Occurrence of tetrodotoxin in the gastropod mollusk Tutufa lissostoma (frog shell)

Paralytic toxicity was detected in gastropod mollusk Tutufa lissostoma (frog shell) specimens collected from Suruga Bay, Shizuoka Prefecture, Japan. Seventeen of the 22 digestive glands removed were toxic; the highest toxicity, expressed as tetrodotoxin (TTX), being as high as 700 mouse units (MU) per gram. Attempts were made to isolate the toxin from pooled digestive glands by activated charcoal treatment and column chromatography on Amberlite IRC-50, CM-Sephadex C-25 and Bio-Rex 70. The toxin showed a specific toxicity (as TTX) of 4200 MU/mg. It exhibited the same thin-layer chromatographic and electrophoretic behaviors and 1H-NMR spectrum as TTX. The toxin gave the same pattern as the TTX standard when alkali-hydrolyzed and analyzed by GC--MS, indicating that it contains the quinazoline skeleton specific to TTX. From these results the frog shell toxin was identified as TTX.     

Toxicities of two freshwater puffers in Taiwan

Forty-two and twelve specimens of puffers Tetraodon ocellatus and T. nigroviridis were, respectively, collected from the aquaria in Taiwan and determined for toxicity by using tetrodotoxin bioassay. It was found that T. ocellatus contained moderate amounts of toxin in skin and viscera, and the highest toxicity scores were 432 MU/g skin and 212 MU/g viscera. The specimens of T. nigroviridus contained weak amounts of toxin in skin, and the highest toxicity score was 124 MU/g. The toxin was partially purified from the toxic specimens of each species by ultrafiltration using a YM-1 membrane, followed by chromatography on Bio-Gel P-2 column. Analyses by electrophoresis, thin layer chromatography, and high performance liquid chromatography showed that the toxin from both species was composed of tetrodotoxin and anhydrotetrodotoxin.     

Tetrodotoxin and derivatives in several species of the gastropod Naticidae

The organs of 195 specimens of 4 species of the gastropod Naticidae, collected from fish markets in Taiwan, were assayed for toxicity. The calf moon shell Natica vitellus contained weak toxicity (10-99 MU/g) in the muscle and digestive gland. The bladder moon shell Polinices didyma contained moderate (100-999 MU/g) and weak toxicity in the muscle and digestive gland, respectively. The digestive gland in 1 out of 20 specimens of the pear-shaped moon shell P. tumidus was toxic (4 MU/g). All tissues of the butterfly moon shell N. alapapilionis were non-toxic. The toxins were partially purified from the toxic specimens of the calf moon shell and the bladder moon shell, and identified to be tetrodotoxin and anhydrotetrodotoxin.     

Possible source of tetrodotoxin in the starfish Astropecten scoparius.

The seasonal variations of toxicity and stomach contents in toxic starfish Astropecten scoparius were detected. The average highest specimen toxicity, expressed as tetrodotoxin (TTX), was 16,821 mouse units (MU). The toxin was composed of TTX only, except April's sample containing mainly TTX along with minor paralytic shellfish poisons. The composition in the stomach of less and more toxic starfish was mainly Veremolpa scabra and Umborium suturale, respectively. The toxicities of U. suturale and V. scabra, found in the starfish stomach were 65 and 33 MU, respectively. For those collected from the coastal waters, U. suturale was toxic with average toxicity value of 77 MU/g, but V. scabra was nontoxic. The toxin in the specimens of U. suturale collected from either the digestive gland of starfish or the coastal waters, was TTX and anhydroTTX only. It indicates that the starfish A. scoparius might mainly accumulate high amount of TTX from U. suturale. Furthermore, both small gastropods U. suturale and Natica psuestes are first reported to contain TTX.     

