Abstract
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-NH2). 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.
Highlights
The neuroendocrine-immune (NEI) regulatory system refers to a complex network formed by the interaction of the nervous system, endocrine system, and immune system [1]
The full length of Sp-Crustacean cardioactive peptide (CCAP) mRNA is 638 bp with a 64-bp 5′ untranslated region (UTR), a 142-bp 3′UTR, and a 432-bp open reading frames (ORFs) encoding a protein of 143 amino acids with a calculated molecular weight of 15.84 kDa and a theoretical isoelectric point of 9.43 (GenBank Accession MN923209)
Phylogenetic analysis of the amino acid sequences of CCAP precursors among different arthropod species showed that Sp-CCAP and other crustacean CCAP clustered into one branch, whereas insect CCAP clustered into another branch (Figure 3)
Summary
The neuroendocrine-immune (NEI) regulatory system refers to a complex network formed by the interaction of the nervous system, endocrine system, and immune system [1]. The nervous and endocrine systems regulate various physiological processes by releasing neuropeptides, neurotransmitters, and hormones [2]. As an extracellular chemical messenger, neuropeptides regulate a range of physiological functions, including immunity, growth, reproduction, metabolism, food intake, and circadian rhythm, by activating specific receptors [3]. Neuropeptide receptors are mostly G protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors. They play a vital role in physiological processes by promoting cellular communication via recognizing various ligands, including bioactive peptides, nucleosides, and amines [4]
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