Trish Moore, Centre for Ecology and Conservation, School of Biosciences, University of Exeter, Cornwall Campus, Penryn, TR10 9EZ, UK. Tel.: +44 1326 371878; fax: +44 1326 253638; e-mail: firstname.lastname@example.org
There is increasing recognition that male–male competition can take many forms, but as yet the form is not predictable a priori. Many recent studies have focused attention on how males in disadvantaged mating roles compensate through sperm competition. However, mating systems in which subordinate males are reproductively suppressed, particularly through the stress of social interactions, may limit the ability of males to respond by increasing investment in sperm quality. We examined the interaction between social status and ejaculate tactics in Nauphoeta cinerea, a cockroach that has a mating system with well-characterized dominance hierarchies. Both social experience with other males and social status influenced aspects of ejaculates. The stress of social interactions reduced the size of the ejaculate and number of sperm inseminated. In ejaculates formed prior to social experience, however, males that go on to become dominant inseminated more sperm than males that go on to become subordinate, suggesting innate differences among males. Our results show that though selection for increased success in sperm competition for subordinate males in a hierarchy can occur, both the traits and the way in which the balance between pre- and post-copulatory strategies is negotiated will depend on specific details of the mating system. These details will include how the physiological effects of social interactions may limit selection through male–male competition.
It is certain that amongst almost all animals there is a struggle between the males for the possession of the female. This fact is so notorious that it would be superfluous to give instances. Darwin, 1871; p. 259.
Although ubiquitous, male–male competition can take many forms. In some species, overt social interactions and male struggles result in the establishment of a social hierarchy where dominant males have preferential access to females. However, how or when do subordinate males realize fitness benefits? If male reproductive success is distributed between pre- and post-copulatory traits, there is the opportunity for selection on both (Shuster & Wade, 2003). Variation in male reproductive success may depend on both the ability of males to compete for copulations and, if females mate with more than one male, the ability of their sperm to compete for fertilization (Parker, 1998; Pizzari & Birkhead, 2002; Snook, 2005; Andersson & Simmons, 2006). Under these conditions subordinate males could adopt less-obvious post-copulatory tactics associated with sperm competition to counteract their initial disadvantage in precopulatory competition (Parker, 1990a,b, 1998;Gage et al., 1995).
There are two opposite relationships between status and ejaculates, with evidence for both. Are both adaptations? Or does the stress of being subordinate result in a physiological constraint that prevents males from investing in reproduction (e.g. Tilbrook et al., 2000)? Do Parker’s models hold only when there is no stress of being subordinate? Male condition is predicted to drive patterns of ejaculate allocation (Harris & Lucas, 2002). Sperm production can be costly (Dewsbury, 1982; Van Voorhies, 1992; Sella & Lorenzi, 2003) and, coupled with the cost of subordination in the form of increased stress, may prohibit subordinate males from evolving superior sperm. Thus, we need to examine how sexual selection mediated through male–male competition influences sperm allocation in a species where the stress of social interactions is documented. If lower social status is stressful, does that constrain the post-reproductive tactics that males adopt?
In this study, we examine the inter-relationship between social status, social experience and sperm quality in the cockroach Nauphoeta cinerea. Males of this cockroach species form linear dominance hierarchies (Ewing, 1972, 1973; Bell & Gorton, 1978) and dominant males monopolize mating opportunities (Schal & Bell, 1983; Moore & Moore, 1999; Moore et al., 2001). Subordinate status reduces male lifespan (Ewing & Ewing, 1973). Thus, N. cinerea offers us an opportunity to examine the interaction between males differing in social status and ejaculate quality in an experimentally tractable species with both pre- and post-copulatory intrasexual selection and large differences in mating success for males of different status.
