DOI: 10.5061/dryad.pzgmsbcqz
title: Thermal phenotypic plasticity of pre- and post-copulatory male harm buffers sexual conflict in wild Drosophila melanogaster

description: Strong sexual selection frequently leads to sexual conflict and ensuing
 male harm, whereby males increase their reproductive success at the
 expense of harming females. Male harm is a widespread evolutionary
 phenomenon with a strong bearing on population viability. Thus,
 understanding how it unfolds in the wild is a current priority. Here, we
 sampled a wild Drosophila melanogaster population and studied male harm
 across the normal range of temperatures under which it reproduces
 optimally in nature by comparing female lifetime reproductive success and
 underlying male harm mechanisms under monogamy (i.e., low male
 competition/harm) vs. polyandry (i.e., high male competition/harm). While
 females had equal lifetime reproductive success across temperatures under
 monogamy, polyandry resulted in a maximum decrease of female fitness at
 24°C (35%), reducing its impact at both 20°C (22%), and 28°C (10%).
 Furthermore, female fitness components and pre- (i.e., harassment) and
 post-copulatory (i.e., ejaculate toxicity) mechanisms of male harm were
 asymmetrically affected by temperature. At 20ºC, male harassment of
 females was reduced, and polyandry accelerated female actuarial ageing. In
 contrast, the effect of mating on female receptivity (a component of
 ejaculate toxicity) was only modulated at 28ºC, where the mating costs for
 females decreased and polyandry mostly resulted in accelerated
 reproductive ageing. We thus show that, across a natural thermal range,
 sexual conflict processes and their effects on female fitness components
 are plastic and complex. As a result, the net effect of male harm on
 overall population viability is likely to be lower than previously
 surmised. We discuss how such plasticity may affect selection, adaptation
 and, ultimately, evolutionary rescue under a warming
 climate.  
description: Fitness assays: quantifying
 male harm To study whether male harm is affected by
 temperature, we established a factorial design to measure survival and
 lifetime reproduction success (LRS) of female flies under monogamy (i.e.,
 one male and one female per vial) vs. polyandry (i.e., three males and one
 female per vial), across three stable temperature treatments typical of
 this population during their reproductively active period in the wild: 20,
 24, and 28°C. Comparison of female fitness at monogamy vs. polyandry is a
 common way to gauge male harm in <em>Drosophila</em> and other
 organisms (Yun, Agrawal,
 & Rundle, 2021),
 and our treatments reflect the low and high-end of the spectrum of sex
 ratios that are typical of <em>D. melanogaster</em> at mating
 patches in the wild (Dukas, 2020). We first randomly divided virgin flies into
 three groups that we allocated to the three different stable temperature
 treatments 48 hours before starting the experiment. Flies remained at
 those temperatures until the end of the experiment. To estimate LRS, we
 transferred flies to fresh vials twice a week using mild CO2 exposure
 anaesthesia. We incubated the vials containing female eggs at 24 ± 4°C for
 15–20 days (~15 days for 28°C, ~17 days for 24°C and ~20 days for 20°C) to
 allow F1 offspring emergence, after which we froze them at -21°C for later
 counting. The differences in incubation time are due to differences in
 developmental time because of temperature differences in the first 3-4
 days of each vial (i.e., before flipping females to new fresh vials). We
 discarded and replaced males by young (2 to 4 days old) virgin males three
 weeks after starting (at the same time for all treatments). We kept a
 stock of replacement males maintained at each of the three temperatures
 (20, 24 and 28°C) to replace dead male flies if needed. We kept flies
 under these conditions during six weeks, after which we discarded males
 and followed females until they died. We started the experiment with 468
 females (78 per each temperature x mating system treatment) and 936 males
 (234 per each temperature x polyandry and 78 per each temperature x
 monogamy). Final sample sizes were: a) at 20°C: 74 females for polyandry
 and 76 females for monogamy; b) at 24°C: 72 females for polyandry and 77
 females for monogamy; c) at 28°C: 70 females for polyandry and 75 females
 for monogamy. Differences between start and final sample sizes across
 treatments are due to discarded/escaped flies since the start of the
 experiment. We estimated the overall degree of male harm by calculating
 the relative harm (H) following Yun et al. (2021):
 <em>H =
 (W<sub>monogamy</sub> - W<sub>polyandry</sub>) /
 W<sub>monogamy</sub><br></em>
 Finally, and using the data
 collected above, we partitioned overall effects on LRS into effects of
 temperature x mating system (i.e., male-male competition level) on early
 reproductive rate (i.e., offspring produced during the first two weeks of
 age) and reproductive (i.e., offspring produced over weeks 1–2 vs. 3–4)
 and actuarial senescence (i.e., survival).
