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Courtship reduces longevity of maleDrosophila melanogaster
Anim. Behav., 1996, 52, 269–278
Courtship reduces longevity of male Drosophila melanogaster RU } DIGER CORDTS & LINDA PARTRIDGE ICAPB, University of Edinburgh (Received 28 July 1994; initial acceptance 29 September 1994; final acceptance 10 November 1995; MS. number: 4704)
Abstract. Male Drosophila melanogaster exposed to virgin females that were experimentally prevented from mating had a higher death rate than males exposed to an equal number of inseminated females. Exposure to virgin females increases the rates of courtship, mating and production of sperm and accessory fluid. The present study attempted to disentangle the relative contributions of these reproductive activities to the elevated male death rate. Males in different experimental groups were induced to perform only parts of sexual activity. Comparison of longevities between these groups showed that courtship alone was sufficient to reduce male life span. Mating itself and production of seminal fluid and sperm, on the other hand, did not seem to be costly but, since any mating costs were confounded with costs of courtship, definite conclusions cannot be drawn. Surprisingly, wild type males kept with females that could not copulate died sooner than males kept with females that could mate. Despite several behavioural differences between these experimental groups, only mounting attempts showed a pattern consistent with that of longevities. Mounting attempts may be an indicator of an altered metabolic rate or hormonal status that renders the males more susceptible to death. ?
Reproduction is costly if it leads to an increase in mortality or a decrease in future fertility. Reproductive costs are an important constraint on life history evolution because they prevent survival and fertility at all ages from being simultaneously maximized by natural selection (e.g. Calow 1979; Partridge & Sibly 1991; Lessells 1992; Stearns 1992). Costs of reproduction have been demonstrated in a variety of species, and considerable diversity exists both in the components of reproduction that are costly and the nature of the cost incurred. Reproductive costs can be ecological in origin, if the impact of external hazards such as disease or predation is increased (e.g. Cade 1975; Tuttle & Ryan 1981). Particularly for males, investment in a current mating opportunity, for instance through parental care, can result in loss of other mating opportunities (e.g. Townsend 1986, 1989). Correspondence and present address: R. Cordts, Arbeitsgruppe für Verhaltensforschung, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany (email: [email protected]). L. Partridge is now at the Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London NW1 3HE, U.K. (email: [email protected]). 0003–3472/96/020269+10 $18.00/0
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1996 The Association for the Study of Animal Behaviour
Costs can also be physiological in origin, and these include costs of production of gametes and associated materials (e.g. Maynard Smith 1958; Lamb 1964), costs of courtship (e.g. Simmons et al. 1992) and costs of parental care (e.g. Clutton-Brock et al. 1982; Gustaffson & Pärt 1990). Although reproductive costs have been frequently demonstrated, few studies have examined the relative costs of different aspects of reproduction. In Drosophila melanogaster reproductive activity reduces longevity and future fertility of both males and females. In females, egg production (Partridge et al. 1987b), non-mating aspects of exposure to males (Partridge et al. 1986; Partridge & Fowler 1990) and mating itself (Fowler & Partridge 1989; Chapman et al. 1995) all incur reproductive costs. For males, the picture is less clear. Exposure to virgin females reduces male longevity (Partridge & Farquhar 198l; Partridge & Andrews 1985) and future fertility (N. Prowse, unpublished data). This manipulation increases male courtship rate, mating rate and production of accessory fluid and sperm; the relative contribution of these processes to reproductive costs has not been measured. In the 1996 The Association for the Study of Animal Behaviour
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present study, we used mutant males that do not produce sperm, and experimentally altered females that could not mate, to obtain evidence about the contribution of different aspects of male reproduction to increased mortality.
1 day after the microcautery. After 24 h the vials were checked for eggs, and only those females that had not laid any eggs were used in the experiment. A pilot experiment had shown this procedure to be sufficient to eliminate all females that were able to copulate in spite of the microcautery.
METHODS Fly Stocks and Maintenance
Experimental Groups
The flies were from a random-bred strain originally collected in Dahomey in 1970 and kept in laboratory culture in population cages since then. All experimental flies were reared at low larval density to ensure phenotypic uniformity, and virgins of both sexes were separated under carbon dioxide anaesthesia not less than 3 h after eclosion. The adult flies were kept at 25)C in a 12:12 h light:dark cycle over food medium made from 100 g flaked yeast, 100 g sucrose, 30 g nipagin, 3 ml propionic acid and 100 ml water, with live yeast sprinkled on the surface. To measure costs of production of accessory fluid it was necessary to obtain males that would court and copulate and would transfer only accessory fluid. These requirements are met by XO-individuals, which are somatic males but which do not produce any motile sperm (Kiefer 1966; Lindsley & Zimm 1992). We bred XO-males by crossing virgin wild type females to males with their Y-chromosome attached to their X-chromosome, and that were Dahomey genetic background for the other three chromosomes. As a marker, this X-chromosome contained the mutations yellow and white. The resulting male progeny carried the Dahomey X-chromosome, had no Y-chromosome and were Dahomey genetic background for their other three chromosomes. We refer to them as ‘XO-males’ in contrast to the wild type ‘XY-males’ in the following. A detailed description of the breeding scheme is given by Chapman (1992). To separate the costs of courtship from the costs of producing and transferring sperm and seminal fluid it was necessary to have females that elicited courtship but that could not copulate. This was achieved by sealing the vaginas of 1-dayold virgin females by microcautery, as described for males in Partridge & Fowler (1990). We refer to these females as mc-females in the following. We checked the success of the microcautery by keeping each female in a vial with a male starting
Our aim was to compare the longevities of wild type and XO-males that courted little and did not mate, that courted a lot and did not mate, and that both courted a lot and mated. The longevities of six different groups of males were therefore compared. Each of the groups consisted of 10 vials. Group 1: XO-males, low courtship, no mating: six XO-males and six y-males (males bearing the mutation yellow). Group 2: XO-males, high courtship, no mating: six XO-males and six mc-females. Group 3: XO-males, high courtship with mating: six XO-males, three virgin females and mc-females. Group 4: XY-males, low courtship, no mating: six XY-males and y-males. Group 5: XY-males, high courtship, no mating: six XY-males and six mc-females. Group 6: XY-males, high courtship with mating: six XY-males, three virgin females and mc-females. In the first block of three groups, the experimental males were XO and could transfer only accessory fluid at mating, while in the second block the males were wild type and could therefore also transfer sperm. Because males of XO genotype are known to court less than wild type males (Åslund et al. 1978; Chapman 1992), and may also differ in other regards, comparisons of male longevity are valid only within each block. Groups 1 and 4 acted as controls with low courtship; the y-males were used to control for competition for food and space with the experimental males, and the yellow mutation allowed them to be distinguished from the experimental males. Microcautery was applied to virgin females, which are more attractive (i.e. elicit more courtship) than mated females (Bastock & Manning 1955; Jallon & Hotta 1979). Males in groups 2 and 5 were exposed to permanently attractive females that could not mate, and they were therefore expected to court more than the males in groups 1 and 4, respectively. Males in groups 3 and 6 were used to detect a cost of mating. We therefore had to make their rates of courtship as similar as possible to
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(a) 1
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(b) 1
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XY-males with y-males XY-males with mc-females XY-males with mc-and virgin females
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Figure 1. Cumulative survival probability plotted against time (a) for XO-males with y-males, mc-females and virgin plus mc-females and (b) for XY-males with y-males, mc-females and virgin plus mc-females.
