Characterization and immune functional analysis of two new type I crustins in the oriental river prawn Macrobrachium nipponense

Crustins are cationic antimicrobial peptides that are categorized into five types and play a key role in the nonspecific immunity of crustaceans. In the present study, full-length cDNA sequences of two Macrobrachium nipponense crustin genes (MnCrustin1 and MnCrustin2) were cloned and identified. The full-length cDNAs of MnCrustin1 and MnCrustin2 were 715 and 881 bp, including 351 and 330 bp open reading frames (ORFs) encoding 117 and 110 amino acids, respectively. A signal peptide, a cysteine (Cys)-rich region and a whey acidic protein (WAP) domain existed in both MnCrustin1 and MnCrustin2. Phylogenetic analysis and multiple sequence alignment indicated that MnCrustin1 and MnCrustin2 belonged to the type I crustin family. The MnCrustin1 and MnCrustin2 mRNAs were expressed in various tissues of M. nipponense and were highly expressed in hemocytes and gills. Recombinant MnCrustin1 and MnCrustin2 inhibited the growth of both gram-positive and gram-negative bacteria in vitro. After Aeromonas hydrophila and white spot syndrome virus (WSSV) infection, MnCrustin1 and MnCrustin2 expression levels were elevated in the hepatopancreas, hemocytes and gills of M. nipponense. Tests of RNA interference (RNAi) and recombinant protein injection were employed to further study the roles of MnCrustin1 and MnCrustin2 in pathogen infection. The results indicated that silencing MnCrustin1 and MnCrustin2 accelerated the mortality rate of M. nipponense after challenge with A. hydrophila or WSSV, while recombinant MnCrustin1 and MnCrustin2 decreased the mortality rate under these conditions. These results suggest that MnCrustin1 and MnCrustin2 both play prominent roles in the immune response of M. nipponense to bacterial and viral infection and are important immune effectors of this prawn against pathogen infection.