Interrupted matings
Virgin male and female pairs of the same strain/population were aspirated into vials without anesthetization. Similarly, sterile hybrid males were paired with females of each population used to generate them. Vials containing a single male and female pair were observed continuously for a period of 3-4 hours. Mating pairs were stopped by freezing at either 2 or 6 minutes into copulation and stored frozen for later dissection. The frozen couples were retrieved from the freezer and allowed to briefly thaw over the course of a few minutes. All dissections were done in a drop of 1× phosphate buffered saline (PBS). The frozen copulating pair was gently separated using forceps and the male was checked for an ejaculate mass on the tip of its aedeagus, in case the separation had pulled it from the female, before being discarded. The removal of the female reproductive tract was done following a protocol described by Adams and Wolfner (2007). Briefly, forceps and pins were used to separate cuticular tissue and open the abdomen. Once the uterus was visible, we gently removed it from the abdomen without disturbing the ejaculate when present. Pins were used to clean excess tissue; no coverslips were placed over samples and images were captured using an inverted Olympus CKX41 microscope. The samples were checked for morphological shifts in the female’s uterus and the presence of a darker mass that denoted an ejaculate. When present, ejaculates were removed from the female’s uterus using dissecting pins and placed on a drop of NucBlue Fixed Cell DAPI stain (Thermo Fisher Scientific). A coverslip was placed over the drop containing the ejaculate and incubated in the dark for 30 minutes. The samples were observed for presence of sperm under both phase contrast and UV light using a Zeiss AX10 microscope.