Direct and correlated responses to chill‐coma recovery selection in Drosophila buzzatii

Chill‐coma recovery (CCR) is an important trait for thermal adaptation in insects. Multiple phenotypes could be affected by selection on CCR if the trait is genetically correlated with other adaptive traits. To test for heritable (co‐)variation in CCR, we examined direct and correlated responses to bi‐directional selection on CCR. Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae) was artificially selected for decreased and increased recovery time following exposure to 0 °C. After 18 selected generations, the selection response in CCR was significant but qualitatively asymmetric, with replicated lines for slow CCR showing the highest response. Knockdown resistance to high temperature was not affected by CCR selection. Starvation resistance in the adult fly showed no clear pattern of correlated responses to CCR selection. Selection on CCR had no impact on developmental time and body size. Chill‐coma recovery shows no apparent genetic trade‐offs with any of the multiple traits included in this study. These results are largely consistent with recent studies on clines in D. buzzatii, which showed that CCR is not across‐population correlated with other clinally varying traits of thermal adaptation. Cold adaptation may evolve toward increased cold resistance independent of upper thermal limits.     

Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster

When ectotherms are exposed to low temperatures, they enter a cold‐induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill‐coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold‐induced perturbations. The metabolites of cold‐hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations.     

Seasonal variation in basal and plastic cold tolerance: Adaptation is influenced by both long‐ and short‐term phenotypic plasticity

Understanding how thermal selection affects phenotypic distributions across different time scales will allow us to predict the effect of climate change on the fitness of ectotherms. We tested how seasonal temperature variation affects basal levels of cold tolerance and two types of phenotypic plasticity in Drosophila melanogaster. Developmental acclimation occurs as developmental stages of an organism are exposed to seasonal changes in temperature and its effect is irreversible, while reversible short‐term acclimation occurs daily in response to diurnal changes in temperature. We collected wild flies from a temperate population across seasons and measured two cold tolerance metrics (chill‐coma recovery and cold stress survival) and their responses to developmental and short‐term acclimation. Chill‐coma recovery responded to seasonal shifts in temperature, and phenotypic plasticity following both short‐term and developmental acclimation improved cold tolerance. This improvement indicated that both types of plasticity are adaptive, and that plasticity can compensate for genetic variation in basal cold tolerance during warmer parts of the season when flies tend to be less cold tolerant. We also observed a significantly stronger trade‐off between basal cold tolerance and short‐term acclimation during warmer months. For the longer‐term developmental acclimation, a trade‐off persisted regardless of season. A relationship between the two types of plasticity may provide additional insight into why some measures of thermal tolerance are more sensitive to seasonal variation than others.     

Cold tolerance in flour beetle species differing in body size and selection temperature

Cold tolerance is an important trait directly related to survival and hence fitness. In the present study, the link is addressed between cold tolerance and body size, which is associated with many key fitness traits, at both the intra‐ and interspecific levels. Specifically, chill coma recovery time, as a metric of cold tolerance, is examined in five related flour beetle species (four of them belonging to the genus Tribolium), two additional Tribolium castaneum Herbst populations selected for higher temperatures, and a mutant showing reduced body size. Recovery times are negatively correlated with the species average body size but not within each species. Females usually recover faster than males, although this difference is significant in only a single species, and is unrelated to body size. Repeating the experimental procedure with the same individuals, after 2 days in isolation with a limited amount of food, results in longer recovery times. Therefore, even if cold acclimation takes place, its influence appears to be diminished by the deleterious effects associated with the experimental procedure. Hence, the findings provide evidence for an association between body size and cold tolerance in the genus Tribolium, with larger species recovering faster from chill than smaller species. By contrast, the smalleyed flour beetle Palorus ratzeburgii Wissmann does not follow this pattern. Additionally, a population of T. castaneum selected for the highest temperature takes longer to recover from chill coma, indicating a trade‐off between cold and heat adaptations and not to a cross‐protection effect, as sometimes demonstrated.     

