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.

Beginning with the larval stages, marine pufferfish such as Takifugu niphobles contain tetrodotoxin (TTX), an extremely potent neurotoxin. Although highly concentrated TTX has been detected in adults and juveniles of these fish, the source of the toxin has remained unclear. Here we show that TTX in the flatworm Planocera multitentaculata contributes to the toxification of the pufferfish throughout the life cycle of the flatworm. A species-specific PCR method was developed for the flatworm, and the specific DNA fragment was detected in the digesta of wild pufferfish adults. Predation experiments showed that flatworm larvae were eaten by the pufferfish juveniles, and that the two-day postprandial TTX content in these pufferfish was 20–50 μg/g. Predation experiments additionally showed flatworm adults were also eaten by pufferfish young, and after two days of feeding, TTX accumulated in the skin, liver and intestine of the pufferfish.

The pufferfish Takifugu niphobles (at present Takifugu alboplumbeus) possesses highly concentrated tetrodotoxin (TTX), an extremely potent neurotoxin that provides effective protection from predators, at least at the larval stages. However, the source of the toxin has remained unclear. Recently, DNA from the toxic flatworm Planocera multitentaculata was detected in the intestinal contents of juveniles and young of the pufferfish, suggesting that the flatworm contributes to its toxification at various stages of its life. In this study, we describe the behavior of the pufferfish in the intertidal zone that appears to contribute to its toxification before and during its spawning period: pufferfish were found to aggregate and ingest flatworm egg plates by scraping them off the surface of rocks. DNA analysis based on 28S rRNA and cytochrome c oxidase subunit I (COI) genes identified the egg plates as those of P. multitentaculata. Liquid chromatography with tandem mass spectrometry analysis revealed that the egg plates contain highly concentrated TTX. The feeding behavior of the pufferfish on the flatworm egg plates was also observed in the aquarium. These results suggest that pufferfish feed on the flatworm egg plate, which enables them to acquire toxicity themselves while providing their offspring with the protective shield of TTX.