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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.

Growing summer season, increased anthropogenic activities poses a continual challenge to resident species Ectotherms like insects are especially vulnerable to rapid climatic changes. High-altitude tropical insect populations have been rarely examined for their responses to high-temperature. We exposed a tropical highland out-bred population of Drosophila melanogaster from the Himalayas to growing summer conditions in outdoor mesocosm units. Population response to thermal changes was tracked over ninety days at phenotypic and genotypic level. Whole genomic resequencing data suggested a clear seasonal allelic shift. Interestingly, the general heat responsive genes were missing in the summer due to monsoon allele shift; an atypical response noted for high-altitude populations. Instead, candidates involved in kinases and phosphorylation emerged as key players. Heat-knockdown time decreased over time indicating a limited ability to handle increasing temperature. Merging data from both allelic shifts and heat-knockdown time indicated a limited capacity for high-altitude insects in handling climate warming.

A dominant perception is that small and motile ectothermic animals must use behavior to avoid exposure to critical or sub-critical temperatures impairing physiological performance. Concomitantly, volunteer exploration of extreme environments by some individuals may promote physiological adjustments and enhance ecological opportunity. Here we introduce to the literature a Thermal Decision System (TDS) which is fully modular, thermally stable, versatile, and adaptable to study navigation through thermal landscapes in insects and other small motile animals. We used a specific setting of the TDS to investigate volunteer navigation through critical cold and hot temperatures in Drosophila melanogaster. We demonstrate that a thermally bold behavior (volunteer crossings through a Critical Temperature Zone, CTZ) characterized a fraction of flies in a sample, and that such a fraction was higher in an outbred population relative to isofemale lines. As set, the TDS generated a thermal gradient within the cold and hot CTZs, and the exploration of this gradient by flies did not relate simply with a tendency to be thermally bold. Mild fasting affected thermal exploration and boldness in complex manners, but thermal boldness was evident in both fasted and fed flies. Also, thermal boldness was not associated with individual critical temperatures. Finally, some flies showed consistent thermal boldness, as flies that performed an extreme thermal cross were more likely to perform a second cross compared with untested flies. We hypothesize that a simple “avoidance principle” is not the only behavioral drive for D. melanogaster facing extreme temperatures over space, and that this pattern may characterize other small motile ectothermic animals with analogous natural history. The physiological correlates, genetic architecture, and interspecific variation of thermal boldness deserve further consideration.

A dominant perception is that small and motile ectothermic animals must use behavior to avoid exposure to critical or sub-critical temperatures impairing physiological performance. Concomitantly, volunteer exploration of extreme environments by some individuals may promote physiological adjustments and enhance ecological opportunity. Here we introduce to the literature a Thermal Decision System (TDS) which is fully modular, thermally stable, versatile, and adaptable to study navigation through thermal landscapes in insects and other small motile animals. We used a specific setting of the TDS to investigate volunteer navigation through critical cold and hot temperatures in Drosophila melanogaster. We demonstrate that a thermally bold behavior (volunteer crossings through a Critical Temperature Zone, CTZ) characterized a fraction of flies in a sample, and that such a fraction was higher in an outbred population relative to isofemale lines. As set, the TDS generated a thermal gradient within the cold and hot CTZs, and the exploration of this gradient by flies did not relate simply with a tendency to be thermally bold. Mild fasting affected thermal exploration and boldness in complex manners, but thermal boldness was evident in both fasted and fed flies. Also, thermal boldness was not associated with individual critical temperatures. Finally, some flies showed consistent thermal boldness, as flies that performed an extreme thermal cross were more likely to perform a second cross compared with untested flies. We hypothesize that a simple “avoidance principle” is not the only behavioral drive for D. melanogaster facing extreme temperatures over space, and that this pattern may characterize other small motile ectothermic animals with analogous natural history. The physiological correlates, genetic architecture, and interspecific variation of thermal boldness deserve further consideration.

