Studies on the resistance of three pathogens isolated from wild <i>Charybdis japonica</i> and their pathogenicity in <i>Portunus trituberculatus</i>

The coagulation property of hemolymph is one of the characteristic indicators of the immune function of crustaceans. This article focuses on the abnormal phenomenon of wild Charybdis japonica, where the hemolymph cannot coagulate in the Qinhuangdao Sea area. The pathogens were isolated and purified, and three dominant strains, N1, N2, and Q1, were obtained. These strains were identified as Vibrio natriegens , V. fortis , and Metschnikowia bicuspidata through morphological observation, physiological and biochemical experiments, and 16S rDNA or ITS sequence analysis. Subsequently, drug sensitivity and probiotic antagonism analyses were performed. Considering the potential for cross-species transmission of crustacean pathogens, the isolated pathogens may bring potential hazards to Portunus trituberculus , an important aquaculture economic species in China. Therefore, artificial infection and histopathological analysis were conducted on P. trituberculatus . The results of drug sensitivity and antagonism assays revealed that stain V. natriegens exhibited susceptibilities towards antibiotics such as streptomycin and norfloxacin, but 10 probiotics had no antagonistic effect on it; strain V. fortis exhibited susceptibilities to antibiotics such as tetracycline and chloramphenicol, moreover, strain V. fortis showed inhibitory effects on Bacillus velezensis and B. amyloliquefaciens . Strain M. bicuspidata exhibited drug susceptibility towards polymyxin B and showed inhibitory effects on B. toyonensis , B. velezensis , and B. amyloliquefaciens . The results of artificial infection tests using V. natriegens , V. fortis , and M. bicuspidata in P. trituberculatus showed cumulative mortality rates of 100%, 75%, and 35%, respectively, after 120 hours of infection. The hemolymph of the deceased P. trituberculatus following injection with strain V. natriegens or V. fortis exhibited a light blue and did not coagulate normally. Conversely, the hemolymph of the deceased crabs following injection with strain M. bicuspidata appeared milky white and normal coagulation. Histopathology examination demonstrated that strains V. natriegens , V. fortis , and M. bicuspidata can all induce lesions in several tissues. This study is the first to isolate V. natriegens and V. fortis from crustaceans, and the infection tests on the P. trituberculatus can replicate the symptoms, such as hemolymph noncoagulation. Based on these findings, it is speculated that the pathogens capable of casing hemolymph noncoagulation syndrome in P. trituberculatus may originate from wild C. japonica . The drug resistance and probiotic antagonism test results can provide a theoretical basis for precise medication and offer insights into the development and application of probiotic preparations targeting the three pathogens.