The evolutionary maintenance of ancient recombining sex chromosomes in the ostrich.

Sex chromosomes have evolved repeatedly across the tree of life and often exhibit extreme size dimorphism due to genetic degeneration of the sex-limited chromosome (e.g. the W chromosome of some birds and Y chromosome of mammals). However, in some lineages, ancient sex-limited chromosomes have escaped degeneration. Here, we study the evolutionary maintenance of sex chromosomes in the ostrich (Struthio camelus), where the W remains 65% the size of the Z chromosome, despite being more than 100 million years old. Using genome-wide resequencing data, we show that the population scaled recombination rate of the pseudoautosomal region (PAR) is higher than similar sized autosomes and is correlated with pedigree-based recombination rate in the heterogametic females, but not homogametic males. Genetic variation within the sex-linked region (SLR) (π = 0.001) was significantly lower than in the PAR, consistent with recombination cessation. Conversely, genetic variation across the PAR (π = 0.0016) was similar to that of autosomes and dependent on local recombination rates, GC content and to a lesser extent, gene density. In particular, the region close to the SLR was as genetically diverse as autosomes, likely due to high recombination rates around the PAR boundary restricting genetic linkage with the SLR to only ~50Kb. The potential for alleles with antagonistic fitness effects in males and females to drive chromosome degeneration is therefore limited. While some regions of the PAR had divergent male-female allele frequencies, suggestive of sexually antagonistic alleles, coalescent simulations showed this was broadly consistent with neutral genetic processes. Our results indicate that the degeneration of the large and ancient sex chromosomes of the ostrich may have been slowed by high recombination in the female PAR, reducing the scope for the accumulation of sexually antagonistic variation to generate selection for recombination cessation.

Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

The role of inter-individual intolerance in group cohesion and the transition to sociality in spiders.

Conspecific tolerance is key for maintaining group cohesion in animals. Understanding shifts from conspecific tolerance to intolerance is therefore important for understanding transitions to sociality. Subsocial species disperse to a solitary lifestyle after a gregarious juvenile phase and display conspecific intolerance as adults as a mechanism to maintain a solitary living. The development of intolerance towards group members is hypothesized to play a role in dispersal decisions in subsocial species. One hypothesis posits that dispersal is triggered by factors such as food competition with the subsequent development of conspecific intolerance, rather than conspecific intolerance developing prior to and potentially driving dispersal. Consistent with this hypothesis, we show that intolerance (inferred by inter-individual distance) developed post-dispersal in the subsocial spider Stegodyphus lineatus. The development of conspecific intolerance was delayed when maintaining spiders in groups showing plasticity in this trait, which is advantageous when trade-offs are not fixed over time. However, major evolutionary transitions, such as the transition to sociality, can permanently modify trade-offs and cause derived adaptations by the evolution of new or modified traits or evolutionary loss of traits that become redundant. Sociality in spiders has evolved repeatedly from subsocial ancestors, and social life in family groups combined with a lack of interaction with competing groups suggests relaxed selection for the development of conspecific intolerance. In the social Stegodyphus sarasinorum we found no evidence for the development of conspecific intolerance, consistent with the loss of this trait. Instead, we found evidence for conspecific attraction, which is likely to govern group cohesion.

Impaired immune function accompanies social evolution in spiders.

An efficient immune system is essential to the survival of many animals. Sociality increases risk of pathogen transmission, which should select for enhanced immune function. However, two hypotheses instead predict a weakened immune function: relaxed selection caused by social immunity/protection, and reduced efficacy of selection due to inbreeding, reproductive skew and female bias in social species that reduce effective population size and accelerate genetic drift. We assessed the effect of social evolution on immune function in a comparative study of two social spider species and their closely related subsocial sister species (genus Stegodyphus). The haemolymph of social species was less efficient in inhibiting bacterial growth of the potentially pathogenic bacteria Bacillus subtilis than that of subsocial species. Reduced efficacy of selection in social species was supported by comparative genomic analysis showing substantially elevated non-synonymous substitutions in immune genes in one of the social species. We propose that impaired immune function results from reduced efficacy of selection because the evolution of sociality in spiders is accompanied by demographic processes that elevate genetic drift. Positive feedback between pathogen-induced local extinctions and the resulting elevation of genetic drift may further weaken responses to selection by pathogens, and threaten species persistence.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Detecting purging of inbreeding depression by a slow rate of inbreeding for various traits: the impact of environmental and experimental conditions.

