SEARCH

SEARCH BY CITATION

Keywords:

  • sexual conflict;
  • sperm depletion;
  • enforced monogamy;
  • Nauphoeta cinerea

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

An evolutionary conflict often exists between the sexes in regard to female mating patterns. Females can benefit from polyandry, whereas males mating with polyandrous females lose reproductive opportunities because of sperm competition. Where this conflict occurs, the evolution of mechanisms whereby males can control female remating, often at a fitness cost to the female, are expected to evolve. The fitness cost to the female will be increased in systems where a few high status males monopolise mating opportunities and thus have limited sperm supplies. Here we show that in the cockroach Nauphoeta cinerea, a species where males enforce female monogamy in the first reproductive cycle, males that have become sperm depleted continue to be able to manipulate female remating behaviour. Although the manipulation severely decreases fecundity in females mated to sperm-depleted males, males benefit, increasing their relative fitness by preventing other males from reproducing. Our results suggest that there is selection on maintaining the mechanism of manipulation rather than maintaining sperm numbers. Taken with previous research on sexual conflict in N. cinerea, this study suggests that the causes and consequences of sexual conflict are complex and can change across the life history of an individual.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Although the fitness benefits of mating with multiple partners seem straightforward for males (Bateman, 1948; Trivers, 1972), it is more difficult to explain the widespread incidence of polyandry in females (Birkhead & Moller, 1998; Snook, 2001). A number of hypotheses have been proposed to explain why females might mate with multiple males, including genetic benefits, material benefits, convenience, correlated evolution and sperm replenishment (Reynolds, 1996; Jennions & Petrie, 2000; Tregenza & Wedell, 2000; Zeh & Zeh, 2001; Hosken & Stockley, 2003; but see Yasui, 1998). Regardless of the form of the benefits, however, there is considerable empirical evidence that females benefit from mating multiply (Arnqvist & Nilsson, 2000; Tregenza & Wedell, 2000).

Although polyandry may benefit females, promiscuity in females that are able to store sperm can reduce male fitness because of sperm competition (Birkhead & Moller, 1998). This divergence between the reproductive interests of males and females gives rise to sexual conflict and the evolution of mechanisms by which males can manipulate female remating behaviour, often at the expense of female fecundity or longevity (Chapman et al., 1995; Snook, 2001). A number of mechanisms for male control of multiple mating in females are known, including behaviours such as mate-guarding, or introducing chemical or physical inhibitors to female mating receptivity (Andersson, 1994; Gillot, 2003). Thus, male control of female remating when polyandry confers reproductive benefits to females initiates an evolutionary conflict between the sexes, suggesting that patterns of polyandry depend on the outcome of sexual conflict.

Recent selection studies utilizing experimentally enforced monogamy in naturally promiscuous species have demonstrated that sexual conflict can lead to the evolution of mechanisms that manipulate patterns of polyandry, despite a cost to female fitness (Rice, 1996; Promislow et al., 1998; Holland & Rice, 1999; Pitnick et al., 2001a). Currently underrepresented in experimental work on sexual conflict are studies of species where opportunities for remating are naturally limited. Such studies would be interesting because predictions differ when female remating opportunities (and therefore sperm competition) are reduced or absent (Rice, 1996; Pitnick et al., 2001b). For example, if males are able to enforce monogamy, the absence of sperm competition should relax selection on sperm quality and quantity (Hunter & Birkhead, 2002) although there would be strong selection on the mechanism of female manipulation. For females unable to avoid male manipulation, and thus limited to a single mating opportunity, it is predicted that there will be selection on mechanisms that allow them to avoid poor quality males (Moore et al., 2003).

In species where male reproductive success is highly skewed, where high status males dominate mating opportunities or lekking species for example, females that mate with a single male face the possibility of having their reproductive success limited by obtaining insufficient sperm to fertilize all their ova (Wedell et al., 2002). Sperm are known to be costly for males to produce (Dewsbury, 1982; Van Voorhies, 1992; Sella & Lorenzi, 2003), with sperm depletion in males having been demonstrated in a number of species (e.g. Nakatsuru & Kramer, 1982; Preston et al., 2001). Sperm limitation in females and resulting fecundity reduction has also been demonstrated in several species (Jones, 2001; Kolodziejczyk & Radwan, 2003). Although female fecundity can be reduced by low male sperm count, a male that can prevent his mate from remating may benefit by avoiding sperm competition, another source of sexual conflict over mating frequency of females (Eberhard, 1998; Snook, 2001).