Simple Summary Most laboratory experiments on insects to date have been conducted using constant temperature settings. Even when the purpose of the study was to investigate effects of temperature, insects have mostly been kept at different but constant temperatures ignoring natural variation in temperature. Here we investigated effects of simple daily temperature fluctuation (22.5/27.5 °C and 20/30 °C) on some development characteristics in five species of fruit flies (Drosophila) originating from areas with different temperature profiles. We demonstrated how species of the same genus can show substantial differences when developing at fluctuating temperatures not always predictable by development at comparable constant temperature (25 °C). Abstract Temperature has profound effects on biochemical processes as suggested by the extensive variation in performance of organisms across temperatures. Nonetheless, the use of fluctuating temperature (FT) regimes in laboratory experiments compared to constant temperature (CT) regimes is still mainly applied in studies of model organisms. We investigated how two amplitudes of developmental temperature fluctuation (22.5/27.5 °C and 20/30 °C, 12/12 h) affected several fitness-related traits in five Drosophila species with markedly different thermal resistance. Egg-to-adult viability did not change much with temperature except in the cold-adapted D. immigrans. Developmental time increased with FT among all species compared to the same mean CT. The impact of FT on wing size was quite diverse among species. Whereas wing size decreased quasi-linearly with CT in all species, there were large qualitative differences with FT. Changes in wing aspect ratio due to FT were large compared to the other traits and presumably a consequence of thermal stress. These results demonstrate that species of the same genus but with different thermal resistance can show substantial differences in responses to fluctuating developmental temperatures not predictable by constant developmental temperatures. Testing multiple traits facilitated the interpretation of responses to FT in a broader context.

1. Insects are susceptible to dehydration, and change in atmospheric humidity could affect their fitness. To understand the impacts of humidity changes on insect's reproductive fitness, we released an outcrossed Drosophila melanogaster population to outdoor mesocosm units (1.5 × 1.5 × 1.5 m in dimension) and tracked their fecundity over 90 days under progressively developing summer season. The study was carried out in a tropical urban garden. 2. The experimental design included tracking an out-crossed D. melanogaster population over 90 days in two sets of cages. One set of population was kept under outdoor conditions. The second set was maintained under laboratory conditions where only temperature was controlled. For 90 days, data on daily egg laying were collected for both indoor and outdoor cages. This design helped us to see temperature and humidity impact on reproductive output both in combination and separately. 3. Often temperature has been found to be the key player in changes in reproductive output in a considerable number of laboratory-based studies. Our work suggested that ‘temperature-humidity’ interactions determine the physiological state of an organism which could untimely impact organismal fitness in a given environmental set-up. This work indicated that under natural conditions fecundity in Drosophila populations was significantly influenced by relative humidity and its interaction with temperature. 4. Our results suggested that while temperature was an important parameter for fecundity, relative humidity individually and in combination with temperature also played an important role in Drosophila reproductive output. Thus, the combination of temperature and relative humidity was a better metric to predict the fecundity in Drosophila populations than considering these parameters individually under natural conditions. It clearly indicated that future warming events could drastically impact insects' reproductive output.

Plastic responses to heat stress have been shown to temporarily increase heat stress tolerance in many small ectotherms. Heat shock proteins (Hsps) have previously been shown to play a role in this induced heat stress tolerance. The heat shock response is fast but short lived, with the cellular Hsp concentration peaking within a few hours after induction. Induced heat stress tolerance, on the other hand, peaks 16–32 h after induction. Therefore, the inducible heat stress response must depend on additional mechanisms. The Turandot gene family has been suggested as a candidate. It contains eight genes that are all upregulated to some degree following heat stress in Drosophila melanogaster. Previously, Turandot A (totA) and Turandot X (totX) have been linked to induced heat stress tolerance. The study presented here aimed to investigate the temporal dynamics of Turandot expression and the functional role of totA and totC for heat stress tolerance. This was done by assaying the temporal heat tolerance and Turandot gene expression after a heat insult, and by exposing Turandot gene knock down flies to a range of heat hardening treatments, and evaluating the effects on heat tolerance. Successful gene knock down was verified by gene expression assays. In addition, expression of hsp70A was included. Both totA, totC, and hsp70A expression increased following a heat hardening treatment, while the results for totX were less clear. The expression of totC temporally co-occurred with and was functionally linked to increased heat tolerance. Expression of totA did not have a significant effect on heat stress tolerance. The complexity of inducible heat tolerance was underlined by the result that knock down of Turandot genes led to increased expression of hsp70. The results suggest that heat tolerance is determined by the interaction between several mechanisms, of which Turandot genes constitute one such mechanism.

