Male perception of female mating status: its effect on copulation duration, sperm defence and female fitness

ANIMAL BEHAVIOUR, 2006, 72, 1259e1268 doi:10.1016/j.anbehav.2006.03.021

Male perception of female mating status: its effect on copulation duration, sperm defence and female fitness URBAN FRIBERG

Animal Ecology, Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University and Department of Ecology and Environmental Science, Section of Animal Ecology, Umea˚ University, Sweden (Received 2 December 2005; initial acceptance 26 January 2006; final acceptance 6 March 2006; published online 2 October 2006; MS. number: 8761)

When females mate with multiple partners, the risk of sperm competition depends on female mating history. To maximize fitness, males should adjust their copulatory investments according to this risk. In the fruit fly, Drosophila melanogaster, the female cuticular hydrocarbon (CH) profile changes when females mate, and males use this to assess female mating status. I tested whether this cue influenced the time males spent copulating with females and if this affected male fertilization success and female fitness. I manipulated female mating status by transferring CHs from either virgin or mated females to virgin females. Males copulated significantly longer with virgin females that had been coated with CHs from mated females (experimental group) than with virgin females coated with CHs from other virgin females (control group). Copulation duration did not differ between females from the experimental group and females that had already mated. To test whether differential investment in copulation affected male sperm defence and female fitness, experimental and control females were mated once to wild-type males and then either housed with males carrying a genetic marker (experiment 1) or alone (experiment 2). In experiment 1 male sperm defence was elevated when males perceived their partner as mated, and this was mainly due to females remating less. Increased male investment in copulation duration also affected female fitness, although this was reversed between experiments 1 and 2. Finally, these results also indicate that copulations are costly to males, since manipulated males copulated for longer with virgin females than they normally would, resulting in higher fertilization success. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Parker’s (1970) proposal that sperm competition should form a potent evolutionary force, shaping many aspects of male reproduction, has now been verified by many studies (reviewed in Birkhead & Møller 1998; Simmons 2001). Success in sperm competition often relies on a whole suite of adaptations (involving behavioural, morphological and physiological traits) that act before, during and/or after copulation (Parker 1970; Simmons 2001). Further success can be gained if investments in different aspects of mating are allocated in accordance with the risk of sperm competition (i.e. cryptic male mate choice or strategic ejaculation), which is especially important when matings are costly to males (Dewsbury 1982; Simmons et al. 1994; Bonduriansky 2001; Wedell et al. 2002). Cryptic male mate choice requires that males can estimate the current risk of sperm competition. One source of Correspondence: U. Friberg, Animal Ecology, Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 18d, SE-752 36 Uppsala, Sweden (email: urban.friberg@ ebc.uu.se; [email protected]). 0003e 3472/06/$30.00/0

information that correlates with the risk of sperm competition is the operational sex ratio, which has been shown to affect male investment in copulation duration (Vepsa¨la¨inen & Savolainen 1995) and ejaculate size (Gage 1991; Gage & Baker 1991; delBarco-Trillo & Ferkin 2004) in insects and a mammal, and males’ choice of oviposition sites in a fish (Smith et al. 2003). Another source of information is female condition, which may correlate negatively with the intensity of sperm competition (Engqvist & Sauer 2001). Finally, and maybe most importantly, the risk of sperm competition will depend on female mating status (Lewis & Iannini 1995; Bonduriansky 2001). Several studies, using a variety of insect taxa, have shown that matings with virgin and mated females differ with respect to copulation duration (e.g. Dickinson 1986; Wedell 1992; Andre´s & Cordero-Rivera 2000; Martin & Hosken 2002; Siva-Jothy & Stutt 2003; Singh & Singh 2004) and ejaculate size and content (e.g. Cook & Gage 1995; Cook & Wedell 1996; see also Wedell et al. 2002), although the direct cues that males use to assess female mating status are largely unknown

1259 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

1260

ANIMAL BEHAVIOUR, 72, 6

(but see Rypstra et al. 2003; Siva-Jothy & Stutt 2003; Carazo et al. 2004). Furthermore, although there are many observations of apparent cryptic male mate choice, no study has tested whether such differential investment results in a net increase in fertilization success for males. To test this empirically would require an experimental design where female mating status was kept constant over two groups of males, while the perception of female mating status was manipulated in one group of males. The fruit fly, Drosophila melanogaster, is a species that has been comprehensively studied with respect to how males assess female mating status. These studies have convincingly shown that males are able to distinguish between mated and virgin females, and that they adjust the intensity of their courtship accordingly (Bastock & Manning 1955; Cook 1975; Cook & Cook 1975; Tompkins & Hall 1981). The key cue used by males is the female cuticular hydrocarbon (CH) profile (Tompkins & Hall 1981; Scott et al. 1988), a trait that is commonly used in sexual interactions in insects (e.g. Chenoweth & Blows 2005; Harris & Moore 2005; Ivy et al. 2005). In D. melanogaster the CH profile changes after females have mated (Tompkins & Hall 1981; Anthony & Jallon 1982; Tompkins 1984; Ferveur et al. 1989). Copulation durations also differ between those involving virgin and mated females (Singh & Singh 2004), although whether this modulation is under male control has not been tested. To test this would, again, require a manipulation that made males perceive females as mated when virgin or vice versa. Coyne et al. (1994) observed that CHs can be transferred between females of different species when kept under crowded conditions. Recently, this technique has also proved successful for transfer of CHs within the species D. melanogaster (Marcillac & Ferveur 2004). This therefore seems to be an ideal species for testing whether cryptic male mate choice is related to female mating status and, if so, if this provides a net fertilization advantage. A topic closely related to cryptic male mate choice, which has received little attention, is how females are affected by differential male copulatory investments. In D. melanogaster, males transfer a cocktail of proteins in their ejaculate that are thought to mediate male fertilization success (reviewed in Chapman 2001). None of these proteins are expressed in females (Chapman 2001) and several of them have been identified as affecting female reproductive traits, including female receptivity, ovulation and oogenesis (reviewed in Wolfner 1997; Arnqvist & Rowe 2005). This, taken together with the fact that matings have proven to be costly for females in this species (Fowler & Partridge 1989) and that this cost is mediated through seminal proteins (Chapman et al. 1995), suggests that differential male investments in copulations may have fitness consequences for females. In this study, I manipulated male perception of female mating status in D. melanogaster and tested whether differential investment in copulation duration results in a net increase in male fertilization success and whether this has any influence on female fitness. More explicitly I tested (1) whether male perception of female mating status affects male sperm defence, (2) through which mechanism(s) this effect would occur and (3) whether female fitness is affected by male differential copulatory investments.

