Decision letter: Thermal phenotypic plasticity of pre- and post-copulatory male harm buffers sexual conflict in wild Drosophila melanogaster

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Materials and methods Results Discussion Data availability References Decision letter Author response Article and author information Metrics Abstract 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 20°C, male harassment of females was reduced, and polyandry accelerated female actuarial aging. In contrast, the effect of mating on female receptivity (a component of ejaculate toxicity) was affected at 28°C, where the mating costs for females decreased and polyandry mostly resulted in accelerated reproductive aging. 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. Editor's evaluation This study has important implications for the impact of sexual conflict on population viability under different temperatures. The authors provide compelling evidence that male harm to females in sexual conflict can be reduced as a function of temperature within the optimal reproductive range of a species. The results have implications for the likelihood of the evolutionary rescue of species facing the climate crisis. https://doi.org/10.7554/eLife.84759.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Females and males share the common goal of siring offspring. This central tenet of sexual reproduction enforces a certain degree of cooperation between the sexes. However, anisogamy frequently leads to distinct sex roles and thus general asymmetries in the reproductive evolutionary interests of females and males, which can in turn result in diverging intensity and form of sexual selection across the sexes (Arnqvist and Rowe, 2005; Chapman et al., 2003a; Janicke et al., 2016; Winkler et al., 2021). This phenomenon, termed sexual conflict, favors traits in one sex that might be costly for the other (Parker, 1979), and can thus lead to antagonistic female-male coevolution (Arnqvist and Rowe, 2005). Sexually antagonistic co-evolution has received much attention and is recognized as a fundamental process in evolution due to its role in shaping male and female adaptations (Bonduriansky et al., 2008), in contributing to drive reproductive isolation and speciation (Arnqvist et al., 2000; Bonduriansky, 2011; Bonduriansky and Chenoweth, 2009; Gavrilets, 2014), and as a major determinant of population demography (Kokko and Brooks, 2003; Bonduriansky and Chenoweth, 2009; Berger et al., 2016). Specifically, sexual conflict has been shown to have profound consequences for female fitness and population growth when it favors male reproductive traits that increase male intra-sexual competitive ability at the expense of harming females (i.e. male harm, Crudgington and Siva-Jothy, 2000; Gómez-Llano et al., 2023; Wigby and Chapman, 2004). Harmful male adaptations are widespread and incredibly diverse and sophisticated across the tree of life (Arnqvist and Rowe, 2005). For example, male harassment of females during pre-copulatory competition for mating has been documented in myriad vertebrate and invertebrate species (Gómez-Llano et al., 2023), driving antagonistic female-male co-evolution in a host of behavioral and morphological traits (Arnqvist and Rowe, 2005). Male harm adaptations in the context of post-copulatory competition are similarly widespread in invertebrates, featuring (amongst others) toxic ejaculates (Wigby and Chapman, 2005), love darts (Koene and Schulenburg, 2005), and a range of male adaptations for traumatic insemination that range from genital ablation to spiny penises (Crudgington and Siva-Jothy, 2000; Lange et al., 2013). Importantly, beyond driving female and male phenotypes and associated diversification processes, male harm generally leads to a ‘reproductive tragedy of the commons’ that can substantially impact population demography by depressing net female productivity (Arnqvist and Tuda, 2010; Berger et al., 2016; Holland and Rice, 1999; Rankin et al., 2011), and even facilitate population extinction (Le Galliard et al., 2005). Understanding what factors underlie male harm evolution, its diversity in form, strength, and outcomes, is thus a main concern in evolutionary biology. Despite a growing number of studies in the field of sexual conflict, most have been conducted under uniform laboratory conditions, frequently in populations adapted to stable environments for hundreds of generations (Chapman et al., 2003b; Hopkins et al., 2020; Wigby and Chapman, 2004). In contrast, recent research has highlighted the role of ecology in shaping the evolution of traits under