Assimilation, Accumulation, and Metabolism of Dinophysistoxins (DTXs) and Pectenotoxins (PTXs) in the Several Tissues of Japanese Scallop Patinopecten yessoensis.

Japanese scallops, Patinopecten yessoensis, were fed with the toxic dinoflagellate Dinophysis fortii to elucidate the relative magnitude of assimilation, accumulation, and metabolism of diarrhetic shellfish toxins (DSTs) and pectenotoxins (PTXs). Three individual scallops were separately exposed to cultured D. fortii for four days. The average cell number of D. fortii assimilated by each individual scallop was 7.7 × 105. Dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2) and their metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the toxin content in individual tissues (digestive gland, adductor muscle, gill, gonad, mantle, and the others), feces and the seawater medium were quantified. Toxins were almost exclusively accumulated in the digestive gland with only low levels being detected in the gills, mantles, gonads, and adductor muscles. DTX1 and PTX2 were the dominant toxins in the D. fortii cells fed to the scallops, whereas the dominant toxins detected in the digestive gland of scallops were PTX6 and esterified acyl-O-DTX1 (DTX3). In other tissues PTX2 was the dominant toxin observed. The ratio of accumulated to assimilated toxins was 21%-39% and 7%-23% for PTXs and DTXs respectively. Approximately 54%-75% of PTX2 and 52%-70% of DTX1 assimilated by the scallops was directly excreted into the seawater mainly without metabolic transformation.

DIFFERENCES IN THE PRODUCTION AND EXCRETION KINETICS OF OKADAIC ACID, DINOPHYSISTOXIN-1, AND PECTENOTOXIN-2 BETWEEN CULTURES OF DINOPHYSIS ACUMINATA AND DINOPHYSIS FORTII ISOLATED FROM WESTERN JAPAN(1).

We established clonal cultures of Dinophysis acuminata Clap. et Lachm. and D. fortii Pavill. isolated from western Japan and examined toxin production in them, focusing on intracellular production and extracellular excretion. At the end of incubations, the total amounts of pectenotoxin-2 (PTX-2), dinophysistoxin-1 (DTX-1), and okadaic acid (OA) in the D. acuminata cultures reached up to 672.7 ± 14.7 (mean ± SD), 88.1 ± 2.8, and 539.3 ± 39.7 ng · mL(-1) , respectively, and the excreted extracellular amounts were equivalent to 5.1, 79.5, and 79.5% of the total amounts, respectively. Similarly, at the end of incubations, the total amounts of PTX-2, DTX-1, and OA in the D. fortii cultures reached up to 526.6 ± 52.6 (mean ±SD), 4.4 ± 0.4, and 135.9 ± 3.9 ng · mL(-1) , respectively, and the excreted extracellular amounts were equivalent to 1.8, 80.1, and 86.6% of the total amounts, respectively. Further, we tested the availability of cell debris and dissolved organic substances that originated from the ciliate prey Myrionecta rubra for growth and toxin production in D. acuminata. Although no significant growth was observed in D. acuminata in the medium containing the cell debris and organic substances originated from M. rubra, the toxicity was significantly greater than that in the control (P < 0.05-0.001); this finding suggested the availability of organic substances for toxin production. However, toxin productivity was remarkably lower than that of Dinophysis species feeding on living M. rubra.

Is yessotoxin the main phycotoxin in Croatian waters?

With the aim of investigating whether yessotoxin (YTX) is responsible for diarrhetic shellfish poisoning (DSP) events in Croatian waters, three different methods were combined: a modified mouse bioassay (MBA) that discriminates YTX from other DSP toxins, the enzyme-linked immunosorbent assay method (ELISA) and liquid chromatography-mass spectrometry (LC-MS/MS). Among 453 samples of mussels and seawater analyzed in 2007, 10 samples were DSP positive. Results obtained by the modified MBA method revealed that most of the samples were positive for YTX, with the exception of samples from Lim Bay (LB 1) The ELISA method also identified the presence of YTX in these samples. DSP toxin profiles showed the presence of okadaic acid (OA) in three, and YTX in four out of nine samples that were analyzed by LC-MS/MS. The phytoplankton community structure pattern revealed Lingulodinium polyedrum (Stein) Dodge, which was present in the water prior to and/or during toxicity events at low concentrations (80 to 1440 cells L(-1)), as a potential YTX producing species. It is proposed that L. polyedrum cells accumulated in mussels and the subsequently observed toxicity may be related to metabolism after ingestion, resulting in carboxy YTX as the major analog in the mussel.

PREDATION BY THE TOXIC DINOFLAGELLATE DINOPHYSIS FORTII ON THE CILIATE MYRIONECTA RUBRA AND OBSERVATION OF SEQUESTRATION OF CILIATE CHLOROPLASTS(1).

This is the first report of the propagation of the toxic dinoflagellate Dinophysis fortii Pavill. under laboratory conditions when fed on the marine ciliate Myrionecta rubra grown with the cryptophyte Teleaulax amphioxeia (W. Conrad) D. R. A. Hill. In contrast, reduced growth of D. fortii (max. of 3-4 divisions) and formation of small cells were observed in the absence of the ciliate or when provided with T. amphioxeia only as prey, showing that D. fortii cannot utilize T. amphioxeia as prey. In the TEM observation of D. fortii cells, which had fully fed on the ciliate prey, well-developed chloroplasts (5-12 µm in length) were seen and three thylakoids were usually arranged in most of the chloroplasts observed, but chloroplasts having two thylakoids were sometimes confirmed. In cells starved for 4 weeks, decrease of chloroplast numbers and disappearance of large chloroplasts were observed, and only a few small chloroplasts (0.5-2 µm in length) remained in the marginal regions. In the observation of the sequestration process of the chloroplasts ingested from M. rubra by D. fortii, within 15 min after D. fortii captured M. rubra, incorporation of almost all of the chloroplasts was observed, while most of the other cell contents still remained in the M. rubra cell. After that, dispersion of the ingested chloroplasts toward the marginal regions was confirmed, suggesting that chloroplasts of M. rubra are ingested and dispersed in D. fortii cells in advance of the ingestion of the other cell contents to prevent them from being digested in food vacuoles. The ingested chloroplasts can also function as kleptoplastids.