Title:

Emergence and Molecular Characterization of Protist Amphibiothecum penneri in American Toads (Anaxyrus americanus) in Northern New England

Emerging infectious diseases in amphibians represent a significant ecological concern, yet many protist pathogens remain poorly characterized. This study examines the emergence of the holozoan protist Amphibiothecum penneri in American toads (Anaxyrus americanus) in Northern New England, where increasing incidence of cutaneous, vesicular lesions has been observed since 2022. A targeted metagenomic workflow was applied to address the challenge of separating co-extracted host and protist DNA from mixed eukaryotic samples. Assembly, taxonomic binning, and BLAST-based host sequence removal reduced host contamination while preserving protist-derived contigs, enabling recovery of a protist-enriched draft genome. Genome evaluation indicated improved completeness following filtering, although the assembly remains fragmented. Comparative protein annotation and phylogenomic analysis support placement of the recovered genome within holozoan protists and are consistent with A. penneri, with additional similarity to related mesomycetozoean taxa. These findings provide strong molecular evidence for the association of A. penneri with disease in A. americanus and establish an effective framework for resolving eukaryotic host–pathogen genomic mixtures.

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