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.

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.

Characterization of Shed genes encoding ecdysone 20-monooxygenase (CYP314A1) in the Y-organ of the blackback land crab, Gecarcinus lateralis.

Molting in decapod crustaceans is controlled by ecdysteroid hormones synthesized and secreted by the molting gland, or Y-organ (YO). Halloween genes encode cytochrome P450 enzymes in the ecdysteroid synthetic pathway. The current paradigm is that YOs secrete an inactive precursor (e.g., ecdysone or E), which is hydroxylated at the #20 carbon to form an active hormone (20-hydroxyecdysone or 20E) by a mitochonrial 20-monooxygenase (CYP314A1) in peripheral tissues. 20-Monooxygenase is encoded by Shed in decapods and Shade in insects. We used eastern spiny lobster Shed sequences to extract six orthologs in the G. lateralis YO transcriptome. Phylogenetic analysis of the deduced amino acid sequences from six decapod species organized the Sheds into seven classes (Sheds 1-7), resulting in the assignment of the G. lateralis Sheds to Gl-Shed1, 2, 4A, 4B, 5A, and 5B. The mRNA levels of the six Gl-Sheds in the YO of intermolt animals were comparable to those in nine other tissues that included hepatopancreas and muscle. qPCR was used to compare the effects of molt induction by multiple leg autotomy (MLA) and eyestalk ablation (ESA) on Gl-Shed mRNA levels in the YO. Molt stage had little effect on Gl-Shed1 and Gl-Shed5B expression in the YO of MLA animals. Gl-Shed5A was expressed at the highest mRNA levels in the YO and was significantly increased during early and mid premolt stages. By contrast, ESA ± SB431542 had no effect on Gl-Shed expression at 1, 3, 5, and 7 days post-ESA. SB431542, which inhibits Transforming Growth Factor-ß/activin signaling and blocks YO commitment, decreased Gl-Shed2 and Gl-Shed4A mRNA levels at 14 days post-ESA. A targeted metabolomic analysis showed that YOs cultured in vitro secreted E and 20E to the medium. These data suggest that the YO expresses 20-monooygenases that can convert E to 20E, which may contribute to the increase in active hormone in the hemolymph during premolt.

Hormonal control of the crustacean molting gland: Insights from transcriptomics and proteomics.

Endocrine control of molting in decapod crustaceans involves the eyestalk neurosecretory center (X-organ/sinus gland complex), regenerating limbs, and a pair of Y-organs (YOs), as molting is induced by eyestalk ablation or multiple leg autotomy and suspended in early premolt by limb bud autotomy. Molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), produced in the X-organ/sinus gland complex, inhibit the YO. The YO transitions through four physiological states over the molt cycle: basal in intermolt; activated in early premolt; committed in mid- and late premolt; and repressed in postmolt. We assembled the first comprehensive YO transcriptome over the molt cycle in the land crab, Gecarcinus lateralis, showing that as many as 23 signaling pathways may interact in controlling ecdysteroidogenesis. A proposed model of the MIH/cyclic nucleotide pathway, which maintains the basal YO, consists of cAMP/Ca2+ triggering and nitric oxide (NO)/cGMP summation phases. Mechanistic target of rapamycin (mTOR) signaling is required for YO activation in early premolt and affects the mRNA levels of thousands of genes. Transforming Growth Factor-ß (TGFß)/Activin signaling is required for YO commitment in mid-premolt and high ecdysteroid titers at the end of premolt may trigger YO repression. The G. lateralis YO expresses 99 G protein-coupled receptors, three of which are putative receptors for MIH/CHH. Proteomic analysis shows the importance of radical oxygen species scavenging, cytoskeleton, vesicular secretion, immune response, and protein homeostasis and turnover proteins associated with YO function over the molt cycle. In addition to eyestalk ganglia, MIH mRNA and protein are present in brain, optic nerve, ventral nerve cord, and thoracic ganglion, suggesting that they are secondary sources of MIH. Down-regulation of mTOR signaling genes, in particular Ras homolog enriched in brain or Rheb, compensates for the effects of elevated temperature in the YO, heart, and eyestalk ganglia in juvenile Metacarcinus magister. Rheb expression increases in the activated and committed YO. These data suggest that mTOR plays a central role in mediating molt regulation by physiological and environmental factors.