METHODS

Fly Stocks I used flies from a large outbred laboratory population of D. melanogaster, called LHM. This population is cultured on a 14-day discrete generation cycle. Each generation is started by 56 ‘juvenile competition vials’ (37-ml vials containing 10 ml of cornmealemolassesekilled yeastmedium), each trimmed to contain 150e200 eggs. Larval, pupal and early adult stages reside in these vials for 11.25 days, at which point a thoroughly mixed sample of 1792 adults from the 56 juvenile competition vials is transferred to ‘adult competition vials’ (16 pairs per vial with 11.6 mg, dry weight, of live yeast added on top of the medium). Eighteen hours before the end of the 14-day generation cycle, the flies are transferred to 56 fresh vials where eggs are laid for the next generation, so that fecundity during these 18 h represents total lifetime fecundity in this population. In addition to LHM, I used a replica of this population, LHM-bw. This population is homozygous for the recessive eye mutation bw, causing flies to have brown instead of red eyes. This population was created by backcrossing the bw allele into the LHM base population nine times. Flies were maintained at 25 C on a 12:12 h light:dark cycle in rearing cabinets. For a detailed description of this population, see Rice et al. (2005). By the start of these experiments, the LHM population had adapted to this constant laboratory environment for more than 330 generations. The laboratory environment can thus be considered to approximate this population’s new natural environment (Rice et al. 2005). The combination of this population’s long history of adaptation to the laboratory and a culturing protocol that is easy to mimic during experiments makes this population an ideal tool for comparing fitness, and other life history traits of manipulated and nonmanipulated individuals.

Transfer of Cuticular Hydrocarbons I used Coyne et al.’s (1994) technique of transferring CHs between flies of different species to transfer CHs from mated to virgin females. Nine, newly eclosed, virgin females (LHM-bw) were kept densely together with either 60 other virgin females (LHM) or 60 (2-day-old) mated females (LHM) for 2 days. The latter females were taken from vials where males and females had interacted for 2 days. The flies were kept in food vials with a cotton plug pushed close to the food surface, leaving a space of approximately 3.3 cm3 for the flies to interact within. This equals a density of 20.8 flies/cm3. Under normal rearing conditions the density is approximately 1.3 flies/cm3. Females in the first group (control females) thus became coated with CHs expressed by the virgin females with which they were housed, whereas females in the second group (experimental females) became coated with CHs from mated females.

Copulation Duration To test whether the method of transferring CHs between flies was successful, in terms of influencing male

FRIBERG: MALE ASSESSMENT OF FEMALE MATING STATUS

perception of female mating status, I carried out a copulation duration experiment. Since copulations with mated females last longer than those with virgin females in D. melanogaster (Singh & Singh 2004), I predicted that males would copulate for longer with females from the experimental group than with females from the control group, assuming that copulation duration is at least partly under male control. Copulation duration was defined as the time from when a male successfully mounted a female until the pair disentangled. After 2 days in dense confinement with either virgin or mated females, the focal virgin females (N ¼ 125 control group; N ¼ 130 experimental group) were separated out under light carbon dioxide anaesthesia and put into individual vials. They remained in these vials for at least 2 h (but no more than 5 h), to recover from the anaesthesia, before three (3-day-old) LHM males were added (without the use of anaesthesia). I recorded copulation duration for females that mated within 1 h. To compare the copulation durations obtained for these two groups of virgin females with that of females that had already mated, I tested a third group of females. These females (N ¼ 74) experienced similar conditions to the other two groups of females (i.e. collected as virgins and stored for 2 days in a 3.3-cm3 compartment of a vial), with the difference that they were stored together with males (45 LHM-bw females with 24 LHM males). Although I did not observe these females mating virtually all females can be assumed to have mated, since nearly all virgin females in this population will mate when exposed to males for only 2 h when 2 days old (Rice 1996). Each of these females was then tested with three (3-day-old) LHM males. Copulation duration was recorded for those that remated within 2 h. The mating period was extended for this group of females, since mated females are more reluctant to mate than virgin females. One of the copulations in the control group lasted considerably longer than the other copulations (more than three times the average copulation and more than 11 standard deviations from the mean). This replicate was excluded from the reported analyses. However, the results reported are robust to its inclusion.

Male Sperm Defence and Female Fitness The method of housing virgin females with mated females was successful in terms of making males perceive virgin females as already mated (see Results below). I therefore used this technique to test whether male perception of female mating status affected male fertilization success and female fitness. Male fertilization success was tested only when the focal males were the first males to mate with a female. All males tested were thus in a sperm defence position. The densities and ages of flies, food composition, amounts of live yeast, and timing of events such as mating and egg production closely matched those experienced by the base population. As a result, the flies were tested in essentially the same environment to which they had adapted for over 330 generations.