Inbreeding depression (ID) has since long been recognized as a significant factor in evolutionary biology. It is mainly the consequence of (partially) recessive deleterious mutations maintained by mutation-selection balance in large random mating populations. When population size is reduced, recessive alleles are increasingly found in homozygous condition due to drift and inbreeding and become more prone to selection. Particularly at slow rates of drift and inbreeding, selection will be more effective in purging such alleles, thereby reducing the amount of ID. Here we test assumptions of the efficiency of purging in relation to the inbreeding rate and the experimental conditions for four traits in D. melanogaster. We investigated the magnitude of ID for lines that were inbred to a similar level, F ≈ 0.50, reached either by three generations of full-sib mating (fast inbreeding), or by 12 consecutive generations with a small population size (slow inbreeding). This was done on two different food media. We observed significant ID for egg-to-adult viability and heat shock mortality, but only for egg-to-adult viability a significant part of the expressed inbreeding depression was effectively purged under slow inbreeding. For other traits like developmental time and starvation resistance, however, adaptation to the experimental and environmental conditions during inbreeding might affect the likelihood of purging to occur or being detected. We discuss factors that can affect the efficiency of purging and why empirical evidence for purging may be ambiguous.

Evidence for Faster X Chromosome Evolution in Spiders.

In species with chromosomal sex determination, X chromosomes are predicted to evolve faster than autosomes because of positive selection on recessive alleles or weak purifying selection. We investigated X chromosome evolution in Stegodyphus spiders that differ in mating system, sex ratio, and population dynamics. We assigned scaffolds to X chromosomes and autosomes using a novel method based on flow cytometry of sperm cells and reduced representation sequencing. We estimated coding substitution patterns (dN/dS) in a subsocial outcrossing species (S. africanus) and its social inbreeding and female-biased sister species (S. mimosarum), and found evidence for faster-X evolution in both species. X chromosome-to-autosome diversity (piX/piA) ratios were estimated in multiple populations. The average piX/piA estimates of S. africanus (0.57 [95% CI: 0.55-0.60]) was lower than the neutral expectation of 0.75, consistent with more hitchhiking events on X-linked loci and/or a lower X chromosome mutation rate, and we provide evidence in support of both. The social species S. mimosarum has a significantly higher piX/piA ratio (0.72 [95% CI: 0.65-0.79]) in agreement with its female-biased sex ratio. Stegodyphus mimosarum also have different piX/piA estimates among populations, which we interpret as evidence for recurrent founder events. Simulations show that recurrent founder events are expected to decrease the piX/piA estimates in S. mimosarum, thus underestimating the true effect of female-biased sex ratios. Finally, we found lower synonymous divergence on X chromosomes in both species, and the male-to-female substitution ratio to be higher than 1, indicating a higher mutation rate in males.

Prey to predator body size ratio in the evolution of cooperative hunting-a social spider test case.

One of the benefits of cooperative hunting may be that predators can subdue larger prey. In spiders, cooperative, social species can capture prey many times larger than an individual predator. However, we propose that cooperative prey capture does not have to be associated with larger caught prey per se, but with an increase in the ratio of prey to predator body size. This can be achieved either by catching larger prey while keeping predator body size constant, or by evolving a smaller predator body size while maintaining capture of large prey. We show that within a genus of relatively large spiders, Stegodyphus, subsocial spiders representing the ancestral state of social species are capable of catching the largest prey available in the environment. Hence, within this genus, the evolution of cooperation would not provide access to otherwise inaccessible, large prey. Instead, we show that social Stegodyphus spiders are smaller than their subsocial counterparts, while catching similar sized prey, leading to the predicted increase in prey-predator size ratio with sociality. We further show that in a genus of small spiders, Anelosimus, the level of sociality is associated with an increased size of prey caught while predator size is unaffected by sociality, leading to a similar, predicted increase in prey-predator size ratio. In summary, we find support for our proposed 'prey to predator size ratio hypothesis' and discuss how relaxed selection on large body size in the evolution of social, cooperative living may provide adaptive benefits for ancestrally relatively large predators.

Genomic signatures of experimental adaptive radiation in Drosophila.

