Current Research
Overview: Genetic dissection of metal resistance
The Goal: I am currently characterizing the genetic factors that influence copper resistance as a part of a broader goal to understand the genetic control of physiological and behavioral responses to heavy metal pollution.
The Rationale: Metal pollution is a ubiquitous and persistent threat to human and environmental health. Copper is a common pollutant and is an excellent “model metal” for studying heavy metal stress response: many of the genes that metabolize and detoxify copper also interact with non-essential heavy metals (e.g. lead and cadmium). Our work has demonstrated that copper resistance is phenotypically correlated with resistance to other metals (lead, cadmium, and others), with evidence of overlap in the genetic control of these resistance traits.
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The Questions:
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What is the genetic basis of copper resistance?
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Do genetic factors that influence copper resistance have life stage-specific effects?
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Is the physiological response to copper influenced by behavioral responses to copper stress?
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What genetic factors influence the dynamic shift in gene expression in response to copper stress?
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Does copper resistance vary in natural populations in response to pollution resulting from mining activities?
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The Support: Specific projects described below were supported by:
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NIH K99 Pathway to Independence grant (2021-23)
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K-INBRE Matching Funds Award (Office of Research, KU) (2021-22)
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NIH NRSA F32 postdoctoral fellowship (2019-2021)
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K-INBRE Postdoctoral Fellowship (2017-2018)
Extensive, life stage-specific allelic variation
Tissue and treatment-specific patterns in gene expression and control
Complex interplay between genetic, physiological, and behavioral variation
What is the genetic basis of copper resistance?
To the point: The physiological response to copper stress is a genetically complex trait that is influenced by naturally occurring allelic variation in genes responsible for metabolism, detoxification, and transport of copper and other more dangerous heavy metals including lead and cadmium.
A little more detail: I used Quantitative Trait Locus (QTL) mapping to identify genes with allelic variation that influence copper resistance (recently published in Genetics). This work was carried out using >1500 unique, genetically stable strains from the Drosophila Synthetic Population Resource (DSPR, King et al. 2012, Genetics). I measured copper resistance in adult female flies following 48 hr exposure to 50mM Copper(II) Sulfate and mapped 12 regions of the Drosophila genome (QTL) that are associated with adult copper resistance (Fig 1). These regions included hundreds of potential candidate genes, several of which are known to be involved in copper metabolism. This result demonstrates that allelic variation for key copper metabolism genes exists in natural populations despite the delicate balance required for proper copper homeostasis. I narrowed down genes implicated by QTL for functional testing and demonstrated that genes involved in copper metabolism such as copper chaperone for superoxide dismutase (Ccs) as well as genes involved in metabolism of other metals (Znt41F, trpl) influence the adult response to copper stress.
Even more detail:
Everman ER et al. 2021. Genetics. PMCID:PMC8045719; DOI:10.1093/genetics/iyaa020
Figure 1. QTL map of regions of the genome with allelic variants assoc-iated with resistance to copper stress in the DSPR. Genes in call-outs are among those functionally tested with RNAi.
Do genetic factors that influence copper resistance have life stage-specific effects?
To the point: The developmental and adult responses to copper stress is not strongly phenotypically correlated, but does share some overlap of QTL associated with each trait. The genetic factors that contribute to copper resistance are therefore likely life stage-specific.
A little more detail: I measured developmental delay and developmental viability in 100 DSPR strains in response to copper stress (exposed to 2mM Copper(II) Sulfate throughout development). The effect of copper on development was generally negative but was also highly variable across the 100 DSPR strains. In organisms with complex life cycles, trait responses are often decoupled such that for a given genotype, individuals who are resistant to a stress or stimulus as adults may be highly susceptible as larvae. I tested for evidence of decoupling by correlating the developmental responses with adult survival. The relationship was not significant, suggesting that copper resistance in developmental stages is not predictive of adult resistance (and vice versa). I used QTL mapping to identify regions of the genome that may influence developmental response to copper and identified one QTL near the gene mekk1, which has been previously linked to response to cadmium. This QTL overlapped with one of the QTL associated with adult copper resistance, and alleles present at this region had correlated estimated effects on copper resistance across life stages. Collectively, my results suggest that there is evidence of some shared genetic architecture for copper resistance across life stages, although this one site does not completely explain variation in the developmental response to copper. I am currently measuring developmental viability in the remaining DSPR strains to more rigorously address these questions and vet these findings.
Even more detail:
Everman ER et al. 2021. Genetics. PMCID:PMC8045719; DOI:10.1093/genetics/iyaa020
Figure 2. Developmental viability under copper stress and adult copper resistance are not correlated; however, overlap in the genetic architecture of these traits suggests they are not completely independent.
Is the physiological response to copper influenced by behavioral responses to copper stress?
To the point: DSPR strains that are highly resistant to copper also tend to consume less copper-contaminated food in 24hr compared to strains that die more quickly when exposed to copper. Allelic variation is responsible for a large amount of variation in copper resistance, but behavioral aversion to copper also plays a role.
