Charybdis japonica is widely distributed along the coasts of China,Japan,Korea and Southeast Asia.Desired for its good taste as well as being rich in protein and low in cholesterol,this crab is currently one of the most important commercially farmed crabs in the world due to its rapid growth and easy culture methods.During the last two decades with the constantly expanding scale of aquaculture and the rapid development of intensive culture techniques in China,the environmental and health problems in C.japonica cultures have resulted in outbreak of infectious diseases.The worldwide shrimp/crab culture industry has suffered significant economic losses due to diseases mainly caused by vibrios,particularly Vibrio alginolyticus,one of the most detrimental pathogens widely disseminated in crabs.In recent years,the treatment of bacterial diseases of aquatic animals has relied on application of large quantities of antibiotics,which not only causes development of drug resistant pathogens but also presents a health risk to humans.Improving immunity and disease resistance in crustaceans is one of the fundamental measures for preventing and controlling disease.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 enzymes related to innate immune function during infection,i.e.phenoloxidase(PO),superoxide dismutase(SOD) and lysozyme(LSZ).Four groups of crabs were tested by intramuscular injection of C.japonica:control group,normal saline; immunity group,0.5% polysaccharide; infected group,normal saline plus V.alginolyticus challenge 24 h later; immunized infected group,0.5% polysaccharide plus V.alginolyticus challenge 24 h later.After 6-12 h of exposure to V.alginolyticus,the activities of PO,SOD and LSZ increased in C.japonica hepatopancreas,but then decreased over longer infection time.Interestingly,enzyme activities in crabs treated with polysaccharides before V.alginolyticus challenge were higher than those in infected group,showing an immunoprotective rate of 72.73% at 7 days after exposure.This phenomenon suggested that the polysaccharides could enhance the organism's antiviral defenses by improving activities of immuno-enzymes.Morphological examination by transmission electron microscopy revealed significant damage in the hepatopancreas of V.alginolyticus- infected crabs,showing parts of the hepatopancreas epithelium and microvilli were reduced in number and size or damaged.Meanwhile,morphology of the mitochondria and rough endoplasmic reticulum(RER) changed.Moreover,heterochromatinized nuclei could be seen in hemocytes with ruptured nuclear membranes.The structure of hepatopancreas nuclei in immunized infected group was normal compared with that in infected group,and the microvilli arranged densely in neat rows.The lysosomes in hepatopancreas cell of immunized infected group increased; however,dropsical endoplasmic reticulum,thin cytoplasm and distorted mitochondria could still be seen.