The toxicity of the Japanese marine pufferfish was extensively studied by Tani, who reported that 14 of the 21 species examined were toxic [18]. Later, 8 pufferfish species were added to the list of toxic species [19–22], and a total of 22 species are presently listed as TTX-bearing marine pufferfish (Table 1), all belonging to the Tetraodontidae family.

Lagocephalus wheeleri and L. gloveri of the same family are usually regarded as non-toxic species, although they occasionally show weak toxicity [23]. All species of the Diodontidae and Ostraciontidae families are TTX-free, though the skin of Ostraciontidae contains an ichthyotoxic and hemolytic substance called pahutoxin [24,25]. Food poisoning cases due to ingestion of the liver of Ostraciontid boxfish sometimes occur in Japan, but the causative agent is a toxin with delayed hemolytic activity, possibly palytoxin or a similar substance, and not TTX or pahutoxin [26]. Thai brackish water pufferfish Tetraodon nigroviridis and T. steindachneri, and freshwater pufferfish T. fangi, T. leiurus, and T. suwatii are also toxic. The toxin of brackish water species was identified as TTX [27,28], but in the freshwater species, saxitoxins (STXs), toxins that belong to the paralytic shellfish poison (PSP) family, were detected as the main toxic principles [29,30]. Freshwater pufferfish, Colomesus asellus from Brazil [31] and T. turgidus from Cambodia [32], also possess PSP. Recently, some marine pufferfish (Arothron spp.) from the Philippines [33], and Arothron firmamentum from Japanese waters [34] were also reported to possess STXs as the major toxins. Furthermore, palytoxin-like substance(s), in addition to a small amount of PSP, were detected in a Bangladeshi freshwater pufferfish belonging to the genus Tetraodon [35].

The generally large individual and regional variations in pufferfish toxicity [20] are too great to be explained by endogenous factors alone, i.e., differences in physiologic conditions among individual pufferfish, suggesting their exogenous intoxication.

The distribution of TTX in pufferfish bodies appears to be species-specific (Table 1). In marine species, the liver and ovary generally have the highest toxicity, followed by intestines and skin. Muscles and/or testis are non-toxic or weakly toxic, except for in Lagocephalus lunaris and Chelonodon patoca, and are regarded as edible by the Japanese Ministry of Health, Labour, and Welfare, even in many toxic species. In contrast, in C. patoca inhabiting coastal or brackish waters of the Okinawa and Amami Islands [36], and in Thai/Bangladeshi/Cambodian brackish water and freshwater pufferfish, the skin has the highest toxicity [27–29,37].

Our recent immunohistochemical investigations using a monoclonal anti-TTX antibody clarified the intratissue microdistribution of TTX in the skin and ovary of TTX-bearing pufferfish [38–40]. Species-specificity was also recognized in the microdistribution, i.e., TTX was localized in the nucleus, yolk vesicles, and yolk granules of oocytes in the ovary of the marine species T. vermicularis, whereas it was detected in the connective tissues and nucleus of some oocytes in C. patoca [39]. TTX-bearing glands were observed in the skin of T. vermicularis, and TTX-bearing secretory cells (succiform cells) were observed in brackish water species such as C. patoca, T. steindachneri, and T. nigroviridis [38–40].

In the toxic marine pufferfish, the liver generally shows very high toxicity throughout the year except in the spawning season, during which the ovary becomes highly toxic by accumulating TTX transferred from the liver [20]. TTX in the eggs spawned from the ovary could be involved in protecting the eggs from predators. On the other hand, when toxic pufferfish encounter enemies, their bodies swell to two or three times their usual size and TTX is excreted from their skin to repel the enemies [41,42]. These observations suggest that the pufferfish accumulate TTX as a biologic defense agent.

TTX was long believed to be present only in pufferfish. Since Mosher et al. detected TTX in the eggs of the California newt Taricha torosa in 1964 [43], however, TTX has been detected in a wide variety of animals, as shown in Table 2 [9].

It is quite unlikely that these TTX-bearers belonging to particular species of different phyla possess a common gene that codes for TTX production. The trumpet shell Charonia sauliae accumulates TTX by ingesting toxic starfish [10], supporting the hypothesis that the TTX of pufferfish is not endogenous, but comes via the food chain. The exact origin of TTX in the food chain, however, remains unknown. Because the ecologic environments of TTX-bearing animals seem to have no common factor other than being closely related to an aquatic system, bacteria, omnipresent organisms that commonly inhabit the aquatic system, are implicated as the primary source of TTX.

