New cell lines derived from larvae of the neotropical fruit fly Drosophila willistoni persistently infected with Wolbachia

Although several hundred continuous cell lines have been generated from Drosophila spp. fruit flies over the past half-century, nearly all are derived from a single species, Drosophila melanogaster, and none are derived from neotropical flies. To address this deficit, a simplified protocol was used to generate three primary cell cultures from larvae of Drosophila willistoni originating from Costa Rica. All three primary cultures developed into continuous cell lines, and all three cell lines, designated DWL/LULS68, DWL/LULS70 and DWL/LULS72, were found to be persistently infected with the bacterial endosymbiont Wolbachia. Sublines free of Wolbachia were generated from all three cell lines by prolonged tetracycline treatment. Molecular analysis, karyotyping and fluorescence in situ hybridization confirmed species origin of the cells as D. willistoni and identified the Wolbachia as the strain wWil. Wolbachia wWil was successfully transferred from D. willistoni cells to heterologous cell lines derived from the sand fly Lutzomyia longipalpis, the biting midge Culicoides sonorensis and the tsetse fly Glossina morsitans, but not to cell lines derived from ticks or triatomine bugs. The new D. willistoni cell lines are expected to facilitate many aspects of research into this species and its bacterial symbionts.