Immune responses of prophenoloxidase in the mud crab Scylla paramamosain against Vibrio alginolyticus infection: in vivo and in vitro gene silencing evidence.

Phenoloxidase (PO) plays an important role in arthropod melanization. In the present study, a proPO gene was obtained from the mud crab Scylla paramamosain, then we localized the proPO mRNA in hemocytes and detected the expression of proPO after bacterial challenge. In vivo and in vitro gene silencing mediated by dsRNA was also used to investigate the function of proPO in innate immune. The full-length of the proPO cDNA was 2600 bp and the predicted ORF encoded a protein of 673 amino acids with a predicted molecular mass of 77.3 kDa. The deduced amino acid and the main functional domain of proPO shared a high similarity to the mud crab Scylla serrata. In situ hybridization assay showed that the proPO mRNA was localized in the granular and semi-granular cells. The expression level of proPO in hemocytes showed a clear time-dependent pattern during the 96 h course after stimulated by Vibrio alginolyticus. In this study, high expression levels were observed at 3, 12, 24 and 48 h, respectively and the highest expression level was observed at 12 h, and this suggested that proPO was induced by bacteria and involved in immune response. In vivo proPO and GFP dsRNA treatment experiments showed that, proPO mRNA transcript was reduced to 39%, but the PO activity showed no significant difference (P > 0.05). Results indicated that the expression of proPO could be inhibited by dsRNA, and the enzyme activity may be influenced by incomplete knockdown of proPO, or hemocyanin, and other proPO isoforms as well. In vitro proPO-silenced experiments showed that the levels of proPO were decreased by 36%, 64% and 77% at 8, 16 and 32 h, respectively. Meanwhile, the quantity of bacteria was significantly larger in proPO dsRNA treatment than that in control at 3 h, calculated by 4,6-diamino-2-phenylindole staining (P < 0.01). These data demonstrated that the proPO gene plays an important role in the control of systemic bacterial infections and could help us to elucidate the defense role of the proPO-activating system in crabs. In addition, in vitro gene silencing operation mediated by dsRNA was expected to be a new tool for investigating the function of genes in crustaceans in the case of lacking cell line.