Elegans Populations Research Articles

Droplet digital PCR as a tool for investigating dynamics of cryptic symbionts.

Interactions among symbiotic organisms and their hosts are major drivers of ecological and evolutionary processes. Monitoring the infection patterns among natural populations and identifying factors affecting these interactions are critical for understanding symbiont–host relationships. However, many of these interactions remain understudied since the knowledge about the symbiont species is lacking, which hinders the development of appropriate tools. In this study, we developed a digital droplet PCR (ddPCR) assay based on apicomplexan COX1 gene to detect an undescribed agamococcidian symbiont. We show that the method gives precise and reproducible results and enables detecting cryptic symbionts in low target concentration. We further exemplify the assay's use to survey seasonally sampled natural host (Pygospio elegans) populations for symbiont infection dynamics. We found that symbiont prevalence differs spatially but does not show seasonal changes. Infection load differed between populations and was low in spring and significantly increased towards fall in all populations. We also found that the symbiont prevalence is affected by host length and population density. Larger hosts were more likely to be infected, and high host densities were found to have a lower probability of infection. The observed variations could be due to characteristics of both symbiont and host biology, especially the seasonal variation in encounter rates. Our findings show that the developed ddPCR assay is a robust tool for detecting undescribed symbionts that are otherwise difficult to quantify, enabling further insight into the impact cryptic symbionts have on their hosts.

Natural variation in fecundity is correlated with species-wide levels of divergence in Caenorhabditis elegans.

Life history traits underlie the fitness of organisms and are under strong natural selection. A new mutation that positively impacts a life history trait will likely increase in frequency and become fixed in a population (e.g., a selective sweep). The identification of the beneficial alleles that underlie selective sweeps provides insights into the mechanisms that occurred during the evolution of a species. In the global population of Caenorhabditis elegans, we previously identified selective sweeps that have drastically reduced chromosomal-scale genetic diversity in the species. Here, we measured the fecundity of 121 wild C. elegans strains, including many recently isolated divergent strains from the Hawaiian islands and found that strains with larger swept genomic regions have significantly higher fecundity than strains without evidence of the recent selective sweeps. We used genome-wide association (GWA) mapping to identify three quantitative trait loci (QTL) underlying the fecundity variation. In addition, we mapped previous fecundity data from wild C. elegans strains and C. elegans recombinant inbred advanced intercross lines that were grown in various conditions and detected eight QTL using GWA and linkage mappings. These QTL show the genetic complexity of fecundity across this species. Moreover, the haplotype structure in each GWA QTL region revealed correlations with recent selective sweeps in the C. elegans population. North American and European strains had significantly higher fecundity than most strains from Hawaii, a hypothesized origin of the C. elegans species, suggesting that beneficial alleles that caused increased fecundity could underlie the selective sweeps during the worldwide expansion of C. elegans.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses. Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Alvaxanthone, a Thymidylate Synthase Inhibitor with Nematocidal and Tumoricidal Activities.

With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.

Evaluation of Carotenoids Protection Against Oxidative Stress in the Animal Model Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a versatile and powerful model organism for animal experimental research and, despite being an invertebrate, displays remarkably similar molecular bases and conserved cellular pathways to those of humans. Oxidative stress is an etiological factor that influences numerous diseases, degenerative processes and aging. C. elegans has revealed as an opportune and feasible organism to investigate the antioxidant effects of different bioactives or complex food matrices, and a number of protocols have been developed by using different oxidative stressors. Carotenoids are recognized as quenchers and scavengers of reactive oxygen species, and many of their related health benefits attributed in the diet are tightly linked to their antioxidant properties. In this chapter, we report a simple and rapid assay to evaluate the protection capacity of pure carotenoids or complex carotenoid extracts against oxidative stress in the model system C. elegans. The protocol describes a representative feeding experiment by adding carotenoids to the nematode growth medium and after an incubation period, the C. elegans populations fed with carotenoids are exposed to an acute oxidative stress by using H2O2 as oxidative agent. The protection against oxidative stress is evaluated as the survival rate of the nematodes fed with the carotenoid prior to receiving oxidative treatment compared with the survival rate of control nematode population. In order to confirm the carotenoid intake by the nematodes during the feeding experiment a bioassimilation experiment is also reported.

Tightly linked antagonistic-effect loci underlie polygenic phenotypic variation in C. elegans.

