Genetic Variation In Male And Female Reproductive Characters

Behavior Genetics, Vol. 35, No. 4, July 2005 (
2005) DOI: 10.1007/s10519-004-1246-8

Genetic Variation in Male and Female Reproductive Characters Associated with Sexual Conflict in Drosophila melanogaster Urban Friberg1–3 Received 20 July 2004—Final 5 Nov. 2004

Recent studies have shown that elevated mating, courtship and seminal substances affect female fitness negatively in Drosophila melanogaster. It has also been shown that males vary with respect to these characters and that male harm to females correlates positively with components of male fitness. These results suggest that there is sexual conflict over the effect of such male characters. An important component of this scenario is that females have evolved counteradaptations to male harm, but so far there is limited evidence for this. Here I define female resistance as the ability to withstand an increased exposure to males. Across 10 genetically differentiated lines of D. melanogaster, I found genetic variation among females in the reduction of lifespan that followed from exposure to males of different durations. There was also genetic variation among males with regards to the degree to which they decrease the lifespan of their mates. These results suggest that genetic variation for female ability to endure male sexually antagonistic adaptations exists and may play an important role in male–female coevolution. KEY WORDS: Drosophila melanogaster; female resistance; male harm; seminal fluid; sexual antagonism; sexual selection.

accessory gland proteins in their ejaculate (Chapman et al., 1995) and male genotypes differentially affect female lifespan after only one mating (Civetta and Clark, 2000; Sawby and Hughes, 2001). Further, fecundity and lifespan of females continuously housed with males vary with male phenotype, where larger males impose higher costs to females primarily as a result of elevated courtship and mating (Friberg and Arnqvist, 2003; Pitnick and Garcı´ a-Gonza´lez, 2002). Male harm to females has also proven to be genetically variable, as shown in several selection experiments with D. melanogaster (Holland and Rice, 1999; Rice, 1996) and Sepsis cynipsea (Martin and Hosken, 2003). Other studies have examined variation in male reproductive characters, believed to correlate with male fitness. In D. melanogaster, these include traits that are employed in both pre- and post-mating interactions since females mate multiply and store sperm from several males (Gromko and Pyle, 1978; Imhof et al., 1998). Success in achieving matings depends on courtship, and several studies have shown that high male courtship activity is associated with short time to first mating (e.g., Bastock, 1956;

INTRODUCTION Females may fare better by limiting their interactions with males after having received enough sperm to fertilize their eggs, simply because male–female interactions involve direct costs to females in many species (e.g., Fowler and Partridge, 1989; Hurst et al., 1995; Magurran and Seghers, 1994; Partridge and Fowler, 1990; von Helversen and von Helversen, 1991; Watson et al., 1998). The fruit fly, Drosophila melanogaster, has been particularly well studied in this respect. Several recent studies of this species have revealed substantial costs to females, in terms of reduced lifespan and lifetime fecundity, imposed by males. For example, females mated to normal males have reduced lifespan compared to females mated to males that lack 1

2

3

Department of Ecology and Environmental Science, Section of Animal Ecology, Umea˚ University, 901 87, Umea˚, Sweden. Department of Animal Ecology, Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 18d, 752 36, Uppsala, Sweden. To whom correspondence should be addressed at Department of Animal Ecology Evolutionary Biology Centre, Uppsala University Norbyva¨gen 18d, 752 36, Uppsala, Sweden.

455 0001-8244/05/0700-0455/0
2005 Springer Science+Business Media, Inc.

