Unraveling bacterial and eukaryotic communities in secondary water supply systems: Dynamics, assembly, and health implications

•SWSS eukaryotes mainly include protists and metazoa, with low abundance of amoebae.•Eukaryotes display higher variations than bacteria during water transportation.•Distinct ecological processes govern bacterial and eukaryotic community assembly.•Tank biofilm emerged as an important source of eukaryotes in tap water using FEAST.•Tank materials affected amoeba community structures. Secondary water supply systems (SWSSs) are crucial water supply infrastructures for high-rise buildings in metropolitan cities. In recent years, they have garnered public attention due to increased microbial risks. However, our understanding of SWSS microbial ecology, particularly concerning the composition of eukaryotes and the underlying mechanisms driving microbial dynamics and assembly in SWSSs, remains elusive. Herein, we conducted a comprehensive investigation on both eukaryotes and bacteria along the water transportation pathway and across various microbial habitats (water, biofilm, and sediment) in SWSSs. Sequencing results revealed that eukaryotes within SWSSs predominantly consist of protists (average abundance: 31.23%) and metazoans (20.91%), while amoebae accounted for 4.71% of the total. During water transportation from the distribution mains to taps, both bacterial and eukaryotic communities exhibited significant community shifts, and higher degrees of variation were observed for eukaryotic community among different locations within SWSSs. The normalized stochasticity ratio (NST) analysis demonstrated that bacterial community assembly was governed by stochastic processes, while eukaryotic community assembly was primarily shaped by deterministic processes. Within SWSS tanks, bacterial communities significantly varied across water, biofilm, and sediment, whereas eukaryotic communities showed minor differences among these habitats. The co-occurrence networks analysis revealed that tank biofilm and sediment harbored more eukaryote-bacterium linkages than water, suggesting biofilm and sediment might be hotspots for inter-kingdom interactions. We also applied FEAST analysis to track the source of tap water microbiota, results of which showed that household-tap bacteria mainly originated from tank water. In contrast, tank biofilm was identified as the primary microbial source to eukaryotes in household tap water. Additionally, engineering factors such as tank materials significantly affected amoeba community, and the SWSS configuration was found to influence Legionella and