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1 Animal Behaviour 78 (2009) Contents lists available at ScienceDirect Animal Behaviour journal homepage: Male house mice do not adjust sperm allocation in response to odours from related or unrelated rivals Steven A. Ramm *, Paula Stockley Mammalian Behaviour & Evolution Research Group, Faculty of Veterinary Science, University of Liverpool article info Article history: Received 4 March 2009 Initial acceptance 9 April 2009 Final acceptance 23 June 2009 Published online 23 July 2009 MS. number: R Keywords: copulatory behaviour copulatory plug house mouse inclusive fitness Mus musculus domesticus relatedness rodent sperm allocation sperm competition Sperm competition theory predicts that males should adjust the number of sperm they ejaculate adaptively, according to sociosexual cues of sperm competition at the time of mating. Specifically, it is predicted that (1) males will respond to an increased risk of sperm competition from rivals by increasing sperm allocation, and (2) the increase in allocation will be lower when rivals are related. In species that use odour for communication, scent-based cues provide information on the presence and identity of conspecifics, and could thus serve as a basis for adjusting sperm allocation. We tested these predictions in the house mouse, Mus musculus domesticus, a species for which scent is critical to many aspects of social and reproductive communication. Sperm allocation was measured for subject males mating immediately following exposure to odour stimuli deriving from (1) themselves (control treatment), (2) a brother or (3) an unrelated male. The behavioural responses of subjects to conspecific odours indicated that males detected the presence of these stimuli in their environment, but contrary to theoretical predictions we found no evidence that they increase the number of sperm ejaculated in response to either of the odour cues indicating an elevated risk of sperm competition. Similarly, we found no significant differences between treatment groups in other traits linked to ejaculate investment, including copulatory plug size and copulatory behaviour. These findings contrast with previously studied rodent species, suggesting that responses to cues of sperm competition risk may display considerable interspecific variability. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. Sperm competition occurs when the sperm of two or more males compete to fertilize a given set of ova (Parker 1970, 1998). Because male success in sperm competition often depends on the number of sperm transferred at mating (e.g. Martin et al. 1974; Preston et al. 2003), but the costs of producing large numbers of sperm are substantial (Dewsbury 1982), evolutionarily stable strategy (ESS) models have sought to predict optimal sperm allocation patterns when the likely occurrence or intensity of sperm competition varies (e.g. Parker 1990a, b, 1998, 2000; Parker et al. 1996, 1997; Parker & Ball 2005). A key prediction of such sperm competition theory is that males should increase the number of sperm transferred in a particular ejaculate in response to an elevated risk of sperm competition at the time of mating (Parker et al. 1997; Parker 1998). In an extension to this general theory, Parker (2000) considered the case in which males know their relatedness to their rivals, which affects the dynamics of sperm allocation because of the inclusive fitness benefits to be gained * Correspondence: S. A. Ramm, Mammalian Behaviour & Evolution Research Group, Faculty of Veterinary Science, University of Liverpool, Leahurst Campus, Neston CH64 7TE, U.K. address: sramm@liv.ac.uk (S.A. Ramm). through the reproductive success of relatives (Hamilton 1964). Here, theory predicts that the increased sperm allocation favoured under an elevated risk of sperm competition should be reduced in direct proportion to the coefficient of relatedness of the rival (e.g. r ¼ 0.5 for a brother; Parker 2000). Consistent with the theoretical predictions outlined above (Parker et al. 1997; Parker 1998), there is now evidence that males of diverse animal taxa are able to adjust sperm allocation facultatively, and often transfer more sperm when mating under conditions associated with an elevated risk of sperm competition (reviewed in Wedell et al. 2002; see also Evans et al. 2003; Pizzari et al. 2003; delbarco-trillo & Ferkin 2004; Pound & Gage 2004; but see Schaus & Sakaluk 2001; Ramm & Stockley 2007). The cues used by males to assess sperm competition risk vary between species, and include information relating to the presence of rival males as well as the mating status of females and of the subject male himself (see Parker et al. 1997; Wedell et al. 2002). In mammals, evidence suggests that males can adjust ejaculate allocation in response to cues including whether or not they have guarded the female prior to copulation (Norway rats, Rattus norvegicus: Bellis et al. 1990), the presence of a rival male (R. norvegicus: Pound & Gage 2004; house mice, Mus musculus domesticus: Preston & Stockley 2006; Ramm & Stockley 2007) and odour cues indicating the number and /$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi: /j.anbehav

