Roles And Mechanism Of Allatostatin In Reproductive Endocrine Regulation Of The Mud Crab,scylla Paramamosain

Allatostatins(ASTs)are the most aboundant neuropeptides in arthropods.Based on their distinct structural differences,ASTs are subdivided into three different families and they are currently known as A-type,B-type,and C-type.In insects,it is well known that they are pleiotropic peptides and involved in many important physiological process,including ecdysis,metamorphosis,feeding,and reproduction etc.However,ASTs are poorly studied in crustaceans;therefore little is known about their functions.The mud crab(Scylla paramamosain)is one of the most inportant aquaculture species in the southeast coastal region in China,which warrants of great value in economic.In this study,we identified AST-B and AST-C and their receptors in the mud crab S.paramamosain,and then described their tissue distribution and experssion patterns during ovarian development.In addition,the endocrine regulation and mechanism of AST-B and AST-C on reproduction in S.paramamosain were subsequently investigated.The main results are offered as following:1)This study reported a B-type allatostatins precursor(SpAST-B)and its receptor(SpAST-BR)in the mud crab Scylla paramamosain,and further investigated a possible role of SpAST-B in vitellogenesis.Firstly,the full length cDNA of both SpAST-B and SpAST-BR were isolated from S.paramamosain.Ligand-receptor binding assay revealed that SpAST-BR was coupled with Gia protein to inhibit the activity of adenylate cyclase that reducing the level of cAMP in cytoplasm.RT-PCR suggested that SpAST-B was confined to the ovary and the nervous tissues whereas SpAST-BR was extensively expressed in the detected tissues except heart.In addition,the expression patterns of both SpAST-B and SpAST-BR in ovary were negatively correlated to vitellogensis.In situ hybridization revealed that SpAST-B was exclusively expressed in oocytes while SpAST-BR was expressed in both oocytes and follicular cells.Finally,an in vitro experiment demonstrated that synthetic SpAST-B significantly reduced the expression of SpVg in ovary and knockdown of SpAST-BR by RNA interference significantly increased the expression of SpVg in ovary.In conclusion,our results suggested that oocyte-derived SpAST-B may inhibit the vitellogenesis that occurs in ovary through autocrine/paracrine signaling.2)This study investigated the regulation of SpAST-B on the endocrine activity that occurs in eyestalk ganglion,Y-organ,and mandibular organ.In situ hybridization showed that SpAST-B-expressing cell was widely present in the cerebral ganglion,whereas SpAST-BR-expressing cell existed in the glandular cells of both Y-organ and mandibular organ.It was demonstrated that 20E biosynthesis in the Y-organ was significantly inhibited by SpAST-B peptides both in vivo and in vitro.Injection of SpAST-B peptides promoted the expression of SpMIH in the eyestalk ganglion,which revealed that SpAST-B inhibits 20E biosynthesis in the Y-organ directly and by MIH indirectly.In addition,SpAST-B significantly reduced the abundance of MF biosynthetic enzyme genes in the mandibular organ in vitro,indicating that SpAST-B had an inhibitory effect on MF biosynthesis.Finally,injection of SpAST-B significantly restrained oocytes growth but had no effect on the expression of SpVg and SpVgR.In summary,these results suggested that SpAST-B may inhibit the 20E biosynthesis by Y-organ to regulate ovary development.Given the localization of SpAST-BR in the oocytes,we believed that SpAST-B might inhibit ovarian development directly or/and through Y-organ indirectly.3)This study investigated the tissue distribution patterns of C-type allatostatin and its receptor in the mud crab S.paramamosain,and further explored its potential role in ovarian development.Firstly,the cDNAs encoding C-type allatostatin precursor and its putative receptor were isolated,respectively.Subsequently,RT-PCR results suggested that,SpAST-C was mainly expressed in the nervous tissue,middle gut and the heart while SpAST-CR had an extensive expression in the detected tissues except the eyestalk ganglion and hepatopancreas.Furthermore,the SpAST-C expressing cells in the cerebral ganglion were detected through in situ hybridization,it showed that SpAST-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 SpAST-C had no effect on the abundance of SpVg in the hepatopancreas and ovary but significantly reduced the expression of SpVgR in the ovary in a dose-dependent manner.Furthermore,it was demonstrated that the SpVgR expression,Vn content,and oocyte diameter in ovary were reduced after 16-days injection of SpAST-C.Finally,the transcripts of SpAST-CR were specifically localized in the oocytes of ovary by in situ hybridization,which further revealed that the oocytes were target cells for SpAST-C.In conclusion,our results suggested that SpAST-C act as a circulating hormone to directly inhibit the absorption of Vg by oocytes and obstruct oocyte growth.4)In this study,the potential roles of SpAST-C and SpAST-CCC in regulating endocrine activites that occur in the Y-organ and mandibular organ were investigated.Firstly,the ligand-receptor binding assay confirmed that SpAST-CR was the receptor of both SpAST-C and SpAST-CCC,which coupled Gi? protein to inhibit the activity of adenylate cyclase that reducing the level of cAMP in cytoplasm.In situ hybridization showed that SpAST-CR was extensively expressed in the glandular cells of Y-organ.Subsequently,both in vivo and in vitro experiments suggested that,SpAST-C had an inhibitory role in the 20E biosynthesis by the Y-organ,while SpAST-CCC had no effect on the 20E biosynthesis.In addtion,the in vitro evidence suggested that both SpAST-C and SpAST-CCC significantly reduced the abundance of expression of MF biosynthetic enzyme genes in the mandibular organ,which indicated that these two peptides might inhibit the MF biosynthesis in mandibular organ.In conclusion,this study reported for the first time that AST-C inhibits 20E biosynthesis and AST-C family peptides might have inhibitory effect on MF biosynthesis in crustaceans.