We tested the hypothesis that the relationship between status and ejaculate quality in N. cinerea is similar to species such as mice and the cichlid fish, where both the opportunities for selection on post-copulatory strategies are limited and the stress of being subordinate is high. We manipulated social experience and measured status, sperm quality, sperm reserves and ejaculate characteristics previously shown to be important in male reproductive success in this species (Montrose et al., 2004). We measured characteristics of individual males prior to and after male–male social competition to ask whether social experience and social status changes male ejaculate tactics or if there are intrinsic differences associated with males of different status.
Materials and methods
Nauphoeta cinerea mass colonies and individual experimental animals were housed under standard conditions of 27 °C, 12/12-light/dark photoperiod, provided with ad libitum rat chow and water. Late-instar nymphs were isolated from mass colonies and separated according to sex. Nymphs were housed in 17 × 12 × 6.5-cm plastic containers with food and water. On the day of adult emergence, adult animals were isolated in individual 11 × 11 × 3-cm plastic boxes with food and water. Males were marked at 5 days post-adult emergence by applying a 3-mm filter paper disc with a numerical ID. Discs were affixed to the individuals with rubber cement, which has no effect on behaviour (Moore et al., 1997). Males were then placed back into their individual containers until they were allocated to one of the experimental treatments described below.
Evaluation of male social status
Males were either isolated for the duration of the experiment (socially naïve) or experienced male–male social interactions between 7 and 12 days post-adult emergence (socially experienced). Males are sexually mature by 7 days of age (Moore et al., 1995). Socially experienced males were characterized by their social status at the end of male–male interactions. In order to assign social status to males in groups, four randomly chosen 7-day-old males were placed into an open plastic container (17 × 12 × 6.5 cm). The social groups were observed for 10 min on the first day and for 10 min every 24 h for a total of 5 days. We recorded all behaviours during agonistic interactions between the four males (Bell & Gorton, 1978; Moore, 1990). These behaviours included the aggressive acts of lunging, butting, biting, grappling, chasing and climbing on top of the other male, along with the submissive acts of crouching, retreating slowly from an interaction and running away. All observations were carried out under photographic red light at 27 °C. At the end of the 5-day observation period, each male was assigned a social ranking based on the number of aggressive or submissive interactions with the other males in his group. Nauphoeta cinerea males form linear dominance hierarchies where the alpha male dominates all other males in the group, the beta male dominates all but the alpha male, and so on until the most subordinate male that is submissive to all other males in the group (Ewing, 1972, 1973; Bell & Gorton, 1978). The most dominant (highest ranking male within his group) or most subordinate (lowest ranking male within his group) were used for further study.
Characterization of ejaculate characteristics
Males were mated to a 10-day-old virgin female to obtain ejaculates for examination. Following copulation, the spermatophore was removed from the female and weighed immediately (Ohaus Explorer analytical balance; Ohause, Pine Brook, NJ, USA). The sperm ampulla, containing the sperm, was then removed from the spermatophore and weighed separately. The ampulla was then placed in 200 μL of cockroach Ringers and ground with a pestle to release sperm. Sperm numbers were assessed as in Moore et al. (2004). Briefly, a 10-μL aliquot was diluted 10×, stained with 5-μL 0.05% Eosin Y, and seven 5-μL subsamples dried onto a slide. All sperm within the seven subsamples were counted. Sperm viability was assessed on the remaining sperm from the ampulla using the LIVE/DEAD sperm viability kit (Molecular Probes, Leiden, The Netherlands) using the manufacturer’s recommended protocol and observed on an Olympus BX51 fluorescent microscope (Olympus UK Ltd, Watford, UK). The sperm viability kit contained two fluorescent dyes, a membrane-permeable nucleic acid stain SYBR 14 and the membrane impermeable dye propidium iodide. SYBR 14 enters living cells and stains the nuclei green. Propidium iodide only enters dead cells, staining them red. The percent living sperm was calculated and the number of sperm with green nuclei divided by the total number of sperm observed over a random selection of 10 fields of view.