Recent work has provided strong empirical support for the classic polygenic model for trait variation. Population‐based findings suggest that most regions of genome harbor variation affecting most traits. Here, we use the approach of experimental genetics to show that, indeed, most genomic regions carry variants with detectable effects on growth and reproduction in Caenorhabditis elegans populations sensitized by nickel stress. Nine of 15 adjacent intervals on the X chromosome, each encompassing ∼0.001 of the genome, have significant effects when tested individually in near‐isogenic lines (NILs). These intervals have effects that are similar in magnitude to those of genome‐wide significant loci that we mapped in a panel of recombinant inbred advanced intercross lines (RIAILs). If NIL‐like effects were randomly distributed across the genome, the RIAILs would exhibit phenotypic variance that far exceeds the observed variance. However, the NIL intervals are arranged in a pattern that significantly reduces phenotypic variance relative to a random arrangement; adjacent intervals antagonize one another, cancelling each other's effects. Contrary to the expectation of small additive effects, our findings point to large‐effect variants whose effects are masked by epistasis or linkage disequilibrium between alleles of opposing effect.

Selection and gene flow shape niche-associated variation in pheromone response

From quorum sensing in bacteria to pheromone signaling in social insects, chemical communication mediates interactions among individuals in a local population. In Caenorhabditis elegans, ascaroside pheromones can dictate local population density, in which high levels of pheromones inhibit the reproductive maturation of individuals. Little is known about how natural genetic diversity affects the pheromone responses of individuals from diverse habitats. Here, we show that a niche-associated variation in pheromone receptor genes contributes to natural differences in pheromone responses. We identified putative loss-of-function deletions that impair duplicated pheromone receptor genes (srg-36 and srg-37), which were shown previously to be lost in population-dense laboratory cultures. A common natural deletion in srg-37 arose recently from a single ancestral population that spread throughout the world and underlies reduced pheromone sensitivity across the global C. elegans population. We found that many local populations harbor individuals with wild-type or a deletion allele of srg-37, suggesting that balancing selection has maintained the recent variation in this pheromone receptor gene. The two srg-37 genotypes are associated with niche diversity underlying boom-and-bust population dynamics. We hypothesize that human activities likely contributed to the gene flow and balancing selection of srg-37 variation through facilitating migration of species and providing favorable niche for recently arose srg-37 deletion.

Partial Selfing Can Reduce Genetic Loads While Maintaining Diversity During Experimental Evolution.

Partial selfing, whereby self- and cross- fertilization occur in populations at intermediate frequencies, is generally thought to be evolutionarily unstable. Yet, it is found in natural populations. This could be explained if populations with partial selfing are able to reduce genetic loads and the possibility for inbreeding depression while keeping genetic diversity that may be important for future adaptation. To address this hypothesis, we compare the experimental evolution of Caenorhabditis elegans populations under partial selfing, exclusive selfing or predominant outcrossing, while they adapt to osmotically challenging conditions. We find that the ancestral genetic load, as measured by the risk of extinction upon inbreeding by selfing, is maintained as long as outcrossing is the main reproductive mode, but becomes reduced otherwise. Analysis of genome-wide single-nucleotide polymorphisms (SNPs) during experimental evolution and among the inbred lines that survived enforced inbreeding indicates that populations with predominant outcrossing or partial selfing maintained more genetic diversity than expected with neutrality or purifying selection. We discuss the conditions under which this could be explained by the presence of recessive deleterious alleles and/or overdominant loci. Taken together, our observations suggest that populations evolving under partial selfing can gain some of the benefits of eliminating unlinked deleterious recessive alleles and also the benefits of maintaining genetic diversity at partially dominant or overdominant loci that become associated due to variance of inbreeding levels.

Mutational Landscape of Spontaneous Base Substitutions and Small Indels in Experimental Caenorhabditis elegans Populations of Differing Size.

Experimental investigations into the rates and fitness effects of spontaneous mutations are fundamental to our understanding of the evolutionary process. To gain insights into the molecular and fitness consequences of spontaneous mutations, we conducted a mutation accumulation (MA) experiment at varying population sizes in the nematode Caenorhabditis elegans, evolving 35 lines in parallel for 409 generations at three population sizes (N = 1, 10, and 100 individuals). Here, we focus on nuclear SNPs and small insertion/deletions (indels) under minimal influence of selection, as well as their accrual rates in larger populations under greater selection efficacy. The spontaneous rates of base substitutions and small indels are 1.84 (95% C.I. ± 0.14) × 10-9 substitutions and 6.84 (95% C.I. ± 0.97) × 10-10 changes/site/generation, respectively. Small indels exhibit a deletion bias with deletions exceeding insertions by threefold. Notably, there was no correlation between the frequency of base substitutions, nonsynonymous substitutions, or small indels with population size. These results contrast with our previous analysis of mitochondrial DNA mutations and nuclear copy-number changes in these MA lines, and suggest that nuclear base substitutions and small indels are under less stringent purifying selection compared to the former mutational classes. A transition bias was observed in exons as was a near universal base substitution bias toward A/T. Strongly context-dependent base substitutions, where 5'-Ts and 3'-As increase the frequency of A/T → T/A transversions, especially at the boundaries of A or T homopolymeric runs, manifest as higher mutation rates in (i) introns and intergenic regions relative to exons, (ii) chromosomal cores vs. arms and tips, and (iii) germline-expressed genes.

Size Sorting in Bonytail Skews the Sex Ratio of Stocked Fish

AbstractFor sexually dimorphic species, size‐based management can cause changes in the sex ratio of the target population. Bonytail Gila elegans populations in the Colorado River basin are augmented annually in an effort to re‐establish wild populations. To increase survival rates of stocked individuals, the Lower Colorado River Multi‐Species Conservation Program's Habitat Conservation Plan implemented a minimum stocking size. Anecdotal reports on hatchery populations suggest that females grow at a faster rate and reach larger sizes than males. If this observation is correct, a minimum stocking size may cause skewed sex ratios of stocked populations. To test this prediction, we compared average male and female lengths in 3‐year‐old fish from two cohorts before and after they were sorted by size. Our data support the observation that size in Bonytail is sexually dimorphic by age 3, with females being the larger sex. This is also the age at which fish begin to reach the minimum size to meet stocking requirements. As predicted, size‐grading produced a significantly skewed sex ratio in the animals slated for release. The consequences of augmenting wild populations with groups of animals having skewed sex ratios are largely unknown but could include increased mate competition, inadvertent artificial selection, or a reduction in genetic diversity.

A multi-parent recombinant inbred line population of C. elegans allows identification of novel QTLs for complex life history traits

BackgroundThe nematode Caenorhabditis elegans has been extensively used to explore the relationships between complex traits, genotypes, and environments. Complex traits can vary across different genotypes of a species, and the genetic regulators of trait variation can be mapped on the genome using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) derived from genetically and phenotypically divergent parents. Most RILs have been derived from crossing two parents from globally distant locations. However, the genetic diversity between local C. elegans populations can be as diverse as between global populations and could thus provide means of identifying genetic variation associated with complex traits relevant on a broader scale.ResultsTo investigate the effect of local genetic variation on heritable traits, we developed a new RIL population derived from 4 parental wild isolates collected from 2 closely located sites in France: Orsay and Santeuil. We crossed these 4 genetically diverse parental isolates to generate a population of 200 multi-parental RILs and used RNA-seq to obtain sequence polymorphisms identifying almost 9000 SNPs variable between the 4 genotypes with an average spacing of 11 kb, doubling the mapping resolution relative to currently available RIL panels for many loci. The SNPs were used to construct a genetic map to facilitate QTL analysis. We measured life history traits such as lifespan, stress resistance, developmental speed, and population growth in different environments, and found substantial variation for most traits. We detected multiple QTLs for most traits, including novel QTLs not found in previous QTL analysis, including those for lifespan and pathogen responses. This shows that recombining genetic variation across C. elegans populations that are in geographical close proximity provides ample variation for QTL mapping.ConclusionTaken together, we show that using more parents than the classical two parental genotypes to construct a RIL population facilitates the detection of QTLs and that the use of wild isolates facilitates the detection of QTLs. The use of multi-parent RIL populations can further enhance our understanding of local adaptation and life history trade-offs.

Extreme allelic heterogeneity at a Caenorhabditis elegans beta-tubulin locus explains natural resistance to benzimidazoles.

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among multiple nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on the genotyping of three variant sites in the orthologs of the β-tubulin gene found to confer resistance in the free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites to identify additional mechanisms that might contribute to BZ resistance in the field. Here, we took an unbiased genome-wide mapping approach in the free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we found that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in the β-tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and segregates within the C. elegans population, suggesting that selection in natural niches could enrich for resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the C. elegans population, including many novel molecular variants. Population genetic analyses indicate that ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence that these alleles likely occurred recently because of local selective pressures. Additionally, we find purifying selection at all five β-tubulin genes, despite predicted loss-of-function variants in ben-1, indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one β-tubulin gene. Furthermore, we used genome-editing to show that the most common parasitic nematode β-tubulin allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent of ben-1 and the other β-tubulin loci, suggesting that there are multiple mechanisms underlying BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for the study of mechanisms that give rise to BZ resistance.

