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).