Experiment 1 In this experiment the influence of male perception of female mating status on male sperm defence was measured. I achieved this by comparing fertilization success of males that perceived their virgin partner as mated with that of males that perceived their virgin partner as virgin. Control and experimental females were produced as above. After 2 days, the focal females from each vial were separated out under anaesthesia and marked by having one of their wings pierced with a sharp needle under a dissecting microscope. The wings were pierced in the middle of the discal cell, which is framed by the wing veins L4 and L5 and by the posterior crossvein. All nine females from the same vial were pierced in the same wing (i.e. left or right). Females from the same vial were then put into a new vial for 2e3 h to recover from the anaesthesia. After the recovery period, I added 14 (3-day-old) LHM males to these vials for 2 h (without the use of anaesthesia). Virtually all virgin female flies of this age, housed with a 50% excess of males, mate once and only once in this population, in this time period (Rice 1996). After 2 h, I put eight females from each treatment group (pierced in the opposite wing, so that they could be distinguished) into an adult competition vial together with 16 LHM-bw males (the remaining ‘spare’ mated females were not used unless needed as a replacement for injured, escaped or dead females). In these adult competition vials the females competed for a limited resource (live yeast, 11.6 mg dry weight), while having the opportunity to remate with the LHM-bw males. Two days later the females were separated out and put into individual oviposition vials for 18 h (Fig. 1). Females were thereafter retained for size measurements. Offspring emerging from these vials were counted and scored for eye colour 11 days after the oviposition. Red-eyed offspring were sired by the focal males and brown-eyed offspring by their competitors (males in the adult competition vials). With this design, several aspects of male defensive fertilization success could be measured. Most important of these was male defence fitness, which is the absolute number of (red-eyed) offspring sired by a male that was the first to mate with a female. Male defence fitness was also partitioned into its two causative components: remating (the fraction of females that remated with a competitor male) and P1 (the fraction of the offspring sired by the focal male when his partner remated). These two traits were also combined into a measure of net defence (the proportion of offspring sired by the focal male, regardless of whether his partner remated). I used total female offspring production (red-eyed plus brown-eyed offspring) as a measure of female fitness. A female was considered as remated if she produced brown-eyed offspring (i.e. offspring fathered by a competitor male). Although the second male’s relative share of the offspring production (P2) is high in D. melanogaster (Simmons 2001; 72.1% among control females and 72.3% among experimental females in the present study), some females that remated might have been classified as nonremated females. Females that produced eggs but no offspring were excluded from analysis in this experiment and in experiment 2.

1261

1262

ANIMAL BEHAVIOUR, 72, 6

Virgin females with virgin females

Virgin females with mated females

Wing piercing of females was alternated between vials for the experimental and control groups. In total, 82 vials with eight females from each treatment were scored in this experiment.

Experiment 2

(a)

Experiment 2 was designed to measure female fitness (offspring production) among experimental and control females, free from any behavioural changes induced by males (such as a differential tendency of females from the two treatment groups to remate). This experiment was similar to the previous in design, but no competitor males were added to the competition vials. Females therefore mated only once. Since both the females and the males included in this experiment mated only once, their fitnesses were identical. In total, 35 vials with eight females from each treatment were scored in this experiment. No difference in fitness was found between females that had been pierced in their left or their right wing (experiment 1: paired t test: t81 ¼ 0.91, P ¼ 0.364; experiment 2: t34 ¼ 0.73, P ¼ 0.471).

Female size (b)

To control for variation in fecundity resulting from variation in female body size, I measured all females included in the fitness assays, except for those that escaped. I used thorax length as a proxy for body size. Thorax length correlates closely with other measures of body size, such as wing length (Robertson & Reeve 1952). I measured thorax length from the midpoint of the anterior margin of the thorax to the distal midpoint of the scutellum. To take measurements I used a digitizing tablet (Summasketch III, CalComp Technology, Inc., Anaheim, CA, U.S.A.) under a sidemounted camera lucida attached to a dissecting microscope (Leica MZ8, Leica AG, Heerbrugg, Switzerland).

Statistical Analyses (c)

(d)

Figure 1. Experimental set-up. (a) Nine virgin females (population LHM-bw) were housed with either 60 other virgin females (population LHM) or 60 mated females (LHM), under crowded conditions for 2 days. (b) One group of the focal females was then pierced in their left wing while the other females were pierced in their right wing. The nine female flies from each group were then housed with 14 males

Effects of the manipulation of female CHs on copulation duration were tested with ANOVA. Multiple post hoc comparisons between the three groups of females (control females, experimental females and mated females) were evaluated with Tukey’s honestly significant difference (HSD) test. Data from experiments 1 and 2 (male and female offspring production, P1, remating and net defence) were analysed with mixed-model ANCOVAs when the assumptions of parametric testing were fulfilled. In those cases maximum likelihood methods, as implemented by the REML algorithm in the JMP statistical software package version. 5.0.1 (SAS Institute, Cary, NC, U.S.A.) were used with treatment (control females, experimental females

(LHM) for 2 h, where females mated. (c) Immediately after mating, eight females from each group were transferred to a new common vial (experiment 1: together with 16 LHM -bw males; experiment 2: without males) where they resided for 2 days while they competed for a limiting resource (live yeast). (d) The females were then separated and placed into individual oviposition vials for 18 h.