2 686 S.A. Ramm, P. Stockley / Animal Behaviour 78 (2009) condition of rival males in the vicinity (meadow voles, Microtus pennsylvanicus: delbarco-trillo & Ferkin 2004, 2006; Vaughn et al. 2008). This latter response to male odours is likely to be adaptive for many species of mammals which rely primarily on odour cues for communication (Brennan & Kendrick 2006; Roberts 2007). Moreover, since scent marks can potentially be used to assess information about individual identity and relatedness (reviewed in Thom & Hurst 2004; Brennan & Kendrick 2006), it is possible that males of some species may also use such cues to discriminate between rivals of differing relatedness when adjusting their sperm allocation strategies. The house mouse is an ideal mammalian model to test predictions concerning sperm competition risk. Dominant male house mice aggressively defend territories against rival males, but are not always successful in monopolizing mating opportunities with resident females in their territory, since females may move between male territories and seek additional copulations (Bronson 1979). Consistent with these behavioural observations, Dean et al. (2006) reported evidence of multiple paternity in around 20% of litters in natural populations of house mice, again placing this species within the sperm competition risk range identified by Parker (1998). Male house mice use urinary scent marks to communicate with conspecific females and rival males (Hurst et al. 2001; Hurst & Beynon 2004), and so, like meadow voles (delbarco- Trillo & Ferkin 2004, 2006), may be sensitive to odour cues indicative of elevated sperm competition risk. Moreover, it is known that urinary cues encode individual identity signals in mice (Hurst et al. 2001; Cheetham et al. 2007) and are used to assess relatedness in the context of mate choice (Sherborne et al. 2007). Such cues might therefore be used as a basis for discriminating between related and unrelated rivals in sperm allocation decisions. We tested whether male house mice adjust sperm allocation in response to odourbased cues of rival males present in the environment, and also whether they vary sperm allocation decisions according to the relatedness of these rival males. METHODS Subjects and Husbandry Male subjects (aged months) were from a large, outbred colony of wild house mice maintained for six or fewer generations in captivity, originally derived from several local populations in Cheshire. Each male was individually housed under standard husbandry conditions for wild house mice, that is, in a cage measuring cm and 12 cm high (M3, North Kent Plastic Cages Ltd., Rochester, U.K.), with Corn Cob Absorb 10/14 substrate and paper wool bedding material, and ad libitum access to food (LabDiet 5002) and water. All subjects were maintained under controlled environmental conditions (20 21 C, relative humidity 45 65%) and a reversed 12:12 h light cycle (lights off at 0800 hours). At the end of the experiment, subject males were returned to the stock of animals maintained for use by other researchers. Females used for each mating assay were from a pool of 44 sexually experienced BALB/c laboratory mice maintained in pairs but otherwise housed identically to males. Laboratory females were used to minimize variation caused by female effects and hence to minimize the number of animals used (Festing et al. 2002), and for their increased propensity to mate under laboratory conditions. To induce oestrus cycling prior to mating assays, females were stimulated with soiled bedding from a nonexperimental male (Marsden & Bronson 1964) and females in oestrus were identified on each morning of the experiment according to vaginal cytology (Bronson et al. 1966). If the pair failed to mate, the female was returned to the pool and used in a new mating assay at least 4 days later. No Home Office licence or local ethical review was required for the study. Experimental Design We aimed to test whether male house mice adjust their investment in ejaculates in response to odour cues of an elevated sperm competition risk, and whether they respond differently to the odours of brothers and unrelated males. Odour cues were introduced to the subject males home cages shortly before they mated, to simulate a situation whereby the subject male detects evidence of a recent intrusion into his territory, indicating an elevated risk of sperm competition with respect to the subsequent copulation. To reduce variation and minimize animal numbers, we used a matched-subject design such that each male was tested in