A little more detail: Toxicity induced by heavy metals is typically caused by consumption of metal-contaminated food and water sources. Bioaccumulation of heavy metals through food webs is a particularly concerning effect of metal pollution that can have far-reaching impacts on ecosystem health. Because my assessments of copper resistance involve consumption of copper, I measured variation in copper consumption in 100 DSPR strains to determine whether aversion influenced resistance. Overall, regardless of genotype and resistance level, flies consume less food when copper is present. Interestingly, more copper sensitive strains tended to consume more copper food compared to copper resistant strains raising the question of whether resistant strains live longer simply because they are consuming less toxin. I was able to rule out behavioral aversion to copper consumption through functional tests of candidate genes associated with adult copper resistance; however, it remains clear that behavioral responses to copper play a role in resistance. Furthermore, there is clear evidence from previous work of negative cognitive and behavioral effects of heavy metal exposure. I am currently following up on this work with the following projects:
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Characterization of copper detection ability in the DSPR. I measured copper consumption at a range of low concentrations to identify the concentration at which flies fed differently on copper food compared to control food. This "discriminating concentration" is variable among the 200 DSPR strains measured so far, and the relationship between adult copper resistance and copper consumption has held. I plan to use QTL mapping to identify regions of the genome that contribute to behavioral discrimination against copper.
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Characterization of copper preference among DSPR strains. Consumption of low copper concentrations under "no choice" conditions may result from inability to detect copper or from higher tolerance for the averse taste of copper. To differentiate between these possibilities I am currently developing a high throughput choice assay for a subset of high and low copper resistance DSPR strains.
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Integration between behavioral and physiological responses to copper. In addition to the feeding-specific experiments outlined above, I am also measuring learning capacity in flies that have developed under copper and control conditions. These three projects will produce multiple behavioral trait measure that I will correlate with adult and developmental physiological responses to copper. This work will allow estimation of genetic correlations between these traits that will clarify the whole organism response to copper stress.
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What genetic factors influence the dynamic shift in gene expression in response to copper stress?
To the point: Variation in gene expression can be treated as a quantitative trait that may be influenced by allelic variants that alter expression. The gene expression response to copper stress is highly variable among high and low copper resistance DSPR strains and is tissue-specific. Nearly a quarter of genes are influenced by QTL that alter expression under copper conditions.
A little more detail: I used RNA sequencing to measure the gene expression response to copper stress in head and gut tissues from 96 DSPR strains (totaling 384 mRNAseq libraries each sequenced to 5M reads). I used differential expression (DE) analysis and expression QTL (eQTL) mapping to characterize treatment- and tissue-specific gene expression variation. DE analysis revealed that gene expression variation is influenced by a tissue and treatment interaction, illuminating new patterns for genes with treatment- and tissue-specific expression. Nearly a quarter of the genome was influenced by allelic variants that altered gene expression under copper and control conditions in each tissue (Fig 3). Many of these variants were near genes and may indicate variants in promoter regions that influence transcription factor binding (cis eQTL Fig 3). A smaller number of eQTL located far from the genes with which they were associated may indicate trans regulatory variants that influence pathways or modules of co-expressed genes (trans eQTL Fig 3). Trans eQTL may be especially important for understanding the shift in gene expression due to copper exposure as trans eQTL outnumbered cis eQTL associated with the response to copper (Fig 3). This work demonstrates variation in copper resistance is driven by alleles that impact gene expression and regulation and is some of the first to characterize patterns of gene x environment (GxE) interaction eQTL that influence gene expression variation associated with stress. I am in the process of finalizing the analysis and writing up the results of this work for publication.
Figure 3. Gene expression in each tissue and treatment was influenced by many cis eQTL (totals above bars). Many trans eQTL underlying copper response suggests GxE interactions are influenced by distant regulatory elements. Most cis and trans eQTL are unique to tissue and treatment.
Does copper resistance vary in natural populations in response to pollution resulting from mining activities?
To the point: Drosophila melanogaster populations near mining sites are more resistant to copper toxicity compared to populations collected from agricultural sites and reference panels used for genetic mapping (DSPR and DGRP). Naturally occurring allelic variants in genes involved in oxidative stress response and response to xenobiotic substances contribute to copper resistance in wild flies.
A little more detail: I collected wild mated fe-male Drosophila melanogaster from four populations. Two of the populations were from mining sites (one active, one inactive for ~50 years) and two populations were from agricultural sites (one berry farm, one fruit farm). Offspring from collected females were used to establish large laboratory populations and copper resistance was assessed in animals taken from the 3rd generation post collection to minimize lab adaptation. Copper resistance varied among and within populations, and individuals from the mining populations were most resistant, indicating that those populations are experiencing selection that influences copper resistance. Individuals with highest copper resistance from each population were pooled, as was a random sample of individuals. I extracted DNA from the 8 pools and sequenced them (~125-200x coverage). I am currently analyzing data from this experiment, but preliminary results from one population indicate significant differentiation in allele frequencies between the average and resistant pools for the Berry Farm population at genes involved in oxidative stress response and resistance to chemotherapy drugs.
