Unraveling the assembly mechanisms and differentiated ecological functions of protist cell-associated and free-living bacterial communities during two <i>Prorocentrum shikokuense</i> blooms

Free-living and cell-associated microbial communities are crucial in the development, maintenance, and decline of harmful algal blooms (HABs), yet little is known about how they differ and change during bloom succession and whether the phycosphere-associated bacterial community of an algal species remains similar in different geographic populations. This study employed 16S rRNA gene amplicon sequencing on size-fractionated samples to investigate community assembly, dynamics, and co-occurrence patterns of free-living and cell-associated bacterial communities during two <i>Prorocentrum shikokuense</i> (Dinophyceae) blooms in the East China Sea. The two communities exhibited distinct compositions and dynamics changes, influenced by varying degrees of assembly processes and environmental factors. Core phyla-Proteobacteria (25.8%-57.1% in cell-associated communities, 37.3%-55.5% in free-living communities), Bacteroidota (12.5%-25.1%; 13.3%-18.5%), Actinobacteriota (6.7%-16.1%; 23.7%-33.3%), and Planctomycetota (3.6%-40.7%; 2.4%-5.6%)-comprised 81.8%-95.8% of observed amplicon sequence variants. The cell-associated community structure changed more evidently during bloom development compared to free-living communities, sharing a similar structure despite spatial and temporal differences. Environmental factors contributed more to the variance in free-living communities. Homogeneous selection predominantly shaped free-living composition, while cell-associated communities were more influenced by stochastic processes, especially dispersal limitation. Cell-associated community diversity was determined by residence space rather than the geographic location or bloom stage, with simpler co-occurrence networks compared with free-living communities. Furthermore, cell-associated communities showed enriched ecological functions in nutrient cycling and cytolysis. The ecological implications of the higher abundance of Rhizobiales in cell-associated communities for nitrogen fixation were also discussed. This work enhances our understanding of algal-bacteria interactions and the distinct dynamics between free-living and cell-associated communities.IMPORTANCEMicrobial communities are crucial in the development of harmful algal blooms (HABs), yet how free-living and cell-associated bacterial communities differ and change during bloom succession remains unclear. Using 16S rRNA gene amplicon sequencing, we investigated the dynamics and assembly mechanisms of these two community types during two <i>Prorocentrum shikokuense</i> blooms in the East China Sea. Our findings reveal distinct structural compositions and dynamics between cell-associated and free-living communities, shaped by varying assembly processes and environmental factors. Cell-associated bacteria in the phycosphere of <i>P. shikokuense</i>, strongly influenced by dispersal limitation due to their close interaction with the algal host, exhibit enriched functions in nutrient cycling and cell lysis. This suggests that cell-associated bacteria may play an essential role in algal bloom development and dissipation. This research broadens our understanding of algae-bacteria interactions and microbial community dynamics during harmful algal blooms, offering valuable information for managing algal blooms and protecting marine ecosystems.