Effects of short-term exposure to ferrous sulfate on bioaccumulation, oxidative stress biomarkers, immunity, and intestinal microbiota in Litopenaeus vannamei.

The accumulation of heavy metals in aquatic environments poses critical threats to aquaculture, with iron (Fe) being one of the most prevalent contaminants from industrial and agricultural effluents. This study evaluated the acute toxicity and mechanistic impacts of ferrous sulfate (FeSO₄) on the Pacific white shrimp (Litopenaeus vannamei). Acute toxicity tests established the 96-h median lethal concentration (LC₅₀) of Fe at 2.52 mg/L, determined across exposure intervals of 0, 24, 48, 72, and 96-h at nominal concentrations ranging from 0.2, 1.0, 5.0, 25.0, 125.0, and 625.0 mg/L. No mortality occurred in control shrimp, whereas mortality increased progressively with both concentration and duration of FeSO₄ exposure. FeSO₄ exposure caused significant Fe accumulation in hepatopancreas and muscle, accompanied by elevated reactive oxygen species and malondialdehyde, and suppression of key antioxidant and immune enzymes; superoxide dismutase (SOD), catalase (CAT), and lysozyme (LZM). Transcriptional analysis revealed strong upregulation of stress proteins (HSP70, HSP90, GSH-Px), apoptotic regulators (caspase-3, p53), and immune effectors (metallothionein), whereas ferritin expression decreased, indicating disruption of Fe homeostasis. Microbiota sequencing demonstrated pronounced dysbiosis: control shrimp maintained balanced commensal taxa, while Fe-exposed groups were enriched in stress-tolerant and opportunistic genera such as Shewanella and Vibrio. Functional prediction (Tax4Fun) indicated that Fe exposure enhanced xenobiotic biodegradation, immune diseases, and cell-death-related pathways, while functions associated with energy, amino-acid and carbohydrate metabolism, and nervous system were comparatively downregulated. Collectively, FeSO₄ exposure impaired antioxidant defences, triggered apoptosis, and induced intestinal dysbiosis, with implications for aquaculture health management and environmental risk assessment.