Mining Of Neuropeptides And Their Receptors,and Studies On Reproductive Mechanism Of SNPF In The Mud Crab,Scylla Paramamosain

Neuropeptides are diverse,ubiquitous signaling molecules that affect many important physiological processes.Most of them commonly achieve their signaling function via interaction with cell membrane-spanning G-protein coupled receptors?GPCRs?.Knowledge on ovarian development of the mud crab,Scylla paramamosain,is critical for aquaculture and resources management of the commercially important species in the coastal area of Southeast China.By using next-generation sequencing?NGS?,a total of 100 distinct mature peptides derived from 32 neuropeptide transcripts were identified.Meanwhile,65 neuropeptide GPCRs were predicted by mining of transcriptome databases.Employing the heterologous cell?HEK293T?expression and dual-luciferase assay system,ligand-receptor pairings of crustacean cardioactive peptide?CCAP?and short neuropeptide F?sNPF?were functionally identified.Furthermore,in situ hybridization,Ca²⁺ mobilization and signal transduction were used to investigate the sNPF proposed function as an intra-ovarian regulatory factor inhibiting ovarian development through an autocrine/paracrine regulatory mechanism.The main research findings of this study are as follows:1)NGS,RT-PCR and quantitative real-time PCR techniques were used to identify neuropeptides that may be involved in ovarian development of S.paramamosain.A total of 32 neuropeptide transcripts from 24 neuropeptide families,100 distinct mature peptides were predicted from the transcriptome database of female mud crab cerebral ganglia.Among them,GSEFLamide and WXXXRamide families were identified from the cerebral ganglia of crustaceans for the first time.Among these neuropeptides,21 transcripts of interest were selected to confirm.All of them were detected in the cerebral ganglia,and they widely distributed in other nervous tissues and the ovary.Most of them also showed differential expression in the cerebral ganglia during vitellogenesis,suggesting they may be involved in regulating ovarian development.Overall,these findings provide a guide for further studies of peptides function in reproduction of S.paramamosain.2)In this study,we adopted a combinatorial bioinformatics analysis to identify S.paramamosain neuropeptide GPCRs?SpGRs?.A total of 65 assembled GPCR sequences were collected from the transcriptome database.Subsequently these SpGRs were identified by comparison to known neuropeptide GPCRs based on the sequence-similarity-based clustering and phylogenetic analysis.Of these SpGRs,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 SpGR-A54 was activated by CCAP peptide in a dose-dependent manner.This is the first CCAPR 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.3)Based on the above bioinformatics analysis,we identified a mud crab candidate sNPF receptor?SpGR-A8?.In this study,we identified sNPF as endogenous ligand for this receptor in mammalian cell line HEK293T.We designated this receptor as sNPFR.RNA in situ hybridization in pre-vitellogenic ovary and RT-PCR on isolated denuded oocytes and follicle layers showed that sNPF was exclusively localized to the follicle cells,whereas sNPFR was detected in both follicle cells and oocytes.We also found that sNPF partly suppressed spontaneous maturation of denuded oocytes,resulting in intracellular cAMP accumulation and Ca²⁺ mobilization.Moreover,injection of synthetic sNPF peptides inhibited the expression of vitellogenin and vitellogenin receptor genes in vivo.These combined results suggest for the first time that sNPF may have inhibitory functions in vitellogenesis and oocyte maturation,possibly via the autocrine/paracrine pathway.