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Direct effects of polyandry on female fitness in Callosobruchus chinensis
ANIMAL BEHAVIOUR, 2006, 71, 539–548 doi:10.1016/j.anbehav.2005.05.017
Direct effects of polyandry on female fitness in Callosobruchus chinensis TOMOHIR O HA RANO* , Y U KI O YA SU I † & TA K AH IS A MI YA TA KE*
*Laboratory of Evolutionary Ecology, Graduate School of Environmental Science, Okayama University yLaboratory of Entomology, Faculty of Agriculture, Kagawa University (Received 13 December 2004; initial acceptance 18 February 2005; final acceptance 1 May 2005; published online 17 February 2006; MS. number: 8389)
In many insect species with high levels of polyandry, females benefit directly from remating. The effects of remating on female fitness have generally been examined by comparing the fitness of females in multiplemating and single-mating treatments. In this standard approach, females in the multiple-mating group that refuse to remate are often excluded from the analysis. We investigated the effects of remating in the adzuki bean beetle, Callosobruchus chinensis, a species with low levels of polyandry. Females that refused to remate when given an opportunity to do so were less fecund and shorter lived than females that accepted remating. In this case, excluding females that refuse to remate from the multiple-mating treatment biases the composition of the two treatment populations, and is thus problematic. When we included such females, we found no difference in fecundity, fertility and longevity between females given an opportunity to remate and those that were not. In addition, when we compared females that were allowed to complete remating naturally and those whose remating was interrupted before sperm transfer we found significantly negative effects of female remating on fecundity, suggesting that remating reduces the fitness of polyandrous females in C. chinensis, which is inconsistent with many studies on polyandrous species. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Females of the majority of animal species are polyandrous (Thornhill & Alcock 1983; Ridley 1988; Birkhead & Møller 1998; Birkhead 2000). For males, reproductive success generally increases with more mates, whereas for females it is determined by the number of offspring produced (Bateman 1948). Therefore, the fitness advantages for males of multiple mating are easily understood, but they are less clear for females. Multiple mating may decrease female fitness because mating involves various costs to females: general time and energy expenditures (Thornhill & Alcock 1983), increased risk of predation (Arnqvist 1989), risk of pathogen transmission (Hurst et al. 1995), physical injury (Crudgington & Siva-Jothy 2000) and toxic effects of substances in male ejaculates (Chapman et al. 1995). On the other hand, multiple mating may increase female fitness through benefits such as replenishing sperm supplies, stimulating egg production and offering materials or resources that contribute to female survival
Correspondence: T. Miyatake, Laboratory of Evolutionary Ecology, Graduate School of Environmental Science, Okayama University, Okayama 700-8530, Japan (email: [email protected]). Y. Yasui is at the Laboratory of Entomology, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan. 0003–3472/05/$30.00/0
and/or reproduction (Thornhill & Alcock 1983; Arnqvist & Nilsson 2000). A review (Ridley 1988) and a meta-analysis (Arnqvist & Nilsson 2000) of studies on the direct effects of multiple mating on female fitness found that in many insect species females benefit from polyandry in terms of increased lifetime offspring production. Arnqvist & Nilsson’s metaanalysis, however, was biased towards species with high levels of polyandry, because most research addressing the direct benefits of polyandry has been carried out on polyandrous species (Torres-Vila et al. 2004). The frequency of polyandrous females varies considerably between species (Ridley 1988; Torres-Vila et al. 2004), so polyandry tends to evolve in species in which females benefit directly from multiple mating, an analysis focused on polyandrous species will bias the outcome towards the positive effects of polyandry on female fitness. Arguments about the adaptive significance of female multiple mating should not ignore species with infrequent polyandry. Increased reproductive output of females through polyandry is not apparent in species where it occurs at low levels (Ridley 1988; Torres-Vila et al. 2004). A marked variation in female remating frequency has been found among strains of the adzuki bean beetle, Callosobruchus chinensis (Miyatake & Matsumura 2004;
539 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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ANIMAL BEHAVIOUR, 71, 3
Harano & Miyatake 2005). In this species, almost all females are monandrous in strains with low remating frequencies, whereas in strains with high remating frequencies both polyandrous and monandrous females exist and neither mating pattern is predominant (Miyatake & Matsumura 2004; Harano & Miyatake 2005). Mating itself reduces female survival (Yanagi & Miyatake 2003), but we have no information about the effects of remating on female fitness in this beetle. We therefore investigated the direct effects of polyandry on female fitness in C. chinensis. The standard approach to examine the effects of remating on female fitness is to allow females assigned to one treatment to mate multiply and females assigned to
another treatment to mate singly, and then compare fitness components such as fecundity, fertility and longevity between the two treatments (e.g. Fox 1993; Wilson et al. 1999; Fedorka & Mousseau 2002). When this experimental method is used, some females assigned to the multiplemating treatment may refuse to remate. When the multiple-mating treatment comprises females that refuse to remate, that is, monandrous females and females that accept remating, that is, polyandrous females, the singlemating treatment is expected to comprise both females that would refuse to remate and females that would accept remating if provided with opportunities for remating (Fig. 1a). Females in the multiple-mating treatment that refuse to remate when given the opportunity are usually
(a) Composition of female groups
Treatments
Monandrous females
Polyandrous females
Not remated
Remated
Monandrous females
Polyandrous females
Not remated
Not remated
Multiple-mating treatment
Single-mating treatment
(b)
(c) Monandrous/ Multiple
Polyandrous/ Multiple
Polyandrous/ Multiple
Monandrous/single + Polyandrous/single
(d)
(e) Monandrous/Multiple + Polyandrous/Multiple
Polyandrous/ Multiple
Monandrous/single + Polyandrous/single
Polyandrous/ single
Figure 1. (a) Outline of the standard approach to investigate the effects of remating on female fitness. The multiple-mating treatment includes females that refuse to remate, that is, monandrous females, and females that accept remating, that is, polyandrous females in a certain proportion. The single-mating treatment is then expected to comprise females that would refuse to remate (‘monandrous’) and females that would accept remating (‘polyandrous’) if they had an opportunity to remate, in the same proportion. It is impossible to distinguish between these females, however, because they do not receive an opportunity to remate. The difference in fitness between females mated multiply and females mated singly has often been tested by (b) comparing polyandrous females in the multiple-mating treatment with all females in the single-mating treatment, but if the monandrous and polyandrous females possess different traits, excluding monandrous females from the multiple-mating treatment biases the composition of females in the two treatments. In the present study, we compared (c) females that refused to remate and females that accepted remating in the multiple-mating treatment, (d) all females in the multiple-mating and single-mating treatments and (e) females allowed to complete remating naturally (equivalent to polyandrous females in the multiple-mating treatment) and females whose remating was interrupted (equivalent to polyandrous females in the single-mating treatment).
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
excluded from the analysis (Torres-Vila et al. 2004). In this case, the fitness of polyandrous females in the multiplemating treatment is compared to that of all females in the single-mating treatment (Fig. 1b). The comparison, however, may involve a methodological problem. If female condition, which results from body size or nutritional status or the effects of costs derived from a previous mating or genetic attributes and so on, vary between females that refuse to remate and females that accept remating then, independent of remating effects, fitness will differ between the two groups. If this is the case, excluding females that refuse to remate from the multiple-mating treatment biases the type of females in both treatments and thus will prevent fair comparison between them (Torres-Vila et al. 2004). In the present study, we considered this problem and designed experiments to examine the direct effects of remating on female fitness. In the first experiment, we compared traits of females that refused to remate and females that accepted remating when they had opportunities to remate (Fig. 1c) to test an association between female receptivity to remating (refuse or accept) and female condition. We also, compared the fitness components of females that had opportunities to remate, whether or not they accepted, with those of females that had no opportunities to remate to examine the direct effects of remating on female fitness in unbiased compositions of the two treatments (Fig. 1d). The second experiment was designed to examine the effects of remating on fitness only in polyandrous females. In one treatment, female remating was completed naturally and in another female remating was immediately interrupted. Females in the former treatment correspond to polyandrous females in the multiple-mating treatment. On the other hand, females in the latter treatment would show no effects of remating even though they had accepted it, and thus correspond to polyandrous females in the singlemating treatment. Therefore we expected to detect the net effects of remating by comparing fitness components of females in the two treatments (Fig. 1e). METHODS
Insects and Culture We used a strain of C. chinensis, referred to as the isC strain, which was established with about 200 adults collected from mung beans, Vigna radiata, in Ishigaki City, Ishigaki Island, Okinawa, Japan, in 1997 (Yanagi & Miyatake 2003). The isC strain has the highest known frequency of female remating among C. chinensis populations (Harano & Miyatake 2005) and thus is appropriate for this study because the effects of remating are difficult to examine in strains in which few females remate. The beetles were reared from eggs laid by parents collected randomly from a stock culture. Virgin beetles were randomly collected and kept in single-sex groups of up to 10 adults in plastic cups (2.8 cm high, 7 cm in diameter). All rearing and subsequent experiments were conducted in a chamber maintained at 25 C and 50% relative humidity under a photoperiod cycle of 14:10 h light:dark. Adult beetles were not given food and water, except as stated below.