sexual conflict (Arbuthnott et al., 2014; García‐Roa et al., 2019; MacPherson et al., 2018; Perry and Rowe, 2018; Yun et al., 2017), including habitat complexity (Malek and Long, 2019; Miller and Svensson, 2014; Myhre et al., 2013), nutritional status (Fricke et al., 2010), or sex ratio and population density (Chapman et al., 2003a). For example, Gomez-Llano et al., 2018 recently showed that conspecific densities and the presence of heterospecifics modify the intensity and outcome of sexual conflict in the banded demoiselle (Calopteryx splendens), and the spatial complexity of the environment in which mate competition occurs mediates how sexual conflict operates in fruit flies (Yun et al., 2018). The incorporation of more realistic ecological scenarios in the study of sexual conflict is thus a key avenue to disentangle the evolution of male harm, and its consequences for population viability (Cornwallis and Uller, 2010; Fricke et al., 2009; Plesnar-Bielak and Łukasiewicz, 2021). Temperature is recognized as a crucial abiotic ecological factor due to its impact on life history traits and physiological and behavioral responses (De Lisle et al., 2018; Kim et al., 2020; Miler et al., 2020; Monteiro et al., 2017). Furthermore, temperature varies in nature widely within and across spatiotemporal scales (e.g. daily, inter-seasonal, and intra-seasonal variation). Consequently, it may have short, medium, and long-term effects on organism phenotypes that can impact many different aspects of its reproductive behavior (e.g. sex-specific potential reproductive rates, operational sex ratios, density, etc.; García-Roa et al., 2020). In fact, a recent meta-analysis suggests that temperature may have a sizeable effect on sexual selection processes even when fluctuations occur well within the normal range of temperature variation for the studied species (García-Roa et al., 2020). This latter finding is particularly relevant given that we know almost nothing about how average temperature fluctuations, such as those experienced by wild populations during their reproductive season, affect male harm and sexual selection at large. Our aim was to contribute to fill this gap in knowledge by studying how male harm responds to temperature variation that mimics average fluctuations that are normal during the reproductive season, using Drosophila melanogaster flies sampled from a wild population. D. melanogaster is an ideal subject for this study because it exhibits high levels of male-male competition, has well-characterized pre- and post-copulatory male harm mechanisms, and is perhaps the main model species in the study of sexual conflict (Arbuthnott et al., 2014; Chapman et al., 2003a; MacPherson et al., 2018; Malek and Long, 2019; Wigby and Chapman, 2005). During male-male pre-copulatory competition over access to mating, males harm females via intense harassment that causes physical injuries, interferes with female behaviors such as egg-laying or feeding, and results in energetically costly resistance to males (Bretman et al., 2019; Partridge and Fowler, 1990; Teseo et al., 2016). In the context of sperm competition over fertilizations, males can also harm females via toxic ejaculates, whereby certain male seminal fluid proteins manipulate female re-mating and egg-laying rates to the male’s advantage, but at a cost to females in terms of lifespan and lifetime reproductive success (Chapman et al., 2003b; Sirot et al., 2009; Wigby and Chapman, 2005). These proteins are secreted by male accessory glands, have been well characterized, and are strategically allocated by males in response to variation in the socio-sexual context (Hopkins et al., 2019; Sirot et al., 2011). Additionally, despite the fact that almost all work on male harm has, to date, been conducted in laboratory strains adapted to stable temperature conditions, D. melanogaster reproduces in the wild under a wide range of temperatures (Dukas, 2020; Kapun et al., 2018). Briefly, we collected flies from a continental wild population in Requena (Spain) that experiences significant fluctuations in temperature even during the mildest months when it is reproductively active (e.g. July: average: 24.9°C, average min: 19.8°C, average max: 30.1°C, Fick and Hijmans, 2017). After acclimation of the resulting population to laboratory conditions under a fluctuating temperature regime mimicking natural conditions, we conducted five different experiments to gauge how temperature variation within a normal range (i.e. 20°C, 24°C, and 28°C) affects: (a) the overall impact of male-male competition on female lifetime reproductive