456 Gromko, 1987; Manning, 1963; Partridge et al., 1987a). The level of additive genetic variation in courtship and mating speed was earlier considered to be low (Gromko, 1987; Stamenkovic-Radak et al., 1992) but more recent and accurate estimates have found both courtship and mating speed to be highly heritable (Hoffmann, 1999). Sperm competition ability also varies among male genotypes, both with regards to displacing sperm from previous matings, stored in females, and with regards to defending sperm when females remate (e.g., Clark et al., 1995, 2000; Hughes, 1997; Prout and Bundgaard, 1977). The very same male traits that cause harm to females thus also seem to contribute to variation in fitness among males. This has also been directly confirmed for sperm competition ability, which was found to correlate with female mortality rate by Civetta and Clark (2000). Further evidence for an association between harm to females and male fitness was documented by Rice (1996), who allowed males to evolve to a static female genotype. Selected males had higher fitness than control males, but also induced higher female mortality rate. Other artificial selection experiments, where males and females have coevolved under monogamy or polyandry, have added support by showing that monandrous males were less harmful to females but also less competitive when tested against a standard male competitor (Holland and Rice, 1999, Pitnick et al., 2001a). The above findings suggest that males cause harm to females because it enhances a male’s relative fertilization success. Sexual conflict theory predicts that such sexually antagonistic traits could evolve when the interests of interacting males and females do not coincide (Rice, 2000). Females are then expected to evolve counteradaptations to neutralize the detrimental effects of these male adaptations, resulting in sexually antagonistic coevolution. Unfortunately, relatively few studies have so far investigated female counteradaptations/resistance (but see Holland and Rice, 1999; Pitnick et al., 2001b; Wigby and Chapman, 2004 for D. melanogaster and Arnqvist and Rowe, 1995, 2002; Morrow and Arnqvist, 2003 for other species). The aim of this study was to test for genetic variation in the deleterious effects that males have on females on the one hand, and in female ability to resist such effects on the other. This was achieved by measuring female lifespan when exposed to males for either a limited time or throughout their life, across 10 genetically differentiated lines of D. melanogaster, extracted from the same population.

Friberg MATERIALS AND METHODS Fly Stock Flies used in this study were originally collected at Mas Canet, France in 1999. They have since been maintained in mass culture in population cages with overlapping generations (see Bangham et al., 2003 for details). From this laboratory population, 30 randomly chosen mated females were used to found 30 inbred lines. Every new generation, following the first, was started using a single female mated to a brother, a procedure repeated for more than 30 generations. All lines were thereafter maintained at a low population density until the start of the experiments. All flies were fed with standard cornmeal food and maintained at 25C at a 12 h:12 h light:dark photoperiod in rearing cabinets. Methods Ten lines were selected for the experiments. All experimental flies were reared in 200 ml bottles, each started with 10 pairs of flies. Virgin males and females were collected from these vials day 10 to 12 within 5 h of eclosion and stored in vials separated by sex and line. All handling of flies were conducted under light anesthesia (CO2), always performed at least 3 h after the files had eclosed. Male and Female Influence on Female Lifespan For the purpose of this study, I regard female resistance to antagonistic adaptations in males as the inverse of the reduction in female lifespan that follows from an increased exposure to males. Ideally, both antagonistic effects and resistance to these should be measured directly in female fitness, but this is logistically problematical. Using lifespan as a proxy for fitness is clearly an approximation, but there are at least two reasons for why it is reasonable in this context. First, in fruit fly populations cultured under these conditions, with overlapping generations, the phenotypic correlation between lifespan and female fitness is very high, ranging from r = 0.67–0.76 (Friberg and Arnqvist, 2003; Kidwell and Malick, 1967; Partridge, 1988; Tantawy and Rakha, 1964). Second, most studies of sexually antagonistic effects of male adaptations in Drosophila have documented effects primarily on female lifespan rather than, for example, age-specific fecundities (Friberg and Arnqvist, 2003; Rice, 1996; Sawby and Hughes, 2001; Wigby and Chapman, 2004).

