Chronic environmentally relevant concentration of copper exposure induces intestinal oxidative stress, inflammation, and microbiota disturbance in freshwater grouper (Acrossocheilus fasciatus)

The influence of chronic environmentally relevant concentration of Cu2+ exposure on fish intestinal health has been rarely studied. In the present study, freshwater grouper (Acrossocheilus fasciatus) was subjected to 0 (control), 0.01 mg/L Cu2+ (Cu0.01), and 0.04 mg/L Cu2+ (Cu0.04) for 30 days. The Cu0.04 group obtained a significantly reduced survival rate, weight gain, and feed intake compared to the control group (P < 0.05). Both levels of Cu2+ exposure induced oxidative stress, evidenced by increased antioxidant enzymes' activities and malondialdehyde (MDA) contents in the intestine and serum. Based on 16S rDNA analysis, both levels of Cu2+ exposure significantly reduced intestinal microbiota community richness. In the Cu2+ exposure groups, Firmicutes/Bacteroidota ratio, and potentially pathogenic bacteria, such as Proteobacteria, genus Pseudomonas, Citrobacter, Shinella, and Aeromonas were enriched. Meanwhile, the richness of probiotic bacteria, such as Fusobacteriota, Planctomycetota, Cetobacterium, Gemmobacter, and Gemmata were significantly reduced by Cu2+ exposure. Both levels of Cu2+ exposure significantly reduced villus length, lamina propria width, and muscular thickness in the foregut and hindgut, but increased intestinal goblet cell numbers. 0.04 mg/L Cu2+ exposure significantly upregulated superoxide dismutase (sod), pro-inflammation genes nuclear factor kappa b subunit 1 (nfκb1) and interleukin 1 beta (il1β) expression, but downregulated anti-inflammation gene transforming growth factor beta 1 (tgfβ1) expression. In summary, chronic environmentally relevant concentrations of Cu2+ exposure induced intestinal oxidative stress, inflammation, prevalence of pathogen and inhibition of probiotic bacteria, and damage intestinal integrity of freshwater grouper.