The research group led by Professor Yuichi Oba of the College of Bioscience and Biotechnology, Chubu University on December 3 announced the recreation of the deep green glow by using the luminescent substance luciferin with a reconstituted genetic sequence thought to be responsible for the luciferase (the enzyme producing the glow) in the fireflies of the Cretaceous period around 100 million years ago. The deep green glow is unlike the yellow-green glow of the Genji firefly (Luciola cruciata) or the Heike firefly (Aquatica lateralis) found in Japan today and is thought to have been a defense against predators.

Test tubes aglow. Unlike the yellow-green glow of the modern Genji firefly (left), the luminescence of the extracted Cretaceous genetic sequence from around 100 million years ago has a deep green coloration (Credit: Chubu University)

There are some 2,000 species of firefly worldwide, their glow ranging in color from green to yellow-green to yellow and orange. The luciferin is common to all fireflies, and it is known that their glow is different depending on the amino-acid sequencing of the luciferase. For this reason, much research has been undertaken on luciferase genes.

The research group focused on the technique of “ancestral sequencing reconstruction.” They calculated the past luciferase genetic sequence based on an amino acid evolution algorithm. This is a method long used in evolutionary biology, achieving the reconstruction of proteins such as ancient hemoglobin and hormone receptors. However, this is the first time that it has directly manifested in a visible phenomenon.

The evolution of firefly glow and the reconstruction experiment (Credit: Chubu University, English words in the figure are provisional translations by JST)

When the luciferase sequence from before fireflies emerged around 100 million years ago was reconstructed, the glow was very weak and red in color. Then, reconstructing sequences from various periods since the Cretaceous, a wide range of glow colors could be seen from deep green to yellow. In this way, the firefly family has revealed a scenario that has evolved from the ancestors of insects that glow red..

Evolution of the firefly glow (Credit: Chubu University, English words in the figure are provisional translations by JST)

Fireflies are poisonous and known to have a very nasty flavor. Oba says, “The dark green color is likely a warning to nocturnal predators that ‘I taste awful, do not eat me.’ This also seems to prove the hypothesis that firefly colors have become diverse because fireflies use their glow in sexual communication.”

The research group is made up of Chubu University, Nagahama Institute of Bio-Science and Technology, and Kagoshima University. These findings were published on December 3 2020 in the online edition of US science journal Science Advances.