Variation and secretion of toxins in gastropod mollusc Niotha clathrata.

Nearly 300 specimens of the gastropod mollusc Niotha clathrata were collected in South Taiwan. All specimens were assayed for toxicity by the official method for tetrodotoxin (TTX). The frequency value of toxic specimens in N. clathrata was 30.0%, 68.0% and 80.4% at Anping, Chiating and Tungkang, respectively. The highest lethal potency of a gastropod specimen was 1900 mouse units (MU). The specimens collected from Tungkang showed the highest frequency of toxic specimens and toxicity, followed by those from Chiating and Anping. The specimens collected in autumn and spring showed higher toxicity than those collected in other seasons. Moreover, another 17 specimens of N. clathrata were collected for testing the toxin secretion by electric shock treatment. It is found that the gastropod did not secrete any additional toxin when electric shock treatment was performed twice at approximately 1-hr intervals. The toxicity of secreted toxin was 206 MU.     

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.     

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.     

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.     

Occurrence of paralytic shellfish toxins in Cambodian Mekong pufferfish Tetraodon turgidus: selective toxin accumulation in the skin.

The toxicity of two species of wild Cambodian freshwater pufferfish of the genus Tetraodon, T. turgidus and Tetraodon sp., was investigated. Tetraodon sp. was non-toxic. The toxicity of T. turgidus was localized mainly in the skin and ovary. Paralytic shellfish toxins (PSTs), comprising saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX), account for approximately 85% of the total toxicity. Artificially reared specimens of the same species were non-toxic. When PST (dcSTX, 50 MU/individual) was administered intramuscularly into cultured specimens, toxins were transferred via the blood from the muscle into other body tissues, especially the skin. The majority (92.8%) of the toxin remaining in the body accumulated in the skin within 48h. When the same dosage of tetrodotoxin (TTX) was similarly administered, all specimens died within 3-4h, suggesting that this species is not resistant to TTX. Toxin analysis in the dead specimens revealed that more than half of the administered TTX remained in the muscle and a small amount was transferred into the skin. The presence of both toxic and non-toxic wild specimens in the same species indicates that PSTs of T. turgidus are derived from an exogenous origin, and are selectively transferred via the blood into the skin, where the toxins accumulate.     

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.     

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.     

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.     

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques--mouse bioassay, ELISA, HPLC and mass spectrometry-we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6+/-12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6+/-0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.     

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.     

Occurrence of tetrodotoxin and paralytic shellfish poison in the Taiwanese crab Lophozozymus pictor

Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in all 15 specimens of the xanthid crab Lophozozymus pictor collected from northern Taiwan in 1993. The average toxicity of crab specimens was 921 ± 231 (mean ± S.E.) mouse units. The toxin of crab was partially purified and then identified. It was found that the crab toxin contained TTX and gonyautoxin. The ratio of TTX to gonyautoxin for crab toxin was about 9:1.     

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.     

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.     

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.     

Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica).

Brevetoxin uptake and elimination were examined in Eastern oyster (Crassostrea virginica) exposed to recurring blooms of the marine alga Karenia brevis in Sarasota Bay, FL, over a three-year period. Brevetoxins were monitored by in vitro assays (ELISA, cytotoxicity assay, and receptor binding assay) and LC-MS, with in vivo toxicity of shellfish extracts assessed by the traditional mouse bioassay. Measurements by all methods reflected well the progression and magnitude of the blooms. Highest levels recorded by mouse bioassay at bloom peak were 157 MU/100g. Oysters were toxic by mouse bioassay at levels >or=20 MU/100g for up to two weeks after bloom dissipation, whereas brevetoxins were measurable by in vitro assays and LC-MS for several months afterwards. For the structure-based methods, summed values for the principal brevetoxin metabolites of PbTx-2 (cysteine and cysteine sulfoxide conjugates), as determined by LC-MS, were highly correlated (r(2)=0.90) with composite toxin measurements by ELISA. ELISA and LC-MS values also correlated well (r(2)=0.74 and 0.73, respectively) with those of mouse bioassay. Pharmacology-based cytotoxicity and receptor binding assays did not correlate as well (r(2)=0.65), and were weakly correlated with mouse bioassay (r(2)=0.48 and 0.50, respectively). ELISA and LC-MS methods offer rapid screening and confirmation, respectively, of brevetoxin contamination in the oyster, and are excellent alternatives to mouse bioassay for assessing oyster toxicity following K. brevis blooms.     

Comparison of paralytic shellfish poisoning toxin between carnivorous crabs (Telmessus acutidens and Charybdis japonica) and their prey mussel (Mytilus galloprovincialis) in an inshore food chain.

Paralytic shellfish poisoning toxin in two shore crab species, Telmessus acutidens and Charybdis japonica, were compared with the toxin in the prey mussel Mytilus galloprovincialis and causative dinoflagellates Alexandrium tamarense, all having been collected at Onahama, Fukushima Prefecture, in the northern part of Japan. When the toxicities were detected in mussels by mouse bioassays, 73.7% of the sampled T. acutidens were toxic in the hepatopancreas. T. acutidens has been found to become toxic for three years, therefore, it can be concluded that the crab commonly and repeatedly accumulate the toxins via the food chain at Onahama. C. japonica was also expected to be a possible vector species, because small quantities of the toxins were detected in eight specimens of the crab by HPLC analysis. By the comparison of the toxin profiles in the dinoflagellates, mussels and the crab T. acutidens, reductive conversions of GTX1 and GTX4 were observed when the toxins passed through the three species in the food chain. But increases of STX and neoSTX by further reductive process were not observed in the crab. The absence of the STX group toxins in the crab suggests that the crab eliminates the toxin before such reductive process occur.     

Toxic marine puffer fish in Thailand seas and tetrodotoxin they contained

A total of 155 puffers caught from two of Thailand's seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.     

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.