three treatments: (1) after exposure to its own odour ( self control treatment); (2) after exposure to the odour of an unrelated male ( unrelated male treatment), and (3) after exposure to the odour of an unfamiliar brother ( brother treatment). The number of sperm transferred in the first ejaculate was then compared for each male across treatments to test for evidence of differential sperm allocation in response to the presence or absence of rival male odour cues (see below). There was an interval of at least a week between treatments, and trials were balanced with respect to treatment order. To ascertain an appropriate sample size for our study, we conducted a power analysis based on results reported by delbarco-trillo & Ferkin (2004), which is the only previous study to have used a comparable experimental design. This study found that meadow voles adjust sperm allocation in response to odour cues from a conspecific male by increasing sperm allocation by 72% on average. Since the two treatments used by delbarco-trillo & Ferkin (2004) are broadly equivalent to our self and unrelated treatment groups, we used the difference in sperm allocation from their study and estimates of between male variability from a previous study of sperm allocation in house mice (Ramm & Stockley 2007) in our power analysis, assuming that the sperm allocation in the brother treatment group would be intermediate between the self and unrelated treatments as predicted by theory (Parker 2000). In a fully paired design, we calculated that to achieve a conventional power of 0.80 (Quinn & Keough 2002) would require a sample size of three and five pairs for the self unrelated and brother unrelated comparisons, respectively, and 0.95 power would be achieved with four and six pairs, respectively. To minimize the total number of animals used while retaining high power to detect predicted effects, we therefore designed the study to achieve at least six males mating in all three treatment groups (Festing et al. 2002). Because male mating propensity under these experimental conditions is low (Ramm & Stockley 2007), we used a total of 22 subject males, 11 of which were excluded during the course of the experiment after failing to mate on three consecutive attempts or because they had failed to mate after two attempts by the end of the experiment. There was no evidence that the treatment group affected male mating propensity, based on whether or not each of the 22 males mated on the first occasion they were paired with a female (Fisher s exact test: P ¼ 0.2). When our goal of six males mating in all three treatments had been achieved, two remaining subjects had mated in two treatments and three had mated in one treatment, giving us sperm allocation data from a total of 25 matings across 11 males. Power to detect predicted relationships of similar magnitude to those observed by delbarco-trillo & Ferkin (2004) should therefore be high (>0.97 in both paired self brother and brother unrelated tests and an overall one-way ANOVA using data from all 25 matings). Moreover, our power to detect an effect in the opposite direction to that predicted by

3 S.A. Ramm, P. Stockley / Animal Behaviour 78 (2009) theory, but similar to that found in our previous experiment for house mice (Ramm & Stockley 2007), was also high (0.85). Odour Stimuli Stimulus donors were either experimental males or nonexperimental males maintained under identical conditions. Each odour stimulus comprised approximately 25 g of soiled bedding from the floor of the donor s cage, which was then sealed in a small plastic bag, coded (so that the experimenter was subsequently blind to its source), and stored at 20 C prior to use (median 37 days, maximum 117 days). Freezing of samples prior to use is routine in studies of scent communication in mammals with no detectable effect on subjects responses to social odours (Smadja & Ganem 2002; Lenochova et al. 2009). In house mice, identity-encoding nonvolatile components of urine remain biologically active after freezing (e.g. Cheetham et al. 2007) and slowly released volatiles remain at high abundance (Armstrong et al. 2005). For each male, we collected three types of odour stimulus: (1) soiled bedding from its own cage (for use in the self treatment); (2) soiled bedding from an unrelated male s cage (for use in the unrelated male treatment); and (3) soiled bedding from a brother s cage (for use in the brother treatment). Unrelated males were identified as being of a similar age to the subject but sharing no grandparents from our database records of the mouse colony. To avoid potential confounding effects of familiarity when comparing subjects responses to related versus unrelated males, we additionally only selected odour donors for the brother treatment group that the subject male had not encountered prior to the odour exposure. To achieve this, brothers were