Search

Adults of the ectoparasitic copepod Caligus fugu found on tetrodotoxin (TTX)-bearing pufferfish such as Takifugu alboplumbeus and Takifugu flavipterus are known to accumulate TTX in body tissues and parts other than the ovaries, oviducts, eggs, and cuticles. This study aimed to demonstrate, using immunoenzymatic staining techniques, that the TTX-free planktonic/infective copepodid stage of C. fugu could accumulate TTX in the tissues after molting into the parasitic stage (chalimus I) and then fed on mucus of host puffers. All the tissues of the planktonic copepodids were completely TTX-free, whereas chalimus I copepods accumulated TTX in parts other than the cuticles, guts, and some muscles. Chalimus IV and adult copepods retained TTX in these body parts but not in the reproductive organs, which were TTX-resistant, indicating that TTX was not vertically transmitted via eggs. Non-cellular TTX-positive contents found in the guts of some chalimi and adults indicated that the copepods potentially accumulated TTX by feeding on host mucus rather than skin tissues and blood. This study revealed that the presence or absence of TTX in some body parts differed among individuals of the parasite.

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 μg/mL TTX for 1?48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine., Toxicon, 174, pp.8-12; 2019

It is known that tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin and had been detected in various taxa. However, the exact function of the toxin in TTX-bearing organisms has remained unclear. In Takifugu pufferfish species, it has been suggested that TTX is utilized to protect larvae from predators but no experimental proof exists. In the present study, we used pufferfish Takifugu alboplumbeus larvae from wild and cultured parents to determine the effects of the maternal TTX on the survival of toxic and non-toxic pufferfish larvae, respectively. TTX contents in the larval pufferfish differed between the larvae derived from wild and cultured parents (1.23 ± 0.20 ng/individual vs. undetectable levels, respectively). Immunohistochemical staining with anti-TTX monoclonal antibody demonstrated that the TTX-specific signals were primarily observed at the body surface of the larvae of wild parents, but not of cultured parents. Predation experiments demonstrated that the juveniles of Girella punctata and Chaenogobius gulosus, used as predator fish, ingested the pufferfish larvae derived from either type of parents, but instantly spat out those from wild parents only. These results indicate that larvae, which are at the most vulnerable stage in the life of pufferfish, are protected by maternal TTX.

Tetrodotoxin (TTX) was intramuscularly administered to nontoxic cultured specimens of the pufferfish Takifugu rubripes to investigate differences in the toxin transfer and accumulation profiles between the skin and liver. Test fish were administered TTX at doses of 30 (Low dose; LD), 100 (Medium dose; MD), and 300 (High dose; HD) μg/individual, respectively. Liquid chromatography/mass spectrometry analysis for TTX revealed that the TTX concentration in both the skin (0.48-1.7 μg/g) and liver (0.43-6.0 μg/g) at 24 h after the toxin administration increased with an increase in the dose. The TTX accumulation ratio (ratio (%) of accumulated TTX in each tissue (μg/tissue) to the administered dose (μg/individual)) of the skin (11.1-38.6) significantly decreased with an increase in the dose, whereas that of the liver (18.4-21.3) was almost constant irrespective of the dose. Immunohistochemical observations of the skin sections revealed TTX-positive signals in basal cells in LD, but as the dose increased, TTX-positive signals were also observed in the epidermis. In the liver sections, TTX-positive signals were rarely observed in LD and MD, but the TTX was distributed throughout the liver tissue in HD.

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., Toxicon : official journal of the International Society on Toxinology, 53(1), pp.99-103; 2009

Maturation-associated changes in the internal distribution of tetrodotoxin (TTX) in the goby Yongeichthys criniger were investigated in 29 and 40 female specimens collected from Okinawa, Japan, from August 2008 to June 2009 (Group I), and from November 2009 to August 2010 (Group II), respectively. In Group I, based on changes in the gonadosomatic index (GSI) and histologic observation of the ovary, the period from October 2008 through January 2009 was estimated to be the ‘previtelline-forming period’, February through March 2009 the ‘vitelline-forming period’, April through June 2009 the ‘spawning period’, and August 2008 the ‘end of spawning period’ of the preceding year. The TTX content (mouse unit [MU] per gram tissue) of each Y. criniger tissue (skin, muscle, liver, and ovary) quantified by liquid chromatography/mass spectrometry (LC/MS) was generally high during the spawning period and continued to rise until the end of spawning period, especially in the ovary. Total TTX per individual increased considerably during the spawning period, most of which located in the ovary, indicating that Y. criniger obtains a high amount of TTX during the spawning period, and accumulates most of it in the ovary. In contrast, the TTX content of the skin was highest at the end of spawning period, and most of the total TTX located in the skin during this period as well as during the previtelline-forming period. In Group II, the maturation stage of the ovaries of all specimens was determined, and the specimens were grouped accordingly. In the perinucleolus stage, yolk vesicle stage, and yolk globule stage I, most of the TTX was localized in the skin, but the TTX in the ovary greatly increased as the maturation stage advanced from yolk globule stage I to yolk globule stage III.

