The retinoid X receptor from mud crab: new insights into its roles in ovarian development and related signaling pathway

In arthropods, retinoid X receptor (RXR) is a highly conserved nuclear hormone receptor. By forming a heterodimeric complex with the ecdysone receptor (EcR), RXR is known to be vital importance for various physiological processes. However, in comparison to EcR, the RXR signaling pathway and its roles in crustacean reproduction are poorly understood. In the present study, the RXR mRNA was detected in the ovarian follicular cells of mud crab Scylla paramamosain (SpRXR) and during ovarian maturation, its expression level was found to increase significantly. In vitro experiment showed hat both SpRXR and vitellogenin (SpVg) mRNA in the ovarian explants were significantly induced by 20-hydroxyecdysone (20E) but not methyl farnesoate (MF). However, differing from the in vitro experiment, injection of MF in in vivo experiment significantly stimulated the expressions of SpRXR and SpVg in female crabs at early vitellogenic stage, but the ecdysone and insect juvenile hormone (JH) signaling pathway genes were not induced. The results together suggest that both MF and SpRXR play significant roles in regulating the expression of SpVg and ovarian development of S. paramamosain through their own specific signaling pathway rather than sharing with the ecdysone or the insect JH

Inhibitory Role of the Mud Crab Short Neuropeptide F in Vitellogenesis and Oocyte Maturation via Autocrine/Paracrine Signaling

Neuropeptides, in addition to their classical role in the nervous system, act on intraovarian factors to regulate reproductive functions in vertebrates. However, this function of neuropeptides has not been characterized in crustaceans. Short neuropeptide F (sNPF), a highly conserved invertebrate neuropeptide, has been reported to be involved in feeding, metabolism, and in differentiation processes including reproduction. Although sNPF and its receptor (sNPFR) have been detected in the ovary in different species, ovarian colocalization of sNPF/sNPFR has not been investigated. In this study, we identified Scylla paramamosain (mud crab) sNPF (Sp-sNPF) as an endogenous ligand for the S. paramamosain orphan G protein-coupled receptor NPY2R in mammalian cell line HEK293T. We designated this receptor as Sp-sNPFR. RNA in situ hybridization in pre-vitellogenic ovary and reverse transcription-PCR on isolated denuded oocytes and follicle layers showed that Sp-sNPF was exclusively localized to the follicle cells, whereas Sp-sNPFR was detected in both follicle cells and oocytes. We also found that Sp-sNPF partly suppressed spontaneous maturation of denuded oocytes and caused intracellular cAMP accumulation and Ca2+ mobilization. Moreover, injection of synthetic Sp-sNPF peptides inhibited the expression of vitellogenin and vitellogenin receptor genes in vivo. These combined results suggest for the first time that Sp-sNPF may have inhibitory functions in vitellogenesis and oocyte maturation possibly via the autocrine/paracrine pathway

Identifying neuropeptide GPCRs in the mud crab, Scylla paramamosain, by combinatorial bioinformatics analysis

Neuropeptides, ubiquitous signaling molecules, commonly achieve their signaling function via interaction with cell membrane-spanning G-protein coupled receptors (GPCRs). In recent years, in the midst of the rapid development of next-generation sequencing technology, the amount of available information on encoded neuropeptides and their GPCRs sequences have increased dramatically. The repertoire of neuropeptides has been determined in many crustaceans, including the commercially important mud crab, Scylla paramamosain; however, determination of GPCRs is known to be more difficult and usually requires in vitro binding tests. In this study, we adopted a combinatorial bioinformatics analysis to identify S. paramamosain neuropeptide GPCRs. A total of 65 assembled GPCR sequences were collected from the transcriptome database. Subsequently these GPCRs were identified by comparison to known neuropeptide GPCRs based on the sequence-similarity-based clustering and phylogenetic analysis, which showed that many of them are closely related to insect GPCR families. Of these GPCRs, most of them were detected in various tissues of the mud crab and some of them showed differential expression by gender, suggesting they are involved in different physiological processes, such as sex differentiation. By employing ligand-receptor binding tests, we demonstrated that the predicted crustacean cardioactive peptide (CCAP) receptor was activated by CCAP peptide in a dose-dependent manner. This is the first CCAP receptor that has been functionally defined in crustaceans. In summary, the present study shortlists candidate neuropeptide GPCRs for ligand-receptor binding tests, and provides information for subsequent future research on the neuropeptide/GPCR signaling pathway in S. paramamosain

Transcriptome profiling of claw muscle of the mud crab (Scylla paramamosain) at different fattening stages.

In crustaceans, muscle growth and development is complicated, and to date substantial knowledge gaps exist. In this study, the claw muscle, hepatopancreas and nervous tissue of the mud crab (Scylla paramamosain) were collected at three fattening stages for sequence by the Illumina sequencing. A total of 127.87 Gb clean data with no less than 3.94 Gb generated for each sample and the cycleQ30 percentages were more than 86.13% for all samples. De Bruijn assembly of these clean data produced 94,853 unigenes, thereinto, 50,059 unigenes were found in claw muscle. A total of 121 differentially expressed genes (DEGs) were revealed in claw muscle from the three fattening stages with a Padj value < 0.01, including 63 genes with annotation. Functional annotation and enrichment analysis showed that the DEGs clusters represented the predominant gene catalog with roles in biochemical processes (glycolysis, phosphorylation and regulation of transcription), molecular function (ATP binding, 6-phosphofructokinase activity, and sequence-specific DNA binding) and cellular component (6-phosphofructokinase complex, plasma membrane, and integral component of membrane). qRT-PCR was employed to further validate certain DEGs. Single nucleotide polymorphism (SNP) analysis obtained 159,322, 125,963 and 166,279 potential SNPs from the muscle transcriptome at stage B, stage C and stage D, respectively. In addition, there were sixteen neuropeptide transcripts being predicted in the claw muscle. The present study provides a comprehensive transcriptome of claw muscle of S. paramamosain during fattening, providing a basis for screening the functional genes that may affect muscle growth of S. paramamosain

Top 20 annotations of <i>S</i>. <i>paramamosain</i> transcriptome with the highest bit score.

<p>Top 20 annotations of <i>S</i>. <i>paramamosain</i> transcriptome with the highest bit score.</p

Putative neuropeptide precursors in the muscle transcriptome of <i>S</i>. <i>paramamosain</i>.

<p>Putative neuropeptide precursors in the muscle transcriptome of <i>S</i>. <i>paramamosain</i>.</p

The classification of unigenes in three GO categories in <i>S</i>. <i>paramamosain</i>.

<p>The x-axis indicated GO process; the y-axis on the left side indicated the percentage of the unigenes of this process in all genes; the y-axis on the right side indicated the number of unigenes in the process.</p

Statistics of DEGs in the muscle transcriptome in <i>S</i>. <i>paramamosain</i>.

<p>Statistics of DEGs in the muscle transcriptome in <i>S</i>. <i>paramamosain</i>.</p

The GO terms with significant DEGs of muscle of <i>S</i>. <i>paramamosain</i>.

<p>The GO terms with significant DEGs of muscle of <i>S</i>. <i>paramamosain</i>.</p