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Crustacean cardioactive peptide (CCAP), a cyclic amidated non-apeptide, is widely found in arthropods. The functions of CCAP have been revealed to include regulation of heart rate, intestinal peristalsis, molting, and osmotic pressure. However, to date, there has not been any report on the possible involvement of CCAP in immunoregulation in crustaceans. In this study, a CCAP precursor (designated as  Sp -CCAP) was identified in the commercially important mud crab  Scylla paramamosain , which could be processed into four CCAP-associated peptides and one mature peptide (PFCNAFTGC-NH 2 ). Bioinformatics analysis indicated that  Sp -CCAP was highly conserved in crustaceans. RT-PCR results revealed that  Sp-CCAP  was expressed in nerve tissues and gonads, whereas the  Sp -CCAP receptor gene ( Sp-CCAPR ) was expressed in 12 tissues of  S. paramamosain , including hepatopancreas.  In situ  hybridization further showed that an  Sp-CCAPR -positive signal is mainly localized in the F-cells of hepatopancreas. Moreover, the mRNA expression level of  Sp-CCAPR  in the hepatopancreas was significantly up-regulated after lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge. Meanwhile, the mRNA expression level of  Sp-CCAPR , nuclear transcription factor NF-κB homologs ( Sp-Dorsal  and  Sp-Relish ), member of mitogen-activated protein kinase (MAPK) signaling pathway ( Sp-P38 ), pro-inflammatory cytokines factor ( Sp-TNFSF  and  Sp-IL16 ), and antimicrobial peptide ( Sp-Lysozyme, Sp-ALF, Sp-ALF4 , and  Sp-ALF5 ) in the hepatopancreas were all up-regulated after the administration of synthetic  Sp -CCAP mature peptide both  in vivo  and  in vitro . The addition of synthetic  Sp -CCAP mature peptide  in vitro  also led to an increase in nitric oxide (NO) concentration and an improved bacterial clearance ability in the hepatopancreas culture medium. The present study suggested that  Sp -CCAP signaling system might be involved in the immune responses of  S. paramamosain  by activating immune molecules on the hepatopancreas. Collectively, our findings shed new light on neuroendocrine-immune regulatory system in arthropods and could potentially provide a new strategy for disease prevention and control for mud crab aquaculture.

A Possible Role of Crustacean Cardioactive Peptide in Regulating Immune Response in Hepatopancreas of Mud Crab