Sequential decline in cyanobacterial, total prokaryotic, and eukaryotic responses to backward flow in a river connected to Lake Taihu

River ecosystems face escalating challenges due to altered flow regimes from human activities, such as urbanization with hydrological modifications. Understanding the role of microbial communities for ecosystems with changing flow regimes is still incomplete and remains at the frontier of aquatic microbial ecology. In particular, influences of riverine backward flow on the aquatic biota remain largely unknown. Therefore, we examined the impact of backward flow on the cyanobacterial, total prokaryotic, and eukaryotic communities in the Changdougang River, which naturally flows into Lake Taihu, through environmental DNA metabarcoding. We analyzed the differences in community diversity, assembly, and ecological network stability among groups under backward, weak, and forward flow direction conditions. Non-metric multidimensional scaling showed higher variations in communities of groups across flow direction conditions than seasonal groups. Variations in alpha and beta diversity showed that cyanobacterial and total prokaryotic communities experienced strong homogenization under backward flow conditions, whereas the ecological uniqueness of the eukaryotic community decreased. Assembly of the three flow-related communities was primarily governed by drift and dispersal limitation in stochastic processes. However, in the cyanobacterial community, homogeneous selection in deterministic processes increased from 22.79% to 42.86% under backward flow, aligning with trends observed in the checkerboard score (C-score). More importantly, the topological properties of ecological networks and the degree of average variation revealed higher stability in the cyanobacterial community compared to total prokaryotic and eukaryotic communities. Considering the variations in cohesion, the network stability in the cyanobacterial community decreased under backward flow. Our findings emphasize the distinct and sequentially diminishing responses of cyanobacterial, total prokaryotic, and eukaryotic communities to backward flowing rivers. This knowledge is crucial for maintaining ecological health of rivers, assessing the complex ecological impacts on hydrological engineering, and formulating sustainable water management strategies.