success (i.e. male harm), (b) how the net effects of harm are accomplished in terms of different female fitness components (i.e. reproductive rate, actuarial aging, and reproductive aging), and (c) underlying male pre-copulatory (i.e. harassment) and post-copulatory (i.e. ejaculate toxicity) harm mechanisms (Figure 1). Figure 1 with 5 supplements see all Download asset Open asset Schematic overview of the study. (A) Our aim was to study how temperature variation, across a range at which reproduction is optimum in the wild, may affect: the net decrease in female fitness resulting from male harm, what female fitness components are mainly affected by male harm, and pre-copulatory (i.e. sexual harassment) and post-copulatory (i.e. ejaculate effects on female receptivity, short-term fecundity, and survival) mechanism of harm. (B) General design of the study: (1) We sampled a wild population of Drosophila melanogaster flies that reproduce optimally between 20°C and 28°C, (2) We setup a population in the lab and left it to acclimate for a few generations under a programmed fluctuating temperature regime that mimics wild conditions in late spring-early summer (20–28°C range with mean at 24°C), (3) We run a series of five experiments (each repeated at 20°C, 24°C, and 28°C) to study temperature effects on net male harm, female fitness components and male pre- and post-copulatory mechanisms of harm. Materials and methods Field collection Request a detailed protocol In October 2018, we used banana traps to sample Drosophila melanogaster flies from three wineries in Requena (Spain): ‘Hispano-Suizas’ (39.466128,–1.149642), ‘Pago de Tharsys’ (39.497834,–1.122781), and ‘Dominio de la Vega’ (39.515079,–1.143757). Traps were setup within the wineries, but in premises that were open to the exterior and to ample surrounding vineyards and/or in the vineyards themselves. After collection, we anesthetized flies using mild CO2 exposure. We then separated and individually distributed field-collected females in vials with standard food, left them to lay eggs for a period of 48 hr, and then incubated their eggs at 24°C, 60% humidity, and a 12:12 dark-light cycle for 14 days to allow adult flies to emerge. We inspected the genital arch of F1 males of each of these female isolines to distinguish D. melanogaster isofemale lines from D. simulans. We then collected three males and three females from each D. melanogaster isofemlae line (total of 276 flies from 46 isofemale lines) and released them into a population cage with a surplus of food medium supplemented with live yeast, setting up the ‘Vegalibre (VG)’ population. In November 2019 and October 2020, to maintain natural variation, we re-sampled the wineries and added 348 and 756 flies from 58 and 126 isofemale lines, respectively, following the same procedure (27 isofemale lines from ‘Pago de Tharsys’ and 31 isofemale lines from ‘Dominio de la Vega’ in November 2019 and 33 isofemale lines from ‘Pago de Tharsys,’ 50 isofemale lines from ‘Dominio de la Vega’ and 43 isofemale lines from ‘Hipano-Suizas’ in October 2020). Stock maintenance and acclimation Request a detailed protocol We carried out all experiments between March 2020 and April 2021, using individuals from the VG field population kept in the laboratory with overlapping generations at an average temperature of 24°C with daily pre-programmed fluctuations (±4°C) mimicking natural daily temperature conditions during the reproductively active season, at ~60% humidity and on a 12:12 hr light:dark cycle (Pol Eko ST 1200 incubator). The lowest temperature was set up 1 hr after sunrise and the highest 1 hr after midday. It is important to note that our stock population of flies was kept under a programmed fluctuating temperature regime that mimics their average circadian rhythm in the field, but temperature fluctuations in nature will be inherently subject to minor stochastic variations whose effects we controlled for (and thus did not capture) in this experiment. We used maize-malt medium (7 g of agar, 72 g of malt, 72 g of maize, 9 g of soya, 15 g of live yeast, 20 g of molasses, and 33 ml of Nipagin mix –3 g of methyl 4-hydroxy-benzoate and 28 ml of ethanol 90%– per 1000 ml of water) as a food source throughout maintenance and experiments. To collect experimental flies, we introduced yeasted grape juice agar plates into stock populations to induce female oviposition. We then collected eggs and placed them in bottles containing ~75 ml of medium to be incubated at 24 ± 4°C at a mean density of 223 ± 14.3 (95% CI) (Clancy and Kennington, 2001). We collected virgin flies within 6 