A monarch caterpillar. Caterpillars eat and store the toxic compound in milkweed to deter predators. The toxins are retained through metamorphosis, making the butterfly toxic as well. [UC Berkeley photo by Noah Whiteman]

An international team of researchers headed by scientists at the University of California (UC), Berkeley, has used CRISPR-Cas9 genome editing to turn fruit flies that might otherwise represent a handy snack for frogs and birds, into potentially poisonous prey that could cause anything that eats them—including humans—to vomit.

The team, led by Noah K. Whiteman, PhD, principal investigator and an associate professor at UC Berkeley, introduced into the flies the same three mutations in a single gene that are carried by the monarch butterfly (Danaus plexippus), which can eat and sequester the poisonous plant milkweed as caterpillars, and then retain some of the toxin as adult butterflies, to deter predators. Milkweed is a highly toxic plant that would kill most animals, including humans. But like the monarch butterfly, the CRISPR-engineered fruit fly larvae were similarly able to eat milkweed and retain the plant’s toxins when they metamorphosed into now-poisonous adult “Monarch flies”. Critically, the mutations had to occur in the right sequence, otherwise the flies would not have survived the three separate mutational events.

The work, reported in Nature, represents the first time that anyone has recreated in a multicellular organism a set of evolutionary mutations leading to a totally new adaptation to the environment, in this case, a new diet and a new way of deterring predators. Whiteman and colleagues in the United States, France, and Germany, describe their studies in a paper titled, “Genome editing retraces the evolution of toxin resistance in the monarch butterfly.”

Milkweed and a variety of other plants, including foxglove, which is the source of digitoxin and digoxin that are used to treat heart failure, contain related, deadly cardiac glycoside toxins. Although these plants have a bitter taste that is in itself a deterrent, a small number of insect species, including the monarch butterfly and its relative the queen butterfly (Danaus gilippus), thrive on milkweed and use it to repel predators. The monarch is descended from a tropical insect that made its way to North America after the last ice age, and carries three mutations that allow it to eat milkweed, which gives it both a survival advantage and a natural defense against predators. “The monarch resists the toxin the best of all the insects, and it has the biggest population size of any of them; it’s all over the world,” Whiteman explained.

Monarch butterfly (Danaus plexippus) adult. [Photograph by Ellen Woods. Copyright Anurag Agrawal, Cornell University]
Whiteman is interested in the evolutionary battle between plants and parasites and particularly in the evolutionary adaptations that have allowed the monarch to survive the milkweed’s toxins. This interest includes whether other insects that also have—albeit less robust than the monarch’s—resistance to milkweed use similar mechanisms to disable the plant’s toxin. “Since plants and animals first invaded land 400 million years ago, this coevolutionary arms race is thought to have given rise to a lot of the plant and animal diversity that we see, because most animals are insects, and most insects are herbivorous: they eat plants,” he said.

Most of the poisons in these toxic plants are a type of cardenolide, which interfere with the sodium/potassium pump (Na+/K+-ATPase) that most of our body cells use to move sodium ions out and potassium ions in. The pump creates an ion imbalance that is critical for the transmission of electrical signals along nerve cells. Digitoxin in foxglove, and the ouabain toxin in milkweed, block the sodium pump and so the ion channel, preventing the cell from establishing this sodium/potassium gradient. This disrupts ion balance, which results in heart cells starting to beat so strongly that the heart fails, and cardiac arrest causes death.

Scientists have identified two specific amino acid mutations in the protein pump that monarchs and other insects have evolved to prevent the toxin from binding. But Whiteman and his colleagues weren’t satisfied that different insects coincidentally developed the same two identical mutations in the sodium pump—14 separate times. To see how the mutations may have evolved they harnessed CRISPR-Cas9 genome editing to engineer these same mutations in fruit flies, to to see if they would also make the flies immune to the toxic effects of cardenolides. The team made three separate CRISPR edits in a single gene in the fruit fly that were identical to those that render the monarch butterflies immune to milkweed and able to sequester its toxin. They found, surprisingly, that these three single-nucleotide substitutions in one gene were sufficient to give fruit flies the same toxin resistance as monarchs. Studies showed that the engineered flies were 1,000 times less sensitive to milkweed toxin than the wildtype fruit flies.

The experiments involved inserting single, double, and triple mutations into the fruit fly’s own sodium pump gene, in various orders. In this way, they could assess which mutations were essential. The results showed that insects having only one of the two known amino acid changes were better at resisting the plant poisons, but they also demonstrated serious nervous system side effects. Interestingly, humans’ sodium pump mutations are often associated with seizures. The fruit fly studies also showed that the third, compensatory mutation somehow reduced the negative effects of the other two mutations. Without this compensatory mutation the fly larvae died.

Interestingly, resistance to milkweed toxin did come at a cost. The “Monarch flies” are not as quick to recover from being shaken—a test known as “bang” sensitivity as wildtype flies. “This shows there is a cost to mutations, in terms of recovery of the nervous system and probably other things we don’t know about,” Whiteman said. “But the benefit of being able to escape a predator is so high … if it’s death or toxins, toxins will win, even if there is a cost.”

Whiteman’s team also showed that 20 other insect groups, including moths, beetles, wasps, flies, aphids, and a weevil, which are able to eat milkweed and related toxic plants have independently evolved mutations in one, two, or three of the same amino acid positions, enabling these animals to overcome to varying degrees, the toxic effects of the plant toxins. Most of these insects have orange coloration as a warning to predators. When the team reconstructed the one, two, or three mutations that led to each of four butterfly and moth lineages, they found that while each mutation conferred some resistance to the toxin, all three mutations were necessary to make the monarch butterfly the most resistant. “One substitution that evolved confers weak resistance, but it is always present and allows for substitutions that are going to confer the most resistance,” commented study first author Marianna Karageorgi, PhD, a geneticist and evolutionary biologist. “This substitution in the insect unlocks the resistance substitutions, reducing the neurological costs of resistance. Because this trait has evolved so many times, we have also shown that this is not random.”

The one compensatory mutation that is required for insects with the most resistant mutations to survive represents an effective constraint on how insects could evolve toxin resistance, and explains why all 21 lineages converged on the same solution, Whiteman said. In other situations, such as where the protein involved is not so critical to survival, animals might find different solutions.

“Our study illuminates how the monarch butterfly evolved resistance to a class of plant toxins, eventually becoming unpalatable, and changing the nature of species interactions within ecological communities,” the authors stated. “Although mutational paths to adaptive peaks have been identified in microorganisms, this is, to our knowledge, the first in vivo validation of a multi-step adaptive walk in a multicellular organism, and illustrates how complex organismal traits can evolve by following simple rules.”

“This helps answer the question, ‘Why does convergence evolve sometimes, but not other times?'” Whiteman said. “Maybe the constraints vary. That’s a simple answer, but if you think about it, these three mutations turned a Drosophila protein into a monarch one, with respect to cardenolide resistance. That’s kind of remarkable.”

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