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We have previously detected tetrodotoxin (TTX) in the brain of the wild toxic torafugu Takifugu rubripes by immunohistochemistry and LC/MS analysis. We have also indicated that TTX is a stress-relieving substance in the brain and reduces agonistic interactions in torafugu juveniles. Although the toxicity of marine pufferfish in the Japanese waters has been extensively examined for food hygiene, whether wild toxic pufferfish generally possess TTX in the brain has not been investigated. In the present study, we examined the presence of TTX in the brain of several wild toxic marine pufferfishes such as kusafugu T. alboplumbeus, komonfugu T. flavipterus, shosaifugu T. snyderi, okinawafugu Chelonodontops patoca, and in wild non-toxic pufferfishes such as shirosabafugu Lagocephalus spadiceus and yoritofugu Sphoeroides pachygaster. We also examined tsumugihaze Yongeichthys criniger, known to possess TTX in the skin, viscera, and gonad. TTX was extracted from the brain, liver, skin, and muscle and was analyzed by LC/MS. TTX was detected in the brain as well as in the liver, skin, and muscle in kusafugu, komonfugu, shosaifugu, okinawafugu, and tsumugihaze. In shirosabafugu, low level of TTX (0.8 mouse unit/g-brain) was detected in the brain in 1 out of 3 individuals. In yoritofugu, no TTX was detected in any of the tissues. We conclude that the brain is also an organ that contains TTX in the wild toxic marine pufferfishes.

A previously unreported heterodetic cyclic peptide, homophymamide A (1), was isolated from a Homophymia sp. marine sponge. The structure of homophymamide A was determined to be a lower homologue of anabaenopeptins by spectroscopic analysis, chemical degradation, and chemical synthesis. Analysis of the acidic hydrolysate showed that the racemization of Lys took place, leading us to pose a cautionary note on the configurational assignment of peptides that contain a ureido bond.

Myrindole A, a bis-indole alkaloid, was isolated from the deep-sea sponge Myrmekioderma sp. The high degree of unsaturation of the molecule complicated the assignment of its structure by standard 2D-NMR experiments but was ultimately achieved by a combination of 1H-15N-HMBC and 1,n-ADEQUATE experiments as well as the comparison of measured and calculated CD spectra. Myrindole A showed antimicrobial activity against Gram-positive and Gram-negative bacteria.

Toxic crabs of the family Xanthidae contain saxitoxins (STXs) and/or tetrodotoxin (TTX), but the toxin ratio differs depending on their habitat. In the present study, to clarify within reef variations in the toxin profile of xanthid crabs, we collected specimens of the toxic xanthid crab Zosimus aeneus and their sampling location within a single reef (Yoshihara reef) on Ishigaki Island, Okinawa Prefecture, Japan, in 2018 and 2019. The STXs/TTX content within the appendages and viscera or stomach contents of each specimen was determined by instrumental analyses. Our findings revealed the existence of three zones in Yoshihara reef; one in which many individuals accumulate extremely high concentrations of STXs (northwestern part of the reef; NW zone), another in which individuals generally have small amounts of TTX but little STXs (central part of the reef; CTR zone), and a third in which individuals generally exhibit intermediate characteristics (southeastern part of the reef; SE zone). Furthermore, light microscopic observations of the stomach contents of crab specimens collected from the NW and CTR zones revealed that ascidian spicules of the genus Lissoclinum were dominant in the NW zone, whereas those of the genus Trididemnum were dominant in the CTR zone. Although the toxicity of these ascidians is unknown, Lissoclinum ascidians are considered good candidate source organisms of STXs harbored by toxic xanthid crabs., Marine Drugs, 19(12), art. no. 670; 2021

We tested whether tetrodotoxin (TTX) functions as a stress relieving substance in puffer fish. We orally administered TTX to the juveniles of hatchery-reared non-toxic tiger puffer Takifugu rubripes and measured the effects of TTX on brain corticotropin-releasing hormone (CRH) mRNA expression and plasma cortisol levels in comparison with effects in non-toxic juveniles. Firstly, the reciprocal connections of CRH and adrenocorticotropic hormone (ACTH) were confirmed by dual-label immunohistochemistry. CRH-immunoreactive (ir) cell bodies were detected in the hypothalamus and CRH-ir fibers were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Next, a TTX-containing diet (2.35 mouse units (517?ng)/g diet) or a non-toxic diet were fed to the fish for 28 days under a recirculating system. Standard length and body weight became significantly larger in the TTX-treated group. The degree of loss of the caudal fin, which is an indicator of the degree of agonistic interactions, where high values show a higher loss of caudal fin of a fish due to nipping by other individuals, was significantly lower in the TTX-treated group. Relative CRH mRNA expression levels in the brain and cortisol levels in the plasma were significantly lower in the TTX-treated group. These results indicate that TTX functions as a stress relieving substance by affecting the CRH-ACTH-cortisol axis and reducing agonistic interactions in tiger puffer juveniles., Toxicon, 171, pp.54-61; 2019

