Immunomodulatory Role Of Crustacean Cardioactive Peptide In The Mud Crab,Scylla Paramamosain

Crustacean cardioactive peptide(CCAP)is a C-terminal amidated cyclic nonapeptide hormone widely found in arthropods.It is involved in the regulation of various physiological and metabolic processes,including heartbeat,ecdysis behavior,gut contraction,osmotic regulation and reproduction.In recent years,a lot of evidence have shown that neuropeptides play an indispensable role in the neuroendocrine immune regulation of invertebrates.However,there has not been any report on the involvement of CCAP in immunoregulation in crustaceans.The mud crab,Scylla paramamosain,is widely distributed in the Indo-Pacific Ocean and mangrove ecosystems,and is one of the important mariculture crabs in China.Farmed crabs are vulnerable to various bacteria,fungi,viruses and other pathogens,which severely restricts the healthy and sustainable development of the crab aquaculture industry.In this study,a CCAP precursor(designated as Sp-CCAP)was identified from S.paramamosain.The tissue distribution and in situ hybridization localization of SpCCAP/Sp-CCAPR were detected.In vivo and in vitro experiments were carried to explore the immunoregulatory effects of Sp-CCAP signaling system in the neuroendocrine-immune regulatory system(NEI)of crustaceans.The main results are as follows:1)Sp-CCAP could be processed into four CCAP associated peptides and one mature peptide(PFCNAFTGC-NH2).Bioinformatics analysis indicated that Sp-CCAP was highly conserved in crustaceans.RT-PCR results showed that Sp-CCAP was expressed in nervous system and gonads,while Sp-CCAPR was expressed in 11 tissues and hemocytes of S.paramamosain.In situ hybridization further showed that Sp-CCAP positive signal mainly localized in all types of cells in the nervous tissue,while SpCCAPR positive signal mainly localized in the semigranular cells of hemocytes and localized in the F-cells of hepatopancreas.2)The Sp-CCAP mRNA expression level in the thoracic ganglion was significantly up-regulated after lipopolysaccharide(LPS)stimulation,while the SpCCAP mRNA expression level was up-regulated first and then down-regulated after polyriboinosinic polyribocytidylic acid(Poly(I:C))stimulation.After injection of SpCCAP synthesis peptide,the phagocytosis ability of hemocytes was significantly higher than that of synchronous control group.Meanwhile,the mRNA expression of phagocytosis related gene(Sp-Rab5),nuclear transcription factor NF-κB homologues(Sp-Relish),C-type lectin(Sp-CTL-B),prophenoloxidase(Sp-proPO),proinflammatory cytokines factor(Sp-TNFSF,Sp-IL16)and antimicrobial peptide(SpALF1 and Sp-ALF5)in the hemocytes were also significantly up-regulated at different time points after the injection of Sp-CCAP synthetic peptide,but Sp-TNFSF,Sp-ALF1 and Sp-ALF5 were down-regulated significantly at 24h.In addition,RNA interference with Sp-CCAP in the thoracic ganglion can inhibit the phagocytic activity of hemocyte and inhibit the mRNA expression of Sp-Rab5,Sp-Relish,Sp-CTL-B,Sp-TNFSF,SpIL16 and Sp-ALF5 in the hemocytes,and ultimately weaken the ability of haemolymph bacteria clearance of mud crab.3)Both LPS and Poly(I:C)stimulation promoted the Sp-CCAPR mRNA expression in the hepatopancreas.Meanwhile,the mRNA expression level of SpCCAPR,Sp-Dorsal,Sp-Relish,Sp-P38,Sp-TNFSF,Sp-IL16 and antimicrobial peptide(Sp-Lysozyme,Sp-ALF,Sp-ALF4 and Sp-ALF5)in the hepatopancreas were all upregulated after the administration of Sp-CCAP synthetic peptide both in vivo and in vitro.The administration of Sp-CCAP synthetic peptide in vitro also increased nitric oxide(NO)concentration,and enhanced bacterial clearance ability in the hepatopancreas culture medium.In summary,Sp-CCAP can act on both the hemocytes and hepatopancreas of mud crab in a neuroendocrine manner,and participate in the regulation of the immune response.These findings provide novel insights into the regulatory mechanism of the CCAP signaling system involved in the immune response.This study could enrich the researches on the immune mechanism mediated by neuropeptides and their receptors in invertebrates.It will provide a new strategy for disease prevention and control for crab aquaculture from the perspective of the neuroendocrine-immune regulatory system.