Experiment 1: Remating Opportunities First, we compared traits of females that refused to remate and females that accepted remating (Fig. 1c) to determine whether receptivity to remating (refuse or accept) was associated with female condition. We then compared fitness components of females that had opportunities to remate, regardless of whether they accepted (multiplemating treatment) with those that did not have opportunities to remate (single-mating treatment; Fig. 1d) to test the effects of the presence of opportunities to remate on female fitness. This experiment was conducted under both no-feeding and feeding conditions, because adults of C. chinensis can reproduce without feeding, but they greatly increase their fecundity and longevity by feeding (Umeya & Shimizu 1968). Under the feeding condition, females were given water and adult food (1:2 yeast extract: sugar). All virgin males used as mates for females were given water and adult food and were aged 2–5 days. On day 1 after it emerged, we placed one virgin female in a glass vial (4.4 cm high, 1.7 cm in diameter) with one virgin male, and observed mating for 1 h. After copulation, the male was removed, and the female was maintained separately in a plastic cup containing 10 adzuki beans, Vigna angularis, as the oviposition substrate. We randomly assigned the mated females to either the multiple-mating treatment or the single-mating treatment. In the multiple-mating treatment, the mated females had contact with males on days 1 and 3 after the first mating under both no-feeding and feeding conditions, and on day 5 after the first mating under the feeding condition because of their prolonged life span. The mated female and a virgin male were placed in a glass vial and observed each day until either the female remated once or 1 h had passed. In these observations, remated females had no opportunity to remate further whereas females that did not remate received another opportunity to remate. In the single-mating treatment, mated females were not allowed contact with males after the first mating. Beans as oviposition substrate were replaced with 10 fresh ones every day under the no-feeding condition, and with 10 every day for the first 5 days, five every day for the next 5 days and three every day for the remaining days under the feeding condition because egg production declines with maternal age (Yanagi & Miyatake 2002). We counted the eggs produced and hatched. A few females (2/77 in the multiple-mating treatment and 1/39 in the single-mating treatment under the no-feeding condition; 0/55 in the multiple-mating treatment and 1/31 in the single-mating treatment under the feeding condition) that did not lay viable eggs after the first mating were excluded from the analysis, because they had probably failed to be inseminated by the first male. All females of both treatments were reared until they died under the no-feeding condition or until 40 days after emerging under the feeding condition. Under the feeding condition, therefore, we recorded the numbers of eggs produced and hatched for 40 days after the female emerged. Longevity of females was also recorded until 40 days after emergence. In C. chinensis, egg size and thus hatching success decrease with maternal age (Yanagi & Miyatake 2002), and therefore
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ANIMAL BEHAVIOUR, 71, 3
the percentage of eggs produced that hatch depends on the temporal pattern of oviposition over the female’s life span. To assess fertility, we therefore calculated the percentage of eggs that hatched on each day. In this calculation, we pooled the number of eggs produced after 4 days from the female’s first mating under the no-feeding condition and after 6 days from the female’s first mating under the feeding condition because the number of eggs was small. As a measure of body size we recorded elytra length after the females died.
Females that refused versus females that accepted remating To test an association between receptivity to remating and female condition, we compared body size, number of eggs produced, number of eggs hatched, percentage of eggs hatched and longevity of females that refused to remate (N ¼ 33 for the no-feeding condition; N ¼ 11 for the feeding condition) and females that accepted remating (N ¼ 42 for the no-feeding condition; N ¼ 44 for the feeding condition) in the multiple-mating treatment (Fig. 1c). The female’s decision to remate is, in part, influenced by male traits in this beetle (T. Harano & T. Miyatake, unpublished data). In the present experiment, we randomly assigned males to females, but if male courtship behaviour changes according to female phenotype, then the change in male behaviour may influence female receptivity to remating. When a male C. chinensis encounters a female, he generally attempts to mate with her (Yanagi & Miyatake 2003). In our experiment, we are certain that a male encountered a female many times at each opportunity to remate and thus the difference in female receptivity to remating did not depend on whether a male attempted to mate with a female, although the intensity of male mating attempts may differ between female phenotypes.
Multiple- versus single-mating treatments To examine the direct effects of remating on female fitness in unbiased compositions of the two treatments, we compared the number of eggs produced, the number of eggs hatched, the percentage of eggs hatched and longevity of females in the multiple-mating treatment (N ¼ 75 for the no-feeding condition; N ¼ 55 for the feeding condition) and females in the single-mating treatment (N ¼ 38 for the no-feeding condition; N ¼ 30 for the feeding condition; Fig. 1d). The lack of difference in the elytra length of females between the multiple-mating and single-mating treatments under the no-feeding condition (multiple-mating treatment: X SE ¼ 1:89 0:01 cm; single-mating treatment: 1.88 0.01 cm; ANOVA: F1,111 ¼ 0.38, P ¼ 0.538) and under the feeding condition (multiple-mating treatment: 1.88 0.01 cm; single-mating treatment: 1.88 0.01 cm; ANOVA: F1,83 ¼ 0.06, P ¼ 0.809) confirmed the unbiased compositions of the two treatment groups, with regard to body size.
Experiment 2: Remating Interruption We established one treatment in which females were allowed to complete remating naturally (control) and
another in which female remating was immediately interrupted (interruption treatment). By excluding monandrous females, we were thus able to examine the direct effects of remating on female fitness only in those females that were receptive to remating. Before this experiment, we observed that 10 virgin females whose copulating was interrupted as soon as they mated (within 5 s) did not lay viable eggs and thus confirmed that the interruption completely prevented sperm transfer. Furthermore, sperm are not transferred to the female spermatheca within 20 s of copulating in the isC strain of C. chinensis, which we used in this study (T. Yamane & T. Miyatake, unpublished data). All females and virgin males received water and adult food and the latter were 2–5 days old. On day 1 after it emerged we placed one virgin female in a glass vial with one virgin male, and observed mating for 1 h. After copulation, the male was removed, and the female was maintained individually in a plastic cup containing 10 adzuki beans as an oviposition substrate. We randomly assigned the mated females to either the control or the interruption treatment. On day 3 after the female had mated once, we placed one mated female and one virgin male in a plastic petri dish (1.5 cm high, 9.1 cm in diameter) and observed them for 1 h. In the interruption treatment, copulating pairs were separated with two very soft writing brushes within 5 s of the female initiating remating. Females that did not remate in 1 h of observation were excluded from both treatments. Beans as an oviposition substrate were replaced with 10 fresh ones every day for the first 5 days, five every day for the next 5 days, and five every 4–7 days for the remaining days. We counted the eggs produced and hatched over 40 days after emergence. A few females (0/32 in the control treatment and 1/32 in the interruption treatment) that did not lay viable eggs after the first mating were excluded from the analysis because they had probably not been inseminated by the first male. To assess fertility, we calculated the percentage of eggs produced that hatched on each day. In this calculation, we pooled the small number of eggs produced after 6 days from the female’s first mating. Female elytra length was measured after death. We compared the number of eggs produced, the number of eggs hatched and the percentage of eggs hatched of females in the control (N ¼ 31) and interruption treatments (N ¼ 32; Fig. 1e). The lack of difference in the elytra length of females between the two treatments (control: X SE ¼ 1:87 0:01 cm; interruption treatment: 1.87 0.01 cm; ANOVA: F1,61 ¼ 0.06, P ¼ 0.814) confirmed the unbiased compositions of the two treatments, with regard to body size.
Statistics Elytra length, the number of eggs produced, the number of eggs hatched and longevity were log transformed and the percentage of eggs hatched was arcsine transformed to be approximated to a normal distribution. We used an analysis of variance (ANOVA) to compare elytra length and the percentage of eggs hatched between female groups. Fecundity and longevity covary with body size in a related species, C. maculatus (Fox 1993), so we used an
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
analysis of covariance (ANCOVA), with elytra length as a covariate to compare the number of eggs produced, the number of eggs hatched and longevity of female groups if the ANCOVA detected a significant effect of elytra length on the traits. If the ANCOVA detected no significant effects of elytra length on the traits we used an ANOVA to compare groups. The log-rank test was used to compare survival of the female groups. For statistical analyses we used SPSS (SPSS Inc. 2001).
