Post-copulatory fitness - sperm competition
Ahead of sperm competition assays, we created 13 spa inbred lines in order to obtain genetically uniform males to compete against our focal males. We did this by mating full-sibling spa originating from our stock population for three successive generations. We then selected the inbred line with the lowest inter-individual variance in reproductive behaviour (mating latency and mating duration; in a monogamous setting), with an average trait value most similar to the ancestral spa population, and with the strongest competitive abilities (i.e. low mating latency and high mating duration; Fig S1, S2). We then examined the effect of sexual perception on sperm-offense abilities (paternity share of a male mating second with a female;P2) , as it is the main sperm competition measure explaining male fitness in D. melanogaster (Fricke et al. , 2010). With this intent, we monogamously housed ca. 700 spa virgin females from stock populations with a genetically uniform spa virgin male for 150 minutes, in order for the couple to mate. After 150 minutes, we discarded spa males and kept females alone in the vial for 48 hours in order to provide a realistic time lag between the two matings (i.e. similar to what D. melanogaster my experience in the wild; Gromko and Markow, 1993; Harshman and Clark, 1998; Imhof et al. , 1998; Jones and Clark, 2003; Giardina, Clark and Fiumera, 2017; Soto-Yéber et al. , 2018; Dukas, 2020). Additionally, this 48h time lag permitted us to adequately assess whether spa females successfully mated with the spa male, via observation of eggs/first instar larvae in the egg laying substrate. We discarded all females that did not produce at least one egg from the pool of standard mated females used for sperm competition assays, which left us with 645 mated spa females. Following these 48 hours, we haphazardly set up 321 females to mate with control males and 324 to mate with female-exposed males, in fresh vials. Due to logistic limitations, we did this in two batches in which we balanced assignation the number of replicate of each treatment (female-exposed and control males). We recorded mating duration and only considered a mating as successful if it lasted longer than 10 minutes. Following a successful mating, males were immediately discarded to prevent remating, and females were left alone in the vial. Remating trials lasted 150 minutes, after which we discarded all females that did not remate. A total of 282 females remated with the male they were offered (136 female-exposed and 146 control males). We allowed isolated females to lay eggs for 4 days, during which we flipped them into fresh yeasted vials every day. We then incubated vials for 15 days to allow F1 offspring emergence (average generation time being ca. 10 days). We then froze them at ca. -20°C for later counting of offspring of each phenotype (wt vs spa ). We pooled the offspring count from the 4 consecutive days in order to score sperm-offense abilities of the focal male. We discarded females that did not produce a single viable offspring during these four days (7 females) from further analyses, as no focal male paternity share could be computed. We also discarded 2 females from further analyses due to human error (e.g. escaped flies, erroneous sex determination following mating trial, etc.). Our final sample size was then 273 (n = 132 treatment females, n = 141 control females).
We computed sperm offense (P2) as the proportion of offspring sired by the focal (wt ) male:
\begin{equation} P2=\frac{N\text{wt}\ }{N\text{spa\ }+\ N\text{wt}}\nonumber \\ \end{equation}
Where Nwt is the absolute number of offspring sired by the focal (wt ) male, and Nspa is the absolute number of offspring sired by the standard competitor (spa ) male.