those of the males in groups 2 and 5, respectively, and so males in groups 3 and 6 were provided with mc-females in addition to the virgin females, thus ensuring that at least half the females present were virgin at all times. The flies were transferred to vials with fresh food every day until all males had died, the non-experimental males and the females were replaced with fresh ones, and the death of experimental males was noted. To prevent differences
in density, males that had survived were, where necessary, re-grouped at transfer to maintain a density as close as possible to six males per vial. Every second day the males were supplied with new, 4-day-old females or y-males. Behavioural Observations To measure accurately the behaviour of the males in the different experimental conditions, we
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Table I. Results of the survival analysis (data in Fig. 1) XO-males with
XO-males with y-males mc-females
y-males
mc-females
—
46.530 <0.0016 —
mc-+virgin females XY-males with y-males mc-females mc-+virgin females
XY-males with mc-+virgin females
44.222 <0.0016 0.019 —
y-males
mc-females
mc-+virgin females
0.470 —
—
—
5.131 —
—
—
—
57.709 <0.0016 —
5.267 17.819 <0.0016 18.045 <0.0016 —
÷2 and P-values (two-sided, after Bonferroni-correction) are shown. — indicates tests were not computed.
set up a replicate of the experiment (‘behavioural experiment’) lasting only 7 days. Observations were conducted every day for 2 h starting 1 h before the lights went on, using a short flash of light from a small torch to allow observations during the dark-phase. On those days when the females/y-males were changed, we transferred the flies as soon as the lights went on. Immediately afterwards, the 1-h light-phase observation was done. We noted the behaviour of the male and female nearest to the observer in a randomly selected half of the vial every 6 min for each vial. We classified behaviour into the following categories: male: motionless (without courting), moving (without courting), licking, wing-vibration, wing-scissoring, mountingattempt, copula; female: motionless (courted and not courted), moving (courted and not courted), rejection (kicking, extruding ovipositor, decamping).
Statistical Analysis All statistical tests were done using BMDP except for Meddis’ factorial analysis which we computed by hand. We compared the longevities of the flies with the log rank (Mantel–Cox) test and analysed the behavioural data with a repeated measures ANOVA. This ANOVA requires a fixed number of repeats so, owing to deaths of exper-
imental males, we only used the data for eight vials (chosen by using random numbers) out of each experimental group. The grouping variables were: (1) male: XO versus XY; (2) female: mc-females versus virgin plus mc-females; (3) light: dark-phase versus lightphase. The control groups with y-males (groups 1 and 4) were not included in this analysis as the large difference in the amount of courtship between the control groups and the others would have masked any of the much smaller differences between the groups with females. The purpose of the control groups 1 and 4 was to compare them with the groups with mc-females, groups 2 and 5, respectively. Therefore we calculated a second ANOVA, this time with the following levels of the grouping variables: (1) male: XO- versus XY-males; (2) female: y-males versus mc-females; (3) light: dark-phase versus light-phase. Dependent variables in both ANOVAS were the amount (in per cent of time) of male motionless, male moving, male wing-vibration, male mounting-attempts, female motionless and female moving. To meet the condition of sphericity, the percentages had to be transformed by either Y Y x*=arcsine (x/n) or x*=arctan (x/n) . For the other behavioural categories (licking, wingscissoring, copulation, rejection) no adequate transformation could be found. Therefore, the results for these categories were analysed by a
Cordts & Partridge: A cost of courtship (b)
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Time spent moving (%)
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XO-males with ymcmales females
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mcymc+ virgin males females females
mc+ virgin females
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XO-males with ymcmales females
XY-males with
mcymc+ virgin males females females
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Figure 2. (a) Time spent motionless; (b) time spent moving; (c) time spent wing-vibrating; and (d) mounting attempts of the experimental males in the six experimental groups. Bars are mean values of the means of eight vials for 6 consecutive days, vertical lines are standard deviations. .: Light-phase; /: dark-phase.
non-parametric factorial analysis using ranks according to Meddis (1984). RESULTS Longevity Experiment Figure 1 shows the results of the longevity experiment. The survival data were compared with the log rank test and the resulting probabilities were corrected for multiple comparisons with the sequential Bonferroni test (Rice 1989; Table I). Males of group 2 (XO-males plus mc-females) and group 5 (XY-males plus mc-females) could not copulate, and lived significantly less long than the respective controls kept with y-males. Some non-mating consequence of exposure to females therefore seems to be an important factor reducing the longevity of sexually active males.
