Popul Ecol (2013) 55:493–498 DOI 10.1007/s10144-013-0380-7
ORIGINAL ARTICLE
Larval competition causes the difference in male ejaculate expenditure in Callosobruchus maculatus Masako Katsuki • Yukihiko Toquenaga Takahisa Miyatake
•
Received: 9 September 2012 / Accepted: 30 April 2013 / Published online: 1 June 2013 Ó The Society of Population Ecology and Springer Japan 2013
Abstract The seed beetle Callosobruchus maculatus larvae exhibit two types of resource competition: scramble, in which a resource is shared, and contest, in which the resource is monopolized. This difference in larval behavior results in different adult densities. Under contest competition, adult density remains constant regardless of larval density, but under scramble competition, adult density increases with larval density. This in turn affects mating frequency during adulthood, and thus, the intensity of sexual selection operating on males. In this study, we examined the relationship between larval competition types and male reproductive investment in mating. We assessed the male ejaculate expenditure per mating across geographic strains of C. maculatus. The male investment (ejaculate expenditure) increased with the degree of scramble competition and decreased with the degree of contest competition. We therefore suggest that males experience different selective pressures depending on the type of larval competition: scramble type males are selected for increased reproductive investment. Keywords Larval competition Seed beetle Sperm competition
M. Katsuki T. Miyatake (&) Graduate School of Environmental Science, Okayama University, Okayama, Japan e-mail:
[email protected] Y. Toquenaga Doctoral Program in Biological Sciences, University of Tsukuba, Ibaraki, Japan
Introduction When a female copulates with two or more males, sperms of these males compete in the female reproductive tract for fertilization (Parker 1970). In a raffle sperm competition (Parker 1990), it is predicted that males who transfer more sperm to the female spermatheca or reproductive duct have increased paternity (Parker 1990; Parker and Pizzari 2010; Kelly and Jennions 2011). Furthermore, in some species the male ejaculate includes seminal substances that restrict female receptivity, thereby delaying female remating with other males (Simmons 2001; Arnqvist and Rowe 2005; McNamara et al. 2009). This delay in female remating increases the chance that sperm competition is avoided altogether. Although this investment in reproductive traits enhances male paternity, these seminal substances are costly (Dewsbury 1982; Paukku and Kotiaho 2005; Brown et al. 2009; Lewis et al. 2011). There is a trade-off between the investment in energetically expensive traits and investment in other traits related to fitness (e.g., Martin and Hosken 2004; Oliver and Cordero 2009; Yamane et al. 2010). Therefore, males may maximize their fitness by optimizing the balance between investment in mating and in other traits, in response to the risk and/or intensity of the sperm competition a male faces (Simmons 2001). Indeed, there is abundant evidence of associations between the intensity of sperm competition and ejaculatory characters in relation to testis and accessory gland size in various taxa (Hunter and Birkhead 2002; Crudgington et al. 2009; Gay et al. 2009). In addition, the intensity and risk of sperm competition is influenced by various selective pressures, such as the frequency of encounter with potential mates and adult density. Among various factors that affect sperm competition, we focused on the difference in larval types in resource acquisition.
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In the developmental stage, larval competition over a resource is inevitable. However, the degree of this competition shows tremendous variability both between and within species (for reviewed see Mideo 2009). The variation reflects the ability to compete for resources and individual aggressiveness (Toquenaga and Fujii 1990a, b; Toquenaga 1993; Mano and Toquenaga 2008a). Contest and scramble competitions are two extremes of the variation (Mano and Toquenaga 2008b). In contest competition, the resource intake of an individual and the number of survivors remain constant, irrespective of an increase in the number of competitors, due to resource monopolization. In contrast, in scramble competition, individuals share the available resources as the number of competitors increases. For example, only one individual can emerge from a mung bean, Vigna radiata, in a bruchid contest species, Callosobruchus analis, whereas more than six individuals can emerge in a scramble species, C. phaseoli (Toquenaga and Fujii 1991). As a result, the proportion of contest and scramble competition types causes a difference in the number of rival males, i.e., higher intensity of sperm competition. Theoretically, differences in sperm competition intensity should cause different selective pressures on ejaculate expenditure and testis size (Parker and Ball 2005). Furthermore, while the possibility that variation in the proportion of competition types is related to reproductive traits in a review was suggested (Isbell 1991), there is little experimental evidence for this. Therefore, we hypothesized that males who exhibit the scramble larval competition type invest more in reproduction than males of the contest larval competition type, and that there is a negative association between the proportion of contest and scramble competition types and the male reproductive investment. The seed beetle Callosobruchus maculatus has a geographic variation in the degree of resource competition in the larval stage, and shows scramble and contest competition types as two extremes (Takano et al. 2001). The scramble individual shares resources; in contrast, the contest individual eliminates competitors and monopolizes the resource itself. The intensity of aggressiveness differs between geographic strains (Takano et al. 2001). Further, in this species, the investment in sperm competition, especially the ejaculate expenditure in one mating, is considerably associated with male fitness (e.g., Eady 1995; Ro¨nn et al. 2011). A larger ejaculate from a male inhibits female remating. In contrast, a female who receives a small ejaculate tends to remate (Eady 1995; Savalli and Fox 1999a). Female remating directly affects male paternity because the second male that mates with a female fathers a larger percentage of the offspring than the first male (Eady 1994). Therefore, the ejaculate expenditure of a C. maculatus male plays an important role on his fitness in sperm competition.
