Yelena Pacheco and Dr. Seth Bybee, Biology
Introduction
Fireflies are well known for their bioluminescent flashes causing them to be a particular group of interest concerning insect vision research. Their flashes are used for both sexual communication and as an aposematic warning to predators. Bioluminescent fireflies are known to use either a yellow-green flash or a yellow-orange flash to communicate between sexes in order to find a mate. There have been many studies concerning flash patterns’ role in sexual communication among fireflies; however, the understanding of firefly vision as a whole allows for many more untouched research possibilities. As a member of the Bybee lab I had the opportunity to travel to country of Rwanda in search of fireflies. While in Rwanda we found a species of fireflies attracted to a red LED light, measuring at a wavelength of 630nm, used in a firefly trap, preliminarily identified as Diaphanes sp. This presented many questions concerning firefly bioluminescence and their visual systems considering fireflies are not known to see or bioluminesce at such long wavelengths of light. The purpose of this project was to further investigate the visual systems of the collected Rwandan fireflies, answer whether or not there is evidence of a red shifted luciferase in the collected specimens and describe the collected specimens as a new species.
Methodology
Our investigation of the collected Diaphanes sp. specimens will require us to identify the possession of visual opsins, which are proteins that influence an organism’s ability to see certain wavelengths of light. Fireflies are known to posses two classes of opsins; long wavelength sensitive (LWS) and ultra-violet sensitive (UVS). Among fireflies there are different possibilities for duplication or expression of opsins that also influence what and organism can see. Current protocols are being developed by the Bybee lab to determine the visual opsins possessed by the Diaphanes sp. specimens. Specimens were prepped in the field for RNA extraction by placing dissected specimens in a solution of RNA-Later allowing the preservation of the RNA necessary to analyze the present visual opsin protiens. This will be done by extracting RNA from each specimen using NucleoSpin columns and reverse-transcribing the RNA into cDNA libraries using the Illumina TruSeq RNA v2 sample preparation. The prepped mRNA libraries will then be sequenced on an Illumina HiSeq 2000 by the Microarray and Genomic Analysis Core Facility at the Huntsman Cancer Institute at the University of Utah (Salt Lake City, UT, USA). From this transcriptomic data, both opsins and luciferase can be searched for and extracted using established bioinformatics pipelines in the Bybee Lab via tools created in the PIA-Galaxy software package. Description of the species will consist of comparing the collected fireflies to specimens from each of the other Diaphanes species and identifying unique morphological characteristics. These morphological characteristics will then be incorporated into a published description of the new species.
Results
Because protocols to identify the visual opsins of Diaphanes sp. are still in the developmental stages we have yet to determine which opsins the Diaphanes sp. specimens posses. The Bybee lab has successfully looked at the visual systems of other firefly groups and has determined the opsins present in these groups; we hope to further modify these protocols in order to obtain the necessary transcriptomic data for opsin and luciferase analysis of Diaphanes sp. Upon further investigation into the genera of Diaphanes we found that the group was much larger than initially expected. First we must compare the collected specimens to all described species of Diaphanes in order to be certain that this is in fact a new species. This will consist of looking at both the species descriptions and type specimens for each species, which will require us to obtain specimen loans from multiple museums across the world.
Discussion
Once this project is completed we will have a have a more clear understanding of firefly vision. If the presence of a red shifted opsin is found this will the first recorded finding for fireflies. Additionally the possibility of a red luciferase in fireflies would also be a new discovery considering there is no record of red bioluminescent fireflies. A red luciferase discovery may also lead to innovations in the medical field considering luciferase gene markers are currently being used in cancer and other medical research.
Conclusion
The collection of Diaphanes sp. in Rwanda led to many new questions about firefly vision. The findings of this project will not only help us to better understand firefly vision but insect vision as a whole. Discovery of a red shifted opsin class and red luciferase would be a significant new finding when it comes to both firefly vision and bioluminescence. We hope to further develop our protocols in order to answer all of our scientific questions presented in this project.