Behavioral Ecology Advance Access originally published online on July 22, 2008
Behavioral Ecology 2008 19(6):1173-1178; doi:10.1093/beheco/arn090
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Dual function of seminal substances for mate guarding in a ground beetle
Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo, Kyoto 606-8502, Japan
Address correspondence to Y. Takami. E-mail: takami{at}terra.zool.kyoto-u.ac.jp.
Received 28 March 2008; revised 17 June 2008; accepted 19 June 2008.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Males of internally fertilizing animals often produce ejaculates consisting of various substances in addition to sperm. Seminal substances can inhibit female remating through physical blocking of the female genitalia or by physiological induction of the female refractory period. We demonstrated that the seminal substances of the ground beetle Leptocarabus procerulus serve as both physical (i.e., mating plugs) and physiological (i.e., substances inducing female refractory behavior) devices for mate guarding. Although females counteract these functions via expulsion of the plug and the delay of the occurrence of refractory behavior, the interplay of these 2 functions can be compensatory and consistently lowers the female remating rate during a postmating period that is important for male fertilization success. Such interplay of 2 defensive strategies may be a male adaptation against female resistance, as predicted by the hypothesis that multiple functions in seminal substances are a historical outcome of an arms race between males and females. Our findings highlight the importance of sexual conflict in the evolution of complex seminal substances.
Key words: accessory gland protein, mating plug, sexual conflict, sexual selection, sperm competition.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
In promiscuous species, male reproductive success in postcopulatory stages is mainly governed by competition with rival males and by cryptic female choice for a certain male. Thus, these mechanisms are strong agents of sexual selection, resulting in various morphological, physiological, and behavioral adaptations (Parker 1970
; Smith 1984; Eberhard 1996; Birkhead and Møller 1998; Simmons 2001). Males of many animals produce ejaculates consisting of various substances in addition to sperm, and many functions of seminal substances in postcopulatory sexual selection have been documented (Eberhard 1996; Simmons 2001; Arnqvist and Rowe 2005; see also Cameron et al. 2007). These seminal substances can improve male fertilization success in sperm competition, and one of the most conspicuous functions is to hinder females from further remating (i.e., defensive adaptation in sperm competition; Parker 1984).
Male–male competitive functions of seminal substances can be achieved in different ways. Seminal substances can coagulate in the female reproductive tract, serving as mating plugs that physically block the female genital opening (Dickinson and Rutowski 1989; Matsumoto and Suzuki 1992; Baker 1994; Shine et al. 2000) or decrease female attractiveness (Orr and Rutowski 1991; Polak et al. 2001). Seminal fluids can also stimulate the female reproductive tract mechanically (Sugawara 1979), or the accessory gland products can pass through the walls of the female reproductive tracts into the hemolymph, thereby physiologically inducing a female refractory period (reviewed in Simmons 2001; Gillott 2003; Chapman and Davies 2004; Poiani 2006). However, the possibility that multiple defensive adaptations are involved within ejaculates has rarely been examined (Simmons 2001) or failed to be detected (e.g., Shine et al. 2000; Sauter et al. 2001; cf., Duvoisin et al. 1999). The exception is ditrysian lepidopterans: female refractory period is induced in response to insemination and the mating plugs are persistent because they do not hinder oviposition (reviewed in Simmons 2001). Molecular mechanisms driving the effects of seminal substances in sperm competition have recently been examined in Drosophila, demonstrating that male accessory gland proteins function in sperm competition defense in multiple ways, both by protecting sperm against rival male sperm displacement and by decreasing female receptivity (Clark et al. 1995; Chapman 2001; Kubli 2003; Chapman and Davies 2004; Poiani 2006; Ravi Ram and Wolfner 2007). Nevertheless, empirical evidence remains fragmented and indirect (Clark et al. 1995; see also Chapman 2001); therefore, further examination for the possibility of multiple defensive adaptations within ejaculates is warranted.
One hypothesis regards multiple defensive adaptations in seminal substances as the historical outcome of an arms race between males and females (Chapman and Davies 2004; Poiani 2006) and predicts that multiple defensive traits are functional in both preventing female remating and in cooperating against female manipulation. For example, if the function of the first trait is reduced by the female, the second trait will compensate for the declined function of the first trait. To examine this hypothesis, research should focus on whether and how individual defensive traits operate and cooperate with each other and how females constrain the functions of male-defensive traits during sperm competition in a single species.