Based on the above suggestion that bacteria might be the primary source of TTX, in the 1980s we investigated the TTX production of marine bacteria. Intestinal bacteria from several TTX-bearing organisms were isolated, cultured, and examined for lethal potency to mice. Vibrio alginolyticus from the toxic starfish Astropecten polyacanthus [73,74], one Vibrio strain from the xanthid crab Atergatis floridus [11,74], and another Vibrio strain from the toxic marine pufferfish Takifugu snyderi [74] had a paralytic toxicity of 213, 30, and 3 MU per 500 ml of medium, respectively. The toxic principles produced peaks identical to those of authentic TTX and anhydrotetrodotoxin (anhydroTTX) in high performance liquid chromatography with post-column fluorescence derivatization (Figure 2)[74]. In addition, gas chromatography/mass spectrometry analyses revealed that they produce a C₉-base, the degradation product of TTX, on alkaline hydrolysis [75], leading us to conclude that the isolated bacteria produce TTX. Shewanella alga and Alteromonas tetraodonis isolated from a red alga Jania sp. [76], Shewanella putrefaciens from a TTX-bearing marine puffer Takifugu niphobles [77], and some other marine bacteria isolated from TTX-bearing organisms [78] also produce TTX.

As mentioned above, many years of studies on TTX have revealed that (1) pufferfish toxicity shows remarkable individual and regional variations, (2) TTX is distributed over various organisms, including food animals of pufferfish, (3) the trumpet shell accumulates TTX by ingesting toxic starfish, and (4) marine bacteria primarily produce TTX, suggesting that pufferfish do not synthesize TTX, but accumulate it through the food chain, which starts from marine bacteria. If this is the case, pufferfish should become non-toxic when fed TTX-free diets in an environment in which the invasion of TTX-bearing organisms has been eliminated.

To test this hypothesis, we collected more than 6000 specimens of the marine pufferfish Takifugu rubripes that were cultured with non-TTX-containing diets in such environments (net cages on the sea or land aquaria located in eight prefectures of Japan) and examined the toxicity of their livers and some other body parts, including gonads and muscle, using an accepted TTX bioassay [14]. All specimens were non-toxic, i.e., their toxicity was under the detection limit (2–10 MU/g) (Tables 3 and 4). Furthermore, liquid chromatography/mass spectrometry analysis demonstrated that none of the 20 liver specimens from Nagasaki and 5 liver specimens from Yobuko contained detectable amounts of TTX, i.e., their TTX content was below 0.1–0.4 MU/g.

When non-toxic cultured pufferfish T. rubripes were reared on TTX-containing diets, however, they became toxic within 40 days when fed 4 MU TTX/g body mass/day, and in 100 days when fed 0.5 MU TTX/g body mass/day. Thereafter, the toxicity continuously increased up to the end of experimental period (Figure 3) [79]. In contrast, three non-pufferfish species were not intoxicated with TTX, even after culturing for 139 days when fed 4 MU TTX/g body mass/day. These species seem not only to exclude, but also to detoxify, TTX.

In a similar feeding experiment using a PSP [consisting of STX and neosaxitoxin (neoSTX)]-containing diet for 60 days, non-toxic pufferfish were also intoxicated with PSP. Their toxicity scores, however, were only 21% to 53% of those of the fish fed with the same dosage of TTX as controls (unpublished data). Thus, the TTX-bearing marine pufferfish tend to selectively accumulate more TTX than PSP.

These results clearly demonstrate that the TTX of toxic pufferfish is not endogenous, but is derived from the food chain (Figure 4). Another possibility is that the TTX is produced by symbiotic or parasitic bacteria that are directly accumulated inside the puffer body and not obtained via the food chain. The amount of TTX produced by bacteria, however, appears to be too little to account for the TTX accumulation in pufferfish, therefore bioconcentration likely has a large role in the accumulation of TTX in pufferfish. Furthermore, based on the observation that 6000 specimens of pufferfish reared over 1 year in net cages or land aquaria were non-toxic, the TTX produced in the intestines of pufferfish by marine bacteria must be a minor contributor, or rather negligible, to account for the TTX accumulation in pufferfish.

Some studies of the intoxication mechanism have been performed in amphibians, especially newts. Shimizu and Kobayashi [80] first observed that when various radioactive precursors were administered to two species of newts, Taricha torosa and T. granulosa, none of the precursors were incorporated into TTX molecules, although the authors did not clearly conclude whether TTX in the newts was endogenous or exogenous. On the other hand, Hanifin et al. claim that newts can biosynthesize TTX by themselves, because TTX levels in the dorsal skin of T. granulosa significantly increased under artificial rearing with non-toxic diets for 1 year [81], and because the newts released a large amount of TTX from their skin after receiving an electric shock, but the TTX levels in the dorsal skin significantly recovered during the 9 months thereafter [82].

During our studies on the toxicity and toxin profiles of the Japanese newt Cynops pyrrhogaster [83,84], we observed that the eggs of the newt retained a certain amount of TTX inherited from their parents, which gradually disappeared in the larval stage. In the juvenile stage in a terrestrial environment, the newt abruptly begins to accumulate TTX again, and becomes highly toxic in the adult stage. When artificially reared with non-toxic diets for 5 years after hatching, however, the newt became non-toxic (< 2 MU/g). The non-toxic newt, when orally administered TTX or 6-epitetrodotoxin (6-epiTTX), accumulated approximately 50% of the administered toxins. These observations strongly suggest that the TTX of C. pyrrhogaster is exogenous, i.e., the newt begins to accumulate TTX after metamorphosis, with their food organisms as its origin.

