Diarrhetic shellfish poisoning (DSP) is a gastrointestinal disorder caused by the consumption of seafood contaminated with okadaic acid (OA) and dinophysistoxins (DTXs). OA and DTXs are potent inhibitors of protein phosphatases 2A, 1B, and 2B, which may promote cancer in the human digestive system. Their expression in dinoflagellates is strongly affected by nutritional and environmental factors. Studies have indicated that the level of these biotoxins is inversely associated with the growth of dinoflagellates at low concentrations of nitrogen or phosphorus, or at extreme temperature. However, the presence of leucine or glycerophosphate enhances both growth and cellular toxin level. Moreover, the presence of ammonia and incubation in continuous darkness do not favor the toxin production. Currently, studies on the mechanism of this biotoxin production are scant. Full genome sequencing of dinoflagellates is challenging because of the massive genomic size; however, current advanced molecular and omics technologies may provide valuable insight into the biotoxin production mechanism and novel research perspectives on microalgae. This review presents a comprehensive analysis on the effects of various nutritional and physical factors on the OA and DTX production in the DSP toxin-producing Prorocentrum spp. Moreover, the applications of the current molecular technologies in the study on the mechanism of DSP toxin production are discussed.

We aimed to study the growth and toxicity responses of non-toxic (CCMP683) and toxic (CCMP2804) strains of Prorocentrum hoffmannianum under various nitrate and phosphate concentrations. The 2 strains were cultured in L1-Si medium with standard, depleted or 10-fold repleted nitrate or phosphate. CCMP683 cultured in standard L1-Si medium exhibited delayed growth. Nitrate or phosphate depletion decreased the cell density of both strains. Repletion of nitrate slightly increased the cell density of both strains. Repletion of phosphate also slightly increased the cell density of CCMP2804 but surprisingly decreased the cell density of CCMP683. Okadaic acid (OA) and its derivatives were not detected in CCMP683. OA was detected only in CCMP2804. Depletion of nitrate or phosphate increased the cellular concentration of OA, and repletion of nitrate or phosphate had no effect on the cellular concentration of OA. Correlation analysis indicated that the cellular concentration of OA was negatively correlated with cell density. Differences in the growth response to phosphate repletion and in the ability to produce OA suggest that the 2 strains may be good candidates for comparative studies related to phosphate metabolism and OA toxicity.

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Academic research on dinoflagellate, the primary causative agent of harmful algal blooms (HABs), is often hindered by the coexistence with bacteria in laboratory cultures. The development of axenic dinoflagellate cultures is challenging and no universally accepted method suit for different algal species. In this study, we demonstrated a promising approach combined density gradient centrifugation, antibiotic treatment, and serial dilution to generate axenic cultures of Karenia mikimotoi (KMHK). Density gradient centrifugation and antibiotic treatments reduced the bacterial population from 5.79 ± 0.22 log10 CFU/mL to 1.13 ± 0.07 log10 CFU/mL. The treated KMHK cells were rendered axenic through serial dilution, and algal cells in different dilutions with the absence of unculturable bacteria were isolated. Axenicity was verified through bacterial (16S) and fungal internal transcribed spacer (ITS) sequencing and DAPI epifluorescence microscopy. Axenic KMHK culture regrew from 1000 to 9408 cells/mL in 7 days, comparable with a normal culture. The established methodology was validated with other dinoflagellate, Alexandrium tamarense (AT6) and successfully obtained the axenic culture. The axenic status of both cultures was maintained more than 30 generations without antibiotics. This efficient, straightforward and inexpensive approach suits for both armored and unarmored dinoflagellate species.