hr of eclosion, under ice anesthesia, and then sexed and kept them in vials with food until their use (~3 days later), at 24 ± 4°C (see below for more details). Net impact of male harm on female fitness and underlying behavioural mechanisms (experiment 1) Fitness assay Request a detailed protocol 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°C, 24°C, and 28 °C. Comparison of female fitness at monogamy vs. polyandry is standard procedure to gauge male harm in Drosophila and other organisms (see Yun et al., 2021 for a review and Gómez-Llano et al., 2023 for a recent meta-analysis). While the sex ratio in this species is typically 1:1, the operational sex ratio is male-biased and frequently reaches a 3:1 (or higher) male-bias in mating patches in the wild (Dukas, 2020). Thus, the 1:1 vs. 3:1 sex ratios used in this study represent biologically relevant scenarios and have actually become standard in Drosophila studies measuring male harm to females (Yun et al., 2021; Gómez-Llano et al., 2023). We first collected virgin flies into same-sex vials of 15 individuals and then randomly allocated them to either of the three temperature treatments 48 hr before starting the experiment, at which temperatures they remained until its end. To estimate LRS, we transferred flies to fresh vials twice a week using mild CO2 anesthesia. We incubated the vials containing female eggs at 24 ± 4°C for 15–20 days (~15 days for vials coming from 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 caused by temperature differences during the first 3–4 days of each vial (i.e. the time eggs remained at their respective temperature treatments before flipping females to new fresh vials and incubation at 24 ± 4°C). We discarded and replaced males with young (2–4 days old) virgin males (receiving the same treatment as described above for original males) three weeks after starting the experiment (at the same time for all treatments). In addition, we kept a stock of replacement males maintained at each of the three temperatures to replace dead male flies if needed. We kept focal female flies under these conditions for six weeks, after which we discarded males and followed females until they died for survival analysis (see Figure 1—figure supplement 1 for an overview of the experimental design). We started the experiment with 468 females (78 per temperature and mating treatment) and 936 males (234 per temperature in polyandry and 78 per temperature in monogamy). Due to discarded (e.g. accidentally damaged during handling) and escaped flies, final (female) sample sizes were: (a) at 20°C, npolyandry = 74 and nmonogamy = 76, (b) at 24°C: npolyandry = 72 and nmonogamy = 77, and (c) at 28°C: npolyandry = 70 and nmonogamy = 75. We estimated the overall degree of male harm by calculating relative harm (H) following Yun et al., 2021: H=Wmonogamy −WpolyandryWmonogamy where W corresponds to female fitness. Thus, H provides an estimate of the relative decrease in female fitness due to male harm. Using the data collected above, we partitioned overall LRS effects into effects on early reproductive rate (i.e. offspring produced during the first two weeks of age), actuarial aging (i.e. lifespan), and reproductive aging (i.e. offspring produced over weeks 1–2 vs. 3–4). We used weeks 3–4 as an estimate of late reproductive rate because mortality was already evident at this point (i.e. reflecting aging) and then was very high from week 5 onwards (Figure 4—figure supplement 1; thus preventing accurate estimation of reproductive success). Finally, we also calculated rate-sensitive fitness estimates for each individual female and treatment population. Rate-sensitive fitness estimates take into account when offspring are produced, not just how many offspring are produced, and thus allow estimating fitness subject to the population growth rate (Edward et al., 2011). It is important to understand how differences in the number and timing of offspring production translate into fitness under different demographic scenarios. For example, early reproduction is particularly favored in increasing populations whereas late reproduction gains in importance in decreasing populations. Thus, while LRS is most suited to estimate individual fitness in stable populations, rate-sensitive estimates are preferred when r ≠ 0 (Brommer et al., 2002). We calculated both individual (ωind) and population (ωpop) rate-sensitive fitness for the following intrinsic rates of population growth: r=–0.1, r=–0.05, r=0, r=0.05, and r=0.1 (see Edward et al., 2011 for a detailed account). We then used ωpop values to calculate the relative