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.由海洋甲藻产生的麻痹性贝类毒素（Paralytic shellfish toxins, PSTs）是一类兼具生态与公共卫生影响的强效神经毒素。尽管学界已对影响PST合成的环境因子开展了充分研究，但细胞发育在其中发挥的作用仍不明晰。本研究针对连续培养30天的斑点梭甲藻（Centrodinium punctatum），探究了其形态变化与PST合成之间的时序协同调控关系。本研究采用成像流式细胞术与柱后氧化高效液相色谱（HPLC）技术，基于细胞性状与毒素谱特征划分出三个阶段：第一阶段（第0~10天）呈现均匀细胞的指数生长状态，细胞直径为30~40 μm，主要合成石房蛤毒素（saxitoxin, STX，36.8±1.2 fmol·cell⁻¹，占总毒素的约70%）与低毒性毒素类似物；第二阶段（第10~20天）出现细胞大小异质性，首次检出高毒性的GTX1与GTX2，至第20天时二者含量分别达到4.1±0.1 fmol·cell⁻¹与1.3±0.1 fmol·cell⁻¹；第三阶段（第20~30天）以大型细胞（直径>50 μm）、叶绿素荧光强度降低以及最高毒素多样性为特征，其中GTX1与GTX2的含量达到峰值，分别为8.7±0.4 fmol·cell⁻¹与11.8±0.3 fmol·cell⁻¹。整体而言，总毒素含量提升了5倍（从52.2±1.7 fmol·cell⁻¹增至269.6±8.9 fmol·cell⁻¹），毒理学毒性提升了4倍（约16 pg·cell⁻¹升至>80 pg·cell⁻¹）。细胞大小、细胞偏心率与毒素水平之间的强相关性证实，毒素生物合成受发育调控而非时间依赖。本研究结果揭示，斑点梭甲藻的毒素合成与形态分化紧密关联，为PST调控机制研究提供了新视角，同时也有助于优化有害藻华风险评估工作。