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Pufferfish of the family Tetraodontidae possess tetrodotoxin (TTX) and/or saxitoxins (STXs), but the toxin ratio differs, depending on the genus or species. In the present study, to clarify the distribution profile of TTX and STXs in Tetraodontidae, we investigated the composition and intra-body distribution of the toxins in Canthigaster valentini. C. valentini specimens (four male and six female) were collected from Amami-Oshima Island, Kagoshima Prefecture, Japan, and the toxins were extracted from the muscle, liver, intestine, gallbladder, gonads, and skin. Analysis of the extracts for TTX by liquid chromatography tandem mass spectrometry and of STXs by high-performance liquid chromatography with post-column fluorescence derivatization revealed TTX, as well as a large amount of STXs, with neoSTX as the main component and dicarbamoylSTX and STX itself as minor components, in the skin and ovary. The toxins were also detected in the other tissues, but in much lower amounts than in the skin and ovary. The TTX/STX ratio varied greatly, depending on the tissue, but TTX was the major toxin component in the whole body, and STXs accounted for 25% and 13% of the total toxin amount in males and females, respectively. Like the marine pufferfish of the genus Arothron, C. valentini should be considered a pufferfish with considerable amounts of both TTX and STXs present simultaneously., Toxins, 12(7), art.no.436; 2020

Natural hybrids among pufferfishes belonging to the genus Takifugu have been reported occasionally, yet very little is known about their toxicity. To study the tetrodotoxin (TTX) distribution in the bodies of natural hybrids, 10 putative hybrids between Takifugu rubripes and Takifugu porphyreus, which differ in the toxicity of the skin (the former is hardly toxic whereas the latter shows high toxicity), were collected from the coastal waters off Yamaguchi and Shimane Prefectures and used for genetic and biochemical analyses. Genetic identification of parental species using amplified fragment length polymorphism and mitochondrial DNA markers revealed that five and four individuals were F1 hybrids between T. rubripes females and T. porphyreus males and those of the opposite cross, respectively. In addition, the remaining hybrid was a first-generation backcross to T. rubripes. Quantitative analysis of TTX by high performance liquid chromatography-fluorescence detection showed that the skin of F1 hybrids has TTX, differing from T. rubripes, suggesting the possibility of a dominant mode of inheritance of toxicity of the skin. TTX was detected in the muscle of F1 hybrids only between T. porphyreus females and T. rubripes males, albeit at a low concentration of

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

To clarify the differences in toxin selectivity between marine and freshwater pufferfish, we conducted experiments in artificially reared nontoxic specimens of Takifugu pardalis (marine) and Pao suvattii (freshwater) using tetrodotoxin (TTX) and paralytic shellfish poison (PSP, decarbamoylsaxitoxin (dcSTX) or saxitoxin (STX)). T. pardalis specimens were administered feed homogenate containing TTX or dcSTX (dose of toxin, 55.2 nmol/fish) and P. suvattii specimens were administered feed homogenate containing TTX + STX (dose of each toxin, 19.2 nmol/fish) by oral gavage. The toxin content in the intestine, muscle, skin, liver, and gonads was quantified after 24 and 48 or 72 h. In T. pardalis, TTX administered into the intestine was absorbed into the body and transferred and retained mainly in the skin and liver, while dcSTX was hardly retained in the body, although it partly remained in the intestine. In strong contrast, in P. suvattii, little TTX remained in the body, whereas STX was absorbed into the body and was transferred and retained in the ovary and skin. The findings revealed that TTX/PSP selectivity differs between the marine species T. pardalis and the freshwater species P. suvattii. T. pardalis, which naturally harbors TTX, selectively accumulates TTX, and P. suvattii, which naturally harbors PSP, selectively accumulates PSP.

The pufferfish saxitoxin- and tetrodotoxin-binding protein 2 (PSTBP2), which is involved in toxin accumulation, was knocked out in Takifugu rubripes embryos by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome-editing technology. Treating the embryos with one of two single-guide RNA (sgRNA) resulted in mutation rates of 57.1% and 62.5%, respectively, as estimated using a heteroduplex mobility assay at 3 days postfertilization. Both sgRNAs might induced frameshift mutations that knocked out the T. rubripes PSTBP2.

Structure analysis was performed on the antibiotic-resistance-gene region of conjugative plasmids of four fish farm bacteria.The kanamycin resistance gene, IS26, and tetracycline resistance gene (tetA(D)) were flanked by two IS26s in opposite orientation in Citrobacter sp. TA3 and TA6, and Alteromonas sp. TA55 from fish farm A. IS26-Inner was disrupted with ISRSB101. The chloramphenicol resistance gene, IS26 and tetA (D) were flanked by two IS26s in direct orientation in Salmonella sp. TC67 from farm C. Structures of tetA (D) and IS26 were identical among the four bacteria, but there was no insertion within the IS26-Inner of Salmonella sp. TC67. Horizontal gene transfer between the strains of two different genera in fish farm A was suggested by the structure homologies of mobile genetic elements and antibiotic resistance genes.

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., Toxicon, 78, pp.35-40; 2014

To investigate the effects of growth (organ development) on tetrodotoxin (TTX) dynamics in the pufferfish body, TTX-containing feed homogenate was administered to 6- and 15-month old non-toxic cultured specimens of the pufferfish Takifugu rubripes at a dose of 40 mouse units (MU) (8.8 μg)/20 g body weight by oral gavage. After 24 h, the specimens were killed and the skin tissues (dorsal and ventral), muscle, liver, digestive tract, and gonads were separated. TTX content (μg/g) in each tissue, determined by liquid chromatography/mass spectrometry, revealed that the TTX distribution profile, particularly the TTX content of the liver, greatly differed between the two ages; the TTX score of 15-month old fish (3.3 μg/g) was nearly 5-fold that of 6-month old fish (0.68 μg/g). The total remaining TTX amount per individual (relative amount to the given dose) was 31% in 6-month old fish, of which 71% was in the skin, and 84% in 15-month old fish, of which 83% was in the liver. The gonadosomatic index (GSI) and hepatosomatic index (HSI) scores, and histologic observations of the gonads and liver suggest that although there is little difference in maturation stage between these two ages, there are clear distinctions in the developmental stage of the liver. The results suggest that the TTX dynamics in T. rubripes are linked to the development of the liver, i.e., the TTX taken up into the pufferfish body via food organisms is eliminated or transferred mainly to the skin in young fish with an undeveloped liver, but as the fish grow and the liver continues to develop, most of the TTX is transferred to and accumulated in the liver.

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.

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.

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 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 (

Four genes of Takifugu rubripes, tentatively designated Tr1-Tr4, encoding homologs of pufferfish saxitoxin- and tetrodotoxin-binding protein, were identified by BLAST search and 3'-RACE. RT-PCR and MALDI-TOF mass spectrometry allowed the identification and discrimination of Tr isoforms from the non-toxically cultured specimens. The expression of Tr1 and Tr3 mRNAs exclusively in the liver and the presence of their products as 120-kDa plasma proteins were confirmed.

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) 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