FRIBERG: MALE ASSESSMENT OF FEMALE MATING STATUS

and mated females) as a fixed factor, vial as a random factor and female size as a covariate. Several of the variables measured departed severely from normality and no transformation could be found that stabilized the residual distribution of the inferential models. In these cases, paired t tests were conducted, where each pair (replicate) consisted of the two means of the eight females per treatment group within each vial. When replicates were based on means of eight females rather than individual females, all these variables fulfilled assumptions for parametric testing. To account for female size variation, I used the residuals from a model with female size as the independent variable before calculating the means. All paired t tests were weighted with the number of observations per vial (normally 16 [8 per treatment group] but sometimes fewer because of dead or escaped females) as the weight. Results from paired t tests are reported also for those variables that were analysed by ANCOVA. All statistical evaluations were performed in JMP, except for calculation of P values for post hoc comparisons which were performed in SYSTAT version 10 (Systat Software Inc., Port Richmond, CA, U.S.A.).

Male Defence and Female Fitness Experiment 1 Male defence fitness was about 10% higher for males mating with experimental females than for males mating with control females (Table 1, Fig. 3). This was mainly explained by females in the experimental group remating less frequently than females in the control group (Table 1, Fig. 4). Consequently, competitor offence fitness (number of brown-eyed offspring) was lower with females from the experimental group (experimental group mean  SE ¼ 14.5  0.6; control group mean  SE ¼ 17  0.6; Table 1). No effect on P1 was detected (experimental group mean  SE ¼ 27.7  1.3%; control group mean  SE ¼ 27.9  1.2%; Table 1). These results were also reflected in net defence, which was about 13% higher among males mating with experimental females than in those mating with control females (Table 1, Fig. 5). Female fitness was generally unaffected by the treatments, although control females had higher fitness than experimental females within the subset of females that did not remate (Table 1).

Experiment 2 RESULTS

Copulation Duration Copulation durations differed significantly across the three treatment groups (control females, experimental virgins and mated females; F2,326 ¼ 9.58, P < 0.0001). Post hoc comparisons revealed that copulations involving control females were significantly shorter than copulations involving experimental females (P ¼ 0.001) and females that had already mated (P ¼ 0.001; Fig. 2). No difference in copulation duration was detected between matings involving experimental females and females that had already mated (P ¼ 0.845; Fig. 2), indicating that males did not differentiate between these two groups of females.

Copulation duration (s)

1150

b b

1100

Table 1. Effects on male fertilization success and female fitness of males mating with virgin females that they perceived as virgin (control females) and virgin females that they perceived as mated (experimental females) t (F )*,y Experiment 1 (with remating) Male defence 2.06 fitness Among 0.43 remated females Among not 2.00 (5.54) remated females Female fitness 1.03 (1.30) Among remated females Among not remated females Competitor’s offence fitness Remating P1x Net defence

1050 a 1000

Virgins with virgins

Virgins with nonvirgins

Nonvirgins

Female mating status and condition Figure 2. Mean copulation duration SE for virgin females coated with other virgin females’ cuticular hydrocarbons, virgin females coated with mated females’ cuticular hydrocarbons and previously mated females. Bars with different letters are significantly different (P ¼ 0.001).

dfy,z

Py

81

0.043

80

0.665

2.96

81

0.050 (0.019) 0.305 (0.254) 0.843 (0.692) 0.050 (0.019) 0.004

73 (1,439)

3.66 0.45 3.01

81 80 81

0.0004 0.652 0.004

34 (1,508)

0.055 (0.014)

81 (1,1204)

0.19 (0.16)

80 (1,681)

2.00 (5.54)

73 (1,439)

Experiment 2 (without remating) Male and female 1.99 (6.13) fitness

950

900

Since females were not allowed to remate in this experiment, male and female fitness was constrained to be equal. Fitness was significantly higher for males that mated with experimental females than for males that mated with control females (Table 1, Fig. 3). Consequently,

*A positive t value, from weighted paired t tests, indicates that the variable had a higher mean value among experimental than control females. yAnalogous statistics from ANCOVAs are reported in parentheses for variables that fulfilled assumptions for parametric testing on an individual level. zDegrees of freedom vary between tests since some variables were not represented in all vials (e.g. all females remated in some vials). xFraction of offspring sired by focal male when his partner remated.

1263

ANIMAL BEHAVIOUR, 72, 6

70

45

*

50 35

Net defence

Male (defence) fitness

**

60

40

30 25

40 30 20

*

10 20 15

1

2 Experiment

Figure 3. Number of offspring sired by focal males SE. In experiment 1, females had the opportunity to remate and focal male offspring production represents male defence fitness. In experiment 2, females mated only once and male offspring production represents male fitness. ,: Offspring of males mating with females from the experimental group which were perceived as mated; G: offspring of males mating with females from the control group which were perceived as virgin. Means and standard errors are based on raw data. *P < 0.05, see text for details of statistical testing.

female fitness was higher among females in the experimental group. This result was thus opposite to that found within the subset of females that did not remate in experiment 1.

DISCUSSION It is typically assumed that differences in copulation duration between mated and virgin females, which have 0.8 0.7 Proportion remating

1264

***

0.6 0.5 0.4 0.3 0.2 0.1 Mated

Virgin

Males’ perception of female Figure 4. Proportion of females remating SE in experiment 1 among experimental females which were initially perceived as mated and among control females which were initially perceived as virgin. Means and standard errors are based on raw data. ***P < 0.001, see text for details of statistical testing.

