Remating and sperm competition experiment
To further examine determinants of male reproductive success we assessed male post-mating competitive ability. We tested male sperm defense ability by first mating females to a temperature challenged focal male and subsequently to a Sb mutant competitor. In this assay, all males and females were five days post-eclosion when mated. Females and the Sb competitor males were grown at 25°C. For the first mating with the focal males, 50 (for the 25°C treatment) or 70 (for the remaining treatments) individual pairs were set up and given three and half hours to mate. We continuously observed pairs and noted the time when pairs were set together, started and ended mating to calculate mating latency and copulation duration. After a successful mating, males were discarded, and females were allowed to lay eggs for 48h. After this time, females were transferred to a new vial containing a virgin heterozygous Sb male. Pairs were allowed to mate for two hours (see Supplementary table S3 for the total number of mated pairs in both matings). We again recorded mating behavior as before and scored how many females remated. Females who remated were kept in the same vial for two days, allowing them to lay eggs, while Sb males were discarded. After these 48h we transferred females to new vials allowing them to lay eggs for two more days. We kept vacated vials from both the intermating interval and the four days after remating at standard conditions for 12 days allowing all the offspring to eclose. Vials were frozen and the offspring counted, whereby for the vials after remating we determined paternity by separately counting offspring scored as presenting the Sb mutation versus the wild type phenotype. As Sb fathers were heterozygous for the mutation, we corrected the counts for paternity scores. Assuming half the offspring fathered by the Sb males present the Sb phenotype and the other half the wild type phenotype, we doubled the Sb counts and corrected the wildtype counts by subtracting the number of Sb offspring counted from the total wild type progeny number.