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

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.

Keywords: arthropod; crustacean cardioactive peptide; hepatopancreas; immunoregulation; neuropeptide.

Figure 1

Nucleotide and deduced-amino acid sequences of Sp-CCAP cDNA. The initiation codon, termination codon, signal peptide, crustacean cardioactive peptide (CCAP) mature peptide, CCAP-associated peptides, amidation site, cleavage sites, and cysteine residues are marked by different symbols.

Figure 2

Multiple alignments of the deduced amino acid sequence of crustacean cardioactive peptides (CCAPs) among various crustacean species. The CCAP mature peptide is indicated by the red line; conserved amino acid residues are marked by asterisks. GenBank accession numbers of CCAPs are as follows: Callinectes sapidus (ABB46290.1); Carcinus maenas (ABB46291.1); Portunus trituberculatus (AVK43051.1); Penaeus vannamei (ALP06206.1); Homarus gammarus (ABB46292.1); and Procambarus clarkia (BAF34910.1).

Figure 3

Phylogenetic analysis of crustacean cardioactive peptides (CCAPs) relative to various crustacean and insect species. The sequences used in evolutionary tree analysis include Callinectes sapidus (ABB46290.1); Carcinus maenas (ABB46291.1); Portunus trituberculatus (AVK43051.1); Homarus gammarus (ABB46292.1); Procambarus clarkia (BAF34910.1); Nephrops norvegicus (QBX89037.1); Cherax quadricarinatus (AWK57511.1); Penaeus vannamei (ALP06206.1); Macrobrachium nipponense (ASH96804.1); Periplaneta Americana (Q75UG5.1); Rhodnius prolixus (ACZ52615.1); Nilaparvata lugens (BAO00946.1); Nicrophorus vespilloides (XP_017778790.1); and Orchesella cincta (ODM98622.1).

Figure 4

Tissue distribution of Sp-CCAP and Sp-CCAPR in S. paramamosain. 1, eyestalk ganglion; 2, cerebral ganglion; 3, thoracic ganglion; 4, gill; 5, hepatopancreas; 6, hemocytes; 7, stomach; 8, midgut; 9, heart; 10, epidermis; 11, muscle; 12, gonad; and 13, the negative control.

Figure 5

Localization of Sp-CCAPR mRNA in the hepatopancreas by in situ hybridization. (A) Positive signals with the antisense probes. (B) Sense probes of Sp-CCAPR served as the negative control. (C) Histological observation of hepatopancreatic tubule epithelial cells: E, E-cell (E: embryonic); F, F-cell (F: fibrillar); B, B-cell (B: blisterlike); and R-cell (R: resorptive).

Figure 6

Changes in mRNA expression of Sp-CCAPR in the hepatopancreas after lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid [Poly (I:C)] injection. (A) After LPS stimulation. (B) After Poly (I:C) stimulation. All data are shown as mean ± SEM (n = 5); statistical analysis was performed using Student's t-test. *indicates significant difference (p < 0.05).

Figure 7

Effects of Sp-CCAP injection on the mRNA expressions of immune-related genes in the hepatopancreas. (A) Sp-CCAPR; (B) Sp-P38; (C) Sp-Dorsal; (D) Sp-Relish; (E) Sp-TNFSF; (F) Sp-IL16; (G) Sp-Lysozyme; (H) Sp-ALF1; (I) Sp-ALF4; (J) Sp-ALF5. All data are shown as mean ± SEM (n = 5); statistical analysis by Student's t-test. * and **on the top of bars indicate significant (p < 0.05) and highly significant differences (p < 0.01), respectively.

Figure 8

Effects of Sp-CCAP addition at different concentrations on mRNA expressions of immune-related genes in in vitro cultured hepatopancreas tissues and NO concentration in the culture media. (A) Sp-CCAPR; (B) Sp-P38; (C) Sp-Dorsal; (D) Sp-Relish; (E) Sp-TNFSF; (F) Sp-IL16; (G) Sp-Lysozyme; (H) Sp-ALF1; (I) Sp-ALF4; (J) Sp-ALF5; and (K) the concentration of NO. All data are shown as mean ± SEM (n = 4); statistical analysis performed by one-way ANOVA followed by Duncan's test. * and **on top of bars indicate significant (p < 0.05) and highly significant differences (p < 0.01), respectively.

Figure 9

Bacterial clearance capacity of hepatopancreas culture medium treated with different concentrations of Sp-CCAP as compared with the no Sp-CCAP addition control. (A) Results with Staphylococcus aureus. (B) S. aureus colonies grown on Luria–Bertani (LB) plates. (C) Results with Vibrio parahaemolyticus. (D) S. aureus colonies grown on 2216E plates. All data are shown as mean ± SEM (n = 4); statistical analysis performed by one-way ANOVA followed by Duncan's test. * and **on top of bars indicate significant (p < 0.05) and highly significant differences (p < 0.01), respectively.