Molt-inhibiting hormone (MIH) gene from the green mud crabScylla paramamosainand its expression during the molting and ovarian cycle

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

Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

Recently, crustacean hyperglycaemic hormone (CHH) has gained wide attention for its diverse physiological functions. In this study, two cDNA sequences encoding CHH, Sp-CHH1 and Sp-CHH2, were cloned from the eyestalk ganglia of the mud crab, Scylla paramamosain. Each conceptually translated precursor is expected to be processed into the signal peptide, the crustacean hyperglycaemic hormone precursor-related peptide (CPRP) and the CHH mature peptide. Sp-CHH1 and SpCHH2 share an identical sequence for the first 40 residues from the amino-terminus of the peptides, but substantially different from each other in the C-terminus. Quantitative real-time PCR (qPCR) showed that Sp-CHH transcripts were mainly detected in the eyestalk ganglia and to a much lesser extent in the brain, thoracic ganglia, stomach, hepatopancreas, heart and muscle. RNA in situ hybridization in the eyestalk ganglia indicated that the Sp-CHH was localized in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis. Moreover, Sp-CHH transcripts were stage-specific changed during the moulting cycle, as it increased significantly from postmoult stage to intermoult stage, then decreased sharply at premoult stage. Sp-CHH expression levels in the eyestalk ganglia were also examined at different ovarian stages (stage I to stage V). It was found that the expression was gradually increased from stage I to stage IV, and then it was decreased at stage V. All the findings together suggested that Sp-CHH should be pleiotropic, which might inhibit the synthesis of ecdysteroids by Y-organs and be involved in promoting vitellogenesis in the mud crab.

Molt-inhibiting hormone from the tropical land crab, Gecarcinus lateralis: Cloning, tissue expression, and expression of biologically active recombinant peptide in yeast

Molt is a critical developmental process in crustaceans. Recent studies have shown that the hepatopancreas is an important source of innate immune molecules, yet hepatopancreatic patterns of gene expression during the molt cycle which may underlie changes in immune mechanism are unknown. In this study, we performed Illumina sequencing for the hepatopancreas of the mud crab, Scylla paramamosain during molt cycle (pre-molt stage, post-molt stage, and inter-molt stage). A total of 44.55 Gb high-quality reads were obtained from the normalized cDNA of hepatopancreas. A total of 70,591 transcripts were assembled; 55,167 unigenes were identified. Transcriptomic comparison revealed 948 differentially expressed genes (DEGs) in the hepatopancreas from the three molt stages. We found that genes associated with immune response patterns changed in expression during the molt cycle. Antimicrobial peptide genes, inflammatory response genes, Toll signaling pathway factors, the phenoloxidase system, antioxidant enzymes, metal-binding proteins and other immune related genes are significantly up-regulated at the post-molt stage and inter-molt stage compared with the pre-molt stage, respectively. These genes are either not expressed or are expressed at low levels at the pre-molt stage. To our knowledge, this is the first systematic transcriptome analysis of genes capable of mobilizing a hepatopancreas immune response during the molt cycle in crustaceans, and this study will contribute to a better understanding of the hepatopancreas immune system and mud crab prophylactic immune mechanisms at the post-molt stage.

Tributyrin enhances growth and intestinal health in green mud crab (Scylla paramamosain) through butyrate-driven metabolic regulation.

Role of red pigment concentrating hormone in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis

Molecular characterization of a cDNA encoding Na+/K+/2Cl− cotransporter in the gill of mud crab (Scylla paramamosain) during the molt cycle: Implication of its function in osmoregulation

Transcriptional silencing of vitellogenesis-inhibiting and molt-inhibiting hormones in the giant freshwater prawn, Macrobrachium rosenbergii, and evaluation of the associated effects on ovarian development

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

Regulation of ecdysteroid secretion from the Y-organ by molt-inhibiting hormone in the American crayfish, Procambarus clarkii

Very little is known regarding the release patterns or circulating titers of neuropeptides in crustaceans, in particular those concerned with regulation of molting hormone (ecdysteroid) synthesis, molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH), which is also an adaptive hormone, centrally important in carbohydrate metabolism. Furthermore, the currently accepted model of molt control is founded on an untested hypothesis suggesting that molting can proceed only after decline in MIH titer. Accordingly, we measured simultaneous circulating neuropeptide profiles for both MIH and CHH by RIA of purified hemolymph during the molt cycle at fine temporal scale during day/night cycles and seasonally. For CHH we additionally determined release patterns after physiologically relevant stress. Results show that both hormones are released exclusively and episodically, rather than continuously, with notably short half-lives in circulation, suggesting dynamic and short-lived variations in levels of both hormones. During the molt cycle, there are no overt changes in MIH titer, except a massive and unprecedented increase in MIH during late premolt, just before ecdysis. The function of this hormone surge is unknown. Treatment with various stressors (hypoxia, temperature shock) showed that CHH release occurs extremely rapidly, within minutes of stress. Release of CHH after stressful episodes during premolt (when gut endocrine cells synthesize large quantities of CHH) is exclusively from the sinus gland: CHH from the gut is never involved in the stress response. The results show a hitherto unsuspected dynamism in release of MIH and CHH and suggest that currently accepted models of molt control must be reconsidered.

Decapod crustaceans regulate molting through steroid molting hormones (ecdysteroids) synthesized by the molting gland (Y-organ, YO). Molt-inhibiting hormone (MIH), a neuropeptide synthesized and secreted by the eyestalk ganglia, negatively regulates YO ecdysteroidogenesis. MIH signaling is mediated by cyclic nucleotide second messengers. cGMP-dependent protein kinase (PKG) is the presumed effector of MIH signaling by inhibiting mechanistic Target of Rapamycin Complex 1 (mTORC1)-dependent ecdysteroidogenesis. Phylogenetic analysis of PKG contiguous sequences in CrusTome, as well as 35 additional species in NCBI RefSeq, identified 206 PKG1 sequences in 108 species and 59 PKG2 sequences in 53 species. These included four PKG1α splice variants in the N-terminal region that were unique to decapods, as well as PKG1β and PKG2 homologs. In vitro assays using YOs from the blackback land crab (Gecarcinus lateralis) and green shore crab (Carcinus maenas) determined the effects of MIH±PKG inhibitors on ecdysteroid secretion. A general PKG inhibitor, Rp-8-Br-PET-cGMPS, countered the effects of MIH, as ecdysteroid secretion increased in PKG-inhibited YOs compared with C. maenas YOs incubated with MIH alone. By contrast, a PKG2-specific inhibitor, AP-C5 {4-(4-[1H-imidazol-1-yl]phenyl)-N-2-propyn-1-yl-2-pyrimidinamine}, enhanced the effects of MIH, as ecdysteroid secretion decreased in G. lateralis and C. maenas YOs incubated with AP-C5 and MIH compared with YOs incubated with MIH alone. These data suggest that both PKG1 and PKG2 are activated by MIH, but have opposing effects on mTORC1-dependent ecdysteroidogenesis. A model is proposed in which the dominant role of PKG1 is countered by PKG2, resulting in low ecdysteroid production by the basal YO during intermolt.

Role of short neuropeptide F in regulating eyestalk neuroendocrine systems in the mud crab Scylla paramamosain