The mixotrophic dinoflagellate Akashiwo sanguinea frequently forms harmful algal blooms around the world and has caused massive deaths of shellfish, finfish and birds, yet its toxic mechanism is still unclear. In this study, toxic effects of A. sanguinea on co-culturing phytoplankton and zooplankton were investigated. The results showed that sonicated cultures of A. sanguinea JX13 and JX14, isolated from the Pearl River Estuary, had a significant lethal effect on the rotifer Brachionus plicatilis, with the highest mortality rate of 80%. The highest inhibition rates of A. sanguinea cultures JX13 (90%) and JX14 (80%) on R. salina were much higher than that of AS2 (20%). Toxicity varied with the growth stage, during which A. sanguinea cells in the exponential stage showed the highest toxicity (40%), while A. sanguinea filtrate had the highest toxicity (10%) in the decline stage. The action mode of A. sanguinea toxicity on plankton was explored through an osmotic membrane culture device. It was found that A. sanguinea JX13 displayed an inhibitory effect on coexisting phytoplankton, whether they had contact or not, but the inhibition rate increased by 25% with contact. A lethal effect of A. sanguinea JX13 on rotifer Brachionus plicatilis was observed only in contact treatment. This study suggests that direct contact is the key action mode to trigger the release of toxins and induce toxic effects of A. sanguinea on co-occurring plankton.

The dinoflagellate Cochlodinium geminatum can form near-monospecific blooms which have occurred at the same location in the South China Sea repeatedly in the last decade. Here, we demonstrate the allelopathic effects of C. geminatum isolated from the bloom waters of the Pearl River Estuary, South China Sea, in August 2011, which inhibited four of six co-occurring phytoplankton species tested. Inhibition increased with rising cell density and reached a maximum in the declining growth stage. Four different C. geminatum culture components negatively affected growth of Akashiwo sanguinea in order of decreasing percent inhibition: sonicated culture, filtrate of sonicated culture, whole-cell culture and filtrate of whole-cell culture. The highest inhibition was at 25 °С and salinities of 30 PSU and light intensities of 100 μmol photons m⁻² s⁻¹. Overall, our study showed that C. geminatum could inhibit growth of phytoplankton competitors during succession of phytoplankton community, achieving nearly monospecific blooms, and the allelopathic effects were regulated by environmental factors.

The mixotrophic dinoflagellate Akashiwo sanguinea 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 A. sanguinea toxins using chromatographic techniques. The acetone extract of A. sanguinea 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 (p < 0.05), suggesting that A. sanguinea 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 c-related precursors. These findings highlight that the photosensitive toxins in A. sanguinea may serve dual roles in stress adaptation and ecological competition, potentially contributing to the formation of the blooms.