Temporal dynamics of cellular differentiation and paralytic shellfish toxin diversification in Centrodinium punctatum

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates are potent neurotoxins with ecological and public health implications. While environmental factors influencing PST production are well studied, the role of cellular development remains unclear. Here we investigated the temporal coordination between morphological changes and PST production in Centrodinium punctatum over 30 days of cultivation. Using imaging flow cytometry and HPLC with post-column oxidation, we identified three stages defined by cell traits and toxin profiles. Stage 1 (days 0–10) showed exponential growth of uniform cells (30–40 µm) producing mainly saxitoxin (STX, 36.8 ± 1.2 fmol cell?¹, ~70% of total) and low-toxicity analogs. Stage 2 (days 10–20) featured size heterogeneity and first appearance of high-toxicity GTX1 and GTX2, reaching 4.1 ± 0.1 and 1.3 ± 0.1 fmol cell?¹ by day 20. Stage 3 (days 20–30) was marked by large cells (>50 µm), reduced chlorophyll fluorescence, and maximum toxin diversity, including peak GTX1 (8.7 ± 0.4 fmol cell?¹) and GTX2 (11.8 ± 0.3 fmol cell?¹). Overall, total toxin content increased five-fold (52.2 ± 1.7 to 269.6 ± 8.9 fmol cell?¹), while toxicological toxicity rose four-fold (~16 to >80 pg cell?¹). Strong correlations between cell size, eccentricity, and toxin levels confirmed that toxin biosynthesis is developmentally regulated rather than time-dependent. These findings reveal that C. punctatum toxin production is tightly linked to morphological differentiation, providing new insights into PST regulation and improving harmful algal bloom risk assessment.