RESULTS
Experiment 1: Remating Opportunities Females that refused versus females that accepted remating
Erlytra length (mm)
Under the no-feeding condition, females that accepted remating had significantly longer elytra than females that refused to remate when females had opportunities to remate (F1,73 ¼ 9.94, P ¼ 0.002; Fig. 2a). An ANCOVA, with elytra length as a covariate, detected significant effects of elytra length on the numbers of eggs produced (F1,72 ¼ 19.26, P < 0.001) and hatched (F1,72 ¼ 22.68, P < 0.001). More eggs were produced by females that
No. of eggs
Feeding condition
No-feeding condition 1.94
(a)
**
1.92
1.94 1.9
1.88
1.88
1.86
1.86
1.84
1.84 (b) **
60
160 **
80
20
40 Produced
0
Hatched
100
(c)
100
80
80
60
60
40
40
20
20
0
1
*
120
40
0
(d)
1.92
1.9
80
% Eggs hatched
accepted remating (ANCOVA: F1,72 ¼ 8.59, P ¼ 0.005; Fig. 2b), but the number of eggs hatched did not differ significantly between groups (ANCOVA: F1,72 ¼ 1.97, P ¼ 0.164; Fig. 2b). There were no significant differences in the percentage of eggs hatched on any day between groups (day 1: F1,73 ¼ 0.87, P ¼ 0.353; day 2: F1,73 ¼ 2.05, P ¼ 0.157; day 3: F1,67 ¼ 0.70, P ¼ 0.405; after 4 days: F1,57 ¼ 0.10, P ¼ 0.751; Fig. 2c). Females that accepted remating had higher survival than females that refused to remate (log-rank test: P ¼ 0.005; Fig. 3a), and an ANCOVA detected a significant effect of elytra length on longevity (F1,72 ¼ 6.02, P ¼ 0.017) and a significant difference in longevity between groups (females that accepted remating: X SE ¼ 7:9 0:2 days; females that refused to remate: 7.1 0.1 days; F1,72 ¼ 4.73, P ¼ 0.033). Under the feeding condition, there was no significant difference in elyta length between the female groups (F1,53 ¼ 0.814, P ¼ 0.371; Fig. 2d). An ANCOVA detected no significant effects of elytra length on the numbers of eggs produced (F1,52 ¼ 2.11, P ¼ 0.153) and hatched (F1,52 ¼ 2.05, P ¼ 0.159). More eggs were produced (ANOVA: F1,53 ¼ 8.43, P ¼ 0.005) and more hatched (ANOVA: F1,53 ¼ 6.77, P ¼ 0.012) by females that accepted remating (Fig. 2e). The percentage of eggs hatched on day 1 was significantly lower for females that accepted
2
3
Produced
Hatched (f)
*
0 4+ 1 2 3 Days after female’s first mating
(e)
4
5
6+
Figure 2. Comparison of body size and fitness components (X þ SE) for females that accepted remating (-) and females that refused to remate (,) when given opportunities to remate. (a) Elytra length, (b) number of eggs produced and hatched and (c) percentage of eggs hatched under the no-feeding condition. N ¼ 42 females that accepted remating and 33 that did not. (d) Elytra length, (e) number of eggs produced and hatched and (f) percentage of eggs hatched under the feeding condition. N ¼ 44 females that accepted remating and 11 that did not. *P < 0.05; **P < 0.01 (a, c, d and f) ANOVA; (b) ANCOVA, with elytra length as a covariate; (e) ANOVA, because of no significant effects of elytra length by ANCOVA, with elytra length as a covariate.
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ANIMAL BEHAVIOUR, 71, 3
1
(a)
0.8 0.6 0.4 Cumulative survival rate
544
0.2 0 0 1
1
2
3
4
5
6
7
8
9
10 11 12
(b)
0.8 0.6 0.4 0.2 0 0
5
10
15
20
25
30
35
40
Days after female emerged Figure 3. Survival of females that accepted remating (d) and females that refused to remate (- - - - -) when given opportunities to remate under (a) the no-feeding condition and (b) the feeding condition.
remating (F1,53 ¼ 4.76, P ¼ 0.034; Fig. 2f), but the difference was extremely small. There were no significant differences in the percentage of eggs hatched on any other day between groups (day 2: F1,53 ¼ 1.15, P ¼ 0.289; day 3: F1,50 ¼ 0.90, P ¼ 0.347; day 4: F1,43 ¼ 0.37, P ¼ 0.544; day 5: F1,41 ¼ 0.02, P ¼ 0.885; after 6 days: F1,39 ¼ 0.10, P ¼ 0.753; Fig. 2f). Thus, we found no clear evidence for a difference in the percentage of eggs hatched between females that accepted remating and those that did not. Female that accepted remating had higher survival (logrank test: P ¼ 0.001; Fig. 3b). An ANCOVA detected no significant effect of elytra length on longevity (F1,52 ¼ 0.46, P ¼ 0.503). Females that accepted remating lived longer (median 20 days) than those that did not (12 days; ANOVA: F1,53 ¼ 9.16, P ¼ 0.004).
Multiple- versus single-mating treatments Under the no-feeding condition, an ANCOVA, with elytra length as a covariate, detected significant effects of elytra length on the numbers of eggs produced (F1,110 ¼ 60.37, P < 0.001) and hatched (F1,110 ¼ 62.71, P < 0.001), and there were no significant differences in the numbers of eggs produced (F1,110 ¼ 0.11, P ¼ 0.742) and hatched (F1,110 ¼ 0.21, P ¼ 0.650) between the multiple- and single-mating treatments (Fig. 4a). There were no
significant differences in the percentage of eggs hatched on any day between the two treatments (day 1: F1,111 ¼ 0.66, P ¼ 0.420; day 2: F1,111 ¼ 1.54, P ¼ 0.217; day 3: F1,103 ¼ 0.38, P ¼ 0.537; after 4 days: F1,83 ¼ 0.10, P ¼ 0.752; Fig. 4b). There was also no significant difference in female survival between the two treatments (log-rank test: P ¼ 0.553; Fig. 5a); an ANCOVA detected a significant effect of elytra length on female longevity (F1,110 ¼ 16.38, P < 0.001) but no significant difference in female longevity between the two treatments (multiple-mating treatment: X SE ¼ 7:5 0:1 days; singlemating treatment: 7.6 0.2 days; F1,110 ¼ 0.33, P ¼ 0.564). Under the feeding condition, the results were similar to those under the no-feeding condition. An ANCOVA detected significant effects of elytra length on the numbers of eggs produced (F1,82 ¼ 6.67, P ¼ 0.012) and hatched (F1,82 ¼ 7.40, P ¼ 0.008), but no significant difference in the numbers of eggs produced (F1,82 ¼ 0.27, P ¼ 0.607) and hatched (F1,82 ¼ 0.07, P ¼ 0.794) between the two treatments (Fig. 4c). There were no significant differences in the percentage of eggs hatched on any day between the two treatments (day 1: F1,83 ¼ 0.70, P ¼ 0.404; day 2: F1,83 ¼ 0.74, P ¼ 0.393; day 3: F1,76 ¼ 0.34, P ¼ 0.563; day 4: F1,68 ¼ 1.19, P ¼ 0.279; day 5: F1,63 ¼ 0.09, P ¼ 0.769; after 6 days: F1,62 ¼ 2.12, P ¼ 0.151; Fig. 4d). Female survival did not differ significantly between treatments (log-rank test: P ¼ 0.244; Fig. 5b). An ANCOVA detected no significant effect of elytra length on female longevity (F1,82 ¼ 0.06, P ¼ 0.809), and an ANOVA detected no significant difference in female longevity between the two treatments (median: multiple-mating treatment: 14 days; single-mating treatment: 25 days; F1,83 ¼ 1.22, P ¼ 0.273).
Experiment 2: Remating Interruption An ANCOVA, with elytra length as a covariate, detected significant effects of elytra length on the numbers of eggs produced (F1,60 ¼ 9.94, P ¼ 0.003) and hatched (F1,60 ¼ 7.85, P ¼ 0.007). The ANCOVA showed that females whose remating was interrupted produced more eggs (F1,60 ¼ 6.26, P ¼ 0.015; Fig. 6a) and hatched more eggs (F1,60 ¼ 6.90, P ¼ 0.011; Fig. 6a) than females allowed to complete remating naturally. There were no significant differences in the percentage of eggs hatched on any day before 5 days between the two treatments (day 1: F1,61 ¼ 0.51, P ¼ 0.477; day 2: F1,61 ¼ 0.06, P ¼ 0.807; day 3: F1,61 ¼ 0.57, P ¼ 0.452; day 4: F1,60 ¼ 0.06, P ¼ 0.804; day 5: F1,60 ¼ 1.97, P ¼ 0.165; Fig. 6b). The hatching success of eggs produced after 6 days was significantly higher in the interruption treatment than in the control (F1,60 ¼ 4.94, P ¼ 0.030; Fig. 6b).
DISCUSSION In the present study, we first compared traits of the C. chinensis females that refused to remate and the females that accepted remating when they had opportunities to remate
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
No-feeding condition
Feeding condition 160
80 No. of Eggs
(a)
(c)
60
120
40
80
20
40
0
Produced
0
Hatched
Produced
Hatched
100
100 % Eggs hatched
(b)
(d)
80
80
60
60
40
40
20
20
0
1
2
3
4+
0
1
2
3
4
5
6+
Days after female’s first mating Figure 4. Comparison of fitness components (X SE) of females given opportunities to remate (multiple-mating treatment; -) and females deprived of opportunities to remate (single-mating treatment; ,). (a) Number of eggs produced and hatched and (b) percentage of eggs hatched under the no-feeding condition (N ¼ 75 for multiple-mating treatment, N ¼ 38 for single-mating treatment); (c) number of eggs produced and hatched and (d) percentage of eggs hatched under the feeding condition (N ¼ 55 for multiple-mating treatment, N ¼ 30 for singlemating treatment). (a and c) ANCOVA, with elytra length as a covariate; (b and d) ANOVA.