In this study we examined the causes and consequences of sexual conflict on patterns of female remating in the cockroach Nauphoeta cinerea, a species with male enforced monogamy on females (Roth, 1964a). We tested the hypothesis that because of selection arising from sexual conflict, male manipulation of female remating would persist even in males with low fertility. We assessed mating behaviour and lifetime reproductive success of females mated with males with manipulated reproductive history. We predicted that frequently mated males would become sperm depleted, but that even sperm depleted males would inhibit remating by females in order to reduce likelihood of encountering sperm competition with another male.

The study organism: Nauphoeta cinerea

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Nauphoeta cinerea is a model system for studying the interaction between sexual selection and sexual conflict in a species with a complex mating system and reproductive cycles of fertilization, pregnancy and birth. Sexual selection in this species involves both male–male competition and female mate choice (reviewed in Moore, 1990a). Males form dominance hierarchies and dominant males monopolize mating opportunities. Females differentiate among males and require significantly less courtship effort from preferred males (Moore, 1990a, b; Clark et al., 1997), although they do not always prefer the most dominant male (Moore, 1988). Sexual selection in N. cinerea is mediated by male-produced sex pheromones. Increased quantities of two components of the three-component pheromone blend, 2-methylthiazolidine and 4-ethyl-2-methoxyphenol, confer dominant status on males (Moore et al., 1997), whereas higher levels of the third component, 3-hydroxy-2-butanone, confers subordinate status. This third component is maintained in the pheromone blend by balancing selection, as it is attractive to females (Moore & Moore, 1999).

Nauphoeta cinerea is ovoviviparous. Females carry developing eggs, packaged into an ootheca, in a brood pouch until the offspring emerge as first-instar nymphs (Roth & Willis, 1954). The presence of the ootheca in the brood pouch activates a stretch receptor and inhibits female receptivity and growth of oocytes for the second clutch of offspring (Roth, 1964b). Oocyte maturation and growth in preparation for the second clutch is initiated after parturition of the first clutch of offspring. Some, but not all, females are receptive to mating for 24–48 h following parturition (Roth, 1964b; Moore et al., 2003).

Sexual conflict occurs in this species because of male manipulation of female mating behaviour (Moore et al., 2001, 2003). Mating and insertion of a spermatophore into the bursa copulatrix inhibits the sexual receptivity centre in the female's brain (Roth, 1964a). Inhibition of receptivity depends only on the physical presence and size of the spermatophore and not on chemical signals from it, as receptivity can be inhibited by insertion of a glass bead into the bursa copulatrix (Roth, 1964a). Male control of female remating behaviour enforces monogamy on females and ensures paternity of the first clutch of offspring.

Animal husbandry

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Nauphoeta cinerea mass colonies and individual experimental animals were housed under standard conditions of 27 °C, 12/12-light/dark photoperiod, and offered 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. On the day of adult emergence, the adult animals were isolated in individual 11 × 11 × 3 cm clear plastic boxes. Adults were used in mating trials at 10 days after adult emergence, which coincides with maximal fertility, attractiveness, and mate discrimination (Moore & Moore, 2001).

Experiment 1: Effect of male multiple mating and recovery time on female fecundity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

To examine the effect of multiple matings and recovery time between matings on male fertility, we allowed males to mate sequentially with four females. We manipulated the recovery time between matings for two groups of males, with males being randomly allowed either 1 or 4 days between matings. Every mating pair was observed to ensure that mating took place, but courtship behaviour was not recorded. Following mating, females were returned to their individual containers and maintained under standard rearing conditions until death. Females were checked daily for parturition of offspring or dropped unfertilized oothecae. We estimated lifetime fertility for each female by recording the date of parturition and number of offspring of each clutch, after which time offspring were removed from the female's container and returned to a mass-rearing colony.

