Not many foods served in a restaurant can kill you, but pufferfish is the exception. Tetrodotoxin, the toxin responsible for such culinary fatalities, reveals a fascinating story of convergent evolution...

Not many foods served in a restaurant can kill you, but pufferfish is the exception. Best known as the Japanese delicacy fugu, great care is employed so that the toxic parts of the fish are excised. Even so, fatalities are not unknown, and historical records, such as those of the great explorer James Cook, reveal narrow escapes and on occasion sudden death. And the toxin tetrodotoxin (TTX) reveals a fascinating story of convergent evolution in at least two ways (as does the similar saxitoxin).

Tetrodotoxin is one of the most powerful neurotoxins known – it is about 1200 times more toxic to humans than cyanide and it has no known antidote. The molecular structure of this alkaloid is relatively simple, forming a cyclic series of carbon atoms, but the active site is derived from guanine (one of those all-purpose molecules that, in addition to being central as a base of DNA, is also employed to gas-proof swim bladders and act as a reflector). With its guanidinium group, TTX binds to voltage-gated sodium channels on nerve membranes, thus blocking nerve conduction and causing motor paralysis and ultimately death from suffocation.

Distribution of tetrodotoxin

For a long time, TTX was believed to occur only in pufferfish (Tetraodontidae), after which it was named upon its discovery in Spheroides rubripes in 1910. Meanwhile, however, the toxin has been found in a wide variety of animals.

In fish

According to a recent review by Noguchi and Arakawa (2008, Marine Drugs, vol. 6, pp. 220-242), a total of 22 pufferfish species in the family Tetraodontidae are currently listed as TTX-bearing, while the closely related porcupinefish (Diodontidae) and boxfish (Ostraciidae) contain no TTX. The distribution of TTX in the bodies of pufferfish varies among species. While the liver (and during the spawning season also the ovary) has the highest toxicity in marine species, the skin seems to be the most toxic part in those inhabiting brackish water and freshwater. Tetrodotoxin is employed in defence, in combination with the rather bizarre inflation behaviour that characterises pufferfish and their relatives. When threatened, puffers inflate their body by swallowing water, while excreting TTX from their skin. Also the eggs can be highly toxic, probably to protect them from predators. Intriguingly, in some marine species such as Fugu niphobles, the females seem to employ TTX as a pheromone to attract males during spawning and thus increase the chances of their eggs being fertilised.

Apart from the well-documented occurrence of TTX in pufferfish, a species of goby (Yongeichthys criniger) has been shown to possess the toxin as well.

In marine invertebrates

In the marine realm, not only fish, but also a large number of different invertebrates contain TTX. Among the best-known examples are the justly feared blue-ringed octopuses (genus Hapalochlaena), which possess TTX in the salivary gland and proboscis, which indicates that it assists in prey capture. Ribbon worms (or nemerteans) such as Lineus fuscoviridis seem to use it as predatory venom as well. TTX has furthermore been isolated from other marine worms of different phyla, namely flatworms (e.g. Planocera spp.), arrow worms (or chaetognaths, e.g. Parasagitta spp.) and annelids (e.g. Pseudopolamilla occelata), various genera of marine snails (e.g. Charonia and Niotha), crustaceans (the horseshoe crab Carcinoscorpius rotundicauda as well as xanthid crabs from different genera) and starfish (genus Astropecten).

In terrestrial vertebrates

In 1964, TTX was first discovered in an amphibian, namely in the eggs of the California newt Taricha torosa. The presence of a toxin in these newts had already been reported in the 1930s, when eye tissue transplanted to the embryos of other species promptly led to their paralysis, but only later was it actually identified as TTX. That TTX occurs outside the oceans is particularly remarkable as bioactive natural products are normally found in either a marine or a non-marine environment. Meanwhile the toxin has been detected in a wide range of amphibians from different families (but the Taricha newts are by far the most toxic and understandably avoided by practically all animals). Almost all members of the family Salamandridae studied so far contain TTX, with the European fire salamander (Salamandra salamandra), which employs the steroidal alkaloid samandarine, being the exception. One study also reported TTX activity from a species belonging to another caudate family, the tiger salamander Ambystoma tigrinum (Ambystomatidae), but this finding could not be confirmed in another analysis.

Frogs generally appear to be less toxic than tailed amphibians. While TTX has been detected in almost all bufonid species in the genus Atelopus examined, some members of three other families contain the toxin as well. The Neotropical dendrobatid or poison dart frogs are justly celebrated for their venomousness (and, of course, their striking convergences with the Madagascan mantellid frogs) and although they usually employ various other alkaloids, at least one species (Colostethus inguinalis) has low levels of TTX in its skin. What presumably is an independent acquisition occurs in a rhacophoridid tree frog from Asia (genus Polypedates), where the highest TTX levels documented actually approach those of Taricha (although there is great individual variation in toxicity). Finally, the tiny, golden pumpkin toadlet (Brachycephalus ephippium) in the family Brachycephalidae has relatively high levels of TTX in its skin and liver.

Origin of tetrodotoxin

In the pufferfish, TTX is produced by endosymbiotic bacteria that often seem to be passed down the food chain. Evidence for this comes from studies on puffers that were raised in a hatchery without any access to TTX-containing food. These individuals were non-toxic but became so when fed either TTX-containing food or TTX-producing bacteria. Furthermore, the large variation in pufferfish toxicity between individuals and regions also supports the idea of an exogenous intoxication rather than a production of the toxin by the fish themselves. A variety of bacteria can produce TTX – mainly those in the genera Vibrio and Pseudomonas, but also an actinomycete (Nocardiopsis dassonvillei), which suggests that TTX synthesis itself may well be convergent.

It seems likely that TTX is always bacterial in origin, also outside the pufferfish, implying that the symbiosis between TTX-producing bacteria and animals has evolved independently numerous times. Harlequin frogs (Atelopus varius) that were raised from eggs in captivity had no detectable levels of TTX in their skin as adults. The Japanese fire belly newt Cynops pyrrhogaster has a certain amount of TTX in the egg (inherited from the parents) that fades away during the larval stage. In a natural environment, juveniles quickly accumulate TTX, but in a laboratory setting with no TTX-containing food available, they were non-toxic as adults.

Resistance to tetrodotoxin

An obvious question is that given TTX is so lethal, and for the most part its possession is evidently defensive, then how do animals like the pufferfish and Taricha newts that accumulate it in large concentrations in their bodies not drop dead themselves? The answer in a sense is obvious, but it is also convergent. The resistance to TTX is centred on the voltage-gated sodium channels, which are normally effectively disabled by the toxin. These channels consist of a large protein with four repeating domains, each of which contains a highly conserved region that contributes to pore formation. Key substitutions of amino acids in the pore region substantially reduce affinity to the toxin and allow the channels to function normally. Intriguingly, in the various species of pufferfish we see striking convergences at this molecular level in one or more of the channel protein domains. However, the changes involve only very few sites, suggesting that selection for normal channel function strongly limits the possibilities for resistance-conveying mutations. Tetrodotoxin resistance thus represents “an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome” (Jost et al. 2008, Molecular Biology and Evolution, vol. 25, p. 1016).

Resistance to TTX does not only mean that pufferfish can employ it as an effective chemical defence, but is also has another advantage – they can selectively feed on TTX-bearing organisms that TTX-sensitive fish have to avoid. Interestingly, garter snakes (Thamnophis sirtalis) that prey on the otherwise invulnerable newt Taricha have convergently acquired TTX resistance. Snakes from populations that do not encounter the newts in the wild, however, are poisoned when fed newts in the laboratory, indicating a co-evolutionary interaction between the predator and its prey.