Daly et al. [85] examined the intoxication mechanism of frogs. They hatched eggs of the Panamanian frog Atelopus varius which possesses both bufadienolides (a type of steroid) and TTX in the skin, and artificially reared them for 25 to 45 months. In the skin of the adult frogs, bufadienolides were detected at the same level as in wild specimens, but TTX was not detected, leading them to conclude that bufadienolides are endogenous and the TTX is exogenous, possibly derived from food organisms or environmental factors such as symbiotic bacteria.

Three species each of toxic pufferfish [T. niphobles, T. pardalis, and T. rubribes (cultured specimens)], non-toxic pufferfish (L. wheeleri, L. gloveri, and Liosaccus cutaneus), and non-toxic general fish (Oplegnathus punctatus, O. fasciatus, and Girella punctata) were examined for their resistance to TTX [86]. The MLD of TTX administered intraperitoneally to the three toxic species of pufferfish was 300 to 750 times greater than that of mice (Table 5). Due to this high resistance, the Takifugu species can accumulate huge amounts of TTX. Non-toxic pufferfish showed 13 to 15 times stronger resistance against TTX than mice. These pufferfish are generally non-toxic, but can become weakly toxic in some habitats [23]. They can accumulate a limited amount of TTX due to the medium level of resistance to TTX. In contrast, general non-toxic fish had the same low resistance levels as mice, i.e., they cannot accumulate TTX. The goby Yongeichthys criniger (formerly Gobius criniger) also has high resistance to TTX, and can accumulate TTX [63]. TTX-bearing xanthid crabs [87] and the Japanese newt [84] exhibit even greater resistance to TTX than pufferfish. Based on these findings, it is clear that for TTX-bearing animals to accumulate a certain level of TTX in their body, they must be endowed with high tolerance to TTX.

The mechanism of TTX resistance in pufferfish and rewts has been explained based mainly on the TTX-resistant sodium channels found in the animals, in which the aromatic amino acid commonly located in the p-loop region of domain I in TTX-sensitive sodium channels is replaced by a nonaromatic amino acid, resulting in their extremely low affinity to TTX [88–91]. Garter snakes and clams can also acquire similar sodium channel mutation-based TTX/PSP resistance by interacting with their toxic food organisms, TTX-bearing newts and PSP-producing dinoflagellates, respectively [92,93]. On the other hand, the shore crab Hemigrapsus sanguineus possesses TTX-binding proteins in its hemolymph, which confer TTX resistance on this non-toxic crab [94,95]. Pufferfish and newts may be equipped not only with TTX-resistant sodium channels, but also with other mechanisms whereby they mask and/or modulate TTX molecules inside their body.

In China and Taiwan, food poisoning incidents attributable to the ingestion of small gastropods such as Niotha clathrata, Natica vitellus, Oliva miniacea, O. hirasei, Nassarius glans, and Zeuxis samiplicutus, occur frequently [108–112]. In China, more than 300 people were poisoned, and 16 died during 1977–2001. In June 2001, a poisoning incident due to ingestion of a small gastropod of the family Nassariidae, Z. samiplicutus occurred in South Zheijiang, Mainland China. About 30 victims were reported to have symptoms, including paralysis, coma, nausea, vomiting, ataxia, and aphasia. The causative agent was identified as TTX by thin-layer chromatography, electrophoresis, and high performance liquid chromatography [113,114].

It is astonishing that there continues to be many victims, as well as deaths, due to the ingestion of commercially cooked small gastropods in China, which may become a serious public health issue. Moreover, this type of poisoning has spread from the coast of Zheijiang to other areas of the East China Sea and Yellow Sea. Very recently (July 2007) in Nagasaki, Japan, a food poisoning incident occurred due to the same type of small gastropod, Alectrion glans. An investigation revealed that a large amount of TTX (600 MU/g) remained in the leftover cooked gastropods, including muscles and digestive glands (unpublished data). Although the TTX accumulation mechanism in the gastropods remains uncertain, we suspect that the gastropods might become toxic by feeding on the toxic viscera of pufferfish that died after spawning.

In December 1979, a 41-year-old man ate a boiled digestive gland (about 60 g) of the trumpet shell Charonia sauliae, which he had caught in the coastal waters of Shimizu, Shizuoka Prefecture. Thirty minutes after ingestion, he was hospitalized and received artificial respiration. Although he remained unconscious for 2 days, he recovered fully in 5 days.

The leftover digestive gland had a toxicity score of 17000 MU, and the responsible toxin was identified as TTX [47]. Thus, it was estimated that the victim might have ingested an amount of TTX almost equivalent to the MLD. TTX was also identified as the causative agent in the leftovers from similar TTX poisoning cases in Wakayama Prefecture in 1982 and Miyazaki Prefecture in 1987 [115].

Poisonings due to the ingestion of eggs of the horseshoe crab Carcinoscorpius rotundicauda have occasionally been reported in Thailand [116]. The symptoms of the victims were mostly similar to those caused by TTX or PSP. C. rotundicauda toxin consists mainly of TTX and anhydroTTX [59,117,118], but sometimes contains STX and neoSTX [119]. Hence, the responsible toxin for horseshoe crab poisonings is considered to be TTX and/or PSP.