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Here, we examined whether variations in ejaculatory expenditure and the degree of resource competition types are negatively correlated. To test this hypothesis using different geographic strains of the seed beetle C. maculatus, we first assessed the proportion of contest competition larvae and male ejaculate expenditure of each geographical strain. Then, we investigated the relationship between them.
Materials and methods Insects Callosobruchus maculatus females lay eggs on the surface of a bean. Hatched larvae enter and grow in the bean. Emerged adults mate outside the bean. We used seven strains of C. maculatus originally derived from a range of different countries (see Takano et al. 2001): iQ (South India), tQ (Tel Aviv, Israel), yQ (Yemen), aoQ (USA), aoQ2 (USA), hQblk (a black phenotypic mutant of the hQ strain that was originally collected in Japan from beans imported from New Zealand), and wQ (Japan). These strains were obtained from Tsukuba University, Japan, and subsequently maintained at Okayama University, Japan. The hQblk is a mutant strain but not extreme in life-history traits (Mano and Toquenaga 2011). All strains used in the present study were kept in a growth chamber at 25 °C with 16L8D light. After emergence, approximately 20 pairs were allowed to mate and lay eggs on about 100 adzuki (Vigna angularis) beans. All strains were maintained in our laboratory for five to ten generations. We used individuals that emerged from beans that were infested by a single larva to exclude the effects of larval competition. They were 2–5 days old at the time of the experiment. Estimating C value of strains To evaluate the degree of contest type competition across the strains, we used the C value, which indicates the proportion of the contest type in a population (Takano et al. 2001). The C value model is based on the following assumptions. Suppose that two eggs are oviposited by females of the same strain on a bean that is large enough for two larvae to complete their development to full size. When both larvae are of the scramble type, two adults emerge. If one of them is of the contest type, only one adult emerges because the contest larva kills the other. Even if both larvae are contest types, we assumed that only one of them emerges. We also assumed that natural mortality occurs before larvae start competing. Thus, a single bean can be assigned to one of the following six emergence patterns: none (N), only a male (M), only a female (F), two males (MM), two females (FF), or one male and one
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female (MF). We assumed that males and females have the same pre-competition mortality, l. The proportion of males is m, and the C values of males and females are p and q, respectively. Each female is assumed to lay eggs randomly on beans. We further assumed that a male and a female have the same probability of winning the contest when both are contest types. We used the model in Takano et al. (2001) and calculated the C values in each strain (see detail Takano et al. 2001). Summing up the obtained probabilities gives us the expected frequencies of the six emergence patterns as follows: N ¼ l2 MM ¼ ð1 lÞ2 m2 ð1 pÞ2
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Measurement of ejaculate expenditure Virgin males were weighed to the nearest 1/100 mg using an electric balance (Excellence Plus XP, Mettler-Toledo, Columbus, OH, USA) just before the following experiment. A male was then placed in a vial (diameter 9 height = 1.4 cm 9 4.0 cm) with a virgin female of the same strain and permitted to mate. After mating, the male’s mass was measured again. We took the difference in male weight before and after mating as an indicator of male reproductive investment, i.e., ejaculate expenditure. We regard ejaculate expenditure as male investment in reproduction but not merely in one mating. We also measured the female’s mass as her size before she mates. Statistics
FF ¼ ð1 lÞ2 ð1 mÞ2 ð1 qÞ2 MF ¼ 2ð1 lÞ2 mð1 mÞð1 pÞð1 qÞ M ¼ ð1 lÞ2 mpð2 q mp þ mqÞ þ 2lð1 lÞm F ¼ ð1 lÞ2 ð1 mÞqð2 q mp þ mqÞ þ 2lð1 lÞð1 mÞ Note that N þ MM þ FF þ MF þ M þ F ¼ 1: The natural mortality can be calculated as the proportion of adults that emerge from beans each of which had a single hatched egg (control treatment). The values from the control data were used as the external hypothesis, and the other three parameters (m, p, and q) were estimated by a maximum likelihood method. If the natural mortality before competition between the two individuals is different from that without any competition, then the l value must be estimated as well. p and q values are not necessarily different, and the two values can be assumed to be equal. The hypotheses just described are not hierarchical, and we used Akaike’s information criterion (AIC) to compare the competing models. There were four types of models: 1. 2. 3. 4.