To assess whether sperm depleted males were able to recover fertility with sufficient time, the set of males with a 1-day recovery period (i.e. mated four times over 3 days) were allowed a recovery period by maintaining them in individual containers for 5 days after the fourth mating. They were then mated to a fifth virgin female. Following mating the female was maintained in an individual container under standard rearing conditions and lifetime fertility data were collected as above.

Male multiple mating and courtship

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

To test the effects of male multiple mating on male and female courtship behaviour, males were mated sequentially to five virgin females with a 1-day recovery period between each mating. Each mating trial was conducted in a 17 × 12 × 6.5 cm clear plastic arena, and courtship and mating behaviour were observed and recorded using the methodology of Clark et al. (1997). Specifically, we recorded the time at which the male initiates courtship and copulation duration (under male control; Clark et al., 1997), and the time at which the female responds to the male courtship display by climbing onto the male's back and the time between response to courtship and the start of copulation (under female control).

Male multiple mating and female remating

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

In order to determine if multiple mating by males influences his ability to inhibit sexual receptivity in females, each of the five females mated in the first part of this experiment was offered a second mating opportunity with a virgin male. One hour after mating with the first male, each female was placed in a mating arena with a second virgin male and observed for 30 min. At the end of this time, both the female and male were placed back into individual containers and returned to the incubator. If re-mating occurred, the female was isolated and maintained under standard rearing conditions. If re-mating did not occur, the animals were separated for 24 h and placed together again the next day and observed for another 30 min. Previous research in our laboratory has shown that receptivity at 24 h post-parturition is an accurate indicator of receptivity prior to fertilization of the second clutch of eggs (Moore et al., 2003). Regardless of whether female re-mated by the end of the second trial with the new male, each female was isolated and maintained as above.

Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

In order to determine the effects of sperm depletion on spermatophore size, structure and sperm viability, males were allowed to mate successively with five females with a 1-day recovery period between each mating using the same protocol as described above. All animals used were maintained as above and mated at 7–10 days after adult emergence. Courtship behaviour was not recorded. Following mating, the first, third and fifth females were isolated and spermatophores were dissected from them immediately after mating (spermatophores from only these three matings were examined because of logistic constraints).

We recorded the mass of the spermatophore. Spermatophores in N. cinerea consist of a sperm sac embedded in a gelatinous, proteinaceous mass called the spermatophylax (Roth, 1964a, b; Buschor et al., 1984). We subsequently removed the sperm sac from the spermatophore and recorded its mass alone. The sperm sac was ground in 200 μL cockroach ringers [150 mm NaCl, 3.1 mm KCl, 5.4 mm CaCl, 2.0 mm MgCl, 5 mmTES (Sigma, St. Louis, MO, USA), 50 mm sucrose, 10 mm glucose, pH 7.0] to isolate sperm. Sperm were stained using a live/dead sperm kit (Molecular Probes, Eugene, OR, USA), using the recommended protocol, and visualized on an Olympus BX51 fluorescent microscope (Olympus UK Ltd., London, UK). A total of 200 sperm were counted per sample and sperm viability was calculated as the percentage of live sperm from the total number counted.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

We used the general linear model in systat to examine how individuals and sperm characteristics changed over time. We report the univariate repeated measures analysis, which has more error degrees of freedom and thus more power than the multivariate analysis, rather than the manova, as our data meet the required assumptions for the univariate tests (Wilkinson et al., 1996), although univariate analyses that were significant were also significant with multivariate analyses. Given the temporal nature of our experiment, we predicted a priori that any change overall would be observed between subsequent matings. Although the repeated measures output provides information on single degree of freedom contrasts, it does not provided focused pair-wise comparisons. As we had a priori specific comparisons we wished to make, contrast analysis of paired comparisons is more appropriate. Therefore, whenever there was a significant overall significance in the anova, we tested specific hypotheses using paired t-tests to examine our a priori pairwise contrasts between subsequent matings (Rosenthal & Rosnow, 1985). All statistical analyses were done using systat 9.0. Where analyses were performed on nonindependent data sets, Bonferroni corrections were applied. However, significant results remained significant after this correction so uncorrected P-values are presented for ease of interpretation by the reader.

