Multi-omics insights into copper sulfate toxicity in American shad (Alosa sapidissima): impacts on oxidative stress, cytoskeletal integrity, immune regulation, and gut–metabolism balance

Copper sulfate is widely used in aquaculture to control parasitic diseases, however, its toxicological effects on American shad (Alosa sapidissima) remain poorly understood. Juvenile shad exposed to 0.7 mg·L −1 copper sulfate for 2–8 h exhibited elevated alkaline phosphatase, acid phosphatase (except at 8 h), polyphenol oxidase, malondialdehyde, and aryl hydrocarbon receptor levels, with peak ethoxyresorufin-O-deethylase and ryanodine receptor activities at 4 h, indicating oxidative stress. To investigate the mechanistic responses, mature shads were exposed for 72–144 h. Histopathology revealed compressed hepatic sinusoids and dark-stained hepatocytes, whereas transcriptome profiling identified enrichment of immune and cytoskeletal pathways (endocytosis, lysosome, phagosome, proteasome, Salmonella infection, and actin cytoskeleton regulation) via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differentially expressed genes included upregulation of arpc1b (actin-related protein 2/3 complex subunit 1b), arpc4 (actin-related protein 2/3 complex subunit 4-like isoform X1), loc121683783 (integrin alpha-V-like isoform X1), loc121703951 (integrin beta-2-like), loc121705416 (integrin beta-3-like), loc121696317 (cathepsin S-like isoform X1), ctss1 (cathepsin K-like), loc121713053 (major histocompatibility complex class I-related gene protein-like), cxcr4b (C-X-C chemokine receptor type 4), and downregulation of loc121718945 (actin-related protein 2/3 complex subunit 1A), arpc5b (actin-related protein 2/3 complex subunit 5b), loc121704963 (integrin alpha-M−like isoform X1), loc121705521 (integrin alpha-X-like isoform X1), itgb5 (integrin beta-5), pfn1 (profilin-1-like) at 144 h. Fecal metagenomics showed decreased Serpula , and S. lacrymans (positive with arpc1b , loc121696317 expressions), while metabolomics revealed altered carbohydrate metabolism, with decreased D-fructose and increased UDP-N-acetyl-alpha-D-glucosamine. Collectively, copper sulphate exposure induced oxidative stress in juvenile fish and disrupted cytoskeletal integrity, immune regulation, gut microbiota imbalance, and energy (lipid) metabolism in mature American shade, highlighting potential ecological and aquaculture risks.