How did evolution produce a firefly?

On one level, we have fireflies figured out. We know the enzyme they use to make light (called luciferase), as well as the chemicals they use in the light-generating reaction. We know them so well that we've turned them into useful tools for studying other aspects of biology, such that lots of people who have never even seen a firefly have used firefly luciferase in the lab.

But on another level, there's a lot we don't understand. Fireflies clearly exercise a level of control over when they light up, and they do so only in specialized organs. And there's nothing like that organ in other species. So, somehow, fireflies evolved an elaborate light-producing organ, and there's no sign of any potential precursors in related species. Which makes it a bit of a mystery.

Now, a pair of researchers from Wuhan, China, (Xinhua Fu and Xinlei Zhu) have started unraveling what's going on at the level of the genes responsible. And, while they haven't produced a complete picture of how evolution built the fireflies, they've brought us a lot closer.

Following the light

We know a fair bit about how fireflies produce light. First off, it's not limited to the adults we're familiar with. The larval stages also light up, and it's thought that this is a way to signal their toxicity to potential predators. One idea is that this was how the light-emitting system initially evolved, and it was later adapted for mating.

In any case, the adult organs form in specific segments of the beetle's abdomen (they form in different segments in males and females). They include a transparent cuticle on the exterior of the insect and specialized light-emitting cells underneath it.

Within those cells, the luciferase enzyme is sent into a compartment called the peroxisome, where various chemicals are normally broken down through oxidation reactions. On the simplest level, this makes sense because the reaction luciferase catalyzes involves oxygen. But there are some indications that the peroxisomes of the light-producing cells are specialized for that and may no longer be able to perform all the other functions of peroxisomes.

One of the easiest ways to study novel features like this is to identify the genes involved in producing them. Here, genetics doesn't work especially well, as fireflies are very difficult to breed in the lab. Another option is to sequence the genome of fireflies and their relatives and look for newly evolved genes or existing genes that have undergone duplications so that there are extra copies. This has been tried in fireflies, and some potentially interesting genes have been identified. But none of these seem to have anything to do with making light.

The genetics of light

Fu and Zhu were working with a species of aquatic firefly, and its genome hadn't been sequenced yet, so they started with that. It turned out to be unusually large and filled with a lot of extraneous junk, such as mobile DNA elements and large introns within genes. Still, they were able to complete roughly 98 percent of the genome and identify most of the genes within it.

Because the light-producing organs develop in specific segments (one in females, a second in males), the researchers focused on genes known to help determine a segment's identity—the ones that tell a segment whether to develop a wing or a leg or some other structure. These are called homeobox genes, and they play roles in establishing the identity of body parts in everything from fruit flies to humans.

By checking the activity of homeobox proteins found in the firefly genome, Fu and Zhu came up with half a dozen that were active at the right time and place to potentially influence light organ formation. They then knocked down the activity of each of these genes using a technology called RNA interference. Based on the loss of activity, three of the genes seem to be involved in coordinating the production of flashes of light from the organ. In the case of two other genes, the loss of their activity disrupted the formation of the light-producing organ.

Since homeobox proteins regulate other genes by binding to DNA near them, the researchers tested for altered gene activity in animals where these two homeobox proteins have been knocked down. They found that one of the genes is the luciferase enzyme itself. A detailed look showed that the two homeobox proteins form a complex on the DNA near the luciferase gene and directly activate it in the light-producing cells.

One of the homeobox proteins also activates genes that are involved in getting the luciferase into the organelle where it catalyzes the light-producing reaction.

Fu and Zhu haven't looked at all aspects of light production; there's no indication here about what is needed to produce the clear cuticle that lets the light out of the abdomen, for example. But the new paper has gone some way toward describing the basic features of the system. For one, it suggests that the production of light-producing cells is separate from the system that produces coordinated flashes of light from these cells. It also indicates that the homeobox proteins that help determine which segments the light-producing organ develops in are directly involved in the production of the luciferase that the organs ultimately rely on.

By identifying the genes that are key players in all these processes, they've also provided new avenues for follow-up to glean more details about how this system works.

However, in terms of evolution, this initial picture tells a familiar story. Fireflies didn't necessarily evolve anything completely new to create a light-producing organ in their abdomen. Instead, once the basic pieces were in place, they came under the control of the homeobox genes that were already active in dictating the identity of different abdominal segments.

Nature Communications, 2024. DOI: 10.1038/s41467-024-45559-7  (About DOIs).