Ecdysteroid Secretion Research Articles

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.

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.

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-β-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.

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.

AKH Signaling in D. melanogaster Alters Larval Development in a Nutrient-Dependent Manner That Influences Adult Metabolism.

Metabolism, growth, and development are intrinsically linked, and their coordination is dependent upon inter-organ communication mediated by anabolic, catabolic, and steroid hormones. In Drosophila melanogaster, the corpora cardiaca (CC) influences metabolic homeostasis through adipokinetic hormone (AKH) signaling. AKH has glucagon-like properties and is evolutionarily conserved in mammals as the gonadotropin-releasing hormone, but its role in insect development is unknown. Here we report that AKH signaling alters larval development in a nutrient stress-dependent manner. This activity is regulated by the locus dg2, which encodes a cGMP-dependent protein kinase (PKG). CC-specific downregulation of dg2 expression delayed the developmental transition from larval to pupal life, and altered adult metabolism and behavior. These developmental effects were AKH-dependent, and were observed only in flies that experienced low nutrient stress during larval development. Calcium-mediated vesicle exocytosis regulates ecdysteroid secretion from the prothoracic gland (PG), and we found that AKH signaling increased cytosolic free calcium levels in the PG. We identified a novel pathway through which PKG acts in the CC to communicate metabolic information to the PG via AKH signaling. AKH signaling provides a means whereby larval nutrient stress can alter developmental trajectories into adulthood.

De novo transcriptome assembly and functional annotation for Y-organs of the blue crab (Callinectes sapidus), and analysis of differentially expressed genes during pre-molt

Blue crabs (Callinectes sapidus) undergo incremental growth involving the shedding (molting) of the old exoskeleton, and subsequent expansion and re-calcification of the newly synthesized one. The cellular events that lead to molting are triggered by steroid hormones termed ecdysteroids released from Y-organs, paired endocrine glands located in the anterior cephalothorax. The regulatory pathways leading to increased synthesis and release of ecdysteroids are not fully understood, and no transcriptome has yet been published for blue crab Y-organs. Here we report de novo transcriptome assembly and annotation for adult blue crab Y-organs, and differential gene expression (DGE) analysis between Y-organs of intermolt and premolt crabs. After trimming and quality assessment, a total of 91,819,458 reads from four cDNA libraries were assembled using Trinity to form the reference transcriptome. Trinity produced a total of 171,530 contigs coding for 150,388 predicted genes with an average contig length of 613 and an N50 of 940. Of these, TransDecoder predicted 31,661 open reading frames (ORFs), and 10,210 produced non-redundant blastx results through Trinotate annotation. Genes involved in multiple cell signaling pathways, including Ca2+ signaling, cGMP signaling, cAMP signaling, and mTOR signaling were present in the annotated reference transcriptome. DGE analysis showed in premolt Y-organs up-regulated genes involved in energy production, cholesterol metabolism, and exocytosis. The results provide insights into the transcriptome of blue crab Y-organs during a natural (rather than experimentally induced) molting cycle, and constitute a step forward in understanding the cellular mechanisms that underlie stage-specific changes in the synthesis and secretion of ecdysteroids by Y-organs.

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.

Role of protein phosphatase 2A in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

In the present study, the roles of a major serine/threonine protein phosphatase 2A (PP2A) in prothoracicotropic hormone (PTTH)-stimulated prothoracic glands (PGs) of Bombyx mori were evaluated. Immunoblotting analysis showed that Bombyx PGs contained a structural A subunit (A), a regulatory B subunit (B), and a catalytic C subunit (C), with each subunit undergoing development-specific changes. The protein levels of each subunit were not affected by PTTH treatment. However, the highly conserved tyrosine dephosphorylation of PP2A C subunit (PP2Ac), which appears to be related to activity, was increased by PTTH treatment in a time-dependent manner. We further demonstrated that phospholipase C (PLC), Ca2+, and reactive oxygen species (ROS) are upstream signaling for the PTTH-stimulated dephosphorylation of PP2Ac. The determination of PP2A enzymatic activity showed that PP2A enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Okadaic acid (OA), a specific PP2A inhibitor, prevented the PTTH-stimulated dephosphorylation of PP2Ac and reduced both basal and PTTH-stimulated PP2A enzymatic activity. The determination of ecdysteroid secretion showed that treatment with OA did not affect basal ecdysteroid secretion but did significantly inhibit PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PP2A activity is involved in ecdysteroidogenesis. Treatment with OA stimulated the basal phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) without affecting PTTH-stimulated ERK and 4E-BP phosphorylation. From these results, we hypothesize that PTTH-regulated PP2A signaling is a necessary component for the stimulation of ecdysteroidogenesis, potentially by mediating the link between ERK and TOR signaling pathways.

