Mixotrophy plays a critical role in the ecology of harmful algal blooms (HABs), yet direct evidence linking prey availability to physiological responses under natural bloom conditions remains limited. During the 2023 ECOHAB research cruise in the Gulf of Mexico, we investigated the grazing activity of the toxic dinoflagellate Karenia brevis using cultured Synechococcus as prey. Natural bloom water was incubated with added Synechococcus alongside Karenia and prey controls. Changes in protist plankton populations were monitored by flow cytometry, while photosynthetic performance was evaluated using pulse-amplitude-modulated (PAM) fluorometry. Cell abundance, respiration, and dissolved organic matter (DOM) metabolomic profiles were also measured to assess physiological and metabolic responses associated with prey addition. Flow cytometric analyses enabled discrimination of Karenia, picoeukaryotes, nanophytoplankton, heterotrophic bacteria and Synechococcus populations throughout the incubation, providing a detailed assessment of prey dynamics and community responses. Metabolomic analyses revealed treatment-specific differences in extracellular metabolic composition following incubation with cyanobacteria prey, complementing physiological measurements of Karenia responses. Together, these complementary approaches provide new insights into the trophic strategy of K. brevis during natural bloom events and demonstrate the value of integrating flowcytometric, physiological, and metabolomic analyses to investigate mixotrophic processes in marine harmful algal blooms.

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