Recently it has been proposed, that the holobiont, i.e., the host and its associated microbiome, constitute a distinct biological entity, on which selection operates. This is a fascinating idea that so far has limited empirical justification. Here Drosophila melanogaster lines from a large-scale artificial selection experiment, where we selected for stress resistance traits and for longevity, were used to test the hologenome hypothesis. We raised flies from all selection regimes, including a regime where flies were kept at benign standard laboratory condition (control regime) throughout the duration of the experiment, under common garden conditions and sequenced the microbiome of the flies. We found abundant differences in microbial communities between control and selection regimes, but not between replicate lines within the regimes, and microbial diversity was higher in selected relative to control lines. Several major core Drosophila bacterial species were differentially abundant in the different selection regimes despite flies being exposed to similar nutritional and general environmental conditions. Our results support the idea that the host and microbiome genomes have evolved in concert and provide experimental support for the hologenome theory of evolution.

Pathogens can alter the thermal tolerances of insects; these changes can have cascading impacts across trophic levels in terrestrial food webs. However, the effects of fungal infections on thermal tolerances and behavioral responses to extreme temperatures (ET) across trophic levels have rarely been studied. We examined how a fungal pathogen, Beauveria bassiana, affects the thermal physiology and behavior of an herbivorous insect, Acyrthosiphon pisum, and a predator beetle, Hippodamia convergens. We compared thermal tolerance limits (CTMin and CTMax), thermal boldness (voluntary exposure to ET), energetic cost (ATP) posed by each response (thermal tolerance and boldness) between healthy and infected insects with two fungal loads. Results showed that fungal infection reduced upper thermal tolerance (CTMax) of aphids and beetles, as well as lower thermal tolerance (CTMin) of beetles. CTMin of aphids was not altered by infection. Healthy aphids and beetles crossed warm ET zones (ETZ). Fungal infection modified the tendency, or boldness, of aphids and predator beetles to either cross warm or cold ETZ. ATP levels increased with pathogen infection in insect species, and the highest ATP levels were found in individuals that crossed cold ETZ. Fungal infection narrows the thermal tolerance range and inhibited thermal boldness behaviors to cross ET which may have implications for predator-pay interactions, food web structures, and species distributions. As environmental temperature rises, response to thermal stress will be asymmetric among members of a food web at different trophic levels.

