The mud crab, Scylla paramamosain is an important marine economically farmed crab in China, widely distributed on the southeast coast of China. The prevalent mode of mixed artificial culture in ponds often leads to outbreaks of several diseases in crabs, among which, fish and crustacean diseases caused by Pseudomonas putida have been reported and concerned. In our previous study, P. putida was also isolated from the testes of S. paramamosain. Interestingly, the expression of two gonadally highly expressed antimicrobial peptides, SCY2 and Scyrepocin, were significantly up-regulated. In this study, we found that P. Putida colonized mainly the hepatopancreas and gills, causing damage to the hepatopancreas basal membrane and inducing vacuolization within the hepatic tubules; in the gills, the pathogefn led to a reduction of connective tissues and a blurring of the septum. We found that the transcript and protein expression levels of both SCY2 and Scyreprocin were significantly altered after bacterial infection, and the positive fluorescence signal intensity of both proteins was remarkably enhanced. To further investigate the immune effects of the two proteins in crabs, the recombinant proteins were first obtained through a prokaryotic expression system. The results showed that the survival rate of crabs in the two protein-treated groups was significantly increased. Meanwhile, the expression levels of some immune-related pathway genes and antimicrobial peptide genes, and the activity of oxidative enzymes were restored to the normal level. The results implied that SCY2 and Scyreprocin had a significant anti-infective effect in male crabs, and could effectively inhibit inflammatory responses and play an immunomodulatory role. Using the antibody-blocking technique, the corresponding polyclonal antibodies to the proteins were injected into female crabs through the germinal pore. Flow cytometry analysis results showed that the fluorescence intensities of the two proteins were significantly inhibited. Subsequent infection with P. putida revealed a dramatic decrease in the survival rate of female crabs, and the bacterial clearance rate in the spermathecae was significantly lower than that of the control group. Moreover, the expression levels of spToll2, spDorsal, and spPelle were significantly upregulated, SOD activity was reduced, and MDA levels were increased. These results indicated that the blockade of SCY2 and Scyreprocin led to elevated levels of inflammation and disturbed immune homeostasis in female crabs.

In summary, this study revealed the immunological role of gonadally highly expressed antimicrobial peptides against P.putida in S.paramamosain, especially exerting an in vivo anti-infective role in the protection of crab during fertilization.