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Insulin-like androgenic gland hormone (IAG) is the most widely known hormone that regulates sexual differentiation in crustaceans. Previously, a transcriptome study described two transcripts of IAGs (Lvit-IAG1 and Lvit-IAG2) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Herein, we explored the function of Lvit-IAG2 via RNA interference (RNAi) experiments, and then compared the functional differences between the two IAGs. We demonstrated that Lvit-IAG2 positively regulated the expression of crustacean hyperglycemic hormone (Lvit-CHH) in eyestalk ganglion but exhibited no significant effect on the expression of gonad-inhibiting hormone (Lvit-GIHs) and crustacean female sex hormone (Lvit-CFSHs). Besides, Lvit-IAG2 gene knockdown caused degeneration in appendices masculinae (AM) and suppressed germ cells at the secondary spermatocyte stage. Moreover, silencing the Lvit-IAG2 gene impeded ovarian development, including smaller previtellogenic oocytes, and lower expression of vitellogenin (Lvit-Vg) gene in hepatopancreas and vitellogenin receptor (Lvit-VgR) gene in the ovarian region. Notably, silencing the Lvit-IAG2 gene impeded individual growth of the species. Collectively, findings from this study demonstrate that Lvit-IAG2 and Lvit-IAG1 coordinatively function to modulate sexual differentiation in L. vittata; meanwhile, Lvit-IAG2 stimulates the growth of the PSH species.

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C-type allatostatins are a family of peptides that characterized by a conserved unblocked-PISCF in C-terminal. In insects, it is well known that C-type allatostatin has a potent inhibitory effect on juvenile hormone biosynthesis by the corpora allata. Recently, C-type allatostatin has been widely identified from crustacean species but little is known about its roles. Therefore, this study described the tissue distribution patterns of C-type allatostatin and its putative receptor in the mud crab Scylla paramamosain, and further explored its potential effect on vitellogenesis. Firstly, the cDNAs encoding C-type allatostatin (Sp-AST-C) precursor and its putative receptor (Sp-AST-CR) were isolated, respectively. Subsequently, RT-PCR results suggested that, Sp-AST-C was mainly expressed in the nervous tissue, middle gut and the heart while Sp-AST-CR had an extensive expression in the detected tissues except the eyestalk ganglion and hepatopancreas. Furthermore, the Sp-AST-C expressing cells in the cerebral ganglion were detected through in situ hybridization, it showed that Sp-AST-C was localized in cluster 6, 8 of protocerebrum, cluster 9, 10, 11 of deutocerebrum, and cluster 14, 15 of tritocerebrum. The whole-mount immunofluorescence gave a similar distribution pattern. An in vitro experiment showed that, the synthetic Sp-AST-C had no effect on the abundance of Sp-Vg in the hepatopancreas and ovary but significantly reduced the expression of Sp-VgR in the ovary in a dose-dependent manner. Furthermore, it was demonstrated that the Sp-VgR expression, Vn content, and oocyte diameter in ovary were reduced after 16-days injection of Sp-AST-C. Finally, the transcripts of Sp-AST-CR were specifically localized in the oocytes of ovary by in situ hybridization, which further revealed that the oocytes were target cells for Sp-AST-C. In conclusion, our results suggested that Sp-AST-C signaling system was involved in the regulation of ovarian development, in which Sp-AST-C might inhibit the uptake of yolk by oocytes directly and obstruct oocyte growth.

Crustacean female sex hormone (CFSH) plays a pivotal role in the development of secondary sex characteristics in dioecious crustaceans. However, until now the knowledge concerning its functions in hermaphroditic species is scanty. Herein, we explored the function of CFSH (Lvit-CFSH1a) in the peppermint shrimp Lysmata vittata, a species characterized by a rare reproductive system of protandric simultaneous hermaphroditism (PSH). Lvit-CFSH1a cDNA was 1,220-bp in length with a 720-bp ORF encoded a polypeptide of 239-aa. RT-PCR showed that Lvit-CFSH1a was exclusively expressed in the eyestalk ganglion. For female physiology, it was found that Lvit-CFSH1a was indispensable for the development of female gonopores, but it might not involve vitellogenesis of the species. For male physiology, Lvit-CFSH1a suppressed Lvit-IAG2 expression in short-term silencing experiment and recombinant protein injection experiment, but did not affect male sexual differentiation in long-term silencing experiment. In addition, silencing the Lvit-CFSH1a gene impeded individual growth in L. vittata.