Estimating l, m, p, and q Estimating l, m, and p, assuming p = q Estimating m, p, and q, using a fixed l estimated from the control data Estimating m and p assuming p = q with fixed l estimated from the control data.
The AIC calculation and parameter estimation were conducted with an R script using the optim function in R ver. 2. 13. 0 (R Development Core Team 2011). The C value ranges from zero to one. A value closer to one indicates a high proportion of the contest type, while a value closer to zero indicates a high proportion of the scramble type. The R script for the C value calculation is available from the authors.
In C. maculatus, there are positive correlations between ejaculate amount and male and female body size (Savalli and Fox 1998, 1999b), so we analyzed ejaculate expenditure using a general linear model (GLM) with male or female body mass as a covariate. To examine whether the C value is associated with relative ejaculate expenditure, we calculated the adjusted ejaculate expenditure for each strain using the least square mean from the GLM and analyzed it by Pearson’s product moment correlation. Because it is a proportion, the C value was arcsine transformed. JMP version 9 was used for the analyses (SAS Institute 2010).
Results The mean ± SE of male ejaculate expenditures on the first mating are shown in Table 1. We found a significant difference in male ejaculate expenditure on the first mating across strains (GLM; strain: F6,191 = 3.6755, P = 0.011, male weight: F1,191 = 4.7650, P = 0.031, female weight: F1,191 = 0.207, P = 0.650). Therefore, we estimated the adjusted mean ejaculate expenditure removing the effect of male body size with GLM. The adjusted means of male ejaculate expenditure on the first mating were aoQ: 0.239 mg (n = 26), tQ: 0.183 mg (n = 19), aoQ2: 0.202 mg (n = 32), hQblk: 0.192 mg (n = 30), iQ: 0.149 mg (n = 29), yQ: 0.219 mg (n = 30), and wQ: 0.232 mg (n = 30). In addition, C values as the degree of larval competition were strongly and negatively correlated with adjusted ejaculate expenditure across the strains (Fig. 1, r = -0.8010, P = 0.0304). Thus, the higher the proportion of scramble resource competition within a strain, the larger the ejaculate expenditure by males of that strain.
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Table 1 Parameters of C value estimation and absolute ejaculate expenditure mean ± SE of all strains
Proportion of males (m)
C value of males (p)
C value of females (q)
Mortality (l)
aoQ
0.47
0.03
0.00
0.14
aoQ2
0.62
0.00
0.00
0.08
hQblk
0.47
0.32
0.32
iQ
0.35
1.00
tQ
0.56
wQ
0.54
yQ
0.53
Ejaculate expenditure Mean
SE
4
0.21
0.02
2
0.18
0.01
0.45
2
0.22
0.02
1.00
0.55
2
0.16
0.02
0.08
0.08
0.00
2
0.18
0.02
0.00
0.00
0.16
2
0.23
0.01
0.10
0.10
0.28
2
0.23
0.01
Fig. 1 Across geographic strains of C. maculatus, there is a negative correlation between the C value (index of the proportion of contest competition type individuals) and adjusted ejaculate expenditure
Discussion The C value, i.e., the proportion of contest and scramble competition types, was different among strains (Table 1), and this difference reflects the number of adults that emerged. Geographic variation in the degree of larval competition was associated with ejaculatory expenditure in C. maculatus: males derived from strains exhibiting scramble type competition invested more in reproduction than males from strains with more contest type competition. Males should be selected to maximize fitness by optimizing the balance between investment in reproduction and other fitness-related traits in response to the intensity and/or risk of sperm competition that they face (e.g., Parker 1990, 1998). Intensities of sperm competition are likely to differ between larval competition types: under contest competition, the number of adults remains constant due to
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Model
the elimination of rivals. However, with scramble competition, high larval density results in high numbers of adults. When adult density is high, the frequency of encounters between the sexes is also high (Gage 1995), and females mate more frequently compared to those at low densities (Gage 1995; Martin and Hosken 2003); thus, the frequency of female mating is liable to be higher under scramble competition. In C. maculatus, testis size decreases in males who are reared under an experimental evolution regime that prevents sperm competition (Gay et al. 2009), suggesting that selective pressures imposed by sperm competition act on not only testis size but also investment in ejaculation. Therefore, male ejaculate expenditure may be affected by the intensification of sperm competition in relation to the degree of larval competition. We regarded the male ejaculate expenditure on one mating as