Ecdysis triggering hormone modulates molt behaviour in the redclaw crayfish Cherax quadricarinatus, providing a mechanistic evidence for conserved function in molt regulation across Pancrustacea.

Molting enables growth and development across ecdysozoa. The molting process is strictly controlled by hormones - ecdysteroids. Ecdysteroidogenesis occurs in theprothoracic glands and stimulated by prothoracicotropic hormone in insects, while it ensues in the Y-organ and regulated by the molt inhibiting hormone in crustaceans. A peak in ecdysteroids in the hemolymph induces a cascade of multiple neuropeptides including Ecdysis Triggering Hormone (ETH) and Corazonin. The role of ETH is well defined in controlling the molt process in insects, but it is yet to be defined in crustaceans. In this study, we investigated the behavioral response of intermolt crayfish to ETH and Corazonin injections as well as the impact of ETH on the molt period using in vivo assays. Injection of Corazonin and ETH resulted in a clear and immediate eye twitching response to these two neuropeptides. The Corazonin injection induced eye twitching in slow and asynchronous manner, while ETH injection caused eye twitching in a relatively fast and synchronous way. A single injection of ETH to crayfish resulted in a remarkable prolong molt period, at twice the normal molting cycle, suggesting that ETH plays a key role in controlling the molt cycle in decapod crustaceans. Given the key significance of ETH in molt regulation and its plausible application in pest control, we characterized ETH across the pancrustacean orders. Bioinformatic analysis shows the mature ETH sequence is identical in all studied decapod species. ETH can be classified into specific groups based on the associated motif in each insect order and shows an insect motif -KxxPRx to be conserved in crustaceans.

Characterization of G-protein coupled receptors from the blackback land crab Gecarcinus lateralis Y organ transcriptome over the molt cycle.

BACKGROUND: G-protein coupled receptors (GPCRs) are ancient, ubiquitous, constitute the largest family of transducing cell surface proteins, and are integral to cell communication via an array of ligands/neuropeptides. Molt inhibiting hormone (MIH) is a key neuropeptide that controls growth and reproduction in crustaceans by regulating the molt cycle. It inhibits ecdysone biosynthesis by a pair of endocrine glands (Y-organs; YOs) through binding a yet uncharacterized GPCR, which triggers a signalling cascade, leading to inhibition of the ecdysis sequence. When MIH release stops, ecdysone is synthesized and released to the hemolymph. A peak in ecdysone titer is followed by a molting event. A transcriptome of the blackback land crab Gecarcinus lateralis YOs across molt was utilized in this study to curate the list of GPCRs and their expression in order to better assess which GPCRs are involved in the molt process. RESULTS: Ninety-nine G. lateralis putative GPCRs were obtained by screening the YO transcriptome against the Pfam database. Phylogenetic analysis classified 49 as class A (Rhodopsin-like receptor), 35 as class B (Secretin receptor), and 9 as class C (metabotropic glutamate). Further phylogenetic analysis of class A GPCRs identified neuropeptide GPCRs, including those for Allatostatin A, Allatostatin B, Bursicon, CCHamide, FMRFamide, Proctolin, Corazonin, Relaxin, and the biogenic amine Serotonin. Three GPCRs clustered with recently identified putative CHH receptors (CHHRs), and differential expression over the molt cycle suggests that they are associated with ecdysteroidogenesis regulation. Two putative Corazonin receptors showed much higher expression in the YOs compared with all other GPCRs, suggesting an important role in molt regulation. CONCLUSIONS: Molting requires an orchestrated regulation of YO ecdysteroid synthesis by multiple neuropeptides. In this study, we curated a comprehensive list of GPCRs expressed in the YO and followed their expression across the molt cycle. Three putative CHH receptors were identified and could include an MIH receptor whose activation negatively regulates molting. Orthologs of receptors that were found to be involved in molt regulation in insects were also identified, including LGR3 and Corazonin receptor, the latter of which was expressed at much higher level than all other receptors, suggesting a key role in YO regulation.

Effects of temperature on survival, moulting, and expression of neuropeptide and mTOR signalling genes in juvenile Dungeness crab (Metacarcinus magister).

