Combined Exposure to Wildfire Ash and Road Salts Reshapes Skin and Gut Microbiota and Reduces Fitness in Amphibians.

Rising wildfire risk and continued winter road maintenance increase the potential for wildfire ash and road salts to enter cold-region amphibian breeding habitats via rainfall or snowmelt runoff, yet their combined effects on skin and gut microbiota remain unclear. Rana dybowskii tadpoles were exposed for 28 days to control (C), low-temperature road salt (D), eco-friendly road salt (H), aqueous extract of wildfire ash (T), T + D (TD), and T + H (TH). Skin and gut microbiota were analyzed using 16S rRNA gene sequencing. The results showed that exposure reduced body mass, developmental proportion, and survival rate, with the strongest reductions in body mass and survival under T + H. Exposure increased the microbial dysbiosis index (MDI), and skin and gut MDI were negatively associated with survival rate and developmental proportion, respectively. Two-way models and permutational multivariate analysis of variance (PERMANOVA) revealed interaction effects on alpha and beta diversity of the microbiota, with synergistic reductions mainly in gut alpha diversity. Non-metric multidimensional scaling (NMDS), redundancy analysis (RDA), and canonical correspondence analysis (CCA) indicated an apparently dominant role of aqueous extract of wildfire ash in microbiota restructuring. The β-nearest taxon index (βNTI)-based analysis further showed altered gut assembly processes. Overall, single and combined exposures to aqueous extract of wildfire ash and road salts can impair tadpole fitness and disrupt skin and gut microbiota homeostasis. This study provides evidence for assessing amphibian risks under co-occurring wildfire ash runoff and road-salt inputs in cold-region breeding habitats.