A previously unreported heterodetic cyclic peptide, homophymamide A (

Myrindole A, a bis-indole alkaloid, was isolated from the deep-sea sponge

The toxins present in poisonous and venomous animals have both offensive and defensive functions. The blue-lined octopus Hapalochlaena fasciata has tetrodotoxin (TTX) in various organs, including the salivary glands, muscles and skin, and TTX is assumed to be used in both offence and defence. In this study, we explore the evolution of toxicity in H. fasciata by measuring the levels and distribution of TTX in H. fasciata specimens collected in Japan and by investigating the TTX allocation within the body. TTX was detected in all 13 adults and 3 paralarvae studied. The concentration of TTX was highest in the anterior and posterior salivary glands, and the total amount of TTX was highest in the muscles and skin. Larger individuals had significantly higher amounts of TTX, but the TTX concentrations in the muscles and skin were not correlated with body weight. There were large individual differences in TTX concentration and some individuals showed extremely low TTX levels. These individual differences may be related to regional, seasonal and sexual differences, although the origin of TTX in this species is still unclear. Furthermore, we found a strong positive correlation between TTX concentrations in the posterior salivary gland and TTX concentrations in muscles and skin, with the former being c. 20 times greater than the latter. This suggests that the allocation ratio may not result from a plastic allocation, reflecting the TTX needs of each organ or the amount of available TTX, but rather may, at least partly, be evolutionarily fixed.

The species classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their toxicity and toxin profile is limited. In the present study, to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie (designated PNH and KTI, respectively).Phylogenetic analysis of partial sequences of three mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2) gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile revealed that both Pao sp. A and Pao sp. B possess saxitoxins (STXs), comprising STX as the main component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding the regulatory limit for STXs set by the Codex Committee,whereas in Pao sp. B, only the skin contained high toxin concentrations. The difference in the STX accumulation ability between the two species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in freshwater pufferfish., Toxins, 12(11), art.no.689; 2020

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

Stellatolide H (1) was isolated from a deep-sea sponge Discodermia sp. as the cytotoxic constituent. The planar structure of 1 was elucidated on the basis of the NMR spectroscopic and mass spectrometric data. The absolute configurations of the constituent amino acid residues were determined by the Marfey’s method. Stellatolide H (1) is a peptide lactone of the callipeltin class with its N-terminus blocked by 3-hydroxy-6,8-dimethyldeca-(4Z,6E)-dienoic acid (Hdda).

The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7–6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish., Toxins, 13(10), art. no. 731; 2021

Tetrodotoxin (TTX), also known as pufferfish toxin, has been detected in marine edible bivalves worldwide. In this study, several bivalve species, Azumapecten farreri subsp. akazara, Patinopecten yessoensis and Mytilus galloprovincialis, collected from the Pacific side of the northern Japanese Islands, were studied for the accumulation of TTX in the presence of toxic planocerid larvae. LC-MS/MS analysis demonstrated that TTX was detected only in the midgut gland of A. farreri subsp. akazara. Toxic flatworm-specific PCR and direct sequencing of the amplicons showed that the DNA fragments of the Planocera multitentaculata COI gene were detected in the gut contents of the toxified bivalves. The planocerid larvae were also detected in the environmental seawaters. Toxification experiments in the aquarium demonstrated that the mussel M. galloprovincialis was also toxified by feeding on the toxic flatworm larvae. These results suggest that the source of TTX accumulation in edible bivalves is toxic flatworm larvae.

SUMMARY The red alga Digenea simplex was cultured with various culture media to clarify the nutritional conditions to produce kainic acid (KA). Unlike the domoic acid-producing red alga Chondria armata, D. simplex was insensitive to excessive manganese, and grew best (mean growth rate approximately 800% for 25 days) in modified PES medium (mPES; seawater + nitrate, phosphate, iron, trace metals, vitamins, and 2-[4-(2-hydroxyethyl)-1-piperazinyl]- ethanesulfonic acid) prepared with autoclaved seawater. Liquid chromatography-mass spectrometry analysis of the algal extracts revealed that the KA content of the explants cultured with mPES or N·P·Fe medium (seawater + nitrate, phosphate, and iron) was somewhat higher than that of wild specimens (1748–2378 μg g−1 vs 1562 μg g−1). The 1H-nuclear magnetic resonance spectrum of the KA extracted and purified from pooled explants was indistinguishable from the previously reported KA spectrum. When D. simplex was cultured for 6 weeks with medium in which NaNO3 of mPES was replaced by Na15NO3, the ratio of 214KA to total measured KA (totalKA = 213KA + 214KA) in the cultured explants (0.1 at the beginning of culture) gradually increased to 2.5, indicating that D. simplex produces KA in proportion to its growth under the condition in which sufficient nitrogen source is available.

