Cloning, identification, and functional characterization of novel prophenoloxidases (ShproPO) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

Prophenoloxidase (proPO) is essential in the prophenoloxidase-activating system (proPO-AS) which is important for defense against foreign infection in crustaceans. However, most studies have focused on expression in the presence of a single pathogenic bacterium, and very few have addressed the presence of environmental contaminants simultaneously, such as cadmium (Cd) and Aeromonas hydrophila. Our study aimed to investigate the function of proPO in the freshwater crab Sinopotamon henanense and the changes in its expression by Cd and infection of A. hydrophila. A novel proPO from the hemocytes of S. henanense (ShproPO) was found in this research, the full-length cDNA of ShproPO was 2620 bp of encoding a protein of 678 amino acids containing three typical hemocyanin domains. The ShproPO protein could be found in both the granular (GHc) and the semi-granular hemocytes (SGHc). The ShproPO mRNA was found to be abundantly expressed in hemocytes and could be influenced by A. hydrophila infection. These results indicate that ShproPO could be involved in the antibacterial process. Further research found that low concentrations of Cd could promote its expression after infection with A. hydrophila. Therefore, it was hypothesized that Cd disrupted the response of crabs to A. hydrophila infection. Subsequently, PO enzyme activity was found to be significantly reduced through in vivo RNA interference with ShproPO, and the results suggested that ShproPO is likely to be a key enzyme in the melanization response. Finally, ShproPO was found to significantly enhance the phagocytosis of A. hydrophila-infected hemocytes by in vitro recombination, confirming that ShproPO is involved in hemocyte-mediated melanization and phagocytosis. Our findings reveal completely new insight into the immunotoxicity of Cd and the immune function of ShproPO in S. henanense.