(Fig. 1c). The females that refused to remate were smaller, less fecund and shorter lived than females that accepted remating (Figs 2, 3). These differences are probably attributable to differences in their viability and reproductive capability rather than to the effects of remating. In other words, larger, more viable and more fecund females tended to accept remating. The tendency for larger females to remate at higher frequencies has been reported in other insects (Torres-Vila et al. 1997; Bergstro¨m et al. 2002). A physiological explanation for the tendency may be that larger and more fecund females can accept additional sperm in their larger spermatheca and use more sperm for egg production at higher rates (Torres-Vila et al. 1997). Another explanation may be that larger and more viable females accept remating because they are less sensitive to the costs of mating, whereas smaller and less viable females refuse to remate because they are more sensitive to these costs. On the other hand, the greater propensity of larger and more fecund females to remate may be attributable to differential courtship behaviour of males. If males are able to perceive the reproductive quality of females, they may attempt to mate with larger and more fecund females more ardently than with smaller and less fecund females. As mentioned above, we found differences in fitness components between polyandrous and monandrous females. This indicates that exclusion of females that refuse to remate results in a nonrandom sample of larger, more viable and more fecund females in the multiple-mating treatment. Comparing the fitness of polyandrous females in the multiple-mating treatment and all females in the
single-mating treatment is thus unfair and problematic (Fig. 1b). When we compared fitness components of all females that had opportunities to remate, regardless of whether they accepted, and all females deprived of opportunities to remate (Fig. 1d), we found no differences between the two groups (Figs 4, 5). However, this comparison may underestimate the effects of remating on fitness in polyandrous females because the group of females provided with opportunities to remate includes not only the females that remated but also those that mated only once. Finally, to examine the direct effects of remating on female fitness only in the females that were receptive to remating, we compared fitness components of females that were allowed to complete remating naturally and those whose remating was interrupted immediately, before sperm was transferred (Fig. 1e). The females whose remating was interrupted had significantly higher fecundity than the females that were allowed to complete remating (Fig. 6a). In experiment 2 we found a significant difference in fecundity between females in the multiple-mating treatment that accepted remating and those in the singlemating treatment that would have remated if they had an opportunity to remate (Fig. 6a). In experiment 1, females in the multiple-mating treatment that refused to remate and the females in the single-mating treatment that would have refused to remate even if they had an opportunity to remate are expected to have similar fitness components. It seems that including both groups in experiment 1 prevented us from finding the significant difference in fecundity between females in the multiple-mating and the
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200
1
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* 160 No. of eggs
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Produced
Hatched
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Cumulative survival rate
546
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* 60
40
20 5
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Days after female emerged Figure 5. Survival of females given opportunities to remate (d) and that were deprived of opportunities to remate (- - - - -) under (a) the no-feeding condition and (b) the feeding condition.
single-mating treatments. Our results thus indicate that remating directly reduces fitness of polyandrous females in C. chinenis. Torres-Vila et al. (2004) indicated that an analysis that excludes monandrous females does not examine the direct effects of remating for the average female. This indication applies to experiment 2 in our study, that is, the comparison between females that were allowed to complete remating naturally and females whose remating was interrupted (Fig. 1e), because the comparison excluded females that were not receptive to remating. We cannot obtain experimental evidence of the direct effects of remating on the fitness of females that were not receptive to remating, although we presume that such females would not benefit directly from remating because they more strongly resist remating than do polyandrous females. We found no positive effects of female remating on the percentage of eggs hatched, suggesting that females of C. chinensis acquire sufficient sperm to fertilize their eggs in a single mating. Female remating had no positive effects on fecundity, suggesting that remating in C. chinensis does not stimulate egg production or provide materials that contribute to increased female fecundity and/or longevity. On the basis of their meta-analysis of 122 studies addressing the direct benefit of multiple mating on female fitness, Arnqvist & Nilsson (2000) have argued that in many insect species females benefit directly from multiple
0
1
2 3 4 5 Days after female’s first mating
6+
Figure 6. Comparison of fitness components (X SE) of females that completed remating (-) and females whose remating was interrupted (,). (a) Number of eggs produced and hatched, (b) percentage of eggs hatched (N ¼ 31 for control, N ¼ 32 for interruption treatment). *P < 0.05; (a) ANCOVA, with elytra length as a covariate; (b) ANOVA.
mating in terms of increased fecundity and that the evolution of polyandry can be understood in terms of the direct benefits. Our finding in C. chinensis is inconsistent with this argument. Arnqvist & Nilsson’s meta-analysis is biased towards polyandrous species (Torres-Vila et al. 2004). In three species related to C. chinensis, C. maculatus (Fox 1993; Wilson et al. 1999), C. analis (Wilson et al. 1999) and C. subinnotatus (Mbata et al. 1997), which were included in Arnqvist & Nilsson’s meta-analysis, fecundity is increased by female remating. In C. maculatus 85% of females remated (Eady 1991), and in C. analis 66% (Wilson et al. 1999). Torres-Vila et al. (2004) have classified species as monandrous if less than 40% of females are polyandrous and as polyandrous if more than 40% are polyandrous. According to their classification, C. maculatus and C. analis are classified as polyandrous species; C. subinnotatus is probably polyandrous, because in one study (Mbata et al. 1997) females mated two, three or four times at intervals of 24 h. In C. chinensis, the frequency of polyandrous females differs between strains (range 6–40%, Harano & Miyatake 2005); C. chinensis is thus classified as a monandrous species according to Torres-Vila et al.’s classification. Therefore, in Callosobruchus, the three polyandrous species show direct benefits
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
of remating, whereas a monandrous species, C. chinensis, did not. This suggests a positive association between the direct benefit of polyandry on female fitness and the frequency of female remating across species. Our study raises the question why some females of C. chinensis remate in spite of suffering disadvantages. Besides the hypothesis that polyandry directly increases female fitness, there is the genetic (indirect) benefit hypothesis that females may benefit indirectly from polyandry through increased fitness of their offspring with genetic superiority or diversity (Thornhill & Alcock 1983; Yasui 1998). In the absence of genetic benefits that entirely offset disadvantages of remating, females may also be coerced into accepting superfluous mating by persistent males (Clutton-Brock & Parker 1995; Arnqvist 1997). When a male C. chinensis encounters a female, he generally rubs her with his antenna and mounts from the rear, and if the female then flees he often runs after her (Yanagi & Miyatake 2003). This persistent courtship behaviour of males may be costly for females (Yanagi & Miyatake 2003), and thus females may benefit directly from remating by decreasing the costs of male harassment. If this is the case, female remating in C. chinensis can be explained by the convenience polyandry hypothesis that females mate multiply to reduce the costs of sexual harassment (Thornhill & Alcock 1983). The interests of males and females often conflict at mating (Parker 1979; Holland & Rice 1998; Partridge & Hurst 1998; Arnqvist & Rowe 2002; Rowe & Arnqvist 2002; Chapman et al. 2003; Pizzari & Snook 2003). Our finding that remating is harmful to females suggests that there is sexual conflict between reluctant females and persistent males over female remating in C. chinensis. Further investigation is needed to test the hypotheses of genetic benefit and convenience polyandry.
Acknowledgments We thank the anonymous referees for valuable comments on the manuscript. This study was supported by a grantin-aid for Scientific Research (KAKENHI 16370013, 16657009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References Arnqvist, G. 1989. Multiple mating in a water strider: mutual benefits or intersexual conflict? Animal Behaviour, 38, 749–756. Arnqvist, G. 1997. The evolution of water strider mating systems: causes and consequences of sexual conflicts. In: The Evolution of Mating Systems in Insects and Arachnids (Ed. by J. C. Choe & B. J. Crespi), pp. 146–163. Cambridge: Cambridge University Press. Arnqvist, G. & Nilsson, T. 2000. The evolution of polyandry: multiple mating and female fitness in insects. Animal Behaviour, 60, 145–164. Arnqvist, G. & Rowe, L. 2002. Antagonistic coevolution between the sexes in a group of insects. Nature, 415, 787–789. Bateman, A. J. 1948. Intra-sexual selection in Drosophila. Heredity, 2, 349–368.
