1027C Poster - 16. Techniques and technology
Saturday April 09, 1:30 PM - 3:30 PM

Authors:
Alexandra Keene; Mark Stenglein

Affiliation: Colorado State University, Fort Collins, CO

Keywords:
k. next-generation sequencing; u. other (virus-host dynamics)

Paleogenomic sequencing has enabled the reconstruction of extinct genomes and shed light on ancient host-pathogen interactions. But sequencing has focused for the most part on ancient DNA rather than ancient RNA, in part because of a belief that RNA is fragile and short-lived. In this study, I experimentally assessed the stability of single and double-stranded RNA over months in dried insects. I also optimized methods for non-destructive extraction of RNA from pinned D. melanogaster museum specimens that had been collected over the past 120 years. I was particularly interested in reconstructing the historical dynamics of D. melanogaster and galbut virus, which is an exceptionally successful persistent virus in current fly populations. Galbut virus is a three-segmented partitivirus that is ubiquitous in wild D. melanogaster populations: its average global prevalence is higher even than that of Wolbachia. This high degree of success is attributable to galbut virus being biparentally vertically transmitted with near 100% efficiency, which should drive the virus to 100% prevalence in wild populations. But multiple lines of evidence indicate that some flies are resistant to infection and this virus and host may be co-existing in an uneasy equilibrium. To understand how long galbut virus has been infecting D. melanogaster and how its sequence might have changed over the past century, I obtained specimens ranging from 15-120 years from entomological collections across the United States. I obtained fragmented but detectable RNA from these old specimens and was able to amplify galbut virus sequences using short-range RT-qPCR from century-old flies. I am extending and confirming this finding using shotgun RNA sequencing to fully reconstruct the virome of these historic specimens. This indicates that this currently successful virus has infected D. melanogaster since the dawn of Drosophila research, and supports the conclusion that ancient dried insect specimens are a source of usable RNA for paleogenomic studies.