Diversity of cutaneous microbiome of Pelophylax perezi populations inhabiting different environments.

Potential synergetic interaction between chemicals, climate change and the emergence of opportunistic diseases is of utmost concern within the amphibian decline scenario. Understand the structure and dynamic of this microbiome and how environmental stressors act on this community is a priority. The present study aimed to: i) characterize the skin microbiome of Pelophylax perezi frog by looking for variations between populations in reference and under stress conditions (one metal contaminated and another with salinity fluctuations) and ii) evaluate the tolerance of skin-isolated bacteria to chemical contamination. Skin swabs were collected from frog populations inhabiting three reference sites (LB, VA and AM), one metal-rich contaminated (TP) and one brackish lentic aquatic system (SL). The frogs' skin microbiome was characterized by culture independent method (DGGE) and by cultivation methods. DGGE showed a characteristic profile in frogs from TP population. Results of recovered communities revealed low morphotype diversity and density (Colony Forming Units per frog) on individuals from TP population, comparatively to the other sampled populations. Isolated bacteria identified based on 16S rRNA gene sequence belong mainly to the classes Actinobacteria and Alphaproteobacteria. Ecotoxicological assays with acid-metal contaminated effluent (ETP) showed that the percentage of tolerant strains was higher in frogs from TP population than in SL and LB populations. In conclusion, results suggest that: a) environmental specific characteristics, as the presence of chemical contaminants, influence the composition of amphibian microbiome, comparing with sites without such contaminants; b) there are differences in microbiome composition between populations; and c) bacteria historically exposed to effluent may evolve tolerance to this kind of contamination.