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.

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.

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.

Marine Takifugu pufferfish, which naturally possess tetrodotoxins (TTXs), selectively take up and accumulate TTXs, whereas freshwater Pao pufferfish, which naturally possess saxitoxins (STXs), selectively take up and accumulate STXs. To further clarify the TTXs/STXs selectivity in pufferfish, we conducted a TTX/STX administration experiment using Chelonodontops patoca, a euryhaline marine pufferfish possessing both TTXs and STXs. Forty nontoxic cultured individuals of C. patoca were divided into a seawater group (SW, acclimated/reared at 33‰ salinity; n = 20) and a brackish water group (BW, acclimated/reared at 8‰ salinity; n = 20). An aqueous TTX/STX mixture was intrarectally administered (both at 7.5 nmol/fish), and five individuals/group were analyzed after 1–48 h. Instrumental toxin analyses revealed that both TTX and STX were taken up, transferred, and retained, but more STX than TTX was retained in both groups. TTX gradually decreased and eventually became almost undetectable in the intestinal tissue, while STX was retained at ~5–10% of the dose level, and only STX showed transient transfer in the liver. The BW group showed a faster decrease/disappearance of TTX, greater STX retention in the intestine, and greater STX transient transfer to the liver. Thus, C. patoca appears to more easily accumulate STXs than TTXs, especially under hypoosmotic conditions.

Several species of crabs from the Xanthidae family are recognized as dangerous marine organisms due to their potent neurotoxins, including paralytic shellfish toxin (PST), tetrodotoxin (TTX), and palytoxin (PLTX). However, the mechanisms of toxin accumulation and transport and the origin of these toxins in toxic xanthid crabs remain unknown. The identification of toxic crab species, their toxicity and toxin composition, and toxin profiles have been studied thus far. To date, more than ten species of xanthid crabs have been confirmed to possess toxins. Recently, several new studies on crabs, including the geographic distribution of toxin profiles and the ecological role of crabs, have been reported. Therefore, this review provides a summary of global research on toxic xanthid crabs, containing new findings and hypotheses on the toxification in and the origins of these crabs. Furthermore, the challenges and future perspectives in this field are also discussed.

Marine pufferfish of the genus Canthigaster living in the vicinity of coral reefs are thought to be toxic in the skin because they are unpalatable to predators, however, very little is known about the toxin profile of most species of this genus. In the present study, to fully clarify the toxin profile of the Canthigaster marine pufferfish, the composition, concentration, and intra-body distribution of toxins contained in marine pufferfish Canthigaster rivulata, collected from three sampling locations in Nagasaki Prefecture, Japan, were investigated using high-performance liquid chromatography with post-column fluorescence derivatization (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Instrumental analyses revealed that marine pufferfish C. rivulata is capable of carrying both tetrodotoxin (TTX) and saxitoxins (STXs) simultaneously, and the skin and ovaries are the most toxic tissues, with other tissues such as the liver being weakly toxic. Overall, TTX was more abundant than STXs in C. rivulata, but there were differences between individuals, with some possessing more STXs than TTX. Higher STXs content was detected in the skin of individuals from Tameshi than in those from Kakidomari. Despite individual and regional differences, C. rivulata has been identified as a Canthigaster marine pufferfish that possesses a large amount of TTX and STXs.