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms.

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode research community, there is a need for an affordable and effective way to maintain large, age-matched populations of C. elegans. Here, we present a methodology for mechanically sorting and cleaning C. elegans. Our aim is to provide a cost-effective, efficient, fast, and simple process to obtain animals of uniform sizes and life stages for their use in experiments. This tool, the Caenorhabditis Sieve, uses a custom-built lid system that threads onto common conical lab tubes and sorts C. elegans based on body size. We also demonstrate that the Caenorhabditis Sieve effectively transfers animals from one culture plate to another allowing for a rapid sorting, synchronizing, and cleaning without impacting markers of health, including motility and stress-inducible gene reporters. This accessible and innovative tool is a fast, efficient, and non-stressful option for maintaining C. elegans populations.

Mutational and transcriptional landscape of spontaneous gene duplications and deletions in Caenorhabditis elegans

Gene duplication and deletion are pivotal processes shaping the structural and functional repertoire of genomes, with implications for disease, adaptation, and evolution. We employed a mutation accumulation (MA) framework partnered with high-throughput genomics to assess the molecular and transcriptional characteristics of newly arisen gene copy-number variants (CNVs) in Caenorhabditis elegans populations subjected to varying intensity of selection. Here, we report a direct spontaneous genome-wide rate of gene duplication of 2.9 × 10-5/gene per generation in C. elegans, the highest for any species to date. The rate of gene deletion is sixfold lower (5 × 10-6/gene per generation). Deletions of highly expressed genes are particularly deleterious, given their paucity in even the N = 1 lines with minimal efficacy of selection. The increase in average transcript abundance of new duplicates arising under minimal selection is significantly greater than twofold compared with single copies of the same gene, suggesting that genes in segmental duplications are frequently overactive at inception. The average increase in transcriptional activity of gene duplicates is greater in the N = 1 MA lines than in MA lines with larger population bottlenecks. There is an inverse relationship between the ancestral transcription levels of new gene duplicates and population size, with duplicate copies of highly expressed genes less likely to accumulate in larger populations. Our results demonstrate a fitness cost of increased transcription following duplication, which results in purifying selection against new gene duplicates. However, on average, duplications also provide a significant increase in gene expression that can facilitate adaptation to novel environmental challenges.

Molecular and ecological signatures of an expanding hybrid zone.

Many species are currently changing their distributions and subsequently form sympatric zones with hybridization between formerly allopatric species as one possible consequence. The damselfly Ischnura elegans has recently expanded south into the range of its ecologically and morphologically similar sister species Ischnura graellsii. Molecular work shows ongoing introgression between these species, but the extent to which this species mixing is modulated by ecological niche use is not known. Here, we (1) conduct a detailed population genetic analysis based on molecular markers and (2) model the ecological niche use of both species in allopatric and sympatric regions. Population genetic analyses showed chronic introgression between I. elegans and I. graellsii across a wide part of Spain, and admixture analysis corroborated this, showing that the majority of I. elegans from the sympatric zone could not be assigned to either the I. elegans or I. graellsii species cluster. Niche modeling demonstrated that I. elegans has modified its environmental niche following hybridization and genetic introgression with I. graellsii, making niche space of introgressed I. elegans populations more similar to I. graellsii. Taken together, this corroborates the view that adaptive introgression has moved genes from I. graellsii into I. elegans and that this process is enabling Spanish I. elegans to occupy a novel niche, further facilitating its expansion. Our results add to the growing evidence that hybridization can play an important and creative role in the adaptive evolution of animals.

Fitness decline under osmotic stress in Caenorhabditis elegans populations subjected to spontaneous mutation accumulation at varying population sizes.

The consequences of mutations for population fitness depends on their individual selection coefficients and the effective population size. An earlier study of Caenorhabditis elegans spontaneous mutation accumulation lines evolved for 409 generations at three population sizes found thatNe = 1 populations declined significantly in fitness whereas the fitness of larger populations (Ne = 5, 50) was indistinguishable from the ancestral control under benign conditions. To test if larger MA populations harbor a load of cryptic deleterious mutations that are obscured underbenignlaboratory conditions, we measured fitness underosmotic stressvia exposure to hypersaline conditions. The fitness ofNe = 1 lines exhibited a further decline under osmotic stress compared to benign conditions. However, the fitness of larger populations remained indistinguishable from that of the ancestral control. The average effects of deleterious mutations inNe = 1 lines were estimated to be 22% for productivity and 14% for survivorship, exceeding values previously detected underbenignconditions. Our results suggest that fitness decline is due to large effect mutations that are rapidly removed via selection even in small populations, with implications for conservation practices. Genetic stochasticity may not be as potent and immediate a threat to the persistence of small populations as other demographic and environmental stochastic factors.

The Local Coexistence Pattern of Selfing Genotypes in Caenorhabditis elegans Natural Metapopulations.

To study the interplay of rare outcrossing and metapopulation structure, we focus on the nematode Caenorhabditis elegans. Its remarkably low outcrossing rate is at the extreme end of the spectrum for facultative selfing organisms. At the demographic level, C. elegans natural populations undergo boom and bust dynamics on ephemeral resources, with the dauer diapause larva acting as the dispersal form. Here we investigate the small-scale genetic structure of C. elegans populations in two localities over several years, using 2b restriction-associated DNA sequencing of nearly 1000 individuals. We find a remarkably small number of genome-wide haplotypes, almost exclusively in the homozygous state, confirming the low effective outcrossing rate. Most strikingly, the major haplotypes in a locality remain intact and do not effectively recombine over several years. From the spatial pattern of diversity, we estimate that each subpopulation or deme is seeded by a mean of 3–10 immigrating individuals. Populations are thus formed by clones that compete at two levels, within a subpopulation and at the metapopulation level. We test for the presence of local phenotypic variation in pathogen resistance and dauer larva nictation, which could possibly explain the maintenance of different genotypes by heterogeneous selection in different local environments or lifecycles. This study is the first to address the local spatiotemporal genetic structure of C. elegans on feeding substrates. We conclude that these animals coexist as competing homozygous clones at the smallest population scale as well as in the metapopulation.

Assessment of Caenorhabditis elegans Competitive Fitness in the Presence of a Bacterial Parasite.

Accurate measurements of an organism's fitness are crucial for measuring evolutionary change. Methods of fitness measurement are most accurate when incorporating an individual's survival and fecundity, as well as accounting for any ecological interactions or environmental effects experienced by the organism. Here, we describe a protocol for measuring the relative mean fitness of Caenorhabditis elegans populations, or strains, through an assay that accounts for individual survival, fecundity, and intraspecific competitive ability in the presence of a bacterial parasite. In this competitive fitness assay nematodes from a focal population or strain are mixed with a GFP-marked tester strain in equal proportions, the mixture of nematodes are then exposed to a parasite, and the relative competitive fitness of the focal strain is determined by measuring the change in the ratio of focal nematodes to GFP-marked nematodes after one generation. Specifically, this protocol can be implemented to measure changes in nematode host fitness after experimental evolution by determining the relative competitive fitness of evolved versus ancestral nematode populations.

Behavioral Assays to Study Oxygen and Carbon Dioxide Sensing in Caenorhabditis elegans.

Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a C. elegans population behavioral assay used to measure locomotory responses to changes in environmental oxygen (O2) and carbon dioxide (CO2) concentrations. These behavioral assays are high-throughput and enable examination of genetic, neuronal and circuit function.

Adaptation costs to constant and alternating polluted environments.

Some populations quickly adapt to strong and novel selection pressures caused by anthropogenic stressors. However, this short‐term evolutionary response to novel and harsh environmental conditions may lead to adaptation costs, and evaluating these costs is important if we want to understand the evolution of resistance to anthropogenic stressors. In this experimental evolution study, we exposed Caenorhabditis elegans populations to uranium (U populations), salt (NaCl populations) and alternating uranium/salt treatments (U/NaCl populations) and to a control environment (C populations), over 22 generations. In parallel, we ran common‐garden and reciprocal‐transplant experiments to assess the adaptive costs for populations that have evolved in the different environmental conditions. Our results showed rapid evolutionary changes in life history characteristics of populations exposed to the different pollution regimes. Furthermore, adaptive costs depended on the type of pollutant: pollution‐adapted populations had lower fitness than C populations, when the populations were returned to their original environment. Fitness in uranium environments was lower for NaCl populations than for U populations. In contrast, fitness in salt environments was similar between U and NaCl populations. Moreover, fitness of U/NaCl populations showed similar or higher fitness in both the uranium and the salt environments compared to populations adapted to constant uranium or salt environments. Our results show that adaptive evolution to a particular stressor can lead to either adaptive costs or benefits once in contact with another stressor. Furthermore, we did not find any evidence that adaptation to alternating stressors was associated with additional adaption costs. This study highlights the need to incorporate adaptive cost assessments when undertaking ecological risk assessments of pollutants.