[Toxicity and toxin profile of the dinoflagellate Alexandrium tamiyavanichii and toxic mussels in Harima-Nada of Seto Inland Sea, Japan].

From October to November 2004, the paralytic shellfish poison (PSP)-producing dinoflagellate Alexandrium tamiyavanichii was observed at Harima-Nada, Seto Inland Sea at a maximum cell density of 4,960 cells/L. The wild cells of the dinoflagellate collected from the same seawaters, and cultured cells derived from them showed toxicity scores of 6.25-15.4 x 10(-4) and 2.7-3.5 x 10(-4) MU/cell, respectively, both of which were much higher than those of previously reported strains. PSP of the wild cells was mainly composed of gonyautoxin (GTX) 5 (40.6-52.4 mol%) and GTX4 (15.6-24.8 mol%), showing a unique composition that was greatly different from those of the previously reported strains, or of the cultured cells, whose main toxin component was GTX3 (average 37.6 mol%). The mussel Mytilus galloprovincialis collected from the same area in the same period accumulated a relatively high level of PSP (13-28 MU/g), suggesting a risk that A. tamiyavanichii may induce high-level PSP contamination of bivalves even at a cell density as low as around 5,000 cells/L.

[Toxicity and toxin profiles of xanthid crabs collected around Nakanoshima in the Tokara Islands, Japan].

A total of 36 specimens of 5 xanthid crab species, Zosimus aeneus (n=16), Xanthias lividus (n=4), Leptodius sanguineus (n=3), Daira perlata (n=10) and Eriphia sebana (n=3), were collected around Nakanoshima Island, which is located at the northeastern part of the Tokara Islands, Kagoshima Prefecture, Japan in May and July 2000, and their toxicity was determined by mouse bioassay. Nine of 16 Z. aeneus specimens and all of 4 X. lividus specimens showed lethal potency to mice (2.1-11 MU/g, 2.8-8.6 MU/g, respectively), whereas all the other species were non-toxic (less than 2.0 MU/g). LC/MS analyses indicated that the toxin of the Z. aeneus specimens was mainly composed of tetrodotoxin (41% of total toxicity), and 11-oxotetrodotoxin contributed to the remaining toxicity. The toxin of the X. lividus specimens, however, was apparently not tetrodotoxin. In HPLC-FLD analyses, no paralytic shellfish poison component was detected in either of the two toxic species.

[Survey of food poisoning incidents in Japan due to ingestion of marine boxfish and their toxicity].

From 1990 to 2008, 9 food poisoning incidents due to ingestion of marine boxfish occurred in Nagasaki, Miyazaki, Mie and Kagoshima Prefectures, Japan, and a total of 13 persons were poisoned. Their main symptom was severe muscle pain arising from rhabdomyolysis, which was usually accompanied by the discharge of black urine and abnormal elevation of serum creatine phosphokinase. Twelve out of the 13 victims recovered in a few days to two months, while one died after approximately 2 weeks. Since the symptoms were very similar to those caused by parrotfish "aobudai" Scarus ovifrons poisoning, the causative substance was considered to be parrotfish toxin, i.e., a palytoxin-like substance. Epidemic surveys after the incidents in Miyazaki and Nagasaki identified the leftovers as "hakofugu" Ostracion immaculatus. During screening tests to clarify the toxicity of boxfish from Western Japan, 47 of 129 specimens (36.4%) of O. immaculatus, and 7 of 18 specimens (38.9%) of "umisuzume" Lactoria diaphana were found to show acute and/or delayed lethal activity to mice (0.5-2.0 mouse unit/g). Among the tissues tested, the frequency of toxicity was highest in the viscera excluding liver (28.6% in O. cubicus, 33.3% in L. diaphana), followed by muscle (10.9%, 5.6%) and liver (6.2%, 5.6%). From the above results, we conclude that O. cubicus and L. diaphana inhabiting the coast of Japan sometimes contain toxic substance(s), which can sporadically cause food poisonings very similar to parrotfish poisoning.

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae).

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua. These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.

Occurrence of PSP-producing dinoflagellate Alexandrium tamiyavanichii in Bingo-Nada, the central coastal water of the Seto Inland Sea, Hiroshima Prefecture, Japan.