Photoperiod is one of the most reliable seasonal cues that organisms can use to prepare for upcoming environmental changes. Evidence suggests that exposure to different photoperiod can activate plastic responses in stress resistance traits, while there is limited evidence on the plastic response induced by daily progressive cumulative changes in photoperiod. In this study, we assayed the effect of within generation daily uni-directional and cumulative changes in photoperiod on stress resistance and life history traits in four Drosophila species. We predicted that daily increasing photoperiod, mimicking upcoming summer conditions, should lead to an increase in heat resistance and establish trade-offs with other fitness related traits. On the other hand, we predicted that daily decreasing photoperiod should reflect upcoming winter conditions leading to an increase in cold resistance. We found that within genreation changes in photoperiod had a significant effect on life history and stress resistance traits in the four Drosophila species. The observed response was different across species, with D. melanogaster showing five out of six studied traits affected, while in D. mercatorum only one trait was significantly affected. The exposure to changing photoperiod led to an increased upper thermal resistance in D. melanogaster and D. mercatorum and a decreased lower thermal resistance in D. melanogaster and D. simulans, as well as a decreased starvation and desiccation resistance in D. virilis. The developmental time was shorter when flies were exposed to the two photoperiod regimes compared to constant day length control in D. melanogaster and D. simulans. A limited effect was observed on egg-to-adult-viability and desiccation resistance. The results of this study show that daily change in photoperiod induced a plastic response in different traits of drosophilids, suggesting that this environmental parameter needs to be carefully considered in evolutionary studies.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last 20 years. A major challenge is the integration of these disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution and population structure 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 population samples from over 100 locations in >20 countries on four continents based on a combination of 121 unpublished and 150 previously published genomic datasets. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. 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 dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, 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 dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. 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 dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail., DrosEU is funded by a Special Topic Networks (STN) grant from the European Society for Evolutionary Biology (ESEB). MK (M. Kapun) was supported by the Austrian Science Foundation (grant no. FWF P32275); JG by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900) and by the Spanish Ministry of Science and Innovation (BFU-2011-24397); TF by the Swiss National Science Foundation (SNSF grants PP00P3_133641, PP00P3_165836, and 31003A_182262) and a Mercator Fellowship from the German Research Foundation (DFG), held as a EvoPAD Visiting Professor at the Institute for Evolution and Biodiversity, University of Münster; AOB by the National Institutes of Health (R35 GM119686); MK (M. Kankare) by Academy of Finland grant 322980; VL by Danish Natural Science Research Council (FNU) grant 4002-00113B; FS Deutsche Forschungsgemeinschaft (DFG) grant STA1154/4-1, Project 408908608; JP by the Deutsche Forschungsgemeinschaft Projects 274388701 and 347368302; AU by FPI fellowship (BES-2012-052999); ET Israel Science Foundation (ISF) grant 1737/17; MSV, MSR and MJ by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200178); AP, KE and MT by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (451-03-68/2020-14/200007); and TM NSERC grant RGPIN-2018-05551.

In the past the European otter Lutra lutra was distributed throughout most of Europe but since the 1980s its distributional range has been reduced. Currently, the otter population is increasing. Conservation efforts have been implemented, however due to the natural elusiveness of the species it is difficult to monitor. Non-invasive sampling has proven to be the most effective method to derive population parameters such as presence/absence, genetic variability and population structure in European otters. The method to collect non-invasive samples is robust and provides reliable data. This study investigates the validity of the present state-of-The-Art method of identifying otter feces, and suggests modifications and improvements of the method. Results from the comparison of field collected data and data derived from a blind test show that the method is applicable in areas abundant with otters, however the method loses its power in the periphery of the distributional range. In these areas, it would be relevant to supplement traditional sampling with DNA analysis to verify the identification of the sample.

