Identification And Functional Analysis Of The Genes Related To Molting And Sexual Regulation In Exopalaemon Carinicauda

Exopalaemon carinicauda is not only an important economic shrimp, but also a good experimental animal for biological studies of the crustaceans. Various physiological processes of crustacean, such as molting, growth etc, are regulated by interaction of multiple hormones. Molting is one basic physiological process of crustacean, which is closely related to growth and development. Knowledge on sex differentiation and sex determination is the basis of sex control in animals. In this paper, we identified several functional genes related to regulation of molting or sex development in Exopalaemon carinicauda and their functions were preliminarily analyzed. The main progresses are as follows:Molting behavior is an important physiological process related to growth, metamorphosis and reproduction in crustacean. Previous studies have elaborated the molting process controlled by 20-hydroxyecdysone (20E) and upstream hormones, peptides and environmental factors which regulate 20E function. However, the downstream genes regulated by 20E and their functions during molting are still largely unknown. Eclosion hormone (EH) is a kind of neuropeptide that is regulated by 20E and triggers ecdysis behavior at the end of molting in insects. In the present study, an eclosion hormone like gene EcEH was identified from Exopalaemon carinicauda. The deduced amino acid sequence of EcEH contained a signal peptide, a typical eclosion domain and six conserved cysteine residues forming three putative disulfide bonds. EcEH was predominantly expressed in the epidermis, gills and eyestalk. In situ hybridization analysis showed that EcEH transcripts were localized in gills and eyestalks. During molting process, EcEH was mainly detected in the premolt stage. The expression level of EcEH in mid premolt stage could be up-regulated by exogenous 20E. Silencing of EcEH using double-stranded RNA delayed both the molting process and ecdysis behavior of E. carinicauda. Furthermore, injection of exogenous at mid premolt stage (D2) remarkably promoted the molting process and ecdysis rate of E. carinicauda. These data revealed that EcEH participated in the molting process in E. carinicauda and its expression might be regulated by 20E. To our knowledge, this was the first time to characterize the function of eclosion hormone in crustacean.Insulin-like androgenic gland hormone (IAG), is secreted by the androgenic gland (AG), which is a specific organ of male crustaceans. AG plays a key role in the sex differentiation of male crustaceans, male sexuality and spermatogenesis. The IAG gene has been described in many crustaceans, including shrimp, prawns and crabs, but knowledge about its function is is still very limited. In this study, An IAG like gene (named as EcIAG) were identified in Exopalaemon carinicauda. The ORF sequence of IAG gene was 531 bp, encoding 176 amino acids. The amino acid sequence includes a hydrophobic signal peptide,6 conserved cysteine residues, which form three disulfide bonds, and with two conserved domain (insulin-like IPRO16179). The tissue distribution of EcIAG was studied by RT-qPCR, which was specially expressed in AG and Testis. In situ hybridization showed that EcIAG was mainly localized in AG cells. The gene expression of IAG gene during different developmental stages was studied by RT-qPCR. The data showed that EcIAG was highly expressed in gastrul stage and postlarval stage P2. In order to know whethter the eyestalk has some hormones regulating the expression of EcIAG, we study the effects of eyestalk ablation on its expression. The results showed that the expression level of EcIAG was significantly up-regulated after eyestalk ablation, indicating that there were hormones in eyestalk regulating the expression of EcIAG. The related studies of in situ hybridization provide some information for understanding the regulation of sex differentiation in crustacean.Sox9 (SRY-box9), a class of transcription factors with a high degree of sequence similarity to the sex determination gene SRY of the animals. In this study, we identified Sox9 gene in Exopalaemon carinicauda (named as EcSox9), and analyzed its function. The ORF sequence of Sox9 gene was 672 bp, encoding 243 amino acids. The deduced amino acid sequence includes conserved HMG BOX domain. Tissue distribution analysis showed that EcSox9 was widely distributed in the detected tissues, but it showed the highest expression level in gill and hepatopancreas. The expression of EcSox9 gene increased with the development of embryos and larvae. The expression of EcSox9 in the androgenic gland was apparently up-regulated after eyestalk ablation, which suggested that there were some endocrine factors negatively regulating the expression of EcSox9. RNAi technology was performed to test the possible regulatory relationship between the genes EcSox9 and EcIAG. Injection of double strand RNA of EcSox9 (dsSox9) reduced the expression of EcIAG in the androgenic gland, which indicated that EcSox9 might positively regulate the expression of ExIAG. Injection of double strand RNA of EcIAG (dsIAG) upregulated the expression of EcSox9, which indicated that the expression level of EcIAG had a feedback regulation on the expression of EcSox9.