Function of the MOB kinase activator-like 1 in the innate immune defense of the oriental river prawn (Macrobrachium nipponense).

The monopolar spindle one binder (MOB) protein, a key signal transducer of the Hippo signaling pathway, is involved in growth control and cancer. In this study, a new MOB kinase activator-like 1 of the oriental river prawns, Macrobrachium nipponense, (MnMOB1) was isolated and characterized. The open reading frame of MnMOB1 consisted of 651 nucleotides that encoded 216 amino acid residues and contained the Mob1_phocein domain. Phylogenetic analysis revealed that MnMOB1 clustered together with the MOB1 from Penaeus vannamei. The distribution of MnMOB1 expression in various tissues of normal prawn revealed that the MnMOB1 expression was highest in the hepatopancreas followed by those in the intestines, gill, heart, stomach, and hemocytes. In prawns challenged with Staphylococcus aureus and Vibrio parahaemolyticus, the expression levels of MnMOB1 in the hepatopancreas, gills, and intestine were upregulated. Furthermore, the expression levels of crustins and anti-lipopolysaccharide factors in prawn injected with S. aureus and V. parahaemolyticus and MnMOB1 knockdown were significantly decreased relative to those in the control group. These findings indicated that MnMOB1 is involved in the regulation of antimicrobial peptide expression and plays a crucial role in the innate immunity of M. nipponense.

Characterization of an immune deficiency (IMD) homolog from the oriental river prawn, Macrobrachium nipponense.

The immune deficiency (IMD) signal pathway mediates innate immunity against Gram-negative bacteria in crustaceans. In the present study, an IMD homolog (MnIMD) from the oriental river prawn, Macrobrachium nipponense was identified. The full-length cDNA of MnIMD was 782bp with an open reading frame of 549 bp that encodes a putative protein of 182 amino acids including a death domain at the C-terminus. Multiple alignment analysis showed that IMDs in prawn M. nipponense and other crustaceans shared high similarity. The recombinant protein of MnIMD was expressed and purified for further functional analyses. Western blot analysis indicated that MnIMD was present in many tissues, but with the highest level in the gills, which was consistent with the qRT-PCR results. After Vibrio parahaemolyticus challenge, MnIMD was significantly induced in gills. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of different antimicrobial peptide (AMP) genes, including Cru4 and Cru6. These results are helpful in promoting research on the innate immunity in M. nipponense.

Single CRD containing lectin from Macrobrachium rosenbergii (MrLec) participates in innate immunity against pathogen infections.

As a type of pattern-recognition proteins, lectins perform important functions in the innate immunity of crustaceans, including prawns. Although several reports showed that C-type lectin domain family (CLEC) importantly functions in host-pathogen interactions, limited research has focused on CLEC in Macrobrachium rosenbergii. In the present study, a new single CRD containing CLEC (designated as MrLec) was reported in freshwater prawns, M. rosenbergii. The full-length cDNA of MrLec consisted of 1027 bp with an open reading frame of 801 bp, which encoded a peptide of 266 amino acid residues. Genomic sequence for MrLec was also obtained from the M. rosenbergii, which contain 4 exons and 3 introns. MrLec was found to contain a single carbohydrate-recognition domain with an EPN motif. MrLec was ubiquitously distributed in various tissues of a normal prawn, particularly in the hepatopancreas and gills. MrLec expression in the gills was significantly upregulated after a challenge with Vibrio parahaemolyticus and downregulated at 24 h after MrLec RNA interference (MrLec-RNAi). The expression levels of some AMPs, including antilipopolysaccharide factor 1 (Alf1) and lysozyme 2 (Lyso2), also markedly decreased after MrLec-RNAi. Recombinant MrLec can agglutinate (Ca(2+)-dependent) and bind both Gram-negative and Gram-positive bacteria. Results suggested that MrLec participates in the recognition of invading pathogens and functions in the immune response of prawn against pathogen infections.

Involvement of Relish gene from Macrobrachium rosenbergii in the expression of anti-microbial peptides.

Relish is an NF-kB transcription factor involved in immune-deficiency (IMD) signal pathway. In this study, a Relish gene (MrRelish) was identified from Macrobrachium rosenbergii. The full length of MrRelish comprises 5072 bp, including a 3510 bp open reading frame encoding a 1169 bp amino acid protein. MrRelish contains a Rel homology domain (RHD), a nucleus localization signal, an IκB-like domain (6 ankyrin repeats), and a death domain. Phylogenetic analysis showed that MrRelish and other Relish from crustaceans belong to one group. MrRelish was expressed in all detected tissues, with the highest expression level in hemocytes and intestines. MrRelish was also upregulated in hepatopancreas at 6 h after Vibrio anguillarum challenge. The over-expression of MrRelish could induce the expression of antimicrobial peptides (AMPs), such as Drosophila Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), and Cecropin (CecA) and shrimp Penaeidin (Pen4). The RNAi of MrRelish in gills showed that the expression of crustin (cru) 2, Cru5, Cru8, lysozyme (Lyso) 1, and Lyso2 was inhibited. However, the expression of anti-lipopolysaccharide factor (ALF) 1 and ALF3 did not change when MrRelish was knocked down. These results indicate that MrRelish may play an important role in innate immune defense against V. anguillarum in M. rosenbergii.