Heat knockdown resistance and chill‐coma recovery as correlated responses to selection on mating success at high temperature in Drosophila buzzatii

Reproduction and related traits such as mating success are strongly affected by thermal stress. We tested direct and correlated responses to artificial selection in replicated lines of Drosophila buzzatii that were selected for mating success at high temperature. Knockdown resistance at high temperature (KRHT) and chill‐coma recovery (CCR) were tested as correlated selection responses. Virgin flies were allowed to mate for four hours at 33°C in three replicated lines (S lines) to obtain the selected flies and then returned at 25°C to lay eggs. Other three replicated lines were maintained at 25°C without any selection as control (C lines). After 15 selection generations, KRHT and CCR were measured. Both traits were assessed in flies that did not receive any hardening pretreatments as well as in flies that were either heat or cold hardened. Thermotolerance traits showed significant correlated responses with higher KRHT in S than in C lines, both with a heat‐hardening pretreatment and without a heat‐hardening pretreatment. CCR time was longer in S than in C lines both with a cold‐hardening pretreatment and without a cold‐hardening pretreatment. Hardening treatments improved both KRHT and CCR in all cases excepting KRHT in C lines. Overall, KRHT and CCR showed an antagonistic pattern of correlated responses to our selection regime, suggesting either pleiotropy or tightly linked trait‐specific genes partially affecting KRHT and CCR.     

QTL mapping of freezing tolerance: links to fitness and adaptive trade‐offs

Local adaptation, defined as higher fitness of local vs. nonlocal genotypes, is commonly identified in reciprocal transplant experiments. Reciprocally adapted populations display fitness trade‐offs across environments, but little is known about the traits and genes underlying fitness trade‐offs in reciprocally adapted populations. We investigated the genetic basis and adaptive significance of freezing tolerance using locally adapted populations of Arabidopsis thaliana from Italy and Sweden. Previous reciprocal transplant studies of these populations indicated that subfreezing temperature is a major selective agent in Sweden. We used quantitative trait locus (QTL) mapping to identify the contribution of freezing tolerance to previously demonstrated local adaptation and genetic trade‐offs. First, we compared the genomic locations of freezing tolerance QTL to those for previously published QTL for survival in Sweden, and overall fitness in the field. Then, we estimated the contributions to survival and fitness across both field sites of genotypes at locally adaptive freezing tolerance QTL. In growth chamber studies, we found seven QTL for freezing tolerance, and the Swedish genotype increased freezing tolerance for five of these QTL. Three of these colocalized with locally adaptive survival QTL in Sweden and with trade‐off QTL for overall fitness. Two freezing tolerance QTL contribute to genetic trade‐offs across environments for both survival and overall fitness. A major regulator of freezing tolerance, CBF2, is implicated as a candidate gene for one of the trade‐off freezing tolerance QTL. Our study provides some of the first evidence of a trait and gene that mediate a fitness trade‐off in nature.     

Elevational trends in life histories: revising the pace‐of‐life framework

Life‐history traits in birds, such as lifespan, age at maturity, and rate of reproduction, vary across environments and in combinations imposed by trade‐offs and limitations of physiological mechanisms. A plethora of studies have described the diversity of traits and hypothesized selection pressures shaping components of the survival–reproduction trade‐off. Life‐history variation appears to fall along a slow–fast continuum, with slow pace characterized by higher investment in survival over reproduction and fast pace characterized by higher investment in reproduction over survival. The Pace‐of‐Life Syndrome (POLS) is a framework to describe the slow–fast axis of variation in life‐history traits and physiological traits. The POLS corresponds to latitudinal gradients, with tropical birds exhibiting a slow pace of life. We examined four possible ways that the traits of high‐elevation birds might correspond to the POLS continuum: (i) rapid pace, (ii) tropical slow pace, (iii) novel elevational pace, or (iv) constrained pace. Recent studies reveal that birds breeding at high elevations in temperate zones exhibit a combination of traits creating a unique elevational pace of life with a central trade‐off similar to a slow pace but physiological trade‐offs more similar to a fast pace. A paucity of studies prevents consideration of the possibility of a constrained pace of life. We propose extending the POLS framework to include trait variation of elevational clines to help to investigate complexity in global geographic patterns.     

