Characterization and expression of SpHsp60 in hemocytes after challenge to bacterial, osmotic and thermal stress from the mud crab Scylla paramamosain

Hsp60 play a crucial role in the process of pathogenic and protective immune responses and is implicated in autoimmune disease. In order to understand the environmental response and immune response of this gene, we cloned a Hsp60 (SpHsp60) gene from the mud crab Scylla paramamosain, localized SpHsp60 in hemocytes by in situ hybridization, and detected the expression of SpHsp60 after stresses in relation to three housekeeping genes (β-actin, 18S rRNA and GAPDH). The full-length of the SpHsp60 cDNA was found to be 2424 bp. The predicted ORF encoded a protein of 576 amino acids with a predicted molecular mass of 61.19 kDa and a theoretical isoelectric point (pI) of 5.46. It shared high scoring identity 95% with the swimming crab Portunus trituberculatus. In situ hybridization assay showed that a higher expression occurred in the granular and semigranular cells when compared to the hyaline hemocytes. It suggested that SpHsp60 was mainly contributed from the granular and semigranular cells in hemolymph. The expression level of SpHsp60 in hemocytes showed a clear time-dependent pattern during the 96 h after stimulated by Vibrio alginolyticus. During this experiment the gene was induced and the highest expression level was observed at 3 h. The significantly up-regulated expression and rapid response of SpHsp60 indicated that the crabs were sensitive to bacterial challenge. After osmotic stress, the expression of SpHsp60 in hemocytes showed that this gene was induced by the high salinity (30‰) and the crabs had its adaptive responses to high salinity, when compared to the normal salinity (15‰). SpHsp60 mRNA expression in hemocytes was analyzed after thermal stress at 6 h, the highest and the lowest expression levels of SpHsp60 were observed at 36 and 32 °C, respectively. This study demonstrated that SpHsp60 was easily induced at the higher temperatures. Based on our research, SpHsp60 participate in innate immune and environmental response of S. paramamosain. It could be used as a biomarker to test the stress caused by the local aquaculture environment.