Sexual Conflict in Drosophila melanogaster Females were housed individually with a single male each in a vial (100 mm depth, 27 mm diameter) containing 10 ml of food medium sprinkled with live yeast. Males were either removed after 25 h (limited exposure) or left to cohabit with the female for her entire life (lifetime exposure). All males and females were approximately 24-h-old at the start of the experiment. Young females were used as most females mate within the first 24 h after they eclose (Ashburner, 1989). To be sure that all flies in the experiments were reproductively functional, all females were checked for larval production after 3 days. Females that failed to produce larvae in that period of time were discarded and new replicates were started to replace them. Every 7th day, the experimental flies were transferred, under light anesthesia, to fresh vials until the death of the female. In the lifetime exposure treatment, males were replaced every 10th day with a new 24-h-old male. If a male died before his 10th day, a 24-h-old male replaced him no later than the following day. Female survival was monitored daily by observations through the glassware. Flies that look dead were examined more carefully by observing them through a microscope. The procedure described above was repeated until all females had died. Each female line was exposed to males from all the 10 lines, giving 100 combinations per male exposure treatment. All combinations were replicated with five females in both exposure treatments (Ntot = 1000 females). Male and Female Mating Activity and Size I also measured mating activity in a separate experiment, for both sexes in all 10 lines. Three-dayold virgin flies (5 females and 7 males) were shaken together, without the use of anesthesia, in a vial (100 mm depth, 27 mm diameter) containing 10 ml food medium. After 15 min, the number of copulating pairs was recorded. Because a mating in D. melanogaster takes approximately 20 min (Ashburner, 1989), no matings should have terminated within this 15-min period. Again, a full factorial design was employed (100 combinations) with five replicates of each combination (Ntot = 500 vials). I measured the thorax length of 20 males and females from each line. The thorax was measured from the midpoint of the anterior margin of the thorax to the distal midpoint of the scutellum, using the ocular micrometer of a binocular microscope. The flies measured were not included in any of the

457 experiments. Thorax length has proven to be a good measure of general size and correlates closely with other measures of body size, such as wing length (Robertson and Reeve, 1952). Statistical evaluations were performed with JMP and SYSTAT, using general linear models (GLM) and Pearson product moment correlations. Residuals from all analyses were checked and found not to depart significantly from normality (Sharpio Wilk test, all p > 0.42), unless otherwise stated.

RESULTS Male and Female Influence on Female Lifespan Differences in lifespan among females exposed to different male genotypes can be interpreted as a result of variation in the detrimental, rather than beneficial, effects males have on females, since male D. melanogaster do not transfer nutrients to females (Chapman and Partridge, 1994; Pitnick et al., 1997). As shown in Table I(A) and (B), male genotypes varied significantly in their effect on female lifespan under both exposure treatments (mean female lifespan under limited and lifetime exposure, respectively: 31.4 S.D. 2.8 days and 21.7 S.D. 4.1 days). Estimating female resistance to male harm is more problematical. Genotypes vary inherently in general viability and a simple measure of variance in female lifespan after an exposure to males would represent a mixture of variation in female viability and in resistance to males. This may be particularly true for differences among inbred genotypes. As expected, female lifespan across female genotypes was highly correlated between the two exposure treatments (r = 0.94; p < 0.001). The effect of female line per se in Table I(A) and (B) is therefore not very informative. Female lifespan for a particular male genotype in one exposure treatment was, however, not correlated with that in the other exposure treatment (r = 0.15; p = 0.69), indicating that inbreeding was unlikely to explain variation among male genotypes. A measure of female resistance that controls for differences in viability across lines is the reduction in lifespan that occurs between the two exposures treatments. I first analyzed variation across genotypes in this reduction in a full three-way analysis of variance (see Table I(C)). Exposure treatment had a major impact on female lifespan, showing that an increased exposure to males indeed reduced female lifespan. Interestingly enough, the effect of male line differed in the two exposure treatments (male

458

Friberg

Table I. Analysis of variance of the effects of male and female genotype, in D. melanogaster, on female lifespan under either (A) limited exposure to males or (B) lifelong exposure to males. (C) Both treatments analyzed in a joint three-way analysis of variance Source

SS

df

MS

F-ratio

p

(A) Male Female Male · Female Error (R2 = 0.403)