Identification of tetrodotoxin in marine gastropods implicated in food poisoning

Attempts were made to identify the toxin in the gastropod Zeuxis samiplicutus that was responsible for the recent food poisoning incidents in southern Zhejiang, Mainland China. Symptoms associated with the poisonings included paralysis, coma, vomiting, and aphasia. The remaining specimens of gastropod were assayed for tetrodotoxin toxicity (TTX). The range of specimen toxicity was found to be 4 - 186 mouse units (MU), and the average toxicity was 111 +/- 45 MU. The toxin was partially purified from the methanol extract of the gastropod by ultrafitration and Bio-Gel P-2 column chromatography. Cellulose acetate membrane electrophoresis, TLC, and HPLC analyses demonstrated that the toxin contained TTX. It was concluded that the causative agent of the above food poisoning was TTX.     

Occurrence of a new toxin and tetrodotoxin in two species of the gastropod mollusk Nassariidae.

The lethalities of 102 specimens of three species of the gastropod mollusk Nassariidae, collected from fish markets in Taiwan, were examined. The frequency of toxicity in Zeuxis scalaris and Z. castus-like specimens was 94 and 41%, respectively. The range of lethal potency in toxic specimens of Zeuxis scalaris and Z. castus-like was 2-140 and 2-13 mouse units, respectively, while all tissues of Z. castus were non-toxic. The toxins were partially purified from the toxic specimens of Z. scalaris and Z. castus-like. Two toxin fractions were obtained from the extract of each species of Nassariidae by using Bio-Gel P-2 column chromatography. Analyses by thin layer chromatography, electrophoresis, high performance liquid chromatography and ultraviolet spectroscopy, showed that toxin fraction I contained tetrodotoxin while toxin fraction II contained a new neurotoxin.     

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus.

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2)mouse unit (MU) per gram of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus.     

Toxicity assessment for the horseshoe crab Carcinoscorpius rotundicauda collected from Cambodia.

In this study, we assessed the toxicity of the horseshoe crab Carcinoscorpius rotundicauda collected from Cambodia within two successive months during rainy (April-May) and dry (December-January) seasons, respectively. Toxicity assessments of the collected specimens by standard mouse bioassay showed marked individual variation, and their toxin profiles by liquid chromatography/mass spectrometry (LC/MS) revealed tetrodotoxin (TTX) was the main toxin while no paralytic shellfish toxins (PSTs) were detected. All specimens were toxic and the highest toxicity values were 315, 113, 60, 47, 44 and 38 mouse units (MU)/g in the tissues of hepatic caecum, egg, viscera, muscle, intestine and testis, respectively. Although the current findings showed that the Cambodian C. rotundicauda was a moderately toxic species, they are not suitable for human consumption due to their toxicity. To the best of our knowledge, this is the first scientific study on toxic marine seafood ever investigated in Cambodian territorial waters.     

Occurrence of tetrodotoxin in the skin of a rhacophoridid frog Polypedates sp. from Bangladesh.

Twenty-three specimens of a tree-frog Polypedates sp. were collected from two locations (Mymensingh and Barisal) of Bangladesh in 1999, and assayed for their toxicity scores and toxin principle. Among the tissues, only the skin of the Mymensingh specimens was found to be toxic in mouse test, with the toxicity scores of 31-923MU/g. The toxin isolated from the skin was analyzed by high-performance liquid chromatography, electrospray ionization-time of flight mass spectrometry and proton nuclear magnetic resonance, and characterized as tetrodotoxin, a toxin principle.     

Survey of paralytic toxins in shellfish in southern Taiwan between 1995 and 1997

To establish the safety data of shellfish in southern Taiwan, a total of 3,074 specimens of 30 shellfish species were seasonally collected from August 1995 to March 1997. These samples were assayed for the presence of tetrodotoxin (TTX) and paralytic shellfish poisons (PSP) by the bioassay methods. It was found that some major shellfish including oyster, clam, ear shell, and purple clam were nontoxic, but four species, Babylonia formosae, Niotha clathrata, Natica lineata, and Natica vitellus, were toxic. The toxic percentages of these four species was 1% in B. formosae, 56% in N. clathrata, 37% in N. lineata, and 23% in N. vitellus. The toxic composition was TTX in B. formosae. In the other three shellfish types the toxic composition was TTX and PSP.     

