Roles Of Insulin-like Androgenic Gland Hormone And Crustacean Female Sex Hormone In Sexual Differentiation In The Peppermint Shrimp,Lysmata Vittata

It is generally believed that both insulin-like androgenic gland hormone(IAG)and crustacean female sex hormone(CFSH)regulate sexual differentiation in decapods.In gonochoristic species,the mechanism by which IAG and CFSH mediate sexual differentiation has been well studied.However,the mechanism underlying sexual differentiation mediated by IAG or CFSH in protandric simultaneous hermaphroditism(PSH)crustaceans remains unclear.Additionally,the receptor for CFSH has not yet been identified.The pepper shrimp Lysmata vittata displays a unique sexual system,PSH,whereby individuals first mature as males;however,with increasing age and size,they acquire reproductive functions for both males and females.In this paper,we used L.vittata as a model organism to explore functions of IAG and CFSH in regulating sexual differentiation of the PSH species.Meanwhile,Glutathione S-transferase(GST)pulldown assays and RNA interference(RNAi)were utilized to identify CFSH receptors.The present results would be helpful to elucidate the molecular mechanism for endocrine regulation of the unique PSH sexual system in crustaceans.The main results are summarized as follows:1)In the present study,we expounded functions of Lvit-IAG1 and Lvit-IAG2 in regulating primary and secondary sexual characteristics of this PSH species.Both Lvit-IAG1 and Lvit-IAG2 gene were exclusively expressed in the androgenic gland(AG)and their expression levels were significantly higher at the male phase than the euhermaphrodite phase during the life cycle of the PSH species.Results of long-term silencing experiments revealed that Lvit-IAG2 and Lvit-IAG1 coordinatively functioned to modulate male sexual differentiation in L.vittata.Lvit-IAG1 was suggested to be closely related to the normal development of both appendix masculina(AM)and male gonopores,and participates in primary-to-secondary spermatocyte transition.However,Lvit-IAG2 was suggested to predominantly acted on the normal development of AM,and participated in spermatocyte-to-spermatid transition.Notably,knockdown of Lvit-IAG1 as well as Lvit-IAG2 impeded the ovarian development.Lvit-IAG1 was suggested to regulate the ovarian development by inhibiting gonad-inhibiting hormone(Lvit-GIH)expression in the eyestalk ganglion;while Lvit-IAG2 might contribute to ovarian development via promotig the individual growth in L.vittata.Moreover,knockdown of the these two Lvit-IAGs induced changes in gene expression of neuropeptides in the eyestalk ganglion,indicating that AG might have negative feedback on eyestalk ganglion of this PSH species.2)In the present study,we also expounded functions of Lvit-CFSHla and Lvit-CFSHlb in regulating primary and secondary sexual characteristics of the PSH species.Both Lvit-CFSH1a and Lvit-CFSHlb gene were exclusively expressed in the eyestalk ganglion.RT-PCR results revealed that the expression levels of Lvit-CFSH1a had no significant changes during gonad development,while the expression levels of Lvit-CFSH1a significantly increased with the gonad development from stage I to stage Ⅲ and then obviously decreased at stage Ⅳ,indicating that only Lvit-CFSHlb was possibly engaged in ovarian development.Results of long-term silencing experiments showed that silencing either Lvit-CFSH1a gene or Lvit-CFSH1b gene hindered development of female gonopores,indicating that Lvit-CFSHla and Lvit-CFSHlb might act in concert,aimed at regulating female sexual differentiation in the PSH shrimp.Results of in vivo studies also demonstrated that Lvit-CFSHs regulated male sexual differentiation by inhibiting Lvit-IAGs expression.Moreover,silencing of both Lvit-CFSH genes suppressed ovarian development.Results of in vivo studies indicated that Lvit-CFSH1a might contribute to ovarian development via promoting the individual growth.On the other hand,in vitro study showed that Lvit-CFSH1b could promote ovarian development by directly stimulating vitellogenine(Lvit-Vg)expression in hepatopancreas and vitellogenin receptor(Lvit-VgR)expression in ovary.3)In the present study,we identified two potential CFSH receptors,namely,Lvit-IL17Rland Lvit-IL17R2,by GST pulldown assays and RNAi.Results of sequence analysis demonstrated that Lvit-IL17Rland Lvit-IL 17R2 were transmembrane proteins with a SEFIR domain.GST pulldown experiments confirmed the interactions between Lvit-CFSH1a/Lvit-CFSH1b and Lvit-IL17R1/Lvit-IL17R2.Meanwhile,results of RNAi revealed that Lvit-IL17R1 and Lvit-IL17R2 displayed similar functions with Lvit-CFSH1b in regulating sexual differentiation as well as gonad development,indicating that Lvit-IL17R1 and Lvit-IL17R2 were receptors to Lvit-CFSH1b.However,results of the present study showed that Lvit-CFSH1a could also regulate female sexual differentiation and regulate male sexual differentiation via inhibiting Lvit-IAG2 expression.Given these findings,we cannot exclude the possibility that Lvit-IL17R1 and Lvit-IL17R2 were receptors to Lvit-CFSH1a.