Experiments manipulating the nutritional environment and the associated microbiome of animals have demonstrated their importance for key fitness components. However, there is little information on how macronutrient composition and bacterial communities in natural food sources vary across seasons in nature and on how these factors affect the fitness components of insects. In this study, diet samples from an orchard compost heap, which is a natural habitat for many Drosophila species and other arthropods, were collected over 9 months covering all seasons in a temperate climate. We developed D. melanogaster on diet samples and investigated stress resistance and life-history traits as well as the microbial community of flies and compost. Nutrient and microbial community analysis of the diet samples showed marked differences in macronutrient composition and microbial community across seasons. However, except for the duration of development on these diet samples and Critical Thermal maximum, fly stress resistance and life-history traits were unaffected. The resulting differences in the fly microbial community were also more stable and less diverse than the microbial community of the diet samples. Our study suggests that when D. melanogaster are exposed to a vastly varying nutritional environment with a rich, diverse microbial community, the detrimental consequences of an unfavourable macronutrient composition are offset by the complex interactions between microbes and nutrients.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses., M.W. was supported by the UK Natural Environmental Research Council through the E3 doctoral training programme (NE/L002558/1), and S.R. was supported by Wellcome Trust PhD programme (108905/Z/15/Z). A.B. received funding from BBSRC (grant number BB/P00685X/1). T.F. received funding from Swiss National Science Foundation (grant numbers 31003A-182262, PP00P3_165836, and PP00P3_133641/1). C.G. received funding from Agence Nationale de la Recherche (grant number ANR-15-CE32-0011-01). J.G. received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (H2020-ERC-2014-CoG-647900) and from the Fundación Española para la Ciencia y la Tecnologia-Ministerio de Economía y Competitividad (FCT-15-10187). S.G. received funding from Deutsche Forschungsgemeinschaft (grant number GR 4495/2). M.K. received funding from Academy of Finland projects (268214 and 322980). M.K. received funding from Austrian Science Fund (FWF; grant number P32275). V.L. received funding from Danish Research council for natural Sciences (FNU; grant number 4002-00113B). B.S.O. received funding from the Scientific and Technological Research Council of Turkey (TUBITAK; grant number 214Z238). J.P. received funding from Deutsche Forschungsgemeinschaft (grant number PA 903/8). M.S.-R., M.S.V., and M.J. received funding from the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 451-03-68/2020-14/200178). F.S. received funding from Deutsche Forschungsgemeinschaft (grant number STA1154/4-1; Projektnummer 408908608). M.T., A.P., and K.E. received funding from the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 451-03-68/2020-14/200007). The DrosEU consortium has been funded by a Special Topics Network (STN) grant by the European Society of Evolutionary Biology (ESEB).

Drosophila melanogasteris an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of theDrosophilavirome. Here we report fourteen DNA viruses detected in a metagenomic analysis of approximately 6500 pool-sequencedDrosophila, sampled from 47 European locations between 2014 and 2016. These include three new Nudiviruses, a new and divergent Entomopox virus, a virus related toLeptopilina boulardifilamentous virus, and a virus related toMusca domesticasalivary gland hypertrophy virus. We also find an endogenous genomic copy of Galbut virus, a dsRNA Partitivirus, segregating at very low frequency. Remarkably, we find thatDrosophilaVesanto virus, a small DNA virus previously described as a Bidnavirus, may be composed of up to 12 segments and represent a new lineage of segmented DNA viruses. Two of the DNA viruses,DrosophilaKallithea nudivirus andDrosophilaVesanto virus are relatively common, found in 2% or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly-available datasets, withDrosophilaKallithea nudivirus andDrosophilaVesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses inD. melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies inDrosophila, and lay the foundation needed to harness the power ofDrosophilaas a model system for the study of DNA viruses.

Organisms are exposed to temperatures that vary, for example on diurnal and seasonal time scales. Thus, the ability to behaviorally and/or physiologically respond to variation in temperatures is a fundamental requirement for long-term persistence. Studies on thermal biology in ectotherms are typically performed under constant laboratory conditions, which differ markedly from the variation in temperature across time and space in nature. Here, we investigate evolutionary adaptation and environmentally induced plastic responses of Drosophila simulans to no fluctuations (constant), predictable fluctuations or unpredictable fluctuations in temperature. We whole-genome sequenced populations exposed to 20 generations of experimental evolution under the three thermal regimes and examined the proteome after short-term exposure to the same three regimes. We find that unpredictable fluctuations cause the strongest response at both genome and proteome levels. The loci showing evolutionary responses were generally unique to each thermal regime, but a minor overlap suggests either common laboratory adaptation or that some loci were involved in the adaptation to multiple thermal regimes. The evolutionary response, i.e., loci under selection, did not coincide with induced responses of the proteome. Thus, genes under selection in fluctuating thermal environments are distinct from genes important for the adaptive plastic response observed within a generation. This information is key to obtain a better understanding and prediction of the effects of future increases in both mean and variability of temperatures.