¨ m, J., Wiklund, C. & Kaitala, A. 2002. Natural variation in Bergstro female mating frequency in a polyandrous butterfly: effects of size and age. Animal Behaviour, 64, 49–54. Birkhead, T. R. 2000. Promiscuity: An Evolutionary History of Sperm Competition and Sexual Conflict. London: Faber & Faber. Birkhead, T. R. & Møller, A. P. 1998. Sperm Competition and Sexual Selection. San Diego: Academic Press. Chapman, T., Liddle, L. F., Kalb, J. M., Wolfner, M. F. & Partridge, L. 1995. Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature, 373, 241–244. Chapman, T., Arnqvist, G., Bangham, J. & Rowe, L. 2003. Sexual conflict. Trends in Ecology and Evolution, 18, 41–47. Clutton-Brock, T. H. & Parker, G. A. 1995. Sexual coercion in animal societies. Animal Behaviour, 49, 1345–1365. Crudgington, H. S. & Siva-Jothy, M. T. 2000. Genital damage, kicking and early death. Nature, 407, 855–856. Eady, P. E. 1991. Sperm competition in Callosobruchus maculatus (Coleoptera: Bruchidae): a comparison of two methods used to estimate paternity. Ecological Entomology, 16, 45–53. Fedorka, K. M. & Mousseau, T. A. 2002. Material and genetic benefits of female multiple mating and polyandry. Animal Behaviour, 64, 361–367. Fox, C. W. 1993. Multiple mating, lifetime fecundity and female mortality of the bruchid beetle, Callosobruchus maculatus (Coleoptera: Bruchidae). Functional Ecology, 7, 203–208. Harano, T. & Miyatake, T. 2005. Heritable variation in polyandry in Callosobruchus chinensis. Animal Behaviour, 70, 299–304. Holland, B. & Rice, W. R. 1998. Chase-away sexual selection: antagonistic seduction versus resistance. Evolution, 52, 1–7. Hurst, G. D. D., Sharpe, R. G., Broomfield, A. H., Walker, L. E., Majerus, T. M. O., Zakharov, I. A. & Majerus, M. E. N. 1995. Sexually transmitted disease in a promiscuous insect, Adalia bipunctata. Ecological Entomology, 20, 230–236. Mbata, G. N., Shu, S. & Ramaswamy, S. B. 1997. Rhythmicity of mating and oviposition in Callosobruchus subinnotatus (Pic) (Coleoptera: Bruchidae). Journal of Insect Behavior, 10, 409–423. Miyatake, T. & Matsumura, F. 2004. Intra-specific variation in female remating in Callosobruchus chinensis and C. maculatus. Journal of Insect Physiology, 50, 403–408. Parker, G. A. 1979. Sexual selection and sexual conflict. In: Sexual Selection and Reproductive Competition in Insects (Ed. by M. S. Blum & N. A. Blum), pp. 123–166. New York: Academic Press. Partridge, L. & Hurst, L. D. 1998. Sex and conflict. Science, 281, 2003–2008. Pizzari, T. & Snook, R. R. 2003. Perspective: sexual conflict and sexual selection: chasing away paradigm shifts. Evolution, 57, 1223–1236. Ridley, M. 1988. Mating frequency and fecundity in insects. Biological Review, 63, 509–549. Rowe, L. & Arnqvist, G. 2002. Sexually antagonistic coevolution in a mating system: combining experimental and comparative approaches to address evolutionary processes. Evolution, 56, 754– 767. SPSS Inc. 2001. SPSS for Windows Version 11.0. Chicago: SPSS Inc. Thornhill, R. & Alcock, J. 1983. The Evolution of Insect Mating Systems. Cambridge, Massachusetts: Harvard University Press. Torres-Vila, L. M., Stockel, J. & Rodrı´guez-Molina, M. C. 1997. Physiological factors regulating polyandry in Lobesia botrana (Lepidoptera: Tortricidae). Physiological Entomology, 22, 387–393. Torres-Vila, L. M., Rodrı´guez-Molina, M. C. & Jennions, M. D. 2004. Polyandry and fecundity in the Lepidoptera: can methodological and conceptual approaches bias outcomes? Behavioral Ecology and Sociobiology, 55, 315–324. Umeya, K. & Shimizu, K. 1968. Studies on the comparative ecology of bean weevils. III. Effect of feeding on the life span and oviposition
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of the adult of three species of bean weevils. Research Bulletin of the Plant Protection Service Japan, 5, 39–49 [In Japanese.]. Wilson, N., Tufton, T. J. & Eady, P. E. 1999. The effect of single, double and triple matings on the lifetime fecundity of Callosobruchus analis and Callosobruchus maculatus (Colepotera: Bruchidae). Journal of Insect Behavior, 12, 295–306. Yanagi, S. & Miyatake, T. 2002. Effects of maternal age on reproductive traits and fitness components of the offspring in the
bruchid beetle, Callosobruchus chinensis (Coleoptera: Bruchidae). Physiological Entomology, 27, 261–266. Yanagi, S. & Miyatake, T. 2003. Costs of mating and egg production in female Callosobruchus chinensis. Journal of Insect Physiology, 49, 823–827. Yasui, Y. 1998. The ‘genetic benefits’ of female multiple mating reconsidered. Trends in Ecology and Evolution, 13, 246– 250.
Direct effects of polyandry on female fitness in Callosobruchus chinensis TOMOHIR O HA RANO* , Y U KI O YA SU I † & TA K AH IS A MI YA TA KE*
*Laboratory of Evolutionary Ecology, Graduate School of Environmental Science, Okayama University yLaboratory of Entomology, Faculty of Agriculture, Kagawa University (Received 13 December 2004; initial acceptance 18 February 2005; final acceptance 1 May 2005; published online 17 February 2006; MS. number: 8389)
In many insect species with high levels of polyandry, females benefit directly from remating. The effects of remating on female fitness have generally been examined by comparing the fitness of females in multiplemating and single-mating treatments. In this standard approach, females in the multiple-mating group that refuse to remate are often excluded from the analysis. We investigated the effects of remating in the adzuki bean beetle, Callosobruchus chinensis, a species with low levels of polyandry. Females that refused to remate when given an opportunity to do so were less fecund and shorter lived than females that accepted remating. In this case, excluding females that refuse to remate from the multiple-mating treatment biases the composition of the two treatment populations, and is thus problematic. When we included such females, we found no difference in fecundity, fertility and longevity between females given an opportunity to remate and those that were not. In addition, when we compared females that were allowed to complete remating naturally and those whose remating was interrupted before sperm transfer we found significantly negative effects of female remating on fecundity, suggesting that remating reduces the fitness of polyandrous females in C. chinensis, which is inconsistent with many studies on polyandrous species. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Females of the majority of animal species are polyandrous (Thornhill & Alcock 1983; Ridley 1988; Birkhead & Møller 1998; Birkhead 2000). For males, reproductive success generally increases with more mates, whereas for females it is determined by the number of offspring produced (Bateman 1948). Therefore, the fitness advantages for males of multiple mating are easily understood, but they are less clear for females. Multiple mating may decrease female fitness because mating involves various costs to females: general time and energy expenditures (Thornhill & Alcock 1983), increased risk of predation (Arnqvist 1989), risk of pathogen transmission (Hurst et al. 1995), physical injury (Crudgington & Siva-Jothy 2000) and toxic effects of substances in male ejaculates (Chapman et al. 1995). On the other hand, multiple mating may increase female fitness through benefits such as replenishing sperm supplies, stimulating egg production and offering materials or resources that contribute to female survival
Correspondence: T. Miyatake, Laboratory of Evolutionary Ecology, Graduate School of Environmental Science, Okayama University, Okayama 700-8530, Japan (email: [email protected]). Y. Yasui is at the Laboratory of Entomology, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan. 0003–3472/05/$30.00/0
and/or reproduction (Thornhill & Alcock 1983; Arnqvist & Nilsson 2000). A review (Ridley 1988) and a meta-analysis (Arnqvist & Nilsson 2000) of studies on the direct effects of multiple mating on female fitness found that in many insect species females benefit from polyandry in terms of increased lifetime offspring production. Arnqvist & Nilsson’s metaanalysis, however, was biased towards species with high levels of polyandry, because most research addressing the direct benefits of polyandry has been carried out on polyandrous species (Torres-Vila et al. 2004). The frequency of polyandrous females varies considerably between species (Ridley 1988; Torres-Vila et al. 2004), so polyandry tends to evolve in species in which females benefit directly from multiple mating, an analysis focused on polyandrous species will bias the outcome towards the positive effects of polyandry on female fitness. Arguments about the adaptive significance of female multiple mating should not ignore species with infrequent polyandry. Increased reproductive output of females through polyandry is not apparent in species where it occurs at low levels (Ridley 1988; Torres-Vila et al. 2004). A marked variation in female remating frequency has been found among strains of the adzuki bean beetle, Callosobruchus chinensis (Miyatake & Matsumura 2004;
539 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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Harano & Miyatake 2005). In this species, almost all females are monandrous in strains with low remating frequencies, whereas in strains with high remating frequencies both polyandrous and monandrous females exist and neither mating pattern is predominant (Miyatake & Matsumura 2004; Harano & Miyatake 2005). Mating itself reduces female survival (Yanagi & Miyatake 2003), but we have no information about the effects of remating on female fitness in this beetle. We therefore investigated the direct effects of polyandry on female fitness in C. chinensis. The standard approach to examine the effects of remating on female fitness is to allow females assigned to one treatment to mate multiply and females assigned to
another treatment to mate singly, and then compare fitness components such as fecundity, fertility and longevity between the two treatments (e.g. Fox 1993; Wilson et al. 1999; Fedorka & Mousseau 2002). When this experimental method is used, some females assigned to the multiplemating treatment may refuse to remate. When the multiple-mating treatment comprises females that refuse to remate, that is, monandrous females and females that accept remating, that is, polyandrous females, the singlemating treatment is expected to comprise both females that would refuse to remate and females that would accept remating if provided with opportunities for remating (Fig. 1a). Females in the multiple-mating treatment that refuse to remate when given the opportunity are usually
(a) Composition of female groups
Treatments
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Figure 1. (a) Outline of the standard approach to investigate the effects of remating on female fitness. The multiple-mating treatment includes females that refuse to remate, that is, monandrous females, and females that accept remating, that is, polyandrous females in a certain proportion. The single-mating treatment is then expected to comprise females that would refuse to remate (‘monandrous’) and females that would accept remating (‘polyandrous’) if they had an opportunity to remate, in the same proportion. It is impossible to distinguish between these females, however, because they do not receive an opportunity to remate. The difference in fitness between females mated multiply and females mated singly has often been tested by (b) comparing polyandrous females in the multiple-mating treatment with all females in the single-mating treatment, but if the monandrous and polyandrous females possess different traits, excluding monandrous females from the multiple-mating treatment biases the composition of females in the two treatments. In the present study, we compared (c) females that refused to remate and females that accepted remating in the multiple-mating treatment, (d) all females in the multiple-mating and single-mating treatments and (e) females allowed to complete remating naturally (equivalent to polyandrous females in the multiple-mating treatment) and females whose remating was interrupted (equivalent to polyandrous females in the single-mating treatment).
