Potential of a mammalian and avian environmental DNA metabarcoding assay as a tool for fecal pollution source tracking in river waters†

Currently, common fecal source identification methods in water environments are based on the detection of host-specific gene markers, such as 16S rRNA gene fractions of Bacteroidales and mitochondrial DNA. However, with this approach, identifying all animals contributing to fecal pollution in a catchment has been a challenge, considering numerous species and populations of animals in the area. In this study, we examined the effectiveness of a metabarcoding approach that comprehensively targets mammalian and avian mitochondrial DNA as an approach to identify animal species potentially contributing to pollution. Surface water samples (n = 108) were collected monthly at five sites in Toyama prefecture, Japan under wet and dry weather conditions for two years. The samples were subjected to the metabarcoding targeting mammalian and avian mitochondrial DNA. Further, human (HF183)-, pig (Pig-2-Bac)-, and ruminant (BacR)-specific gene markers were quantified. Human-like DNA (74 samples) and livestock-like DNA, namely, pig-(66 samples), cattle-(23 samples), and chicken-(52 samples) like DNA were frequently observed. Additionally, DNA associated with wild terrestrial animals, waterfowls, and urban birds commonly found in the study area were observed regardless of the weather conditions. Human- and livestock-like DNAs exhibited similar detection trends to their corresponding markers across sites, though the presence of mitochondrial DNA from non-fecal sources was also suggested. The metabarcoding assay was effective for simultaneously and comprehensively evaluating animal species' potential contributions to fecal pollution. Comparing the stability of animal markers and their DNA in the environment would help to further validate the effectiveness of the assay.