This week’s BEACON Researchers at Work blog post is by MSU graduate student Patric Vaelli.

Few animals can strut around the woodlands of the Pacific Northwest with as much poise and confidence as the rough-skinned newt (Taricha granulosa). While opportunistic predators lurk in every bush and stream, most avoid encountering this intrepid salamander as it ambles from one pond to the next. Fearless and unwavering, this small amphibian possesses one of the most deadly poisons found in nature: tetrodotoxin or TTX. While the newt does not use the toxin as venom, that is, it does not incorporate the poison into its bite, it does exude TTX when faced with predators as a defense mechanism.

In antiquity, TTX was most widely known as the poison of toxic puffer fishes within the family Tetrodontidae, from which the toxin derives its name. However, in recent decades TTX has been detected in an enormous diversity of animals including flatworms, nematodes, crabs, starfishes, octopuses, toads, and newts (see photos below). The wide distribution of TTX among evolutionarily unrelated animal lineages has led to the hypothesis that animals accumulate TTX from their environment, as the repeated evolution of the biosynthetic pathway that produces such a structurally complex molecule (see figure below at left) in all of these organisms is highly unlikely.

Three representative tetrodotoxic animals. Clockwise from top: Rough-skinned newt, puffer fish, and blue-ringed octopus.

Molecular structure of tetrodotoxin (TTX). (source)

Indeed, in nearly all tetrodotoxic animals, the accumulation of TTX has been associated with toxin-synthesizing bacteria present within their organs or upon their skin. These relationships are generally referred to as mutualisms, as the bacteria are provided with nutrients and a nice place to live, and the animal gains protection from the toxin secreted by the bacteria. In other cases, animals can accumulate TTX through their diet, but even then the fundamental source of the toxin is still attributed to toxic bacteria.

However, the ultimate origin of TTX in our intrepid salamander, the rough-skinned newt, remains to be elucidated, and this is the subject of my graduate research at Michigan State University.

Witch’s Brew: Don’t forget the Eye of Newt

Even though Shakespeare was likely referring to a seed or fruit when the witches added eye of newt to their mysterious potion in The Tragedy of Macbeth, the real thing would have been just as suitable. Present in every organ of the newt including the eyes, TTX is a potent neurotoxin that inhibits the electrical signaling underlying neurotransmission in the nerves and muscles of nearly all animals. Specifically, TTX binds to the voltage-gated sodium channels in excitable cells, preventing the influx of positive Na⁺ ions necessary to generate an action potential. The consequences of ingesting TTX are severe: numbness, nausea, paralysis, convulsions, and death—and there is no antidote. Fortunately, death can be avoided if the victim is placed on a respirator to prevent suffocation, as is the strategy implemented when a restaurant patron ingests improperly prepared puffer fish sushi, or Fugu.

So why spend my time investigating the production of TTX in newts? Well, they don’t float like a butterfly or sting like a bee, but rough-skinned newts are the heavy weight champions of tetrodotoxicity in nature, and they are easily the most poisonous vertebrate on the North American continent. They possess high enough concentrations of the toxin to make a puffer fish nauseous, and easily enough to kill several adult humans. One half milligram of toxin can kill a 165 lb. human, and the highest total TTX measured in a newt was fourteen milligrams. Okay, wait, what’s the deal with that? A newt is never going to encounter a predator as large as one human, so why would they possess enough toxin to kill 28 people?

Newts do have one natural enemy slithering in the woodlands, a predator that has evolved the ability to withstand the neurotoxic effects of TTX. In populations that overlap with newts, garter snakes in the genus Thamnophis have overcome the toxic defense of newts. In most cases, the levels of TTX in newts are matched by the levels of resistance in their snake predators, as determined by mutations in their sodium channels. Consequently, newts and snakes are locked in a coevolutionary arms race, where increasing toxicity in the newt prey is matched by increasing resistance in the predator, leading to an asymmetric escalation of extreme traits in these lineages.

Poison or Perfume?

Electro-olfactogram recording of newt olfactory neurons in the nose. Electrical response to positive control amino acid solution (top) and TTX (bottom).

The Eisthen lab at MSU originally became interested in TTX and rough-skinned newts because of a claim that male puffer fishes could smell TTX released from females, and that the toxin acted as a pheromone in these animals. Furthermore, later reports found that larval newts seek shelter when low concentrations of TTX are present, suggesting that larvae use TTX as an olfactory cue for the presence of adult newts that may potentially cannibalize them. Our lab has since conducted behavioral and electrophysiological experiments to decipher the roles that TTX might play in chemical communication. The figure at right shows an electro-olfactogram recording, made by former graduate student Justin Schroeder, from a newt nose presented with amino acids (positive control) and TTX. The recordings show that an electrical signal is generated in olfactory neurons when presented with these two stimuli, demonstrating that newts are capable of smell

ing the otherwise deadly neurotoxin.

We Are Not Alone

Cultured bacterial isolates from newt skin.

For nearly 600 million years, animals have evolved in a world dominated by microorganisms, and through this time we have remained in close association with microbial life. In humans, for example, nearly 100 trillion microbes inhabit our skin and gastrointestinal tract, where they help digest our food and produce vitamins that we cannot synthesize ourselves. In tetrodotoxic animals, mutualistic relationships with toxic bacteria have resulted in enormous benefits to the hosts, who become unpalatable to virtually all predators. Newts present an interesting situation, because the source of TTX remains unknown despite the fact that newts possess the highest concentrations of the toxin, and TTX is central to their coevolutionary arms race with garter snakes. There is tenuous evidence that newts may be synthesizing the toxin themselves, and have thus evolved biosynthesis of the neurotoxin in parallel with the various genera of bacteria that produce it. However, these data do not reject a microbial origin. Through BEACON, our lab has established collaborations with Dr. Kevin Theis at MSU, and the Foster and Harmon labs at the University of Idaho to investigate if the newt microbiome contains TTX-producing bacteria (see photo above). The elucidation of this elusive microbe(s) may provide early evidence that the microbiota inhabiting an animal can respond to natural selection upon the host (in this case predation), suggesting that in the evolution of animals, including ourselves, we are not alone.

For more information about Patric’s work, you can contact him at pvaelli at gmail dot com.