(Owen McMillan/STRI)

Engineered mutant butterflies give a glimpse deep into the genetic roots of wing patterns, an international team reported Monday in the Proceedings of the National Academy of Sciences. The authors of the new study rearranged colors on butterfly wings by deleting a single gene using a genome editing tool called CRISPR. The gene's absence had a dramatic effect in seven butterfly species, including some that aren't closely related.

“It’s extraordinary that it works so broadly,” said Owen McMillan, a staff scientist at the Smithsonian Tropical Research Institute and an author of the study. “It's getting rewired across nymphalids [a family of butterflies] in really fundamentally different ways.”

Flight is a neat trick. But flying is not a butterfly wing's only purpose. The wings are covered in scales, arrayed into patterns like ceramic tiles in a mosaic.

These scaled mosaics play many roles. Several butterflies use the patterns as warnings, like the orange and black of a monarch butterfly that tell predators they are poisonous. For some, such as the dead leaf butterfly, which looks like you might expect, wings are camouflage. Eyespots on wings fool predators into attacking from the rear rather than the head. Other bright butterflies communicate with sex symbols in scaly flashes.

But Arnaud Martin, an evolutionary geneticist at George Washington University who studies wing patterns, says he doesn't care much about what butterfly wings do. He's hunting after the pattern itself.

A normal painted lady butterfly wing (left) next to a mutant wing. (Arnaud Martin/George Washington University)

“We use butterfly wing patterns as a proxy to understand fundamental rules about the function of genes,” said Martin, an author of the new study.

Butterfly patterns appeal to him because, though they're ornate, they exist on a simple canvas: Unlike the architecture of our hearts or brains, butterfly markings are confined to essentially two dimensions. But if there are basic rules for butterfly patterns, it stands to reason they might hold for skeletons, too.

Butterflies and moths were also appealing in their sheer diversity. Pluck a random animal from a museum and, if the hypothetical institution had collected one of every known species alive, there's a 1-in-10 chance that you're holding a butterfly or moth. Butterflies and moths account for nearly 200,000 species. Each species has its own wing pattern.

Martin and his colleagues had previously identified a gene called WntA (pronounced “wint-ay”) in the genome of Heliconius butterflies. They knew it was important for wing patterns.

At the time of WntA's discovery the biologists had no way to verify the function of the gene. “Our job as geneticists is to do reverse engineering — to understand that black box that is butterfly wing pattern,” Martin said.

CRISPR, a bacterial system that has been likened to cellular cut-and-paste, allowed them to crack open the black box. For asking questions, CRISPR is “extraordinary,” McMillan said. “If it's used well, it is an incredibly powerful tool.” (CRISPR-tweaked insects are far less ethically fraught than CRISPR-tweaked humans. Biologists also recently used the tool to create the world's first mutant ants.)

Researchers had used CRISPR previously to change genes of monarch butterflies. But this study was the first to use CRISPR to scrub out a gene in a broad stroke of butterflies. The study authors deleted WntA in common buckeye butterflies, speckled wood butterflies, painted lady butterflies, monarch butterflies, two species of Heliconius butterflies and Gulf fritillary butterflies.

Butterflies showing mutated wings on their right sides. (Nathalie Vessillier)

There's a popular misconception that, during metamorphosis, the entire caterpillar liquefies in its cocoon. And then this larval smoothie congeals into a butterfly. But that's not how it works, Martin said. Some of the animal's body parts begin growing as caterpillars and stay intact through the metamorphosis process.

In fact, the study authors found, WntA turns on while the animals are caterpillars. WntA, a signaling gene, begins coordinating specific parts of the wing pattern even before the limbs are visible.

“In the larva you have miniature baby wings that are growing trapped inside the body,” Martin said. “We were blown away to see that the WntA gene was already providing the spatial information necessary to make patterns.”

When the butterflies reached adulthood, WntA's flexibility came into focus. It had switched from penciler to inker to painter and back again, its purpose written and rewritten over millions of years of evolution, persisting across butterfly species.

“That flexibility is really surprising, especially given that it is such a conserved gene,” said Carolina Concha, biogenomics researcher at the Smithsonian Tropical Research Institute and an author of the study.  “This study highlights how flexible this regulation is, to be involved in so many aspects of patterning. It's extraordinary!”

In Heliconius butterflies, what was a red stripe in wild butterflies became a large red spill in mutant ones, like watercolor bleeding outside the lines. In others eyespots disappeared. Color splotches moved. Mutant monarch butterflies grew white scales along the edges of their wings.

This means that, where the gene controls vertical stripes in other wild butterflies, WntA traces the veins of monarch wings in black, Martin said. “It's a subtle role,” he said, “but it has departed from its ancestral one.”

McMillan said the gene must have been rapidly rewired to be used in so many different ways. Perhaps other genes have been redeployed so quickly in other species, too. “That’s pretty cool, to a biologist like me,” he said, “when you think about the variation being generated in nature.”

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