Understanding the genetic underpinnings of complex traits requires knowledge of the genetic variants that contribute to phenotypic variability. Reliable statistical approaches are needed to obtain such knowledge. In genome-wide association studies, variants are tested for association with trait variability to pinpoint loci that contribute to the quantitative trait. Because stringent genome-wide significance thresholds are applied to control the false positive rate, many true causal variants can remain undetected. To ameliorate this problem, many alternative approaches have been developed, such as genomic feature models (GFM). The GFM approach tests for association of set of genomic markers, and predicts genomic values from genomic data utilizing prior biological knowledge. We investigated to what degree the findings from GFM have biological relevance. We used the Drosophila Genetic Reference Panel to investigate locomotor activity, and applied genomic feature prediction models to identify gene ontology (GO) categories predictive of this phenotype. Next, we applied the covariance association test to partition the genomic variance of the predictive GO terms to the genes within these terms. We then functionally assessed whether the identified candidate genes affected locomotor activity by reducing gene expression using RNA interference. In five of the seven candidate genes tested, reduced gene expression altered the phenotype. The ranking of genes within the predictive GO term was highly correlated with the magnitude of the phenotypic consequence of gene knockdown. This study provides evidence for five new candidate genes for locomotor activity, and provides support for the reliability of the GFM approach. Understanding the genetic underpinnings of complex traits requires knowledge of the genetic variants that contribute to phenotypic variability. Reliable statistical approaches are needed to obtain such knowledge. In genome-wide association studies, variants are tested for association with trait variability to pinpoint loci that contribute to the quantitative trait. Because stringent genome-wide significance thresholds are applied to control the false positive rate, many true causal variants can remain undetected. To ameliorate this problem, many alternative approaches have been developed, such as genomic feature models (GFM). The GFM approach tests for association of set of genomic markers, and predicts genomic values from genomic data utilizing prior biological knowledge. We investigated to what degree the findings from GFM have biological relevance. We used the Drosophila Genetic Reference Panel to investigate locomotor activity, and applied genomic feature prediction models to identify gene ontology (GO) cate- gories predictive of this phenotype. Next, we applied the covariance association test to partition the genomic variance of the predictive GO terms to the genes within these terms. We then functionally assessed whether the identified candidate genes affected locomotor activity by reducing gene expression using RNA interference. In five of the seven candidate genes tested, reduced gene expression altered the phenotype. The ranking of genes within the predictive GO term was highly correlated with the magnitude of the phenotypic consequence of gene knockdown. This study provides evidence for five new candidate genes for locomotor activity, and provides support for the reliability of the GFM approach

Climatic conditions can be very heterogeneous even over small geographic scales, and are believed to be major determinants of the abundance and distribution of species and populations. Organisms are expected to evolve in response to the frequency and magnitude of local thermal extremes, resulting in local adaptation. Using replicate yellow dung fly (Scathophaga stercoraria; Diptera: Scathophagidae) populations from cold (northern Europe) and warm climates (southern Europe), we compared 1) responses to short-term heat and cold shocks in both sexes, 2) heat shock protein (Hsp70) expression in adults and eggs, and 3) female reproductive traits when facing short-term heat stress during egg maturation. Contrary to expectations, thermal traits showed minor geographic differentiation, with weak evidence for greater heat resistance of southern flies but no differentiation in cold resistance. Hsp70 protein expression was little affected by heat stress, indicating systemic rather than induced regulation of the heat stress response, possibly related to this fly group's preference for cold climes. In contrast, sex differences were pronounced: males (which are larger) endured hot temperatures longer, while females featured higher Hsp70 expression. Heat stress negatively affected various female reproductive traits, reducing first clutch size, overall reproductive investment, egg lipid content, and subsequent larval hatching. These responses varied little across latitude but somewhat among populations in terms of egg size, protein content, and larval hatching success. Several reproductive parameters, but not Hsp70 expression, exhibited heritable variation among full-sib families. Rather than large-scale clinal geographic variation, our study suggests some local geographic population differentiation in the ability of yellow dung flies to buffer the impact of heat stress on reproductive performance.

textcopyright 2017 John Wiley Sons Ltd. The effective population size (N e ) is a central factor in determining maintenance of genetic variation. The neutral theory predicts that loss of variation depends on N e , 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 N e 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. (Less)

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.

