Integrated analysis of trichlorfon-induced intestinal toxicity in Micropterus salmoides: Barrier Impairment, microbiome disruption, and metabolic reprogramming

Trichlorfon is widely used to control parasites but may also induce sublethal effects in fish. However, its chronic effects on intestinal metabolism and toxicity remain unclear. Here, we investigated the effects of chronic trichlorfon exposure (0.01, 0.1, and 1 mg/L) on juvenile largemouth bass through integrated histological, physiological, microbiological, and metabolomic analyses. Trichlorfon exposure disrupted neurotransmitter balance, significantly reducing excitatory neurotransmitters (acetylcholine, glutamate, dopamine) and increasing gamma-aminobutyric acid. Mitochondrial dysfunction induced energetic stress and triggered a metabolic shift from oxidative phosphorylation to anaerobic glycolysis, supported by ATP depletion, accumulation of glycolytic and tricarboxylic acid intermediates, and downregulation of AMPKα1, PGC-1α, SDHa, and SDHb. Oxidative stress caused lipid peroxidation, decreasing linoleic and gamma-linolenic acids, and increasing malondialdehyde. Antioxidant defenses were suppressed, with impaired glutathione metabolism, decreased peroxidase and superoxide dismutase activities. Mucosal barrier was compromised, with disrupted glycosylation precursors, reduced expression of barrier genes (Occludin, ZO-1, Claudin-1), increased of proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), and marked histopathological alterations. Altered polyunsaturated fatty acid metabolism promoted inflammatory eicosanoids (prostaglandin E₂, 5(S), 14(R)-lipoxin B₄). In addition, digestion and nutrient absorption were impaired, reflected by reduced conjugated bile acids, essential amino acids, and digestive enzyme activities. Gut microbial dysbiosis was observed, with increased opportunistic pathogens and reduced beneficial lactic acid bacteria. Overall, chronic trichlorfon exposure disrupted intestinal homeostasis. This study offers new insights into the intestinal metabolic alterations in fish exposed chronically to trichlorfon and highlights the need for prudent pesticide management in aquaculture.