Evolutionary Biology Unlocks Secret Toxins Hidden Inside Poison Dart Frogs

There is a group of colorful amphibians that are known for storing defensive alkaloids in glands on their skin. These species, known as poison dart frogs, possess mechanisms that capture toxins obtained from the arthropods that make up their diet, transport them, and store them. The frogs use these toxins to defend themselves against predators and pathogens. Some species even transform certain substances into more toxic ones before storing them.

Scientists have long debated how this ability evolved, considering the vast majority of amphibians do not possess it and some sequester and store only small amounts of alkaloids. This type of toxin can be produced by plants and arthropods and is mainly found in ants and mites that serve as food for the frogs.

A study supported by FAPESP and published in the journal Proceedings of the Royal Society B suggests that evolution may have occurred gradually in this case.

The big question in our work was how this ability evolved. To sequester the alkaloids, a resistance mechanism is needed to ingest them without becoming poisoned. In addition, transport mechanisms are required to carry the toxins from the digestive system to the skin. And also some mechanism to prevent the degradation of the alkaloids and keep them intact in the skin. Therefore, there must have been several steps in the evolution from a phenotype that doesn't sequester alkaloids to one that sequesters and modifies them."

Adriana Jeckel, University of São Paulo (IB-USP)

The study is part of the project "A Multidisciplinary Approach to the Study of Amphibian Diversification", and Phase 2 of it, both of which are supported by FAPESP and coordinated by Taran Grant, a professor at IB-USP and also an author of the study.

In addition to the USP researchers, the study includes authors from the Butantan Institute and universities in the United States and Japan. The results confirm the existence of at least four phenotypes of frogs with regard to their relationship with alkaloids.

While poison dart frogs – that is, anuran amphibians of the Dendrobatidae family – merely sequester alkaloids or sequester and modify them, species from other closely related groups can sequester and store only small amounts. Other, more distantly related species sequester nothing at all, even though they can metabolize the toxins and are not poisoned.

Daily Doses of Toxin

In the experiments, the researchers administered synthetic versions of alkaloids to various species of frogs. All but one of the frogs had been raised in captivity since the larval stage. The one collected in the wild was compared to other individuals from the same location to ensure that its skin did not contain alkaloids.

When the frogs reached adulthood, the researchers administered a 50% ethanol solution containing the alkaloids directly into their mouths for 14 days. The control group received only the alcohol solution.

After this period, the animals went seven days without receiving the toxins. Then, the alkaloids were isolated from their skin and analyzed using gas chromatography and mass spectrometry. This made it possible to determine the amount of compounds stored and which ones had changed.

Poison dart frogs of the species Adelphobates galactonotus and Dendrobates tinctorius accumulated 10 times more alkaloids in their skin than Allobates femoralis and 100 times more than Dryophytes cinereus. Interestingly, one alkaloid was not detected in any A. femoralis tissue. This species of poison dart frog was collected in the wild.

No significant amounts of alkaloids were found in the feces or liver of any species. "The absence of alkaloids in the tissues of Allobates and in the feces of all species shows that they're somehow metabolizing these toxins. Even when they don't accumulate them, they possess mechanisms that are very efficient, from an evolutionary standpoint, to prevent self-poisoning," says Jeckel.

Two phenotypes were identified among poison dart frogs. Species such as Phyllobates vittatus and Epipedobates anthonyi, which have been studied in other research, accumulate alkaloids in the same form in which they ingest them.

However, two other species from the same group that were analyzed in this study – Dendrobates auratus and Ranitomeya ventrimaculata – modified the alkaloids through biochemical processes such as hydroxylation and methylation and accumulated them in these forms.

"These modifications may be necessary for them to accumulate the alkaloids. Otherwise, the original ingested substances could be toxic or degrade within the body," says Grant.

Another difference among poison dart frogs is their ability to accumulate different proportions of alkaloids. In the experiments, Dendrobates auratus sequestered between 11.8% and 86.4% of ingested alkaloids, while Phyllobates vittatus sequestered between 7.9% and 24.5%.

"This is evidence that the ability to sequester alkaloids did not arise alongside anuran amphibians, nor did it take an evolutionary leap and appear suddenly. There are intermediate categories that don't necessarily evolve toward capture and modification but that were selected to deal with the presence of these toxins in their food – the insects," Jeckel concludes.