Experiment 1: Effect of male multiple mating and recovery time on female fecundity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Males mated sequentially to females with 24-h recovery time between matings became sperm depleted and reduced female fecundity as the number of mating partners increased (Fig. 1). The number of offspring was significantly reduced in both the first clutch (Fig. 1a; F3,72 = 4.933, P = 0.004) and over the lifetime of the female (Fig. 1b; F3,72 = 5.836, P = 0.001). Males provided with 5 days of rest following these four matings regain full fertility (Fig. 1a and b), with no significant difference between the number of offspring produced by the first female mated on day 0 and the fifth female mated on day 8 in the first clutch (t24 = −0.352, P = 0.728) or over the lifetime of the female (t24 = −0.285, P = 0.778). Male fertility was not a simple function of numbers of matings as it did not decline with sequential matings when males were provided with 4 days of recovery time between matings. There was no significant difference in the number of offspring produced either in the first clutch (Fig. 1c; F3,75 = 0.678, P = 0.568) or over the lifetime of the female (Fig. 1d; F3,75 = 2.244, P = 0.090).

image

Figure 1. Male fertility decreases with multiple matings when males are not provided with sufficient recovery time between matings. Males mated sequentially to four females with 24-h recovery had reduced fertility in both the first clutch (a) and over the lifetime of his mating partner (b). If these sperm depleted males are provided with a 5-day rest period, however, they are able to regain full fertility (a) and (b). When provided with a 4-day recovery period between matings males are able to maintain fertility both in the first clutch (c) and over the lifetime of the female (d). Fertility data was collected on 26 males mated sequentially in both the 24-h and 4-day recovery groups.

Download figure to PowerPoint

Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Males responded to all female mating partners equally, regardless of the number of his previous mating encounters. Male response time was unaffected with no significant difference in the latency for a male to initiate courtship with a female regardless of his mating history (F2,74 = 0.195, P = 0.824). In contrast, male mating history had a significant effect on female behaviour during courtship. Females took longer to respond to male courtship displays of males as the number of previous matings a male engaged in increased (Fig. 2a; F2,74 = 0.3.877, P = 0.025). Latency of female response to the male courtship display increased marginally as the numbers of previous partners increased (Fig. 2b; F2,74 = 2.846, P = 0.064). Total courtship time required by a female prior to accepting copulation was significantly increased as the number of previous mating partners of the male increased (Fig. 2c; F2,74 = 3.973, P = 0.023).

image

Figure 2. Females delay courtship as the number of previous mating partners a male has had increases. Females take longer to respond to the male courtship display (a), and take longer to assess the male (b). A longer total courtship time is required for females to accept copulation with an experienced male (c). Courtship behaviour of 38 males mated to five females each was observed and analysed.

Download figure to PowerPoint

Females re-mated in only two of 40 mate trials. Both females that remated were the fourth female out of five to mate with the male. The first female to remate occurred early in the trials and was left to give birth as outlined in the methods. However, given that remating by females appeared to be a rare occurrence by the time that the second female remated, we chose to investigate further by dissecting the female to look for the presence of a spermatophore. We found only a single spermatophore in the bursa indicating that only one male had completed a successful copulation.

As observed in experiment 1, male sperm depletion increased with the number of matings. The decrease in offspring number was evident both in a given female's first clutch of offspring (Fig. 3a; F4,144 = 6.329, P < 0.001) and in the number of offspring produced by the female over her lifetime (Fig. 3b; F4,144 = 10.500, P < 0.001). In order to examine the pattern of female fecundity reduction due to sperm depletion in detail, pair-wise contrast analysis was used. There was no significant difference between the number of offspring produced in the first clutch of a female that mated to a virgin male or a male that had mated only once previously (t36 = 1.136, P = 0.264) or one or two previous matings (t36 = 0.742, P = 0.463). There is, however, a reduction in female fecundity in the first clutch between males with two and three previous matings (t36 = 2.234, P = 0.032). There is no significant difference in the number of offspring in the first clutch between males that had three and four previous matings (t36 = 0.289, P = 0.774).

image

Figure 3. Fertility of males (number of offspring born to mating partners) decreases as the number of mating partners increases. There is an overall reduction in the number of offspring in the first clutch (a) and produced over the lifetime (b) of females mated to males that have mated previously. The average number of offspring per clutch is also reduced (c). Pairwise contrast analyses that showed significant differences between samples are marked with an asterisk. Courtship behaviour of 38 males mated to five females each was observed and analysed.