full sibs of the subject, but came from a different litter. Mating Assays Mating assays were conducted under low-level red lighting in a high-sided enclosure 60 cm 60 cm containing food and water. Initially, each male subject was introduced into the enclosure inside its home cage. After removing all excess bedding and behavioural enrichment from the male s cage, we commenced remote video recording and introduced the odour stimulus into the cage in a standardized position (at the end furthest from the food and water dispensers). After 30 min, the lid of the cage was removed to enable the male to enter the larger enclosure, and bedding material from the subject female was introduced into the cage. After a further 10 min, the female was introduced into the enclosure. To reduce the risk of aggression from the male, the female was initially constrained within a plastic cylinder 20 cm long 5 cm in diameter with wire mesh covering one end and the other end lodged against the enclosure wall. After ensuring the male was in the enclosure, we removed the male s home cage. After a further 10 min the female was released, and remote monitoring for mating activity commenced. All trials were continuously monitored remotely, using a CCTV video stream relayed to an adjoining laboratory. Trials were terminated if no mating activity was observed within the first 60 min after the female was released. Trials were also terminated in the rare cases when the male persistently chased the female around the enclosure, to avoid the risk of aggression and injury to the female (four trials). No injuries to females were observed. Terminated trials were repeated atleast 4 days later with the same odour stimulus and either the same or a different female. When the pair did mate, the trial was terminated after the male s first ejaculation. Sperm Counts Females were killed 10 min after ejaculation with an overdose of halothane, weighed and, after a further 10 min, dissected to recover sperm from the reproductive tract as described previously (Ramm & Stockley 2007). After removal, the reproductive tract was cut immediately rostral to the copulatory plug and quickly placed in a Sterilin tube containing 2 ml of 1% citrate solution, at which point the two uterine horns were also cut immediately caudal to the haemostats to collect all sperm contained within the region of the tract bounded by the plug and clamps. To aid sperm dispersal, the separated female tract was first cut in half (i.e. into two uterine horns) and then a longitudinal incision made along each horn. The mixture was agitated gently for 5 min, mixed thoroughly with a pipette, and then a small amount pipetted out into the two chambers of an improved Neubauer haemocytometer. The haemocytometer was then placed inside a sealed box containing moist cotton wool and allowed to stand for 15 min before sperm were counted under a 40 microscope objective based on standard techniques (European Society of Human Reproduction and Embryology 2002). We weighed a second ejaculate component, copulatory plug mass, immediately after each dissection (0.001 g). Quantifying Male Behaviour We analysed the remote DVD recordings of each trial to quantify male investigatory behaviour after the introduction of the odour stimulus and male copulatory behaviour after the introduction of the female. Investigation of the odour stimulus was assessed by recording the total amount of time spent in the vicinity of the odour stimulus for the first 10 min after its introduction. This was standardized by measuring the amount of time the nose of the mouse was within a defined region at the end of the cage where the stimulus was introduced (delineated by the cage walls on three sides and a metal bar on the cage lid on the fourth side, and equal to approximately one-third of the total cage area). For copulatory behaviour, we measured three parameters: intromission latency (the time from female release to the first intromission), ejaculation latency (the time from the first intromission to the first ejaculation) and intromission number (the total number of intromissions prior to the first ejaculation). Statistical Analyses We conducted power analyses (all for two-tailed tests) using UnifyPow.sas version (O Brien 1998), a macro for SAS (implemented in SAS version 9.1; SAS Institute Inc., Cary, NC, U.S.A.) or the power.t.test function in the stats package for R version (The R Foundation for statistical computing, Vienna, Austria). Appropriate transformations of the raw data for parametric analysis were identified using the box.cox.powers function in the car package for R version 2.4.0: sperm counts and copulatory behaviour parameters were square-root transformed (l z 0.5), whereas no transformation was applied for time spent in the vicinity of the odour stimulus, since the estimated l did not differ significantly from one. All parametric statistical analyses were conducted using general linear models (to test for an overall treatment effect on sperm allocation) and paired t-tests (to compare specific treatment groups) in SPSS version 14.0 (SPSS Ltd, Chicago, IL, U.S.A.) using two-tailed significance tests. RESULTS Odour Stimulus Investigation When odours were introduced into subject males home cages, we found that they responded differently depending on the source of the odour stimulus ( self, brother or unrelated male ). That is,

