Bacterial load in infected males
To examine how fast D. melanogaster males clear the P. entomophila infection, we infected 1-2 day old virgin males in groups of 20 individuals as described above. We then measured bacterial load of individual flies at 4, 8 and 24 h from the onset of the infection treatment, randomly choosing 2 infection vials to sample at each timepoint. We carefully removed survivors by light anaesthesia and randomly selected 5 individuals.
Each individual fly was then placed in an Eppendorf tube containing small glass beads and 100μL of 70% ethanol to surface sterilize the fly cuticle. The tube was inverted a few times to ensure proper mixing after which the 70% ethanol was removed and replaced by 100μL of Luria broth (LB). We then placed the Eppendorf tubes on a Precellys bead ruptor for 30 seconds at 6000rpm in order to homogenize the flies. The homogenate was then serially diluted to obtain concentrations of 1:10, 1:100, 1:1000, 1:10,000 and 1:100,000. We plated 3 μL of each of these dilutions in 5 replicates on a single LB plate containing 1% milk. The plates were left for 50 hours at room temperature and colonies from each dilution and replicate were counted. For each dilution and time point combination, we calculated an average count of the number of colonies for the 5 technical replicates (from each sample) followed by calculating the total colony forming units using the formula below:
\begin{equation} Total\ Colony\ Forming\ Units=\ Number\ of\ colonies\ for\ a\ given\ dilution\times Dilution\ factor\nonumber \\ \end{equation}