Characterization of a novel immune deficiency gene of Macrobrachium rosenbergii reveals antibacterial and antiviral defenses.

OBJECTIVE: We sought to identify and characterize an immune deficiency (IMD) homolog from the giant freshwater prawn (also known as the giant river prawn) Macrobrachium rosenbergii. The IMD is a death-domain-containing protein that plays a crucial role as an adaptor protein in the IMD pathway-one of the most important response mechanisms to viral and bacterial invasion of invertebrates. METHODS: An IMD homolog gene from M. rosenbergii (MrIMD) was isolated using rapid amplification of complementary DNA ends. The tissue distribution and response to immune challenge of MrIMD were analyzed by real-time reverse transcription polymerase chain reaction to understand the regulatory mechanism of MrIMD messenger RNA (mRNA) expression in M. rosenbergii. RESULT: The open reading frame of MrIMD comprised 555 nucleotides encoding a protein consisting of 184 amino acids, with a conserved death domain at the C-terminus. The MrIMD protein demonstrated 53-74% similarity with IMDs from other crustaceans; the highest similarity was with the IMD from the oriental river prawn M. nipponense. Gene expression analysis revealed that MrIMD mRNA levels were highest in gill tissues. After Aeromonas hydrophila stimulation, MrIMD was significantly upregulated in the muscle, gills, and intestine, whereas there was no significant difference in the hemocytes and hepatopancreas. In the case of Macrobrachium rosenbergii nodavirus stimulation, MrIMD was dramatically upregulated in the muscle and hepatopancreas, whereas downregulation was observed in the gills. CONCLUSION: These results suggest that the MrIMD gene may play different roles in response to gram-negative bacteria and viral infection and plays a crucial role in innate immunity as an important key molecule in the defense against bacterial and viral infections.

A novel tumor necrosis factor receptor-associated factor 6 (TRAF6) gene from Macrobrachiumrosenbergii involved in antibacterial defense against Aeromonas hydrophila.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein that triggers downstream cascades mediated by both TNFR and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF6 is involved in various biological processes, including innate and adaptive immunity. In the present study, a homolog of TRAF6 from Macrobrachium rosenbergii (MrTRAF6) was identified and characterized. The full-length cDNA of MrTRAF6 consisted of 2,114 nucleotides with an open reading frame (ORF) of 1,695 nucleotides encoding a 564-amino acid protein that contained a conserved TRAF family motif including two RING-type zinc fingers and a C-terminal meprin and TRAF homology (MATH) domain. The putative amino sequence of MrTRAF6 shared 45.5-97.3% identity with TRAF6s from other crustacean species with the highest identity to Macrobrachium nipponense TRAF6. Phylogenetic analysis revealed that MrTRAF6 was closely related to TRAF6 of invertebrates and clustered with crustaceans. According to gene expression analysis, the MrTRAF6 transcript demonstrated broad expression in all tissues tested, with the highest expression level in gill and the lowest in muscle tissues. Upon immune challenge with Aeromonas hydrophila, significant upregulation of MrTRAF6 expression was found in the gill, hepatopancreas, hemocyte, and muscle. Furthermore, an RNA interference assay showed that silencing MrTRAF6 by dsRNA could reduce the expression of mannose-binding lectin (MBL) and crustin, but no significant change was detected in anti-lipopolysaccharide factor 5 (ALF5) levels. In addition, the cumulative mortality rate of MrTRAF6-silenced M. rosenbergii was significantly increased after A. hydrophila infection. These findings indicated that MrTRAF6 is involved in antibacterial activity and plays a critical role in the innate immune response of M. rosenbergii.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.