The costs of mating and producing seminal fluid, on the other hand, appeared to be negligible. Group 3 males (XO-males plus virgin females plus mc-females) could court, copulate and transfer seminal fluid (but no sperm), whereas group 2 males could only court. A difference in longevity would therefore reflect a cost of the production of seminal fluid (plus the cost of mating). Yet the males of these groups did not differ significantly in their survival rate. Comparison of the longevities of males of group 5 (XY-males plus mc-females) and group 6 (XY-males plus virgin females plus mc-females) reveals an unexpected result. Males of these groups differed in that males of group 5 could only court while males of group 6 could court, copulate and transfer sperm and seminal fluid. Different longevities would therefore be a measure of the costs of producing sperm and seminal fluid. Surprisingly, males of group 6 survived
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Table II. Results of the ANOVA with repeated measures and Meddis’ factorial analysis using ranks, respectively, for the behavioural experiment with the factor ‘female’ comparing groups with mc-females versus virgin (plus mc) females Behaviour
Main effects
Interaction effects
Motionless*
Male (88.73; <0.0002) Light (5.68; 0.041)
Male–Light (8.62; 0.0096)
Moving*
Male (47.55; <0.0002) Light (8.36; 0.0108)
Wing-vibration*
Male (167.22; <0.0002) Female (15.19; 0.0006) Light (10.72; 0.0036)
Male–Female (14.60; 0.0006)
Mounting attempts*
Male (77.90; <0.0002) Female (59.51; <0.0002) Light (25.43; <0.0002)
Male–Female (22.64; <0.0002)
Licking‡
Male–Female (4.07; 0.0438) Female–Light (4.07; 0.0438)
Wing-scissoring‡
Male–Female (6.19; 0.0129) Male–Light (6.11; 0.0134) Female–Light (6.64; 0.01)
Copulation‡
Female (20.45; <0.0001) Light (20.45; <0.0001)
Female (or y-male) motionless*
Male (19.02; 0.0002)
Female (or y-male) moving*
Male (22.06; <0.0002)
Female (or y-male) rejection‡
Male (12.03; 0.0005)
Male–Female (41.19; <0.0001) Male–Light (41.19; <0.0001) Female–Light (20.84; <0.0001)
Male–Female (5.8; 0.016) Male–Light (4.69; 0.03) Female–Light (16.14; <0.0001)
Values in parentheses are F-ratio with df=1,52 and two-sided P or H with df=1, respectively. Y *Transformed by x*= arctane (x/n) . †Transformed by x*=arcsine (x/n)Y. ‡Analysed with Meddis’ factorial analysis.
significantly longer than males of group 5 despite the fact that they could mate. Behavioural Experiment The greatest single difference observed was between the behaviour of XO- and XY-males with XO-males being less active and courting less than XY-males (Fig. 2, Tables II and III). Furthermore, the females (or y-males) were more active when exposed to XY- than to XO-males (Tables II, III, Fig. 3). Comparison of the behaviour of males exposed to y-males with that of males exposed to females that could not mate (Table III, Fig. 3) shows that the males exposed to females both spent significantly less time still and courted more (wing-vibration and mounting attempts).
Among the groups of males that had access to females (Table II, Fig. 2), the type of female (mcfemales versus virgin plus mc-females) as well as an interaction of female type and male genotype influenced male behaviour. Male activity was not significantly affected by the type of females present. XO-males kept with mc-females showed more wing-vibration than XO-males with virgin plus mc-females while there seemed to be no such difference in XY-males. For mounting attempts it was the opposite. While there was no or just a small difference in the mounting attempts of XO-males exposed to different types of females (Fig. 2d), XY-males with virgin plus mc-females attempted significantly fewer copulations than XY-males kept with mc-females alone (Fig. 2d). Both XO- and XY-males moved significantly more in the dark phase and showed significantly
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Table III. Results of the ANOVA with repeated measures and Meddis’ factorial analysis using ranks, respectively, for the behavioural experiment with the factor ‘female’ comparing groups with y-males versus mc-females Behaviour
Main effects
Interaction effects
Motionless*
Male (153.94; <0.0002) Female (59.07; <0.0002) Light (7.17; 0.0194)
Moving†
Male (77.15; <0.0002) Light (13.44; 0.0084)
Male–Female (8.93; 0.0084)
Wing-vibration*
Male (26.10; <0.0002) Female (330.07; <0.0002) Light (10.72; 0.0036)
Male–Female (24.72; <0.0002)
Mounting attempts*
Male (93.95; <0.0002) Female (323.14; <0.0002) Light (11.90; 0.0022)
Male–Female (85.92; <0.0002)
Licking‡
Male–Female (5.00; 0.0254) Male–Light (5.00; 0.0254) Female–Light (5.00; 0.0254)
Wing-scissoring‡
Male–Female (6.31; 0.012) Male–Light (4.76; 0.0292) Female–Light (4.08; 0.0434)
Female (or y-male) motionless*
Male (7.52; 0.0164) Female (44.94; <0.0002)
Female (or y-male) rejection‡
Female (7.74; 0.0055)
Male–Female (4.58; 0.0324) Male–Light (10.9; 0.001) Female–Light (4.58; 0.0324)
Values in parentheses are F-ratio with df=1,52 and two-sided P or H with df=1, respectively. Y *Transformed by x*=arcsine (x/n) . Y †Transformed by x*= arctan (x/n) . ‡Analysed with Meddis’ factorial analysis.
more wing-vibration and mounting attempts during the light phase.
