Phosphorus rather than nitrogen driving biosynthesis of diarrhetic shellfish toxins in Prorocentrum caipirignum via ATP

Abstract

Okadaic acid and its analogues such as dinophysisitoxins-1, -2 (DTX-1, -2) are potent inhibitors of protein phosphatase and causative toxins of diarrhetic shellfish poisoning (DSP). These toxins are produced by dinoflagellate genera Dinophysis and Prorocentrum. Numerous studies have reported that the cellular content of these toxins increased under macronutrient limitation or other stress conditions in genus Prorocentrum. However, our recent study demonstrated positive linear or exponential relationships between toxin production rate (R_tox) and phosphate consumption rate in five strains of P. lima complex/P. caipirignum. To further clarify macronutrients roles in OA production, P. caipirignum SE10 selected for its extremely low DTX-1 content due to potential competitive relationship with OA, was exposed to nitrogen (N) or phosphorus (P) limitation via batch or semi-batch cultures, after which the depleted nutrients were replenished to assess OA production dynamics. The R_tox of OA peaked initially before declining under both N and P-limited treatments. Notably, R_tox increased only upon P replenishment rather than N replenishment, confirming phosphorus's critical role in OA production. In P-addition experiments, R_tox stagnated in the P-deficient condition but rose proportionally with increasing P concentration. Meanwhile, ATP and NADPH levels surged 7.5-fold and 1.5-fold, respectively, within 1 h of P-addition compared to P-deficient treatment. To probe how P affects OA production, inhibitors targeting ATP and NADPH synthesis were applied. OA production was specifically suppressed by ATP inhibitors, such as N, N-dicyclohexylcarbodiimid (DCCD), rotenone (ROT), and 2,4-dintrophenol (DNP). The highest inhibition occurred with 20 μM DCCD, reducing OA cellular content by 90 % after 48 h. Moreover, increasing ATP inhibitor concentration shift R_tox from positive to negative values. These finding demonstrated that phosphorus drives OA production primarily by modulating ATP levels, which directly regulate toxin synthesis.