cost (Cr) of polyandry for each temperature treatment across different values of r as: Cr=ωpop (polyandry)ωpop(monogamy) Behavioral measures Request a detailed protocol Immediately after the fitness experiment started, we conducted behavioral observations on the first day of the experiment across all treatments (Figure 1—figure supplement 1). Our aim was to investigate the behavioral mechanisms that might underlie the potential fitness effects evaluated above. Due to logistic limitations, we conducted behavioral observations in the same temperature control room, so we had to conduct trials at 20°C, 24°C, and 28°C over three consecutive days (with both monogamy and polyandry treatments evaluated at the same time for each temperature), in a randomized order (i.e. 20°C, 28°C, and 24°C). Note that we collected virgin flies over three consecutive days to ensure all flies were 5 days-old at the start of the experiment. We recorded the following behaviors: (a) courtship intensity (number of courting males per female per hour), (b) male-male aggression rate (i.e. number of aggressions per hour), and (c) female rejection (i.e. number of rejections per hour; see Bastock and Manning, 1955; Connolly and Cook, 1973 for behavioral descriptions). 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 behaviors 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 the recording of all matings (see below). Scans consisted in observing all vials in succession for ca. 3 s each and recording all occurrences of the behaviors listed above (i.e. all-occurrences recording of target behaviors combined with scan sampling). We interspersed behavioral scans with very quick (<1 min) mating scans where we rapidly swept all vials for copulas at the beginning, in the middle, and at the end of each complete scan. This strategy ensured that we recorded all successful matings (>10 min), which typically last between 15 and 25 min in our population of D. melanogaster. We obtained a total of 49 scans per vial. Behavioral observations were conducted only once, on day 1 of the fitness experiment, as prior experiments have shown that courtship, aggressive and female rejection behaviors in D. melanogaster are sufficiently stable over time so that our behavioral indexes are representative of long-term treatment differences (e.g. Carazo et al., 2015; Carazo et al., 2014). In contrast to courtship, aggression, and rejection indexes, note that total mating frequency over the first day cannot be taken as a reliable measure of mating rate (Wolfner, 1997). Thus, our rationale in recording this variable was just to ensure that early mating ensued normally across treatments (which was the case, see Figure 5—figure supplement 2), but we did not include this variable in our statistical analyses. Mating effect on female reproduction and survival (experiments 2 to 5) To examine post-mating mechanisms that might underlie the fitness effects observed in our first experiment, we conducted four additional experiments to test whether temperature modulates the well-documented effects that mating with a male has on female receptivity, short-term fecundity, and survival. In D. melanogaster, males manipulate female reproduction via their ejaculate, which increases male fitness but frequently decreases female lifespan and lifetime reproductive success (Chapman et al., 1995). Briefly, males transfer seminal fluid proteins (SFPs) produced by their accessory glands that stimulate female short-term fecundity, decrease female receptivity, and ensure sperm storage, thus generally promoting male success in sperm competition (Chapman et al., 1995; Wigby and Chapman, 2005). In addition, prior studies have shown that males are able to tailor investment into SFPs according to perceived sperm competition risk (SCR) and intensity (Hopkins et al., 2019). Thus, we set-up a factorial design where we manipulated the temperature (i.e. 20°C, 24°C, and 28°C) and perceived SCR levels (i.e. males kept alone vs. with 7 more males in a vial) at which adult focal males were kept prior to mating. Then, we measured how the reception of a treated male’s ejaculate after a single mating in a common garden environment (i.e. all matings at 24 °C) affected female fecundity, survival, and reproduction, following standard assays to gauge male ejaculate effects on females in D. melanogaster (e.g. Chapman et al., 1995; Perry et al., 2013; Wigby and Chapman, 2005; Wigby et al., 2009). We conducted separate experiments implementing two different temperature treatment durations (i.e. 48 hr and 13 days), to include two potential different scenarios. Our 