DISCUSSION The most important finding of the present study is that courtship alone is sufficient to reduce males’ life span; males exposed to microcauterized females died significantly earlier than those kept with y-males. This difference in survival tallies with the differences in behaviour. XO- and XY-males kept with mc-females showed more wing-vibration and attempted more mountings, and spent correspondingly less time motionless, than the longer living control groups kept with y-males. These results do not show either which aspects of courtship reduce longevity, or by what physiological mechanisms they exert their effect. Sexual activity has been shown to elevate the
metabolic rate of female D. simulans (Giesel et al. 1989), and increased metabolic rate reduces life span in D. melanogaster (Miquel et al. 1976). The metabolic costs of the increased activity associated with male courtship may therefore be important in reducing life span. The highly significant effect of light on wing-vibration and mounting attempts was probably due to new females being introduced every second day at the beginning of the light phase. Most of the courtship therefore took place during the following 1-h light-phase observation. In contrast, the results showed no evidence for a cost of mating. The mortalities of XO-males with mc-females and XO-males with virgin plus mc-females did not differ significantly. Therefore, production of seminal fluid and copulation appeared to have negligible costs for longevity. However, some caution in interpretation of this result may be needed, because XY-males showed
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(b) Time spent moving (%)
Time spent motionless (%)
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XO-males with ymcmales females
XY-males with
mcymc+ virgin males females females
mc+ virgin females
100 80 60 40 20 0
XO-males with ymcmales females
XY-males with
mcymc+ virgin males females females
mc+ virgin females
Figure 3. (a) Time spent motionless and (b) time spent moving by the females in the six experimental groups. Bars are mean values of the means of eight vials for 6 consecutive days, vertical lines are standard deviations. .: Light-phase; /: dark-phase.
an apparent negative cost of mating; males that could court, copulate and transfer both seminal fluid and sperm survived significantly better than males that could only court. One type of behaviour, mounting attempts, showed a pattern of variation that could explain this pattern of longevities. XO-males exposed to mc- and mc- plus virgin females did not differ significantly in both their levels of mounting attempts and their survival rates. In contrast, XY-males exposed to mc-females attempted far more copulations than those exposed to mc- plus virgin females, and correspondingly suffered from a higher mortality. How mounting attempts might affect survival, however, remains unclear. It seems unlikely that energetic costs of the movements involved in a mounting attempt alone are responsible for the drop in longevity. Mounting attempts could rather be an indicator of an altered metabolic rate or hormonal status. These results again suggest the importance of costs of courtship, but conclusions about costs of mating cannot be drawn, because any costs were confounded by courtship costs. Female D. melanogaster exposed to males incur costs to longevity as a result of both non-mating effects (Partridge & Fowler 1990; Chapman 1992) and mating itself (Fowler & Partridge 1989; Chapman et al. 1995). It therefore seems that costs of reproduction in both sexes of D. melanogaster are not only a consequence of diversion of nutrients into gamete production. Costs of producing sperm and seminal fluid may have gone undetected because opportunities
for successful copulations in the two groups with virgin females were relatively rare (0.25 copulations per day per male if one assumes equal competitive abilities, which seems plausible as the males were bred under relaxed larval competition). Production of seminal fluid and sperm might be costly, but the low rate of copulations could have been insufficient to reveal such costs under the experimental conditions. Other studies have shown costs of sexual activity in male fruit flies allowed much higher mating opportunities (one and eight virgins per day per male in Partridge & Farquhar 1981; three virgins per day per male in Partridge & Andrews 1985). Costs seen there might be of a different kind to the ones seen in the present study. Since most females in the wild are inseminated (Boulétreau 1978) males will probably have to court many females before actually mating, and so courtship costs are likely to be of real importance in the field. It is not known how often females re-mate in nature; the figure of once per day has been suggested (Partridge et al. 1987c ; van Vianen & Bijlsma 1993). It will be important to determine if costs of sexual activity other than costs of courtship occur at this level of mating. In addition to any negative effect on life span, mating can be costly to future fertility in male D. melanogaster (Lefevre & Jonsson 1962). These costs should be the subject of future work. As previously reported (Åslund et al. 1978; Chapman 1992) XO-males moved and courted less often than XY-males. This might explain the significant influence of male genotype on female
Cordts & Partridge: A cost of courtship behaviour. Females courted by larger males that courted more were more likely to move (Partridge et al. 1987a). Similarly females kept with XY-males may have moved more often because they were courted more often. There were no significant differences in longevity between XOand XY-males when exposed to the same kinds of females or to control y-males.
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Kiefer, B. I. 1966. Ultrastructural abnormalities in developing sperm of X/O Drosophila melanogaster. Genetics, 54, 1441–1452. Lamb, M. J. 1964. The effects of radiation on the longevity of female Drosophila subobscura. J. Insect Physiol., 10, 487–497. Lefevre G. & Jonsson, V. B. 1962. Sperm transfer, storage and utilization in Drosophila melanogaster. Genetics, 47, 1719–1736. Lessels, C. M. 1992. The evolution of life histories. In: Behavioural Ecology: An Evolutionary Approach. 3rd edn (Ed. by J. R. Krebs & N. B. Davies), pp. 32–65. London: Blackwell Scientific Publications. Lindsley, D. L. & Zimm, G. G. 1992. The Genome of Drosophila melanogaster. San Diego: Academic Press. Maynard Smith, J. 1958. The effects of temperature and of egg-laying on the longevity of female Drosophila subobscura. J. exp. Biol., 35, 832–842. Meddis, R. 1984. Statistics Using Ranks. Oxford: Blackwell. Miquel, J., Lundgren, K. G., Bensch, K. G. & Atlan, H. 1976. Effects of temperature on the life span, vitality and fine structure of Drosophila melanogaster. Mech. Ageing Devel., 5, 347–370. Partridge, L. & Andrews, R. 1985. The effect of reproductive activity on the longevity of male Drosophila melanogaster is not caused by an acceleration of ageing. J. Insect Physiol., 31, 393–395. Partridge, L. & Farquhar, M. 1981. Sexual activity reduces lifespan of male fruitflies. Nature, Lond., 294, 580–582. Partridge, L. & Fowler, K. 1990. Non-mating costs of exposure to males in female Drosophila melanogaster. J. Insect Physiol., 36, 419–425. Partridge, L. & Sibly, R. 1991. Constraints in the evolution of life histories. Phil. Trans. R. Soc. Lond. Ser. B., 332, 3–13. Partridge, L., Fowler, K., Trevitt, S. & Sharp, W. 1986. An examination of the effects of males on the survival and egg-production rates of female Drosophila melanogaster. J. Insect Physiol., 32, 925–929. Partridge, L., Ewing, A. & Chandler, A. 1987a. Male size and mating success in Drosophila melanogaster: the roles of male and female behaviour. Anim. Behav., 35, 555–562. Partridge, L., Green, A. & Fowler, K. 1987b. Effects of egg-production and of exposure to males on female survival in Drosophila melanogaster. J. Insect Physiol., 33, 745–749. Partridge, L., Hoffmann, A. & Jones, J. S. 1987c. Male size and mating success in Drosophila melanogaster and D. pseudoobscura under field conditions. Anim. Behav., 35, 468–476. Rice, W. R. 1989. Analyzing tables of statistical tests. Evolution, 43, 223–225. Simmons, L. W., Teale, R. J. Maier, M., Standish, R. J. Bailey, W. J. & Withers, P. C. 1992. Some costs of reproduction for male bushcrickets, Requena verticalis (Orthoptera: Tettigoniidae): allocating resources to mate attraction and nuptial feeding. Behav. Ecol. Sociobiol., 31, 57–62.
