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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.

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

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

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.

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.

From October 2006 to December 2007, wild specimens of the pufferfish Takifugu poecilonotus (93 females, 45 males) were collected from the Ariake Sea. Tissue toxicity was examined by mouse bioassay, and tetrodotoxin (TTX) content in the blood plasma by enzyme-linked immunosorbent assay. The relationship between toxicity and maturation was investigated based on changes in the gonadosomatic index: December-March in females and November-March in males, the 'maturation period'; April, 'just after spawning'; and the other months, the 'ordinary period'. Toxicity of both sexes was high throughout the year, but sharply declined in April. In all tissues examined (skin, liver, and ovary) other than testis, toxicity exceeded 1000MU/g or 10,000MU/individual in many individuals. Seasonal profiles of tissue toxicity differed markedly between sexes. In females, liver toxicity was high during the ordinary period, and ovary toxicity was high during the maturation period. In males, little maturation-associated change in the toxin distribution was observed. Plasma TTX levels were similar between the sexes (1.59-15.1MU/ml), and fluctuated largely throughout the year without corresponding changes in tissue toxicity. The percentage of TTX binding to high molecular-weight substances in the plasma varied in association with maturation; the binding ratio fluctuated at relatively low levels during the ordinary period, and stabilized at a high level during the maturation period., Toxicon, 55(2-3), pp.289-297; 2010

In this study, we assessed the toxicity of the horseshoe crab Carcinoscorpius rotundicauda collected from Cambodia within two successive months during rainy (April-May) and dry (December-January) seasons, respectively. Toxicity assessments of the collected specimens by standard mouse bioassay showed marked individual variation, and their toxin profiles by liquid chromatography/mass spectrometry (LC/MS) revealed tetrodotoxin (TTX) was the main toxin while no paralytic shellfish toxins (PSTs) were detected. All specimens were toxic and the highest toxicity values were 315, 113, 60, 47, 44 and 38 mouse units (MU)/g in the tissues of hepatic caecum, egg, viscera, muscle, intestine and testis, respectively. Although the current findings showed that the Cambodian C. rotundicauda was a moderately toxic species, they are not suitable for human consumption due to their toxicity. To the best of our knowledge, this is the first scientific study on toxic marine seafood ever investigated in Cambodian territorial waters.

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella (Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4 x 10(7)cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.

The caligid copepod Pseudocaligus fugu, a common parasite on the body surface of both toxic (Takifugu pardalis) and cultured, non-toxic (Takifugu rubripes) puffer fishes, was isolated and analyzed for the presence of tetrodotoxin (TTX) using a monoclonal anti-TTX antibody-based immunohistochemical technique. Histological sections of female P. fugu from Takifugu pardalis explicitly revealed that positive brown staining was visible in regions of gut and appendages, and also in the general body tissues from the prosome to the urosome. It is absent from the epicuticle, ovary, oviduct, uterus and egg sacs, where clear negative blue color reaction was obtained. In contrast, the caligids on cultured, non-toxic Takifugu rubripes, considered as negative control, had no sign of TTX. The results indicate that there is no vertical transmission of TTX in the parasitic caligids, which could acquire TTX by feeding on the toxic mucus and skin tissues of host puffer fish.

Tetrodotoxin (TTX) was localized as brown color in different tissues of an undescribed species of the nemertean genus Cephalothrix (phylum Nemertea) and a turbellarian Planocera reticulata (phylum Platyhelminthes) on light microscopy by means of a monoclonal anti-TTX antibody. In the Cephalothrix sp., TTX was recognized in the vesicles apically arranged in the bacillary cells in the epidermis, basal lamina, the granular cells in the proboscis epithelium, rhynchocoel epithelium, and the vesicles in the basal portion of the intestinal wall near the blood vessels and rhynchocoel. The excretory system and the ovum also showed positive reaction of TTX antigen-antibody. On the other hand, the hermaphrodite flatworm P. reticulata exhibited TTX antigen-antibody complex only in their ovum. To our knowledge, this is the first experimental effort on micro-distribution of TTX in invertebrates.

Intra-tissue distribution of tetrodotoxin (TTX) in the skin of larva, juvenile, and adult Japanese newt Cynops pyrrhogaster was investigated by means of a monoclonal antibody-based immunoenzymatic technique. In the investigation, TTX was localized at immature glands in juvenile, and at the granular cells composing of granular and mixed glands in adult specimens under a light microscope. No specific stain was recognized in larval section. A duct like structure extending from the granular gland towards super epithelial layer was visualized in the toluidine blue treated skin section of adult newt. When stimuli by wiping with gauze ('handling stimulus') were given, C. pyrrhogaster was found to secrete an applicable amount of TTX and 6-epiTTX from the skin, suggesting that the newt has a granular gland of TTX to secrete it towards the body surface possibly as a biological defensive agent.

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.