Virgin Mated Males’ perception of female Figure 5. Percentage of offspring sired by males SE summing over females that remated as well as those that did not (i.e. net defence) in experiment 1, for males mated to experimental females which were initially perceived as mated and for males mated to control females which were initially perceived as virgin. Means and standard errors are based on raw data. **P < 0.01, see text for details statistical testing.

been noted in many insect species, reflect males adjusting their copulatory investment according to the risk of sperm competition (see Introduction and Simmons & Siva-Jothy 1998 for a review). However, it is often difficult to confirm that a shift in copulation duration is under male control, because it is rarely known what cues males use to assess female mating status and how to manipulate them experimentally (but see Siva-Jothy & Stutt 2003). I found that male perception of female mating status could be manipulated in D. melanogaster, by coating virgin females with CHs expressed by mated females. In response to this manipulation, males mated with virgin females for longer when they perceived them as already mated than when they perceived them as virgin. Furthermore, the average length of copulation in the former group was very similar to that seen in females that had mated. These findings are consistent with previous results showing that copulations with mated D. melanogaster females are longer than those with virgin females (Singh & Singh 2004), but also demonstrate that this shift in copulation duration is, at least to a large extent, under male control. A crucial assumption of sperm competition theory is that copulatory investment relates to fertilization success. Plenty of evidence supporting this assumption exists on an observational level, in terms of studies that show that natural phenotypic variation in copulation duration relates to fertilization success (e.g. McLain 1980; Rubenstein 1989; Thornhill & Sauer 1991; Hadrys et al. 1993; Arnqvist & Danielsson 1999). This assumption has also received support from studies where copulation duration has been experimentally interrupted (e.g. Michiels 1992; Harshman & Prout 1994). However, there are no previous direct tests of whether male modulation of copulation duration according to female mating status results in the predicted adaptive effects on male fertilization success. In the present study, male D. melanogaster prolonged their copulation duration when mating with females they

FRIBERG: MALE ASSESSMENT OF FEMALE MATING STATUS

perceived as mated (i.e. high sperm competition risk) and subsequently increased their fertilization success. However, two different adaptive scenarios, with opposite results, could be predicted from these tests. If males alter copulations qualitatively (e.g. by altering the content of the seminal fluid) when mating with virgin and mated females (because defending sperm from being displaced and replacing stored sperm are conducted by different means), manipulated males would be predicted to have lower fertilization success than unmanipulated males, since manipulated males would not perform in relation to their partner’s mating status. Conversely, if males merely alter copulations quantitatively (e.g. by altering the amount of seminal fluid) then manipulated males would always be predicted to have higher fertilization success when they increase their investments. My results suggest that male D. melanogaster change their copulatory investment quantitatively, rather than qualitatively, since manipulated males prolonged copulations and subsequently increased their fertilization success. The positive effect on fertilization success was manifested in different ways in the two experiments. In experiment 1, the gain in offspring production was mainly indirect, and resulted from experimental females remating to a lesser extent and thus, on average, producing more offspring sired by the focal males. In experiment 2 (where females were not allowed to remate) the positive effect was direct, since experimental females were induced to produce more offspring. This positive effect on female fitness was, however, reversed among the subset of females that did not remate in experiment 1. It is thus not entirely clear from these results whether long copulations have a positive or negative net effect on female fitness. The observation that males inducing higher sperm defence also elevate female fitness is consistent with a previous study that used the same population (Friberg et al. 2005). That study showed that males that were genetically superior sperm defenders also had a positive impact on offspring production among the subset of females that did not remate. In contrast, Gilchrist & Partridge (2000) did not find any relation between lengths of interrupted copulations with virgin females and offspring production. In defensive sperm competitiveness (P1), males mating with experimental and control females did not differ. At least to some extent, this result may reflect males investing in qualitatively different aspects of mating when copulating with virgin and mated females. The mating effort of manipulated males may therefore have been partly misplaced, in the context of sperm competition, and might have affected their offensive sperm competitiveness (P2, the fraction of offspring sired by the second male to mate with a female) occurring in its correct context. Earlier research into the processes of sperm competition in this species does not help resolve whether this hypothesis is true. In D. melanogaster a mating can be divided into two parts. During the first part, males seem to displace sperm that is already present with seminal fluids (Gilchrist & Partridge 2000) or influence females to eject some of the stored sperm (Snook & Hosken 2004). Towards the end of this first part the male then transfers his own sperm. Sperm transfer is rapid and probably does not last longer than a minute

(Gilchrist & Partridge 2000). The remainder of the copulation appears to be devoted entirely to transferring seminal fluids, aimed at reducing the female’s future remating propensity (Gilchrist & Partridge 2000). Since the prolonged matings with the females from the experimental group strongly reduced female remating propensity, it seems likely that males mating with these females put most of their extra investment into prolonging the second part of the mating. This hypothesis receives some support from a study by Morrow et al. (2005). They showed that P1 was not affected by the female’s mating history, since males that were first and second to mate with a female that mated three times had comparable P1 values. Alternatively, if the composition of the seminal fluid is similar throughout a mating, males mating with females from the experimental group may have prolonged the period of displacement before sperm transfer. This would potentially affect P2 occurring in its correct context, but would also reduce female remating propensity. The latter of these two hypotheses could be tested by estimating when sperm are delivered in manipulated and nonmanipulated males. The results described above suggest that prolonged copulations may have a positive effect on female fitness. This result is puzzling since remating, which could conceivably be compared with a single copulation of twice the duration of a normal copulation, is generally costly to female D. melanogaster (Fowler & Partridge 1989; Chapman 1992; Chapman et al. 1993, 1995). However, the fitness cost of remating once is not substantial (about 6%) in the present population (Kuijfer et al. In press). Thus, prolonged copulations may not result in noticeable additional cost to females, especially since the difference in copulation duration between the two treatments was only about 10%. The cost of remating in D. melanogaster is mediated by seminal fluids (Chapman et al. 1995). These seminal fluids do, however, also include some substances that promote female egg production after insemination (reviewed in Wolfner 1997). The presence of seminal fluid components with opposing effects on female fitness may therefore make female fitness peak when some intermediate amount of seminal fluid is received. This amount may be somewhat more than what is received from a single mating but less than what is received from mating twice. Finally, these experiments also indicate that prolonged copulations are costly to male D. melanogaster. This conclusion is based on the result that the manipulated group of males copulated for longer with virgin females than they normally would have and subsequently gained a fertilization benefit from doing so. If prolonging copulation is not costly to males, there would be no trade-off between copulation duration and, for example, searching for or copulating with additional mates, and males would be predicted to mate for longer with virgin females than they normally do. All studies that show male modulation of copulation duration and ejaculate components and/or amounts in relation to the risk of sperm competition point to males being sperm limited or that matings being costly to males in some other dimension (Bonduriansky 2001; Simmons 2001; Wedell et al. 2002). These findings,