At the end of the experiment, male body size was measured by capturing an image using an Olympus SZX9 stereomicroscope and a Nikon coolpix digital camera (Nikon, Kingston upon Thames, UK). A 1 mm standard was included in all photographs. The length of the pronotum was measured on the photomicrographs with ImageJ 1.29× (http://rsb.info.nih.gov/ij/). Males were then dissected and their seminal vesicles removed and weighed (Ohaus Explorer analytical balance) to determine potential sperm reserves. All males were 12 days post-adult emergence on the day of dissection.
We first compared spermatophores from three groups of males mated for the first time at 12 days of age to examine the effects of social status on ejaculate characteristics independently of age. We compared spermatophores from socially naïve males, males that were socially experienced and characterized as the most dominant of their group, and males that were socially experienced and characterized as the most subordinate of their group.
We next compared spermatophores from socially experienced males both before and after social experience to determine if ejaculate characteristics changed as a result of social experience. Males were mated immediately before being put into male social groups, the spermatophores were then recovered and characterized as described. At the end of male–male social interactions, males were mated for a second time and the spermatophore recovered and characterized. Because these males had been assigned a ranking within their group, we could compare the spermatophores formed before and after social experience by individual males ranked as dominant or subordinate.
Finally, we examined the effect of social experience and social status on male sperm reserves. All males were dissected at the end of the experiment to characterize their seminal vesicle. Preliminary results indicated that the number of matings a male undertakes prior to dissection affects the size of the seminal vesicle, therefore we controlled for the number of matings in all treatments. Thus, socially naïve males were either dissected as virgins (zero matings), after mating at 12 days of age (one mating) or after mating at 7 days of age and then again at 12 days of age (two matings). Socially experienced males were either dissected as virgins (zero matings), or after mating at the end of social experience at 12 days of age (one mating), or after mating before social experience at 7 days of age and then again following social experience at 12 days of age (two matings). The socially experienced males were further characterized by their ranking within their group.
We analysed all data with anova, using Fisher’s LSD for specific pair-wise comparisons as we had specific a priori hypotheses regarding differences among groups. We used systat 9.0 for all analyses.
In all of our experiments we were interested in the effects of social experience independent of age. However, social experiences occur over time and age did effect ejaculate characteristics. In specific pair-wise comparisons between socially naïve 7-day-old males and socially naïve 12-day-old males, the mass of the spermatophore and sperm ampulla were smaller in the 7-day-old males (P < 0.001, P = 0.021, respectively). Sperm viability of socially naïve 7-day-old males was lower than that of socially naïve 12-day-old males (P = 0.029) and socially naïve 7-day-old males inseminated fewer sperm than socially naïve 12-day-old males (P = 0.008). Thus, in all our analyses we controlled for age either by only comparing the ejaculate characteristics of males at the same age or by using repeated measure anovas to specifically analyse changes over time in ejaculate characteristics as a function of social experience.
Association between male body size and social status
Male body size did not influence male status. There was no significant difference between the size of dominant and subordinate males (t = −0.364, d.f. = 27, P = 0.719).
Association between social status and ejaculates
We compared the first ejaculates of socially naïve 12-day-old males to those of socially experienced dominant or subordinate males. Social experience, but not status, influenced spermatophore size (Fig. 1a; F2,71 = 3.186, P = 0.047). In specific pair-wise comparisons isolated males produced bigger spermatophores than males that have been in a hierarchy. Thus, the spermatophore of isolated males was significantly larger than both dominant (P = 0.035) and subordinate (P = 0.038) males. However, there was no significant difference between dominant and subordinate males (P = 0.675). There was no effect of social experience or status on the sperm ampulla size (F2,71 = 0.061, P = 0.941).
Males that have experienced male–male competition inseminated fewer sperm than socially naïve males of the same age (Fig. 1b; F2,62 = 4.446, P = 0.016). This effect was further influenced by status. Isolated males inseminated more sperm than dominant males (P = 0.006), but subordinate males were not significantly different than either socially naïve males (P = 0.052) or dominant males (P = 0.231). There was an effect of social experience on sperm viability (Fig. 1c; F2,71 = 66.689, P < 0.001). Socially naïve males had lower sperm viability than both dominant males (P < 0.001) and subordinate males (P < 0.001), but dominant and subordinate males did not differ (P = 0.609).