Here, we address multiple defensive adaptation in seminal substances in the ground beetle Leptocarabus procerulus, which possesses 2 potential defensive devices for sperm competition: a large spermatophore that serves as a mating plug and seminal substances that induce female refractory behavior. Males of L. procerulus deposit a particularly large spermatophore within the female bursa copulatrix (Figure 1A) compared with a related species, Carabus insulicola (Takami 2002). Sperm is placed in the most anterior portion of the spermatophore, near the opening of the common oviduct and the spermatheca, whereas most of the posterior portion consists of a gelatinous mass secreted from the male accessory gland that forms a mating plug (Figure 1A). Thus, the spermatophore likely serves other functions in addition to holding sperm in the bursa copulatrix. Previous studies of C. insulicola, which shares similar female genital anatomy with L. procerulus, revealed that sperm is transferred into the spermatheca within several hours after copulation and the spermatophore is dissolved and removed from the female genitalia a few days after copulation (Takami 2002, 2007). These processes of insemination and spermatophore removal are also expected in L. procerulus. In addition, to reject males that attempt to copulate, once-mated females of ground beetles often exhibit refractory behavior by bending the abdominal tip to avoid genital insertion (Takami 2002). Thus, the mating plug and its removal as well as female refractory behavior have the potential to influence the rate of female remating in ground beetles. Our goal was to examine the effectiveness of the 2 possible male sperm-defensive devices used during sperm competition in response to female-induced constraints. The results of our experiments demonstrate that these 2 male strategies are effective in both preventing female remating and cooperating to compensate for functions reduced by the female. We discuss the evolutionary mechanisms of multiple functions in seminal substances in the context of sperm competition and sexual conflict.
|
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Study organism
We collected adult beetles during sexually inactive seasons (May to early June; Sota 1985
) in 2006 (and in 2007 for the experiment of female refractory behavior) using pitfall traps at several sites (within 10 km of each other) in Suzuka-shi and Yokkaichi-shi, Mie Prefecture, Japan. To prevent sexual activity until experiments, males and females were housed separately in plastic containers (20 x 15 x 8 cm) with moistened moss in an incubator under long-day conditions (16:8 h light:dark) at 20 °C for approximately 3 months (until mid-September). Beetles were fed minced beef ad libitum. Then, incubator conditions were shifted to short-day lighting (12:12 h light:dark) at 20 °C to induce sexual activity, and beetles were maintained under these conditions for at least 3 weeks prior to experiments. The collected beetles had never mated during the year of collection prior to experiments because most adults are thought to eclose in the spring and confirmed to be reproductively immature until the autumn (Sota 1984, 1985). Nonetheless, some may not have been virgins because adults reproduced in the autumn can overwinter and survive until the next autumn to reproduce again (Sota 1984, 1985, 1987).
Female refractory behavior
To examine whether female refractory behavior was physiologically induced by male seminal substances, we conducted an artificial injection experiment using beetles maintained for 21–29 days under short-day lighting. The testes and accessory glands were removed from males euthanized with CO2. Tissues were weighed and homogenized in 1.5-ml Eppendorf tubes with insect saline (0.75% NaCl, 0.05% KCl, 0.015% CaCl2, 0.005% NaHCO3, 0.004% NaH2PO4, and 0.02% glucose) on ice (0.1 mg µl–1). After centrifuging (2000 x g, 30 s), 20 µl of supernatant was injected into the abdomen of CO2-anesthetised females using a syringe (1-ml scale, 27-gage needle). Experimental females were randomly assigned to 3 treatments: injecting 1) the extract of the accessory gland, 2) extract of the testis or 3) insect saline only. Because we did not intend to examine possible interaction between substances from the different male organs, there was no treatment for the accessory gland plus testis.
After 24-h recovery, we examined the occurrence of female refractory behavior and its effectiveness to reduce mating. In the evening, under room lighting at 23 ± 2 °C, 1 male and 1 experimental female were chosen arbitrarily, placed in an empty plastic box (12.5 x 12.5 x 9 cm), and allowed to mate. The occurrences of female refractory behavior and male genital insertion success were recorded. Mated females were frozen after the experiment and dissected to determine whether a spermatophore had been deposited. Males that did not attempt to mount the female within 5 min were discarded and replaced with a new male (the same procedure was followed in the experiments described below).
