Isolation and Characterization of Photosensitive Hemolytic Toxins from the Mixotrophic Dinoflagellate Akashiwo sanguinea

The mixotrophic dinoflagellate <i>Akashiwo sanguinea</i> is known to have acute toxic effects on multiple marine organisms, while the composition and chemical properties of its toxins remain unclear. In this study, we established a method for separation and purification of <i>A. sanguinea</i> toxins using chromatographic techniques. The acetone extract of <i>A. sanguinea</i> exhibited higher hemolytic activity and shorter incubation time compared to methanol and ethyl acetate extracts. Five fractions were obtained by solid-phase extraction (SPE), of which SPE3 (acetone/water ratio 3:2) and SPE4 (acetone/water ratio 4:1) exhibited the highest hemolytic activities and allelopathic effects. Further purification on SPE3 and SPE4 using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD) resulted in 11 subfractions, among which Fr4-5 displayed the strongest hemolytic activity. Nearly all active subfractions exhibited higher hemolytic activities incubated under light than those in the dark (<i>p</i> < 0.05), suggesting that <i>A. sanguinea</i> can produce both photosensitive and non-photosensitive toxins, with the former being the primary contributors to its hemolytic activity. Molecular characterization by UV-Vis, FTIR, and HRMS/MS analysis revealed that the structural features of Fr4-5 were highly consistent with porphyrin analogs and could be derived from chlorophyll <i>c</i>-related precursors. These findings highlight that the photosensitive toxins in <i>A. sanguinea</i> may serve dual roles in stress adaptation and ecological competition, potentially contributing to the formation of the blooms.