Download figure to PowerPoint

The lifetime fertility pattern was similar to that of the first clutch alone. Lifetime fecundity began to drop in females mated to males with two previous matings (t36 = 2.265, P = 0.030). Lifetime fecundity of females mated to males with three previous matings was also reduced compared with males with two previous partners (t36 = 2.899, P = 0.006), but there was no significant difference between the fecundity of females mated to males with three or four previous matings (t36 = 0.127, P = 0.899), nor between the lifetime fecundity of females mated to either virgin males or males with one previous mating (t36 = 0.095, P = 0.925).

There was a significant overall reduction in the number of offspring per clutch (Fig. 3c; F4,144 = 10.259, P < 0.001). Pair-wise contrasts between mating partner numbers indicated that fecundity reduction occurs sharply between females mated to males with two and three previous matings (t36 = 3.163, P < 0.003). There were no significant pair-wise differences in average offspring per clutch in other comparisons (virgins males and males with one previous mating: t36 = 0.168, P = 0.867; males with one or two previous matings: t36 = 1.631, P = 0.112; males with three or four previous matings: t36 = −0.109, P = 0.914). The difference in lifetime fecundity between females mated to males with one and two previous matings is thus not a result of fewer offspring per clutch but is likely to be due to a reduction in the number of clutches a female produced as the number of previous mates her partner had increased (F4,144 = 3.821, P = 0.006).

Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Spermatophore mass decreased as the number of mating partners increased (Fig. 4a; F2,58 = 61.853, P < 0.001). For the series of five mates, pair-wise contrast analysis confirmed an initial large reduction in spermatophore mass between virgin males and males with two previous matings (t29 = 9.413, P < 0.001) but no difference between males with two or four previous matings (t29 = 1.045, P = 0.305). The sperm sac showed a similar decrease in mass as the number of mating partners increased (Fig. 4b; F2,56 = 68.292, P < 0.001). Pair-wise contrast analysis again showed sperm sac mass pattern similar to that of total spermatophore mass, with a decrease between virgin males and males with two previous matings (t29 = 16.277, P < 0.001), but not between males with two or four previous matings (t28 = 1.875, P = 0.071).

image

Figure 4. Spermatophore characteristics change as the number of mating partners a male had increased. Total spermatophore weight decreased (a). The weight of the sperm sac also decreased (b). The ratio of the weight of the sperm sac to total spermatophore weight also decreased (c), indicting that males are maintaining spermatophore size by increasing the proportion of spermatophylax material relative to sperm produced. Pairwise contrast analyses that showed significant differences are marked with an asterisk. Sample size was 30 males, spermatophores recovered from three females for each male.

Download figure to PowerPoint

In order to determine if the change in total spermatophore size is proportional to the change in sperm sac to spermatophore mass. The ratio of sperm sac size to total spermatophore weight decreased as the number of mating partners increased (Fig. 4c; F2,56 = 11.503, P < 0.001). Pair-wise contrast analysis again showed a significant difference between the spermatophore produced by virgin males and males with two previous matings (t29 = 7.472, P < 0.001), indicating that the sperm sac decreased to a greater degree than the spermatophylax, but not between males with two or four previous matings (t28 = 1.089, P = 0.285).

Sperm viability decreased as the number of mates increased (Fig. 5; F2,58 = 15.302, P < 0.001). This was a linear change, with significant differences between sperm from the first and third matings (t29 = 2.483, P = 0.019) and between sperm from the third and fifth matings (t29 = 2.986, P = 0.006).

image

Figure 5. The percent viable sperm produced by males decreased as the number of mating partners increased. Pairwise contrast analyses that showed significant differences are marked with an asterisk. Sample size was 30 males, sperm recovered from three females for each male.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