1265

1266

ANIMAL BEHAVIOUR, 72, 6

however, have rarely been tested in a fitness context (but see Martin & Hosken 2004). There is more general support for a cost of reproduction to males, including mating, mate searching and courtship (e.g. Partridge & Farquhar 1981; Clutton-Brock & Langley 1997; Prowse & Partridge 1997; Kotiaho et al. 1998; Kotiaho & Simmons 2003). Cordts & Partridge (1996) tested for but failed to detect any cost of mating in D. melanogaster males in terms of decreased survival, and they concluded that courtship alone explains the cost of reproduction to males of this species. However, as stated by the authors themselves, their experimental design allowed only a low male mating frequency (once every fourth day) and their test might therefore have been too conservative to detect male mating costs. In addition to my experiments, it would have been interesting to test for effects on male fertilization success in relation to female mating status from a sperm offence perspective (i.e. by having all females mated in the first place and then converting half of them to be perceived as virgins). However, although it may be possible to make males perceive mated females as virgin, by housing mated with virgin females, it would be difficult to get the majority of these females to remate within a short period of time. Since it is difficult to draw conclusions, on a population level, when only a minor subset of the individuals (possibly genotypes) tested can be included in the analyses, this experiment was not attempted here. Although my study does not provide any chemical evidence for CHs being transferred to the experimental females and for males using this particular cue to assess female mating status, this is by far the most parsimonious explanation. The method I used to transfer CHs between females is known to ‘rub off’ substantial amounts of CHs from ‘donor’ to ‘target’ females (e.g. Coyne et al. 1994, 1999; Coyne & Charlesworth 1997; Savarit et al. 1999; Marcillac & Ferveur 2004), in similar experimental protocols to the one used here. These studies have furthermore shown that this technique is successful in manipulating male perception of females in several other contexts related to courtship and mating, both within and between species of the genus Drosophila. In conclusion, this study shows that male D. melanogaster can assess female mating status and that they adjust their copulatory investments accordingly. Males gained higher fertilization success when investing more in copulations, and this was reached primarily through reducing the risk of sperm competition rather than increasing sperm competitiveness.

Acknowledgments I thank William Rice for kindly providing the LHM and LHM-bw populations, Jean-Franc¸ois Ferveur for providing valuable suggestions on how to manipulate male perception of female mating status, Josefin Friberg and Claudia Fricke for technical assistance and Damian Dowling ¨ ran Arnqvist, Damian Dowfor statistical discussions. Go ling, Josefin Friberg, Claudia Fricke, Mari Katvala, Alexei

Maklakow, Ted Morrow, Johanna Ro¨nn, Andrew Stewart, Allen Moore and two anonymous referees provided helpful comments on the manuscript. This study was supported by grants from Helge Ax:son Johnsons Stiftelse to U.F. and the Swedish Research Council to Go¨ran Arnqvist.

References Andre´s, J. A. & Cordero-Rivera, A. 2000. Copulation duration and fertilization success in a damselfly: an example of cryptic female choice? Animal Behaviour, 59, 695e703. Anthony, C. & Jallon, J. 1982. The chemical basis of sex recognition in Drosophila melanogaster. Journal of Insect Physiology, 28, 873e880. Arnqvist, G. & Danielsson, I. 1999. Postmating sexual selection: the effects of male body size and recovery period on relative paternity success and female egg production rate in a water strider. Behavioral Ecology, 10, 358e365. Arnqvist, G. & Rowe, L. 2005. Sexual Conflict. Princeton, New Jersey: Princeton University Press. Bastock, M. & Manning, A. 1955. The courtship of Drosophila melanogaster. Behaviour, 8, 85e111. Birkhead, T. R. & Møller, A. P. 1998. Sperm Competition and Sexual Selection. London: Academic Press. Bonduriansky, R. 2001. The evolution of male mate choice in insects: a synthesis of ideas and evidence. Biological Reviews, 76, 305e339. Carazo, P., Sanchez, E., Font, E. & Desfilis, E. 2004. Chemosensory cues allow male Tenebrio molitor beetles to assess the reproductive status of potential males. Animal Behaviour, 68, 123e129. Chapman, T. 1992. A cost of mating with males that do not transfer sperm in female Drosophila melanogaster. Journal of Insect Physiology, 38, 223e227. Chapman, T. 2001. Seminal fluid-mediated fitness traits in Drosophila. Heredity, 87, 511e521. Chapman, T., Hutchings, J. & Partridge, L. 1993. No reduction in the cost of mating for Drosophila melanogaster females mating with spermless males. Proceedings of the Royal Society of London, Series B, 253, 211e217. Chapman, T., Liddle, L. F., Kalib, J. M., Wolfner, M. F. & Partridge, L. 1995. Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature, 373, 241e244. Chenoweth, S. F. & Blows, M. W. 2005. Contrasting mutual sexual selection on homologous signal traits in Drosophila serrata. American Naturalist, 165, 281e289. Clutton-Brock, T. & Langley, P. 1997. Persistent courtship reduces male and female longevity in captive tsetse flies Glossina morsitans Westwood (Diptera: Glossinidae). Behavioral Ecology, 8, 392e395. Cook, P. A. & Gage, M. J. G. 1995. Effects of risk of sperm competition on the number of apyrene and eupyrene sperm ejaculated in the moth Plodia interpunctella (Lepidoptera, Pyralidae). Behavioral Ecology and Sociobiology, 36, 261e268. Cook, P. A. & Wedell, N. 1996. Ejaculate dynamics in butterflies: a strategy for maximizing fertilization success? Proceedings of the Royal Society of London, Series B, 263, 1047e1051. Cook, R. 1975. Courtship of Drosophila melanogaster: rejection without extrusion. Behaviour, 52, 155e171. Cook, R. & Cook, A. 1975. The attractiveness to males of female Drosophila melanogaster: effects of mating, age and diet. Animal Behaviour, 23, 521e526. Cordts, R. & Partridge, L. 1996. Courtship reduces longevity of male Drosophila melanogaster. Animal Behaviour, 52, 269e278.

