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.