a parameter of male reproductive investment in this study. Males should allocate their resources for investment in current and future reproductions (e.g., ejaculate size and number of sperm) to order to maximize their fitness (e.g., Cook and Gage 1995; Gage 1995). Because the net fitness consequences for a male may differ between single and multiple matings, we must pay attention not only to one mating but also subsequent matings in this beetle. In addition, we found only one contest type strain in this experiment; thus it is necessary to find and include other strains of the contest type or establish a strain that was artificially selected for the type of larval resource competition, scramble or contest. When a female receives a small ejaculation from a male, the female tends to remate (Eady 1995; Savalli and Fox 1999a). Therefore, a larger ejaculation decreases the female’s remating receptivity, and thus a male, that invests more in reproduction, is able to increase the rate of paternity. On the other hand, in C. maculatus, the P2 value (i.e., the proportion of eggs fertilized by the second male to mate with a female) is relatively high; for example, when the interval between mating is 24 h, P2 = 0.84 in the scramble type strain (Eady 1994). Therefore, when a male mates with a non-virgin female, the male can sire more
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offspring by transferring a larger ejaculate to the female (Eady 1995). In any situation, a larger reproductive investment increases the rate of paternity. A positive correlation between the intensity of sperm competition and testis size has been documented in various taxa (Hosken and Ward 2001; Firman and Simmons 2008; Dziminski et al. 2010). In polyandrous species with sperm competition, the survival rate of sperm is higher than in monogamous species (Hunter and Birkhead 2002). Furthermore, males exhibit decreased testis size (Gay et al. 2009) and accessory gland size (Crudgington et al. 2009) when the level of sperm competition is decreased by experimental manipulation. In this species, males transfer seminal substances derived from the accessory gland into females to decrease female receptivity (Yamane et al. 2008). Females also absorb nutrients (Fox and Moya-Larano 2009) and water (Edvardsson 2007; Ursprun et al. 2009) from the seminal fluid. A male may prevent female remating by making up for a nutritional deficit in the female. Intensification of sperm competition can select for increased ejaculate expenditure by this male, resulting in the negative correlation between ejaculate expenditure and larval competition degree. An alternative explanation for the correlation between larval competition and ejaculatory expenditure is thought to be allocation of resources to larval competition and reproduction. The degree of larval competition is likely to reflect the investment in larval competition, such as attack and defense behavior against competitors (Toquenaga and Fujii 1990a; Mano and Toquenaga 2008a). Previous work on this species suggests that male reproductive efforts lead to costs in male life history traits (Tuda 1998; Tuda and Iwasa 1998; Paukku and Kotiaho 2005; Brown et al. 2009). Thus, investments in larval competition and reproduction are thought be constrained by the respective allocation of resources to each. To date, less attention has been paid to trade-offs between different developmental stages, larva and adult. However, regardless of the mechanism, the patterns of the evolutionary change seen in male reproduction and the competition-related traits seem to make sense, even if between different stages. In the present study, we found a correlation between male ejaculate expenditure and degree of larval competition. This relationship is thought to reflect a difference in the intensity of sperm competition during adulthood. In C. maculatus, male ejaculatory expenditure is exposed to stronger selective pressures from sperm competition during adulthood under low larval resource competition; in contrast, the evolution of larval competition may relax sexual selection through sperm competition. This beetle provides an excellent model to examine the relationships between larval resource competition, ejaculate expenditure, sperm competition, and sexual conflict.
497 Acknowledgments We thank Tomohiro Harano and Zenobia Lewis for valuable comments, and the Kobe Plant Protection Office for permitting the insects used to be transported both internationally and within Japan. All transportation was conducted under the Japanese Plant Protection Law. This study was partially supported by KAKENHI 23570027 Grant-in-Aid for Scientific Research, JSPS and MEXT to T.M., 224565 to M.K. and 23405008 to Y.T.
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