FITNESS COSTS OF RAPID COLD-HARDENING IN CERATITIS CAPITATA

Authors


Abstract

Rapid cold-hardening (RCH) is a unique form of phenotypic plasticity which confers survival advantages at low temperature. The fitness costs of RCH are generally poorly elucidated and are important to understanding the evolution of plastic physiology. This study examined whether RCH responses, induced by ecologically relevant diel temperature fluctuations, carry metabolic, survival, or fecundity costs. We predicted that potential costs in RCH would be manifested as differences in metabolic rate, fecundity, or survival in flies which have hardened versus those which have not, or flies that have experienced more RCH events would show greater costs than those which have experienced fewer events. One group of flies cooled to 10°C for 2 h for 11 consecutive days experienced daily RCH (Hardened), whereas the other group exposed to 15°C for the same 2-h period each day formed a Control group. Hardened flies had higher survival at –5°C for 2 h than control flies (69 ± 9% vs. 44 ± 19%, P = 0.04). Hardened flies showed no metabolic or fecundity costs, but had reduced average survival (P = 0.0403). Thus, a major cost to repeated low temperature exposures in Ceratitis capitata is through direct mortality caused by chilling injury, although this appears not to be a direct cost of RCH.

For ectotherms living in terrestrial environments, variable thermal regimes occur at daily, seasonal, and annual timescales. In some cases, temperature variability may also be associated with marked unpredictability (Vasseur and Yodzis 2004; Chown and Terblanche 2007). If the environment changes in a manner that organisms cannot avoid or cope using behavioral responses, they will have to adapt to the new conditions or probably face extinction (Williams et al. 2008; Chevin et al. 2010). The ability for an individual organism to modify its phenotype to increase fitness through physiological, behavioral, or morphological modifications, typically taking place within a single generation, is termed “phenotypic plasticity” (West-Eberhard 2003; Ghalambor et al. 2007; Whitman and Agrawal 2009), and is a crucial aspect of species perseverance in the face of climate change (see Huey and Berrigan 1996; Kingsolver and Huey 1998; Gabriel 2005; Chevin et al. 2010). However, plastic physiological responses typically depend on the magnitude and duration of exposure to an environmental stressor and on the ability of the insect to alter its phenotype (Chown and Terblanche 2007; Whitman and Agrawal 2009). Phenotypic plasticity generally occurs as a response to changes in a single or a few key environmental cues, such as shortening of photoperiod and declining temperatures, but the induced trait changes can only be beneficial if environmental signals are reliable and traits match the projected scenarios (see e.g., Coulson and Bale 1990; Larsen and Lee 1994; Deere and Chown 2006; Kristensen et al. 2008; reviewed in Huey and Berrigan 1996; Chown and Terblanche 2007; see also discussions of plasticity more broadly in e.g., Tufto 2000; Kingsolver et al. 2002; Reed et al. 2010).

To understand the evolution of plastic physiological responses and responses to selection, one therefore needs to examine both the spatial and temporal factors acting on a population (e.g., migration and fluctuating thermal regimes) and the costs and benefits for species’ fitness (Hoffmann 1995; Tufto 2000; Sultan and Spencer 2002; DeWitt and Scheiner 2004; Ghalambor et al. 2007; Lalouette et al. 2007). The interaction of genes and environment plays an important evolutionary role as phenotypic plasticity is ultimately a gene-by-environment interaction (Gienapp et al. 2008; Whitman and Agrawal 2009; Overgaard et al. 2010), but responses to environmental conditions may also be set by constraints at either the genetic or phenotypic level (see e.g., Kellermann et al. 2009 and discussions in Kingsolver 2009). Although many studies in physiological acclimation examine the benefits accrued (reviewed in e.g., Chown and Terblanche 2007; Angilletta 2009; Whitman and Agrawal 2009), far fewer consider the fitness costs that arise from thermal stress (Hoffmann 1995; Thomson et al. 2001). Alhough explored primarily in studies of high temperature tolerance, mainly undertaken using Drosophila (Scott et al. 1997; Thomson et al. 2001; Loeschcke and Hoffmann 2007; and see Hoffmann 1995; Janowitz and Fischer 2011), the relative costs and benefits of physiological acclimation responses are now increasingly focused upon, especially in the context of field performance and implications for fitness, using a broader range of thermal conditions and species (e.g., Loeschcke and Hoffmann 2007; Kristensen et al. 2008; Kingsolver et al. 2009; Fischer et al. 2010; Chidawanyika and Terblanche 2011a). One important point to emerge from this work is that the costs of a particular acclimation response have to be carefully separated from the general fitness cost of being exposed to unsuitable temperatures (Hoffmann and Hewa-Kapuge 2000). However, with enough time and genetic variability, insects can adapt to novel thermal regimes, sometimes accompanied by increased plasticity (Kingsolver et al. 2009; Huey 2010; but see Cooper et al. 2011).

Some insects possess a unique form of thermal tolerance plasticity, termed “rapid cold-hardening” (RCH) (Lee et al. 1987). RCH has received much attention since it is a stress-induced, reversible phenotypic response that takes place within a matter of minutes of exposure to new thermal conditions. Simply put, RCH is a process whereby an insect can rapidly increase its low temperature tolerance after a brief pre-exposure to a mild nonlethal low temperature (reviewed in Lee and Denlinger 2010), although improvements in low temperature survival can also occur in response to high-temperature treatments (e.g., Sinclair and Chown 2003; Chidawanyika and Terblanche 2011b). In some species, RCH can be the difference between none of the population surviving a low temperature event and all of the population surviving this same event (Lee and Denlinger 2010), and RCH could therefore be a trait strongly selected for evolutionary fitness benefits. Furthermore, the response is phylogenetically widespread (Lee and Denlinger 2010) and likely ecologically relevant (Kelty and Lee 1999; Powell and Bale 2005; Nyamukondiwa et al. 2010). The mechanisms underlying RCH responses are the focus of much research and include increases in carbohydrate cryoprotectants, membrane remodeling, and the inhibition of apoptosis in at least some species (Lee and Denlinger 2010). Notably, heat shock proteins (Hsps) and gene transcription do not appear to be immediately associated with the RCH response (Kelty and Lee 1999, 2001; Sinclair et al. 2007; but see also Colinet et al. 2010a,b). Although the mechanisms underlying RCH and the benefits gained are increasingly well examined (e.g., MacMillan et al. 2009; Teets et al. 2011), far fewer studies have examined the relative fitness costs of this response, except perhaps with a few obvious exceptions (e.g., Coulson and Bale 1990; Powell and Bale 2004; Shreve et al. 2004; Overgaard et al. 2007).

The costs involved for insects that undergo RCH may vary and are generally poorly established (Lee and Denlinger 2010). Since metabolic demands, but not necessarily overall whole-organismal rates, change under different thermal conditions and perhaps also depending on the number of stressful events encountered within an organisms lifetime (e.g., Nedvěd et al. 1998; Pétavy et al. 2001; Sinclair and Chown 2005; Marshall and Sinclair 2010; and see Gutschick and BassiriRad 2003), this can potentially lead to trade-offs in resource allocation that affects various life-history traits or strategies (e.g., growth-rate, body size, egg-laying, survival, tissue or cell maintenance, mating or courtship rituals, as well as decreased heat tolerance), essentially affecting long-term survival of populations (e.g., Coulson and Bale 1990, 1992; Shreve et al. 2004; Overgaard and Sørensen 2008; Marshall and Sinclair 2010; Janowitz and Fischer 2011). However, maintaining a plastic response such as RCH, which could potentially result in consumption of valuable body resources, could allow the insect to exploit resources available later on as a result of increased acute survival at low temperatures. Nevertheless, the evolution of RCH remains complex and unresolved (Sinclair et al. 2003; Lee and Denlinger 2010; Strachan et al. 2011) perhaps at least partly owing to poor understanding of the relative fitness costs of such a response. RCH responses also appear to be traded-off against greater low temperature tolerance, at least among Drosophila species (Nyamukondiwa et al. 2011). Cold-tolerance and acclimation strategies, and in some cases their associated costs, have been theoretically investigated in various models (e.g., Sultan and Spencer 2002; Voituron et al. 2002; Régnière and Bentz 2007). However, fitness costs of plasticity are generally poorly examined from an empirical perspective and especially in the case of acute physiological responses such as RCH.

The aim of this study is to examine the relative fitness costs and benefits of the RCH response and fluctuating low temperatures. Specifically, we examined whether ecologically relevant diel temperature fluctuations, which induce typical RCH responses, carry a metabolic, survival, or fecundity cost in the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae). Previous work has shown that C. capitata has a classic RCH response, similar in magnitude, speed of development of response, and duration of RCH survival benefits to other Diptera (Nyamukondiwa et al. 2010). We predicted that any potential costs in RCH would be manifested as either (1) a difference in metabolic rate, fecundity, or survival in flies that have hardened versus those that have not hardened, or (2) flies that have experienced more hardening events over time would show greater costs than those which have hardened on fewer occasions relative to similar-aged control flies. Specifically, we measured the direct survival benefits of RCH on acute low temperature survival in Hardened versus Control flies. In addition, we investigated whether there is a metabolic cost to RCH. Finally, we investigated the survival and fecundity of flies in Hardened and Control groups under benign conditions to estimate the potential fitness costs associated with RCH.

Materials and Methods

STUDY ANIMALS AND TREATMENT CONDITIONS

Larvae of C. capitata were obtained by shipping in insulated containers from Citrus Research International, Nelspruit, South Africa. The culture conditions are a reasonable reflection of wild populations as it is a large, regularly supplemented population of outbred flies kept in conditions which are similar to ecological conditions of wild populations (see Terblanche et al. 2010). The larvae were randomly split into two treatment groups (Control and Hardened) of equal density and kept in two incubators set at 25°C (±1°C) until eclosion. Relative humidity (RH) was regulated to 76% by means of saturated salt (NaCl) solutions located within each container. Container temperatures and humidity were monitored with temperature and humidity loggers (resolution: 0.5°C, DS1923L-F5, Maxim iButton Logger, Fairbridge Technologies, Essex, UK). Once emerged, the flies in both groups were fed honey–water and sugar and given bananas for oviposition, and maintained at 25°C for at least three days before treatments started. Thereafter, once a day, the temperatures in the incubators were rapidly dropped for 2 h to 15°C at a rate of 0.0833°C/min or 10°C at a rate of 0.1667°C/min for the Control and Hardened groups, respectively. After the 2-h period, the incubators returned to 25°C (Fig. S1). Diel temperature fluctuations continued for 11 consecutive days and flies were exactly the same age within each treatment group at the start of Hardening and Control treatments. However, owing to equipment constraints (a maximum of seven flies per respirometry run) and to ensure that similar-aged flies were used in all respirometry experiments, the trials of Control and Hardened flies were offset by a day. Controlling for age in MR and thermal tolerance is critical since ageing (independent from mass) and flight muscle development can influence MR and thermal tolerance estimates in insects (Bowler and Terblanche 2008; Piiroinen et al. 2010). Thus, day 1 for the Hardened treatment commenced one day prior to the Control group. Only fed male flies were used for metabolic rate estimation to eliminate confounding effects of reproductive status in female flies. The adult flies were weighed before and after each respirometry trial using a calibrated electronic balance (to 0.1 mg; NewClassic MF, MS104S, Mettler-Toledo International Inc., Greifensee, Switzerland). Experiments were repeated in two blocks for each of the main parameters being estimated: metabolic costs and acute survival at –5°C in one block, and survival and fecundity estimated in a separate experimental block.

RAPID COLD-HARDENING

To determine whether RCH takes place in the 25–10°C (Hardened) group relative to the 25–15°C (Control) group, n = 10 male flies (× 5 replicates, total n = 50) were placed inside a water bath at –5°C for 2 h directly after being exposed to 10°C or 15°C for 2 h in the programmed diel thermal fluctuations. Flies were then returned to 25°C for 24 h (with food and water) and then scored for survival in each of the treatment and control groups. This was repeated after 1, 5, 8, and 11 RCH treatments to verify that typical RCH responses were induced in C. capitata during the experiments.

METABOLIC RATE MEASUREMENTS

Briefly, a flow-through respirometry system was used to determine resting metabolic rates (RMRs) of C. capitata in both the Hardened and Control groups indirectly from the adult flies’ CO2 production (Fig. S1). Groups of seven flies per group per RCH event (i.e., at a given age) were used for MR measurements, that is, n = 7 per control group per RCH event and n = 7 per hardened group per RCH event (total of 56 respirometry recordings). The system is described in detail in the Supporting information.

SURVIVAL AND FECUNDITY ESTIMATES

Flies were kept under the same conditions as previously described for both Control and Hardened groups as above. Here, 400 flies were divided into five replicates at each acclimation temperature per group, that is, five replicates of five females and five males per container for day 1 to 11 in the Hardened group (25–10°C) and five replicates of five females and five males per container for day 1 to 11 in the Control group (25–15°C). Each container was supplied with a whole banana for oviposition and sugar-water was provided for feeding ad libitum. After 11 days, the net survival of flies within each container was scored as well as the summed number of pupae and larvae produced per container. In these experiments, rearing containers were treated as replicates, that is, independent units, but all individuals within a container were pooled for statistical analyses.

STATISTICS

Survival data of adult C. capitata following RCH events and exposure to –5°C were investigated using a generalized linear model (GLZ) assuming a binomial distribution and a logit link function in SAS statistical software (Proc Genmod), with corrections for overdispersion (following e.g., Nyamukondiwa et al. 2010).

To assess the effects of RCH treatments on the metabolic rate of C. capitata, we first used a full factorial analysis of variance (ANOVA) with metabolic rate as the dependant variable and acclimation temperature and days of RCH as the categorical variables. In a second model, we investigated the effects of mass on MR using a general linear model (GLM, ANCOVA) with MR as the dependant variable, acclimation temperature and number of RCH events as the categorical variables and mass as the covariate. Key assumptions of ANOVA were tested including normality of data distribution (Shapiro–Wilks) and equality of variance (using Levene's test) followed by a post-hoc Tukey HSD test to identify homogeneous groups. In all cases, RMR data are presented in mlCO2.h−1.

Tests of the effects of treatment temperature and RCH events on survival and fecundity were performed using GLZ in SAS. Fecundity was scored as the number of pupae and larvae produced per five pairs of adult flies after 1, 5, 8, and 11 RCH events. Fecundity analysis was undertaken using a poisson distribution and a log link function with fecundity as the dependent variable, and acclimation temperature and number of RCH events as independent categorical variables. Mortality was scored as the number of dead flies after 1, 5, 8, and 11 days of RCH. Mortality analysis was undertaken using a binomial distribution and a logit link function with proportion mortality as the dependent variable (n dead/ total n), RCH events and treatment temperature as categorical predictor variables. Data are presented as means ± SEM unless otherwise stated and tested by comparison of overlap in 95% confidence intervals among groups.

Results

ACUTE SURVIVAL AFTER EXPOSURE TO –5°C

As expected, the RCH treatment (10°C for 2 h) resulted in a significant improvement in survival of C. capitata exposed to –5°C for 2 h relative to those which did not have a pretreatment at 10°C for 2 h (69 ± 9% vs. 44 ± 19%; df = 1, Wald χ2= 4.21, P = 0.04). However, there was no significant effect of the number of RCH events on survival (df = 2, Wald χ2= 2.49, P = 0.2873), suggesting a lack of acclimation induced by these treatments and a lack of any cumulative effects. In addition, there was no significant effect of the interaction between RCH events and treatment group (Control vs. Hardened) (df = 2, Wald χ2= 1.67, P = 0.4340) indicating that both groups remained similar in basal low temperature tolerance and hardening ability over time (Table S1).

RESPIROMETRY

The only variable that significantly affected absolute RMR was the number of hardening events (equivalent to days of treatment), but the acclimation temperature to which the flies were exposed for 2 h per day (10 or 15°C) had no significant effect on RMR measured at 25°C (Table S2). However, mass had a significant effect on RMR (P < 0.005) and we therefore repeated these analyses adjusting for size among days in analysis of covariance (ANCOVA). Once body mass was accounted for, neither the number of days of exposure to hardening conditions, nor the treatment group (Hardened vs. Control) had any significant effect on the mass-specific RMR of the flies recorded at 25°C (Table S2 and Fig. 1). Moreover, the interaction between the number of RCH events and hardening treatment was not significant for either absolute RMR or mass-specific RMR (Table S2).

Figure 1.

(A) Mean unadjusted VCO2 (i.e., not mass-corrected resting metabolic rate [RMR]) (mlCO2/h, means ± 95% CLs) for Ceratitis capitata adults measured at 25°C in the Hardened (i.e., rapidly cold-hardened) for 1, 5, 8 and 11 days by thermal fluctuations from 25 to 10°C. The Control group was subjected to 15°C for 2 h and the Hardened group was exposed to 10°C for 2 h for 11 consecutive days. (B) Average mass (mg, means ± 95% CLs) of flies acclimated over 11 days for both Control and Hardened groups. (C) Mass-adjusted RMR (mlCO2/g/h, means ± 95% CLs.) for C. capitata adults at 25°C after 1, 5, 8, and 11 RCH events. Mass was the only variable to significantly affect MR (P < 0.005), whereas treatment and RCH events showed no significant effect, or the interaction of the two variables significantly affect RMR over 11 RCH events (see Table S2).

There was no significant effect of treatment on RMR (Fig. 1A). There was no significant difference in size between the Hardened and the Control flies over the 11-day period (Fig. 1B). However, flies increased in size over the experimental duration (average mass at day 11, Hardened: 6.42 ± 0.15 mg, Control: 5.96 ± 0.17 mg, P < 0.001), probably as a consequence of maturation and adult development, but this pattern is seen in both the Hardened and the Control groups and thus not likely a consequence of the main treatment (Fig. 1B and Table S2, P > 0.05). Mass-specific metabolic rate did not differ among treatment groups (Fig. 1C; P = 0.262).

SURVIVAL AND FECUNDITY ESTIMATES

On average, fewer pupae and larvae were produced in the Hardened group (average number of larvae and pupae produced over 11 days: Hardened: 7.70 ± 1.63; Control: 17.2 ± 3.96). However, fecundity was not significantly affected by the number of RCH events (P = 0.659) or the interaction of acclimation temperature and RCH events (P = 0.082) (Fig. 2A and Table S3). There was considerable variation in the number of pupae and larvae produced at each day and only after 11 RCH events (days) was there an obvious difference in fecundity between Hardened and Control flies (Fig. 2A). Mortality was significantly higher in the Hardened group than the Control group (average mortality across 11 days: Hardened: 4.45 ± 0.63; Control: 2.25 ± 0.39 flies, P < 0.005) and number of RCH events (P < 0.001), but not by their interaction as mortality was scored per RCH event and was not a cumulative count (P = 0.1291) (Fig. 2B and Table S3). After five RCH events, there was already a significant increase in mortality of Hardened compared to Control flies and this trend continued over the course of the 11 RCH events.

Figure 2.

(A) Mean (±95% CLs) number of pupae and larvae (measures of fecundity) produced following 1, 5, 8, and 11 RCH events. (B) Mean (%, ±95% CLs) mortality scored as n dead flies/n total flies after 1, 5, 8, and 11 RCH events in both the Control and Hardened groups.

Discussion

There is dramatic variation in the magnitude of RCH responses even among closely related species (e.g., Lee and Denlinger 2010; Nyamukondiwa and Terblanche 2010; Strachan et al. 2011) suggesting that such a response may have evolved differentially among terrestrial insect species. RCH could be associated with freeze-intolerant insects living in temperate environments with unpredictable climates, especially during autumn and spring, where RCH likely enhances survival during sudden cold spells (Sinclair and Chown 2006; Terblanche et al. 2007). In addition, the magnitude of RCH is lower in Drosophila species with greater cold tolerance, suggesting that increased RCH may be a trade-off against basal cold tolerance (Nyamukondiwa et al. 2011). This acute plasticity may also be especially beneficial when the timing of key behaviors (e.g., mating or foraging) coincide with cold events (Shreve et al. 2004; Powell and Bale 2006; Terblanche et al. 2008). Many activities are enhanced by RCH including mating and reproductive success (e.g., Shreve et al. 2004), yet a major benefit of RCH in insects is likely immediately increased survival at lower, potentially lethal temperatures. For example, Sarcophaga crassipalpis flesh flies given 10 min at 0°C increase cold tolerance by 50% at –10°C compared to flies that did not experience this pretreatment (Chen et al. 1987). Indeed, it is these types of direct survival benefits at thermal extremes that are most frequently reported for RCH responses (Lee and Denlinger 2010).

In agreement with previous studies of RCH in these (Nyamukondiwa et al. 2010) and other insects that show RCH responses (e.g., Shreve et al. 2004; Overgaard et al. 2005; Powell and Bale 2005; Nyamukondiwa et al. 2011) C. capitata responds to brief low temperature pretreatments with increased survival relative to a control group not given the low temperature (hardening) pre-treatment. Thus, acute low temperature survival is enhanced, likely through a range of biochemical responses (discussed in Lee and Denlinger 2010) and perhaps involving Hsps (Colinet et al. 2010a,b) and membrane lipid composition changes (Overgaard et al. 2005 reviewed in Lee and Denlinger 2010). Improvements in acute low temperature survival could also be replicated in the present study through the use of diel temperature fluctuations differing principally in their magnitude of severity (although also slightly in rate of temperature change) of low temperature exposure. This allowed us to dissect the potential costs of RCH in greater detail than perhaps a single exposure could and, moreover, for a substantial portion of this flies’ adult (reproductive) life span. Consequently, Hardened flies, having been exposed to 10°C for only 2 h on a single occasion, have greater low temperature survival at –5°C for 2 h compared with the Control flies that experienced only 15°C for 2 h before exposure to –5°C for 2  h. Although not well explored in C. capitata, the RCH responses observed are probably accompanied by improvements in a range of other traits, perhaps including preservation of various mating or reproductive behaviors.

Given the limited effects of treatment or number of RCH events on whole-animal metabolic rate in C. capitata, there is little evidence suggesting RCH is metabolically expensive. Our results therefore support Shreve et al.'s (2004) hypothesis, based on results from Drosophila melanogaster, that RCH is not an energetically expensive process, or at least metabolic costs are not easily detectable at the whole-organism level. The same outcome was seen here in C. capitata assessed over longer experimental periods (i.e., more time in the respirometer) and more RCH events. However, RMR of the Hardened flies is perhaps significantly higher than that of the Control flies after one RCH event at 10°C. This is probably due to some physiological adjustment made after the initial sudden drop from 25°C to 10°C. Overall, RMR adjustments do not seem to be common and, therefore, RCH is probably not particularly energetically demanding.

Despite the lack of an obvious metabolic cost, for RCH to have evolved differentially and not simply be ubiquitous across terrestrial ectotherms, it seems reasonable to expect a fitness cost. Indeed, theoretical models of plastic or evolved low temperature responses specifically require some sort of cost function to be integrated to produce realistic results (e.g., Voituron et al. 2002; Régnière and Bentz 2007). It is possible that the costs involve trade-offs within the organism, which are undetectable at the whole-organism level (e.g., re-allocation of energy resources away from somatic tissue maintenance or enzyme activity). However, the latter costs should therefore be detectable either as fitness cost in terms of survival at benign temperatures, overall reproductive lifespan, or net reproductive output.