Toxins extracted from Australian specimens of the crab, Eriphia sebana (Xanthidae)

Aqueous extracts of 75 specimens of the xanthid crab, Eriphia sebana, collected from coral reefs in the Capricorn Group, Great Barrier Reef, Australia, were assayed for toxicity to mice. Thirty-five extracts contained detectable toxicity. Toxicity was quantified by i.p. injection of mice where 1 mouse unit (MU) was defined as the amount of toxin required to kill a 20 g mouse in 15 min. Twenty-four of the extracts induced signs in mice similar to signs expected if paralytic shellfish toxins (PSTs) or tetrodotoxin were the predominant toxins. The highest toxicity found was 887.4 MU from a 98.6 g crab (= 9.0 MU/g). Purification of five combined groups of extracts from crabs collected at the same time and area revealed for the first time the presence in E. sebana of toxins similar to PSTs. TLC and electrophoretic studies showed that these toxins behaved like saxitoxin, neosaxitoxin, gonyautoxin-1 and gonyautoxin-2. Two of these groups of extracts contained only one of these PST-like toxins and the remainder contained two. Eleven of the extracts caused signs in injected mice dissimilar to signs induced by PSTs or tetrodotoxin. Foregut contents of 33 crabs contained fish, crustacean and algal remains. Crustacean remains in one gut content sample included carapace fragments bearing distinctive surface features found on the smaller toxic xanthids, Actaeodes tomentosus and Pilodius areolatus. The combined gut contents of these crabs weighed 10.4 g and the aqueous extract of which contained 4.4 MU/g of toxin, supporting the suggestion of a probable dietary origin of the toxins present in the crabs.     

Paralytic toxins in three new gastropod (Olividae) species implicated in food poisoning in southern Taiwan.

The toxins in the new gastropods Oliva miniacea, O. mustelina and O. nirasei implicated in a food paralytic poisoning incident in South Taiwan in February 2002 were studied. It was found that the three species of gastropods contained moderate amounts of toxin in edible portion only, and the highest toxicity score was 18 MU/g for O. miniacea, 10 MU/g for O. mustelina, and 27 MU/g for O. nirasei. The toxin was partially purified from the toxic specimens of each species by ultrafiltration using a YM-1 membrane, followed by chromatography on Bio-Gel P-2 column. Analyses by HPLC, GC-MS and LC-MS showed that the toxin from O. miniacea, O. nirasei and O. mustelina contained TTX, and related compounds 4-epi TTX and anhydro-TTX. The paralytic shellfish poisons were not found.     

Chelonodon patoca, a highly toxic marine puffer in Japan

Toxicity of a Japanese marine puffer Chelonodon patoca ("okinawafugu") was examined by mouse assay from 1996 to 1999. Frequency of the toxic specimens was found to be 100% with high toxicity scores. Among the tissues tested, toxicity in the skin ranged from 60 to 6,700 MU/g, in the ovary from 25 to 670 MU/g, in the testis from 45 to 550 MU/g, in the muscle from 2 to 390 MU/g, and in the liver from 5 to 380 MU/g. The liver, which is known as one of the most toxic organs in Japanese marine puffer in general, showed lower toxicity in the present study. Thus, the anatomical distribution of toxicity was unique in C. patoca, in comparison with that of other Japanese puffers. C. patoca toxin was characterized as tetrodotoxin (TTX), 4-epiTTX and anhydroTTX by HPLC.     

Accumulation of paralytic shellfish poisoning toxins in the edible shore crab Telmessus acutidens.

Several shellfishes including the crab Telmessus acutidens and its prey bivalve Mytilus galloprovincialis were collected at Onahama in Japan to investigate the accumulation of paralytic shellfish poisoning (PSP) toxins during the blooming season of toxic dinoflagellates. The toxicity of the viscera of T. acutidens collected in 1999 was 30.0 and 80.0 MU/g, and that of M. galloprovincialis was 9.6 MU/g by mouse bioassay. PSP toxins in the crab viscera were identified by HPLC-FLD and ESI-MS, so this is the first observation of PSP toxins in T. acutidens. Carbamate toxins (GTX1-4, and STX) were the major component in the crab as well as in the mussels, and accounted for over 60% on a molar basis. However, the ratio of the N1-OH toxin to N1-H toxin of the crabs were largely different from that of the mussels, and a reductive conversion of the toxins in T. acutidens is concluded as the probable cause. In 2000, PSP toxins were also detected in both crabs and mussels, though the contents were very low compared with the levels observed in 1999. The difference in the toxin abundance suggests that the toxin content in the crab was affected by those of the prey.     

