We used BLAST to compare transcriptome sequences with other arthropod sequences to identify similar transcripts. Our results indicate that the Halloween genes, neverland and ecdysone receptor transcripts are present in the ovaries of R. prolixus. We have quantified, by qPCR, Halloween gene transcript expression in the ovary following a blood meal. Most of the Halloween genes are upregulated during the first 3 days PBM. Knockdown of EcR, USP and shade transcripts, using RNA interference, results in a significant reduction in the number of eggs produced and a severe reduction in egg laying and hatching rate. Furthermore, knockdown of the EcR or shade transcripts altered the expression of the chorion gene transcripts Rp30 and Rp45 at day 3 and 6 PBM. These results indicate that ecdysteroids play critical roles in reproduction of female R. prolixus.

Effects of triazole plant growth regulators on molting mechanism in Chinese mitten crab (Eriocheir sinensis).

Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-ß-d-aminoglucosidase (ß-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis.

Regulation of juvenile hormone and ecdysteroid analogues on the development of the predatory spider, Pardosa pseudoannulata, and its regulatory mechanisms.

Insecticides harm the beneficial organisms, such as predatory spiders, through direct killing or regulation of the development and reproduction. In this study, the bioassay showed that the treatment of juvenile hormone (JH) analogue fenoxycarb delayed the moulting of Pardosa pseudoannulata, a dominant predatory spider in paddy fields. In order to figure out the regulatory mechanism of fenoxycarb on the spider development, we systematically analyzed JH biosynthesis in P. pseudoannulata. All genes involved in JH biosynthesis pathway were retrieved from the genome of P. pseudoannulata, except for CYP15A1. The absence of CYP15A1 was in agreement with the identification of methyl farnesoate (MF) rather than JH III in the spider. The delayed moulting and decreased expression of JH biosynthesis-related genes in the MF-applied spiderlings supported that MF was an active JH. Fenoxycarb treatment significantly upregulated the transcriptional level of JH biosynthesis-related genes and consequently delayed the spiderling moulting. In the spider development, ecdysteroid played the opposite role, in contrast to MF, to accelerate the development, as our previous study. Here we found that the treatment of ecdysteroid analogue tebufenozide accelerated P. pseudoannulata spiderling moulting, which resulted from the expressional suppression of ecdysteroid biosynthesis-related genes. In total, the JH and ecdysteroid analogues affected the development of P. pseudoannulata by the expressional regulation of biosynthesis-related genes, which would be helpful for the evaluation of hormone analogue insecticides in environmental safety, and useful for the protection and application of P. pseudoannulate and related spider species.

Crosstalk between Nutrition, Insulin, Juvenile Hormone, and Ecdysteroid Signaling in the Classical Insect Model, Rhodnius prolixus.

The rigorous balance of endocrine signals that control insect reproductive physiology is crucial for the success of egg production. Rhodnius prolixus, a blood-feeding insect and main vector of Chagas disease, has been used over the last century as a model to unravel aspects of insect metabolism and physiology. Our recent work has shown that nutrition, insulin signaling, and two main types of insect lipophilic hormones, juvenile hormone (JH) and ecdysteroids, are essential for successful reproduction in R. prolixus; however, the interplay behind these endocrine signals has not been established. We used a combination of hormone treatments, gene expression analyses, hormone measurements, and ex vivo experiments using the corpus allatum or the ovary, to investigate how the interaction of these endocrine signals might define the hormone environment for egg production. The results show that after a blood meal, circulating JH levels increase, a process mainly driven through insulin and allatoregulatory neuropeptides. In turn, JH feeds back to provide some control over its own biosynthesis by regulating the expression of critical biosynthetic enzymes in the corpus allatum. Interestingly, insulin also stimulates the synthesis and release of ecdysteroids from the ovary. This study highlights the complex network of endocrine signals that, together, coordinate a successful reproductive cycle.