Contrary to predictions that females should only mate once or a few times to maximize their reproductive success, females of many species mate multiply (Keller & Reeve, 1995; Birkhead & Moller, 1998). In insects there is an average direct fitness gain of multiple mating of 30–70%, despite a negative effect of remating on female longevity (Arnqvist & Nilsson, 2000). When females are promiscuous, males have two mechanisms by which they can increase their relative fitness: fertilize more eggs than other males in the population (through success in gaining access to mating opportunities or in sperm competition) or prevent other males from fertilizing eggs (e.g. by preventing access to females). The incompatibility between the optimal reproductive strategies for females, involving multiple mating partners, and for males, involving monopolizing female mating and thus ensuring paternity of her offspring, results in sexual conflict. Our results demonstrate that N. cinerea males can manipulate female remating behaviour even when they lack sufficient sperm to fertilize a full complement of eggs. Thus, male mating history can exacerbate the fitness effects arising through sexual conflict in N. cinerea, an effect that may be common in species where mate competition interacts with mate choice to produce a system where a few males monopolize matings (Jones, 2001; Preston et al., 2001).

The continued ability of males to manipulate female remating while sperm depleted suggests that selection on the mechanism of male manipulation has been greater than on the ability to replenish sperm supplies. Nauphoeta cinerea males that mated multiply over short periods of time were unable to fertilize the full complement of eggs that a female was able to produce. Males provided with sufficient recovery time between matings were able to recover full fertility. Given this, we predicted that males might delay mating when sperm stores are low. However, males did not alter their courtship behaviour as fertility declined with mate number. The relative benefit of preventing other males from reproducing evidently outweighed the potential benefit of waiting to mate with another female at a later time. The optimal strategy for males may be to pass sufficient sperm to any receptive female to fertilize her lifetime complement of eggs. Males that, by chance, encounter and mate with more than two receptive females continue to increase their relative fitness because of the ability to inhibit female remating and thus preventing other males from reproducing.

Given that the inhibition to remating by females depends solely on the size of the spermatophore (Roth, 1964a) and the importance of remating behaviour to male reproductive success, there should be strong selection on males to maintain spermatophore size over multiple mating episodes for N. cinerea. The mass of the spermatophore did decrease as the number of mating partners increased. However the size of the spermatophore levelled off between the third and fifth spermatophore produced. Males appeared to be maintaining spermatophore size by compensating for the lack of sperm with an increase in the spermatophylax. Thus, sperm depleted males appeared to maintain spermatophore size above a certain threshold required to prevent remating by the female. Because males were able to enforce monogamy on females, there might be reduced selection on sperm quality (Hunter & Birkhead, 2002), a hypothesis supported by the observed decrease in the viability of the sperm in males as the number of mating partners increased.

Selection studies of sexual conflict in Drosophila under experimentally enforced monogamy have demonstrated co-evolution of male manipulation of female reproduction and female resistance to male manipulation (Pitnick et al., 2001a, b). In Drosophila males manipulate female remating through the introduction of seminal proteins (Chapman, 2001). In selection lines where sexual selection was experimentally removed, females from monogamous lines remating less frequently, indicating a lowered resistance to ejaculate manipulation (Pitnick et al., 2001a). Thus, females are free to evolve resistance mechanisms given the requisite variation in the population.

In N. cinerea, as opposed to Drosophila, males may have exploited the system that evolved to regulate sexual receptivity during pregnancy in order to enforce monogamy in the first reproductive cycle. Both the ootheca in the brood pouch and the spermatophores activate a stretch receptor to inhibit female receptivity (Roth, 1964a, b). Given the anatomical juxtaposition of the bursa copulatrix and the brood pouch it is not unreasonable to expect that the males are exploiting the same stretch receptor that evolved to control female receptivity during pregnancy. If males are co-opting a physiological mechanism for sexual receptivity during pregnancy, the option of escaping male manipulation through the recovery of receptivity may not be available to females in this species as it is in Drosophila.

If females are not free to evolve resistance to male manipulation, what possibilities are open to females to optimize fitness in the context of male suppression of receptivity? One possibility would be for females to suppress the fitness cost of mating with potentially sperm limited males by increasing the efficiency of sperm utilization. Thus females would respond to male manipulation with sexual cooperation rather than sexual conflict in the first reproductive cycle. The suggestion that ‘sexual dialectics’ (Gowaty, 1997) can play a role in the evolution of female utilization of sperm and thus influence the results of sperm competition has been made before (Jones et al., 2002). Female rough-skinned newts (Taricha granulosa) exercise free female mate choice, with the mating system providing little opportunity for males to manipulate or undermine female choice. Thus, although females mate with multiple males, the free expression of precopulatory mate choice leads to females accepting sperm only from high quality males. Thus all sperm in their reproductive tracts are of equal value and there is little selective pressure for cryptic mate choice. As predicted, the patterns of sperm utilization in this species are simple (Jones et al., 2002). Similar reasoning can be used in systems in which females are unable to resist male manipulation such as N. cinerea; because females are restricted to a single mate in the first reproductive cycle, all the sperm she received in that copulation would be valuable, particularly if her mate was sperm limited. Thus, we predict that selection would be for efficient sperm utilization, regardless of which male has gained the copulation.