Proteomic analysis of the crustacean molting gland (Y-organ) over the course of the molt cycle

Molting in crustaceans is a highly complex physiological process involving regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone, which negatively regulates synthesis of molting hormones, ecdysteroids, by the YO. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle induced by multiple leg autotomy in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle. Protein abundance differed most notably between intermolt and the three premolt stages, indicative of a biological ‘on-off’ switch. Changes in hemolymph proteins were correlated with stage-specific processes of sclerotization and melanization that facilitate cuticle hardening and support immune reactions. An abundance of cytoskeletal proteins were identified, which corresponded with glandular hypertrophy associated with synthesis and secretion of ecdysteroids. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one‑carbon metabolism changed in abundance in response to increasing energy demands and the requirement for precursors of macromolecular synthesis. Several proteins involved in immune, proteostasis, and oxidative stress responses were correlated with the dynamic and demanding cellular changes associated with ecdysteroidogenesis. These changes in diverse physiological pathways represent the complexity involved with molecular regulation of the YO in decapod crustaceans.

Serotonin induces ecdysteroidogenesis and methyl farnesoate synthesis in the mud crab, Scylla serrata

In the current study, we have examined the role of serotonin in regulating the levels of methyl farnesoate and ecdysteroids in the giant mud crab Scylla serrata and validated that serotonin indeed is a reproductive hormone. Administration of serotonin elevated circulatory levels of methyl farnesoate and ecdysteroids in crabs. Since methyl farnesoate and ecdysteroid act through retinoid X receptor (RXR) and ecdysteroid receptor (EcR) respectively and these receptors are involved in the regulation of reproduction in crustaceans, we have determined the mRNA levels of RXR and EcR in hepatopancreas and ovary after serotonin administration. The expression levels of both RXR and EcR increased significantly in the hepatopancreas and ovary of serotonin injected crabs when compared to the controls. In vitro organ culture studies revealed that incubation of Y-orgas and mandibular organ explants in the presence of serotonin resulted in a significant increase in the secretion of ecdysteroids by Y-organs, but without alterations in MF synthesis in mandibular organs. From the above studies it is evident that serotonin stimulates Y organs resulting in increased ecdysteroidogenesis. Though the circulatory levels methyl farnesoate elevated after serotonin administration, organ culture studies revealed serotonin mediated methyl farnesaote synthesis is indirect probably by inhibiting release of mandibular organ inhibiting hormone from eyestalks.

Myosuppressin is involved in the regulation of pupal diapause in the cabbage army moth Mamestra brassicae

Diapause, a programmed developmental arrest, is common in insects, enabling them to survive adverse seasons. It is well established that pupal diapause is regulated by ecdysteroids secreted by the prothoracic glands (PGs), with cessation of ecdysteroid secretion after pupal ecdysis leading to pupal diapause. A major factor regulating the gland activity is prothoracicotropic hormone (PTTH) secreted from the brain. In our previous study, we demonstrated that the cessation of PTTH release after pupal ecdysis resulted in the inactivation of the PGs, leading to pupal diapause in the cabbage army moth Mamestra brassicae. Here we show that a neuropeptide myosuppressin also contributes to the inactivation of PGs at the initiation of diapause. Myosuppressin suppresses PTTH-stimulated activation of the PGs in vitro. Concentrations of myosuppressin in the hemolymph after pupal ecdysis are higher in diapause pupae than in nondiapause pupae.

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

Roles of mechanistic target of rapamycin and transforming growth factor-β signaling in the molting gland (Y-organ) of the blackback land crab, Gecarcinus lateralis