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
excluded from the analysis (Torres-Vila et al. 2004). In this case, the fitness of polyandrous females in the multiplemating treatment is compared to that of all females in the single-mating treatment (Fig. 1b). The comparison, however, may involve a methodological problem. If female condition, which results from body size or nutritional status or the effects of costs derived from a previous mating or genetic attributes and so on, vary between females that refuse to remate and females that accept remating then, independent of remating effects, fitness will differ between the two groups. If this is the case, excluding females that refuse to remate from the multiple-mating treatment biases the type of females in both treatments and thus will prevent fair comparison between them (Torres-Vila et al. 2004). In the present study, we considered this problem and designed experiments to examine the direct effects of remating on female fitness. In the first experiment, we compared traits of females that refused to remate and females that accepted remating when they had opportunities to remate (Fig. 1c) to test an association between female receptivity to remating (refuse or accept) and female condition. We also, compared the fitness components of females that had opportunities to remate, whether or not they accepted, with those of females that had no opportunities to remate to examine the direct effects of remating on female fitness in unbiased compositions of the two treatments (Fig. 1d). The second experiment was designed to examine the effects of remating on fitness only in polyandrous females. In one treatment, female remating was completed naturally and in another female remating was immediately interrupted. Females in the former treatment correspond to polyandrous females in the multiple-mating treatment. On the other hand, females in the latter treatment would show no effects of remating even though they had accepted it, and thus correspond to polyandrous females in the singlemating treatment. Therefore we expected to detect the net effects of remating by comparing fitness components of females in the two treatments (Fig. 1e). METHODS
Insects and Culture We used a strain of C. chinensis, referred to as the isC strain, which was established with about 200 adults collected from mung beans, Vigna radiata, in Ishigaki City, Ishigaki Island, Okinawa, Japan, in 1997 (Yanagi & Miyatake 2003). The isC strain has the highest known frequency of female remating among C. chinensis populations (Harano & Miyatake 2005) and thus is appropriate for this study because the effects of remating are difficult to examine in strains in which few females remate. The beetles were reared from eggs laid by parents collected randomly from a stock culture. Virgin beetles were randomly collected and kept in single-sex groups of up to 10 adults in plastic cups (2.8 cm high, 7 cm in diameter). All rearing and subsequent experiments were conducted in a chamber maintained at 25 C and 50% relative humidity under a photoperiod cycle of 14:10 h light:dark. Adult beetles were not given food and water, except as stated below.
Experiment 1: Remating Opportunities First, we compared traits of females that refused to remate and females that accepted remating (Fig. 1c) to determine whether receptivity to remating (refuse or accept) was associated with female condition. We then compared fitness components of females that had opportunities to remate, regardless of whether they accepted (multiplemating treatment) with those that did not have opportunities to remate (single-mating treatment; Fig. 1d) to test the effects of the presence of opportunities to remate on female fitness. This experiment was conducted under both no-feeding and feeding conditions, because adults of C. chinensis can reproduce without feeding, but they greatly increase their fecundity and longevity by feeding (Umeya & Shimizu 1968). Under the feeding condition, females were given water and adult food (1:2 yeast extract: sugar). All virgin males used as mates for females were given water and adult food and were aged 2–5 days. On day 1 after it emerged, we placed one virgin female in a glass vial (4.4 cm high, 1.7 cm in diameter) with one virgin male, and observed mating for 1 h. After copulation, the male was removed, and the female was maintained separately in a plastic cup containing 10 adzuki beans, Vigna angularis, as the oviposition substrate. We randomly assigned the mated females to either the multiple-mating treatment or the single-mating treatment. In the multiple-mating treatment, the mated females had contact with males on days 1 and 3 after the first mating under both no-feeding and feeding conditions, and on day 5 after the first mating under the feeding condition because of their prolonged life span. The mated female and a virgin male were placed in a glass vial and observed each day until either the female remated once or 1 h had passed. In these observations, remated females had no opportunity to remate further whereas females that did not remate received another opportunity to remate. In the single-mating treatment, mated females were not allowed contact with males after the first mating. Beans as oviposition substrate were replaced with 10 fresh ones every day under the no-feeding condition, and with 10 every day for the first 5 days, five every day for the next 5 days and three every day for the remaining days under the feeding condition because egg production declines with maternal age (Yanagi & Miyatake 2002). We counted the eggs produced and hatched. A few females (2/77 in the multiple-mating treatment and 1/39 in the single-mating treatment under the no-feeding condition; 0/55 in the multiple-mating treatment and 1/31 in the single-mating treatment under the feeding condition) that did not lay viable eggs after the first mating were excluded from the analysis, because they had probably failed to be inseminated by the first male. All females of both treatments were reared until they died under the no-feeding condition or until 40 days after emerging under the feeding condition. Under the feeding condition, therefore, we recorded the numbers of eggs produced and hatched for 40 days after the female emerged. Longevity of females was also recorded until 40 days after emergence. In C. chinensis, egg size and thus hatching success decrease with maternal age (Yanagi & Miyatake 2002), and therefore
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the percentage of eggs produced that hatch depends on the temporal pattern of oviposition over the female’s life span. To assess fertility, we therefore calculated the percentage of eggs that hatched on each day. In this calculation, we pooled the number of eggs produced after 4 days from the female’s first mating under the no-feeding condition and after 6 days from the female’s first mating under the feeding condition because the number of eggs was small. As a measure of body size we recorded elytra length after the females died.
Females that refused versus females that accepted remating To test an association between receptivity to remating and female condition, we compared body size, number of eggs produced, number of eggs hatched, percentage of eggs hatched and longevity of females that refused to remate (N ¼ 33 for the no-feeding condition; N ¼ 11 for the feeding condition) and females that accepted remating (N ¼ 42 for the no-feeding condition; N ¼ 44 for the feeding condition) in the multiple-mating treatment (Fig. 1c). The female’s decision to remate is, in part, influenced by male traits in this beetle (T. Harano & T. Miyatake, unpublished data). In the present experiment, we randomly assigned males to females, but if male courtship behaviour changes according to female phenotype, then the change in male behaviour may influence female receptivity to remating. When a male C. chinensis encounters a female, he generally attempts to mate with her (Yanagi & Miyatake 2003). In our experiment, we are certain that a male encountered a female many times at each opportunity to remate and thus the difference in female receptivity to remating did not depend on whether a male attempted to mate with a female, although the intensity of male mating attempts may differ between female phenotypes.
Multiple- versus single-mating treatments To examine the direct effects of remating on female fitness in unbiased compositions of the two treatments, we compared the number of eggs produced, the number of eggs hatched, the percentage of eggs hatched and longevity of females in the multiple-mating treatment (N ¼ 75 for the no-feeding condition; N ¼ 55 for the feeding condition) and females in the single-mating treatment (N ¼ 38 for the no-feeding condition; N ¼ 30 for the feeding condition; Fig. 1d). The lack of difference in the elytra length of females between the multiple-mating and single-mating treatments under the no-feeding condition (multiple-mating treatment: X SE ¼ 1:89 0:01 cm; single-mating treatment: 1.88 0.01 cm; ANOVA: F1,111 ¼ 0.38, P ¼ 0.538) and under the feeding condition (multiple-mating treatment: 1.88 0.01 cm; single-mating treatment: 1.88 0.01 cm; ANOVA: F1,83 ¼ 0.06, P ¼ 0.809) confirmed the unbiased compositions of the two treatment groups, with regard to body size.
Experiment 2: Remating Interruption We established one treatment in which females were allowed to complete remating naturally (control) and
another in which female remating was immediately interrupted (interruption treatment). By excluding monandrous females, we were thus able to examine the direct effects of remating on female fitness only in those females that were receptive to remating. Before this experiment, we observed that 10 virgin females whose copulating was interrupted as soon as they mated (within 5 s) did not lay viable eggs and thus confirmed that the interruption completely prevented sperm transfer. Furthermore, sperm are not transferred to the female spermatheca within 20 s of copulating in the isC strain of C. chinensis, which we used in this study (T. Yamane & T. Miyatake, unpublished data). All females and virgin males received water and adult food and the latter were 2–5 days old. On day 1 after it emerged we placed one virgin female in a glass vial with one virgin male, and observed mating for 1 h. After copulation, the male was removed, and the female was maintained individually in a plastic cup containing 10 adzuki beans as an oviposition substrate. We randomly assigned the mated females to either the control or the interruption treatment. On day 3 after the female had mated once, we placed one mated female and one virgin male in a plastic petri dish (1.5 cm high, 9.1 cm in diameter) and observed them for 1 h. In the interruption treatment, copulating pairs were separated with two very soft writing brushes within 5 s of the female initiating remating. Females that did not remate in 1 h of observation were excluded from both treatments. Beans as an oviposition substrate were replaced with 10 fresh ones every day for the first 5 days, five every day for the next 5 days, and five every 4–7 days for the remaining days. We counted the eggs produced and hatched over 40 days after emergence. A few females (0/32 in the control treatment and 1/32 in the interruption treatment) that did not lay viable eggs after the first mating were excluded from the analysis because they had probably not been inseminated by the first male. To assess fertility, we calculated the percentage of eggs produced that hatched on each day. In this calculation, we pooled the small number of eggs produced after 6 days from the female’s first mating. Female elytra length was measured after death. We compared the number of eggs produced, the number of eggs hatched and the percentage of eggs hatched of females in the control (N ¼ 31) and interruption treatments (N ¼ 32; Fig. 1e). The lack of difference in the elytra length of females between the two treatments (control: X SE ¼ 1:87 0:01 cm; interruption treatment: 1.87 0.01 cm; ANOVA: F1,61 ¼ 0.06, P ¼ 0.814) confirmed the unbiased compositions of the two treatments, with regard to body size.