The effect of selection for desiccation resistance on cold tolerance of Drosophila melanogaster

Abstract.  Low temperature and desiccation stress are thought to be mechanistically similar in insects, and several studies indicate that there is a degree of cross-tolerance between them, such that increased cold tolerance results in greater desiccation tolerance and vice versa . This assertion is tested at an evolutionary scale by examining basal cold tolerance, rapid cold-hardening (RCH) and chill coma recovery in replicate populations of Drosophila melanogaster selected for desiccation resistance (with controls for both selection and concomitant starvation) for over 50 generations. All of the populations display a RCH response, and there is no effect of selection regime on RCH or basal cold tolerance, although there are differences in basal cold tolerance between sampling dates, apparently related to inter-individual variation in development time. Flies selected for desiccation tolerance recover from chill coma slightly, but significantly, faster than control and starvation-control flies. These findings provide little support for cross-tolerance between survival of near-lethal cold and desiccation stress in D. melanogaster .     

Effects of acclimation and latitude on the activity thresholds of the aphid Myzus persicae in Europe

Activity thresholds were measured in nine anholocyclic clones of the peach‐potato aphid Myzus persicae collected along a latitudinal cline of its European distribution from Sweden to Spain. The effects of collection origin and intra‐ and intergenerational acclimation on these thresholds were investigated. Low‐temperature (10°C) acclimation for one generation depressed the movement threshold and chill coma temperatures, with the largest reduction in movement threshold recorded for clone UK 1 (8.8–2.5°C) and in chill coma for UK 2 (4.8–2.0°C). High‐temperature (25°C) acclimation for one generation increased the heat movement threshold and heat coma temperature with the largest increase in the movement threshold (40.1–41.1°C) and heat coma (41.4–42.3°C) recorded for clone Swed 1. There was no further intergenerational acclimation over three generations. High‐temperature activity thresholds were less plastic than low‐temperature thresholds, and, consequently, thermal activity ranges were expanded following low‐temperature acclimation. No constant affect of acclimation was observed on chill coma recovery, although clonal differences were observed with Swed 1 and 3 requiring some of the longest complete recovery times. There was no relationship between latitude and activity thresholds with the exception of heat coma data where Scandinavian clones Swed 2 and 3 consistently displayed some of the lowest heat coma temperatures (e.g. 41.3°C for both clones at 20°C) and Mediterranean clones Span 1, 2 and 3 displayed some of the highest (e.g. 42.1, 41.9 and 42.5°C, respectively, at 20°C). These data suggest that clonal mixing could occur over a large scale across Europe, limiting local adaptation to areas where conditions enable long‐term persistence of populations, e.g. adaptation to higher temperatures in the Mediterranean region. It is suggested that aphid thermal tolerance could be governed more by clonal type than the latitudinal origin.     

Intraspecific variation in thermal acclimation and tolerance between populations of the winter ant,Prenolepis imparis

Thermal phenotypic plasticity, otherwise known as acclimation, plays an essential role in how organisms respond to short‐term temperature changes. Plasticity buffers the impact of harmful temperature changes; therefore, understanding variation in plasticity in natural populations is crucial for understanding how species will respond to the changing climate. However, very few studies have examined patterns of phenotypic plasticity among populations, especially among ant populations. Considering that this intraspecies variation can provide insight into adaptive variation in populations, the goal of this study was to quantify the short‐term acclimation ability and thermal tolerance of several populations of the winter ant, Prenolepis imparis. We tested for correlations between thermal plasticity and thermal tolerance, elevation, and body size. We characterized the thermal environment both above and below ground for several populations distributed across different elevations within California, USA. In addition, we measured the short‐term acclimation ability and thermal tolerance of those populations. To measure thermal tolerance, we used chill‐coma recovery time (CCRT) and knockdown time as indicators of cold and heat tolerance, respectively. Short‐term phenotypic plasticity was assessed by calculating acclimation capacity using CCRT and knockdown time after exposure to both high and low temperatures. We found that several populations displayed different chill‐coma recovery times and a few displayed different heat knockdown times, and that the acclimation capacities of cold and heat tolerance differed among most populations. The high‐elevation populations displayed increased tolerance to the cold (faster CCRT) and greater plasticity. For high‐temperature tolerance, we found heat tolerance was not associated with altitude; instead, greater tolerance to the heat was correlated with increased plasticity at higher temperatures. These current findings provide insight into thermal adaptation and factors that contribute to phenotypic diversity by revealing physiological variance among populations.     

Plasticity and cross‐tolerance to heterogeneous environments: divergent stress responses co‐evolved in an African fruit fly

Plastic adjustments of physiological tolerance to a particular stressor can result in fitness benefits for resistance that might manifest not only in that same environment but also be advantageous when faced with alternative environmental stressors, a phenomenon termed ‘cross‐tolerance’. The nature and magnitude of cross‐tolerance responses can provide important insights into the underlying genetic architecture, potential constraints on or versatility of an organism's stress responses. In this study, we tested for cross‐tolerance to a suite of abiotic factors that likely contribute to setting insect population dynamics and geographic range limits: heat, cold, desiccation and starvation resistance in adult Ceratitis rosa following acclimation to all these isolated individual conditions prior to stress assays. Traits of stress resistance scored included critical thermal (activity) limits, chill coma recovery time (CCRT), heat knockdown time (HKDT), desiccation and starvation resistance. In agreement with other studies, we found that acclimation to one stress typically increased resistance for that same stress experienced later in life. A more novel outcome, however, is that here we also found substantial evidence for cross‐tolerance. For example, we found an improvement in heat tolerance (critical thermal maxima, CT_max) following starvation or desiccation hardening and improved desiccation resistance following cold acclimation, indicating pronounced cross‐tolerance to these environmental stressors for the traits examined. We also found that two different traits of the same stress resistance differed in their responsiveness to the same stress conditions (e.g. HKDT was less cross‐resistant than CT_max). The results of this study have two major implications that are of broader importance: (i) that these traits likely co‐evolved to cope with diverse or simultaneous stressors, and (ii) that a set of common underlying physiological mechanisms might exist between apparently divergent stress responses in this species. This species may prove to be a valuable model for future work on the evolutionary and mechanistic basis of cross‐tolerance.     

CHILL-COMA TOLERANCE, A MAJOR CLIMATIC ADAPTATION AMONG DROSOPHILA SPECIES

Abstract.— Most drosophilid species can be classified either as temperate or tropical. Adults of species were submitted to a cold treatment (0°C) and then brought back to ambient temperature. They generally exhibited a chill coma and the time needed to recover was measured. We found in a set of 26 temperate species that recovery was rapid (average 1.8 min, range 0.15–4.9). In contrast, a long recovery time (average 56 min, range 24–120) was observed for 48 tropical species. A few species, like Drosophila melanogaster, are cosmopolitan and can proliferate under temperate and tropical climates. In 9 of 10 such species, slight genetic differences were found: a shorter recovery in temperate than in tropical populations. Comparing physiological data to phylogeny suggests that chill‐coma tolerance has been a recurrent adaptation that is selected for in cold climates but tends to disappear under a permanently warm environment. This major climatic adaptation, evidenced in drosophilids, seems to occur in other insect groups also.     

