Using an acidic vesicle tracer to identify potential bacterivores in coastal waters of the subtropical Western Pacific Ocean

Bacterivores play a key role in transferring energy through microbial food webs and are broadly classified as heterotrophic or mixotrophic based on the presence of inherent chloroplasts. Mixotrophs, especially in low-latitude regions, have recently gained attention due to their dual roles as primary producers and bacterial grazers, which enable them to thrive in nutrient-poor, stratified waters where traditional autotrophs or heterotrophs may be less competitive. This study investigated the composition of bacterivores in the coastal waters of northeastern Taiwan during the warm season (July and August 2022). A combination of LysoTracker staining, flow cytometry sorting, and 18S rRNA gene amplicon sequencing was used to rapidly identify heterotrophic and mixotrophic nanoflagellates (HNFs and MNFs, respectively) based on chloroplast autofluorescence. The dominant eukaryotic lineages included Alveolata, Stramenopiles, Opisthokonta, Chlorophyta, and Rhizaria. HNFs were primarily composed of MAST clades, choanoflagellates (Opisthokonta), Telonemia, and Radiolaria, while MNFs included cryptophytes, haptophytes, and chlorophytes such as Mamiella , Mantoniella and Tetraselmis , as well as stramenopiles like Dictyochophyceae and Chrysophyceae. Notably, several non-motile chlorophytes, including Chloropicon and members of Pycnococcaceae, were identified as potential bacterivores based on Lysotracker signals; however, their phagotrophic capability requires further confirmation in future studies. Despite the limitations of LysoTracker-based methods, this study reveals previously overlooked mixotrophic diversity and highlights the need for further validation. These findings advance our understanding of carbon flow and microbial dynamics in subtropical marine ecosystems under changing environmental conditions.