Courtship assays

For all behavior experiments, males were isolated prior to eclosion in culture tubes with food. Naive day 4 adult male flies were placed in a 1 cm diameter courtship chamber with same-aged wild type Canton-S male or virgin female subject and observed. All courtship scoring was performed single-blind.

Male-Male Courtship: To assess male-male courtship, 4-day-old test males were placed in 1 cm courtship chambers with virgin Canton-S males and time performing stereotypical courtship behaviors (Orientation, licking, tapping, chasing and wing extension) was totaled during a 10 minute window after a gate bisecting the chamber and separating the males was removed. Seconds engaging in courtship behaviors over the 600 second total was converted to a percentage termed courtship index (CI).

Male-Female Courtship: For male-female courtship, 4-day-old males were placed into 1 cm courtship chambers with Canton-S virgin females and recorded until 10 minutes after the final male from the last mating pair in the 14-chamber apparatus dismounted. For pairs that did mate, courtship index (see above) during the time to copulation was tabulated when relevant. The time where the male mounted the female to the time of dismount was recorded in all cases and reported where relevant. At the moment of dismount, recently-mated males were scored for courtship behaviors during the following 10 minutes, and courtship index was reported as with males, but labeled post-copulation courtship index to distinguish it in this context. For pre-copulation/post-copulation comparisons, courtship index (tabulated as a percentage of time performing stereotypical courtship behaviors as a percentage of total time) during the five minutes (or fewer if males mated earlier) before mating was compared to only the first five minutes after males and females broke copulation. Time between mounting and dismounting was recorded as copulation duration.

Mate-and-switch: Inka cell-ablated (ETH-Gal4;Tubulin-Gal80^ts>UAS-Reaper) and Canton-S males isolated as before at eclosion were isolated until day 4, and simultaneously placed in three identical courtship chambers with Canton-S virgin females simultaneously. An experimenter watched the chambers, and immediately after completion, aspirated the male and female out and moved the recently-mated male subject and female partner to a courtship chamber, either empty or containing a counterpart from a desired pair which had completed mating just prior to the transport pair. The new pair were on either side of a gate, and when the 14-well chamber was full of desired partners, the video recording began and gates were retracted. As 3 times as many pairs were being mated as were needed in the final chamber, the entire loading sequence was completed within 3 minutes after the first male dismounted. Conceptual schematic is displayed in Fig 2A.