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Stearns, S. C. 1992. The Evolution of Life Histories. Oxford: Oxford University Press. Townsend, D. S. 1986. The costs of male parental care and its evolution in a neotropical frog. Behav. Ecol. Sociobiol., 19, 187–195. Townsend, D. S. 1989. Sexual selection, natural selection and a fitness trade-off in a tropical frog with male parental care. Am. Nat., 133, 266–272.
Tuttle, M. D. & Ryan, M. J. 1981. Bat predation and the evolution of frog vocalizations in the Neotropics. Science, 214, 677–678. van Vianen, A. & Bijlsma, R. 1993. The adult component of selection in Drosophila melanogaster: some aspects of early remating activity of females. Heredity, 71, 269–276.
Courtship reduces longevity of male Drosophila melanogaster RU } DIGER CORDTS & LINDA PARTRIDGE ICAPB, University of Edinburgh (Received 28 July 1994; initial acceptance 29 September 1994; final acceptance 10 November 1995; MS. number: 4704)
Abstract. Male Drosophila melanogaster exposed to virgin females that were experimentally prevented from mating had a higher death rate than males exposed to an equal number of inseminated females. Exposure to virgin females increases the rates of courtship, mating and production of sperm and accessory fluid. The present study attempted to disentangle the relative contributions of these reproductive activities to the elevated male death rate. Males in different experimental groups were induced to perform only parts of sexual activity. Comparison of longevities between these groups showed that courtship alone was sufficient to reduce male life span. Mating itself and production of seminal fluid and sperm, on the other hand, did not seem to be costly but, since any mating costs were confounded with costs of courtship, definite conclusions cannot be drawn. Surprisingly, wild type males kept with females that could not copulate died sooner than males kept with females that could mate. Despite several behavioural differences between these experimental groups, only mounting attempts showed a pattern consistent with that of longevities. Mounting attempts may be an indicator of an altered metabolic rate or hormonal status that renders the males more susceptible to death. ?
Reproduction is costly if it leads to an increase in mortality or a decrease in future fertility. Reproductive costs are an important constraint on life history evolution because they prevent survival and fertility at all ages from being simultaneously maximized by natural selection (e.g. Calow 1979; Partridge & Sibly 1991; Lessells 1992; Stearns 1992). Costs of reproduction have been demonstrated in a variety of species, and considerable diversity exists both in the components of reproduction that are costly and the nature of the cost incurred. Reproductive costs can be ecological in origin, if the impact of external hazards such as disease or predation is increased (e.g. Cade 1975; Tuttle & Ryan 1981). Particularly for males, investment in a current mating opportunity, for instance through parental care, can result in loss of other mating opportunities (e.g. Townsend 1986, 1989). Correspondence and present address: R. Cordts, Arbeitsgruppe für Verhaltensforschung, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany (email: [email protected]). L. Partridge is now at the Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London NW1 3HE, U.K. (email: [email protected]). 0003–3472/96/020269+10 $18.00/0
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Costs can also be physiological in origin, and these include costs of production of gametes and associated materials (e.g. Maynard Smith 1958; Lamb 1964), costs of courtship (e.g. Simmons et al. 1992) and costs of parental care (e.g. Clutton-Brock et al. 1982; Gustaffson & Pärt 1990). Although reproductive costs have been frequently demonstrated, few studies have examined the relative costs of different aspects of reproduction. In Drosophila melanogaster reproductive activity reduces longevity and future fertility of both males and females. In females, egg production (Partridge et al. 1987b), non-mating aspects of exposure to males (Partridge et al. 1986; Partridge & Fowler 1990) and mating itself (Fowler & Partridge 1989; Chapman et al. 1995) all incur reproductive costs. For males, the picture is less clear. Exposure to virgin females reduces male longevity (Partridge & Farquhar 198l; Partridge & Andrews 1985) and future fertility (N. Prowse, unpublished data). This manipulation increases male courtship rate, mating rate and production of accessory fluid and sperm; the relative contribution of these processes to reproductive costs has not been measured. In the 1996 The Association for the Study of Animal Behaviour
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present study, we used mutant males that do not produce sperm, and experimentally altered females that could not mate, to obtain evidence about the contribution of different aspects of male reproduction to increased mortality.
1 day after the microcautery. After 24 h the vials were checked for eggs, and only those females that had not laid any eggs were used in the experiment. A pilot experiment had shown this procedure to be sufficient to eliminate all females that were able to copulate in spite of the microcautery.
