Characterization of hemocyanin from the mud crab Scylla paramamosain and its expression analysis in different tissues, at various stages, and under Vibrio parahaemolyticus infection.

Hemocyanin is an important respiratory protein in many arthropod and mollusk species. Here, four cDNAs (SpHc1, SpHc2, SpHc3, and SpHc4), encoding distinct hemocyanin subunits from Scylla paramamosain were cloned using EST analyses and the rapid amplification of cDNA ends. The four full-length cDNA fragments (SpHc1-4) were 2281, 2002, 2184, and 2069 bp, respectively, and they encoded four putative proteins (570-676 amino acids) with a molecular mass of ~65.0-76.8 kDa. Quantitative real-time PCR analyses revealed that the four genes were mainly expressed in the hepatopancreas, testis, and hemocytes. SpHc mRNA expression during continuous developmental stages in zoeal phases (Z1, Z2, Z3, Z4, and Z5), megalopa, and juvenile crab I stages were also detected. The expression levels of SpHc3 and SpHc4 were higher than that of SpHc1 and SpHc2 during the first six stages, and they sharply declined during the juvenile stage. After infection with Vibrio parahaemolyticus, the temporal expression of both the four SpHc mRNAs in the megalopa stage rapidly declined during the first 3 h, followed by upregulation and peak expression at 12 h after the challenge. The expression levels of the four SpHc subunits were upregulated at 48 h after the challenge, and were then gradually downregulated. These findings suggest that hemocyanin may potentially be involved in the crab immune response, and that the role of the four subunits may differ in different tissues and during various developmental stages.