Insects are known to selectively balance their intake of protein and carbohydrate to optimize reproduction and survival. For insects who feed on decomposing fruit, fluctuations in macronutrient composition occur as fruits ripe and decomposition progresses which may challenge optimal resource allocation. Using Drosophila melanogaster, we tested the effect of macronutrient fluctuations and the variability of these fluctuations on starvation resistance and components of reproductive output; traits known to be sensitive to different protein to carbohydrate (P:C) ratios in the diet. For 8 days, flies were fed the same protein to carbohydrate (P:C) ratio (constant feeding), or fed diets with fluctuations in P:C ratio on each day; these fluctuations being regular (predictably fluctuating) or irregular (unpredictably fluctuating). The three feeding regimes yielded the same average P:C ratio across the duration of the experiment. We found no difference in starvation resistance across the feeding regimes. Interestingly, there was a sexual dimorphism in the effect on reproductive output with males performing worst in the unpredictable feeding regime, and with no effect of feeding regime on female performance. Our study provides evidence for means of adapting to fluctuating macronutrient composition and suggests females are more tactful than males in storing and allocating resources for reproduction.

Environmental stresses such as extreme temperatures, dehydration and food deprivation may have distinct consequences for different age-classes and for males and females across species. Here we investigate a natural population of the model organism Drosophila melanogaster. Males and females at ages 3, 19 and 35 days were tested for stress resistance; i.e. the ability of flies to cope with starvation and both cold and hot temperatures. Further, we tested a measure of metabolic efficiency, namely mitochondrial DNA copy number (mtDNA CN) in both sexes at all three age-classes. We hypothesize that stress resistance is reduced at old age and more so in males, and that mtDNA CN is a biomarker for sex- and age-dependent reductions in the ability to cope with harsh environments. We show that: (1) males exhibit reduced starvation tolerance at old age, whereas older females are better in coping with periods without food compared to younger females, (2) heat tolerance decreases with increasing age in males but not in females, (3) cold tolerance is reduced at old age in both sexes, and (4) old males have reduced mtDNA CN whereas mtDNA CN slightly increases with age in females. In conclusion, our data provide strong evidence for trait and sex specific consequences of aging with females generally being better at coping with environmental stress at old age. The reduced mtDNA CN in old males suggests reduced metabolic efficiency and this may partly explain why males are less stress tolerant at old age than females. We suggest that mtDNA CN might be a suitable biomarker for physiological robustness. Our findings likely extend to other taxa than Drosophila and therefore we discuss the observations in relation to aging and sex specific lifespan across species.

The thermal biology of ectotherms is often used to infer species' responses to changes in temperature. It is often proposed that temperate species are more cold-tolerant, less heat-tolerant, more plastic, have broader thermal performance curves (TPCs) and lower optimal temperatures when compared to tropical species. However, relatively little empirical work has provided support for this using large interspecific studies. In the present study, we measure thermal tolerance limits and thermal performance in 22 species of Drosophila that developed under common conditions. Specifically, we measure thermal tolerance (CT min and CT max ) as well as the fitness components viability, developmental speed and fecundity at seven temperatures to construct TPCs for each of these species. For 10 of the species, we also measure thermal tolerance and thermal performance following developmental acclimation to three additional temperatures. Using these data, we test several fundamental hypotheses about the evolution and plasticity of heat and cold resistance and thermal performance. We find that cold tolerance (CT min ) varied between the species according to the environmental temperature in the habitat from which they originated. These data support the idea that the evolution of cold tolerance has allowed species to persist in colder environments. However, contrary to expectation, we find that optimal temperature ( T opt ) and the breadth of thermal performance ( T breadth ) are similar in temperate, widespread and tropical species and we also find that the plasticity of TPCs was constrained. We suggest that the temperature range for optimal thermal performance is either fixed or under selection by the more similar temperatures that prevail during growing seasons. As a consequence, we find that T opt and T breadth are of limited value for predicting past, present and future distributions of species. This article is part of the theme issue ‘Physiological diversity, biodiversity patterns and global climate change: testing key hypotheses involving temperature and oxygen’.