FRIBERG: MALE ASSESSMENT OF FEMALE MATING STATUS

Coyne, J. A. & Charlesworth, B. 1997. Genetics of a pheromonal difference affecting sexual isolation between Drosophila mauritiana and D. sechellia. Genetics, 145, 1015e1030. Coyne, J. A., Crittenden, A. P. & Mah, K. 1994. Genetics and pheromonal difference contributing to reproductive isolation in Drosophila. Science, 265, 1461e1464. Coyne, J. A., Wicker-Thomas, C. & Jallon, J. M. 1999. A gene responsible for a cuticular hydrocarbon polymorphism in Drosophila melanogaster. Genetical Research, 73, 189e203. delBarco-Trillo, J. & Ferkin, M. H. 2004. Male mammals respond to a risk of sperm competition conveyed by odours of conspecific males. Nature, 431, 446e449. Dewsbury, D. A. 1982. Ejaculate cost and male choice. American Naturalist, 119, 601e610. Dickinson, J. L. 1986. Prolonged mating in the milkweed leaf beetle Labidomera clivicollis clivicollis (Coleoptera: Chrysomedlidae): a test of the ‘sperm loading’ hypothesis. Behavioral Ecology and Scociobiology, 18, 331e338. Engqvist, L. & Sauer, K. P. 2001. Strategic male mating effort and cryptic male choice in a scorpionfly. Proceedings of the Royal Society of London, Series B, 268, 729e735. Ferveur, J. F., Cobb, M. & Jallon, J. M. 1989. Complex chemical messages in Drosophila. In: Neurobiology of Sensory Systems (Ed. by R. N. Singh & N. J. Strausfeld), pp. 397e409. New York: Plenum. Fowler, K. & Partridge, L. 1989. A cost of mating in female fruitflies. Nature, 338, 760e761. Friberg, U., Lew, T. A., Byrne, P. G. & Rice, W. R. 2005. Assessing the potential for an ongoing arms race within and between the sexes: selection and heritable variation. Evolution, 59, 1540e1551. Gage, M. J. G. 1991. Risk of sperm competition directly affects ejaculate size in the Mediterranean fruit fly. Animal Behaviour, 42, 1036e1037. Gage, M. J. G. & Baker, R. R. 1991. Ejaculate size varies with sociosexual situation in an insect. Ecological Entomology, 16, 331e337. Gilchrist, A. S. & Partridge, L. 2000. Why it is difficult to model sperm displacement in Drosophila melanogaster: the relation between sperm transfer and copulation duration. Evolution, 54, 534e542. Hadrys, H., Schierwater, B., Dellaporta, S. L., Desalle, R. & Buss, L. W. 1993. Determination of paternity in dragonflies by random amplified DNA fingerprinting. Molecular Ecology, 2, 79e87. Harris, W. E. & Moore, P. J. 2005. Female mate preferences and sexual conflict: females prefer males that have had few consorts. American Naturalist, 165 (supplement), S64eS71. Harshman, L. & Prout, T. 1994. Sperm displacement without sperm transfer. Evolution, 48, 758e766. Ivy, T. M., Weddle, C. B. & Sakaluk, S. K. 2005. Females use self-referent cues to avoid mating with previous mates. Proceedings of the Royal Society of London, Series B, 272, 2475e2478. Kotiaho, J. S. & Simmons, L. W. 2003. Longevity cost of reproduction for males but no longevity cost of mating or courtship for females in the male-dimorphic dung beetle Onthophagus binodis. Journal of Insect Physiology, 49, 817e822. Kotiaho, J. S., Alatalo, R. V., Mappes, J., Nielsen, M. G., Parri, S. & Rivero, A. 1998. Energetic cost of size and sexual signalling in a wolf spider. Proceedings of the Royal Society of London, Series B, 265, 2203e2209. Kuijfer, B. Stewart, A. D. & Rice, W. R. In press. The cost of mating rises nonlinearly with copulation frequency in a laboratory population of Drosophila melanogaster. Journal of Evolutionary Biology. Lewis, S. M. & Iannini, J. 1995. Fitness consequences of differences in male mating behaviour in relation to female reproductive status in flour beetles. Animal Behaviour, 50, 1157e1160.

