Combined effects of temperature and salinity on growth, intracellular toxin content and copepod responses of marine dinoflagellate <i>Protoceratium reticulatum</i>

Harmful algal blooms (HABs) are increasing under climate change. The harmful algal blooming dinoflagellate Protoceratium reticulatum is a major producer of yessotoxins (YTXs) that can damage biological tissues. While climate change alters multiple environmental conditions, the combined impacts on P. reticulatum growth and toxin production remain poorly understood. In this study, we first applied an orthogonal experimental design to determine the effects of environmental factors among temperature, salinity, initial pH value and eutrophication related nutrients. We then conducted a full factorial experiment to examine the interactive effects of the two most essential factors, temperature (ranging from 15 to 27.5 °C) and salinity (ranging from 21 to 33 ppt) on growth (measured as cell density) and intracellular homoyessotoxin (hYTX) content of P. reticulatum K-1477. Furthermore, we evaluated how these factors influence the response on two key copepods species, Acartia tonsa and Nitokra spinipes. Our results showed statistically significant interactive effects of temperature and salinity on cell density and intracellular hYTX content. Salinity only caused significant differences at 30 and 33 ppt under 20 and 22.5 °C, suggesting temperature might be the dominant driver of growth and toxin production. No significant relationship between cell density and intracellular hYTX content was found. Finally, exposure assays revealed reduced swimming speed of A. tonsa and increased swimming speed of N. spinipes after exposure to P. reticulatum. These findings demonstrate that warming and salinity fluctuations can modulate P. reticulatum growth and toxicity, underscoring the importance of multi-stressor approaches for predicting harmful algal bloom dynamics under future climate scenarios.