Statistics Elytra length, the number of eggs produced, the number of eggs hatched and longevity were log transformed and the percentage of eggs hatched was arcsine transformed to be approximated to a normal distribution. We used an analysis of variance (ANOVA) to compare elytra length and the percentage of eggs hatched between female groups. Fecundity and longevity covary with body size in a related species, C. maculatus (Fox 1993), so we used an
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
analysis of covariance (ANCOVA), with elytra length as a covariate to compare the number of eggs produced, the number of eggs hatched and longevity of female groups if the ANCOVA detected a significant effect of elytra length on the traits. If the ANCOVA detected no significant effects of elytra length on the traits we used an ANOVA to compare groups. The log-rank test was used to compare survival of the female groups. For statistical analyses we used SPSS (SPSS Inc. 2001).
RESULTS
Experiment 1: Remating Opportunities Females that refused versus females that accepted remating
Erlytra length (mm)
Under the no-feeding condition, females that accepted remating had significantly longer elytra than females that refused to remate when females had opportunities to remate (F1,73 ¼ 9.94, P ¼ 0.002; Fig. 2a). An ANCOVA, with elytra length as a covariate, detected significant effects of elytra length on the numbers of eggs produced (F1,72 ¼ 19.26, P < 0.001) and hatched (F1,72 ¼ 22.68, P < 0.001). More eggs were produced by females that
No. of eggs
Feeding condition
No-feeding condition 1.94
(a)
**
1.92
1.94 1.9
1.88
1.88
1.86
1.86
1.84
1.84 (b) **
60
160 **
80
20
40 Produced
0
Hatched
100
(c)
100
80
80
60
60
40
40
20
20
0
1
*
120
40
0
(d)
1.92
1.9
80
% Eggs hatched
accepted remating (ANCOVA: F1,72 ¼ 8.59, P ¼ 0.005; Fig. 2b), but the number of eggs hatched did not differ significantly between groups (ANCOVA: F1,72 ¼ 1.97, P ¼ 0.164; Fig. 2b). There were no significant differences in the percentage of eggs hatched on any day between groups (day 1: F1,73 ¼ 0.87, P ¼ 0.353; day 2: F1,73 ¼ 2.05, P ¼ 0.157; day 3: F1,67 ¼ 0.70, P ¼ 0.405; after 4 days: F1,57 ¼ 0.10, P ¼ 0.751; Fig. 2c). Females that accepted remating had higher survival than females that refused to remate (log-rank test: P ¼ 0.005; Fig. 3a), and an ANCOVA detected a significant effect of elytra length on longevity (F1,72 ¼ 6.02, P ¼ 0.017) and a significant difference in longevity between groups (females that accepted remating: X SE ¼ 7:9 0:2 days; females that refused to remate: 7.1 0.1 days; F1,72 ¼ 4.73, P ¼ 0.033). Under the feeding condition, there was no significant difference in elyta length between the female groups (F1,53 ¼ 0.814, P ¼ 0.371; Fig. 2d). An ANCOVA detected no significant effects of elytra length on the numbers of eggs produced (F1,52 ¼ 2.11, P ¼ 0.153) and hatched (F1,52 ¼ 2.05, P ¼ 0.159). More eggs were produced (ANOVA: F1,53 ¼ 8.43, P ¼ 0.005) and more hatched (ANOVA: F1,53 ¼ 6.77, P ¼ 0.012) by females that accepted remating (Fig. 2e). The percentage of eggs hatched on day 1 was significantly lower for females that accepted
2
3
Produced
Hatched (f)
*
0 4+ 1 2 3 Days after female’s first mating
(e)
4
5
6+
Figure 2. Comparison of body size and fitness components (X þ SE) for females that accepted remating (-) and females that refused to remate (,) when given opportunities to remate. (a) Elytra length, (b) number of eggs produced and hatched and (c) percentage of eggs hatched under the no-feeding condition. N ¼ 42 females that accepted remating and 33 that did not. (d) Elytra length, (e) number of eggs produced and hatched and (f) percentage of eggs hatched under the feeding condition. N ¼ 44 females that accepted remating and 11 that did not. *P < 0.05; **P < 0.01 (a, c, d and f) ANOVA; (b) ANCOVA, with elytra length as a covariate; (e) ANOVA, because of no significant effects of elytra length by ANCOVA, with elytra length as a covariate.
543
ANIMAL BEHAVIOUR, 71, 3
1
(a)
0.8 0.6 0.4 Cumulative survival rate
544
0.2 0 0 1
1
2
3
4
5
6
7
8
9
10 11 12
(b)
0.8 0.6 0.4 0.2 0 0
5
10
15
20
25
30
35
40
Days after female emerged Figure 3. Survival of females that accepted remating (d) and females that refused to remate (- - - - -) when given opportunities to remate under (a) the no-feeding condition and (b) the feeding condition.
remating (F1,53 ¼ 4.76, P ¼ 0.034; Fig. 2f), but the difference was extremely small. There were no significant differences in the percentage of eggs hatched on any other day between groups (day 2: F1,53 ¼ 1.15, P ¼ 0.289; day 3: F1,50 ¼ 0.90, P ¼ 0.347; day 4: F1,43 ¼ 0.37, P ¼ 0.544; day 5: F1,41 ¼ 0.02, P ¼ 0.885; after 6 days: F1,39 ¼ 0.10, P ¼ 0.753; Fig. 2f). Thus, we found no clear evidence for a difference in the percentage of eggs hatched between females that accepted remating and those that did not. Female that accepted remating had higher survival (logrank test: P ¼ 0.001; Fig. 3b). An ANCOVA detected no significant effect of elytra length on longevity (F1,52 ¼ 0.46, P ¼ 0.503). Females that accepted remating lived longer (median 20 days) than those that did not (12 days; ANOVA: F1,53 ¼ 9.16, P ¼ 0.004).
Multiple- versus single-mating treatments Under the no-feeding condition, an ANCOVA, with elytra length as a covariate, detected significant effects of elytra length on the numbers of eggs produced (F1,110 ¼ 60.37, P < 0.001) and hatched (F1,110 ¼ 62.71, P < 0.001), and there were no significant differences in the numbers of eggs produced (F1,110 ¼ 0.11, P ¼ 0.742) and hatched (F1,110 ¼ 0.21, P ¼ 0.650) between the multiple- and single-mating treatments (Fig. 4a). There were no
significant differences in the percentage of eggs hatched on any day between the two treatments (day 1: F1,111 ¼ 0.66, P ¼ 0.420; day 2: F1,111 ¼ 1.54, P ¼ 0.217; day 3: F1,103 ¼ 0.38, P ¼ 0.537; after 4 days: F1,83 ¼ 0.10, P ¼ 0.752; Fig. 4b). There was also no significant difference in female survival between the two treatments (log-rank test: P ¼ 0.553; Fig. 5a); an ANCOVA detected a significant effect of elytra length on female longevity (F1,110 ¼ 16.38, P < 0.001) but no significant difference in female longevity between the two treatments (multiple-mating treatment: X SE ¼ 7:5 0:1 days; singlemating treatment: 7.6 0.2 days; F1,110 ¼ 0.33, P ¼ 0.564). Under the feeding condition, the results were similar to those under the no-feeding condition. An ANCOVA detected significant effects of elytra length on the numbers of eggs produced (F1,82 ¼ 6.67, P ¼ 0.012) and hatched (F1,82 ¼ 7.40, P ¼ 0.008), but no significant difference in the numbers of eggs produced (F1,82 ¼ 0.27, P ¼ 0.607) and hatched (F1,82 ¼ 0.07, P ¼ 0.794) between the two treatments (Fig. 4c). There were no significant differences in the percentage of eggs hatched on any day between the two treatments (day 1: F1,83 ¼ 0.70, P ¼ 0.404; day 2: F1,83 ¼ 0.74, P ¼ 0.393; day 3: F1,76 ¼ 0.34, P ¼ 0.563; day 4: F1,68 ¼ 1.19, P ¼ 0.279; day 5: F1,63 ¼ 0.09, P ¼ 0.769; after 6 days: F1,62 ¼ 2.12, P ¼ 0.151; Fig. 4d). Female survival did not differ significantly between treatments (log-rank test: P ¼ 0.244; Fig. 5b). An ANCOVA detected no significant effect of elytra length on female longevity (F1,82 ¼ 0.06, P ¼ 0.809), and an ANOVA detected no significant difference in female longevity between the two treatments (median: multiple-mating treatment: 14 days; single-mating treatment: 25 days; F1,83 ¼ 1.22, P ¼ 0.273).
Experiment 2: Remating Interruption An ANCOVA, with elytra length as a covariate, detected significant effects of elytra length on the numbers of eggs produced (F1,60 ¼ 9.94, P ¼ 0.003) and hatched (F1,60 ¼ 7.85, P ¼ 0.007). The ANCOVA showed that females whose remating was interrupted produced more eggs (F1,60 ¼ 6.26, P ¼ 0.015; Fig. 6a) and hatched more eggs (F1,60 ¼ 6.90, P ¼ 0.011; Fig. 6a) than females allowed to complete remating naturally. There were no significant differences in the percentage of eggs hatched on any day before 5 days between the two treatments (day 1: F1,61 ¼ 0.51, P ¼ 0.477; day 2: F1,61 ¼ 0.06, P ¼ 0.807; day 3: F1,61 ¼ 0.57, P ¼ 0.452; day 4: F1,60 ¼ 0.06, P ¼ 0.804; day 5: F1,60 ¼ 1.97, P ¼ 0.165; Fig. 6b). The hatching success of eggs produced after 6 days was significantly higher in the interruption treatment than in the control (F1,60 ¼ 4.94, P ¼ 0.030; Fig. 6b).