Deposition and persistence of the mating plug
To characterize the dynamics of deposition and persistence of the mating plug within females, we examined variation in the size of the plug at 0 and 24 h after copulation using beetles maintained under short-day conditions for 21–56 days. Males and females were allowed to mate as described above. The duration of copulation, from insertion to withdrawal of the aedeagus, was measured. Mated females were randomly assigned to 2 groups: frozen immediately (0 h) or frozen 24 h later (24 h). In the second group, mated females were individually housed within a plastic jar (12 cm diameter x 5 cm deep) with moistened moss. Neither food nor oviposition substrate (soil) was provided, and jars were kept in an incubator until females were preserved. Females were dissected to remove the deposited plug, which was weighed to the nearest 0.01 mg using an electronic balance.
Female remating rate and male-defensive ability
To determine the rate of female remating and the effectiveness of male-defensive devices (mating plug and seminal substances inducing female refractory behavior), we conducted double-mating experiments using 2 males (defensive and offensive roles) and 1 female, which had all been maintained under short-day conditions for 21–50 days. The first male (defensive role) and a female were allowed to mate, as described above. Immediately after the first mating, the female was moved into a new box and allowed to mate with the second male (offensive role) (0 h). If a pair mated successfully during the second mating (copulation duration >15 min), the pairs were frozen immediately after the experiment. If a pair did not mate properly in the second trial (no genital insertion or copulation duration <1 min; no cases of copulation lasting between 1 and 15 min were observed), the same pair was allowed to mate again 24 h later (24 h) and was frozen after the trial. During the intertrial interval, the male and female were housed individually in plastic jars provided only with moistened moss. For every mating trial, the occurrence of female refractory behavior and male genital insertion as well as copulation duration were recorded.
Males were dissected to remove the aedeagus and testis. The length of the aedeagus was measured to the nearest 0.01 mm using the ocular micrometer of a binocular microscope. Females were also dissected to remove deposited plugs and ovaries, which were weighed using an electronic balance. The plug and testis were stored in absolute ethanol for DNA extraction. The carcasses of beetles were pinned and dried, and the body length (defined as the distance from the anterior margin of the labrum to the apices of the elytra) was measured to the nearest 0.01 mm using digital calipers.
Spermatophore deposition success of the first and second males was determined using microsatellite DNA markers (Takami and Katada 2001). This was necessary for the cases of doubly "inserted" females that revealed one spermatophore, showing 2 possibilities: the spermatophore of the first male was expelled or that of the second male was not deposited. The anterior portion of the spermatophore contains sperm mass, from which we can extract DNA. We examined the utility of 11 loci and chose 3 loci that were successfully amplified (Cins15: 7 alleles in 202–222 bp; Cins33: 3 alleles in 208–222 bp; Cins36: 11 alleles in 290–308 bp). In some cases, we detected more than 2 bands of amplified DNA fragments, probably due to ambiguity of cross-species polymerase chain reaction amplification. However, by recognizing these genotypes as fingerprints, we could determine all individual genotypes and thus assess the spermatophore deposition success of the first and second males. Detailed analytical procedures were described in Takami (2007).
We analyzed the defensive ability of the first male by constructing generalized linear models (GLMs) of genital insertion success and spermatophore deposition success of the second male. The explanatory variables were the plug weight of the first male and the occurrence of female refractory behavior (binary variable); the body length and ovary weight of the female as well as the body and aedeagus lengths of the second male, were included as possible covariates. All measurements were log-transformed before analyses, and logit-link functions with binomial error distributions were applied in GLM analyses for binomial data. Best models were determined using backward stepwise selection, in which nonsignificant terms at P > 0.1 were removed from the final models. Statistical analyses were performed using JMP version 6 (SAS Institute 2006).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Female refractory behavior
The artificial injection experiment confirmed that female refractory behavior was induced by male-derived substances. Female refractory behavior was induced more frequently by injection of extracts from the accessory gland (90.0%; 9/10) or the testis (72.7%; 8/11) than by saline controls (10%; 1/10). The saline controls significantly differed from the 2 other treatments (Fisher's exact probability, P < 0.05 after Bonferroni correction), but the 2 treatments did not differ (P = 0.59). All females showing refractory behavior (18/31 in total) avoided the insertion of the male genitalia, whereas all females without refractory behavior (13/31) experienced insertion of the genitalia and deposition of a spermatophore (Fisher's exact probability, P < 0.00001). These results suggest that female refractory behavior served to definitively avoid mating.
