Early Metabarcoding Detection of Eukaryotic Putative Pathogens Nearby Wastewater Effluents of Ría de Vigo (NW Spain)

From a One Health perspective, the Ría de Vigo (NW Spain) represents a complex socio-ecological system where urban, industrial, and aquaculture activities converge, creating vulnerabilities due to the interconnection between human, animal, and environmental health. This study evaluates the utility of a multi-marker environmental DNA (eDNA) metabarcoding approach as an early warning tool to detect potentially harmful eukaryotic pathogens near wastewater discharge points in three distinct municipalities of the Ría de Vigo. Water and sediment samples were analyzed using the V4 and V9 regions of the 18S rRNA gene and the ITS-2 region for fungi. Bioinformatics analysis was performed with DADA2 and taxonomic assignments were based on SILVA and UNITE databases. Eukaryotic diversity varied by site and marker, with the highest richness found in inner estuary sediments. The V9 region provided broader taxonomic coverage, while V4 produced higher read abundances. Putative pathogens, including human, zoonotic, and aquaculture-related taxa, were more prevalent in inner estuarine zones. Pathogens such as Rhodotorula mucilaginosa, Acanthamoeba sp., Cryptosporidium sp., and harmful algae (HA) were detected. The findings emphasize the importance of a multi-marker strategy, sediment inclusion, and landscape-scale variables to improve early pathogen detection, contributing to effective eDNA-based surveillance systems for coastal health management.