Toxic effects of combined exposure to cadmium and nitrate on intestinal morphology, immune response, and microbiota in juvenile Japanese flounder (Paralichthys olivaceus)

Cadmium (Cd2+) and nitrate (NO3-) are important environmental pollutants in the offshore marine ecological environment. However, limited research has explored their combined effects, particularly regarding their impact on the microbiota and intestinal health of marine fish. In this study, juvenile Japanese flounders (P. olivaceus) were immersed in seawater samples with different combinations of Cd2+ (0, 0.2, and 2 mg/L) and NO3- (0 and 80 mg/L NO3-N) for 30 days to explore their toxic impacts on intestinal morphology, tight junction (TJ) barrier, immune response, and microbiota. Our results showed that Cd2+ or NO3- exposure alone led to histopathological damage of the gut, while their co-exposure aggravated intestinal damage. Moreover, co-exposure substantially decreased TJ-related gene expression, including occludin, claudin-10, and ZO-2, suggesting increased TJ permeability in the gut. Regarding the immune response, we observed upregulated expression of immune-related markers such as HSP40, IL-1 beta, TNF-alpha, and MT, suggesting the onset of intestinal inflammation. Furthermore, Cd2+ and NO3- exposure led to changes in intestinal microflora, characterized by decreased the abundance of Sediminibacterium and NS3a_marine_group while increasing the prevalence of pathogens or opportunistic pathogens such as Ralstonia, Proteus, and Staphylococcus. This alteration in microbiota composition increased network complexity and alpha-diversity, ultimately causing dysbiosis in the fish gut. Additionally, combined exposure resulted in metabolic disorders that affected the predicted functions of the intestinal microbiota. Overall, our study demonstrates that Cd2+-NO3- co-exposure amplifies the deleterious effects compared to single exposure. These findings enhance our understanding of the ecological risks posed by Cd2+-NO3- co-exposure in marine ecosystems.