Abiotic environmental factors play a fundamental role in determining the distribution, abundance and adaptive diversification of species. Empowered by new technologies enabling rapid and increasingly accurate examination of genomic variation in populations, researchers may gain new insights into the genomic background of adaptive radiation and stress resistance. We investigated genomic variation across generations of large-scale experimental selection regimes originating from a single founder population of Drosophila melanogaster, diverging in response to ecologically relevant environmental stressors: heat shock, heat knock down, cold shock, desiccation and starvation. When compared to the founder population, and to parallel unselected controls, there were more than 100,000 single nucleotide polymorphisms (SNPs) displaying consistent allelic changes in response to selective pressures across generations. These SNPs were found in both coding and noncoding sequences, with the highest density in promoter regions, and involved a broad range of functionalities, including molecular chaperoning by heat-shock proteins. The SNP patterns were highly stressor-specific despite considerable variation among line replicates within each selection regime, as reflected by a principal component analysis, and co-occurred with selective sweep regions. Only ~15% of SNPs with putatively adaptive changes were shared by at least two selective regimes, while less than 1% of SNPs diverged in opposite directions. Divergent stressors driving evolution in the experimental system of adaptive radiation left distinct genomic signatures, most pronounced in starvation and heat-shock selection regimes.

Comparing thermal performance curves across traits: how consistent are they?

Thermal performance curves (TPCs) are intended to approximate the relationship between temperature and fitness, and are commonly integrated into species distributional models for understanding climate change responses. However, TPCs may vary across traits because selection and environmental sensitivity (plasticity) differ across traits or because the timing and duration of the temperature exposure, here termed time scale, may alter trait variation. Yet, the extent to which TPCs vary temporally and across traits is rarely considered in assessments of climate change responses. Using a common garden approach, we estimated TPCs for standard metabolic rate (SMR), and activity in Drosophila melanogaster at three test temperatures (16, 25 and 30°C), using flies from each of six developmental temperatures (16, 18, 20, 25, 28 and 30°C). We examined the effects of time scale of temperature exposure (minutes/hours versus days/weeks) in altering TPC shape and position, and commonly used descriptors of the TPC: thermal optimum (Topt), thermal limits (Tmin and Tmax) and thermal breadth (Tbr). In addition, we collated previously published estimates of TPCs for fecundity and egg-to-adult viability in D. melanogaster We found that the descriptors of the TPCs varied across traits (egg-to-adult viability, SMR, activity and fecundity), but variation in TPCs within these traits was small across studies when measured at the same time scales. The time scale at which traits were measured contributed to greater variation in TPCs than the observed variance across traits, although the relative importance of time scale differed depending on the trait (activity versus fecundity). Variation in the TPC across traits and time scales suggests that TPCs using single traits may not be an accurate predictor of fitness and thermal adaptation across environments.

Expression of thermal tolerance genes in two Drosophila species with different acclimation capacities.

Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. We predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, we compared basal levels of gene expression at different developmental temperatures within each of the two species. Furthermore, we explored the heat hardening dynamics by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster we found an indication of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively.

Linking developmental diet to adult foraging choice in Drosophila melanogaster.

Rather than maximizing intake of available macronutrients, insects increase intake of some nutrients and restrict intake of others. This selective consumption influences, and potentially optimizes, developmental time, reproduction and lifespan of the organism. Studies so far have focused on discriminating between protein and carbohydrate uptake and the consequences on fitness components at different life stages. However, it is largely unknown whether and how the developmental diets, which may entail habitat-specific nutrient restrictions, affect selective consumption in adults. We show that adult female D. melanogaster opt for the same protein to carbohydrate (P:C) ratio regardless of their developmental diet (P:C ratio of 1:1, 1:4 or 1:8). In contrast, males choose a diet that makes up for deficiencies; when protein is low during development, males increase protein consumption despite this being detrimental to starvation resistance. The sexual dimorphism in foraging choice could be due to the different energetic requirements of males and females. To investigate the effect of developmental diet on lifespan once an adult nutritional environment has been established, we also conducted a no-choice experiment. Here, adult lifespan increased as P:C ratio decreased, irrespective of developmental diet, thus demonstrating a 'cancelling out' effect of the nutritional environment experienced during early life stages. Our study provides novel insights into how developmental diet is linked to adult diet by presenting evidence for sexual dimorphism in foraging choice as well as life-stage dependency of diet on lifespan.

Nucleotide diversity inflation as a genome-wide response to experimental lifespan extension in Drosophila melanogaster.