3739.2 32,554.7 13,269.9 73,568.5

9 9 81 388

415.5 3617.2 163.8 189.6

2.19 19.08 0.86

0.022 <0.001 0.786

(B) Male Female Male · Female Error (R2 = 0.458)

7313.7 17,107.7 9974.7 40,633.5

9 9 81 393

812.6 1900.9 123.1 103.4

7.86 18.38 1.19

<0.001 <0.001 0.143

22786.0 5249.4 703.7 142.8 299.0 533.8 144.4 781

155.83 35.90 4.81 0.98 2.04 3.65 0.99 146.2

<0.001 <0.001 <0.001 0.539 0.032 <0.001 0.513

(C) Treatment Female Male Female · Male Treatment · Female Treatment · Male Treatment · Female · Male Error (R2 = 0.483)

22,786.0 47,244.5 6334.1 11,569.9 2690.9 4804.4 11,692.9

line · treatment interaction), suggesting that male genotypes differ in their relative lifespan reducing effects when kept with females for a very short period versus their entire lifetime. The absolute decline in female lifespan between treatments differed across female genotypes, revealing

Lifespan (days)

40

30

20

Limited exposure

Lifetime exposure

Fig. 1. Mean female lifespan for the 10 lines of D. melanogaster, in the two different exposure levels. Female lines differed significantly in the amount to which their lifespan was reduced when continuously exposed to males.

1 9 9 81 9 9 81 11,4202.0

genetic variation in this aspect of female resistance (see female · treatment interaction in Table I(C) and Figure 1). To assess genetic variation in relative decline in female lifespan, I first estimated the expected lifespan from predicted values for each female and male line under limited exposure to males (i.e., model given in Table I(A), but without the interaction term). For all females, the observed lifespan under lifetime exposure was then divided with that predicted from their own and their mates’ genotype under limited exposure, yielding the proportion of lifespan in the lifetime exposure treatment in comparison to the lifespan in the limited exposure treatment. A one-way analysis of variance showed a significant effect of female line on this relative reduction in lifespan (F9,483 = 2.104, p = 0.028), but no transformation of data fully normalized the residuals of this model. However, a resampling test of this one-way analysis of variance, involving bootstrapping the residuals of the original model (see Manly, 1997), confirmed an effect of female line (p = 0.036, based on 9999 bootstrap replicates). Hence, there was genetic variation across lines in both absolute and relative aspects of female resistance. Male and Female Mating Activity and Size Both male and female line influenced the number of flies that copulated within 15 min in the mating

Sexual Conflict in Drosophila melanogaster

459

Table II. Analysis of variance of the effects of male and female genotype, in D. melanogaster, on the number of pairs in copula after 15 min, where each replicate consisted of 5 females and 7 males Source Male Female Male · Female Error (R2 = 0.585)

