Transformation of the Drosophila Sex-Manipulative Endosymbiont Spiroplasma poulsonii and Persisting Hurdles for Functional Genetic Studies

Insects are frequently infected by bacterial symbionts that greatly affect their physiology and ecology. Most of these endosymbionts are, however, barely tractable outside their native host, rendering functional genetics studies difficult or impossible. Spiroplasma poulsonii is a facultative bacterial endosymbiont of Drosophila melanogaster that manipulates the reproduction of its host by killing its male progeny at the embryonic stage. S. poulsonii, although a very fastidious bacterium, is closely related to pathogenic Spiroplasma species that are cultivable and genetically modifiable. In this work, we present the transformation of S. poulsonii with a plasmid bearing a fluorescence cassette, leveraging techniques adapted from those used to modify the pathogenic species Spiroplasma citri. We demonstrate the feasibility of S. poulsonii transformation and discuss approaches for mutant selection and fly colonization, which are persisting hurdles that must be overcome to allow functional bacterial genetics studies of this endosymbiont in vivo.