2018 Volume 6 Issue 2 Pages 83-87
An adequate supply of standard reference material for paralytic shellfish toxins (PSTs) is critical for the accurate chemical quantification using high performance liquid chromatography (HPLC) with fluorescent detection, liquid chromatography-tandem mass spectrometry (LC-MS/MS), biological analysis of these toxins using enzyme-linked immunosorbent assay (ELISA), and immunochromatography. Large batch cultivation for the chain forming species G. catenatum, producers of PSTs of N-sulfocarbamoyl-11-hydroxysulfate toxins (C1 and C2), gonyautoxin 5 (GTX5) and gonyautoxin 6 (GTX6), was investigated using 10 L round-bottom flasks with aeration for the production of GTX5 and GTX6. Aeration rates of 200 mL/min and 500 mL/min were compared, demonstrating that the 500 mL/min aeration rate was adequate to eliminate aggregation of cells. The highest cell density of G. catenatum in 500 mL/min aeration treatment was 9,878 ± 2,617 cells/ml on day 28. Total toxin yield during 10 L cultivation with 500 mL/min aeration was calculated at 30.9 ± 3.6 µmol on day 25, with GTX5 and GTX6 calculated at 3.9 ± 0.7 µmol and 11.4 ± 1.4 µmol, respectively. This simple aeration method will contribute to the more efficient production of PST reference materials.
Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins produced by several species of dinoflagellates in marine environments1,2,3,4). The accumulation of PSTs in bivalves is a serious threat to public health and adversely impacts the economics of bivalve aquaculture1,2,3,4). The standard regulatory method for PST analysis in Japan is the mouse bioassay5), but a desire to move away from animal bioassays has been recognized in recent years. High performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are promising alternatives to animal bioassays6,7,8,9). In addition, enzyme-linked immunosorbent assay (ELISA) and immunochromatography were officially introduced in 2015 as PST screening methods in Japan10). These analytical and screening methods require reference materials to quantify or identify PSTs, but these reference materials are currently available only from sources outside of Japan. Therefore, domestic production of this reference material is an important issue to ensure a stable supply. Alexandrium tamarense, Alexandrium catenella and Gymnodinium catenatum are major PST-producing dinoflagellates distributed around Japanese coastal waters11,12,13,14,15,16), and the cultivation of these toxic dinoflagellates provides the raw materials for extraction of PSTs. Large-scale cultivation of A. tamarense and A. catenella has already been reported17,18,19), but, as far as we know, large scale cultivation of G. catenatum has not been reported. The profiles of PSTs are different among species; GTX5 and GTX6 are the major toxin components produced by G. catenatum20,21). Therefore, it is important to develop a method of large-scale cultivation of G. catenatum to produce reference materials of GTX5 and GTX6.
In this communication, cultivation methods for G. catenatum were investigated using 10 L round-bottom flasks. The paralytic shellfish poisoning (PSP) toxin yield from cultures was analyzed using high performance liquid chromatography with fluorescent detection (HPLC-FLD)6).
G. catenatum (strain GC-123) isolated from Kamae Bay, Oita prefecture was used in this study. This strain was maintained in modified f/2 medium reported by Matsuyama22) with selenious acid added (1.0 µg/L). Cells were inoculated into a commercially available round-bottom 10 L flask (flat flange; Schott AG, Germany) until the cell density of about 200 cells/mL was obtained. During cultivation, filtered air was delivered to the bottom of the flask using a glass tube to generate an upward flow of air to circulate the cells. Aeration rates of 200 mL/min and 500 mL/min were compared. Cultures were maintained at 15°C, 100 µmol photons m-2s-1 and a 16:8h L:D cycle.
2.2. Cell Count during CultivationAn aliquot of culture medium was collected on days 0, 2, 4, 7, 10, 14, 18, 21, 25 and 28 for quantification of G. catenatum cell density using microscopy. In addition, the number of cells within each chain were grouped as 1-4 cells, 5-8 cells and >9 cells and the proportion of each chain to the total cell count was calculated.
