Third-generation RNA Amplicon Sequencing Reveals the Dynamics of Microbial Communities in the Tidal Reach of a Subtropical Estuary

Estuarine ecosystems are characterized by periodic tidal cycles that induce dynamic salinity fluctuations, yet how these dynamic salinity changes affect microbial diversity, community composition, and network structure remains poorly understood. Answering this question would enhance our understanding of the effects of periodic tides on microbial communities in estuaries. To address this knowledge gap, we collected hourly water samples over a 24-h time series from the tidal reach of the Pearl River Estuary (PRE) and explored the dynamics of both prokaryotic and eukaryotic microbial diversity and their co-occurrence networks during a complete tidal cycle by utilizing RNA-derived cDNA combined with third-generation (PacBio) amplicon sequencing. This approach targets the active microbial community (by using RNA) while providing high taxonomic resolution (by full-length sequencing of 16S rRNA gene / ITS regions). Our results revealed that salinity was the dominant driver of the changes of microbial diversity and community compositions in the tidal reach, with a significant decrease in microbial alpha diversity as salinity increased. Furthermore, we observed a non-linear relationship between salinity and microbial network complexity and stability. Notably, network stability in both single- and cross-domain networks was positively correlated with connectance and P/N (the positive-to-negative edge ratio). Additionally, cross-domain networks exhibited higher complexity (node numbers, total links, and average node degree), but lower network stability (robustness and vulnerability) compared to single-domain co-occurrence networks. Overall, our findings provide comprehensive insights into how periodic salinity fluctuations regulate microbial community structure and cross-domain interactions in dynamic estuarine environments.