Experimental evolution for generalists and specialists reveals multivariate genetic constraints on thermal reaction norms

Theory predicts the emergence of generalists in variable environments and antagonistic pleiotropy to favour specialists in constant environments, but empirical data seldom support such generalist–specialist trade‐offs. We selected for generalists and specialists in the dung fly Sepsis punctum (Diptera: Sepsidae) under conditions that we predicted would reveal antagonistic pleiotropy and multivariate trade‐offs underlying thermal reaction norms for juvenile development. We performed replicated laboratory evolution using four treatments: adaptation at a hot (31 °C) or a cold (15 °C) temperature, or under regimes fluctuating between these temperatures, either within or between generations. After 20 generations, we assessed parental effects and genetic responses of thermal reaction norms for three correlated life‐history traits: size at maturity, juvenile growth rate and juvenile survival. We find evidence for antagonistic pleiotropy for performance at hot and cold temperatures, and a temperature‐mediated trade‐off between juvenile survival and size at maturity, suggesting that trade‐offs associated with environmental tolerance can arise via intensified evolutionary compromises between genetically correlated traits. However, despite this antagonistic pleiotropy, we found no support for the evolution of increased thermal tolerance breadth at the expense of reduced maximal performance, suggesting low genetic variance in the generalist–specialist dimension.     

The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.     

Does increased heat resistance result in higher susceptibility to predation? A test using Drosophila melanogaster selection and hardening

Heat resistance of ectotherms can be increased both by plasticity and evolution, but these effects may have trade‐offs resulting from biotic interactions. Here, we test for predation costs in Drosophila melanogaster populations with altered heat resistance produced by adult hardening and directional selection for increased heat resistance. In addition, we also tested for genetic trade‐offs by testing heat resistance in lines that have evolved under increased predation risk. We show that while 35/37 °C hardening increases heat resistance as expected, it does not increase predation risk from jumping spiders or mantids; in fact, there was an indication that survival may have increased under predation following a triple 37 °C compared to a single 35 °C hardening treatment. Flies that survived a 39 °C selection cycle showed lower survival under predation, suggesting a predation cost of exposure to a more severe heat stress. There was, however, no correlated response to selection because survival did not differ between control and selected lines after selection was relaxed for one or two generations. In addition, lines selected for increased predation risk did not differ in heat resistance. Our findings suggest independent evolutionary responses to predation and heat as measured in laboratory assays, and no costs of heat hardening on susceptibility to predation.     

HORMESIS RESULTS IN TRADE‐OFFS WITH IMMUNITY

Many have argued that we may be able to extend life and improve human health through hormesis, the beneficial effects of low‐level toxins and other stressors. But, studies of hormesis in model systems have not yet established whether stress‐induced benefits are cost free, artifacts of inbreeding, or come with deleterious side effects. Here, we provide evidence that hormesis results in trade‐offs with immunity. We find that a single topical dose of dead spores of the entomopathogenic fungus, Metarhizium robertsii, increases the longevity of the fruit fly, Drosophila melanogaster, without significant decreases in fecundity. We find that hormetic benefits of pathogen challenge are greater in lines that lack key components of antifungal immunity (Dif and Turandot M). And, in outbred fly lines, we find that topical pathogen challenge enhances both survival and fecundity, but reduces ability to fight off live infections. The results provide evidence that hormesis is manifested by stress‐induced trade‐offs with immunity, not cost‐free benefits or artifacts of inbreeding. Our findings illuminate mechanisms underlying pathogen‐induced life‐history trade‐offs, and indicate that reduced immune function may be an ironic side effect of the “elixirs of life.”     