Deposition and persistence of the mating plug
Mating plugs were deposited in all 31 cases at 0 h, indicating that plugging failure is quite rare in matings with unmated females (Figure 1B). In contrast, 24 h after copulation, the mean plug weight was half that of plugs at 0 h (mean ± 1 standard deviation—0 h: 6.29 ± 0.86 mg, n = 31; 24 h: 3.43 ± 3.57 mg, n = 42; analysis of variance [ANOVA], F1,71 = 25.2, P < 0.0001). The distribution of plug weights at 24 h were bimodal, with one peak at <1 mg (n = 21), indicating plugs were virtually absent, and the other peak at 6–7 mg (n = 21; Figure 1C). At the latter peak, the mean weight of the plug was 6.79 ± 1.54 mg and did not differ from that at 0 h (ANOVA, F1,50 = 1.24, P = 0.27). These results suggest that deposited plugs may be expelled by half of the females within 24 h after copulation (in contrast to related species in which plugs gradually dissolve; Takami 2002), and plug weight was nearly unchanged within the female genitalia prior to being expelled.
Mean copulation duration was 24.1 ± 3.5 min (n = 73) and was not a good predictor of plug weight (0 h: β = 0.004 ± 0.008, R2 = 0.01, F1,29 = 0.29, P = 0.60; 24 h: β = –7.41 ± 3.70, R2 = 0.09, F1,40 = 4.00, P = 0.052).
Female remating rate and male-defensive ability
Of 40 sets of double-mating trials, in which all of the first males completed copulation, 4 of the second males at 0 h successfully inserted the genitalia and copulated for >15 min. Accordingly, a second mating trial with a second male was necessary at 24 h in the remaining 36 cases. In 5 of those 36 cases, the second male was inactive and was replaced with a third male. Because exclusion of these 5 cases did not alter our conclusions, we report the results including all 40 mating sets. In 13 of the 40 total sets, the plug of the first male was not found in the female genitalia when dissected after the remating trial at 24 h and was likely expelled during the interval between 0 and 24 h (light gray column in Figure 2B). These 13 cases were regarded as spermatophore deposition failure in the following statistical analyses (i.e., conservative treatment).
|
If plugs were the sole functional device for female remating avoidance, we predicted that female remating would be avoided immediately after the first mating (0 h), but the rate of female remating would increase after the plug was expelled (24 h). In accordance with the first prediction, the female remating rate at 0 h was significantly lower than the first mating with respect to the genital insertion and spermatophore deposition success of the second male (Figure 2A,B; Fisher's exact probability, P < 0.001 after Bonferroni correction). At 0 h, female refractory behavior seldom occurred (Figure 2C). The GLM constructed with the 27 cases, for which the plug of the first male could be weighed, revealed that genital insertion by the second male was more frequently hindered when females had a larger plug and smaller ovaries (GLM: R2 = 0.19,
P = 0.0276; plug weight: β = –5.22 ± 2.90, 
P = 0.0449; ovary weight: β = 3.21 ± 1.44, 
P = 0.0074). In addition, larger plugs more often prevented spermatophore deposition by the second male, although the effect was not significant (β = –4.57 ± 2.62, 
P = 0.0560).
In contrast to the second prediction, the female remating rate did not significantly increase at 24 h, despite the finding that the plug of the first male was expelled by 36% of females (13/36); that is, neither the genital insertion success nor the spermatophore deposition success of the second male was significantly different between 0 and 24 h (Figure 2A,B; P = 0.77 and 0.057 in pairwise comparisons, respectively). Note that the second males that were successful at 0 h (n = 4), which may have had relatively higher remating abilities, were removed from 24-h samples (n = 36). However, female refractory behavior was the principal cause of the observed low remating rate because this behavior occurred frequently at 24 h (Figure 2C). The GLM analyses for 24-h data revealed that genital insertion by the second male was effectively prevented by female refractory behavior (β = –1.10 ± 0.41, 
P = 0.0037; we assigned zero to the plug weight for the 13 cases in which the plug could not be found). Female refractory behavior also hindered the spermatophore deposition of the second male, but there was no significant effect of the plug (GLM: R2 = 0.22, 
P = 0.0121; female refractory behavior: β = –1.10 ± 0.47, 
P = 0.0116; plug weight: β = –0.22 ± 0.13, 
P = 0.0803). In addition, the plug size of the first male was not a good predictor of female refractory behavior (GLM:0 h: 
P = 0.31; 24 h: 
P = 0.98), suggesting that female refractory behavior was not induced by physical stimulation of the plugs within the female reproductive tract (cf., Sugawara 1979).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
The seminal substances of L. procerulus served 2 defensive purposes in sperm competition (i.e., as a mating plug and source of physiological activities inducing female refractory behavior) that were both functional in hindering female remating, thus demonstrating the multiple adaptation in seminal substances for sperm competition defense. These data, together with those for Drosophila and ditrysian lepidopterans, confirm multiple devices for sperm competition defense achieved by an ejaculate.
