Unraveling the disease pyramid: the role of environmental micro-eukaryotes in amphibian resistance to the deadly fungal pathogen Batrachochytrium dendrobatidis.

The disease pyramid conceptualizes the predictors of host infection risk, linking the host, the pathogen, environmental conditions, and both host and environmental microbiomes. However, the importance of the interaction between environmental and host-associated microbiomes in shaping infectious disease dynamics remains poorly understood. While the majority of studies have focused on bacteria, the role of micro-eukaryotes has been seldom investigated. Here, we explore three axes of the disease pyramid using an 18S rRNA gene metabarcoding approach to analyze the micro-eukaryotic assemblages of biofilm, water, and skin samples from three European amphibian species. Skin bacterial communities of the investigated amphibian populations have already been shown to be impacted by the presence of the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd), with a higher abundance of protective bacteria in infected populations and a greater environmental microbial contribution to the skin microbiota in Bd-positive lakes. Here, we explored the relationships between the micro-eukaryotic skin communities of these tadpole populations with their surrounding environment. Tadpoles were sampled at 22 mountain lakes located in the Pyrenees (France), 8 of which harbored amphibian populations infected by Bd. We found that biofilms from Bd-negative lakes had higher environmental micro-eukaryotic diversity and a greater abundance of putative anti-Bd fungi, both in the environment and on the skin microbiota of Bufo spinosus and Rana temporaria, but not of Alytes obstetricans. Bayesian SourceTracker analysis further showed that the environmental contribution from biofilms to amphibian skin micro-eukaryotic assemblages was higher in Bd-positive lakes for B. spinosus and R. temporaria, but not for A. obstetricans.IMPORTANCEResearch on host-associated microbiomes and infectious diseases has mostly focused on bacteria, overlooking the potential contributions of micro-eukaryotes to infection dynamics. Here, we show that environmental and skin-associated micro-eukaryotes-especially putative anti-Batrachochytrium dendrobatidis (Bd) fungi-differ between Bd-positive and Bd-negative amphibian populations in mountain lakes. Our results suggest that micro-eukaryotes influence disease resistance and microbiome assembly, similarly to bacteria. Importantly, environmental reservoirs of micro-eukaryotes appear to contribute differently across infection contexts. These findings demonstrate the importance of adopting a broader microbiome perspective that includes micro-eukaryotes when investigating the ecological mechanisms underlying infectious disease risk.