4 688 S.A. Ramm, P. Stockley / Animal Behaviour 78 (2009) Table 1 Summary statistics for male investigative, ejaculate allocation and copulatory behaviour responses following exposure to conspecific odour stimuli from themselves ( Self ), a brother ( Brother ) or an unrelated male ( Unrelated ) Parameter Self Brother Unrelated Overall GLM F 2,22 P Odour investigation (s) Sperm allocation (x10 6 ) Copulatory plug mass (g) Intromission latency (s) Ejaculation latency (s) Intromission number Results of general linear models (GLMs) reported are based on log-transformed data. Means are given SE. the duration of time spent in the vicinity of the different odours during the first 10 min after their introduction into the male s home cage differed significantly according to odour source, both during the first trial in which the male was presented with each odour (Table 1, Fig. 1a) and during the trial in which the male subsequently copulated after being presented with the odour (F 2,21 ¼ 4.54, P ¼ 0.023; note that males mated following the first presentation of the odour in 17/25 [68%] of all trials; one trial was excluded from the second analysis owing to problems with DVD playback). This significant overall effect appeared to result mainly from a significant difference between the time spent by subject males investigating self and unrelated male odours (paired t-test: based on first trials: t 5 ¼ 3.54, P ¼ 0.017; based on mating trials: t 5 ¼ 3.63, P ¼ 0.015). Time spent in the vicinity of the brother stimulus was intermediate between the other two treatment groups; it did not differ significantly from the unrelated male stimulus (first trials: t 6 ¼ 1.50, P ¼ 0.18; mating trials: t 5 ¼ 1.94, P ¼ 0.11) whereas evidence in comparison to the self stimulus was equivocal (first trials: t 6 ¼ 2.47, P ¼ 0.048; mating trials: t 5 ¼ 1.54, P ¼ 0.19). Ejaculate Allocation and Copulatory Behaviour Although male mice were apparently sensitive to the presence of odour cues from rival males (see above), we found no evidence that their sperm allocation was influenced as a result. The effect of treatment group was not significant in a general linear model with the number of sperm recovered from the female s reproductive tract as the dependent variable (Table 1, Fig. 1b). Hence, although results based on the relatively small sample of 25 matings should be interpreted with caution, we found no evidence that sperm allocation either increases or decreases in the presence of rival male odours, despite high predicted power to detect such effects had they been present (see Methods). Paired tests of the same male s response in each of two treatment groups confirmed this general pattern. There was no detectable difference in sperm allocation between the control self treatment and either the brother treatment (t 6 ¼ 0.54, P ¼ 0.61) or the unrelated male treatment (t 5 ¼ 0.68, P ¼ 0.53). Consistent with the results for sperm allocation, there were no significant overall differences according to treatment group in either copulatory plug mass (Table 1) or any of the three copulatory behaviour parameters investigated: intromission latency, ejaculation latency or intromission number (Table 1). In general, paired tests confirmed these patterns, since six of eight tests were nonsignificant. The two exceptions (ejaculation latency in the self and brother treatments: X SE: self : s; brother : s; t 6 ¼ 2.69, P ¼ 0.036; copulatory plug mass in the self and unrelated male treatments: X SE: self : g; unrelated male : g; t 5 ¼ 4.02, P ¼ 0.01) were not consistent across the three treatment groups and, given their size and direction, appear unlikely to be functionally relevant to the outcome of sperm competition. DISCUSSION We found no evidence that male house mice adjust the number of sperm allocated to an ejaculate in response to odour-based cues of 350 (a) 7 (b) Investigation (s) Sperm allocation (x 10 6 ) S B U 0 Rival stimulus S B U Figure 1. The effect of odour stimulus origin on (a) time spent in the vicinity of the odour stimulus for 10 min after its introduction and (b) subsequent sperm allocation in wild house mice exposed to an odour stimulus deriving from one of three sources: self (S), brother (B) or an unrelated male (U). Effect of stimulus origin on time spent in the vicinity of the stimulus: F 2,22 ¼ 4.91, P ¼ 0.017; effect on sperm allocation: F 2,22 ¼ 0.55, P ¼ 0.58.