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. Summary statement. Drosophila melanogaster and D. subobscura differ in heat shock protein expression levels, likely mediated by evolved differences in promoter regions. This corroborates differences in acclimation capacities between the two species.

Aging is a multifactorial trait caused by early as well as late-life circumstances. A society trend that parents deliberately delay having children is of concern to health professionals, for example as advanced parental age at conception increases disease risk profiles in offspring. We here aim to study if advanced parental age at conception affects mitochondrial DNA content, a cross-species biomarker of general health, in adult human twin offspring and in a model organism. We find no deteriorated mitochondrial DNA content at advanced parental age at conception, but human mitochondrial DNA content was higher in females than males, and the difference was twofold higher at advanced maternal age at conception. Similar parental age effects and sex-specific differences in mitochondrial DNA content were found in Drosophila melanogaster. In addition, parental longevity in humans associates with both mitochondrial DNA content and parental age at conception; thus, we carefully propose that a poorer disease risk profile from advanced parental age at conception might be surpassed by superior effects of parental successful late-life reproduction that associate with parental longevity.

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.

Heat-induced hormesis, the beneficial effect of mild heat-induced stress, increases the average lifespan of many organisms. Yet little is known about the mechanisms underlying this effect. We used nuclear magnetic resonance spectroscopy to investigate the long-term effects of repeated mild heat treatments on the metabolome of male Drosophila melanogaster. 10 days after the heat treatment, metabolic aging appears to be slowed down, and a treatment response with 40 % higher levels of alanine and lactate and lower levels of aspartate and glutamate were measured. All treatment effects had disappeared 16 days later. Metabolic reprogramming has been associated with the life extending effects of dietary restriction. The metabolite changes induced by the hormetic treatment suggest that the positive effects might not be limited to the repair pathways induced, but that there also is a change in energy metabolism. A possible direct link between changes in energy metabolism and heat induced increase in Hsp70 expression is discussed.

Heat tolerance is commonly assessed as the critical thermal maximum (CTmax) using the dynamic method exposing organisms to a gradually increasing (ramping) temperature until organisms fall into a coma. The CTmax estimate is dependent on the ramping rate, with decreased rates leading to longer treatments and ultimately lower CTmax estimates. There is a current discussion surrounding the physiological dynamics of the effect of the time of exposure by temperature interaction on these estimates. Besides temperature the time of exposure to limited food (starvation), desiccation, and reduced levels of oxygen or increased levels of CO2 may, in interaction with ramping rate, act as confounding factors when assessing upper thermal limits using the dynamic method. Here we test the role of the different potentially confounding factors for assaying thermal tolerance using a ramping assay under four different ramping rates, varying from 0.01 °C/min to 0.2 °C/min. We find that CTmax values are higher at faster ramping rates and that oxygen or CO2 concentration does not show any negative effect on the CTmax values obtained within the experimental pre-treatment period (32 h). Both water (up to 6 h) and food (up to 42 h) deprivation prior to assay showed a negative correlation with thermal tolerance of the flies. For both traits, we found a significant interaction with ramping rate, most likely due to prolonged assays at lower rates. However, as little water was lost during the ramping assay and as food deprivation only modestly affected CTmax values, results were very robust to the conditions experienced during the assay (even at slow rates) and mainly affected by the conditions experienced prior to performing the assay. Thus, for the most commonly applied experimental conditions CTmax estimates are unlikely to be biased or confounded by ramping rate, starvation, desiccation or deteriorating atmospheric composition.