Molting in decapod crustaceans is controlled by molt-inhibiting hormone (MIH), an eyestalk neuropeptide that suppresses production of ecdysteroids by a pair of molting glands (Y-organs or YOs). Eyestalk ablation (ESA) activates the YOs, which hypertrophy and increase ecdysteroid secretion. At mid premolt, which occurs 7–14days post-ESA, the YO transitions to the committed state; hemolymph ecdysteroid titers increase further and the animal reaches ecdysis ~3weeks post-ESA. Two conserved signaling pathways, mechanistic target of rapamycin (mTOR) and transforming growth factor-β (TGF-β), are expressed in the Gecarcinus lateralis YO. Rapamycin, an mTOR antagonist, inhibits YO ecdysteroidogenesis in vitro. In this study, rapamycin lowered hemolymph ecdysteroid titer in ESA G. lateralis in vivo; levels were significantly lower than in control animals at all intervals (1–14days post-ESA). Injection of SB431542, an activin TGF-β receptor antagonist, lowered hemolymph ecdysteroid titers 7 and 14days post-ESA, but had no effect on ecdysteroid titers at 1 and 3days post-ESA. mRNA levels of mTOR signaling genes Gl-mTOR, Gl-Akt, and Gl-S6k were increased by 3days post-ESA; the increases in Gl-mTOR and Gl-Akt mRNA levels were blocked by SB431542. Gl-elongation factor 2 and Gl-Rheb mRNA levels were not affected by ESA, but SB431542 lowered mRNA levels at Days 3 and 7 post-ESA. The mRNA level of an activin TGF-β peptide, Gl-myostatin-like factor (Mstn), increased 5.5-fold from 0 to 3days post-ESA, followed by a 50-fold decrease from 3 to 7days post-ESA. These data suggest that (1) YO activation involves an up regulation of the mTOR signaling pathway; (2) mTOR is required for YO commitment; and (3) a Mstn-like factor mediates the transition of the YO from the activated to the committed state.

Endocrine Mechanisms Regulating Post-Diapause Development in the Cabbage Armyworm, Mamestra brassicae.

Diapause, a programmed developmental arrest at a specific stage, is common in insects and is regulated by hormones. It is well established that in pupal diapause, cessation of ecdysteroid secretion from the prothoracic glands (PGs) after pupal ecdysis leads to diapause initiation, while resumption of its secretion induces post-diapause development. However, what regulates the activity of the glands is poorly understood, especially for the glands of diapause-terminated pupae. In the present study, we investigate the mechanisms by which post-diapause development is regulated in the cabbage armyworm Mamestra brassicae. We demonstrate that the brain is necessary for the initiation of post-diapause development and that the factor in the brain responsible for the activation of the PGs is the prothoracicotropic hormone (PTTH). Further, through measuring the hemolymph PTTH titers by time-resolved fluoroimmunoassay, we show that PTTH is actually released into the hemolymph prior to the activation of the PGs. Although its peak titer is much lower than expected, this low concentration of PTTH is most likely still effective to activate the PGs of post-diapause pupae, because the responsiveness to PTTH of the glands at this stage is very high compared to that of nondiapause pupal PGs. These results strongly suggest that in M. brassicae, PTTH serves as a trigger to initiate pupa-adult development after diapause termination by stimulating the PGs to secrete ecdysteroid.

Direct effects of hypoxia and nitric oxide on ecdysone secretion by insect prothoracic glands

Insect molting and metamorphosis are controlled by the molt stimulating hormone ecdysone. A recent study suggests that reduced tissue oxygenation correlates with the size-sensing mechanism responsible for triggering molting. When reared in hypoxia, larvae of Manduca sexta and Drosophila melanogaster initiate molting at lower weights than do larvae reared in normoxia. Furthermore, in Drosophila, the signaling gas nitric oxide (NO) appears to be required for normal developmental timing. As observed in Drosophila, NO signaling targets the nuclear hormone receptor beta fushi tarazu transcription factor 1 (βFTZ-F1) through activation of Drosophila hormone receptor 3 (DHR3), two key regulators of ecdysone production and metamorphic tissue progression. We set out to directly examine the effects of hypoxia and NO on ecdysone secretion using prothoracic glands from feeding fifth (last) larval stage M. sexta. Our results indicate that in vitro treatment of prothoracic glands with hypoxia (2% oxygen) or the NO donor DETA-NONOate significantly inhibit ecdysone secretion. Protein markers of glandular activity were also in keeping with an initial inhibition, measured a decrease in phosphorylated ERK (extracellular signal regulated kinase) and an increase in non-phosphorylated 4EBP (eukaryotic initiation factor 4E binding protein). Additionally, gene expression levels of Manduca hormone receptor 3 (mhr3), βftz-f1, nitric oxide synthase (nos), and the PTTH receptor torso, were quantified using real-time PCR. NO treatment increased mhr3 expression and decreased nos expression. Hypoxia increased mhr3 transcription after 2hr, but decreased transcription after 12hr, with no effect on nos expression. Both NO and hypoxia had small effects on βftz-f1 expression, yet strongly increased torso transcription. Our results demonstrate that, in isolated prothoracic glands, hypoxia and NO signaling directly inhibit ecdysteroid secretion, but at the same time alter aspects of prothoracic gland function that may enhance secretory response.

