DOI: 10.5061/dryad.5qfttdz7g
title: Quantifying and comparing male harm between two Drosophila serrata lab populations

description: Male harm arises when traits that increase reproductive success in
 competition with other males also harm females as a side-effect. The
 extent of harm depends on male and female phenotypes, both of which can
 diverge between populations. Within a population, harm is inferred when
 increased exposure to males reduces female fitness, but studies of the
 divergence of male harm rarely manipulate male exposure. Here, we quantify
 male harm and compare its magnitude between two lab populations of
 Drosophila serrata that were derived from a common ancestor seven years
 earlier and subsequently held under conditions that minimized
 environmental differences. We manipulate female exposure to males in a
 factorial design involving all four combinations of males and females from
 these populations, providing insight into divergence in both sexes. Our
 results reveal substantial harm to females and provide stronger evidence
 of divergence in males than in females. Using these and other published
 data, we discuss conceptual issues surrounding the quantification and
 comparison of harm that arise because it involves a comparison of multiple
 quantities (e.g., female fitness under varying male exposure), and we
 demonstrate the increased insight that is gained by manipulating male
 exposure to quantify these.
description: We compared male harm and female resistance between the two
 popuations of <em>Drosophila serrata</em> (the
 '2006' and '2013' University of Ottawa populations).
 The experiment measured the fitness of 2006 and 2013 females when held
 under conditions of high or low exposure to 2006 or 2013 males. Exposure
 was manipulated by altering the frequency with which females were exposed
 to males (high = continuous exposure; low = 6h exposure every 3-4 days).
 All combinations of male exposure (high vs. low), male population (2006
 vs. 2013), and female population (2006 vs. 2013) were performed, yielding
 a 2 × 2 × 2 factorial design involving eight unique combinations of
 treatment levels. The experiment was performed in two blocks over two
 consecutive generations of the populations during the summer of 2014, with
 30 replicates of each of the eight treatment combinations within each
 block, yielding 480 replicates in total.A few individuals were lost due to
 experimental error, resulting in 463 replicates (average of 28.9 per
 treatment combination, range 24-30). Flies for use in
 the assay were collected as virgins using light CO<sub>2</sub>
 anaesthesia and then stored, separately by sex, in vials in groups of 10
 individuals. Two days later, females were marked (to help distinguish them
 from males) by dusting them with red formaldehyde-free fluorescent pigment
 (Brilliant Group, LLC, San Francisco, CA). We then created replicate vials
 with one virgin female and six virgin males per vial from the appropriate
 populations. In the low exposure treatment, after 6 h males were removed
 via aspiration and discarded, while in the high exposure treatment males
 remained. Females were transferred to new vials with 10 mL of fresh media
 every 3-4 days from this point on until the last female died. Males from
 the high exposure treatment were discarded at each transfer and new
 sexually mature males from the appropriate population were added, while in
 the low exposure treatment new males were placed with females for 6 h
 before once again being discarded. Vials were checked at least once daily
 for dead flies; female deaths were recorded and dead males were replaced
 with live ones from the appropriate population. Following transfer of
 females to new vials, the old vials were retained and the number of adult
 offspring produced per vial was counted 14 days later.
description: Rows are replicates (i.e. single females). Female_Last.Alive and
 Female_First.Dead are the days at which the female was last observed alive
 and first observed dead, respectively, with fractional values denoting the
 approximate time of day that check was performed. Num_offspring columns
 give the number of offspring the female produced in a given vial (i.e.
 females were transferred to a new vail every 3-4 days). Zero values
 indicate that the female was alive at least some of the time while in that
 vial but produced no offspring, while missing values (".")
 indicate that that female was dead at that point. Total_Num_Offspring is
 simply the sum of all these values for a given female (i.e.
 replicate).