The mechanical suppression of receptivity in females by the presence of the spermatophore is only active during the first reproductive cycle. Male manipulation of female receptivity continues into the second reproductive cycle, but utilizing a chemically mediated mechanism, the male sex pheromones correlated with male social status (Moore et al., 1997). Exposure of females to the two components of the pheromone blend that confer dominant status during mating results in more rapid offspring development which is correlated with reduced receptivity following parturition of the first clutch (Moore et al., 2003). Females appear to be unable to respond to male manipulation in the first clutch of offspring, but that does not appear to be the case for the second reproductive cycle. Although exposure to male pheromone influences female reproductive physiology (Moore & Moore, 2003), the effect may not be as critical to the female as the inhibition to remating during pregnancy. Thus, as is the case in Drosophila where females vary in their response to ejaculate manipulation (Pitnick et al., 2001a), N. cinerea females vary in their response to exposure to male pheromone. Nauphoeta cinerea females therefore have evolved a mechanism to resist male manipulation. Females chose to expose themselves to the third component of the pheromone blend that confers subordinate status on males (Moore et al., 1997) yet is required to make them attractive to females (Moore & Moore, 1999). Exposure to this component reduces the fertility of females (Moore et al., 2001) yet results in lengthened offspring development time and is correlated with increased receptivity prior to fertilization of the second clutch (Moore et al., 2003). Thus, although female mate choice appears to be maladaptive in N. cinerea, it reflects attempts by females to avoid male manipulation (Moore et al., 2001, 2003), as predicted by models of sexual conflict (Gowaty, 1996, 1997; Holland & Rice, 1998).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References

The interactions between sexual selection and sexual conflict are likely to be complex (see e.g. Pizzari & Snook, 2003). Studies of simple, experimentally tractable systems such as Drosophila provide excellent evidence for the evolution of sexual traits in response to sexually antagonistic, rather than sexually mutualistic, selection (Chapman et al., 2003; Pizzari & Snook, 2003). Studies of species that utilise novel mechanisms of sexual manipulation, as well as species in which the causes and consequences of sexual conflict change across the life history of the individual (Hosken & Stockley, 2003), will be important to fully define how differing reproductive interests of the sexes influence reproductive decisions by both mating partners.

Here we have demonstrated extreme sexual conflict in a mating system in which a few high status, sperm limited males monopolize mating opportunities. Sperm limitation is critical to female fertility in that sperm depleted males continue to be able to manipulate female receptivity. Female resistance to male manipulation may depend on the mechanism utilized by males to control female reproductive behaviour. Female mate choice in this system seems to have evolved as a mechanism for resisting male manipulation to remating prior to fertilization of the second clutch of oocytes (Moore et al., 2001, 2003). Given the inability of females to resist male manipulation in the first reproductive cycle we suggest that females may respond to male manipulation through cooperation rather than conflict. Further research will allow us to further define female response under naturally enforced male monogamy and thus expand our understanding of the complex interaction between the reproductive interests of males and females.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. The study organism: Nauphoeta cinerea
  6. Animal husbandry
  7. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  8. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  9. Male multiple mating and courtship
  10. Male multiple mating and female remating
  11. Male multiple mating and female fecundity
  12. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  13. Statistical analysis
  14. Results
  15. Experiment 1: Effect of male multiple mating and recovery time on female fecundity
  16. Experiment 2: Effect of male multiple mating on courtship behaviour, female mating inhibition, and female fecundity
  17. Experiment 3: Effect of male multiple mating on spermatophore characteristics, and sperm viability
  18. Discussion
  19. Conclusions
  20. Acknowledgments
  21. References