Knockdown of the Halloween Genes spook, shadow and shade Influences Oocyte Development, Egg Shape, Oviposition and Hatching in the Desert Locust.

Ecdysteroids are widely investigated for their role during the molting cascade in insects; however, they are also involved in the development of the female reproductive system. Ecdysteroids are synthesized from cholesterol, which is further converted via a series of enzymatic steps into the main molting hormone, 20-hydoxyecdysone. Most of these biosynthetic conversion steps involve the activity of cytochrome P450 (CYP) hydroxylases, which are encoded by the Halloween genes. Three of these genes, spook (spo), phantom (phm) and shade (shd), were previously characterized in the desert locust, Schistocerca gregaria. Based on recent sequencing data, we have now identified the sequences of disembodied (dib) and shadow (sad), for which we also analyzed spatiotemporal expression profiles using qRT-PCR. Furthermore, we investigated the possible role(s) of five different Halloween genes in the oogenesis process by means of RNA interference mediated knockdown experiments. Our results showed that depleting the expression of SchgrSpo, SchgrSad and SchgrShd had a significant impact on oocyte development, oviposition and hatching of the eggs. Moreover, the shape of the growing oocytes, as well as the deposited eggs, was very drastically altered by the experimental treatments. Consequently, it can be proposed that these three enzymes play an important role in oogenesis.

Involvement of RSK phosphorylation in PTTH-stimulated ecdysone secretion in prothoracic glands of the silkworm, Bombyx mori.

It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis.

Midgut-derived neuropeptide F controls germline stem cell proliferation in a mating-dependent manner.

Stem cell maintenance is established by neighboring niche cells that promote stem cell self-renewal. However, it is poorly understood how stem cell activity is regulated by systemic, tissue-extrinsic signals in response to environmental cues and changes in physiological status. Here, we show that neuropeptide F (NPF) signaling plays an important role in the pathway regulating mating-induced germline stem cell (GSC) proliferation in the fruit fly Drosophila melanogaster. NPF expressed in enteroendocrine cells (EECs) of the midgut is released in response to the seminal-fluid protein sex peptide (SP) upon mating. This midgut-derived NPF controls mating-induced GSC proliferation via ovarian NPF receptor (NPFR) activity, which modulates bone morphogenetic protein (BMP) signaling levels in GSCs. Our study provides a molecular mechanism that describes how a gut-derived systemic factor couples stem cell behavior to physiological status, such as mating, through interorgan communication.

Induction of diapause by clock proteins period and timeless via changes in PTTH and ecdysteroid titer in the sugar beet moth, Scrobipalpa ocellatella (Lepidoptera: Gelechiidae).

The sugar beet moth, Scrobipalpa ocellatella (Boyd), one of the most severe sugar beet pests, causes quantitative and qualitative yield losses late in the autumn. Previously, it was shown that low temperature and short-day photoperiod together cause diapause induction in pupae. Here, the interaction of the critical elements of the diapause induction, including the period (PER), timeless (TIM), prothoracicotropic hormone (PTTH), and ecdysteroid titer, were investigated. Immunohistochemistry results showed that the number of period immunoreactivity (PER-ir) and TIM-ir cells in nondiapause pupae (NDP) was lower than in the brain of the diapause pupae (DP). Moreover, the number of PER-ir and TIM-ir cells in the protocerebrum and optic lobe (OL) of NDP was lower than DP. Moreover, lower PTTH content in the brain and hemolymph of DP was confirmed by competitive enzyme-linked immunosorbent assay. Enzyme immunoassay showed a lower 20-hydroxyecdysone (20E) titer in the hemolymph of the DP compared with the NDP. Within a short-day condition, PER and TIM titers increased in the brain leading to decreasing PTTH titers in the brain and hemolymph that caused decreasing 20E titer in the hemolymph, leading to the induction of diapause. This study suggests that PER and TIM could be one of the brain factors that play an essential role in regulating diapause in S. ocellatella.