Positive feedback regulation of prothoracicotropic hormone secretion by ecdysteroid – A mechanism that determines the timing of metamorphosis

When insect larvae have fully grown, prothoracicotropic hormone (PTTH) is released from the brain, triggering the initiation of metamorphic development through stimulation of ecdysteroid secretion by the prothoracic glands. The present study analyzes the mechanism that regulates the occurrence of this PTTH surge. In the silkworm Bombyx mori, the PTTH surge occurs on day 6 of the fifth instar and is preceded by a small rise in hemolymph ecdysteroid titer, which occurs late on day 5. We therefore hypothesized that this rise of ecdysteroid titer is involved in the induction of the PTTH surge. To test this hypothesis, two experiments were conducted. First, a small amount of 20-hydroxyecdysone was injected on day 4, two days before the expected day of the PTTH surge, to simulate the small rise in hemolymph ecdysteroid titer on day 5. This injection led to a precocious surge of PTTH the next day. Next, the hemolymph ecdysteroid titer on day 5 was artificially lowered by injecting ecdysteroid-22-oxidase, which inactivates 20-hydroxyecdysone. After this treatment, the PTTH surge did not occur on day 6 in 80% of the animals. These results indicate that a small rise of the hemolymph ecdysteroid titer plays a critical role in the induction of the PTTH surge. Since basal ecdysteroidogenic activity of the prothoracic glands increases with larval growth, a circulating level of ecdysteroids may convey information about larval maturity to the brain, to coordinate larval growth and metamorphosis. This is the first report in invertebrates to demonstrate positive feedback regulation of the surge of a tropic hormone by a downstream steroid hormone.

Cadmium inhibits molting of the freshwater crab Sinopotamon henanense by reducing the hemolymph ecdysteroid content and the activities of chitinase and N-acetyl-β-glucosaminidase in the epidermis

Molting is an essential process during the growth of crustaceans, which is coordinated by ecdysteroids secreted by the Y-organ, molting inhibiting hormone secreted by the X-organ sinus-gland complex, as well as chitinase and N-acetyl-β-glucosaminidase synthesized by the epidermis. Cadmium is one of the toxic metals in the aquatic environment. However, the endocrine effects of cadmium on the molting of freshwater crabs and the underlying mechanisms are unknown. To investigate these, freshwater crabs (Sinopotamon henanense) were acutely exposed to 0, 7.25, 14.5 and 29mg/l Cd for 3, 4, 5days or in some experiments for 4days after eyestalk-ablation. The concentration of hemolymph ecdysone and the activities of the molting enzymes chitinase and NAG were measured. Histological changes in the epidermal tissues were documented. Our results showed that eyestalk ablation increased the ecdysteroid content as well as the activities of chitinase and NAG, which were inhibited by cadmium in a concentration-dependent manner; histological examinations demonstrated that eyestalk ablation produced storage particles in the epidermal tissues, which was also reduced by cadmium in a concentration-dependent manner. Our data suggest that cadmium disrupts endocrine function through inhibiting the secretion of ecdysteroids by the Y-organ and altering with the regulation of chitinase and NAG activity in the epidermis. This work provides new insights into the mechanisms underlying the molting inhibition effect of cadmium on the crabs.

Changes of seasonal morph development induced by surgical operations in pupae of the large map butterfly Araschnia burejana Bermer (Lepidoptera: Nymphalidae)

The nymphalid butterfly Araschnia burejana and the papilionid butterfly Papilio xuthus exhibit seasonal diphenism comprising spring-morphs that develop from diapause pupae and summer-morphs that develop from non-diapause pupae. The development of seasonal morphs in A. burejana is regulated by the timing of secretion of ecdysteroids for adult development, whereas that in P. xuthus is regulated by the secretion of summer-morph-producing hormone, which is present in the brains and is under control of the photoperiod. We investigated whether a cerebral factor derived from brains plays a significant role in the regulation of seasonal morph development in A. burejana using surgical operations. Pairs of chilled diapause pupae that had been chilled for more than 3 months at 4°C were joined surgically to each other and then developed into spring-morph or spring-like-morph adults. Chilled diapause pupae that were joined with 1-day-old non-diapause pupae developed into summer-morph or summer-like-morph adults. When the brains of non-diapause pupae were removed surgically 6-8 hr after pupation with and without injection of 20-hydroxyecdysone, a large portion of them developed into spring-morph or spring-like-morph adults, respectively. Furthermore, 90% of non-diapause pupae developed into spring-morph or spring-like-morph adults when the neck was ligated within 5 min after pupation. These results indicated that a cerebral factor showing summer-morph-producing hormone activity, which is secreted from the brain in the early pupal stage, in addition to 20-hydroxyecdysone for adult development, play a significant role in the determination of summer-morph development in non-diapause pupae of A. burejana.

