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The content and composition of paralytic shellfish toxins (PSTs) in Japanese basket clam (Corbicula japonica) and mussels (Mytilus galloprovincialis) from Osaka Bay, Japan, were investigated using a mouse bioassay (MBA) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), and the association between toxicity values of MBA and HILIC-MS/MS was verified based on research data. The overall toxicity in Japanese basket clam was lower than that in the mussel. The PSTs of Japanese basket clam and mussel consisted mainly of C1, C2, and gonyautoxins 1–4 (GTX1–4) taking toxins compositional differences as mol%. When multiplying the content of different toxins by the toxic equivalent factor (TEF), C2 and GTX1–4 accounted for more than 90% of total toxicity (MU TEF/g) based on the MU TEF score converted by TEF for the two species. The total content of C2 and GTX1–4 converted to toxicity was significantly correlated with the toxicity determined by MBA for the two species (r2 > 0.983). This study provides a suitable and ethical monitoring method to investigate toxicity in bivalves contaminated with A. tamarense by analysis of only predominant toxins, along with reducing use of MBA. [ABSTRACT FROM AUTHOR]

Abstract: 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 38.7×10−6 and 111.5×10−6 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 4.5×10−6 and 4.1×10−6 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 β-epimers (C2, GTX3, GTX4), which accounted for 72.7mol%. The toxin profiles of this strain are featured by the presence of a large amount of GTX3 (59.1mol%) 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 β-epimers (C2, GTX3, GTX4), which accounted for 81.8 and 56.5mol%, 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 46.3mol%) 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. [Copyright &y& Elsevier]

During surveillance on the toxicity of invertebrates such as bivalves inhabiting the coasts of Hiroshima Bay in 2001 and 2002, the carnivorous gastropod rapa whelk Rapana venosa collected in the estuary of Nikoh River, was found to contain toxins which showed paralytic actions in mice; the maximum toxicities (as paralytic shellfish poison, PSP) were 4.2 MU/g (May 2001) and 11.4 MU/g (April 2002). Their total toxicities were 224 and 206 MU/viscera of one specimen throughout the monitoring period. Attempts were made to identify the toxic principle in the gastropod. The viscera were extracted with 80% ethanol acidified with acetic acid, followed by defatting with dichloromethane. The aqueous layer obtained was treated with activated charcoal and then applied to a Sep-Pak C18 cartridge. The unbound toxic fraction was analyzed by high-performance liquid chromatography techniques. The gastropod toxin was rather unexpectedly identified as PSP. It was comprised of high toxic component (gonyautoxin-3; GTX3, GTX2, saxitoxin; STX) as the major components, which accounted for approximately 91 mol% of all components along with C1 and C2, which are N-sulfocarbamoyl derivatives.Judging from their toxin patterns, it is suggested that the PSP toxification mechanism of the gastropod that PSP toxins produced by phytoplankton such as Alexandrium tamarense, are transferred to and accumulated in plankton feeders such as the short-necked clam, and then transfered to this carnivorous rapa whelk R. venosa through predation. [Copyright &y& Elsevier]

ABSTRACT: Sediment samples were collected from 135 stations in the western part of the Seto Inland Sea (Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada) to determine the horizontal distribution and abundance of resting cysts of Alexandrium spp. (A. tamarense + A. catenella ). Enumeration of the cysts was performed using the primuline-staining direct count method. Cysts of Alexandrium spp. were rarely found in Iyo Nada, Suo Nada and Beppu Bay, but were widely distributed in Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada. Cyst concentrations ranged from not detected (ND) to 14, ND to 17, ND to 4, 93 to 8137, 8 to 4454, ND to 6, ND to 18 and 4–29 cysts/cm3 wet sediment in Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada, respectively. The majority of cysts occurred in Tokuyama Bay and Hiroshima Bay, where higher densities were observed in the inner bay and along the coastal margin. Relatively higher cyst concentrations were observed at stations with a higher mud content. The abundance of Alexandrium spp. cysts in western Seto Inland Sea is lower than in the eastern Seto Inland Sea, except for Tokuyama Bay and Hiroshima Bay. However, because sporadic blooms of Alexandrium have been observed, continuing monitoring is necessary to prevent paralytic shellfish poisoning outbreaks in this area. [ABSTRACT FROM AUTHOR]

The 5.8Sribosomal RNA gene (rDNA) and flanking internal transcribed specers 1 and 2 (TTS1 and (TTS2) from 7 isolates of Alexandrium castenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fikuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudgonyaulax (Biecheler) Horiguchi exYuki et Fukyo comb, nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reactin (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent "ITS types" destignated as follows; 1) catenella type,2) tamarense type, 3) WKS-1 type,4) Thai tupe, 5) affine type,6) insuetum typem and 7) pseudogonyaulax type, Isolates of the tamarensse type from various locatins in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent form isolates of A. tamarense WKS-1 (WKS-1 type) or A. tamarense CU-15 (That tupe). These latter two strains carried unique ITS types, although they were not distinguishable form isolates of the tamarense tupe by morphological criteria. Distance values between isolates of the tamarense tupe and the WKS-1 for That type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distamce values between isolates of the catenella tupe were extremely low (<0.01). Whereas ditance values of ITS between the catenella type and the tamarense. WKS-1, or That tupe were 0.17,0.18 and 0.40, respectively, isolates of A. affine, A. insuetum. And A. pseudogonyaulax all carried unique ITS types. The ITS of the tamarense type exhibited two distinct ITS sets, the "A gene" and the "B gene," The two sequences accurred in a 1:1 ratin in PCR products. In contrast the ITS of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3' end of ITS1(150-177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28-bp region is more than 10 High diversity of the region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium. [ABSTRACT FROM AUTHOR]

Paralytic shellfish toxins (PSTs) are the major neurotoxic contaminants of edible bivalves in Japan. Tetrodotoxin (TTX) was recently detected in bivalve shellfish around the world, drawing widespread attention. In Japan, high levels of TTX were reported in the digestive gland of the scallop, Patinopecten yessoensis , in 1993; however, no new data have emerged since then. In this study, we simultaneously analyzed PSTs and TTX in scallops cultured in a bay of east Japan using hydrophilic interaction chromatography (HILIC)-MS/MS. These scallops were temporally collected from April to December 2017. The highest concentration of PSTs (182 µmol/kg, total congeners) in the hepatopancreas was detected in samples collected on May 23, lined to the cell density of the dinoflagellate, Alexandrium tamarense , in seawater around the scallops, whereas the highest concentration of TTX (421 nmol/kg) was detected in samples collected on August 22. Contrary to the previous report, temporal variation of the PSTs and TTX concentrations did not coincide. The highest concentration of TTX in the entire edible tissues was 7.3 µg/kg (23 nmol/kg) in samples obtained on August 22, which was lower than the European Food Safety Authority (EFSA)-proposed threshold, 44 µg TTX equivalents/kg shellfish meat. In addition, 12β-deoxygonyautoxin 3 was firstly identified in scallops., Competing Interests: The authors declare no conflict of interest.