In order to examine whether differences in ejaculate characteristics due to social status reflected an innate characteristic of males, we compared males before and after social experience (Fig. 2). In the ejaculate formed prior to social experience, though there was no difference in spermatophore size (F1,57 = 1.573, P = 0.215), males that went on to become dominant inseminated significantly more sperm prior to social experience than males that went on to become subordinate, (Fig. 2a; F1,38 = 4.133, P = 0.0491). The number of sperm inseminated decreased following social experience (time repeated measures anova; F1,38 = 6.0651, P = 0.0142). Though the interaction between time and status was not significant (time * status F1,38 = 3.0314, P = 0.0898), the trend was for dominant males to experience a greater drop in sperm numbers (Fig. 2a).
There was no difference in sperm viability in the ejaculate formed prior to social experience between males that went on to become dominant and those that went on to become subordinante (Fig. 2b; F1,58 = 0.185, P = 0.669). Sperm viability did not change following social experience (time repeated measures anova; (F1,58 = 1.729, P = 0.194), and there was no significant interaction between time and status (F1,58 = 0.051, P = 0.823).
Effects of social experience and social status on sperm storage
To examine the effect of social experience and social status on sperm stores, we compared the sizes of the seminal vesicles of 12-day-old males following different social experiences. We tested for the effect of the number of mates as well as social status (Fig. 3). The number of times a male mated had a significant effect on seminal vesicle size (F2,240 = 45.289, P < 0.001). Virgin males had larger seminal vesicles than males that had mated either once (P < 0.001) or twice (P < 0.001). In addition, males mated once had significantly smaller seminal vesicles than males mated twice (P = 0.027).
There was no effect of status on seminal vesicle size (F2,240 = 0.072, P = 0.931). However, there was a significant interaction between the number of matings and status (F4,234 = 4.957, P < 0.001). Dominant males that had never mated had seminal vesicles that were significantly larger than subordinate males (F1,57 = 6.661, P = 0.013) and approach significance compared with isolated males (F1,48 = 3.528, P = 0.066). Subordinate males and isolated males that had never mated were not significantly different (F1,47 = 0.142, P = 0.708). Males that had mated one time showed a different pattern. Dominant males that mated once had significantly smaller seminal vesicles than subordinate males (F1,57 = 12.704, P < 0.001) and isolated males (F1,45 = 5.327, P = 0.026). Subordinate males and isolated males that had mated once were not significantly different (F1,45 = 1.269, P = 0.266). The effects of status disappeared in males that mated twice. Dominant males were not significantly different from either subordinate or isolated males (F1,57 = 0.062, P = 0.805 and F1,58 = 0.000, P = 1.000 respectively) and subordinate males were not different from isolated males (F1,30 = 0.449, P = 0.641).
Parker (1990a,b) suggests that males should adjust their ejaculate depending on their social role. Other models suggest that sperm allocation will depend on male condition (Harris & Lucas, 2002). We tested the prediction that in N. cinerea the reproductive potential of subordinate males is constrained by the stress of social interactions and thus subordinate males produce poor quality ejaculates. We found that post-copulatory strategies as an alternative to precopulatory roles are possible in this species, and that both flexibility and intrinsic differences occur, but that the stress of social competition also sets limits. Thus, the balance between pre- and post-copulatory reproductive strategies depends on developmental constraints and other life history trade-offs.
