Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea.

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH) expression in the blue crab, Callinectes sapidus.

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.

Changes in ecdysteroid levels and expression patterns of ecdysteroid-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus.

Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus.

Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: cloning and their expression patterns in eyestalks and Y-organs during the molt cycle.

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3-5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5-4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5' UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.

Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD).

Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed ß subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O(2)- and Fe(2+)-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis.