Size Effects on Mating and Egg Production in the Miami Blue Butterfly
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- Trager, M.D. & Daniels, J.C. J Insect Behav (2011) 24: 34. doi:10.1007/s10905-010-9234-8
Body size is often related to reproductive success in insects, but the direction and strength of this relationship differs greatly among systems. We studied the effects of adult size on probability of mating and egg production in the Miami blue butterfly. We found that likelihood of mating was invariant with respect to size. Larger females lived longer, and both size and lifespan positively influenced egg production. However, neither the number of copulations nor the size of male mates had any effect on female fecundity. We discuss these results in the context of butterfly mating systems, larval growth strategies and the possible effects of captive conditions on reproductive behavior.
KeywordsCyclargus thomasi bethunebakerimating systemMiami blue butterflypotential fecundityreproduction
Reproductive success within many arthropod species is positively correlated with body size (Honek 1993). For females, large body size generally indicates high potential fecundity and may increase females’ ability to attract higher-quality males (Blanckenhorn 2000; Tammaru et al. 2002; Gotthard 2008). For males, larger size may confer advantages in male-male contests, higher probability of acceptance by choosy females or greater quantity or quality of sperm or associated nuptial gifts (Iyengar and Eisner 2004; Sutherland et al. 2007). Such size-dependent variation in mate quality may manifest in positive size-biased mate preference, although competition for larger mates within one or both sexes can also result in apparent size-assortative mating (Brown 1990; Harari et al. 1999; Bollache and Cezilly 2004; Bel-Venner et al. 2008). Body size is also correlated with longevity in some systems (e.g., Elgar and Pierce 1988), thereby indirectly influencing fitness because lifespan often positively affects reproductive output (Leather 1988; Bauerfeind and Fischer 2008).
Despite evidence from many systems showing positive effects of body size on various measures of reproduction, including potential fecundity and probability of mating, bigger is not always better (contra Tammaru et al. 2002). In particular, large body size may be associated with costs (e.g., higher predation risk or longer developmental time) that offset apparently strong selective pressure for large phenotypes (Bernays 1997; Blanckenhorn 2000; Gotthard 2000; Berger et al. 2006). For example, in female Pararge aegeria, the longer development time required to attain a large size comes at a cost to fecundity due to the higher predation risk during the prolonged larval stage and limited time for oviposition as an adult due to temperature constraints at the end of the flight season (Gotthard 2004; Berger et al. 2006, 2008; Gotthard et al. 2007). Such life history trade-offs can result in inconsistent relationships between body size and different measures of reproductive performance under different study conditions (e.g., Visser 1994). Additionally, morphological or behavioral characteristics unrelated to size are more important determinants of mating success in some insect taxa (Klingenberg and Spence 1997; Langellotto et al. 2000; Pureswaran and Borden 2003; Kemp 2008). Finally, stochasticity in reproductive opportunities can nullify theoretically optimal growth and reproduction strategies in heterogeneous habitats (e.g., Ellers et al. 2000). Although measuring potential fecundity in controlled laboratory conditions is rarely adequate for assessing full range of fitness effects associated with body size (Leather 1988), investigating these relationships can elucidate the potential advantages and disadvantages of variation in size, with implications for growth strategies during the immature stage and mating behavior in the imaginal stage (Gotthard 2004; Gotthard 2008).
We tested the effects of adult size on multiple components of reproductive performance for both males and females in a captive colony of the Miami blue butterfly (Cyclargus thomasi bethunebakeri). Females of this species pupate at a slightly higher mass than males (Trager and Daniels 2009), but the former appear to allocate relatively more resources to body size since the sexes are similar in wingspan. The relative duration of the larval and pupal stadia differs between sexes, but the total developmental time is similar between males and females and there is no evidence of protandry in this system (Trager and Daniels 2009). Qualitative observations of wild Miami blue butterfly behavior suggest that males exhibit both perching and patrolling mate-searching strategies and commonly chase other males (personal observations). However, preliminary capture-mark-recapture studies found no evidence that they guard well-defined territories (unpublished data). Females may mate more than once, but captive females commonly reject matings by courting males. Eggs are laid singly on new growth of either gray nickerbean (Caesalpinia bonduc) or blackbead (Pithecellobium keyense). The larvae feed on the host plant for the duration of the larval stage; the later instars are commonly tended by ants that consume a sugary solution secreted by the larvae and presumably protect them from predators and parasitoids (Saarinen and Daniels 2006; Trager and Daniels 2009). Miami blue butterflies are currently restricted to small populations in the Florida Keys and is listed by the state of Florida as endangered (Florida Fish and Wildlife Conservation Commission 2003), so any information regarding the factors that contribute to successful mating and population growth could contribute to developing more effective programs for in situ conservation, captive colony propagation and reintroduction.
Based on findings from similar systems, we expected relatively fewer males than females to copulate due to female acceptance of only high-quality males and/or intrasexual competition among males. We tested two alternative hypotheses regarding patterns in size-mediated mate selection in the Miami blue butterfly. First, we tested for size-biased mating with analyses to detect higher probability of mating for smaller-, larger- or average-sized individuals among both males and females. We also tested for size-assortative mating, indicated by copulating pairs being more similar in size to each other than would be expected by chance. Overall, we expected to find selection for large mates among both sexes, resulting in either positive-size biased mating or apparent assortative mating depending on the degree of competition within each sex for high-quality mates (e.g., Brown 1990). We then tested the effects of size and mating history on egg production of female Miami blue butterflies. We expected that larger females would produce more eggs regardless of mate identity. However, male Miami blue butterflies transfer a spermatophore to the female during copulation containing both sperm and nutrients, so we tested if mating history—both number of copulations observed and male size—affected egg production.
We conducted a series of observational studies to assess the effects of adult size on probability of mating and subsequent reproductive performance. We individually marked and measured the forewing chord length of adult Miami blue butterflies that emerged on each of 3 days in a captive colony housed at the Florida Museum of Natural History (Gainesville, Florida, USA). The first trial comprised 150 individuals (83 males, 67 females), the second trial comprised 124 individuals (51 males, 73 females) and the final trial comprised 90 individuals (39 males, 51 females). Although we had no a priori hypotheses for differences among the three trials, we included this factor in the analyses described below. To assess the effects of size on probability of mating, we placed all butterflies in each trial in an outdoor screen flight cage (2 × 2 × 1 m) with ample natural nectar sources and recorded copulations every 30–45 min from 0800 to 1800 h over three consecutive days.
We tested for potential differences in the probability of mating multiple times with a χ2 test on a contingency table containing the frequency of single- and multiple-mated individuals for both sexes. We then conducted a series of tests to detect potential selection of mates based on size. First, we analyzed the effects of forewing chord length on the probability of mating using logistic regression. This model would detect either positive or negative size bias in mating if such patterns were present. We constructed a similar logistic regression model to test if probability of mating was affected by deviation from the mean forewing chord length on (calculated as the absolute value of the standardized difference between the mean and individual forewing chord lengths). If smaller deviation from the mean wing chord length was associated with higher probability of mating, this model would indicate mating preference for average-sized individuals (i.e., perhaps indicating stabilizing selection for size). We included sex and trial as factors in these analyses to account for potential differences between males and female and among the three trials. We initially included the interaction terms in these analyses but they never explained a significant amount of variation so we removed them and present only results from additive models that more accurately estimate the main effects. To test for size-assortative mating, we conducted correlation analysis of the relative forewing lengths of males and females (standardized across trials) that were observed copulating.
For the first trial, we also tested the potential determinants of female reproductive success by removing all surviving females from the cage and placing them individually in small cups in the laboratory with cuttings of the host plant and a readily-accepted artificial nectar source (cotton swabs soaked with sports drink). Females were maintained under incandescent lights with a 14:10 light:dark schedule and temperatures ranging from 20–25C. We counted the eggs laid by each female every day for the remainder of its life and replaced the host plant and nectar daily. To identify the determinants of reproductive output, we tested the effects of female size and the number of observed copulations on female lifespan with multiple linear regression. We then tested the effects of female size, the number of observed copulations and lifespan on total egg production with similar models. For those females that we observed copulating, we then tested the effects of male size on female lifespan and egg production to assess potential male contribution to female fecundity. For those tests of models with multiple predictor variables, we report significance tests from marginal sums of squares. Thus, the P-values reported for these analyses indicate the additional explanatory power of the variable when included in a model already containing the other predictor variables.
We observed 70 copulations overall, with 43 of these occurring in the first of the three trials. Six males and four females mated twice and two males mated three times; we observed all other individuals mating only once. For those individuals that we observed copulating at least once, the probability of mating multiple times did not vary according to sex (χ2 = 0.92, df = 1, P = 0.34).
Parameter estimates and significance tests from logistic regression examining the effects of size (i.e., forewing chord length), sex and trial on probability of mating
Sex = Male
Parameter estimates and significance tests from logistic regression examining the effects of deviation from mean forewing chord length, sex and trial on probability of mating
Deviation from mean forewing length
Sex = Male
Results from multiple regression testing for the effects of size (forewing chord length) and lifespan on lifetime fecundity of female Miami blue butterflies
Results from linear model (ANCOVA) testing for differences female Miami blue butterfly egg production due to size (forewing chord length), lifespan, number of copulations and size of male partners
Forewing chord length
No. of copulations
Insect mating systems are diverse and often complex, but there are some determinants of reproductive success that are fairly consistent across taxa (Honek 1993). Among the Lepidoptera, phenological and morphological predictors of mating success and fecundity vary according to the type of mating system (Fischer and Fiedler 2000; Gotthard et al. 2000). In general, male traits, including size, are thought to be influenced most by sexual selection whereas female traits evolve primarily through differential production of offspring associated with heritable components of body size (Fischer and Fiedler 2000; Gotthard 2008). As such, sex-related differences in larval growth strategies and the predictors of adult reproduction are common, often resulting from differential selection on body size, higher probability of mating associated with early emergence for males, number and timing of generations and number of mates (Gotthard 2008). For example, the lycaenid Jalmenus evagoras is strongly protandrous and females only mate once, usually immediately after emerging from the pupae (Elgar and Pierce 1988). Consequently, it is unsurprising that male mating success in that system was predicted by early emergence, longevity (related to number of encounters with receptive females) and relative size compared to other males in mating aggregations. For female J. evagoras, mean daily egg production was strongly correlated with body size but was unaffected by longevity or size of the male mate.
We developed several predictions regarding the effect of body size on mating probability and the effects of body size and mate identity on both male and female components of fitness for the Miami blue butterfly. Specifically, we expected larger individuals to have higher reproductive success, manifested as both higher probability of mating and higher egg production. Life history trade-offs and non-random mortality could offset such intrinsic relationships in natural conditions (Bernays 1997; Gotthard 2000), but we expected that high larval growth rate resulting in large adult size would have few disadvantages in captivity with no predators and abundant resources.
Despite the sound theoretical basis for our predictions and supporting empirical evidence from related systems (Honek 1993), we found no evidence that size affected probability of mating for either sex of Miami blue butterflies. There was no difference between males and females in the probability of mating multiple times, perhaps indicating that sexes were equally choosy (or not) about their partners. Additionally, we found no evidence that male size affected female reproductive output either directly through higher egg production or indirectly through longer lifespan of the female due to nutritive nuptial gifts transferred in the spermatophore. In contrast, female size had both indirect and direct positive effects on egg production: forewing chord length was positively correlated with lifespan, and both female size and lifespan had additional positive effects on egg production even when considering the other variable (Table 3). These findings cumulatively suggest that mating frequency may be random with respect to body size for both sexes and that there is a strong positive relationship between size and reproductive output for female, but not male, Miami blue butterflies.
The relationship we found between female body size and egg production was consistent with results from many other systems (Honek 1993; Fischer and Fiedler 2000; Tammaru et al. 2002; Gotthard 2008). However, the lack of a relationship between male size and mating frequency or female fecundity in our study was unexpected and difficult to understand given the potential for female selectivity for high-quality males in the Miami blue butterfly mating system. It is possible that female Miami blue butterflies select males based on behavioral, chemical or morphological cues not associated with size (Pureswaran and Borden 2003; Kemp 2008). Alternatively, the environment of the flight cage in which the adults copulated may not have allowed full expression of behaviors such as male-male contests, courting flights and perching displays that can influence mate selection in nature (Bergman et al. 2007). If some of these behaviors are correlated with male size, the conditions of our study may have constrained the benefits of large phenotypes.
There are many reasons why determinants of mating probability or fecundity under laboratory conditions may not reliably predict reproductive success in the field (Leather 1988; Blanckenhorn 2000). For example, the adult Miami blue butterflies in this experiment were from larvae raised with abundant high-quality food and no predators (thus allowing largely unconstrained immature development) and the adults were housed in artificially crowded conditions with ample nectar and no predators. Together, these aspects of captive rearing may have eliminated some of the trade-offs associated with larval growth and adult body size (Bernays 1997; Gotthard 2000). Furthermore, by housing females individually in the laboratory under conditions conducive to both survival and oviposition, we may have obscured some trade-offs between body size, longevity and fecundity (Visser 1994). That is, in the protected environment of the oviposition cages with abundant resources, realized total egg production may have been closer to potential egg production than would be found in habitats with heterogeneous resources and predation risk (Leather 1988; Berger et al. 2006; Gotthard et al. 2007; Gotthard 2008).
Future studies of courting behavior, life history trade-offs and the physical characteristics associated with reproductive success in the wild could dramatically improve our understanding of the Miami blue butterfly mating system, with potential implications for management of the captive colony. For example, it is likely that mechanisms of mate selection differ between natural and artificial habitats. If the traits that determine mating frequency in the wild are heritable, it is possible that relaxed selection in the captive colony could produce individuals with low fitness upon reintroduction (Fleming et al. 1996; Bryant and Reed 1999; Lomnicki and Jasienski 2000). Despite the limitations of studying behavior in artificial conditions, studies like ours can nevertheless identify important relationships between insect body size and reproductive performance that can then be further tested under field conditions or manipulative experiments in captivity. Additionally, for highly endangered taxa such as the Miami blue butterfly, controlled laboratory studies may provide the only information regarding these life history patterns.
We thank Bret Boyd and Matt Standridge for assistance with maintaining the captive colony and performing the experiments. We thanks the Florida Fish and Wildlife Conservation Commission, E.O. Dunn Foundation, U.S. Fish and Wildlife Service and the National Fish and Wildlife Foundation for funding; additionally, M.D. Trager was funded by a National Science Foundation Graduate Research Fellowship. Research for this study was conducted under Florida Fish and Wildlife Conservation Commission permit WX02525f. We thank two anonymous reviewers for helpful comments on a previous version of this paper.