SS

df

MS

F-ratio

p

563.2 150.4 212.2 656.8

9 9 81 400

62.6 16.7 2.6 1.6

38.11 10.18 1.60

<0.001 <0.001 0.002

activity experiment (Table II). Interestingly enough, the specific combination of male and female line was also of importance for the mating activity seen, as revealed by the interaction term in Table II. I also assessed whether male and female mating activities correlated to their effects upon female lifespan. One hypothesis is that the quicker a male genotype is to initiate matings, the more that genotype would reduce female lifespan under lifetime exposure to males simply because females may suffer higher mating rates. Conversely, female genotypes that were faster to mate might also mate more frequently and thus be less resistant. Mating activity was scored as the average number of matings a given genotype was involved in after 15 min (see Materials and methods) with distinct values for male and female genotypes. However, male mating activity did not significantly correlate with their lifespan reducing effects in females (r = 0.19 for absolute and r = 0.07 for relative reduction) and female mating activity was not significantly correlated with their resistance (r =)0.35 for absolute and r = 0.02 for relative reduction, n = 10, p > 0.3 in all cases). Male as well as female size varied between the lines (males: F9,199 = 8.378, p < 0.0001; females: F9,199 = 15.297, p < 0.0001), but the coefficients of variation were small (males 0.88, females 3.02). Earlier contributions have shown that male size correlates positively with how fast males achieve matings (Ewing, 1961, 1964; Markow, 1986, 1988; Partridge et al., 1987a, b; Partridge and Farquhar, 1983; Pitnick, 1991; Wilkinson, 1987) and harm to females (Friberg and Arnqvist, 2003; Pitnick and Garcı´ a-Gonza´lez, 2002). This was, however, not apparent among these lines (male size versus mating activity r = 0.06, p = 0.87; male size versus absolute female lifespan reduction r = 0.13, p = 0.72). I also tested to see if female size was associated with female resistance, as larger females may be better able to cope with courting males and/or seminal fluid. However, no such association was

found (absolute reduction; r = 0.29, p = 0.42). All correlations using relative reductions gave similar results. DISCUSSION I have documented genetic variation (1) among males in the extent to which males reduce female lifespan and (2) among females for resistance to this lifespan reducing effect of males. Here, I discuss each of these main results. It is worth noting that the female lifespan reducing effect that males had (henceforth, male harm to females) differed in the two exposure treatments, since the effects across male genotypes were uncorrelated in these two environments. I suggest that the effects seen in the limited exposure treatment were, at least on a relative scale, primarily due to the deleterious effects of the seminal fluid (see Chapman et al., 1995; Civetta and Clark, 2000; Sawby and Hughes, 2001). In the lifetime exposure treatment, in contrast, effects of the seminal fluid per se were no doubt augmented to a much higher degree by the costs of remating and male harassment (Fowler and Partridge, 1989; Partridge and Fowler, 1990). Vigorously courting males are likely to shorten female lifespan by frequently disturbing them while males successful in mating should induce higher mating rates with the result of more frequent transfer of seminal fluid. I tested for the latter of these two possibilities, but found no significant correlation between male harm and male mating activity. This might, however, simply be because male mating activity with virgin females is a poor predictor of their lifetime mating rate. In any case, it is clear that there was genetic variation among males in the degree of harm they impose upon females, and there seem to be distinct and uncorrelated genetic components of harm possibly relating to seminal fluid versus behavioral traits. I also documented genetic variation in female resistance to male harm. To the extent that a lifespan reduction in these experiments translates into a true fitness cost to females (see Materials and methods), female resistance could be evolvable. This was recently confirmed in a selection experiment, where females selected under high male density lived longer when continuously housed with males compared to females selected under low male density (Wigby and Chapman, 2004). It is more difficult to elucidate which male trait(s) the detected resistance is directed towards, but it could potentially include counteradaptations to any or all of male traits with deleterious

460 effects to females (e.g., seminal fluid, remating or courtship). That variation in female mating rate should be associated with variation in resistance was not supported here, as female mating activity did not correlate with any measure of resistance. Again, I note that female mating activity as measured here might be a poor estimate of a particular female genotype’s remating frequency. Although males apparently cause harm to females in several distinct ways, some are likely to be more costly than others. No study has so far tried to compare the costs from different sources of male traits to female fitness. An experiment addressing this question would provide valuable information of which male traits should bring about the strongest selection for resistance in females. Female D. melanogaster run out of sperm to fertilize their eggs with, if sperm stores are not replenished (Pyle and Gromko, 1978). In the limited exposure treatment females had no access to males after the first 25 h and therefore had emptied their sperm stores before the end of their lives. If female genotypes varied in their ability to withstand an increased cost of producing fertile eggs, in the lifetime compared to the limited male exposure treatment, differences in cost of reproduction could be an alternative explanation to female resistance, for the patterns found. This is however not likely to be the case here, since females of this species do not cease to produce eggs when they run out of sperm (Partridge et al., 1986), but continue egg production at the same rate. Differences among genotypes in ability to resist male harm is hence a more plausible explanation to the interaction between female genotype and exposure treatment. A few studies have reported that premating (Casares et al., 1993) as well as post-mating (Clark et al., 1999) success of a male genotype is dependent on the female genotype. The results from the mating experiments in this study largely support those of Casares et al., (1993) in the sense that male mating activity was dependent upon the specific combination of the male and female genotype. Additive effects of male and female genotypes were, however, stronger than the interaction, suggesting that genetic interactions are of subordinate importance in determining mating speed. I did not find any significant genetic interactions between the sexes concerning male harm and/or female resistance. Genetic interactions between the sexes are expected when the variation in male and female traits is either qualitative or is the cumulated result of quantitative variation in many different traits (see Andre´s and Arnqvist, 2001). Since

