We used genetic crosses with the mixed brown stock to create ST and SR flies that were genetically similar except for the X-chromosome (electronic supplementary material, figure S2). Me personally and SRNyc) and four wild-type stocks (MI, Ny1, New york2, PEI). Males used in the experiment carry an ST or SR X-chromosome from the tester stock, and females are heterozygous at the X-chromosome, with one b X-chromosome and the other from the tester ST or SR stock (electronic supplementary material, figure S2). Flies were collected as virgins and stored 10 flies vial ?1 on regular culture food. At one week of age, flies were transferred to vials containing a blended mushroom-agar food. Survival was assayed every 1–2 days, and the flies were transferred to fresh food every 4 days. Survival analyses used a Cox proportional hazard model with P roc P hreg in SAS v. 9.3 (SAS Institutes), with SR carrier status and line nested within SR carrier status as fixed effect variables, and the vial as a random effect variable. Analyses were completed separately for females and males.
(d) Ladies lover discrimination
We tested for virgin female discrimination against mating with SR males using both no-choice and choice assays. First, we conducted no-choice trials with SRMe and ST males. Using a series of genetic crosses, we created morphologically indistinguishable males that were either SRMe personally or ST with an otherwise similar genetic background (electronic supplementary material, figure S3). Mate trials of a female from the mixed wild-type stock with either a single SRMyself or ST male were observed for 2 h, with mating latency noted. After the trial, males were genotyped as SRMe personally or ST as described above. Analyses used Fisher’s exact tests (FET) and non-parametric Wilcoxon rank-sum tests.
Second, we used a mass mating cage experiment to test whether females discriminate against mating with male SR carriers under high male–male competition conditions. Each cage included 40 females from the dark mixed stock, 40 ST males from the dark mixed stock, and 40 tester males that were wild-type for eye colour and carried ST, SRMyself or SRNY. Females were placed in the cage first, and after 5 min, all the males were added; copulating pairs were removed by aspiration and each male was scored for eye colour. Each cage was observed until half of the females mated. A total of 35 cage experiments were carried out, which included 16 with SRMyself males, 14 with SRNY males and five controls with wild-type ST males from the mixed stock. We used a Cochran–Mantel–Haenszel test within each male type to test for a deviation from 50 : 50 random mating.
(e) Cum battle assay
We used cage assays to examine the rate of female remating and whether SR males suffer reduced fertility in conditions of potentially high sperm competition. We used small cages (10 cm diameter ? 15 cm tall) that contained instant Drosophila food and a mushroom cap. In each cage, we placed 20 dark females, 20 dark ST males, and 20 of either wild-type ST males from the mixed stock or SRMyself males. All flies were 7-day-old virgins when placed in the cage. Each cage was left for 1, 2, 3, 4, 6 or 10 days, after which the flies were removed and females were placed individually in culture vials for 10 days, after which they were discarded. The offspring of each female were scored for eye colour to determine paternity. Between 4 and 10 cages were set up for each day and male SR status (n = 65 cages total).
People that introduced one another dark and you will wild-type of kids was in fact inferred for mated that have both kind of people. Girls that delivered only dark or nuts-type of offspring could have mated with one men, several people of the same phenotype, otherwise guys of various phenotypes then again only made use of the cum of just one type of men. Ergo, it test cannot disentangle pre- and post-mating females discrimination up against SR boys; our objective was to guess if lady mate proliferate, and if from inside the conditions from one another companion competition and you will girls alternatives, whether SR people sire fewer kiddies than ST people.