To reveal the sensing of tetrodotoxin (TTX) by tiger puffer Takifugu rubripes juveniles and its action in the central nervous system (CNS), we conducted transcriptome analysis using next-generation sequencing for the olfactory system and brain of non-toxic cultured juveniles administered TTX. Sixty-seven million reads from the nasal region (olfactory epithelium and skin) and the brain of each of three individuals of the control, TTX-sensing and TTX-administered juveniles were assembled into 153,958 contigs. Mapping raw reads from each sample onto the nucleotide sequences of predicted transcripts in the T. rubripes genome (FUGU version 4) and the de novo assembled contigs to investigate their frequency of expression revealed that the expression of 21 and 81 known genes significantly changed in TTX-sensing and TTX-administered juveniles in comparison with control juveniles, respectively. These genes included those related to feeding regulation and a reward system, and indicated that TTX ingestion of T. rubripes juveniles is controlled in the feeding center in the brain, that T. rubripes may sense TTX as a reward, and that accumulated TTX directly acts on the central nervous system to adjust TTX ingestion., Fisheries Science, 83(3), pp.401-412; 2017

Tetrodotoxin (TTX), a potent neurotoxin, is found in various phylogenetically diverse taxa. In marine environments, the pufferfish is at the top of the food chain among TTX-bearing organisms. The accumulation of TTX in the body of pufferfish appears to be of the food web that begins with bacteria. It is known that toxic pufferfishes possess TTX from the larval/juvenile stage. However, the source of the TTX is unknown because the maternally sourced TTX is extremely small in quantity. Therefore, the TTX has to be obtained from other organisms or directly from the environment. Here, we report evidence that the source of TTX for toxic fish juveniles including the pufferfish (Chelonodon patoca) and the goby (Yongeichthys criniger) is in the food organisms, as seen in their gut contents. Next generation sequencing analysis for the mitochondrial COI gene showed that the majority of the sequence recovered from intestinal contents of these toxic fishes belonged to the flatworm Planocera multitentaculata, a polyclad flatworm containing highly concentrated TTX from the larval stage. PCR specific to P. multitentaculata also showed that DNA encoding the planocerid COI gene was strongly detected in the intestinal contents of the goby and pufferfish juveniles. Additionally, the planocerid specific COI sequence was detected in the environmental seawater collected from the water around the sampling locations for TTX-bearing fish. These results suggest that planocerid larvae are the major TTX supplier for juveniles of TTX-bearing fish species.

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.

We examined the effects of tetrodotoxin (TTX) administration on artificially raised tiger puffer Takifugu rubripes juvenile survival after release into a mesocosm with predators to clarify the ecological significance of TTX. Pellets containing three different concentrations of TTX [0 as the control, 7 and 14 mouse units (MU)/g diet] were fed to non-toxic artificially raised T. rubripes juveniles for 10 days. TTX accumulation in the various tissues of fish was detected except for in the control diet group. TTX administration did not affect survival or growth of the fish. One hundred fish from each diet group were released together after TTX administration into a salt pond mesocosm (2650 m2) with predators (Lateolabrax sp.) for 5 days. Survival after release was significantly higher in both the fish fed with the 7 MU TTX/g diet (62%) and the 14 MU TTX/g diet (74%) than in the control fish (32%)., Fisheries Science, 83(2), pp.191-197; 2017

Paralytic shellfish toxins (PSTs) produced by Alexandrium catenella (formerly A. tamarense) in Korean coastal waters caused the deaths of four people (in 1986 and 1996) who consumed contaminated mussels (Mytilus edulis). This led to more detailed consideration of the risks of PST outbreaks and incidents in Korea, including the introduction of shellfish collection bans. In this study, we investigated the relationships between A. catenella population dynamics and PST accumulation in the mussel M. galloprovincialis. Discharges from the Nakdong River affect the environmental conditions along the Geoje coast, resulting in low salinity and high nutrient levels that trigger blooms of A. catenella. At the toxin peak on 24 April 2017, the toxins detected in A. catenella cells were C1, gonyautoxin (GTX)1 and GTX2, whereas the concentrations of PSTs in M. galloprovincialis were high and in the order of GTX4 &gt, GTX1 &gt, GTX3 &gt, saxitoxin (STX) &gt, GTX2 &gt, neoSTX &gt, decarbamoylgonyautoxin (dcGTX)2 &gt, dc GTX3. The PST level in mussels was also high. At 15 &deg, C, the PSTs are constantly found to be higher (10-fold higher in 2017 and 30-fold higher in 2018) than safe levels for human consumption (80 &mu, g STX diHCl equivalents 100 g&minus, 1).