McLain, D. K. 1980. Female choice and the adaptive significance of prolonged copulation in Nezara viridula (Hemiptera: Pentatomidae). Psyche, 87, 325e336. Marcillac, F. & Ferveur, J. F. 2004. A set of pheromones affects reproduction before, during and after mating in Drosophila. Journal of Experimental Biology, 207, 3927e3933. Martin, O. Y. & Hosken, D. J. 2002. Strategic ejaculation in the common dung fly Sepsis cynipsea. Animal Behaviour, 63, 541e546. Martin, O. Y. & Hosken, D. J. 2004. Copulation reduces male but not female longevity in Saltella sphondylli (Diptera: Sepsidae). Journal of Evolutionary Biology, 17, 357e362. Michiels, N. K. 1992. Consequences and adaptive significance of variation in copulation duration in the dragonfly Sympetrum danae. Behavioral Ecology and Sociobiology, 29, 429e435. Morrow, E. H., Stewart, A. D. & Rice, W. R. 2005. Patterns of sperm precedence are not affected by female mating history in D. melanogaster. Evolution, 59, 2608e2615. Parker, G. A. 1970. Sperm competition and its evolutionary consequences in the insects. Biological Reviews, 45, 525e567. Partridge, L. & Farquhar, M. 1981. Sexual activity reduces lifespan of male fruitflies. Nature, 294, 580e582. Prowse, N. & Partridge, L. 1997. The effects of reproduction on longevity and fertility in male Drosophila melanogaster. Journal of Insect Physiology, 43, 501e512. Rice, W. R. 1996. Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature, 381, 232e234. Rice, W. R., Linder, J. E., Friberg, U., Lew, T. A., Morrow, E. H. & Stewart, A. D. 2005. Inter-locus antagonistic coevolution as an engine of speciation: assessment with hemiclonal analysis. Proceedings of the National Academy of Sciences, U.S.A., 102, 6527e6534. Robertson, F. S. & Reeve, E. 1952. Studies in quantitative inheritance. I. The effects of selection of wing and thorax length in Drosophila melanogaster. Journal of Genetics, 50, 414e448. Rubenstein, D. I. 1989. Sperm competition in the water strider Gerris remigis. Animal Behaviour, 38, 631e636. Rypstra, A. L., Wieg, C., Walker, S. E. & Persons, M. H. 2003. Mutual mate assessment in wolf spiders: differences in the cues used by males and females. Ethology, 109, 315e325. Savarit, F., Sureau, G., Cobb, M. & Ferveur, J. F. 1999. Genetic elimination of known pheromones reveals the fundamental chemical bases of mating and isolation in Drosophila. Proceedings of the National Academy of Sciences, U.S.A., 96, 9015e9030. Scott, D., Richmond, R. C. & Carlson, D. A. 1988. Pheromones exchanged during mating: a mechanism for mate assessment in Drosophila. Animal Behaviour, 36, 1164e1173. Simmons, L. W. 2001. Sperm Competition and Its Evolutionary Consequences in the Insects. Princeton, New Jersey: Princeton University Press. Simmons, L. W. & Siva-Jothy, M. T. 1998. Sperm competition in insects: mechanisms and the potential for selection. In: Sexual Selection and Sperm Competition (Ed. by T. Birkhead & A. Møller), pp. 341e434. London: Academic Press. Simmons, L. W., Llorens, T., Schinzig, M., Hosken, D. & Craig, M. 1994. Sperm competition selects for male mate choice and protandry in the bushcricket, Requena verticalis (Orthoptera: Tettigoniidae). Animal Behaviour, 47, 117e122. Singh, R. S. & Singh, B. N. 2004. Female remating in Drosophila: comparison of duration of copulation between first and second mating in six species. Current Science, 86, 465e470. Siva-Jothy, M. T. & Stutt, A. D. 2003. A matter of taste: direct detection of female mating status in the bedbug. Proceedings of the Royal Society of London, Series B, 270, 649e652.

1267

1268

ANIMAL BEHAVIOUR, 72, 6

Smith, C., Reichard, M. & Jurajda, P. 2003. Assessment of sperm competition by European bitterling, Rhodeus sericeus. Behavioral Ecology and Sociobiology, 53, 206e213. Snook, R. R. & Hosken, D. J. 2004. Sperm death and dumping in Drosophila. Nature, 428, 939e941. Thornhill, R. & Sauer, K. P. 1991. The notal organ of the scorpionfly (Panorpa vulgaris): an adaptation to coerce mating duration. Behavioral Ecology, 2, 156e164. Tompkins, L. 1984. Genetic analysis of sex appeal in Drosophila. Behavior Genetics, 14, 411e440. Tompkins, L. & Hall, J. C. 1981. The different effects on courtship of volatile compounds from mated and virgin Drosophila females. Journal of Insect Physiology, 27, 17e21.

Vepsa¨la¨inen, K. & Savolainen, R. 1995. Operational sex ratios and mating conflicts between the sexes in the water strider Gerris lacustris. American Naturalist, 146, 869e880. Wedell, N. 1992. Protandry and mate assessment in the wartbiter Decticus verrucivorus (Orthoptera, Tettigonidae). Behavioral Ecology and Sociobiology, 31, 301e308. Wedell, N., Gage, M. J. D. & Parker, G. A. 2002. Sperm competition, male prudence and sperm-limited females. Trends in Ecology and Evolution, 17, 313e320. Wolfner, M. F. 1997. Tokens of love: functions and regulation of Drosophila male accessory gland products. Insect Biochemistry and Molecular Biology, 27, 179e192.