Gonyautoxin-3 as a minor toxin in the gastropod Niotha clathrata in Taiwan

Paralytic toxicity was detected in the gastropod mollusc Niotha clathrata collected from South Taiwan in April and November 1993. Each seasonal toxin was partially purified from toxic specimens of N. clathrata by ultrafiltration using a membrane (Diaflo YM-2), followed by chromatography on a column (Bio-Gel P-2). Two toxin fractions (I and II) were then obtained for each seasonal shell toxin. The ratio of fraction I to fraction II for each seasonal shell toxin was about 4:1 according to tetrodotoxin bioassay. Based on analyses by TLC, electrophoresis, and HPLC, fraction I toxin contained tetrodotoxin and its derivative anhydrotetrodotoxin, and fraction II toxin contained gonyautoxin-3 for each seasonal shell toxin.     

Tetrodotoxin and paralytic shellfish poisons in gastropod species from Vietnam analyzed by high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry

Among marine toxins, tetrodotoxin (TTX) and paralytic shellfish poisons (PSPs) are known as notorious neurotoxins that induce serious food poisoning incidents in the Southeast Asia region. The aim of this study was to investigate whether TTX and PSP toxins are important issues of seafood safety. Paralytic toxicity was observed in mice exposed to 34 specimens from five species of gastropods using a PSP bioassay. Five species of gastropods, Natica vitellus, Natica tumidus, Oliva hirasei, Oliva lignaria, and Oliva annulata, were collected from the coastal seawaters in Nha Trang City, Vietnam, between August 2007 and October 2007. The average lethal potency of gastropod specimens was 90 ± 40 (mean ± standard deviation) mouse units (MU) for N. vitellus, 64 ± 19 MU for N. tumidus, 42 ± 28 MU for O. hirasei, 51 ± 17 MU for O. lignaria, and 39 ± 18 MU for O. annulata. All toxic extracts from the sample species were clarified using a C18 Sep-Pak solid-phase extraction column and a microcentrifuge filter prior to analysis. High-performance liquid chromatography coupled with fluorescence detection indicated that the toxins of the olive shell (O. hirasei, O. lignaria, and O. annulata) were mainly composed of saxitoxin (STX) (73–82%), gonyautoxin (GTX) 2, 3 (12–22%), and minor levels of TTX (5–6%). The toxins of N. vitellus and N. tumidus were mainly composed of STX (76–81%) and GTX 1, 4 (19–24%). Furthermore, liquid chromatography–tandem mass spectrometry analysis was used to verify the identity of the PSPs and TTX. Our evidence shows that these gastropods have novel toxin profiles.     

Tetrodotoxin and paralytic shellfish poisons in gastropod species from Vietnam analyzed by high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry

Among marine toxins, tetrodotoxin (TTX) and paralytic shellfish poisons (PSPs) are known as notorious neurotoxins that induce serious food poisoning incidents in the Southeast Asia region. The aim of this study was to investigate whether TTX and PSP toxins are important issues of seafood safety. Paralytic toxicity was observed in mice exposed to 34 specimens from five species of gastropods using a PSP bioassay. Five species of gastropods, Natica vitellus, Natica tumidus, Oliva hirasei, Oliva lignaria, and Oliva annulata, were collected from the coastal seawaters in Nha Trang City, Vietnam, between August 2007 and October 2007. The average lethal potency of gastropod specimens was 90 ± 40 (mean ± standard deviation) mouse units (MU) for N. vitellus, 64 ± 19 MU for N. tumidus, 42 ± 28 MU for O. hirasei, 51 ± 17 MU for O. lignaria, and 39 ± 18 MU for O. annulata. All toxic extracts from the sample species were clarified using a C18 Sep-Pak solid-phase extraction column and a microcentrifuge filter prior to analysis. High-performance liquid chromatography coupled with fluorescence detection indicated that the toxins of the olive shell (O. hirasei, O. lignaria, and O. annulata) were mainly composed of saxitoxin (STX) (73–82%), gonyautoxin (GTX) 2, 3 (12–22%), and minor levels of TTX (5–6%). The toxins of N. vitellus and N. tumidus were mainly composed of STX (76–81%) and GTX 1, 4 (19–24%). Furthermore, liquid chromatography–tandem mass spectrometry analysis was used to verify the identity of the PSPs and TTX. Our evidence shows that these gastropods have novel toxin profiles.     