During surveillance of the distribution of the paralytic shellfish poison (PSP)-producing dinoflagellate in 2003, 2004 and 2005 along the coastlines of the Seto Inland Sea, Hiroshima Prefecture, Japan, some species of toxic phytoplankton were isolated from the eastern coasts, Bingo-Nada, the central regions of the Seto Inland Sea. It was rather unexpectedly revealed from the basis of the morphological characteristics that they were unambiguously identified as Alexandrium tamiyavanichii and Alexandrium catenella. Two strains (ATY041106, ATY051018) of A. tamiyavanichii showed a specific toxicity of 38.7 x 10(-6) and 111.5 x 10(-6)MU/cell, respectively. These values seemed to be several times or much higher than that of A. catenella (AC030816, AC040614), having a specific toxicity of 4.5 x 10(-6) and 4.1 x 10(-6)MU/cell, respectively, isolated in the same area. From the results of HPLC-furuorometric analysis, it revealed that the toxins in ATY041106 exist almost exclusively as beta-epimers (C2, GTX3, GTX4), which accounted for 72.7 mol%. The toxin profiles of this strain are featured by the presence of a large amount of GTX3 (59.1 mol%) and a small amount (20.6%) of C1 and 2 in comparison with the PSP compositions of A. tamarense, which is isolated as the main responsible species in Hiroshima Bay, a western part of coastal sea in Hiroshima Prefecture. On the other hand, it revealed that the toxin profiles of two strains (AC030816, AC040614) of A. catenella exist almost exclusively as beta-epimers (C2, GTX3, GTX4), which accounted for 81.8 and 56.5 mol%, as the same manner. The toxin profiles of these two strains are featured by the presence of a large amount of C2 (80.5 and 46.3 mol%) in comparison with the PSP compositions of A. tamiyavanichii. To our knowledge, this is the first record to show the distribution and harmful influence of A. tamiyavanichii and A. catenella in Bingo-Nada in Hiroshima Prefecture. Though contamination of bivalves with these PSP-producing planktons in this area has not occurred yet so far, attention should be paid to this species as well as the other causative dinoflagellate from the stand point of public health and food hygiene.

Ostreopsis sp., a possible origin of palytoxin (PTX) in parrotfish Scarus ovifrons.

A clone of toxic dinoflagellate Ostreopsis sp. and six specimens of a parrotfish Scarus ovifrons were collected in October 1997 at Tokushima Prefecture, Japan. Ostreopsis sp. was cultured in ESM medium for 16 days, and after rearing the cell pellet (about 4.0x10(5) cells) was extracted with 50% methanol, partitioned between an aqueous layer and 1-butanol layer, and biochemically tested. Similarly, the crude toxin from S. ovifrons was extracted, and tested. The mice injected with each 1-butanol layer from Ostreopsis sp. and S. ovifrons showed the common symptoms of convulsion, drowsiness and collapse, and died within 48 h. The lethal potency of Ostreopsis sp. was calculated to be 1.0x10(-4) MU/cell. All specimens of S. ovifrons were found to be toxic, where the highest potency was determined as 2 MU/g in muscle of one specimen. After being injected with toxins, the serum creatine phosphokinase levels of mice were found to be elevated. Toxins from Ostreopsis sp. and S. ovifrons showed delayed haemolytic activity with mouse and human erythrocytes, which was inhibited by an anti-palytoxin (PTX) antibody antibody and ouabain. Toxins from Ostreopsis sp. and S. ovifrons thus resembled each other, and strongly suggested to be PTX or its akin substance. Additionally, a considerable number of adherent Ostreopsis sp. was found in the gut contents of S. ovifrons during the heavy occurrence of Ostreopsis sp. in October 1997 at Tokushima Prefecture. From the above results, it can be strongly postulated that the dinoflagellate Ostreopsis sp. is the origin of PTX which is sequestered by the parrotfish S. ovifrons through food chain.

A new analytical method for gonyautoxins based on postcolumn HPLC.

A new ion-pairing high-performance liquid chromatography (HPLC) method on a C30 column with a volatile mobile phase was developed to separate the gonyautoxin group (GTXs) from contaminants, allowing the utilization of liquid chromatography/mass spectrometry (LC/MS) with higher performance. A mobile phase consisting of 5 mmol/L heptafluorobutyric acid and 2% acetonitrile in 10 mmol/L ammonium acetate was adopted for separation of GTXs because the C30 column strongly retains GTXs under acidic conditions. The newly adopted method could efficiently separate GTXs from contaminants, especially in the toxic short-necked clam, whereas the routine HPLC so far used has poor resolution to separate GTXs from unknown interfering substances. In our method, GTXs were eluted in the order of GTX5, GTX3, GTX4, GTX2 and GTX1 from the C30 column, and were successfully determined by sonic spray ionization mass spectrometry (SSI-MS) with high sensitivity. This method is characterized by the combination of HPLC using a fluorescence detection system for PSP, and SSI-MS for measurement of the mass number.

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan.

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.