Trade-offs between pre- and post-copulatory reproductive tactics
Although social experience, but not social status, influenced spermatophore size and sperm viability, both influenced the number of sperm produced. In the spermatophore formed prior to social experience, males that were later scored as dominant after social interactions packaged more sperm into their ejaculates than males that were later scored as subordinate. These results indicate dominant males are intrinsically different, something that is ignored in all models to date. These results are consistent with previous work showing that the roles that males adopt (dominant or subordinate) is at least partly influenced by genetic factors (Moore, 1990, 1997; Moore et al., 2002). After social interactions, however, both dominant and subordinate males have reduced numbers of sperm, albeit of higher quality. We have thus documented a rather novel pattern of ejaculate strategy in N. cinerea; overall, all males experiencing social interactions seem to invest in fewer sperm of higher quality. Variation in these traits may be influenced by inherited differences along with other traits associated with male status (Moore, 1997; Moore et al., 2004). We have also shown that males that are raised until they first achieve sexual maturity in the presence of the odour of a female inseminate more sperm than isolated males (Harris & Moore, 2005a). Males can become sperm limited (Montrose et al., 2004) and females prefer males that have had fewer mating partners (Harris & Moore, 2005b). Perhaps high status males, which are often also preferred by females, are of higher quality to females because of the greater investment in sperm numbers. However, these males are also likely to have a greater stake in investing in precopulatory success than in investing in the future reproductive potential of their mates.
Patterns of sperm storage were also associated with social status. Virgin males dominant after social interactions had larger seminal vesicles than subordinate males. Dominant males appear to be more capable of investing resources in sperm even when under the stress of intersexual conflict. The change in seminal vesicle size upon mating was also greatest in dominant males, suggesting that dominant males are able to mobilize sperm faster than subordinate males. We did not find any effect of social experience or social status on other measures of sperm quality.
Conclusions – pre- and post-copulatory behaviour as alternative mating strategies
The use of superior sperm competitive ability as an alternative strategy by subordinate males has intuitive appeal, but may not be possible in all species. In some species the dominant male is able to almost completely monopolize females. Nauphoeta cinerea accomplishes this through physiological manipulation of female receptivity (Roth, 1964; Montrose et al., 2004). In laboratory mice, dominant males prevent subordinate males from leaving their cages and encountering females resulting in almost all offspring being sired by dominant males (DeFries & McClearn, 1970). These types of mate guarding behavioural tactics are very successful and mating opportunities, and the opportunity to engage in post-copulatory competition, therefore can be rare for subordinate males. In addition, subordinate males can be under extreme stress and this may further limit their ability to produce good quality sperm (Ewing & Ewing, 1973; Hoffmann et al., 1999;Koyama & Kamimura, 1999; Faulkes & Bennett, 2001). Thus, even though species may have the same category of mating system, dominance hierarchies, there can be considerable differences with regard to the relationship between status and sperm.
It is clear that males vary in pre- and post-copulatory strategies. However, the relationship between pre- and post-copulatory strategies in response to social environments may not be simple. As pointed out by Pizzari et al. (2007), even in systems where phenotypic correlations between male social status and ejaculate quality have been demonstrated, many questions remain regarding the relationship between status and sperm. How to differentiate between constraints and adaptations? What is the causal relationship between status and sperm? Is the trade-off a result of phenotypic plasticity or genetic polymorphisms? These questions deserve our attention. However, focusing only on species in which variation in reproductive success is partitioned between pre- and post-copulatory events will not provide a complete picture. We also need to examine species in which males can affect the risk and intensity of sperm competition through behaviour such as territorial defence or mate guarding (Alonzo & Warner, 2000). Precopulatory competition can influence post-copulatory sperm competition and result in both alternative, facultative, changes in behaviour or in fixed tactics. There can also be constraints on the flexibility arising from both proximate and ultimate influences. Evolutionary outcomes are likely to depend on detailed characteristics of the mating system, the nature and extent of social interactions and the specific mechanisms of sperm competition that are employed.
We appreciate helpful discussions with Tommaso Pizzari and, once again, the insights into sperm competition shared by Dave Hosken as well as his comments on this manuscript. We also thank the two anonymous reviewers for very helpful comments. This research was supported by a grant from the National Environmental Research Council to P.J.M. and A.J.M.