Work on the common housefly Musca domestica by Coulson and Bale (1990, 1992) showed a decrease in the number of eggs oviposited by cold-hardened M. domestica, along with a reduced life span of the adults and decreased emergence rate of eggs (Coulson and Bale 1992) suggesting multiple costs, both in terms of reproduction and survival. Shreve et al. (2004) found that cold-hardened D. melanogaster preserved reproductive behaviors, that is, courting and mating. Similarly, our results indicate that although flies exposed to several RCH events pay some minor reproductive penalty, this is likely not a consequence of RCH per se but rather overall reduced average temperature in Hardened flies. This implies that diurnal temperature variation, at least for an extended period of time, perhaps comes at a marginal reproductive cost, but there is great variation among days even within the Control group. A lack of reproductive costs has been reported by Kelty and Lee (1999) in short term exposures (five days) in D. melanogaster. However, the most likely explanation of the fitness cost of the difference in the variable temperatures between treatment groups in C. capitata is the highly significant increase in mortality at benign temperatures. This is probably a consequence of direct, chilling injury as opposed to indirect effects on reduced fecundity, given the manner in which we scored these traits. Given that mortality did not increase over time (i.e., no RCH event × treatment group effect was detected), it seems more likely that this is an effect of reduced temperatures, rather than a cost of RCH. Nevertheless, recent work has shown repeated exposure to chilling events in fluctuating thermal regimes could also result in life-history trade-offs and, ultimately, negative population growth rates (e.g., Marshall and Sinclair 2010). Sarcophaga crassipalpis only shows injury from cold-shock three days after treatment due to damage to the neuromuscular system (Kelty et al. 1996) and thus our speculation of chilling damage is not unfounded (and see Yi and Lee 2003).

Phenotypic plasticity of the kind discussed here (i.e., acute, reversible plasticity), resulting in RCH in adult C. capitata likely comes with both evolutionary fitness benefits and costs, as might be expected for plastic responses more broadly (see discussion in e.g., Gilchrist 1995; Hoffmann 1995; Sultan and Spencer 2002; Angilletta 2009; Kingsolver et al. 2009). Even though the flies do not expend extra metabolic energy on the process, fitness penalties are incurred. The costs involved are not necessarily acclimation or plasticity costs per se, but might be a result of chill injury due to exposure to suboptimal temperatures. Although the flies had hardened at 10°C, which likely minimizes chill injury (Yi and Lee 2003), repeated exposure to fluctuating low temperatures has an obvious detrimental effect on population survival under benign conditions, an effect that was not compensated for (or if so, at least not completely) by the RCH response itself.

In conclusion, it is clear that mortality at benign temperatures, although likely influenced in opposite directions by both low temperature and RCH responses, is a major effect caused by the absolute temperature difference in the two treatments (see Woods and Harrison 2002) and probably carries significant evolutionary fitness costs at the population level. By contrast, metabolic costs, and perhaps also to a lesser extent, reproductive costs, appear relatively insignificant to RCH and diurnally fluctuating low temperatures more generally (and see Terblanche et al. 2010). Moreover, the number of RCH events did not improve C. capitata's ability to survive suggesting limited cumulative effects on plastic responses and a lack of acclimation induced by these treatments. Regardless, cold-hardened flies do not appear to gain major benefits from increased fecundity although they likely offset immediate survival benefits against long-term reproductive penalties. In sum, the present study has identified the fitness costs of fluctuating low temperatures in C. capitata. Population modeling of evolved plastic responses can now incorporate realistic, empirically determined costs into demographic models for determining the evolution of RCH and perhaps also plasticity more broadly, and likely improves forecasting of global invasions and climate change responses for this major agricultural pest. These results are of broad significance to understanding the evolution of rapid, reversible phenotypic plasticity in thermal tolerance traits of terrestrial ectotherms.


Associate Editor: D. Carrier

ACKNOWLEDGMENTS

This project was funded by a NRF Blue Skies Grant BS 2008090800006, and DFPT and CRI funding to JST. An NRF THRIP project to P. Addison and Stellenbosch University Sub-Committee B funding to JST further supported equipment and infrastructure used in this project. We are grateful to two anonymous referees and D. Carrier for constructive comments on an earlier version of this article.

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