Neuropeptides play a critical role in regulating animal reproduction. In vertebrates, GnRH, GnIH and kisspeptin are the key neuropeptide hormones of the reproductive axis, however, the reproductive axis for invertebrates is vague. Knowledge on ovarian development of the mud crab, Scylla paramamosain, is critical for aquaculture and resources management of the commercially important species. This study employed Illumina sequencing, reverse transcription-polymerase chain reaction and quantitative real-time PCR techniques to identify neuropeptides that may be involved in ovarian development of S. paramamosain. A total of 32 neuropeptide transcripts from two dozen neuropeptide families, 100 distinct mature peptides were predicted from the transcriptome data of female S. paramamosain cerebral ganglia. Among them, two families, i.e. GSEFLamide and WXXXRamide, were first identified from the cerebral ganglia of crustaceans. Of these neuropeptides, 21 transcripts of interest were selected for further confirmation and all of them were detected in the cerebral ganglia, as well as in other nervous tissues and the ovary. Most of them also had differential expression in the cerebral ganglia during various vitellogenic stages, suggesting their likely involvement in regulating vitellogenesis and ovarian maturation. Overall, these findings provide an important basis for subsequent studies on peptide function in reproduction of S. paramamosain.

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In arthropods, it is known that ecdysteroids regulate molting, limb regeneration, and reproduction through activation of the ecdysone receptor (EcR). However, the ecdysteroid signaling pathway for promotion of ovarian development in crustaceans is still unclear. In this study, three cDNA isoforms of EcR were cloned from the mud crab Scylla paramamosain. qRT-PCR revealed that the SpEcR mRNA was abundant in the eyestalk, ovary and epidermis. During ovarian development, the SpEcR transcripts increased from stage I (undeveloped stage) and reached a peak at stage IV (late vitellogenic stage) before dropping to a lower level at stage V (mature stage). Meanwhile, levels of 20-hydroxyecdysone (20E) in the hemolymph, detected by HPLC-MS, displayed a similar pattern of increase with ovarian development. Results from in situ hybridization indicated that SpEcR mRNA was present in the follicular cells during vitellogenesis. Results from in vivo experiments revealed that 20E at 0.2 mg/g body weight significantly stimulated the expression of SpEcR and vitellogenin (SpVg) in female crabs during the early vitellogenic stage but not during the previtellogenic stage. This was confirmed by results from in vitro experiments which indicated that SpEcR and SpVg expression levels were significantly upregulated in early vitellogenic ovarian explants incubated with 5.0 mM 20E at 3 and 6 h but not in previtellogenic ovarian explants. Finally, results from in vitro gene silencing experiments indicated that the expression of SpEcR and SpVg in the ovary was significantly inhibited by SpEcR dsRNA. All these results together indicated that in S. paramamosain, 20E, and SpEcR, located in the follicular cells, play important roles in the promotion of ovarian development via regulating the expression of SpVg.

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Mud crab is a group of commercially important species that are found throughout the Indo-Pacific region. In this study, the full-length cDNA of molt-inhibiting hormone (Scp-MIH) was cloned from the green mud crab, Scylla paramamosain. The conceptually translated peptide precursor consists of a 78-residue mature peptide, preceded by a 35-residue signal peptide. The mature peptide shares a high identity with the other known MIHs, and contains six conserved cysteine residues that have been proposed to be functionally critical for MIH activity. Analysis of genomic sequence revealed that Scp-MIH gene was organized in a 3 exons/2 introns manner. Quantitative real-time PCR showed that Scp-MIH transcripts were detected mainly in the eyestalk ganglion and brain. During the molting cycle, Scp-MIH mRNA increased significantly from postmolt stage to intermolt stage, then decreased sharply at premolt stage. It implies that MIH is closely related to ecdysteroid production in S. paramamosain. In the ovarian cycle, Scp-MIH transcripts in some neural tissues (eyestalk ganglion and brain) increased from stage I to stage II, and reached the peak value at stage III, then declined at later stage. It suggests that MIH might be involved in the ovarian maturation in S. paramamosain.