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 major ecological and public health impacts. Although environmental drivers of PST production are well studied, the role of cellular development remains unclear. We examined the coordination between morphology and toxin synthesis in Centrodinium punctatum over 30-day cultivation period using imaging flow cytometry and HPLC with post-column oxidation. Three developmental stages were defined. Stage 1 (days 0-10) comprised uniform cells (30-40 μm) in lag and early exponential growth, dominated by production of saxitoxin (STX; 44.33 ± 2.80 fmol cell⁻¹, ∼70% of total) and low-toxicity analogs. Stage 2 (days 10-20) showed greater size variability and appearance of high-toxicity analogs GTX1 and GTX2 (4.09 ± 0.03 and 1.29 ± 0.08 fmol cell⁻¹, respectively), coinciding with sxtN (sulfotransferase) upregulation. Stage 3 (days 20-30) featured enlarged cells (>50 μm), reduced chlorophyll fluorescence, and maximum toxin diversity, with peak GTX1 and GTX2 (28.25 ± 0.25 and 11.83 ± 0.29 fmol cell⁻¹, respectively). Total toxin content and toxicological potency increased by approximately five-fold (52.23 ± 1.69-269.61 ± 8.94 fmol cell⁻¹; ∼16 to >80 pg cell⁻¹). Strong correlations between morphology, fluorescence, and toxin levels indicate that PST biosynthesis is developmentally regulated rather than time-dependent. Targeted gene expression confirmed stage-specific activation of toxin genes. Comparisons with ocean-scale omics datasets support the ecological relevance of these developmental controls. Because high-toxicity PST analogs are preferentially produced at specific developmental stages, toxin risk cannot be reliably inferred from cell abundance alone, highlighting the importance of incorporating developmental state into harmful algal bloom monitoring and risk assessment.