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At long last, we know how the firefly glows

John Hart/Wisconsin State Journal via AP

The quest to understand how fireflies produce light began in very public fashion. In the late 1950s a biologist at Johns Hopkins University named William McElroy paid Baltimore kids to trap tens of thousands of fireflies and bring them to his lab. There, McElroy conducted experiments on substances extracted from the “lantern” at the rear of the firefly’s body. In less than a decade, he and his colleagues had established the basic chemistry of the insect’s luminescence.

Yet even after that, mysteries remained about a reaction that’s familiar to anyone who’s ever sat outside on a summer evening. But now, thanks to unconventional thinking by firefly expert Bruce Branchini at Connecticut College and a last-minute collaboration with chemists at Yale University, the final details of the chemical equation have been nailed down. The results were published in the Journal of the American Chemical Society in June.

All bioluminescent reactions — in fireflies, jellyfish, glow worms, beetles — require three basic ingredients: a substrate on which the reaction occurs (luciferin in the case of fireflies), a protein (usually an enzyme) that orchestrates the reaction, and oxygen that ignites the process. It’s this last step — just what happens when oxygen flows into the firefly’s lantern — that’s remained an open question. For the last 40 years biochemists have had a theory about what goes on, but for a while Branchini had doubted its accuracy.

“There had been a proposal since the 1970s about how [that step] occurred. It was generally accepted. We questioned it, reexamined it, looked at alternative mechanisms,” he says.

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The accepted assumption was that oxygen from the air mixes directly with the luciferin in the lantern to produce a highly energetic substance called oxyluciferin that emits light. Branchini was skeptical of this model because he realized it seemed to imply a process that went against a basic rule of chemistry. Based on more than two decades of experience studying fireflies, he imagined there had to be some sort of intermediate ingredient involved — a negatively charged form of oxygen known as superoxide.

So, in 2011, he began experiments to try and prove that superoxide was lurking in the midst of the whole process. Hard evidence was difficult to come by. The bioluminescent reaction happens extremely fast, in 300 milliseconds from start to finish, which was faster than Branchini and his team could test for the existence of superoxide. “We couldn’t prove it,” Branchini says. “We couldn’t support our contention to the satisfaction of the scientific community and we were stuck.”

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This is where Yale comes in. Branchini knew a chemist there named Gary Brudvig who uses a technique called EPR (for “electron paramagnetic resonance”) in his research on solar energy. Branchini saw that EPR would be a perfect way to essentially trap the products of the firefly’s chemical reaction long enough to see what was really happening.

He and Brudvig ran the firefly reaction using EPR, such that the superoxide ion — if it was really there — would react with another substance and settle into a form that was easier to measure. “You take something transient and make it more stable,” says Branchini.

They had only about a minute to measure the reaction, but using EPR, the hypothesized superoxide quickly came into view. “It worked like a charm the first time,” Brudvig says. “[Branchini] came back a few more times to follow up, reproduce the results, and everything worked very well.”

For 40 years biochemists had held a notion about how fireflies produce light, and for more than four years Branchini had tried without success to prove that it was wrong. Then thanks to an astute hypothesis and the right method for testing it, the exact chemical steps behind that familiar pulsing glow ceased to be a mystery.

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Kevin Hartnett is a writer in South Carolina. He can be reached at kshartnett18@gmail.com.

Watch: Implications of the research

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