METHODS Fly Stocks and Maintenance
Experimental Groups
The flies were from a random-bred strain originally collected in Dahomey in 1970 and kept in laboratory culture in population cages since then. All experimental flies were reared at low larval density to ensure phenotypic uniformity, and virgins of both sexes were separated under carbon dioxide anaesthesia not less than 3 h after eclosion. The adult flies were kept at 25)C in a 12:12 h light:dark cycle over food medium made from 100 g flaked yeast, 100 g sucrose, 30 g nipagin, 3 ml propionic acid and 100 ml water, with live yeast sprinkled on the surface. To measure costs of production of accessory fluid it was necessary to obtain males that would court and copulate and would transfer only accessory fluid. These requirements are met by XO-individuals, which are somatic males but which do not produce any motile sperm (Kiefer 1966; Lindsley & Zimm 1992). We bred XO-males by crossing virgin wild type females to males with their Y-chromosome attached to their X-chromosome, and that were Dahomey genetic background for the other three chromosomes. As a marker, this X-chromosome contained the mutations yellow and white. The resulting male progeny carried the Dahomey X-chromosome, had no Y-chromosome and were Dahomey genetic background for their other three chromosomes. We refer to them as ‘XO-males’ in contrast to the wild type ‘XY-males’ in the following. A detailed description of the breeding scheme is given by Chapman (1992). To separate the costs of courtship from the costs of producing and transferring sperm and seminal fluid it was necessary to have females that elicited courtship but that could not copulate. This was achieved by sealing the vaginas of 1-dayold virgin females by microcautery, as described for males in Partridge & Fowler (1990). We refer to these females as mc-females in the following. We checked the success of the microcautery by keeping each female in a vial with a male starting
Our aim was to compare the longevities of wild type and XO-males that courted little and did not mate, that courted a lot and did not mate, and that both courted a lot and mated. The longevities of six different groups of males were therefore compared. Each of the groups consisted of 10 vials. Group 1: XO-males, low courtship, no mating: six XO-males and six y-males (males bearing the mutation yellow). Group 2: XO-males, high courtship, no mating: six XO-males and six mc-females. Group 3: XO-males, high courtship with mating: six XO-males, three virgin females and mc-females. Group 4: XY-males, low courtship, no mating: six XY-males and y-males. Group 5: XY-males, high courtship, no mating: six XY-males and six mc-females. Group 6: XY-males, high courtship with mating: six XY-males, three virgin females and mc-females. In the first block of three groups, the experimental males were XO and could transfer only accessory fluid at mating, while in the second block the males were wild type and could therefore also transfer sperm. Because males of XO genotype are known to court less than wild type males (Åslund et al. 1978; Chapman 1992), and may also differ in other regards, comparisons of male longevity are valid only within each block. Groups 1 and 4 acted as controls with low courtship; the y-males were used to control for competition for food and space with the experimental males, and the yellow mutation allowed them to be distinguished from the experimental males. Microcautery was applied to virgin females, which are more attractive (i.e. elicit more courtship) than mated females (Bastock & Manning 1955; Jallon & Hotta 1979). Males in groups 2 and 5 were exposed to permanently attractive females that could not mate, and they were therefore expected to court more than the males in groups 1 and 4, respectively. Males in groups 3 and 6 were used to detect a cost of mating. We therefore had to make their rates of courtship as similar as possible to
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(a) 1
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5
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Figure 1. Cumulative survival probability plotted against time (a) for XO-males with y-males, mc-females and virgin plus mc-females and (b) for XY-males with y-males, mc-females and virgin plus mc-females.
those of the males in groups 2 and 5, respectively, and so males in groups 3 and 6 were provided with mc-females in addition to the virgin females, thus ensuring that at least half the females present were virgin at all times. The flies were transferred to vials with fresh food every day until all males had died, the non-experimental males and the females were replaced with fresh ones, and the death of experimental males was noted. To prevent differences
in density, males that had survived were, where necessary, re-grouped at transfer to maintain a density as close as possible to six males per vial. Every second day the males were supplied with new, 4-day-old females or y-males. Behavioural Observations To measure accurately the behaviour of the males in the different experimental conditions, we
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Table I. Results of the survival analysis (data in Fig. 1) XO-males with
XO-males with y-males mc-females
y-males
mc-females
—
46.530 <0.0016 —
mc-+virgin females XY-males with y-males mc-females mc-+virgin females
XY-males with mc-+virgin females
44.222 <0.0016 0.019 —
y-males
mc-females
mc-+virgin females
0.470 —
—
—
5.131 —
—
—
—
57.709 <0.0016 —
5.267 17.819 <0.0016 18.045 <0.0016 —
÷2 and P-values (two-sided, after Bonferroni-correction) are shown. — indicates tests were not computed.
set up a replicate of the experiment (‘behavioural experiment’) lasting only 7 days. Observations were conducted every day for 2 h starting 1 h before the lights went on, using a short flash of light from a small torch to allow observations during the dark-phase. On those days when the females/y-males were changed, we transferred the flies as soon as the lights went on. Immediately afterwards, the 1-h light-phase observation was done. We noted the behaviour of the male and female nearest to the observer in a randomly selected half of the vial every 6 min for each vial. We classified behaviour into the following categories: male: motionless (without courting), moving (without courting), licking, wing-vibration, wing-scissoring, mountingattempt, copula; female: motionless (courted and not courted), moving (courted and not courted), rejection (kicking, extruding ovipositor, decamping).