The spermatophore of L. procerulus served a physical function as a mating plug, which was demonstrated by the pattern that 55% (22/40) of the 0-h second males could not insert their genitalia, despite their aggressive behavior. Even if the second males could insert their genitalia, 83% (15/18) of these males could not inseminate the female, likely because the endophallus could not be properly everted in the bursa copulatrix occupied by the spermatophore of the first male. Mating plugs are often only temporarily useful in most insects, as they are eventually dissolved or expelled by females, occasionally in response to oviposition. Plugs that are too robust would hinder oviposition and would not be beneficial to either sex (Simmons 2001). Thus, the utility of mating plugs for preventing female remating may be limited. The mating plug of L. procerulus was also short lived because it could be expelled by the female; however, the plug was effective in preventing female remating prior to being expelled. This mate-guarding function may confer a strong selective advantage in the context of sperm competition because previous studies in a related ground beetle species suggested that the several hours postmating are important for ensuring the fertilization success of the male (Takami 2002, 2007). During this time period, sperm are gradually transferred from the spermatophore into the spermatheca, and female remating shortly after mating results in higher fertilization success by the second male.
Several studies have reported that seminal substances influence female physiological conditions to decrease their rate of remating (reviewed in Gillott 2003; Chapman and Davies 2004; Poiani 2006). However, the mechanisms responsible for this decreased remating rate are not necessarily clear because the female physiological response to seminal substances often cryptically reduces female attractiveness. For example, in Drosophila, mated females are less mobile than virgins, and changes in the amount of volatile compounds and the composition of cuticular hydrocarbons result in reduced female attractiveness (Tompkins and Hall 1981; Scott and Jackson 1990; Gillott 2003). However, in L. procerulus, the female physiological response to seminal substances was a refractory behavior that was highly effective in avoiding subsequent mating. The nature of this response helped elucidate the proximal mechanism of decreased female remating rate in L. procerulus. The refractory behavior was induced by the extracts from both male accessory glands and testes, similar to other beetle species (Yamane et al. 2008), which suggests the effect of a single general molecule secreted from both organs (e.g., Baer et al. 2001) or multiple molecules secreted from different organs (e.g., Yamane et al. 2008). It remains to be examined whether the injected extracts of the male organs include substances that are actually secreted into the seminal fluid and transferred to the female. Further investigation of the physiological and biochemical processes in inducing female refractory behavior is needed.
The mating plug of L. procerulus was effective immediately after copulation, whereas induced female refractory behavior was effective 24 h postmating. Thus, the 2 male-defensive devices operate in a temporally structured manner as the female remating rate was consistently lowered during the postmating period (Figure 2). If females were likely to encounter males from soon after mating to 24 hours later, these 2 functions could be favored separately because this postmating period is crucial for ensuring male fertilization success (see above). However, this may not be a sole mechanism that results in multiple defensive adaptation in an ejaculate. Our experiments also revealed the female roles that underlie this process: mating plugs were often expelled by females (Figure 1C) and the occurrence of female refractory behavior was delayed (Figure 2C). These female behaviors decreased the effectiveness of each male-defensive device at different times, but the decreased utility of one device was compensated for by the other. This compensatory interplay of the male-defensive devices may be the result of a male adaptation against female manipulation and is consistent with the hypothesis that multiple functions in seminal substances is a historical outcome of an arms race between males and females. Male adaptations for sperm competition are frequently assumed to be costly for females, resulting in sexual conflict and subsequent intersexual arms races (Parker 1979, 1984, 2006; Gavrilets and Hayashi 2006). However, in L. procerulus, whether the functions of seminal substances (plugging the female genitalia and inducing female refractory behavior) are costly for females or whether female-derived effects (expelling plugs and delaying the occurrence of refractory behavior) are counteradaptations to functions exerted by the male have not yet been determined. These questions warrant further experimental analyses.
