Unusual labmates: Lighting up the lab

July 7, 2019

Unusual Labmates is a series that explores some of the more unusual models used for research at Whitehead Institute. From rare plants to luminescent beetles to regenerative starfish and worms, these organisms and their unusual traits provide insights into the underlying biology and incredible diversity of living things.

Massachusetts Institute of Technology (MIT) graduate student Tim Fallon is standing in a field in New Jersey, holding a net and waiting for the last glimmers of sunlight to disappear. As the trees surrounding the field fade into shadow, Fallon watches the ground intently. The air is still and then, hovering above the grass, a small point of light appears. It floats through the air in a concave upwards arc and winks out. Soon, more lights follow suit. These are fireflies, unassuming beetles by day, but at night they put on a dazzling luminescent display in the hope of attracting a mate. Fallon sweeps his net through the air, capturing some of them. He has traveled to New Jersey with other members of Whitehead Institute Member and associate professor of biology at MIT Jing-Ke Weng’s lab to collect specimens of Photinus pyralis, the big dipper firefly, in order to sequence a firefly genome for the very first time.

Fireflies have been around for more than one hundred million years, and in that time have diverged into more than 2,000 species and spread to every continent except Antarctica. The beetles (despite their name, fireflies are actually beetles) are widely known, and often beloved, for their enchanting courtship rituals, but they have also piqued the interest of scientists, who have harnessed the gene behind their light emitting capability for use in research. However, for all of fireflies’ appeal, they are a difficult animal to work with in the lab, and much of their biology remains shrouded in mystery. In the hopes of improving that situation, Weng, Fallon, and collaborators—including Sarah Lower, assistant professor of biology at Bucknell University, and Yuichi Oba, professor at Chubu University in Japan—have been investigating fireflies, primarily by sequencing and analyzing their genomes. This research is providing insights into the evolution of fireflies’ light-producing ability, the biomolecular pathways the fireflies use to luminesce, and could perhaps even inform how the use of firefly-based luminescence in research can be improved.

The chemistry of light

Fireflies produce light using two main ingredients: an enzyme called luciferase and the small molecule luciferin. Luciferase facilitates a chemical reaction that oxidizes the luciferin, and one product of the reaction is light. In the 1980s, researchers recreated the process in plants and plant cells by cloning the firefly gene responsible for the luciferase enzyme from big dipper fireflies and then inserting it into the genomes of their specimens in the lab.[1] When they injected luciferin into the specimens, they began to glow – just like fireflies. Researchers now use this approach in both plant and animal models to track various aspects of biology. They can link the luminescence to a trait or process, and then measure the level of light emitted. For example, researchers can fuse the luciferase gene to a gene of interest such that the two genes will be expressed as one. Then they introduce luciferin to the system and measure the light output using very sensitive equipment that can sense minute changes. The more light that is emitted, the higher the activity level of both luciferase and the gene of interest. This is a widely used assay that, Fallon says, every biologist uses or at least learns about during their training. Luciferase-luciferin has many applications, and along with tracking gene expression it has also been used to track cancer metastasis, monitor medical treatment efficacy, and check for microbial contamination—including on space vehicles such as the Mars Curiosity Rover.[2] The extreme sensitivity of luciferin-luciferase tests makes them an attractive choice in many experiments.

In spite of the widespread use of firefly luminescence, a lot about it still isn’t known, especially when it comes to luciferin. While the gene encoding luciferase has been identified, luciferin is thought to be created through a process involving multiple genes, and the complete set of those genes is unknown, as are the steps involved in the production of luciferin: the intermediate molecules produced and how they are modified to reach the final product. Although Weng’s lab generally studies plants and focuses on understanding how plants evolved biochemical pathways to produce unique small molecules with traits of interest, particularly those with medicinal value, when Fallon joined the lab, he convinced Weng that investigating the unknowns of the small molecule luciferin was a similar and suitable project.

Fireflies in the wild

When Fallon joined the Weng lab, there were not a lot of research tools available for investigating fireflies, starting with the lack of a sequenced genome. Only a handful of firefly genes had even ever been identified. Weng and his collaborators crowdfunded the money to sequence the first firefly genome, and then received funding to sequence a second firefly species and a bioluminescent click beetle, providing a wealth of new data to explore.

The lack of tools for firefly research was due in part to how difficult it is to rear fireflies in the lab, which makes them tricky animals to study. Fireflies are very sensitive to changes in their environments, and in the lab it’s difficult to mimic the right vegetation, climate, seasonal shifts, and other factors that the beetles rely on to time their metamorphoses and thrive.

Unpredictable environmental changes are becoming a common challenge for fireflies beyond the lab as well, due to the impact of humans on their habitats. Fireflies’ sensitivity to these changes may be causing their disappearance. Anecdotally, many people have fond memories of seeing fireflies every summer when they were younger and can attest to the absence of fireflies in those same places now. A large citizen science research project called Firefly Watch is currently underway to figure out the extent to which firefly populations are declining across the United States (U.S.).[3] And in a case indicative of a larger problem, conservation groups recently submitted an urgent petition to recognize the Bethany Beach firefly, whose key habitat in Delaware is at risk due to human development, as the first endangered firefly species in the U.S.[4]

A significant manmade disruption to the fireflies’ habitats is light pollution. Fireflies find each other by signaling with light, but their lanterns are no match for electricity; a firefly trying to outshine a streetlamp might as well be facing off with the sun. For species that evolved faint glows to flash in the dead of night, in the dark of forests, finding a place where their light can be seen is getting harder and harder. Big dipper fireflies have fared better than many others species. They’re large, with bright lanterns, and they tend to flash at twilight, so they are used to competing with some ambient light. Unsurprisingly, big dipper fireflies remain abundant in the wild, including in and near cities. They can be spotted all over the eastern and midwestern U.S., where they are easily identifiable thanks to the distinctive J-shape—resembling the curve of a dipper—that they make as they flash.

Big dipper fireflies have been important in biology research—it is from them that the firefly luciferase gene was first cloned—and so they were the first species that Fallon and his collaborators chose for their genome sequencing project. However, big dipper fireflies fare no better than other firefly species in a lab setting. No one has ever successfully reared big dipper fireflies through a full lifecycle (from egg to egg) in the lab, Fallon says. So, in spite of the ease of collecting big dipper fireflies in the eastern U.S., in order to get a population of fireflies going in the lab, Fallon had to look elsewhere: to Japan.