Statistical Analysis All statistical tests were done using BMDP except for Meddis’ factorial analysis which we computed by hand. We compared the longevities of the flies with the log rank (Mantel–Cox) test and analysed the behavioural data with a repeated measures ANOVA. This ANOVA requires a fixed number of repeats so, owing to deaths of exper-
imental males, we only used the data for eight vials (chosen by using random numbers) out of each experimental group. The grouping variables were: (1) male: XO versus XY; (2) female: mc-females versus virgin plus mc-females; (3) light: dark-phase versus lightphase. The control groups with y-males (groups 1 and 4) were not included in this analysis as the large difference in the amount of courtship between the control groups and the others would have masked any of the much smaller differences between the groups with females. The purpose of the control groups 1 and 4 was to compare them with the groups with mc-females, groups 2 and 5, respectively. Therefore we calculated a second ANOVA, this time with the following levels of the grouping variables: (1) male: XO- versus XY-males; (2) female: y-males versus mc-females; (3) light: dark-phase versus light-phase. Dependent variables in both ANOVAS were the amount (in per cent of time) of male motionless, male moving, male wing-vibration, male mounting-attempts, female motionless and female moving. To meet the condition of sphericity, the percentages had to be transformed by either Y Y x*=arcsine (x/n) or x*=arctan (x/n) . For the other behavioural categories (licking, wingscissoring, copulation, rejection) no adequate transformation could be found. Therefore, the results for these categories were analysed by a
Cordts & Partridge: A cost of courtship (b)
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Figure 2. (a) Time spent motionless; (b) time spent moving; (c) time spent wing-vibrating; and (d) mounting attempts of the experimental males in the six experimental groups. Bars are mean values of the means of eight vials for 6 consecutive days, vertical lines are standard deviations. .: Light-phase; /: dark-phase.
non-parametric factorial analysis using ranks according to Meddis (1984). RESULTS Longevity Experiment Figure 1 shows the results of the longevity experiment. The survival data were compared with the log rank test and the resulting probabilities were corrected for multiple comparisons with the sequential Bonferroni test (Rice 1989; Table I). Males of group 2 (XO-males plus mc-females) and group 5 (XY-males plus mc-females) could not copulate, and lived significantly less long than the respective controls kept with y-males. Some non-mating consequence of exposure to females therefore seems to be an important factor reducing the longevity of sexually active males.
The costs of mating and producing seminal fluid, on the other hand, appeared to be negligible. Group 3 males (XO-males plus virgin females plus mc-females) could court, copulate and transfer seminal fluid (but no sperm), whereas group 2 males could only court. A difference in longevity would therefore reflect a cost of the production of seminal fluid (plus the cost of mating). Yet the males of these groups did not differ significantly in their survival rate. Comparison of the longevities of males of group 5 (XY-males plus mc-females) and group 6 (XY-males plus virgin females plus mc-females) reveals an unexpected result. Males of these groups differed in that males of group 5 could only court while males of group 6 could court, copulate and transfer sperm and seminal fluid. Different longevities would therefore be a measure of the costs of producing sperm and seminal fluid. Surprisingly, males of group 6 survived
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Table II. Results of the ANOVA with repeated measures and Meddis’ factorial analysis using ranks, respectively, for the behavioural experiment with the factor ‘female’ comparing groups with mc-females versus virgin (plus mc) females Behaviour
Main effects
Interaction effects
Motionless*
Male (88.73; <0.0002) Light (5.68; 0.041)
Male–Light (8.62; 0.0096)
Moving*
Male (47.55; <0.0002) Light (8.36; 0.0108)
Wing-vibration*
Male (167.22; <0.0002) Female (15.19; 0.0006) Light (10.72; 0.0036)
Male–Female (14.60; 0.0006)
Mounting attempts*
Male (77.90; <0.0002) Female (59.51; <0.0002) Light (25.43; <0.0002)
Male–Female (22.64; <0.0002)
Licking‡
Male–Female (4.07; 0.0438) Female–Light (4.07; 0.0438)
Wing-scissoring‡
Male–Female (6.19; 0.0129) Male–Light (6.11; 0.0134) Female–Light (6.64; 0.01)
Copulation‡
Female (20.45; <0.0001) Light (20.45; <0.0001)
Female (or y-male) motionless*
Male (19.02; 0.0002)
Female (or y-male) moving*
Male (22.06; <0.0002)
Female (or y-male) rejection‡
Male (12.03; 0.0005)
Male–Female (41.19; <0.0001) Male–Light (41.19; <0.0001) Female–Light (20.84; <0.0001)
Male–Female (5.8; 0.016) Male–Light (4.69; 0.03) Female–Light (16.14; <0.0001)
Values in parentheses are F-ratio with df=1,52 and two-sided P or H with df=1, respectively. Y *Transformed by x*= arctane (x/n) . †Transformed by x*=arcsine (x/n)Y. ‡Analysed with Meddis’ factorial analysis.
significantly longer than males of group 5 despite the fact that they could mate. Behavioural Experiment The greatest single difference observed was between the behaviour of XO- and XY-males with XO-males being less active and courting less than XY-males (Fig. 2, Tables II and III). Furthermore, the females (or y-males) were more active when exposed to XY- than to XO-males (Tables II, III, Fig. 3). Comparison of the behaviour of males exposed to y-males with that of males exposed to females that could not mate (Table III, Fig. 3) shows that the males exposed to females both spent significantly less time still and courted more (wing-vibration and mounting attempts).