Paralytic toxicity in the ribbon worm Cephalothrix species (Nemertea) in Hiroshima Bay, Hiroshima Prefecture, Japan and the isolation of tetrodotoxin as a main component of its toxins.

Paralytic toxicity of ribbon worms ("himomushi" in Japanese), identified as undescribed species of the genus Cephalothrix, found on the surface of the shells of cultured oysters in Hiroshima Bay, Hiroshima Prefecture was examined between April 1998 and December 2001. The toxicity study showed that all of specimens were found to contain toxins with strong paralytic action in mice; the highest toxicity (as tetrodotoxin, TTX) was 25,590 mouse units (MU) per gram for whole body throughout the monitoring period.The main toxic component of this himomushi toxin (HMT) was isolated from a pooled specimen (390 g; total toxicity 2,897,000MU) by a method that consisted of treatment with activated charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H+ form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3520MU/mg. This toxin showed (M+H)+ and (M+H-H(2)O)+ ion peaks at m/z 320 and 302, respectively, by electrospray ionization-mass spectrometry (ESI-MS). The absorption band at 3353, 3235, 1666, 1612 and 1076 cm(-1) were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX. The 1H-NMR spectrum for the recrystallized toxin was the same as that for TTX. The pair of doublets centered at 2.33 (J=10.0Hz) and 5.48 ppm (J=10.0Hz) which are characteristic of TTX, were shown to be coupled by double irradiation. Furthermore, by gas chromatography-mass spectrometry (GC-MS) of the alkali-hydrolyzate of this toxin indicated the presence of quinazoline skeleton (C9-base) specific to TTX.     

Occurrence of saxitoxins as a major toxin in the ovary of a marine puffer Arothron firmamentum.

Eleven male and 14 female specimens of a marine puffer Arothron firmamentum were collected from Oita and Iwate Prefectures, Japan. The toxicity assay using mouse showed that only ovary and skin of the female specimens were toxic, the toxicity scores being 5-740 as paralytic shellfish poison and <5-30 MU/g as tetrodotoxin (TTX), respectively. The toxin extracts from the both tissues were then treated with cartridge columns, and subjected to high performance liquid chromatography and liquid chromatography-mass spectral analyses. In the analyses, saxitoxin (STX) and decarbamoylSTX (dcSTX) were identified as the major toxins in the ovary, while the skin contained only TTX.     

Toxicity and toxin profiles of the newt, Cynops pyrrhogaster from western Japan

A total of 382 specimens of a Japanese newt, Cynops pyrrhogaster, were collected from western Japan during 1996 to 1999, and assayed for their individual, geographical, sexual, seasonal variations, and anatomical distribution of toxicity by mouse. Most of the specimens tested showed toxicity scores ranging from 5 to 370 MU/g, where no seasonal, but large individual, sexual, and regional variations of toxicity were clearly recognized. Among the parts, skin and muscle showed higher toxicity scores (56 MU/g) than liver, stomach, intestine and gonad, whose toxicity ranged from less than 2 to 33 MU/g. The C. pyrrhogaster toxin was purified by several steps of column chromatography and was shown to consist of tetrodotoxin (TTX) and 6-epiTTX as main components, and 4-epiTTX, 4,9-anhydro-6-epiTTX, and 4,9-anhydroTTX as minor ones by means of HPLC and 1H-NMR analyses.