It is often claimed that studies of extant species will be unlikely to distinguish among competing coevolutionary models of sexual selection (Chapman et al. 2003; Arnqvist and Rowe 2005). Sexual conflict would not occur if the mating plug were beneficial to females by providing nutrients (Arnqvist and Nilsson 2000). Although we have no evidence for consumption of the plug by female L. procerulus, the nutritional value of the plug appears to be low because it is relatively small in size (0.83% of male body weight; Takami Y, unpublished data) compared with that of other animals with nuptial feeding of ejaculates (e.g., 20% in dobsonflies, Hayashi 1992; 3–25% in orthopterans, Gwynne 1997). In addition, the consumption of seminal substances with strong physiological activities is of questionable benefit to females (Vahed 2007). Therefore, sexual conflict is unlikely to be mitigated by nutritional benefits to females in L. procerulus.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Research Fellowships for Young Scientists from Japan Society for the Promotion of Science (JSPS) (2003–2005, 04747) and a Grant-in-aid from JSPS (15207004) to Y.T.; Biodiversity Research of the 21st Century COE (A14) from the Ministry of Education, Culture, Sports, Science and Technology to Kyoto University.
|
ACKNOWLEDGEMENTS |
|---|
Y.T. thanks R. Ishikawa for his constant encouragement.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Arnqvist G, Nilsson T. The evolution of polyandry: multiple mating and female fitness in insects. Anim Behav (2000) 60:145–164.[CrossRef][Web of Science][Medline]
Arnqvist G, Rowe L. Sexual conflict (2005) Princeton: Princeton University Press.
Baer B, Morgan ED, Schmid-Hempel P. A nonspecific fatty acid within the bumblebee mating plug prevents females from remating. Proc Natl Acad Sci USA (2001) 98:3926–3928.
Baker DM. Copulatory plugs and paternity assurance in the nematode Caenorhabditis elegans. Anim Behav (1994) 48:147–156.[CrossRef][Web of Science]
Birkhead TR, Møller AP. Sperm competition and sexual selection (1998) London: Academic Press.
Cameron E, Day T, Rowe L. Sperm competition and the evolution of ejaculate composition. Am Nat (2007) 169:E158–E172.[CrossRef][Web of Science][Medline]
Chapman T. Seminal fluid-mediated fitness traits in Drosophila. Heredity (2001) 87:511–521.[CrossRef][Web of Science][Medline]
Chapman T, Arnqvist G, Bangham J, Rowe L. Sexual conflict. Trends Ecol Evol (2003) 18:41–47.[CrossRef]
Chapman T, Davies SJ. Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies. Peptides (2004) 25:1477–1490.[CrossRef][Web of Science][Medline]
Clark AG, Aguadé M, Prout T, Harshman LG, Langley CH. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster. Genetics (1995) 139:189–201.[Abstract]
Dickinson JL, Rutowski RL. The function of the mating plug in the chalcedon checkerspot butterfly. Anim Behav (1989) 38:154–162.[CrossRef][Web of Science]
Duvoisin N, Baer B, Schmid-Hempel P. Sperm transfer and male competition in a bumblebee. Anim Behav (1999) 58:743–749.[CrossRef][Web of Science][Medline]
Eberhard WG. Female control: sexual selection by cryptic female choice. (1996) Princeton: Princeton University Press.
Gavrilets S, Hayashi TI. The dynamics of two- and three-way sexual conflicts over mating. Philos Trans R Soc Lond B Biol Sci (2006) 361:345–354.
Gillott C. Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annu Rev Entomol (2003) 48:163–184.[CrossRef][Web of Science][Medline]
Gwynne DT. The evolution of edible ‘sperm sacs’ and other forms of courtship feeding in crickets, katydids and their kin (Orthoptera: Ensifera). In: The evolution of mating systems in insects and arachnids—Choe JC, Crespi BJ, eds. (1997) Cambridge: Cambridge University Press. 110–129.
Hayashi F. Large spermatophore production and consumption in dobsonflies Protohermes (Megaloptera, Corydalidae). Jpn J Entomol (1992) 60:59–66.