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.

To clarify the production mechanisms and biologic functions of domoic acid (DA) by the red alga Chondria armata, we established a laboratory culture of C. armata. The alga grew better in modified PES medium (mPES) without trace metals or manganese than in unmodified mPES (seawater + nitrate, phosphate, iron, trace metals, vitamins, and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), suggesting that C. armata is especially hypersensitive to the toxicity of excessive manganese. C. armata cultured in N・P・Fe medium (seawater + nitrate, phosphate, and iron) grew best (mean growth rate 828.4%) at a relative nutrient concentration of 50%. Liquid chromatography-mass spectrometry analysis of the algal extracts revealed that the DA content of the cultured explants (2273-3308 ppm) was 4-5 fold higher than that of wild specimens. The extract of pooled explants (60 g) was purified by activated charcoal treatment and several types of column chromatography to afford ca. 10 mg DA. The 1H-nuclear magnetic resonance spectrum of the preparation was indistinguishable from the previously reported spectrum of DA, indicating that C. armata itself has an ability to produce DA., Toxicon, 92, pp.1-5; 2014

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

Behavioral observation was conducted to test whether olfaction is functional to detect tetrodotoxin (TTX) in tiger puffer Takifugu rubripes using Y-maze. We placed either agarose carrier or one agarose and one agarose containing TTX (200 MU) at each head of the channel of Y-maze. Then three non-toxic hatchery-reared juveniles (body length, 5.6 ± 0.4 cm; n = 18) were released into the Y-maze and pecking behavior to carrier was observed for 3 h. The same procedure was tested for olfactory-ablated juveniles and for juveniles taht received sham operation. Juveniles showed significant selectivity to TTX, except for olfactory-ablated juveniles. These results indicate that pufferfish detects TTX by olfactory organ., Ichthyological Research, 60(4), pp.386-389; 2013

To reveal the accumulation profile of tetrodotoxin (TTX) in pufferfish Takifugu rubripes juveniles, we compared the localization of TTX in various tissues among wild juveniles and hatchery-reared juveniles with or without TTX administration using immunohistochemical technique with anti-TTX monoclonal antibody. Immuno-positive reaction was observed in hepatic tissue, basal cell of skin and olfactory, olfactory epithelium, optic nerve and brain (optic tectum, cerebellum, medulla oblongata) of wild juveniles (body length: BL, 4.7-9.4cm). TTX was detected in the same tissues as wild juveniles and epithelial cell layer of intestine of hatchery-reared juveniles (BL, 5.0-5.3cm) to which TTX was orally administrated. No positive reaction was observed from the tissues of hatchery-reared juveniles without TTX administration. These results suggest that orally administrated TTX to the non-toxic cultured juveniles is accumulated in the same manner of wild juveniles. In addition, our study revealed that pufferfish accumulates TTX in the central nervous system., Toxicon, 71, pp.128-133; 2013

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.

From January to June 2009, a total of 64 gastropod specimens of 15 species were collected from the coastal waters of Okinawa Prefecture, Japan, and examined for toxicity by means of mouse bioassay. Among the specimens tested, 5 species, Nassarius glans, Nassariuscoronatus, Olivaannulata, Oliva concavospira and Zeuxis sp., were toxic. The toxicity scores of N. glans were very high; 39.6-461 MU/g in muscle, and 98.6-189 MU/g in viscera including digestive gland, followed by Zeuxis sp. (12.7 MU/g in whole body), N. coronatus (5.64-11.1 MU/g in whole body), O. annulata (10.8 MU/g in the whole body), O. concavospira (6.65 MU/g in the muscle). Liquid chromatography/mass spectrometry (LC-MS) revealed that the major toxic component was tetrodotoxin (TTX), which accounting for 13-82% of the total toxicity. As for the remaining toxicity in the case of N. glans, 4,9-anhydroTTX, 4-epiTTX and 11-oxoTTX were contributors. Moreover, Niotha albescens showed no toxicity (less than 10 MU/g) in mouse bioassay, but TTX (5.08 MU/g) was detected by LC-MS. Paralytic shellfish poison was not detected in any of the specimens by high-performance liquid chromatography with fluorometric detection (HPLC-FLD).

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.

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan. Language: en

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.