Mechanistic target of rapamymcin (mTOR) is a highly conserved protein kinase that controls cellular protein synthesis and energy homeostasis. We hypothesize that mTOR integrates intrinsic signals (moulting hormones) and extrinsic signals (thermal stress) to regulate moulting and growth in decapod crustaceans. The effects of temperature on survival, moulting and mRNA levels of mTOR signalling genes (Mm-Rheb, Mm-mTOR, Mm-AMPKα, Mm-S6K and Mm-AKT) and neuropeptides (Mm-CHH and Mm-MIH) were quantified in juvenile Metacarcinus magister Crabs at different moult stages (12, 19 or 26 days postmoult) were transferred from ambient temperature (∼15°C) to temperatures between 5 and 30°C for up to 14 days. Survival was 97-100% from 5 to 20°C, but none survived at 25 or 30°C. Moult stage progression accelerated from 5 to 15°C, but did not accelerate further at 20°C. In eyestalk ganglia, Mm-Rheb, Mm-AMPKα and Mm-AKT mRNA levels decreased with increasing temperatures. Mm-MIH and Mm-CHH mRNA levels were lowest in the eyestalk ganglia of mid-premoult animals at 20°C. In the Y-organ, Mm-Rheb mRNA levels decreased with increasing temperature and increased during premoult, and were positively correlated with haemolymph ecdysteroid titre. In the heart, moult stage had no effect on mTOR signalling gene mRNA levels; only Mm-Rheb, Mm-S6K and Mm-mTOR mRNA levels were higher in intermoult animals at 10°C. These data suggest that temperature compensation of neuropeptide and mTOR signalling gene expression in the eyestalk ganglia and Y-organ contributes to regulate moulting in the 10 to 20°C range. The limited warm compensation in the heart may contribute to mortality at temperatures above 20°C.

Localization and expression of molt-inhibiting hormone and nitric oxide synthase in the central nervous system of the green shore crab, Carcinus maenas, and the blackback land crab, Gecarcinus lateralis.

In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca2+-dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~105-fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iß) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca2+-dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt.

Elevated expression of neuropeptide signaling genes in the eyestalk ganglia and Y-organ of Gecarcinus lateralis individuals that are refractory to molt induction.

Molting is induced in decapod crustaceans via multiple leg autotomy (MLA) or eyestalk ablation (ESA). MLA removes five or more walking legs, which are regenerated and become functional appendages at ecdysis. ESA eliminates the primary source of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), which suppress the production of molting hormones (ecdysteroids) from the molting gland or Y-organ (YO). Both MLA and ESA are effective methods for molt induction in Gecarcinus lateralis. However, some G. lateralis individuals are refractory to MLA, as they fail to complete ecdysis by 12weeks post-MLA; these animals are in the "blocked" condition. Quantitative polymerase chain reaction was used to quantify mRNA levels of neuropeptide and mechanistic target of rapamycin (mTOR) signaling genes in YO, eyestalk ganglia (ESG), thoracic ganglion (TG), and brain of intact and blocked animals. Six of the seven neuropeptide signaling genes, three of four mTOR signaling genes, and Gl-elongation factor 2 (EF2) mRNA levels were significantly higher in the ESG of blocked animals. Gl-MIH and Gl-CHH mRNA levels were higher in the TG and brain of blocked animals and levels increased in both control and blocked animals in response to ESA. By contrast, mRNA levels of Gl-EF2 and five of the 10 MIH signaling pathway genes in the YO were two to four orders of magnitude higher in blocked animals compared to controls. These data suggest that increased MIH and CHH synthesis in the ESG contributes to the prevention of molt induction by MLA in blocked animals. The up-regulation of MIH signaling genes in the YO of blocked animals suggests that the YO is more sensitive to MIH produced in the ESG, as well as MIH produced in brain and TG of ESA animals. Both the up-regulation of MIH signaling genes in the YO and of Gl-MIH and Gl-CHH in the ESG, TG, and brain appear to contribute to some G. lateralis individuals being refractory to MLA and ESA.

Roles of mechanistic target of rapamycin and transforming growth factor-ß 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-ß (TGF-ß), 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-ß 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-ß 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.

Nitric oxide production and sequestration in the sinus gland of the green shore crab Carcinus maenas.