48 hr treatment aimed to mimic short-term temperature effects on adult males whereas our 13 day treatment aimed to mimic longer-term effects on adult males that span a complete spermatogenesis cycle. The period from the synthesis of deoxyribonucleic acid in the spermatocyte to successful insemination is approximately 10 days long in D. melanogaster (Chandley and Bateman, 1962), so we treated males for 13 days after sperm depleting them (see below) to ensure that males experienced treatment temperatures across the whole spermatogenesis cycle. Receptivity assays (experiments 2 and 3) Request a detailed protocol We first collected focal males as virgins (i.e. within 6 hr of eclosion) under ice anesthesia and randomly placed them either individually (low SCR) or in a same-sex group of eight (high SCR) in plastic vials with food. Next, we randomly divided them into three groups that we allocated to the different stable temperature treatments for either 48 hr (i.e. short treatment duration, experiment 2, Figure 1—figure supplement 2) or 13 days (i.e. long treatment duration, experiment 3, Figure 1—figure supplement 3) immediately before the beginning of each experiment. For experiment 3, we depleted the sperm and seminal fluid of focal males before allocating them to different temperature treatments by housing them with four standard virgin females for 24 hr, given that three successive matings are enough to deplete the accessory glands of male D. melanogaster (Linklater et al., 2007; Macartney et al., 2021). We collected all females and competitor males (i.e. standard males without any previous treatment) used in receptivity assays as virgins and held them in same-sex groups of 15–20 flies at 24 ± 4°C. Experiments started by exposing all virgin females to single focal males for 2.5 hr at 24°C. After a successful copulation, we separated the mated females from the focal males and isolated them until the remating trial. We discarded unmated females and focal males. 72 hr after this first mating with the focal treated male, we individually exposed females to single virgin competitor males for 12 hr. After each trial, we transferred unmated females into a new vial with food, until the next remating trial on the next day (Figure 1—figure supplements 2 and 3). We repeated remating trials for three consecutive days, which allowed us to calculate the cumulative percentage of remated females (and associated re-mating latencies; see below) for each of the three days of each experiment. Due to a large number of vials/flies involved, we conducted the experiments in two blocks each: with n=390 females per batch in experiment 2 (n=436 rematings) and n=420 females per batch in experiment 3 (n=676 rematings). We also recorded mating duration for the first mating (i.e. with the focal treated male), the remating latency (i.e. the time lapse between males being introduced into the female-containing vial and copulation), and mating duration for re-matings. Females and focal and competitor males were 4 days old for experiment 2. In experiment 3, females and competitor males were 4 days old, while focal males were 18 days old. Fecundity and survival assays (experiments 4 and 5) Request a detailed protocol To study the effects of a single mating on female short-term fecundity and long-term survival, we performed two experiments (experiments 4 and 5, Figure 1—figure supplements 4 and 5, respectively) where we compared female fecundity and F1 egg-to-adult viability of females mated with male flies subject to the same factorial design imposed in receptivity experiments (here experiment 4 had a treatment duration of 48 hr while experiment 5 had a treatment duration of 13 days). We collected and treated all focal males as in the receptivity assays described above, and then proceeded to mate virgin females in single pairs with focal males for 2.5 hr at 24°C. After copulation, we separated mated females from focal males and kept them individually in single vials. We discarded unmated females and focal males. We then transferred females to fresh vials every 24 hr for 4 days, and then every 3 days twice. Finally, we followed females until they died by combining them into same-treatment vials of 10 females that were flipped once a week. We removed dead flies at each flip and scored deaths on a daily basis. We counted eggs laid during the first 3 days and incubated vials from days 1, 2, 3, 4, 5, and 8 until adults emerged to count progeny and determine egg-to-adult viability (Figure 1—figure supplements 4 and 5). Sample sizes were 545 females for experiment 4, and 480 females for exper