Intestinal Responses to Short-Term Hexaflumuron Exposure in Grass Carp (Ctenopharyngodon idella): A Combined Physiological, Transcriptomic, and Gut Microbiota Perspective

Hexaflumuron is a benzoylurea insecticide with potential application against arthropod ectoparasites in aquaculture, but its toxicity to non-target fish remains insufficiently characterized. This study evaluated acute toxicity and intestinal responses to sublethal concentrations of hexaflumuron in juvenile grass carp (Ctenopharyngodon idella). The 48- and 96-h LC50 values were 8.80 and 7.16 mg/L, respectively. Fish were subsequently exposed to nominal concentrations of 0.07 or 0.72 mg/L hexaflumuron for 96 h. Intestinal histopathology, antioxidant biomarkers, 16S rRNA sequencing, and transcriptomics were investigated. Both exposure groups showed focal intestinal alterations. Malondialdehyde levels increased, total superoxide dismutase activity exhibited a biphasic response, and catalase activity and reduced glutathione content declined, indicating altered redox homeostasis. Overall gut microbial structure showed no significant difference, whereas taxon-specific shifts occurred, including an increase in Aeromonas to 15.40% at 0.72 mg/L. Transcriptomic analysis identified 1226 and 5496 differentially expressed genes at 0.07 and 0.72 mg/L, respectively. Both concentrations suppressed ribosome biogenesis, protein homeostasis, mitochondrial bioenergetic programs, and complement-associated functions, while the higher concentration induced broader enrichment of cytokine, phagolysosomal, antigen-presentation, and leukocyte-migration pathways. Overall, these results indicated that hexaflumuron perturbs intestinal homeostasis through synergistic impacts on cellular biosynthesis, mitochondrial bioenergetic programs, redox balance, immune regulation, tissue remodeling, and microbial composition.