Among the groups of males that had access to females (Table II, Fig. 2), the type of female (mcfemales versus virgin plus mc-females) as well as an interaction of female type and male genotype influenced male behaviour. Male activity was not significantly affected by the type of females present. XO-males kept with mc-females showed more wing-vibration than XO-males with virgin plus mc-females while there seemed to be no such difference in XY-males. For mounting attempts it was the opposite. While there was no or just a small difference in the mounting attempts of XO-males exposed to different types of females (Fig. 2d), XY-males with virgin plus mc-females attempted significantly fewer copulations than XY-males kept with mc-females alone (Fig. 2d). Both XO- and XY-males moved significantly more in the dark phase and showed significantly
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Table III. Results of the ANOVA with repeated measures and Meddis’ factorial analysis using ranks, respectively, for the behavioural experiment with the factor ‘female’ comparing groups with y-males versus mc-females Behaviour
Main effects
Interaction effects
Motionless*
Male (153.94; <0.0002) Female (59.07; <0.0002) Light (7.17; 0.0194)
Moving†
Male (77.15; <0.0002) Light (13.44; 0.0084)
Male–Female (8.93; 0.0084)
Wing-vibration*
Male (26.10; <0.0002) Female (330.07; <0.0002) Light (10.72; 0.0036)
Male–Female (24.72; <0.0002)
Mounting attempts*
Male (93.95; <0.0002) Female (323.14; <0.0002) Light (11.90; 0.0022)
Male–Female (85.92; <0.0002)
Licking‡
Male–Female (5.00; 0.0254) Male–Light (5.00; 0.0254) Female–Light (5.00; 0.0254)
Wing-scissoring‡
Male–Female (6.31; 0.012) Male–Light (4.76; 0.0292) Female–Light (4.08; 0.0434)
Female (or y-male) motionless*
Male (7.52; 0.0164) Female (44.94; <0.0002)
Female (or y-male) rejection‡
Female (7.74; 0.0055)
Male–Female (4.58; 0.0324) Male–Light (10.9; 0.001) Female–Light (4.58; 0.0324)
Values in parentheses are F-ratio with df=1,52 and two-sided P or H with df=1, respectively. Y *Transformed by x*=arcsine (x/n) . Y †Transformed by x*= arctan (x/n) . ‡Analysed with Meddis’ factorial analysis.
more wing-vibration and mounting attempts during the light phase.
DISCUSSION The most important finding of the present study is that courtship alone is sufficient to reduce males’ life span; males exposed to microcauterized females died significantly earlier than those kept with y-males. This difference in survival tallies with the differences in behaviour. XO- and XY-males kept with mc-females showed more wing-vibration and attempted more mountings, and spent correspondingly less time motionless, than the longer living control groups kept with y-males. These results do not show either which aspects of courtship reduce longevity, or by what physiological mechanisms they exert their effect. Sexual activity has been shown to elevate the
metabolic rate of female D. simulans (Giesel et al. 1989), and increased metabolic rate reduces life span in D. melanogaster (Miquel et al. 1976). The metabolic costs of the increased activity associated with male courtship may therefore be important in reducing life span. The highly significant effect of light on wing-vibration and mounting attempts was probably due to new females being introduced every second day at the beginning of the light phase. Most of the courtship therefore took place during the following 1-h light-phase observation. In contrast, the results showed no evidence for a cost of mating. The mortalities of XO-males with mc-females and XO-males with virgin plus mc-females did not differ significantly. Therefore, production of seminal fluid and copulation appeared to have negligible costs for longevity. However, some caution in interpretation of this result may be needed, because XY-males showed
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(b) Time spent moving (%)
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mcymc+ virgin males females females
mc+ virgin females
100 80 60 40 20 0
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mc+ virgin females
Figure 3. (a) Time spent motionless and (b) time spent moving by the females in the six experimental groups. Bars are mean values of the means of eight vials for 6 consecutive days, vertical lines are standard deviations. .: Light-phase; /: dark-phase.
an apparent negative cost of mating; males that could court, copulate and transfer both seminal fluid and sperm survived significantly better than males that could only court. One type of behaviour, mounting attempts, showed a pattern of variation that could explain this pattern of longevities. XO-males exposed to mc- and mc- plus virgin females did not differ significantly in both their levels of mounting attempts and their survival rates. In contrast, XY-males exposed to mc-females attempted far more copulations than those exposed to mc- plus virgin females, and correspondingly suffered from a higher mortality. How mounting attempts might affect survival, however, remains unclear. It seems unlikely that energetic costs of the movements involved in a mounting attempt alone are responsible for the drop in longevity. Mounting attempts could rather be an indicator of an altered metabolic rate or hormonal status. These results again suggest the importance of costs of courtship, but conclusions about costs of mating cannot be drawn, because any costs were confounded by courtship costs. Female D. melanogaster exposed to males incur costs to longevity as a result of both non-mating effects (Partridge & Fowler 1990; Chapman 1992) and mating itself (Fowler & Partridge 1989; Chapman et al. 1995). It therefore seems that costs of reproduction in both sexes of D. melanogaster are not only a consequence of diversion of nutrients into gamete production. Costs of producing sperm and seminal fluid may have gone undetected because opportunities
for successful copulations in the two groups with virgin females were relatively rare (0.25 copulations per day per male if one assumes equal competitive abilities, which seems plausible as the males were bred under relaxed larval competition). Production of seminal fluid and sperm might be costly, but the low rate of copulations could have been insufficient to reveal such costs under the experimental conditions. Other studies have shown costs of sexual activity in male fruit flies allowed much higher mating opportunities (one and eight virgins per day per male in Partridge & Farquhar 1981; three virgins per day per male in Partridge & Andrews 1985). Costs seen there might be of a different kind to the ones seen in the present study. Since most females in the wild are inseminated (Boulétreau 1978) males will probably have to court many females before actually mating, and so courtship costs are likely to be of real importance in the field. It is not known how often females re-mate in nature; the figure of once per day has been suggested (Partridge et al. 1987c ; van Vianen & Bijlsma 1993). It will be important to determine if costs of sexual activity other than costs of courtship occur at this level of mating. In addition to any negative effect on life span, mating can be costly to future fertility in male D. melanogaster (Lefevre & Jonsson 1962). These costs should be the subject of future work. As previously reported (Åslund et al. 1978; Chapman 1992) XO-males moved and courted less often than XY-males. This might explain the significant influence of male genotype on female
Cordts & Partridge: A cost of courtship behaviour. Females courted by larger males that courted more were more likely to move (Partridge et al. 1987a). Similarly females kept with XY-males may have moved more often because they were courted more often. There were no significant differences in longevity between XOand XY-males when exposed to the same kinds of females or to control y-males.
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