Longitudinal dynamics and cross-domain interactions of eukaryotic populations in wastewater treatment plants

Activated sludge is a large reservoir of novel microorganisms and microbial genetic diversity. While much attention has been given to the profile and functions of prokaryotes, the eukaryotic diversity remains largely unexplored. In this study, we analysed longitudinal activated sludge samples spanning 13 years from the largest secondary wastewater treatment plants in Hong Kong, unveiling a wealth of eukaryotic taxa and 681 856 non-redundant protein-coding genes, the majority (416 044) of which appeared novel. Ciliophora was the most dominant phylum with a significant increase after a transient intervention (bleaching event). Our metagenomic analysis reveals close linkage and covariation of eukaryotes, prokaryotes, and prokaryotic viruses (phages), indicating common responses to environmental changes such as transient intervention and intermittent fluctuations. Furthermore, high-resolution cross-domain relationships were interpreted by S-map, demonstrating a predatory role of Arthropoda, Ascomycota, Mucoromycota, and Rotifera. This high-resolution profile of microbial dynamics expands our knowledge on yet-to-be-cultured populations and their cross-domain interactions and highlights the ecological importance of eukaryotes in the activated sludge ecosystem. © The Author(s) 2025. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.