Mads Fristrup Schou

A Drosophila laboratory evolution experiment points to low evolutionary potential under increased temperatures likely to be experienced in the future

The ability to respond evolutionarily to increasing temperatures is important for survival of ectotherms in a changing climate. Recent studies suggest that upper thermal limits may be evolutionary constrained. We address this hypothesis in a laboratory evolution experiment, encompassing ecologically relevant thermal regimes. To examine the potential for species to respond to climate change, we exposed replicate populations of Drosophila melanogaster to increasing temperatures (0.3 °C every generation) for 20 generations, whereas corresponding replicate control populations were held at benign thermal conditions throughout the experiment. We hypothesized that replicate populations exposed to increasing temperatures would show increased resistance to warm and dry environments compared with replicate control populations. Contrasting replicate populations held at the two thermal regimes showed (i) an increase in desiccation resistance and a decline in heat knock‐down resistance in replicate populations exposed to increasing temperatures, (ii) similar egg‐to‐adult viability and fecundity in replicate populations from the two thermal regimes, when assessed at high stressful temperatures and (iii) no difference in nucleotide diversity between thermal regimes. The limited scope for adaptive evolutionary responses shown in this study highlights the challenges faced by ectotherms under climate change.

Strong Costs and Benefits of Winter Acclimatization in Drosophila melanogaster

Studies on thermal acclimation in insects are often performed on animals acclimated in the laboratory under conditions that are not ecologically relevant. Costs and benefits of acclimation responses under such conditions may not reflect costs and benefits in natural populations subjected to daily and seasonal temperature fluctuations. Here we estimated costs and benefits in thermal tolerance limits in relation to winter acclimatization of Drosophila melanogaster. We sampled flies from a natural habitat during winter in Denmark (field flies) and compared heat and cold tolerance of these to that of flies collected from the same natural population, but acclimated to 25 °C or 13 °C in the laboratory (laboratory flies). We further obtained thermal performance curves for egg-to-adult viability of field and laboratory (25 °C) flies, to estimate possible cross-generational effects of acclimation. We found much higher cold tolerance and a lowered heat tolerance in field flies compared to laboratory flies reared at 25 °C. Flies reared in the laboratory at 13 °C exhibited the same thermal cost-benefit relations as the winter acclimatized flies. We also found a cost of winter acclimatization in terms of decreased egg-to-adult viability at high temperatures of eggs laid by winter acclimatized flies. Based on our findings we suggest that winter acclimatization in nature can induce strong benefits in terms of increased cold tolerance. These benefits can be reproduced in the laboratory under ecologically relevant rearing and testing conditions, and should be incorporated in species distribution modelling. Winter acclimatization also leads to decreased heat tolerance. This may create a mismatch between acclimation responses and the thermal environment, e.g. if temperatures suddenly increase during spring, under current and expected more variable future climatic conditions.

No trade-off between high and low temperature tolerance in a winter acclimatized Danish Drosophila subobscura population.

Coping with cold winter conditions is a major challenge for many insects. In early spring we observed newly emerged Drosophila subobscura, which had overwintered as larvae and pupae. As temperatures increase during spring these flies are faced with higher minimum and maximum temperatures in their natural microhabitat. Thus, there is a potential costly mismatch between winter and early spring acclimatization and the increased ambient temperatures later in adult life. We obtained individuals from a natural Danish population of D. subobscura and acclimated them in the laboratory to 20 °C for one generation, and compared critical thermal maximum (CTmax) and minimum (CTmin) to that of individuals collected directly from their natural microhabitat. The two populations (laboratory and field) were subsequently both held in the laboratory at 20 °C and tested for their CTmax and CTmin every third day for 28 days. At the first day of testing, field acclimatized D. subobscura had both higher heat and cold resistance compared to laboratory flies, and thereby a considerable larger thermal scope. Following transfer to the laboratory, cold and heat resistance of the field flies decreased over time relative to the laboratory flies. Despite the substantial decrease in thermal tolerances the thermal scope remained larger for field acclimatized individuals for the duration of the experiment. We conclude that flies acclimatized to their natural microhabitat had increased cold resistance, without a loss in heat tolerance. Thus while a negative correlation between cold and heat tolerance is typically observed in laboratory studies in Drosophila sp., this was not observed for field acclimatized D. subobscura in this study. We suggest that this is an adaptation to juvenile overwintering in temperate cold environments, where developmental (winter) temperatures can be much lower than temperatures experienced by reproducing adults after emergence (spring). The ability to gain cold tolerance through acclimatization without a parallel loss of heat tolerance affects thermal scope and suggests that high and low thermal tolerance act through mechanisms with different dynamics and reversibility.

Metabolic and functional characterization of effects of developmental temperature in Drosophila melanogaster.