 Behavioural assays:
 quantifying male-male aggression and harassment of
 females Immediately after the fitness experiment
 started, we conducted behavioural observations on the first day of the
 experiment across all treatments, to investigate the behavioural
 mechanisms that might underlie the potential fitness effects evaluated
 above. Due to logistic limitations, we conducted behavioural observations
 in the same temperature control room, so we had to conduct trials at 20,
 24 and 28°C in three consecutive days (with both monogamy and polyandry
 treatments evaluated at the same time for each temperature), in randomized
 order (i.e., 20, 28 and 24°C). However, all flies were 5 days old at the
 start of the experiment. We measured the following behaviours: (a)
 courtship intensity (number of courtships experienced by a female per
 hour), (b) male-male aggression behaviours and (c) female rejection
 behaviours (Bastock
 & Manning, 1955; Connolly & Cook,
 1973). We also recorded
 the number of total matings during the observation period.
 Observations started at
 lights‐on (10 a.m.) and lasted for 8 hr, during which
 time we continuously recorded reproductive behaviours using scan sampling
 of vials. Each complete scan lasted approximately 8 min, so that we always
 conducted one complete scan every 10 min to ensure recording of all
 matings (see below). Scans consisted in observing all vials in succession
 for ca. 3s each and recording all occurrences of the behaviours listed
 above. We interspersed these behavioural scans with very quick (&lt;1
 min) mating scans where we rapidly swept all vials for copulas at the
 beginning, in the middle and at the end of each behavioural scan. This
 strategy ensured that we recorded all successful matings (&gt;10min),
 which typically last between 15 and 25min in our population
 of <em>D. melanogaster</em>, during our 8‐hr observation. We obtained a total of 49 scans
 per vial. Behavioural observations were conducted only once, on day 1 of
 the fitness experiment, as prior experiments have shown that courtship,
 aggressive and female rejection behaviours are stable over time in
 <em>D. melanogaster</em>, so that our behavioural indexes are
 representative of long-term treatment differences (e.g.,
 Carazo, Perry, Johnson,
 Pizzari, & Wigby, 2015; Carazo, Tan, Allen, Wigby, &
 Pizzari, 2014). In
 contrast to courtship and aggression indexes, note that total mating
 frequency over the first day cannot be taken as a reliable measure of
 mating rate (Wolfner,
 1997), and thus our
 rationale in recording this variable was just to ensure that early mating
 ensued normally across treatments (which was the case, see
 SI2). Female reproduction and survival assays:
 quantifying male ejaculate “toxicity” 
 To examine post-mating
 mechanisms that might underlie the fitness effects observed in our first
 experiment, we conducted four additional assays to test whether
 temperature modulates the previously well-documented effects of male
 ejaculates on female reproduction and survival in D. melanogaster.
 Briefly, males of this species transfer seminal fluid proteins (SFPs)
 produced by their accessory glands that increase short-term female
 fecundity, as well as decrease female receptivity and survival
 (Chapman, Liddle, Kalb,
 Wolfner, & Partridge, 1995; Wigby & Chapman,
 2005). In addition, prior
 studies have shown that males are able to tailor investment into SFPs
 according to the expected sperm competition risk and intensity
 (Hopkins et al.,
 2019). Thus, we set up a
 factorial design where we manipulated the temperature (i.e., 20, 24 and
 28ºC) and perceived sperm competition risk levels (i.e., males kept alone
 vs. with 7 more males in a vial) at which adult males were kept prior to
 mating, for two temperature treatment durations (i.e., 48 hours or 13
 days), and then measured how reception of their ejaculate in a common
 garden environment (i.e., 24ºC) affected female fecundity, survival and
 reproduction. Receptivity assays
 We first collected
 experimental males as virgins (i.e., within 6 h of eclosion) under ice
 anesthesia and randomly placed them either individually or in a same-sex
 group of 8 in medium containing plastic vials. Next, we randomly divided
 them into three groups that we allocated to the different stable
 temperature treatments for either 48 hours (experiment 1) or 13 days
 (experiment 2) immediately before the beginning of each experiment. In
 experiment 2 (treatment duration of 13 days), we emptied the seminal fluid
 of experimental males before we allocated them to the different
 temperature / competition treatments. To do so, we
 housed individually experimental males with four standard virgin
 females for 24 hours. This strategy ensured that spermatogenesis took
 place over the 13 days under the temperature / competition
 treatments. We collected all females and competitor males
 used in receptivity assays as virgins and held them in groups of 15 to 20
 flies at 24 ± 4°C. Experiments started by exposing all virgin females to
 single experimental males for 2.5 h at 24°C. After a successful
 copulation, we separated the mated females from the males and kept them
 individually in medium-containing vials until the next mating trial. We
 discarded unmated females and experimental males. 72 h after the first
 mating, we individually exposed females to single virgin competitor males
 for 12 h. After each trial, we transferred unmated females into a new
 medium containing vial, until the next mating assay 24 h later. We
 repeated remating trials every 24 h for three consecutive days. We calculated the
 cumulative percentage of remated females for each of the three days of
 each experiment. We conducted the experiments in two blocks each: with n =
 390 females for each batch in experiment 1 (n = 436 rematings) and n = 420
 females for each batch in experiment 2 (n = 676 rematings). We also
 recorded mating duration for the first mating and mating latency (i.e.,
 time between males being introduced into the female-containing vial and
 copulation) and mating duration for re-matings. Females that did not
 remate within those three days were right-censored. Females and all
 experimental males were 4 days old at the start of experiment 1. In
 experiment 2, females and “re-mating” competitor males were 4 days old,
 while experimental males were 18 days old.