5 S.A. Ramm, P. Stockley / Animal Behaviour 78 (2009) immediate sperm competition risk, at least not to the extent observed in a previous rodent study (delbarco-trillo & Ferkin 2004). Male mice are highly responsive to odour cues of rival males in other competitive contexts (e.g. Hurst et al. 2001), and odour-based cues are critical for many aspects of social and sexual behaviour in this (e.g. Cheetham et al. 2007; Sherborne et al. 2007; Thom et al. 2008) and other rodent species (see Roberts 2007 and references therein). Accordingly, our subject males showed a significantly heightened level of investigation of odour stimuli from rivals (compared to self ), indicating that a foreign odour was detected, even though it did not influence subsequent sperm allocation. These findings for house mice contrast with evidence from a second rodent species, the meadow vole in which males increase sperm allocation in response to an odour cue from one rival male (i.e. high sperm competition risk; delbarco-trillo & Ferkin 2004) but then decrease sperm allocation when presented with odour cues from several other males (i.e. high sperm competition intensity; delbarco-trillo & Ferkin 2006), in accordance with sperm competition theory (Parker 1998). Our results thus suggest that the propensity to adjust sperm allocation to immediate sperm competition level is not necessarily common to all rodents, and that adaptations to sperm competition in this group can be variable (see below). The absence of a detectable response to odour cues from rival males, in terms of both sperm allocation and copulatory behaviour, contrasts with how male mice respond to the actual presence of a rival male. When a rival is present, male house mice significantly reduce the number of intromissions performed prior to ejaculation, and ejaculate fewer sperm (Preston & Stockley 2006; Ramm & Stockley 2007; see also Pound & Gage 2004). Since no similar response was observed here to the presence of a rival male odour, our results support the idea that altered copulatory behaviour and reduced sperm allocation observed previously in the presence of rival males should be interpreted as a response to the risk of take-over, with adaptive changes in copulatory behaviour to hasten ejaculation acting as a constraint on sperm allocation. Since male behavioural responses to the presence of a rival may potentially constrain sperm allocation decisions, rival male odours may often be a more appropriate cue of sperm competition risk for testing predictions of sperm competition theory in this, and possibly many other, species in which males compete aggressively (see also delbarco-trillo & Ferkin 2004). Hence, although we found no evidence for facultative adjustment of sperm allocation in response to the presence of rival male odours in the present study, we would argue that our findings none the less indicate that the response to rival male odours is probably a more useful test of sperm allocation theory than is the response to rival males per se. More broadly, this emphasizes the importance of selecting a species-appropriate cue of sperm competition level when testing predictions of sperm competition games (see also Parker et al. 1997; Engqvist & Reinhold 2005). We also found no evidence that male house mice responded differently according to the relatedness of potential sperm competition rivals. Rival male relatedness is predicted to affect male sperm allocation (Parker 2000), but other than the present study, we know of only one previous attempt to test this theory. Similar to results shown here, Thomas & Simmons (2008) found that male Australian field crickets, Teleogryllus oceanicus, do not respond differently to related and unrelated rivals, although they do adjust sperm viability in a manner implying greater investment in copulations involving a heightened risk of sperm competition (Simmons et al. 2007). Given the high costs of dispersal and social structure of house mice (Bronson 1979), and the risk of sperm competition experienced by males (Dean et al. 2006), it seems reasonable that brothers will sometimes be in competition for fertilizations