DISCUSSION In the present study, we first compared traits of the C. chinensis females that refused to remate and the females that accepted remating when they had opportunities to remate
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
No-feeding condition
Feeding condition 160
80 No. of Eggs
(a)
(c)
60
120
40
80
20
40
0
Produced
0
Hatched
Produced
Hatched
100
100 % Eggs hatched
(b)
(d)
80
80
60
60
40
40
20
20
0
1
2
3
4+
0
1
2
3
4
5
6+
Days after female’s first mating Figure 4. Comparison of fitness components (X SE) of females given opportunities to remate (multiple-mating treatment; -) and females deprived of opportunities to remate (single-mating treatment; ,). (a) Number of eggs produced and hatched and (b) percentage of eggs hatched under the no-feeding condition (N ¼ 75 for multiple-mating treatment, N ¼ 38 for single-mating treatment); (c) number of eggs produced and hatched and (d) percentage of eggs hatched under the feeding condition (N ¼ 55 for multiple-mating treatment, N ¼ 30 for singlemating treatment). (a and c) ANCOVA, with elytra length as a covariate; (b and d) ANOVA.
(Fig. 1c). The females that refused to remate were smaller, less fecund and shorter lived than females that accepted remating (Figs 2, 3). These differences are probably attributable to differences in their viability and reproductive capability rather than to the effects of remating. In other words, larger, more viable and more fecund females tended to accept remating. The tendency for larger females to remate at higher frequencies has been reported in other insects (Torres-Vila et al. 1997; Bergstro¨m et al. 2002). A physiological explanation for the tendency may be that larger and more fecund females can accept additional sperm in their larger spermatheca and use more sperm for egg production at higher rates (Torres-Vila et al. 1997). Another explanation may be that larger and more viable females accept remating because they are less sensitive to the costs of mating, whereas smaller and less viable females refuse to remate because they are more sensitive to these costs. On the other hand, the greater propensity of larger and more fecund females to remate may be attributable to differential courtship behaviour of males. If males are able to perceive the reproductive quality of females, they may attempt to mate with larger and more fecund females more ardently than with smaller and less fecund females. As mentioned above, we found differences in fitness components between polyandrous and monandrous females. This indicates that exclusion of females that refuse to remate results in a nonrandom sample of larger, more viable and more fecund females in the multiple-mating treatment. Comparing the fitness of polyandrous females in the multiple-mating treatment and all females in the
single-mating treatment is thus unfair and problematic (Fig. 1b). When we compared fitness components of all females that had opportunities to remate, regardless of whether they accepted, and all females deprived of opportunities to remate (Fig. 1d), we found no differences between the two groups (Figs 4, 5). However, this comparison may underestimate the effects of remating on fitness in polyandrous females because the group of females provided with opportunities to remate includes not only the females that remated but also those that mated only once. Finally, to examine the direct effects of remating on female fitness only in the females that were receptive to remating, we compared fitness components of females that were allowed to complete remating naturally and those whose remating was interrupted immediately, before sperm was transferred (Fig. 1e). The females whose remating was interrupted had significantly higher fecundity than the females that were allowed to complete remating (Fig. 6a). In experiment 2 we found a significant difference in fecundity between females in the multiple-mating treatment that accepted remating and those in the singlemating treatment that would have remated if they had an opportunity to remate (Fig. 6a). In experiment 1, females in the multiple-mating treatment that refused to remate and the females in the single-mating treatment that would have refused to remate even if they had an opportunity to remate are expected to have similar fitness components. It seems that including both groups in experiment 1 prevented us from finding the significant difference in fecundity between females in the multiple-mating and the
545
ANIMAL BEHAVIOUR, 71, 3
200
1
(a)
(a)
* 160 No. of eggs
0.8 0.6 0.4 0.2 0 0
* 120 80 40
1
2
3
4
5
6
7
8
9
0
10 11 12
Produced
Hatched
100
1 (b)
(b)
0.8
80 % Eggs hatched
Cumulative survival rate
546
0.6 0.4 0.2 0 0
* 60
40
20 5
10
15
20
25
30
35
40
Days after female emerged Figure 5. Survival of females given opportunities to remate (d) and that were deprived of opportunities to remate (- - - - -) under (a) the no-feeding condition and (b) the feeding condition.
single-mating treatments. Our results thus indicate that remating directly reduces fitness of polyandrous females in C. chinenis. Torres-Vila et al. (2004) indicated that an analysis that excludes monandrous females does not examine the direct effects of remating for the average female. This indication applies to experiment 2 in our study, that is, the comparison between females that were allowed to complete remating naturally and females whose remating was interrupted (Fig. 1e), because the comparison excluded females that were not receptive to remating. We cannot obtain experimental evidence of the direct effects of remating on the fitness of females that were not receptive to remating, although we presume that such females would not benefit directly from remating because they more strongly resist remating than do polyandrous females. We found no positive effects of female remating on the percentage of eggs hatched, suggesting that females of C. chinensis acquire sufficient sperm to fertilize their eggs in a single mating. Female remating had no positive effects on fecundity, suggesting that remating in C. chinensis does not stimulate egg production or provide materials that contribute to increased female fecundity and/or longevity. On the basis of their meta-analysis of 122 studies addressing the direct benefit of multiple mating on female fitness, Arnqvist & Nilsson (2000) have argued that in many insect species females benefit directly from multiple
0
1
2 3 4 5 Days after female’s first mating
6+
Figure 6. Comparison of fitness components (X SE) of females that completed remating (-) and females whose remating was interrupted (,). (a) Number of eggs produced and hatched, (b) percentage of eggs hatched (N ¼ 31 for control, N ¼ 32 for interruption treatment). *P < 0.05; (a) ANCOVA, with elytra length as a covariate; (b) ANOVA.
mating in terms of increased fecundity and that the evolution of polyandry can be understood in terms of the direct benefits. Our finding in C. chinensis is inconsistent with this argument. Arnqvist & Nilsson’s meta-analysis is biased towards polyandrous species (Torres-Vila et al. 2004). In three species related to C. chinensis, C. maculatus (Fox 1993; Wilson et al. 1999), C. analis (Wilson et al. 1999) and C. subinnotatus (Mbata et al. 1997), which were included in Arnqvist & Nilsson’s meta-analysis, fecundity is increased by female remating. In C. maculatus 85% of females remated (Eady 1991), and in C. analis 66% (Wilson et al. 1999). Torres-Vila et al. (2004) have classified species as monandrous if less than 40% of females are polyandrous and as polyandrous if more than 40% are polyandrous. According to their classification, C. maculatus and C. analis are classified as polyandrous species; C. subinnotatus is probably polyandrous, because in one study (Mbata et al. 1997) females mated two, three or four times at intervals of 24 h. In C. chinensis, the frequency of polyandrous females differs between strains (range 6–40%, Harano & Miyatake 2005); C. chinensis is thus classified as a monandrous species according to Torres-Vila et al.’s classification. Therefore, in Callosobruchus, the three polyandrous species show direct benefits
HARANO ET AL.: DIRECT EFFECTS OF POLYANDRY
of remating, whereas a monandrous species, C. chinensis, did not. This suggests a positive association between the direct benefit of polyandry on female fitness and the frequency of female remating across species. Our study raises the question why some females of C. chinensis remate in spite of suffering disadvantages. Besides the hypothesis that polyandry directly increases female fitness, there is the genetic (indirect) benefit hypothesis that females may benefit indirectly from polyandry through increased fitness of their offspring with genetic superiority or diversity (Thornhill & Alcock 1983; Yasui 1998). In the absence of genetic benefits that entirely offset disadvantages of remating, females may also be coerced into accepting superfluous mating by persistent males (Clutton-Brock & Parker 1995; Arnqvist 1997). When a male C. chinensis encounters a female, he generally rubs her with his antenna and mounts from the rear, and if the female then flees he often runs after her (Yanagi & Miyatake 2003). This persistent courtship behaviour of males may be costly for females (Yanagi & Miyatake 2003), and thus females may benefit directly from remating by decreasing the costs of male harassment. If this is the case, female remating in C. chinensis can be explained by the convenience polyandry hypothesis that females mate multiply to reduce the costs of sexual harassment (Thornhill & Alcock 1983). The interests of males and females often conflict at mating (Parker 1979; Holland & Rice 1998; Partridge & Hurst 1998; Arnqvist & Rowe 2002; Rowe & Arnqvist 2002; Chapman et al. 2003; Pizzari & Snook 2003). Our finding that remating is harmful to females suggests that there is sexual conflict between reluctant females and persistent males over female remating in C. chinensis. Further investigation is needed to test the hypotheses of genetic benefit and convenience polyandry.
Acknowledgments We thank the anonymous referees for valuable comments on the manuscript. This study was supported by a grantin-aid for Scientific Research (KAKENHI 16370013, 16657009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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