Phylogeny and Toxin Profile of Freshwater Pufferfish (Genus Pao) Collected from 2 Different Regions in Cambodia.

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.

Transfer profile of intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes.

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.

Occurrence of paralytic shellfish toxins in Cambodian Mekong pufferfish Tetraodon turgidus: selective toxin accumulation in the skin.

The toxicity of two species of wild Cambodian freshwater pufferfish of the genus Tetraodon, T. turgidus and Tetraodon sp., was investigated. Tetraodon sp. was non-toxic. The toxicity of T. turgidus was localized mainly in the skin and ovary. Paralytic shellfish toxins (PSTs), comprising saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX), account for approximately 85% of the total toxicity. Artificially reared specimens of the same species were non-toxic. When PST (dcSTX, 50 MU/individual) was administered intramuscularly into cultured specimens, toxins were transferred via the blood from the muscle into other body tissues, especially the skin. The majority (92.8%) of the toxin remaining in the body accumulated in the skin within 48h. When the same dosage of tetrodotoxin (TTX) was similarly administered, all specimens died within 3-4h, suggesting that this species is not resistant to TTX. Toxin analysis in the dead specimens revealed that more than half of the administered TTX remained in the muscle and a small amount was transferred into the skin. The presence of both toxic and non-toxic wild specimens in the same species indicates that PSTs of T. turgidus are derived from an exogenous origin, and are selectively transferred via the blood into the skin, where the toxins accumulate.

Toxicity assessment for the horseshoe crab Carcinoscorpius rotundicauda collected from Cambodia.

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.

Maturation-associated changes in toxicity of the pufferfish Takifugu poecilonotus.

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 1000 MU/g or 10,000 MU/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 (1.59-15.1 MU/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.

Distribution of tetrodotoxin in pufferfish collected from coastal waters of Sihanouk Ville, Cambodia.

In Cambodia, fatal food poisonings associated with the consumption of pufferfish have occurred for decades, but the causative species or toxins have never been documented. Herein, we investigated the toxicity of three pufferfish species of the genus Lagocephalus collected from the coastal waters of Sihanouk Ville, one of the main regions where poisonings have occurred. L. wheeleri and L. spadiceus were non-toxic, whereas L. lunaris was toxic and all of its body tissues exhibited toxicity levels exceeding the safety limit for human consumption (10 mouse units/g). Tetrodotoxin (TTX) was identified as the main toxin in this species; no paralytic shellfish poison(s) were detected. Consequently, we can confirm pufferfish to be a hazardous reservoir of TTX in Sihanouk Ville. It is likely that L. lunaris is one of the causative species of past pufferfish poisonings that have occurred in Cambodia.