Composition and concentration of road salts alter amphibian microbial ecosystems: Insights from Ranadybowskii

Amphibians are globally threatened, and road salts pollution is a major threat. Dissolved salts alter water salinity and chemistry and can disrupt the diversity, structure, and health of skin and gut microbiota; however, the effects of salt concentration and composition remain poorly studied. Using Rana dybowskii tadpoles, this study exposed individuals to two road salt formulations-low-temperature salts (LTS, LT groups) and high-temperature salts (HTS, HT groups)-across a conductivity gradient (166, 1,500, 3,500, and 7500 μS/cm). Microbiota from skin and gut were analyzed by 16S rRNA sequencing, with community structure assessed across 6 effective treatment groups (LHT groups). Elemental assays showed contrasting chemistries (LTS: higher K and Mg; HTS: higher Na, Ca, and Mn). Alpha diversity declined with salinity in both formulations, with stronger losses in the gut. Microbiota health index (MHI) decreased and microbiota dysbiosis index (MDI) increased dose-dependently, accompanied by higher instability. Beta diversity separated treatments (Bray-Curtis and unweighted UniFrac), indicating composition-specific responses to concentration and formulation. Neutral community model (NCM) explained approximately 55 % of abundance-occurrence patterns for skin communities and 49 % for gut communities; using Infer Community Assembly Mechanisms by phylogenetic-bin-based null model analysis (iCAMP), community assembly was attributed predominantly to ecological drift (DR), with homogeneous selection (HoS) increasing at higher salinity. Linear discriminant analysis effect size (LEfSe) identified formulation-specific biomarkers (e.g., Enterococcus and Carnobacterium at moderate LT groups; Rhodococcus and Leucobacter at high LT groups; Mycobacterium under HT groups). BugBase suggested shifts in aerobic, facultative, and biofilm-forming phenotypes. Collectively, both concentration and ionic composition (Na⁺/Ca²⁺/K⁺/Mg²⁺, Mn) regulate amphibian microbiota, with the gut more sensitive than skin. Monitoring and mitigation should therefore address chloride/electrical conductivity (EC) as well as ionic profiles to protect amphibian health and freshwater ecosystem stability.