Tomohiro Takatani

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.

TTX accumulation in pufferfish

Tetrodotoxin (TTX) has been detected in a variety of animals. The finding of TTX in the trumpet shell Charonia sauliae strongly suggested that its origin was its food, a TTX-bearing starfish Astropecten polyacanthus. Since then, the food chain has been consistently implicated as the principal means of TTX intoxication. To identify the primary producer of TTX, intestinal bacteria isolated from several TTX-bearers were investigated for their TTX production. The results demonstrated that some of them could produce TTX. Thus the primary TTX producers in the sea are concluded to be marine bacteria. Subsequently, detritus feeders and zooplankton can be intoxicated with TTX through the food chain, or in conjunction with parasitism or symbiosis. The process followed by small carnivores, omnivores or scavengers, and by organisms higher up the food chain would result in the accumulation of higher concentrations of TTX. Finally, pufferfish at the top of the food chain are intoxicated with TTX. This hypothesis is supported by the fact that net cage and land cultures produce non-toxic pufferfish that can be made toxic by feeding with a TTX-containing diet.

Intra-tissue distribution of tetrodotoxin in two marine puffers Takifugu vermicularis and Chelonodon patoca

Micro distribution pattern of tetrodotoxin (TTX) in several tissues of marine puffers Takifugu vermicularis and Chelonodon patoca was investigated by means of monoclonal antibody-based immunoenzymatic technique under light microscope. In the investigation TTX was visualized at glands in the skin of T. vermicularis, while in C. patoca TTX was detected in succiform cells of the skin section. Similarly, in the ovary section of T. vermicularis TTX was recognized at late peri nucleolus stage, yolk granule stage-I, and yolk granule stage-II of oocytes. The oocytes of late peri nucleolus stage and yolk granule stage-I showed TTX antigen at their nucleus and yolk vesicles, while in yolk granule stage-II TTX was visualized at yolk granules and yolk vesicles. In the ovary of C. patoca TTX was detected in the connective tissues and in the nucleus of some perinucleolar oocytes. In the liver and muscle of C. patoca TTX was found to be distributed in parenchymal hepatocytes and muscle fiber, respectively. This study, however, reveals that intra-tissue distribution of TTX varies in respect of species.

Localization of tetrodotoxin in the skin of a brackishwater puffer Tetraodon steindachneri on the basis of immunohistological study

A monoclonal antibody-based immunoassay for localization of tetrodotoxin (TTX) in the skin of a brackishwater puffer Tetraodon steindachneri is described in this paper. TTX was recognized in the undifferentiated basal cells and succiform cells in the skin under light microscope. Malpighian cells of the skin did not exhibit any TTX antigen. Neither gland nor enclosed gland-like apparatus possessing TTX was apparent in the skin.

Intracellular visualization of tetrodotoxin (TTX) in the skin of a puffer Tetraodon nigroviridis by immunoenzymatic technique

Micro-distributions of tetrodotoxin (TTX) in the skin of a brackish-water puffer Tetraodon nigroviridis were investigated by means of a monoclonal anti-TTX antibody under light and transmission electron microscope. In light microscopy TTX antigen was visualized as brown color in undifferentiated basal cells and succiform cells of the skin, while in electron microscopy TTX was detected as black dots of immunogold in lysosomes of basal cells. From the results, it can be inferred that when TTX from the blood plasma of T. nigroviridis enters into the undifferentiated basal cells through diffusion, it is taken to the lysosomes by phagocytosis and exists there through binding with internal constituent(s) of the organelles.

Delayed haemolytic activity by the freshwater puffer Tetraodon sp. toxin.

In order to elucidate the toxin composition of the freshwater puffer in Bangladesh, about 230 specimens of Tetraodon sp. were collected from 1997 to 1999 and extracted. After partitioning the toxins between an aqueous layer and a 1-butanol layer, the toxin in the aqueous layer was characterized as paralytic shellfish poison (PSP) (data not shown), while the toxin in the 1-butanol layer was identified as palytoxin (PTX) or PTX-like substance based on the delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). This is the first report on the occurrence of PTX or PTX-like substance(s) in puffer fish.

Transfer profile of intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes.

Tetrodotoxin (TTX) was intramuscularly administered to non-toxic cultured specimens of the pufferfish Takifugu rubripes to investigate TTX transfer/accumulation profiles in the pufferfish body. In two groups of test fish administered either 50MU/individual of TTX standard (purified TTX; PTTX) or crude extract of toxic pufferfish ovary (crude TTX; CTTX), TTX rapidly transferred from the muscle via the blood to other organs. The toxin transfer profiles differed between groups, however, from 4 to 72h. In the PTTX group, little TTX was retained in the liver, and most (>96%) of the toxin remaining in the body transferred/accumulated in the skin after 12h, whereas in the CTTX group, a considerable amount of toxin (15%-23% of the administered toxin or 28%-58% of the remaining toxin) was transferred/retained in the liver for up to 24h, despite the fact that 89% of the remaining toxin transferred/accumulated in the skin at the end of rearing period (168h). The total amount of toxin remaining in the entire body at 1-4h was approximately 60% of the administered toxin in both groups, which decreased at 8-12h, and then increased again to approximately 60%-80% at 24-168h. Immunohistochemical observation revealed that the toxin accumulated in the skin was localized at the basal cells of the epidermal layer.

Larval pufferfish protected by maternal tetrodotoxin

Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae.

Transfer profile of intramuscularly administered tetrodotoxin to artificial hybrid specimens of pufferfish, Takifugu rubripes and Takifugu niphobles

Tetrodotoxin (TTX) was intramuscularly administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu niphobles to investigate toxin accumulation in hybrids, and TTX transfer/accumulation profiles in the pufferfish body. In the test fish administered 146 MU TTX in physiologic saline, TTX rapidly transferred from the muscle via the blood to other organs. Toxin transfer to the ovary rapidly increased to 53.5 MU/g tissue at the end of the 72-h test period. The TTX content in the liver and skin was, at most, around 4-6 MU/g tissue, and in the testis it was less than 0.01 MU/g tissue. On the other hand, based on the total amount of toxin per individual (% of the administered toxin), the skin and the liver contained higher amounts (20-54% and 2-24%, respectively), but the amount in the liver rapidly decreased after 8-12 h, and fell below the level in the ovary after 48 h. These findings suggest that part of the TTX is first taken up in the liver and then transferred/accumulated in the skin in male specimens and in the ovary in female specimens.

Difference in the localization of tetrodotoxin between the female and male pufferfish Takifugu niphobles, during spawning

In order to understand the sexual differences in TTX-usage in the pufferfish, Takifugu niphobles, localization of TTX and toxin amount in tissues of mature male and female specimens were investigated by immunohistochemical methods using anti-TTX antibody and LC/MS analysis. Subsequently, differences in the immunohistochemical signals were compared with the amount of TTX. The paraffin-embedded sections of the skin, muscle, liver, gonad and intestinal tract were subjected to anti-TTX monoclonal antibody based on the fluorescent immunohistochemical techniques. Immuno-positive reaction was observed in the skin and liver in males, and the skin and ovary in females. In the skin, TTX was localized at the epidermis, the basal cell layer, the mucous cells and the sacciform cells, and with intense immunoreaction at the flat epithelial cell layer and the sacciform cells. The signal from the liver cells was stronger in males than in females. The intensity of the signal from the tissues correlated with the toxin amounts therein. These results suggest that tissue distributions of TTX and toxin amount in the pufferfish were sex-dependent.