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CRISPR Cuts Through Layers of Butterfly Wing-Pattern Evolution

Heliconius sp. butterflies

The bright wing patterns in butterflies in the genus Heliconius make for a highly visible palette to use CRISPR gene-editing techniques to investigate the evolution of wing patterns and the genes that influence them, say researchers at the Smithsonian Institute of Tropical Research. (Photo credit: Sebastian Menas/Instagram @mena_sebas)

To help shed light on some of these largest questions remaining in evolutionary biology, the authors of a paper published recently in Current Biology paper narrowed in on one evolutionary story—Müllerian mimicry in Heliconius butterflies.

Laura Kraft

Laura Kraft

Heliconius butterflies synthesize compounds from the family of the cyanides and also feed on plants that have these compounds. So, they are very distasteful for predators, especially birds,” says Carolina Concha, Ph.D., Biodiversity Genomics Fellow at the Smithsonian Institute of Tropical Research. In order to advertise their toxicity, these butterflies have evolved bright red, orange, yellow and blue warning colors with strong black banding. Unfortunately for the butterflies, birds typically have to bite and kill a few butterflies before learning that bright colors mean bad tastes. So over time, distantly related yet equally distasteful species of these butterflies have converged to have the exact same wing color and pattern so that both convergent species can benefit from an increased warning signal. This causes birds to learn faster by feeding on fewer individuals from either species.

These bright color patterns in Heliconius butterflies have evolved and diverged into a number of species found in Central and South America over the last 12 to 14 million years, but it was only within very recent evolutionary history—the last 2.5-4.5 million years—that some distantly related distasteful butterflies converged to have the same patterns. Says Concha, “The wing patterns have diversified so much in such a short time, so you have these really diverse forms within a single species and at the same time, you have very distantly related species that converge in a single phenotype.”

Carolina Concha with butterfly display boxes

Carolina Concha, Ph.D., shows off the stack of display boxes of CRISPR-mutation butterflies that she and her collaborators have created in the Smithsonian Institute of Tropical Research in Gamboa, Panama. (Photo credit: Laura Kraft)

“CRISPR allows us to make the same butterflies, with one gene missing. It’s a way to interrogate nature and just ask ‘What is the function of this gene?’ and ‘Did it change during evolution?'” says Arnaud Martin, author on the Current Biology paper and Assistant Professor at George Washington University. One of the earliest genes to be expressed during butterfly wing development is called WntA (pronounced WIN- tah), and it is thought to be a major gene controlling the paint by number system used by butterflies to color and pattern their wings. WntA was once expressed during embryonic development of many different types of organisms, including vertebrates, but has been lost in most. Perhaps after its role in embryonic development had become downplayed in butterflies, WntA developed a new role in wing patterning, defining the boundaries of black and color banding patterns on adult Heliconius butterfly wings.

The researchers proposed to use CRISPR as scissors to precisely cut out the section of DNA that codes for the WntA gene in a few Müllerian co-mimic pairs. They had two complimentary hypotheses: If mimetic wing patterns developed in different butterflies from using a highly similar gene regulatory mechanism involving WntA, then knocking out the gene should result in the same wing pattern changes in mutant butterflies of the different species. If, on the other hand, identical wing patterns were caused by highly different pathways involving WntA, then the wings of two species of mutant butterflies may exhibit different patterns.

CRISPR injection into Heliconius butterfly eggs

Carolina Concha, Ph.D., carefully injects CRISPR into Heliconius butterfly eggs. If she can inject the eggs within two hours after they are laid, she gets less mosaicism in the CRISPR mutation than with three- or four-hour-old eggs. (Photo credit: Luca Livraghi)

After injecting thousands of eggs, Concha finally got full CRISPR knockout of three co-mimetic pairs of Heliconius butterflies. “I honestly thought that co-mimetic Heliconius species were generated by similar tweaks of the same developmental pathways. I was wrong,” says Owen McMillan, Ph.D., Dean of Academic Programs at STRI. In all three co-mimetic pairs, the resulting mutant butterflies had dramatically different wing color patterning after removing the WntA gene, supporting the second hypothesis that even highly different pathways can lead to the same wing pattern.

“CRISPR allowed us to push our basic understanding of the pathways underlying wing pattern formation in entirely new directions. We have made great progress in identifying the key genes underlying pattern formation, but CRISPR allows us, for the first time, to understand how they work. It is a remarkably cool tool for discovery,” says McMillan.

While major changes have been made to the regulatory pathway of WntA, resulting in these wing patterning differences, they do all focus on the same gene. But the really surprising part of this paper is that despite these butterflies developing under different conditions and experiencing different evolutionary histories over millions of years, they still converged upon the same phenotype with a completely different network of gene regulation. “This really highlights that the genome has a few favorite genetic tools that drive the evolution of specific parts of the anatomy, but the way these tools can be used is flexible,” says Martin

If you’d like to learn more, here is a video showing the method of using CRISPR to change butterfly wing patterning:

Laura Kraft is a Ph.D. student at North Carolina State University and a National Science Foundation Graduate Research Fellow. When she isn’t traveling the world, she spends her time making science more accessible through science writing and outreach. Email: ljkraft@ncsu.edu.

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