Abstract

Chattonella marina is an ichthyotoxic, bloom-forming raphidophyte known for its hemolytic activity. However, the mechanisms by which nitrogen (N) and phosphorus (P) limitation influence this hemolytic toxicity remain poorly understood. In this study, both N and P limitation reduced growth, photosynthetic efficiency (F v /F m , Y II , rETR max ), and the expression of nutrient-uptake, tetrapyrrole/chlorophyll biosynthesis genes. Nevertheless, the two nutrients produced opposite effects on toxicity: N limitation lowered hemolytic activity and ROS levels to near zero, whereas P limitation kept both relatively high, similar to nutrient-replete controls. The addition of the antioxidant NAC (N-Acetyl-L-cysteine) reduced hemolytic activity, confirming that ROS contributes to toxicity. Transcriptome data showed that under N limitation, genes for nitrogen uptake and initial reduction ( NRT , NR , glnA ) were upregulated, while downstream assimilation genes ( nirA , GLT1 ) were downregulated. In contrast, under P limitation, all the nitrogen-metabolism-related genes ( NRT , NR , glnA , nirA , GLT1 ) were downregulated. In the tetrapyrrole pathway, most genes were downregulated under both nutrient-limited conditions, except for HemD , suggesting a bottleneck that may result in the accumulation of porphyrin intermediates within the tetrapyrrole/chlorophyll biosynthesis pathway. Together, the secondary products derived primarily from the reaction of ROS with tetrapyrrole-based compounds appear to be the main contributors to hemolytic toxicity. Consequently, high levels of both ROS and porphyrin intermediates under P-limited conditions, as well as high ROS levels but low porphyrin intermediates under nutrient-sufficient conditions, may both contribute to the high hemolytic toxicity of C. marina . In contrast, under N limitation, despite the accumulation of porphyrin intermediates, the strong suppression of photosynthetic electron transport limits both ROS production an