Title: Co-exposure to polystyrene microplastics and permethrin induces gill injury in Gobiocypris rarus involving oxidative stress and microbiota dysbiosis

Microplastics and pesticides often coexist in freshwater environments; however, their combined effects on teleost branchial tissues remain unclear. In this study, adult Gobiocypris rarus were exposed to polystyrene microplastics (PS-MPs, 1 mg/L), permethrin (PM, 0.5 μg/L), or their combination for 21 days. Gill toxicity was evaluated using histopathology, scanning electron microscopy, biochemical assays, immunofluorescence staining, transcriptomics, and 16S rRNA sequencing. Our results revealed that both single and co-exposure manifested as sublethal toxicity in gill tissues, with co-exposure generally inducing more pronounced and widespread adverse effects. Histological and ultrastructural observations showed that co-exposure exacerbated lamellar deformation, epithelial vacuolation, microridge disruption, and epithelial surface damage. 8-OHdG staining revealed the most severe oxidative DNA damage in the PM group. Biochemical analyses showed decreased superoxide dismutase (SOD) activity in all exposure groups and increased malondialdehyde (MDA) levels in PM-containing groups. TUNEL analysis showed the most intense apoptotic signal in the co-exposure group. Transcriptomic analysis revealed that co-exposure induced the largest number of differentially expressed genes and mainly affected lipid transport, membrane-related metabolism, programmed cell death, immune regulation, and PPAR signaling. In addition, 16S rRNA sequencing showed that co-exposure disrupted the gill-associated microbiota, with a loss of dominant commensal bacteria and an increase in opportunistic taxa. Combined analysis further showed that microbial changes were closely associated with oxidative injury, apoptosis-related responses, and immune-related indicators. Overall, these findings indicate that PS-MPs and PM co-exposure exacerbates gill toxicity through multi-level injury processes, providing a theoretical basis for evaluating the ecological risks of microplastic-pesticide co-contamination in freshwater fish.