Effects of Vibrio alginolyticus infection on immune-related enzyme activities and ultrastructure of Charybdis japonica gills

• Vibrio alginolyticus induces innate immune enzyme activities in Charybdis japonica.
• Polysaccharides show immunoprotection against V. alginolyticus in C. japonica.
• V. alginolyticus infection damages ultrastructure of gills in C. japonica.

In this study, the pathogenic mechanism of a Gram-negative bacterium Vibrio alginolyticus in the stone crab Charybdis japonica was explored by investigating activities of innate immune enzymes during infection, including phenoloxidase (PO), superoxide dismutase (SOD) and lysozyme (LSZ). Four groups of crabs were tested by intramuscular injection of C. japonica: control group, normal saline; (Group I) 0.5% polysaccharides; (Group II) normal saline plus V. alginolyticus challenge 24 h later; (Group III) 0.5% polysaccharides plus V. alginolyticus challenge 24 h later. After 6–24 h of exposure to V. alginolyticus, activities of PO, SOD and LSZ increased in C. japonica gills but then sharply decreased as infection progressed. Except for SOD, activities of the other enzymes in the two Vibrio-infected groups (II and III) were significantly lower than those of controls at 120 h post-exposure (P < 0.05). Interestingly, enzyme activities in crabs treated with polysaccharides before challenge (Group III) were higher than those in the non-PS challenged infected crabs (Group II), resulting in an immunoprotective rate of 72.73% at 7 days post-exposure. This phenomenon suggested that the polysaccharides could enhance the organism's antiviral defenses by improving activities of immune-related enzymes. Morphological examination by transmission electron microscopy revealed significant damage in the gill epithelium of V. alginolyticus-infected crabs (Group II), including altered structures of the mitochondria and rough endoplasmic reticulum (RER), as well as electron dense deposits. Although heterochromatinized nuclei in hemocytes with ruptured nuclear membranes due to infection could still be seen in Group III, PS challenged in these crabs ameliorated the damage to a certain extent as the structure of gill cuticles was normal, and the granules in gill hemocytes were increased. This study furthers our understanding of the biochemical alterations induced by vibrio infections, including changes in the antioxidant status, oxidative stress and LSZ activity, which may help to advance strategies for control of V. alginolyticus in crabs.