Tetrodotoxin (TTX) and its analogue 6-epiTTX had been detected in the red-spotted newt, Notophthalmus viridescens. Thirty specimens of a population from Pennsylvania, USA were histologically examined for the presence of intestinal parasites. More than 50% were found to be infected with nematodes (Trichocephalidae), trematodes or cestodes (Pseudophyllidae). The mean values of TTX and 6-epiTTX in parasitized and in non-parasitized newts were not significantly different. Using a monoclonal antibody-based immunoenzymatic technique, TTX was localized in the intestinal tissue as well as in the parasites indicating that they accumulate the toxin and suggesting that TTX and 6-epiTTX are not providing protection from parasites to the newts.

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 146MU TTX in physiologic saline, TTX rapidly transferred from the muscle via the blood to other organs. Toxin transfer to the ovary rapidly increased to 53.5MU/g tissue at the end of the 72-h test period. The TTX content in the liver and skin was, at most, around 4-6MU/g tissue, and in the testis it was less than 0.01MU/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 8-12h, and fell below the level in the ovary after 48h. 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., Toxicon, 58(6-7), pp.565-569; 2011

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 4.0×10 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 1.0×10 −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.

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

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.

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.

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 31–923 MU/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.

Screening tests were carried out on the toxicity of freshwater puffers Tetraodon leiurus complex and Tetraodon suvatii collected from Udonthani province, north-eastern Thailand. Toxicity was highest in the liver and varied according to the location and season of fish catch. Fish which were reared in tap water for 3 months reduced the toxicity substantially. Partial purification was achieved by an ultrafiltration technique. Toxin components were consequently identified by high-performance liquid chromatography. It was found that toxins separated from the eggs, liver, skin and muscle of these puffers were composed of saxitoxin, neosaxitoxin and decarbamoylsaxitoxin.

Two species of freshwater puffer fish, Tetraodon cutcutia and Chelonodon patoca, collected from several locations in Bangladesh, showed lethal potency in mice ranging from 2.0 to 40.0 MU/g tissue as paralytic shellfish poison. In both species, toxicity of the skin was generally higher than the other tissues examined (muscle, liver and ovary). Water-soluble toxins from T. cutcutia were partially purified by activated charcoal treatment followed by column chromatographies using Bio-Gel P-2 and Bio-Rex 70. Analyses by cellulose acetate membrane electrophoresis and high-performance liquid chromatography with fluorometric detection demonstrated that the toxins were composed of saxitoxin, decarbamoylsaxitoxin, gonyautoxins 2 and 3, decarbamoylgonyautoxins 2 and 3, and three unidentified components which are possibly related to paralytic shellfish poison.

Isodomoic acids G and H, two new isomers of the neurotoxin domoic acid, along with isodomoic acids A, B, E and F, were isolated from a red alga, Chondria armata , collected at the southern tip of Kyushu Island. The structures of two of these were deduced to be ( E , E ) and ( Z , E ) isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrro-lidineacetic acid, based on electrospray ionization mass and [ 1 H]nuclear magnetic resonance spectral analyses including [ 1 H- 1 H]correlation spectroscopy and nuclear Overhauser effect correlation spectroscopy.

A ribbon worm Cephalothrix linearis (Nemertean) showed a high toxicity, of up to 22,000 MU/g proboscis and 13,600 MU/g body, in terms of tetrodotoxin. This organism secreted the toxin from the skin when wiped with gauze. The toxin was partially purified from the secretion as well as the wiped body by ultrafiltration and Bio-Gel P-2 column chromatography. Thin-layer chromatographic, electrophoretic, high performance liquid chromatographic, and gas chromatography-mass spectrometric analyses showed that the secreted toxin was composed almost exclusively of a tetrodonic acidic-like substance, whereas the remaining toxin in the wiped body consisted of the said substance and tetrodotoxin.

Extracts of two species of ribbon worm, Lineus fuscoviridis and Tubulanus punctatus, had lethal potencies in mice from 15-503 MU/g of worm and from less than 10-540 MU/g, respectively. The toxins were partially purified by ultrafiltration and column chromatography using Bio-Gel P-2 and Bio-Rex 70 (H+ form). Thin-layer chromatographic, high performance liquid chromatographic and gas chromatography-mass spectrometric analyses revealed the presence of tetrodotoxin and anhydrotetrodotoxin plus some unidentified compounds.

T. Noguchi , O. Arakawa , K. Daigo and K. Hashimoto . Local differences in toxin composition of a xanthid crab Atergatis floridus inhabiting Ishigaki Island, Okinawa. Toxicon24, 705 – 711, 1986.—Specimens of a xanthid crab Atergatis floridus were collected from two adjacent areas in Kabira Bay, Ishigaki Island, Okinawa and compared with respect to toxicity and toxin composition. ‘Reef specimens’, which were collected from the reefs of Kabira Bay, showed an average toxicity score of 380 MU/g as paralytic shellfish poison, and a toxin composition consisting mainly of saxitoxin and a neosaxitoxin-related substance. On the other hand, ‘Kojima specimens’, which were collected from a small island in the bay, showed an average toxicity of 38 MU/g as tetrodotoxin, and a toxin composition consisting mainly of tetrodotoxin and related substance(s).