Molting in decapod crustaceans is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced in the X-organ (XO)/sinus gland (SG) complex of the eyestalk ganglia (ESG). Pulsatile release of MIH from the SG suppresses ecdysteroidogenesis by the molting gland or Y-organ (YO). The hypothesis is that nitric oxide (NO), a neuromodulator that controls neurotransmitter release at presynaptic membranes, depresses the frequency and/or amount of MIH pulses to induce molting. NO synthase (NOS) mRNA was present in Carcinus maenas ESG and other tissues and NOS protein was present in the SG. A copper based ligand (CuFL), which reacts with NO to form a highly fluorescent product (NO-FL), was used to image NO in the ESG and SG and quantify the effects of NO scavenger (cPTIO), NOS inhibitor (l-NAME), and sodium azide (NaN3) on NO production in the SG. Pre-incubation with cPTIO prior to CuFL loading decreased NO-FL fluorescence ~30%; including l-NAME had no additional effect. Incubating SG with l-NAME during pre-incubation and loading decreased NO-FL fluorescence ~40%, indicating that over half of the NO release was not directly dependent on NOS activity. Azide, which reacts with NO-binding metal groups in proteins, reduced NO-FL fluorescence to near background levels without extensive cell death. Spectral shift analysis showed that azide displaced NO from a soluble protein in SG extract. These data suggest that the SG contains NO-binding protein(s) that sequester NO and releases it over a prolonged period. This NO release may modulate neuropeptide secretion from the axon termini in the SG.

Calcium, calpain, and calcineurin in low-frequency depression of transmitter release.

Low-frequency depression (LFD) of transmitter release occurs at phasic synapses with stimulation at 0.2 Hz in both isolated crayfish (Procambarus clarkii) neuromuscular junction (NMJ) preparations and in intact animals. LFD is regulated by presynaptic activity of the Ca(2+)-dependent phosphatase calcineurin (Silverman-Gavrila and Charlton, 2009). Since the fast Ca(2+) chelator BAPTA-AM inhibits LFD but the slow chelator EGTA-AM does not, the Ca(2+) sensor for LFD may be close to a Ca(2+) source at active zones. Calcineurin can be activated by the Ca(2+)-activated protease calpain, and immunostaining showed that both proteins are present at nerve terminals. Three calpain inhibitors, calpain inhibitor I, MDL-28170, and PD150606, but not the control compound PD145305, inhibit LFD both in the intact animal as shown by electromyograms and by intracellular recordings at neuromuscular junctions. Analysis of mini-EPSPs indicated that these inhibitors had minimal postsynaptic effects. Proteolytic activity in CNS extract, detected by a fluorescent calpain substrate, was modulated by Ca(2+) and calpain inhibitors. Western blot analysis of CNS extract showed that proteolysis of calcineurin to a fragment consistent with the constitutively active form required Ca(2+) and was blocked by calpain inhibitors. Inhibition of LFD by calpain inhibition blocks the reduction in phosphoactin and the depolymerization of tubulin that normally occurs in LFD, probably by blocking the dephosphorylation of cytoskeletal proteins by calcineurin. In contrast, high-frequency depression does not involve protein phosphorylation- or calpain-dependent mechanisms. LFD may involve a specific pathway in which local Ca(2+) signaling activates presynaptic calpain and calcineurin at active zones and causes changes of tubulin cytoskeleton.

Molt regulation in green and red color morphs of the crab Carcinus maenas: gene expression of molt-inhibiting hormone signaling components.