Friberg male genotypes varied in harm in distinct ways in this study, the lack of an interaction between male harm and female resistance suggests that females mainly varied in resistance with respect to a single aspect of male harm. The lack of interactions between male and female genotypes for harm and/or female resistance do not support models where genetic variation in female resistance and male harm is based on frequency dependent selection for male harm and female resistance alleles (Gavrilets, 2000; Haygood, 2004). Classical sexual selection theory focuses on the various benefits females might gain from choosing one male over the other. Sexual conflict theory, on the contrary, sometimes assumes males to vary in the direct costs they impose upon females (Gavrilets et al., 2001). In response to these two different modes of sexual selection, females are predicted to evolve preference for benefits in the first case and resistance to costs in the second. Preference for benefits will result in a fertilization bias towards males that offer the greatest benefits. One might therefore conclude that resistance should cause females to preferentially engage with males that are least costly to them. This is, however, not necessarily true. Sexual conflict will result in male reproductive success being biased towards the most harmful males. This follows from the fact that harmful male traits exist only because they give males that express them an advantage in reproductive competition with other males. Female resistance can only reduce costs from interacting with males and may not affect how males are ranked based on their manipulative qualities (but see Gavrilets, 2000; Haygood, 2004). Needless to say, reducing female fitness does not per se benefit males. Harm to females seems to arise as an indirect pleitropic effect of genes which are otherwise beneficial to males (see Morrow et al., 2003). An interesting question concerning the coevolution of male manipulative traits and female resistance traits is if they will become genetically correlated. A simple model of premating sexual conflict over mating rate predicts a positive genetic correlation between male manipulative traits and female resistance traits (Holland and Rice, 1998). By this, resistant females achieve two-fold advantages as they both avoid the direct costs associated with excessive mating and may receive indirect benefits from mating with the most manipulative males (see Chapman et al., 2003). Whether a positive correlation between male and female traits involved in postmating sexual conflict will result is, however, less obvious. I note that the genetic correlation between male harm and female resistance across genotypes

Sexual Conflict in Drosophila melanogaster was not significant in my experiments (r = )0.01 for absolute harm/resistance and r = 0.27 for relative harm/resistance; p > 0.3 and n = 10 in both cases), although the power of this test was obviously limited. In conclusion, this study confirmed earlier results that males vary in their harm to females but also showed that females vary in their resistance to this harm. These findings suggest that sexually antagonistic coevolution may indeed play an important part in shaping the interactions between the sexes.

ACKNOWLEDGMENTS I thank Jenny Bangham and Tracy Chapman for providing the flies and Annika Karlsson, Claudia Fricke, Martin Edvardsson and Tina Nilsson for assisting in the laboratory. Go¨ran Arnqvist provided much help with writing and statistical analyses. I also thank Edward H. Morrow, William R. Rice for many illuminating discussions and Lars Gustafsson and Edward H. Morrow for helpful comments on an earlier draft of the manuscript. This study was financially supported by the Swedish Research Council (grant to G. Arnqvist) and by Helge Ax:son Johnsons Stiftelse and J.C. Kempes Minnes Stipendiefond to U.F.

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