Survival assays at generation 14
To assess adaptation to pathogen, two blocks of survival assays were done on males and females after 14 generations of experimental evolution. To avoid parental effects, we first reared individuals from all populations for one generation in a common garden. To establish the common garden, collected virgins were housed together in vials containing 20 males and 20 females. These individuals were allowed to mate for 72 hours, after which males were discarded. Females (N = 120 per block) from these mating vials were then collected and housed together for 72 hours in groups of 20 to lay eggs. After discarding the females, larvae were allowed to develop and emerging virgin males and females were collected and housed in single-sex groups of 20 each. Virgins (age at infection: Batch 1 – 5-7 days, Batch 2 – 4-5 days) were exposed to P. entomophila in the same manner as described above in single-sex groups of 20. After exposure to the pathogen for 24 hours (OD600nm of infections: Batch 1 – 280, Batch 2 – 300) individuals from each vial were transferred to fresh vials and per vial deaths were scored at 2, 4, 20, 24, 28 (the first time point after transfer to new vials), 44, 52 and 72 hours after pathogen exposure. Alongside the infections, two vials were sham-treated for each of the populations to serve as controls. In each block, we again used flies with a relish mutation to ensure that the pathogen was virulent (Vallet-Gely et al. , 2010).