In decapod crustaceans, regulation of molting is controlled by the X-organ/sinus gland complex in the eyestalks. The complex secretes molt-inhibiting hormone (MIH), which suppresses production of ecdysteroids by the Y-organ (YO). MIH signaling involves nitric oxide and cGMP in the YO, which expresses nitric oxide synthase (NOS) and NO-sensitive guanylyl cyclase (GC-I). Molting can generally be induced by eyestalk ablation (ESA), which removes the primary source of MIH, or by multiple leg autotomy (MLA). In our work on Carcinus maenas, however, ESA has limited effects on hemolymph ecdysteroid titers and animals remain in intermolt at 7 days post-ESA, suggesting that adults are refractory to molt induction techniques. Consequently, the effects of ESA and MLA on molting and YO gene expression in C. maenas green and red color morphotypes were determined at intermediate (16 and 24 days) and long-term (~90 days) intervals. In intermediate-interval experiments, ESA of intermolt animals caused transient twofold to fourfold increases in hemolymph ecdysteroid titers during the first 2 weeks. In intermolt animals, long-term ESA increased hemolymph ecdysteroid titers fourfold to fivefold by 28 days post treatment, but there was no late premolt peak (>400 pg µl(-1)) characteristic of late premolt animals and animals did not molt by 90 days post-ESA. There was no effect of ESA or MLA on the expression of Cm-elongation factor 2 (EF2), Cm-NOS, the beta subunit of GC-I (Cm-GC-Iß), a membrane receptor GC (Cm-GC-II) and a soluble NO-insensitive GC (Cm-GC-III) in green morphs. Red morphs were affected by prolonged ESA and MLA treatments, as indicated by large decreases in Cm-EF2, Cm-GC-II and Cm-GC-III mRNA levels. ESA accelerated the transition of green morphs to the red phenotype in intermolt animals. ESA delayed molting in premolt green morphs, whereas intact and MLA animals molted by 30 days post treatment. There were significant effects on YO gene expression in intact animals: Cm-GC-Iß mRNA increased during premolt and Cm-GC-III mRNA decreased during premolt and increased during postmolt. Cm-MIH transcripts were detected in eyestalk ganglia, the brain and the thoracic ganglion from green intermolt animals, suggesing that MIH in the brain and thoracic ganglion prevents molt induction in green ESA animals.

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.

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.

Evaluating conserved domains and motifs of decapod gonadotropin-releasing hormone G protein-coupled receptor superfamily.

G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation.

CrusTome: a transcriptome database resource for large-scale analyses across Crustacea.

Transcriptomes from nontraditional model organisms often harbor a wealth of unexplored data. Examining these data sets can lead to clarity and novel insights in traditional systems, as well as to discoveries across a multitude of fields. Despite significant advances in DNA sequencing technologies and in their adoption, access to genomic and transcriptomic resources for nontraditional model organisms remains limited. Crustaceans, for example, being among the most numerous, diverse, and widely distributed taxa on the planet, often serve as excellent systems to address ecological, evolutionary, and organismal questions. While they are ubiquitously present across environments, and of economic and food security importance, they remain severely underrepresented in publicly available sequence databases. Here, we present CrusTome, a multispecies, multitissue, transcriptome database of 201 assembled mRNA transcriptomes (189 crustaceans, 30 of which were previously unpublished, and 12 ecdysozoans for phylogenetic context) as an evolving and publicly available resource. This database is suitable for evolutionary, ecological, and functional studies that employ genomic/transcriptomic techniques and data sets. CrusTome is presented in BLAST and DIAMOND formats, providing robust data sets for sequence similarity searches, orthology assignments, phylogenetic inference, etc. and thus allowing for straightforward incorporation into existing custom pipelines for high-throughput analyses. In addition, to illustrate the use and potential of CrusTome, we conducted phylogenetic analyses elucidating the identity and evolution of the cryptochrome/photolyase family of proteins across crustaceans.

In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates.

Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab, Gecarcinus lateralis, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth, Bombyx mori. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designated CHH Family Receptor Candidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. In G. lateralis, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24ß1, -A24ß2, -A34α2, -A34ß1, and -A34ß2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded ß-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34ß1/ß2 had an additional two-stranded ß-sheet. We hypothesize that this second ß-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays.

Rheb, an activator of target of rapamycin, in the blackback land crab, Gecarcinus lateralis: cloning and effects of molting and unweighting on expression in skeletal muscle.