Evolutionary adaptation to environmental stressors: a common response at the proteomic level

Mechanistic trade‐offs between traits under selection can shape and constrain evolutionary adaptation to environmental stressors. However, our knowledge of the quantitative and qualitative overlap in the molecular machinery among stress tolerance traits is highly restricted by the challenges of comparing and interpreting data between separate studies and laboratories, as well as to extrapolating between different levels of biological organization. We investigated the expression of the constitutive proteome (833 proteins) of 35 Drosophila melanogaster replicate populations artificially selected for increased resistance to six different environmental stressors. The evolved proteomes were significantly differentiated from replicated control lines. A targeted analysis of the constitutive proteomes revealed a regime‐specific selection response among heat‐shock proteins, which provides evidence that selection also adjusts the constitutive expression of these molecular chaperones. Although the selection response in some proteins was regime specific, the results were dominated by evidence for a “common stress response.” With the exception of high temperature survival, we found no evidence for negative correlations between environmental stress resistance traits, meaning that evolutionary adaptation is not constrained by mechanistic trade‐offs in regulation of functional important proteins. Instead, standing genetic variation and genetic trade‐offs outside regulatory domains likely constrain the evolutionary responses in natural populations.     

The influence of temperature and photoperiod on the reproductive diapause and cold tolerance of spotted‐wing drosophila,Drosophila suzukii

Knowledge regarding the reproductive status of spotted‐wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is of critical importance in predicting potential infestations of this invasive pest, as eggs are laid in ripe or ripening fruit of several commercially important small‐fruit crops. Token environmental stimuli for the induction of reproductive diapause and improved cold tolerance were identified for D. suzukii. Diapause induction was evaluated by assessing, via dissection, the number of mature eggs carried by field‐captured D. suzukii and laboratory‐reared D. suzukii held under various temperature and photoperiod regimes. Egg load decreased over time in females trapped from July to December at sites in Benton County, OR, and Ontario County, NY, both USA, and reached zero eggs by December at all sites. Photoperiods below 14 h of day length led to reduced egg maturation in laboratory‐reared flies held at moderate temperatures (15 or 20 °C). Whereas very few mature eggs were found in females held at 10 °C under short‐ or long‐day photoperiods for several weeks after eclosion, a spontaneous return to ovarian maturity was observed in short‐day‐entrained females after 7 weeks. Diapause termination was investigated by evaluating fecundity in diapausing females returned to optimal environmental conditions. Whereas long‐day‐entrained flies began producing offspring immediately upon return to optimal conditions, short‐day‐entrained flies returned after 1 and 6 weeks at 10 °C were slower to produce offspring than colony flies or short‐day‐entrained flies returned after 7 weeks. Cold tolerance was evaluated by observing chill coma recovery rates after 24 h exposure to ?1 °C. Cold‐acclimated (diapausing) females recovered from chill coma faster than cold‐hardened or unacclimated females.     

Evolution of male age‐specific reproduction under differential risks and causes of death: males pay the cost of high female fitness

Classic theories of ageing evolution predict that increased extrinsic mortality due to an environmental hazard selects for increased early reproduction, rapid ageing and short intrinsic lifespan. Conversely, emerging theory maintains that when ageing increases susceptibility to an environmental hazard, increased mortality due to this hazard can select against ageing in physiological condition and prolong intrinsic lifespan. However, evolution of slow ageing under high‐condition‐dependent mortality is expected to result from reallocation of resources to different traits and such reallocation may be hampered by sex‐specific trade‐offs. Because same life‐history trait values often have different fitness consequences in males and females, sexually antagonistic selection can preserve genetic variance for lifespan and ageing. We previously showed that increased condition‐dependent mortality caused by heat shock leads to evolution of long‐life, decelerated late‐life mortality in both sexes and increased female fecundity in the nematode, Caenorhabditis remanei. Here, we used these cryopreserved lines to show that males evolving under heat shock suffered from reduced early‐life and net reproduction, while mortality rate had no effect. Our results suggest that heat‐shock resistance and associated long‐life trade‐off with male, but not female, reproduction and therefore sexually antagonistic selection contributes to maintenance of genetic variation for lifespan and fitness in this population.