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A feeding experiment of TTX-containing diet was conducted using the small scavenging marine snails Pliarcularia globosa and Reticunassa festiva. Seventy-five specimens of each species were divided into 15 groups of 5 individuals, of which 3 groups were directly submitted, without feeding, to toxin quantification as described below. TTX was not detected. Each of the remaining 12 groups was accommodated in a plastic case (80×70×40 mm) filled with seawater, and fed for 24 hours with ovary tissue (0.1 g) of the pufferfish Takifugu vermicularis, whose TTX content had previously been determined. Then the seawater was exchanged for fresh seawater, the snails were reared for 4 days without feeding, and then the seawater was changed again. This feeding/rearing cycle (5 days) was repeated 8 times, and 3 groups were sampled every 2 cycles. The combined viscera and combined muscle of each group were each extracted with 0.1% aqueous acetic acid, and then TTX was quantified by liquid chromatography-mass spectrometry. The estimated amount of ingested TTX was calculated by multiplying the difference between the amounts of ovary tissue supplied and remaining by the toxin content (122-126 MU/g). Similar mean values of 5.1 MU/group/cycle in P. globosa and 5.3 MU/group/cycle in R. festiva were obtained. Toxin content (TTX amount per gram of tissue) and toxin amount (TTX amount per group) during the experimental period were 0.23-2.85 MU/g and 0.05-0.96 MU/group, respectively, in P. globosa viscera. Both values increased markedly from the 2nd cycle to the 6th cycle. In contrast, no such increase in toxin content/amount was observed throughout the experimental period in P. globosa muscle (

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.

Tetrodotoxin (TTX) was administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu porphyreus to investigate toxin accumulation in hybrids and TTX transfer/accumulation profiles in the pufferfish body. In test fish administered TTX-containing feed homogenate at a dose of ∼400 MU/fish by oral gavage using a syringe (OGA group), the toxin content (MU/g tissue) of the digestive tract rapidly decreased and that of the liver increased from 1 to 24 h after administration. From 24 to 120 h, the toxin content of the liver decreased gradually, and the toxin appeared in the skin. On the other hand, intramuscularly administered TTX (400 MU/fish) was rapidly transferred to the liver and skin via the blood, and only a little toxin remained in the muscle even at 1 h (IMA group). The total amount of toxin remaining in the whole body (% of administered toxin) was 31-45% in the OGA group, and 42-74% in the IMA group; the scores in the OGA group were generally lower than those in the IMA group. In both OGA and IMA groups, the greatest amount of toxin accumulated in the liver (23-52%) after 8 h, followed by the skin (11-21%) after 72 h. The TTX administration experiment, especially using the oral gavage administration method, revealed that skins and livers of 'torama' pufferfish hybrid are endowed with TTX-accumulating ability, but the muscles are not, and that TTX taken up from toxic feed to the pufferfish body is transferred first to the liver and then to the skin via the blood.

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.

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.

From September 2007 to January 2008, a total of 66 specimens of 7 gastropod species, Nassarius (Alectrion) glans (n=22), Bufonaria rana (n=11), Ficus subintermedia (n=10), Stellaria (Onustus) exutus (n=8), Tonna luteostoma (n=7), Hemifusus tuba (n=4) and Semicassis bisulcata persimilis (n=4), were collected from Tachibana Bay, Nagasaki Prefecture, Japan, and their toxicity was determined by mouse bioassay. Among the gastropods tested, all N. glans specimens were toxic, whereas no other species showed toxicity of more than 5 MU/g. The toxicity scores of N. glans were very high; 48-2,730 MU/g(775+/-615 MU/g)in the muscle, and 16-10,200 MU/g(1,490+/-2,530 MU/g)in the viscera, including digestive gland. Interestingly, toxin was localized in the muscle in 13 of 22 specimens, where the total toxicity of the muscle (725-9,860 MU/individual) was 5.9-110 times higher than that of the viscera. LC/MS analysis demonstrated that the toxin of N. glans consisted mainly of TTX, which accounting for about 60-65% of the total toxicity. As for the remaining toxicity, participation of 11-oxoTTX was suggested. No paralytic shellfish poison was detected in HPLC-FLD analysis.

In Cambodia, fatal food poisonings associated with the consumption of pufferfish have occurred for decades, but the causative species or toxins have never been documented. Herein, we investigated the toxicity of three pufferfish species of the genus Lagocephalus collected from the coastal waters of Sihanouk Ville, one of the main regions where poisonings have occurred. L. wheeleri and L. spadiceus were non-toxic, whereas L. lunaris was toxic and all of its body tissues exhibited toxicity levels exceeding the safety limit for human consumption (10 mouse units/g). Tetrodotoxin (TTX) was identified as the main toxin in this species; no paralytic shellfish poison(s) were detected. Consequently, we can confirm pufferfish to be a hazardous reservoir of TTX in Sihanouk Ville. It is likely that L. lunaris is one of the causative species of past pufferfish poisonings that have occurred in Cambodia.

The paralytic shellfish poison (PSP)-producing dinoflagellate Gymnodinium catenatum (Gc) was fed to the short-necked clam Tapes japonica, and the accumulation, transformation and elimination profiles of PSP were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested most of the Gc cells (4 x 10(6) cells) supplied as a bolus at the beginning of the experiment, and accumulated a maximal amount of toxin (181 nmol/10 clams) after 12 hr. The rate of toxin accumulation at that time was 16%, which rapidly decreased thereafter. During the rearing period, a variation in toxin composition, derived presumably from the transformation of toxin analogues in the clams, was observed, including a reversal of the ratio of C2 to C1, and the appearance of carbamate (gonyautoxin (GTX) 2, 3) and decarbamoyl (dc) derivatives (decarbamoylsaxitoxin (dcSTX) and dcGTX2, 3), which were undetectable in Gc cells. The total amount of toxin contained in clams and residue (remaining Gc cells and/or excrement in the rearing tank) gradually declined, and only about 1% of the supplied toxin was detected at the end of the experiment.