Molt-induced claw muscle atrophy in decapod crustaceans facilitates exuviation and is coordinated by ecdysteroid hormones. There is a 4-fold reduction in mass accompanied by remodeling of the contractile apparatus, which is associated with an 11-fold increase in myofibrillar protein synthesis by the end of the premolt period. Loss of a walking limb or claw causes a loss of mass in the associated thoracic musculature; this unweighting atrophy occurs in intermolt and is ecdysteroid independent. Myostatin (Mstn) is a negative regulator of muscle growth in mammals; it suppresses protein synthesis, in part, by inhibiting the insulin/metazoan target of rapamycin (mTOR) signaling pathway. Signaling via mTOR activates translation by phosphorylating ribosomal S6 kinase (s6k) and 4E-binding protein 1. Rheb (Ras homolog enriched in brain), a GTP-binding protein, is a key activator of mTOR and is inhibited by Rheb-GTPase-activating protein (GAP). Akt protein kinase inactivates Rheb-GAP, thus slowing Rheb-GTPase activity and maintaining mTOR in the active state. We hypothesized that the large increase in global protein synthesis in claw muscle was due to regulation of mTOR activity by ecdysteroids, caused either directly or indirectly via Mstn. In the blackback land crab, Gecarcinus lateralis, a Mstn-like gene (Gl-Mstn) is downregulated as much as 17-fold in claw muscle during premolt and upregulated 3-fold in unweighted thoracic muscle during intermolt. Gl-Mstn expression in claw muscle is negatively correlated with hemolymph ecdysteroid level. Full-length cDNAs encoding Rheb orthologs from three crustacean species (G. lateralis, Carcinus maenas and Homarus americanus), as well as partial cDNAs encoding Akt (Gl-Akt), mTOR (Gl-mTOR) and s6k (Gl-s6k) from G. lateralis, were cloned. The effects of molting on insulin/mTOR signaling components were quantified in claw closer, weighted thoracic and unweighted thoracic muscles using quantitative polymerase chain reaction. Gl-Rheb mRNA levels increased 3.4-fold and 3.9-fold during premolt in claw muscles from animals induced to molt by eyestalk ablation (ESA) and multiple leg autotomy (MLA), respectively, and mRNA levels were positively correlated with hemolymph ecdysteroids. There was little or no effect of molting on Gl-Rheb expression in weighted thoracic muscle and no correlation of Gl-Rheb mRNA with ecdysteroid titer. There were significant changes in Gl-Akt, Gl-mTOR and Gl-s6k expression with molt stage. These changes were transient and were not correlated with hemolymph ecdysteroids. The two muscles differed in terms of the relationship between Gl-Rheb and Gl-Mstn expression. In thoracic muscle, Gl-Rheb mRNA was positively correlated with Gl-Mstn mRNA in both ESA and MLA animals. By contrast, Gl-Rheb mRNA in claw muscle was negatively correlated with Gl-Mstn mRNA in ESA animals, and no correlation was observed in MLA animals. Unweighting increased Gl-Rheb expression in thoracic muscle at all molt stages; the greatest difference (2.2-fold) was observed in intermolt animals. There was also a 1.3-fold increase in Gl-s6k mRNA level in unweighted thoracic muscle. These data indicate that the mTOR pathway is upregulated in atrophic muscles. Gl-Rheb, in particular, appears to play a role in the molt-induced increase in protein synthesis in the claw muscle.

De novo transcriptome assemblies of red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) molting gland and eyestalk ganglia - Temperature effects on expression of molting and growth regulatory genes in adult red king crab.

Red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) are deep-sea crustaceans widely distributed in the North Pacific and Northwest Atlantic Oceans. These giant predators have invaded the Barents Sea over the past decades, and climate-driven temperature changes may influence their distribution and abundance in the sub-Arctic region. Molting and growth in crustaceans are strongly affected by temperature, but the underlying molecular mechanisms are little known, particularly in cold-water species. Here, we describe multiple regulatory factors in the two high-latitude crabs by developing de novo transcriptomes from the molting gland (Y-organ or YO) and eye stalk ganglia (ESG), in addition to the hepatopancreas and claw muscle of red king crab. The Halloween genes encoding the ecdysteroidogenic enzymes were expressed in YO, and the ESG contained multiple neuropeptides, including molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), and ion-transport peptide (ITP). Both crabs expressed a diversity of growth-related factors, such as mTOR, AKT, Rheb and AMPKα, and stress-responsive factors, including multiple heat shock proteins (HSPs). Temperature effects on the expression of key regulatory genes were quantified by qPCR in adult red king crab males kept at 4 °C or 10 °C for two weeks during intermolt. The Halloween genes tended to be upregulated in YO at high temperature, while the ecdysteroid receptor and several growth regulators showed tissue-specific responses to elevated temperature. Constitutive and heat-inducible HSPs were expressed in an inverse temperature-dependent manner, suggesting that adult red king crabs can acclimate to increased water temperatures.