in this species. None the less, there also exist more stable competitive situations involving related males in other species, for example coalitions between brothers to attain copulations occur regularly in lions, Panthera leo, and cheetahs, Acinonyx jubatus (Parker 2000), and it would be of particular interest to explore whether sperm allocation and production are affected under such conditions. In all cases, however, the model assumes that males will be able to identify closely related individuals, which may not always be easy to achieve reliably (Sherbourne et al. 2007). In the present study, levels of odour investigation of the brother stimulus were intermediate between those of the self and unrelated stimuli, and more variable. Closer inspection of the data reveals that this pattern was driven by a greater variance in male response to the brother stimulus, which may relate to the mechanism of kin recognition in this species. Specifically, three males showed a low level of investigation of the brother odour stimulus comparable to their investigation of the self stimulus, whereas the other males tended to show similar levels of investigation to the brother and unrelated stimuli (results not shown). Such a pattern is consistent with genetically encoded kin recognition based on major urinary proteins (MUPs), which may lead to odours from brothers not being recognized as nonself if they share both MUP haplotypes (Hurst et al. 2001; see also Sherborne et al. 2007). Increasing the number of sperm in their ejaculate is not the only way in which males may respond to an elevated risk of sperm competition. In house mice, there is evidence that males might instead adjust total sperm allocation by altering the number of ejaculates delivered to each female (Preston & Stockley 2006). Factors other than the total sperm numbers could also influence the outcome of sperm competition, for example sperm morphology (Firman & Simmons 2008). Moreover, rather than (or in addition to) responding to immediate cues of sperm competition level (delbarco-trillo & Ferkin 2004, 2006; Pound & Gage 2004), males may adjust sperm production and allocation in response to variation in the population average level of sperm competition (Parker et al. 1997). Although our results indicate that male house mice do not adjust their sperm allocation in response to the immediate risk of sperm competition, we have recently found evidence that they are able to adjust overall investment in sperm production according to cues reflecting sperm competition risk at a population level (Ramm & Stockley 2009). In conclusion, although evidence for facultative adjustment of ejaculate size in response to sperm competition level is found among diverse animal taxa (reviewed in Wedell et al. 2002; see also Evans et al. 2003; Pizzari et al. 2003; Pound & Gage 2004), and may often be mediated by odour-based cues (delbarco-trillo & Ferkin 2004, 2006; Carazo et al. 2007; Thomas & Simmons 2009), we have found no evidence that male house mice increase sperm allocation per ejaculate in response to odour-based cues of heightened sperm competition risk, either from related or unrelated males. This reinforces our earlier conclusion (Ramm & Stockley 2007) that the facultative adjustment of ejaculate size is not necessarily common to all rodent species, despite clear evidence that it occurs in some cases (delbarco-trillo & Ferkin 2004, 2006; Pound & Gage 2004; see also Pound 1999). Given the range of sperm allocation patterns observed so far in rodents (delbarco-trillo & Ferkin 2004, 2006; Pound & Gage 2004; Ramm & Stockley 2007; this study), as well as well-documented variation in related reproductive traits (Stockley & Preston 2004; Ramm et al. 2005), this group may prove a particularly useful model for exploring diversity in adaptations to both immediate and average levels of sperm competition. Acknowledgments We thank J. Hurst and members of the Mammalian Behaviour & Evolution group for their support and constructive feedback, L. Burgess, F. Fair, J. Fick, R. Humphries, S. Jopson, J. F. Lemaître and

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