Fly stocks and culturing
We used a Drosophila melanogaster wild type stock collected in
Portugal by Prof. Élio Sucena in Azeitão, in 2007. It was established as
an outbred population from 160 wild caught fertilized females with an
ample degree of genetic variation within the population (Martins et al.,
2014). Flies were cultured in our laboratory at standard conditions:
25°C and 60% humidity at a 12h light-dark cycle. Stocks were kept in
glass bottles filled with 70mL of standard yeast-sugar (SYA) food (Bass
et al., 2007). Once a week, three glass bottles with about 250 recently
eclosed flies each were started, and we mixed flies across bottles
regularly to maintain genetic diversity. We used a temperate population
as we expect them to maintain higher phenotypic variation as they were
initially adapted to exist within a broad thermal range compared with
tropical populations (Hoffmann et al., 2003). Species from temperate
areas are expected to maintain higher phenotypic plasticity even after
adaptation to laboratory conditions and provide a more promising way to
test thermal responses under a broader range of experimental
temperatures and hence, give a more powerful estimation of the species
ability to cope with increasing temperatures (Mathur and Schmidt, 2017).
Although laboratory adaptation may alter some life-history traits,
previous research has shown that some plastic responses are maintained
(Trotta et al., 2006) and previous research in thermal responses still
find ample variation (Parratt et al., 2021; Sales et al., 2018).
For paternity analysis in a sperm competition experiment we used flies
bearing the stubble (Sb ) mutation as a tractable phenotypic
marker. The Sb gene was back-crossed multiple times into the
wild-type Dahomey genetic background. Sb is a dominant mutation
that causes a short, thick bristle phenotype (Lees et al., 1945) that is
visible by eye and can be easily distinguished from the wild type
bristle structure. As the recessive phenotype is lethal, we used males
heterozygous for this mutation in the subsequent sperm competition
assay. The stock was kept under the same standard conditions as
described above.
Throughout all assays, in order to obtain experimental flies, we allowed
parental flies to mate for 24h and oviposit on grape-juice-agar plates
[50 g agar, 600 mL red grape juice, 42.5 mL Nipagin (10% w/v
solution) and 1.1 L water] with a semi-liquid baker’s yeast paste
distributed all around the plate to promote egg laying. We incubated
plates for 24 hours and collected first instar larvae at a density of
100 larvae per vial containing 7mL of SYA food. For all experiments,
flies were collected within 8 hours after eclosion as virgins on ice.
Adult flies were kept in separate sex groups 20 per vial. Throughout the
experiments, females were grown at 25°C, while males were exposed to
different temperature treatments during development. We exposed males to
two challenging temperatures, one moderate (29°C) and one severe (31°C)
challenge, the latter near to the lethal threshold for D.
melanogaster of 32°C (Petavy et al., 2001). We first tested how
developmental temperature affects male fertility and whether males can
recover fertility when placed at a benign temperature after eclosion.
Control males were raised at the standard temperature of 25°C. We
observed changes from day one to six after eclosion. As we were
interested in the fitness consequences of the recovery process and the
underlying mechanisms, we allowed half of the males to recover from the
heat stress (denoted with an R) after eclosion, while keeping the other
half at the stressful temperature. All assays described below were done
under these conditions. In order to maintain temperatures precisely for
our different treatments, incubators with ±0.5°C accuracy were used
(INCU-Line® IL 10). Accuracy was monitored by placing a temperature
logger (NOVUS®; accuracy of ±0.5°C) in each incubator throughout the
course of the experiments to record temperature.