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 SR_{New york}) and four wild-type stocks (MI, New york1, 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) Females companion 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 SR_Myself and ST males. Using a series of genetic crosses, we created morphologically indistinguishable males that were either SR_Me 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 SR_{Me personally} or ST male were observed for 2 h, with mating latency noted. After the trial, males were genotyped as SR_Myself 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, SR_Me or SR_NY. 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 SR_Myself males, 14 with SR_NY 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) Spunk competition 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 SR_Myself 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).

Female one to introduced each other dark and you can wild-type girls and boys were inferred to possess mated having both particular boys. Females one to brought merely black or wild-style of young children have mated having one to men, several men of the identical phenotype, or males of different phenotypes however only made use of the cum of 1 brand of men. Ergo, so it try out cannot disentangle pre- and you may blog post-mating females discrimination facing SR males; all of our goal were to estimate whether female partner multiply, incase during the standards away from both spouse competition and you will lady choice, whether or not SR men sire a lot fewer youngsters than ST guys.