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.