Insulin signaling pathways in lepidopteran ecdysone secretion

Molting and metamorphosis are stimulated by the secretion of ecdysteroid hormones from the prothoracic glands. Insulin-like hormones have been found to enhance prothoracic gland activity, providing a mechanism to link molting to nutritional state. In silk moths (Bombyx mori), the prothoracic glands are directly stimulated by insulin and the insulin-like hormone bombyxin. Further, in Bombyx, the neuropeptide prothoracicotropic hormone (PTTH) appears to act at least in part through the insulin-signaling pathway. In the prothoracic glands of Manduca sexta, while insulin stimulates the phosphorylation of the insulin receptor and Akt, neither insulin nor bombyxin II stimulate ecdysone secretion. Involvement of the insulin-signaling pathway in Manduca prothoracic glands was explored using two inhibitors of phosphatidylinositol-3-kinase (PI3K), LY294002 and wortmannin. PI3K inhibitors block the phosphorylation of Akt and 4EBP but have no effect on ecdysone secretion, or on the phosphorylation of the MAPkinase, ERK. Inhibitors that block phosphorylation of ERK, including the MEK inhibitor U0126, and high doses of the RSK inhibitor SL0101, effectively inhibit ecdysone secretion. The results highlight differences between the two lepidopteran insects most commonly used to directly study ecdysteroid secretion. In Bombyx, the PTTH and insulin-signaling pathways intersect; both insulin and PTTH enhance the phosphorylation of Akt and stimulate ecdysteroid secretion, and inhibition of PI3K reduces ecdysteroid secretion. By contrast, in Manduca, the action of PTTH is distinct from insulin. The results highlight species differences in the roles of translational regulators such as 4EBP, and members of the MAPkinase pathway such as ERK and RSK, in the regulation of insect ecdysone secretion, and in the impact of nutritionally-sensitive hormones such as insulin in the control of ecdysone secretion and molting.

Mechanistic target of rapamycin (mTOR) signaling genes in decapod crustaceans: Cloning and tissue expression of mTOR, Akt, Rheb, and p70 S6 kinase in the green crab, Carcinus maenas, and blackback land crab, Gecarcinus lateralis

Mechanistic target of rapamycin (mTOR) controls global translation of mRNA into protein by phosphorylating p70 S6 kinase (S6K) and eIF4E-binding protein-1. Akt and Rheb, a GTP-binding protein, regulate mTOR protein kinase activity. Molting in crustaceans is regulated by ecdysteroids synthesized by a pair of molting glands, or Y-organs (YOs), located in the cephalothorax. During premolt, the YOs hypertrophy and increase production of ecdysteroids. Rapamycin (1μM) inhibited ecdysteroid secretion in Carcinus maenas and Gecarcinus lateralis YOs in vitro, indicating that ecdysteroidogenesis requires mTOR-dependent protein synthesis. The effects of molting on the expression of four key mTOR signaling genes (mTOR, Akt, Rheb, and S6K) in the YO was investigated. Partial cDNAs encoding green crab (C. maenas) mTOR (4031bp), Akt (855bp), and S6K (918bp) were obtained from expressed sequence tags. Identity/similarity of the deduced amino acid sequence of the C. maenas cDNAs to human orthologs were 72%/81% for Cm-mTOR, 58%/73% for Cm-Akt, and 77%/88% for Cm-S6K. mTOR, Akt, S6K, and elongation factor 2 (EF2) in C. maenas and blackback land crab (G. lateralis) were expressed in all tissues examined. The two species differed in the effects of molting on gene expression in the YO. In G. lateralis, Gl-mTOR, Gl-Akt, and Gl-EF2 mRNA levels were increased during premolt. By contrast, molting had no effect on the expression of Cm-mTOR, Cm-Akt, Cm-S6K, Cm-Rheb, and Cm-EF2. These data suggest that YO activation during premolt involves up regulation of mTOR signaling genes in G. lateralis, but is not required in C. maenas.