2.3. Toxin Yield from the CultureAn aliquot of the culture medium was collected on days 0, 2, 4, 7, 10, 14, 18, 21, 25 and 28 for analysis of PSTs. The culture medium was centrifuged at 600 g for 3 min, and the toxin content in the pellets was analyzed by HPLC-FLD6). The toxin standards: GTX1, GTX2, GTX3, GTX4, dcGTX2, dcGTX3, neosaxitoxin (neoSTX), C1, C2, used for HPLC-FLD analysis, were supplied by Ministry of Agriculture, Forestry and Fisheries of Japan (MAFF). GTX5, GTX6 and dcSTX were kindly provided by Dr. Oshima, former Prof. of Tohoku University. The saxitoxin (STX) standard used in this study was purified at one of our branch laboratories (National Research Institute of Fisheries and Environment of Inland Sea) and kindly quantified by Dr. Asakawa at Hiroshima University.
The cell density of G. catenatum during large-scale cultivation is shown in Fig. 1. The highest cell density in the 200 mL/min aeration treatment reached 7,828 ± 916 cells/mL on day 18, but quickly decreased after that. In the case of the 500 mL/min aeration treatment, the cell density was 8,869 ± 1,229 cells/mL on day 18. The highest cell density in the 500 mL/min aeration treatment was 9,878 ± 2,617 cells/ml on day 28. These results indicate that the 500 mL/min aeration is a more favorable treatment compared to the 200 mL/min aeration. The proportion of chains of 1-4 cells, 5-8 cells and >9 cells relative to the total cell count are shown in Fig. 2. The proportion of long chains >9 cells in the 200 mL/min aeration treatment was highest on day 18 accounting for 58.4% of the total (Fig. 2a). The proportion of >9 cells then quickly decreased after day 18, corresponding to the decrease in cell density (Fig. 1). Large aggregations of cells were observed in the 200 mL/min aeration treatment during the later period of cultivation. On the other hand, the proportion of long chain >9 cells in the 500 mL/min aeration treatment was low compared to those in 200 mL/min aeration accounting for 23.4% on day 18. In addition, no cell aggregates were found in the 500 mL/min aeration treatment. We assume that the long chains caused an aggregation of cells in 200 mL/min aeration, and that the aggregation of cells led to a lower cell density.
The changes in cell density during the cultivation of Gymnodinium catenatum using 10 L round-bottom flasks using different aeration rates: 500 mL/min (closed circle) and 200 mL/min (open triangle). Values are mean ± standard deviation (n = 3).
The proportion of cells observed as chains of 1-4 cells, 5-8 cells and >9 cells (see legend) during the cultivation of Gymnodinium catenatum using 10 L round-bottom flask with aeration.
The toxin yield in the 10 L culture at 500 mL/min aeration is shown in Fig. 3. The total toxin yield reached a maximum of 30.9 ± 3.6 µmol on day 25, with GTX5 and GTX6 calculated at 3.9 ± 0.7 µmol and 11.4 ± 1.4 µmol, respectively. GTX5 and GTX6 certified reference materials are currently available only from National Research Council Canada (NRC), and those products are ampules of 0.5 mL solution at a concentration of 55.7 μM. The products contain 0.02785 μmol of GTX5 and GTX6 in each ampule. This suggests that a sufficient amount of GTX5 and GTX6 will be obtained from G. catenatum cells cultivated by the method developed in this study for use as reference material.
Toxin yield from 10 L culture of Gymnodinium catenatum with 500 mL/min aeration. The closed circles indicate the total toxin analyzed, the open triangles indicate GTX5 and the open diamonds are GTX6. Values are mean ± standard deviation (n = 3).
The large-scale cultivation of G. catenatum was described here to complement the methods already reported for A. tamarense and A. catenella.17,18,19). It is known that PSTs produced by dinoflagellates are transformed into other PST analogs through metabolism by shellfish23,24). Several of the analogs therefore are not produced directly by the toxic dinoflagellates. However, we already have reported the method of chemical conversion of PST to obtain various PST analogs, including those that are not produced directly by the toxic dinoflagellates25,26). We expect that the methods of large-scale cultivation of PST-producing dinoflagellates and the chemical conversion of PSTs will contribute to the production and supply of reference material of PST.
We express our gratitude to Dr. Oshima, professor emeritus at Tohoku University and Animal Products Safety Division, Food Safety and Consumer Affairs Bureau in MAFF, for providing PSP toxin standards. We also thank Dr. Asakawa at Hirohsima University for quantifying saxitoxin prepared in our laboratory. This study was partially supported by the Regulatory research projects for food safety, animal health and plant protection funded by the Food Safety and Consumer Affairs Bureau in MAFF.
The authors have no conflict of interest.