Kubli E. Sex-peptides: seminal peptides of the Drosophila male. Cell Mol Life Sci (2003) 60:1689–1704.[CrossRef][Web of Science][Medline]
Matsumoto K, Suzuki N. Effectiveness of the mating plug in Atrophaneura alcinous (Lepidoptera: Papilionidae). Behav Ecol Sociobiol (1992) 30:157–163.[CrossRef][Web of Science]
Orr AG, Rutowski RL. The function of the sphragis in Cressida cressida (Fab.) (Lepidoptera, Papilionidae): a visual deterrent to copulation attempts. J Nat Hist (1991) 25:703–710.[CrossRef]
Parker GA. Sperm competition and its evolutionary consequences in the insects. Biol Rev (1970) 45:525–567.
Parker GA. Sexual selection and sexual conflict. In: Sexual selection and reproductive competition in insects—Blum MS, Blum NA, eds. (1979) London: Academic Press. 123–166.
Parker GA. Sperm competition and the evolution of animal mating strategies. In: Sperm competition and the evolution of animal mating systems—Smith RL, ed. (1984) San Diego (CA): Academic Press. 2–60.
Parker GA. Sexual conflict over mating and fertilization: an overview. Philos Trans R Soc Lond B Biol Sci (2006) 361:235–259.
Poiani A. Complexity of seminal fluid: a review. Behav Ecol Sociobiol (2006) 60:289–310.[CrossRef][Web of Science]
Polak M, Wolf LL, Starmer WT, Baker JSF. Function of the mating plug in Drosophila hibisci Bock. Behav Ecol Sociobiol (2001) 49:196–205.[CrossRef][Web of Science]
Ravi Ram K, Wolfner MF. Seminal influences: Drosophila Acps and the molecular interplay between males and females during reproduction. Integr Comp Biol (2007) 47:427–445.
SAS Institute. JMP version 6 (2006) Cary (NC): SAS Institute.
Sauter A, Brown MJF, Baer B, Schmid-Hempel P. Males of social insects can prevent queens from multiple mating. Proc R Soc Lond B Biol Sci (2001) 268:1449–1454.[Medline]
Scott D, Jackson LL. The basis for control of post-mating sexual attractiveness by Drosophila melanogaster females. Anim Behav (1990) 40:891–900.[CrossRef][Web of Science]
Shine R, Olsson MM, Mason RT. Chastity belts in gartersnakes: the functional significance of mating plugs. Biol J Linn Soc (2000) 70:377–390.[CrossRef][Web of Science]
Simmons LW. Sperm competition and its evolutionary consequences in the insects (2001) Princeton: Princeton University Press.
Smith RL. Sperm competition and the evolution of animal mating systems (1984) San Diego (CA): Academic Press.
Sota T. Long adult life span and polyphagy of a carabid beetle, Leptocarabus kumagaii in relation to reproduction and survival. Res Popul Ecol (1984) 26:389–400.[CrossRef]
Sota T. Life history patterns of carabid beetles belonging to the subtribe Carabina (Coleoptera, Carabidae) in the Kinki District, western Japan. Kontyû (1985) 53:370–378.
Sota T. Mortality pattern and age structure in two carabid populations with different seasonal life cycles. Res Popul Ecol (1987) 29:237–254.[CrossRef]
Sugawara T. Stretch reception in the bursa copulatrix of the butterfly, Pieris rapae crucivora, and its role in behavior. J Comp Physiol (1979) 130:191–199.[CrossRef]
Takami Y. Mating behavior, insemination and sperm transfer in the ground beetle Carabus insulicola. Zool Sci (2002) 19:1067–1073.[CrossRef][Web of Science][Medline]
Takami Y. Spermatophore displacement and male fertilization success in the ground beetle Carabus insulicola. Behav Ecol (2007) 18:628–634.
Takami Y, Katada S. Microsatellite DNA markers for the ground beetle Carabus insulicola. Mol Ecol Notes (2001) 1:128–130.[CrossRef][Web of Science]
Tompkins L, Hall JC. The different effects on courtship of volatile compounds from mated and virgin Drosophila females. J Insect Physiol (1981) 27:17–21.[CrossRef][Web of Science]
Vahed K. All that glisters is not gold: sensory bias, sexual conflict and nuptial feeding in insects and spiders. Ethology (2007) 113:105–127.[CrossRef][Web of Science]
Yamane T, Kimura Y, Katsuhara M, Miyatake T. Female mating receptivity inhibited by injection of male-derived extracts in Callosobruchus chinensis. J Insect Physiol (2008) 54:501–507.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||