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Chitin deacetylases have been identified and studied in several fungi and insects but not in crustaceans. These glycoproteins function in catalyzing the conversion of chitin to chitosan by the hydrolysis of N-acetamido bonds of chitin. Here, for the first time, the full length cDNA of chitin deacetylase (CDA) gene from crustaceans was fully cloned using a partial fragment obtained from a transcriptome database of the gills of black tiger shrimp Penaeus monodon that survived White Spot Syndrome Virus (WSSV) infection employing Rapid Amplification of cDNA Ends (RACE) PCR. The shrimp CDA, named PmCDA1, was further characterized by in silico analysis, and its constitutive expression determined in apparently healthy shrimp through reverse transcription PCR (RT-PCR). Results revealed that the P. monodon chitin deacetylase (PmCDA1) is 2176 bp-long gene with an open reading frame (ORF) of 1596 bp encoding for 532 amino acids. Phylogenetic analysis revealed that PmCDA1 belongs to Group I CDAs together with CDA1 and CDA2 proteins found in insects. Moreover, PmCDA1 is composed of a conserved chitin-binding peritrophin-A domain (CBD), a low-density lipoprotein receptor class A domain (LDL-A) and a catalytic domain that is part of CE4 superfamily, all found in group I CDAs, which are known to serve critical immune function against WSSV. Finally, high expression of PmCDA1 gene in the gills of apparently healthy P. monodon was observed suggesting important basal function of the gene in this tissue. Taken together, this is a first report of the full chitin deacetylase 1 (CDA1) gene in crustaceans particularly in shrimp that exhibits putative immune function against WSSV and is distinctly highly expressed in the gills of shrimp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pattern recognition receptors (PRRs) are involved in the effective innate defense against several microbes. Here, we identified a nucleotide-oligomerization domain (NOD)-like receptor subfamily C (NLRC) from Japanese flounder (Paralichthys olivaceus). Full-length transcript of JfNLRC is composed of 3976 bp encoding a protein of 1175 deduced amino acid residues. The presence of a signature nucleotide-binding domain (NACHT) and leucine-rich repeated domain (LRR) suggested that the protein is a member of the NLR family. Interestingly, its C-terminus presents an extra PRY/SPRY (B30.2) domain similar to fish in the Trim (finTrim) family. A phylogenic tree of JfNLRC revealed that full-length JfNLRC diverged from the NOD1 and NOD2 clusters, and the NACHT domain in JfNLRC was clustered within the NLRC3 group. Stimulation by formalin-killed Edwardsiella tarda, Streptococcus iniae, and lipopolysaccharide (LPS) showed that the JfNLRC expression was raised a few hours after stimulation, suggesting this novel protein is involved in the immediate response against both Gram-positive and Gram-negative bacteria. Furthermore, the IL-1β mRNA expression level in JfNLRC-over-expressing HINAE cells was significantly increased, when compared to a control, after LPS-stimulation and E. tarda infection. These results suggested that JfNLRC probably induced IL-1β gene expression mediated by LPS-stimulation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cell and determinant markers are important in fish immunology and have vast applications in aquaculture but the availability of such markers are quite limited. Hence, there is a need to identify and also further improve existing markers in fish. Here, we developed effective polyclonal antibodies (pAbs) targeting specific parts of the Japanese flounder (Paralichthys olivaceus) IgM constant (C) region. Recombinant proteins from the CHmu2 and CHmu3 termed IgM fragment 1 (rIgM1) and from CHmu4 termed IgM fragment 2 (rIgM2) were expressed and used to construct mouse pAb-IgM1 and pAb-IgM2, respectively. pAb-IgM1 detected both the approximately 77 kDa and the approximately 72 kDa heavy chains detected while pAb-IgM2 marked only the approximately 77 kDa heavy chain of Japanese flounder. Both pAbs detected IgM heavy chain in immune-related tissues, heart and serum. pAb-IgM2, but not pAb-IgM1, revealed cross reactions with other fish species detecting pronounced multiple IgM bands suggesting that the CHmu4 is an important functional region in the teleost IgM molecules. Finally, the pAb-IgMs detected surface IgM+ (sIgM+) and cytoplasmic IgM+ (cIgM+) B cells in Japanese flounder kidney in vivo.

Members of the interleukin 6 (IL6)-cytokine subfamily of proteins are involved in numerous physiological processes including cellular development, inflammatory function, and acute phase and immune responses. Previously, a cytokine-like gene named M17, which is closely associated with the IL6 subfamily, has been identified in fish with no apparent orthologue in higher vertebrates. Here, we cloned a novel cDNA from Japanese flounder, Paralichthys olivaceus, which had significant identity but exhibited contrasting expression with fish M17s, named here as M17 Homologue (MSH). With subsequent in silico search and full annotation of the M17 orthologue in zebrafish (Danio rerio), MSH orthologues in tiger puffer (Takifugu rubripes), green spotted pufferfish (Tetraodon nigroviridis) and stickleback (Gastorosteus aculeatus), as well as structural, synteny comparisons and phylogenetic analysis with known IL6-cytokines, we determined the novelty of the fish MSH. Japanese flounder MSH was observed to be highly expressed in immune-related tissues and are induced by immune stimulants, lipopolysaccharide (LPS), polyI:C and peptidoglycan (PG) in vitro suggesting that it is involved in fish immunity particularly against viral and bacterial agents, a functional feature exhibited by previously reported fish cytokines.