Emamectin Benzoate-Induced Gut Dysbiosis in Asian Stinging Catfish: An Integrated Culture-Dependent and 16S rRNA Gene Sequencing Approach

The gut microbiota of fish plays a crucial role in nutrition, metabolism and immune regulation, and is highly sensitive to environmental stressors such as pesticide exposure. This study investigated the effects of emamectin benzoate (EMB) exposure on Asian stinging catfish (Heteropneustes fossilis) gut microbiota using an integrated culture-dependent and culture-independent approach to assess functional and taxonomic dysbiosis. Gut smear samples from control and EMB-treated fish at two sublethal concentrations (0.5 µg/L and 5 µg/L) were analyzed for major functional bacterial groups, including heterotrophic, lipid-hydrolysing, starch-hydrolysing, spore-forming, and Gram-negative bacteria and Pseudomonas spp., using standard plate count techniques. In parallel, microbial community composition and diversity were examined through 16S rRNA (V3–V4 region) gene amplicon sequencing followed by bioinformatic analysis. Culture-based results showed a significant decline in total heterotrophic bacteria and key functional groups in EMB-treated fish, indicating suppression of microbial metabolic activity and functional imbalance. Lipid-hydrolysing and starch-hydrolysing bacteria showed pronounced sensitivity to pesticide exposure, while spore-forming bacteria exhibited a marked reduction, suggesting compromised microbial resilience. Although Gram-negative bacteria declined overall, Pseudomonas spp. displayed a non-linear response, with an initial decrease, followed by partial recovery under higher exposure. Culture-independent analysis demonstrated reduced alpha diversity, altered community structure, and taxonomic shifts in EMB-treated fish. Pseudomonadota exhibited a distinct pattern characterized by decline at 0.5 µg/L and partial recovery at 5 µg/L, reflecting adaptive tolerance rather than restoration of microbial homeostasis. Overall, the combined evidence indicates pronounced EMB-induced gut dysbiosis at both functional and compositional levels. This study highlights the fish gut microbiome as a sensitive biomarker of stress and underscores the ecological risks associated with EMB in aquatic environments.