Puffer Fish Gut Microbiota Studies Revealed Unique Bacterial Co-Occurrence Patterns and New Insights on Tetrodotoxin Producers.

Tetrodotoxin (TTX) is a potent neurotoxin isolated mainly from toxic puffer fish. To date, the TTX biosynthetic mechanism inside its hosts remains unresolved. Here, we hypothesize the TTX synthesis relies on the host gut microbiota, including the neglected non-culturable bacteria. In these studies, we collected the gut contents from 5 puffer fish species of the genus Takifugu including one suspected hybrid species for gut microbiota study by 16S rRNA amplicon metagenomics approach. Their gut samples were divided into toxic and non-toxic groups based on the TTX concentrations in the livers detected by LC-MS/MS. Bacterial diversity studies showed that gut microbiota structures were significantly different between toxic and non-toxic species. Vibrio and Cyanobacteria centered at the gut bacterial co-occurrence network, suggesting their importance in TTX biosynthesis. The results of PICRUSt2 metagenomic prediction and gene set enrichment analysis provided new support of arginine-precursor required in TTX biosynthesis. This is the first study to profile the gut microbiota in toxic and non-toxic puffer fish species by 16S rRNA amplicon metagenomic approach, defining significant microbial co-occurrence patterns in their gut environment. Our data supported the proposed biosynthesis of TTX inside the hosts by their gut bacterial symbionts using arginine as a precursor.

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction.

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 +/- 4.0 and 87.1 +/- 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.

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.

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.

Two novel species of tetrodotoxin-producing bacteria isolated from toxic marine puffer fishes.

Out of eight dominant discrete bacterial colonies isolated and purified from the toxic marine puffer fishes collected in Hong Kong waters, two novel species of non-sporing, non-acid-fast and chemoorganotrophic bacteria capable of producing tetrodotoxin (TTX, a potent non-protein neurotoxin), as well as one previously reported and confirmed TTX-producing bacterium. They were identified as Microbacterium arabinogalactanolyticum, Serratia marcescens and Vibrio alginolyticus, respectively, all of which are widely distributed in soils, sewage or marine environments. Each bacterial isolate (500 ml broth medium cultured in darkness without aeration for 10 days at 25 degrees C) could produce an amount of toxicity, after extraction and purification, ranging from 78.3 to 105.3 mouse units (MU) in 500 ml of broth medium by mouse bioassay. The principal toxic component in the bacterial cultures was determined to be TTX by thin layer chromatography and mass spectrometry.

The annual toxicological profiles of two common puffer fish, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected along Hong Kong coastal waters.

The toxicological profiles of two local common puffer fish, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected in Hong Kong waters were investigated continuously for 14 months (June 1997-August 1998). Their annual spawning seasons (as evident by the enlargement of gonads and presence of eggs in the ovary) were found to be from October to February (four consecutive months) and December to February (two consecutive months), respectively. The toxicities of their internal organs were determined by standard mouse bioassay and expressed in terms of mouse units (MU). The ovary (in both species) and liver (only in T. niphobles) were discovered to be moderately toxic (100-1000 MU/g) in their non-spawning seasons. Whilst all the intestine and skin were weakly toxic (between 10 and 100 MU/g), their flesh, however, was basically non-toxic (less than 10 MU/g) throughout the whole year. The testes, which were only developed in their spawning seasons, were also non-toxic. This study showed that the toxicological profiles of both of the two species had prominent seasonal patterns which were highly related to their spawning seasons. Contrary to the most common belief, both species were discovered to be relatively less toxic during their spawning seasons.