BACKGROUND: Evolutionary theory predicts that antagonistically selected alleles, such as those with divergent pleiotropic effects in early and late life, may often reach intermediate population frequencies due to balancing selection, an elusive process when sought out empirically. Alternatively, genetic diversity may increase as a result of positive frequency-dependent selection and genetic purging in bottlenecked populations. RESULTS: While experimental evolution systems with directional phenotypic selection typically result in at least local heterozygosity loss, we report that selection for increased lifespan in Drosophila melanogaster leads to an extensive genome-wide increase of nucleotide diversity in the selected lines compared to replicate control lines, pronounced in regions with no or low recombination, such as chromosome 4 and centromere neighborhoods. These changes, particularly in coding sequences, are most consistent with the operation of balancing selection and the antagonistic pleiotropy theory of aging and life history traits that tend to be intercorrelated. Genes involved in antioxidant defenses, along with multiple lncRNAs, were among those most affected by balancing selection. Despite the overwhelming genetic diversification and the paucity of selective sweep regions, two genes with functions important for central nervous system and memory, Ptp10D and Ank2, evolved under positive selection in the longevity lines. CONCLUSIONS: Overall, the 'evolve-and-resequence' experimental approach proves successful in providing unique insights into the complex evolutionary dynamics of genomic regions responsible for longevity.

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.

Unexpected high genetic diversity in small populations suggests maintenance by associative overdominance.

The effective population size (Ne ) is a central factor in determining maintenance of genetic variation. The neutral theory predicts that loss of variation depends on Ne , with less genetic drift in larger populations. We monitored genetic drift in 42 Drosophila melanogaster populations of different adult census population sizes (10, 50 or 500) using pooled RAD sequencing. In small populations, variation was lost at a substantially lower rate than expected. This observation was consistent across two ecological relevant thermal regimes, one stable and one with a stressful increase in temperature across generations. Estimated ratios between Ne and adult census size were consistently higher in small than in larger populations. The finding provides evidence for a slower than expected loss of genetic diversity and consequently a higher than expected long-term evolutionary potential in small fragmented populations. More genetic diversity was retained in areas of low recombination, suggesting that associative overdominance, driven by disfavoured homozygosity of recessive deleterious alleles, is responsible for the maintenance of genetic diversity in smaller populations. Consistent with this hypothesis, the X-chromosome, which is largely free of recessive deleterious alleles due to hemizygosity in males, fits neutral expectations even in small populations. Our experiments provide experimental answers to a range of unexpected patterns in natural populations, ranging from variable diversity on X-chromosomes and autosomes to surprisingly high levels of nucleotide diversity in small populations.

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

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.

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

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 melanogasterat 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.

Preservation of potassium balance is strongly associated with insect cold tolerance in the field: a seasonal study of Drosophila subobscura.

There is interest in pinpointing genes and physiological mechanisms explaining intra- and interspecific variations in cold tolerance, because thermal tolerance phenotypes strongly impact the distribution and abundance of wild animals. Laboratory studies have highlighted that the capacity to preserve water and ion homeostasis is linked to low temperature survival in insects. It remains unknown, however, whether adaptive seasonal acclimatization in free-ranging insects is governed by the same physiological mechanisms. Here, we test whether cold tolerance in field-caught Drosophila subobscura is high in early spring and lower during summer and whether this transition is associated with seasonal changes in the capacity of flies to preserve water and ion balance during cold stress. Indeed, flies caught during summer were less cold tolerant, and exposure of these flies to sub-zero temperatures caused a loss of haemolymph water and increased the concentration of K(+) in the haemolymph (as in laboratory-reared insects). This pattern of ion and water balance disruption was not observed in more cold-tolerant flies caught in early spring. Thus, we here provide a field verification of hypotheses based on laboratory studies and conclude that the ability to maintain ion homeostasis is important for the ability of free-ranging insects to cope with chilling.

Inbreeding affects locomotor activity in Drosophila melanogaster at different ages.

The ability to move is essential for many behavioural traits closely related to fitness. Here we studied the effect of inbreeding on locomotor activity (LA) of Drosophila melanogaster at different ages under both dark and light regimes. We expected to find a decreased LA in inbred lines compared to control lines. We also predicted an increased differentiation between lines due to inbreeding. LA was higher in the dark compared to the light regime for both inbred and outbred control lines. As expected, inbreeding increased phenotypic variance in LA, with some inbred lines showing higher and some lower LA than control lines. Moreover, age per se did not affect LA neither in control nor in inbred lines, while we found a strong line by age interaction between inbred lines. Interestingly, inbreeding changed the daily activity pattern of the flies: these patterns were consistent across all control lines but were lost in some inbred lines. The departure in the daily pattern of LA in inbred lines may contribute to the inbreeding depression observed in inbred natural populations.