Effects of pathogenic yeast Metschnikowia bicuspidata infection on immune function and intestinal microflora of the Chinese mitten crab Eriocheir sinensis

Abstract

Chinese mitten crab (Eriocheir sinensis) aquaculture has been significantly impeded by “milky disease,” which is caused by the pathogenic yeast Metschnikowia bicuspidata. To investigate the effects of infection by this parasite on the Chinese mitten crab, a suspension of M. bicuspidata (1 × 10⁷ CFU/mL) was injected into healthy crabs at the membrane of the fourth dactyl joint. The changes of non-specific immune-related enzyme activities and the expression levels of related genes in the hemolymph and hepatopancreas of crabs were monitored after infection, as were the effects on the structure of intestinal microflora. In the serum and hepatopancreas, activities of superoxide dismutase and catalase gradually decreased; those of glutathione peroxidase, alkaline phosphatase, and acid phosphatase gradually increased; that of lysozyme increased first and then decreased; and total antioxidant capacity fluctuated over time. Post-infection, the expression levels of several Toll signaling pathway-related genes, including Toll1, Toll2, MyD88, and Dorsal, in the hemocytes and hepatopancreas of crabs were mainly upregulated between 6 and 96 h, with significant downregulation at 192 h. The expression of LZM and Crustin was induced by M. bicuspidata and reached peak levels at 48 or 96 h. The proPO gene was continuously upregulated throughout the experimental period and still exhibited a high expression level at 192 h. The expression trends of Toll1, Toll2, and proPO were similar in hemocytes and the hepatopancreas. The intestinal microbial community composition also changed. At the phylum level, the relative abundance of Firmicutes decreased and the relative abundance of Proteobacteria increased after infection. At the genus level, Gram-negative bacteria such as Aeromonas and Bacteroides were abundant at 96 h post-infection. As infection time increased, the abundance of conditional pathogens such as Shewanella, Vibrio, and Mycobacterium increased. Overall, the results showed that M. bicuspidata induced oxidative stress and inflammatory immune responses in crabs. Infection also caused changes in the intestinal microflora structure, which affected the host's intestinal microecological balance and immune function. These results provided a reference for understanding the pathogenesis of “milky disease” in E. sinensis and devising prevention strategies.