The ability of ectotherms to respond to changes in their thermal environment through plastic mechanisms is central to their adaptive capability. However, we still lack knowledge on the physiological and functional responses by which ectotherms acclimate to temperatures during development, and in particular, how physiological stress at extreme temperatures may counteract beneficial acclimation responses at benign temperatures. We exposed Drosophila melanogaster to 10 developmental temperatures covering their entire permissible temperature range. We obtained metabolic profiles and reaction norms for several functional traits: egg-to-adult viability, developmental time, and heat and cold tolerance. Females were more heat tolerant than males, whereas no sexual dimorphism was found in cold tolerance. A group of metabolites, mainly free amino acids, had linear reaction norms. Several energy-carrying molecules, as well as some sugars, showed distinct inverted U-shaped norms of reaction across the thermal range, resulting in a positive correlation between metabolite intensities and egg-to-adult viability. At extreme temperatures, low levels of these metabolites were interpreted as a response characteristic of costs of homeostatic perturbations. Our results provide novel insights into a range of metabolites reported to be central for the acclimation response and suggest several new candidate metabolites. Low and high temperatures result in different adaptive physiological responses, but they also have commonalities likely to be a result of the failure to compensate for the physiological stress. We suggest that the regulation of metabolites that are tightly connected to the performance curve is important for the ability of ectotherms to cope with variation in temperature.

Proteomic data reveal a physiological basis for costs and benefits associated with thermal acclimation

ABSTRACT Physiological adaptation through acclimation is one way to cope with temperature changes. Biochemical studies on acclimation responses in ectotherms have so far mainly investigated consequences of short-term acclimation at the adult stage and focussed on adaptive responses. Here, we assessed the consequences of rearing Drosophila melanogaster at low (12°C), benign (25°C) and high (31°C) temperatures. We assessed cold and heat tolerance and obtained detailed proteomic profiles of flies from the three temperatures. The proteomic profiles provided a holistic understanding of the underlying biology associated with both adaptive and non-adaptive temperature responses. Results show strong benefits and costs across tolerances: rearing at low temperature increased adult cold tolerance and decreased adult heat tolerance and vice versa with development at high temperatures. In the proteomic analysis, we were able to identify and quantify a large number of proteins compared with previous studies on ectotherms (1440 proteins across all replicates and rearing regimes), enabling us to extend the proteomic approach using enrichment analyses. This gave us detailed information on individual proteins, as well as pathways affected by rearing temperature, pinpointing potential mechanisms responsible for the strong costs and benefits of rearing temperature on functional phenotypes. Several well-known heat shock proteins, as well as proteins not previously associated with thermal stress, were among the differentially expressed proteins. Upregulation of proteasome proteins was found to be an important adaptive process at high-stress rearing temperatures, and occurs at the expense of downregulation of basal metabolic functions. Summary: By investigating flies maintained at cold, benign and high temperatures, we provide a physiological basis for why ectotherms raised at low and high temperatures are more cold and heat resistant, respectively.

Thermal fluctuations affect the transcriptome through mechanisms independent of average temperature

Terrestrial ectotherms are challenged by variation in both mean and variance of temperature. Phenotypic plasticity (thermal acclimation) might mitigate adverse effects, however, we lack a fundamental understanding of the molecular mechanisms of thermal acclimation and how they are affected by fluctuating temperature. Here we investigated the effect of thermal acclimation in Drosophila melanogaster on critical thermal maxima (CTmax) and associated global gene expression profiles as induced by two constant and two ecologically relevant (non-stressful) diurnally fluctuating temperature regimes. Both mean and fluctuation of temperature contributed to thermal acclimation and affected the transcriptome. The transcriptomic response to mean temperatures comprised modification of a major part of the transcriptome, while the response to fluctuations affected a much smaller set of genes, which was highly independent of both the response to a change in mean temperature and to the classic heat shock response. Although the independent transcriptional effects caused by fluctuations were relatively small, they are likely to contribute to our understanding of thermal adaptation. We provide evidence that environmental sensing, particularly phototransduction, is a central mechanism underlying the regulation of thermal acclimation to fluctuating temperatures. Thus, genes and pathways involved in phototransduction are likely of importance in fluctuating climates.

Trait-specific consequences of inbreeding on adaptive phenotypic plasticity

Environmental changes may stress organisms and stimulate an adaptive phenotypic response. Effects of inbreeding often interact with the environment and can decrease fitness of inbred individuals exposed to stress more so than that of outbred individuals. Such an interaction may stem from a reduced ability of inbred individuals to respond plastically to environmental stress; however, this hypothesis has rarely been tested. In this study, we mimicked the genetic constitution of natural inbred populations by rearing replicate Drosophila melanogaster populations for 25 generations at a reduced population size (10 individuals). The replicate inbred populations, as well as control populations reared at a population size of 500, were exposed to a benign developmental temperature and two developmental temperatures at the lower and upper margins of their viable range. Flies developed at the three temperatures were assessed for traits known to vary across temperatures, namely abdominal pigmentation, wing size, and wing shape. We found no significant difference in phenotypic plasticity in pigmentation or in wing size between inbred and control populations, but a significantly higher plasticity in wing shape across temperatures in inbred compared to control populations. Given that the norms of reaction for the noninbred control populations are adaptive, we conclude that a reduced ability to induce an adaptive phenotypic response to temperature changes is not a general consequence of inbreeding and thus not a general explanation of inbreeding–environment interaction effects on fitness components.

Inbreeding depression across a nutritional stress continuum

Many natural populations experience inbreeding and genetic drift as a consequence of nonrandom mating or low population size. Furthermore, they face environmental challenges that may interact synergistically with deleterious consequences of increased homozygosity and further decrease fitness. Most studies on inbreeding–environment (I-E) interactions use one or two stress levels, whereby the resolution of the possible stress and inbreeding depression interaction is low. Here we produced Drosophila melanogaster replicate populations, maintained at three different population sizes (10, 50 and a control size of 500) for 25 generations. A nutritional stress gradient was imposed on the replicate populations by exposing them to 11 different concentrations of yeast in the developmental medium. We assessed the consequences of nutritional stress by scoring egg-to-adult viability and body mass of emerged flies. We found: (1) unequivocal evidence for I-E interactions in egg-to-adult viability and to a lesser extent in dry body mass, with inbreeding depression being more severe under higher levels of nutritional stress; (2) a steeper increase in inbreeding depression for replicate populations of size 10 with increasing nutritional stress than for replicate populations of size 50; (3) a nonlinear norm of reaction between inbreeding depression and nutritional stress; and (4) a faster increase in number of lethal equivalents in replicate populations of size 10 compared with replicate populations of size 50 with increasing nutritional stress levels. Our data provide novel and strong evidence that deleterious fitness consequences of I-E interactions are more pronounced at higher nutritional stress and at higher inbreeding levels.

Fast egg collection method greatly improves randomness of egg sampling in Drosophila melanogaster

When obtaining samples for population genetic studies, it is essential that the sampling is random. For Drosophila, one of the crucial steps in sampling experimental flies is the collection of eggs. Here an egg collection method is presented, which randomizes the eggs in a water column and diminishes environmental variance. This method was compared with a traditional egg collection method where eggs are collected directly from the medium. Within each method the observed and expected standard deviations of egg-to-adult viability were compared, whereby the difference in the randomness of the samples between the two methods was assessed. The method presented here was superior to the traditional method. Only 14% of the samples had a standard deviation higher than expected, as compared with 58% in the traditional method. To reduce bias in the estimation of the variance and the mean of a trait and to obtain a representative collection of genotypes, the method presented here is strongly recommended when collecting eggs from Drosophila.When obtaining samples for population genetic studies, it is essential that the sampling is random. For Drosophila, one of the crucial steps in sampling experimental flies is the collection of eggs. Here an egg collection method is presented, which randomizes the eggs in a water column and diminishes environmental variance. This method was compared with a traditional egg collection method where eggs are collected directly from the medium. Within each method the observed and expected standard deviations of egg-to-adult viability were compared, whereby the difference in the randomness of the samples between the two methods was assessed. The method presented here was superior to the traditional method. Only 14% of the samples had a standard deviation higher than expected, as compared with 58% in the traditional method. To reduce bias in the estimation of the variance and the mean of a trait and to obtain a representative collection of genotypes, the method presented here is strongly recommended when collecting eggs from Drosophila.

Reversibility of developmental heat and cold plasticity is asymmetric and has long-lasting consequences for adult thermal tolerance

ABSTRACT The ability of insects to cope with stressful temperatures through adaptive plasticity has allowed them to thrive under a wide range of thermal conditions. Developmental plasticity is generally considered to be a non-reversible phenotypic change, e.g. in morphological traits, while adult acclimation responses are often considered to be reversible physiological responses. However, physiologically mediated thermal acclimation might not follow this general prediction. We investigated the magnitude and rate of reversibility of developmental thermal plasticity responses in heat and cold tolerance of adult flies, using a full factorial design with two developmental and two adult temperatures (15 and 25°C). We show that cold tolerance attained during development is readily adjusted to the prevailing conditions during adult acclimation, with a symmetric rate of decrease or increase. In contrast, heat tolerance is only partly reversible during acclimation and is thus constrained by the temperature during development. The effect of adult acclimation on heat tolerance was asymmetrical, with a general loss of heat tolerance with age. Surprisingly, the decline in adult heat tolerance at 25°C was decelerated in flies developed at low temperatures. This result was supported by correlated responses in two senescence-associated traits and in accordance with a lower rate of ageing after low temperature development, suggesting that physiological age is not reset at eclosion. The results have profound ecological consequences for populations, as optimal developmental temperatures will be dependent on the thermal conditions faced in the adult stage and the age at which they occur. Summary: Using Drosophila melanogaster, we show how developmental plasticity of cold tolerance is completely reversible in the adult stage, while adult acclimation in heat tolerance is constrained by developmental temperature.