Sperm competition experiments

In each experiment, knockdown females were generated by crossing UAS-RNAi lines for each candidate gene to a GAL4 driver. To obtain control females with wild-type gene expression, flies from the appropriate background stock (attP or w¹¹¹⁸) were crossed with flies from the same GAL4 driver. Control and knockdown females were mated to cn bw males in single-pair matings on day 0 in vial 1. Copulations were observed. Males were removed after copulation ended and mated females were retained in the individual vials. In the evening of day 1, two bw^D males were added to each vial and left with the female overnight. Both bw^D males were removed in the morning of day 2, and each female was transferred to vial 2. Each female was transferred again every 48 hr to vials 3, 4, and 5 (on days 4, 6, and 8, respectively). All females were discarded on day 10. Progeny from eggs laid in vials 1–5 were reared to adulthood and the paternity of F1 female progeny was scored based on eye color: female offspring of cn bw males had red eyes and female offspring of bw^D males had brown eyes. Male progeny were not scored because they were w, making it impossible to use eye color to assess their paternity. On average, each experiment consisted of 71.8 ± 25.1 control females and 65.9 ± 24.3 knockdown females who had mated at least once (mean ± SD). Of these females, 51.9 ± 21.7 control females and 46.9 ± 21.0 knockdown females in each experiment had mated with both males. Sample sizes for each experiment can be found in Table S2.

Since each female was paired with two bw^D males and left overnight for the second mating, there was a chance for multiple remating events to occur, which would affect sperm competition. Nonetheless, in a separate experiment, we found that 0 out of 275 mated females remated twice within a 16-hr period.