Mum... I don’t like it... *spits out food*

Tetrodotoxin (TTX) is known as one of the most potent neurotoxins and is a specific blocker of voltage-gated sodium channels of excitable membranes of muscle and nerve tissues. It was originally believed that it occurred exclusively in pufferfish, however, it has been detected in an array of other species since; in the eggs of the California newt Taricha torosa, other fish such as gobies, and invertebrates including octopuses, crabs, shellfish, flat and ribbon worms. TTX is produced primarily by marine bacteria, such as Pseudoalteromonas tetraodonis, certain species of Pseudomonas and Vibrio, and it appears that it finds its way into pufferfish through the food chain. Tissue-specific distribution of TTX has been widely investigated with food hygiene as the main viewpoint, using mainly the genus Takifugu. It has been revealed that it is commonly distributed in the liver and ovaries, however, localisation in other tissues is species-specific; i.e., besides finding TTX in the liver and ovaries of Takifugu rubripes, it was found to be concentrated in the skin and intestine and marginally present in the testes and skeletal muscle in Takifugu niphobles. Previous work by Itoi et al., has revealed that tissue-specific distribution and the amount of TTX in the mature pufferfish T. niphobles were sex-dependent; female gonads and male liver showed the highest concentrations of TXT followed by male skin. Following this, suggestions that TTX may act as a chemical defence against predators has surfaced, additionally, it has been suggested that it may be used as a pheromone during spawning giving larvae an advantage for survival.

In this study, Itoi et al., (2014) conducted predation experiments, measurement, and immunohistochemical analysis to reveal the effect of TTX as a chemical defence in pufferfish larvae. Predation behaviour was observed using larvae of up to four days old post-hatch; juveniles of Japanese flounder Paralichthys olivaceus and sea bass Lateolabrax sp. were used as the predatory fish against T. rubripes larvae. For T. niphobles larvae, juveniles of fish such as Yatabe blennies (Parablennius yatabei and Omobranchus elegans), gobies (Chaenogobius annularis and Tridentiger trigonocephalus) and Smallscale blackfish (Girella punctata) were used as the predators. Juveniles of G. punctata were also used as predators against T. niphobles eggs. Adult brine shrimp (Artemia sp.) and medaka larvae were used as negative control for the prey. For TTX quantification, the method sounds quite complex and I can insert it is the comments field if anybody wants to review in more detail. However, the overall gist of it was quantifying the positive charged TTX via Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) from 60–100 specimens of pufferfish larvae. Immunohistochemistry followed, by labelling sections of pufferfish larvae with fluorescence and observing under an all-in-one fluorescence microscope (again, if anyone would like the complete method in detail, I can insert it in the comments). Difference in responses of predators (expelling vs swallowing) to TTX- pufferfish and to nontoxic organisms (medaka and Artemia sp.) was tested by the Pearson’s Chi-square test with Yates’ continuity correction.

In the predation experiments for T. rubripes larvae both of the predators ingested the pufferfish larvae but spat them out immediately; similar behaviour was observed in the predators for T. niphobles larvae. Negative control prey (Artemia sp. and medaka larvae) revealed significant differences between the responses of predators to that of the TTX-pufferfish.  LC-MSMS analysis revealed very small amounts of TTX in the egg and larvae of T. niphobles, and T. rubripes, suggesting that the amount of TTX in the pufferfish larvae does not constitute a lethal dose to the juvenile predator fish. However, the results suggest that the predators can sense even trace amounts of TTX in the larval pufferfish. Itoi et al., (2014) summarised that the female parent transfers TTX vertically to the eggs and larvae from the ovaries suggesting beneficial strategies for increasing the survival of egg and larvae in pufferfish. Itoi et al., (2014) went on to conclude that in a natural environment, it is easy to imagine rapid speciation of Takifugu could be a consequence of TTX, however, despite the toxicity of the pufferfish; overfishing is still an issue and can cause major decline in stocks.

Itoi, S., Yoshikawa, S., Asahina, K., Suzuki, M., Ishizuka, K., Takimoto, N., Mitsuoka, R., Yokoyama, N., Detake, A., Takayanagi, C., Eguchi, M., Tatsuno, R., Kawane, M., Kokubo, S., Takanashi, S., Miura, A., Suitoh, K., Takatani, T., Arakawa, O., Sakakura, Y., Sugita, H., 2014. Larval puffer fish protected by maternal tetrodotoxin. Toxicon. 78, 35-40.