 Fecundity and survival
 assays To gauge effects on female short-term fecundity
 and long-term survival, we performed two experiments (experiments 3 and 4)
 where we compared the oviposition and egg fertility of females mated with
 male flies subject to the same factorial design imposed in receptivity
 experiments. We collected and treated all experimental males as in the
 receptivity assays described above, and then proceeded to mate ~4-day-old
 virgin females in single pairs to either 4- (experiment 3, 48h temperature
 treatment duration) or 18-day-old experimental males (experiment 4, 13d
 temperature treatment duration) for 2.5h at 24°C. After copulation, we separated
 mated females from males and kept them individually in medium-containing
 vials. We discarded unmated females. We then transferred females to fresh vials every
 24h for 4 days, and then every 3 days twice. Later, we transferred female
 flies to fresh vials once a week, and we combined vials to maintain
 density at ten flies per vial. We kept female flies under these conditions
 until they died. We removed dead flies at each transfer and recorded
 female deaths. We counted eggs laid the first 3 days and vials from days
 1, 2, 3, 4, 5, and 8 after mating were retained to count progeny to
 determine egg viability. We tested 545 females at the starting point of
 the experiment 3, and 480 females at the starting point of the experiment
 4. Statistical analyses
 We performed all statistical
 analyses using R statistical software (version 3.5.2). In all models, we
 assessed fit by visual inspections of diagnostic plots on raw and residual
 data (Zuur, Ieno,
 & Elphick, 2010).
 To examine temperature effects on male harm, we evaluated the interaction
 between mating system and temperature on female fitness (LRS), survival
 and male and female reproductive behaviours. We fitted generalized linear
 models (GLMs) with temperature, mating system and their interaction as
 fixed effects. We also used Cox proportional hazards survival model to
 analyse potential differences in mortality risk across treatments, using
 the <em>survival</em> and <em>survminer</em>
 packages, including the females lost during manipulations as “right
 censored” individuals (i.e., individuals that are taken into account for
 demographic analysis until the day they disappear, Kleinbaum & Klein,
 2012). We also censored
 any remaining females after the six weeks of the experiment. Graphical
 inspection of LRS and reproductive behaviours (courtship intensity,
 intersexual aggression behaviours and female rejection) revealed that the
 normality assumption was apparently violated, as well as the independence
 assumption for LRS. Box–Cox transformation (Quinn & Keough,
 2002) solved these
 problems and allowed us to run a GLM with a Gaussian error distribution.
 In all these afore mentioned cases, fitted models were subsequently
 validated. In addition, we compared GLMs with their corresponding null
 GLMs using likelihood ratio test. In all models, we used ANOVA type III to
 compute p-values. We detected a problem of collinearity between mating
 system and the interaction in the LRS model. We fitted the model again
 without the main mating system effect (which was not our main interest)
 and also ran models separately for each temperature level. As a
 complementary analysis, we ran a model with temperature as factor with a
 quadratic contrast table predetermined (note that the relation between LRS
 and temperature does not seem to be linear, Fig 1), obtaining similar
 results. For the total matings recorded across the 8h observation period
 (i.e., mating rate), we initially used a GLM with Poisson errors. However,
 the dataset contained many zero values. We therefore analysed the data
 using a Hurdle model, in which the zero values are modelled separately
 from the nonzero values (Zuur et al., 2010). Additionally, we compared GLM and Hurdle
 models using an information-theoretic approach based on Akaike’s
 Information Criterion (AIC). The minimum AICc value indicates the
 best-supported model given the trade-off between fit to the data and model
 complexity (Konishi
 & Kitagawa, 2008).
 To estimate how male harm impact estimated population growth across
 varying demographic scenarios (i.e., decreasing, stable and growing
 populations), based on our LRS measures, we calculated rate-sensitive
 fitness estimates (Edward,
 Fricke, Gerrard, & Chapman, 2011) against different population background growth
 rates of r = -0.1, r = -0.05, r = 0, r = 0.05 and r = 0.1.
 For
 receptivity (mating duration and remating latency) and fecundity (total
 adults) analyses, we fitted generalized linear models (GLMs) with
 temperature, sperm competition risk level, treatment duration and their
 interaction as fixed effects. For egg production data, we fitted a
 generalized linear mixed model (GLMM) with temperature, sperm competition
 risk level, treatment duration and their interaction as fixed effects and
 day number as a random effect, with a zero inflated distribution, in which
 the zero values are modelled separately from the nonzero values
 (Zuur et al.,
 2010). We assessed
 significance of factors by dropping individual terms from the full model
 using the “drop1” function, refitting where the interaction was
 nonsignificant. We also used Cox proportional hazards survival model to
 analyse potential differences in mortality risk across treatments, using
 the <em>survival</em> and <em>survminer</em>
 packages, including the females lost during manipulations as “right
 censored” individuals. Finally, we also ran models separately for each
 temperature level for the cases in which a significant interaction between
 sperm competition risk level and temperature treatment was detected.     
  
description: Data files included are .xlsx. We performed
 all statistical analyses using R statistical software.