Even more detail: I am planning to present this project in 2022 at the Evolution meeting.
Figure 4. Copper resistance varies within and among wild populations. DSPR = Drosophila Synthetic Population resource, DGRP = Drosophila Genetic Reference Panel. All populations are significantly different from each other except DGRP and the Berry Farm population (Tukey HSD P << 0.001, exp.-wide alpha = 0.05).
Past Research and Side Projects
Plasticity in Gene Expression Across Pupal Stages
To the point: Development involves dynamic shifts in gene expression. I measured gene expression in early, middle, and late stage pupae and found distinct patterns of gene expression corresponding to each stage.
A little more detail: Quantitative traits are influenced by genetic variation, expression variation, and environmental variation. With this project, I am focusing on variation in gene expression across three developmental stages and two temperatures. Pupal development in Drosophila melanogaster can be staged based on visual characteristics of the developing fly, and the time between each stage is influenced by rearing temperature. Flies reared at cooler temperatures typically take longer to develop compared to flies reared at warmer temperatures. My key question is whether temperature influences gene expression at developmental stages and whether temperature-dependent developmental timing is independent of levels of gene expression.
Evolution and Genetic Control of Stress across Age
Natural selection varies in effectiveness across age, generally becoming less and less effective as organisms become older. This age-specific change in natural selection allows polymorphisms with negative effects on fitness to be maintained in the population, whether through the accumulation of late-acting polymorphisms across generations (mutation accumulation) or through changes in the additive effects of polymorphisms across age (antagonistic pleiotropy). I developed a novel approach to addressing this question that leverages genomic information available from the Drosophila melanogaster Genetic Reference Panel (DGRP) to calculate the effects of age- and phenotype-associated polymorphisms and demonstrated that, while mutation accumulation primarily drives age-related change within phenotypes across age, both mutation accumulation and antagonistic pleiotropy influence age-related change between phenotypes across age.
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More detail: Everman ER and Morgan TJ 2017. Evolution. DOI: 10.1111/evo.13408
Seasonality, Thermal Tolerance, and Plasticity
Drosophila melanogaster
Variation in seasonal temperature is known to influence allele frequencies associated with thermal tolerance in natural Drosophila melanogaster populations, and it is also known that long -term and short-term variation in temperature can result in increased capacity to survive thermal stress. I study the interaction between variation in thermal tolerance and phenotypic plasticity due to seasonal variation in temperature in a natural population of Drosophila melanogaster collected from orchards in Topeka, KS. Interestingly, it appears that increased selection for cold tolerance reduces capacity for phenotypic plasticity--flies collected during warmer periods of the year tend to be much more more plastic compared to flies collected during cooler parts of the year. This raises interesting questions about trade-offs that may constrain the evolution of phenotypic plasticity and thermal tolerance in natural populations.
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More detail: Noh S and Everman ER et al. 2017. Ecol Evol. PMCID: PMC5528237; DOI: 10.1002/ece3.3112
Hemidactylus turcicus
As an introduced species invades a novel environment, it comes into contact with a suite of potentially new drivers of selection. If that species is ectothermic, a fundamental component of its success relates to the capacity of the species to respond to a novel thermal regime. In North America, the introduced Mediterranean gecko, Hemidactylus turcicus, has been expanding north since 1910 and has become established in thermal environments that are both similar to (Galveston, TX; warm and relatively constant) and different from (Oklahoma City, OK; cool and more variable) those it encounters in its native range. Average differences in temperature can lead to selection on both physiological thermotolerance (genetic effects) and thermoregulation (behavioral plasticity). When coupled with seasonal variability, thermal selection can also increase acclimation capacity (physiological plasticity). This theoretical model illustrates how geographic variation leading to differences in the thermal environment (both in terms of average temperature and seasonal variability) can influence the thermal physiology and performance of these geckos. I suspect that these responses will be influenced by a combination of genetic adaptation and phenotypic plasticity. In collaboration with Paul Klawinski at William Jewell College, we examine this theoretical model using 30 individuals collected from each population (Galveston, TX and Oklahoma City, OK) and each season (winter and summer).
Behavioral and Physiological Plasticity
Fitness is determined by the ability of an organism to survive and to reproduce. However, the mechanisms that produce increased survival may not be identical to those that increase reproductive success. In collaboration with Jennifer Gleason at the University of Kansas, I used nineteen natural Drosophila melanogaster genotypes from the Drosophila Genetic Reference Panel to determine if adaptive plasticity following short-term acclimation through rapid cold-hardening (RCH) affects mating behavior and success. We confirmed that exposure to the acclimation temperature is beneficial to survival following cold stress; however, we found that this same acclimation temperature exposure led to less efficient male courtship and a significant decrease in the likelihood of mating. Genotypic variation in RCH capacity was correlated with variation in courtship duration of males not exposed to the acclimation temperature, indicating that the capacity to acclimate can positively influence reproductive fitness, but only in constant environmental conditions. Finally, we tested if the exposure of males to the acclimation temperature influenced courtship song. While exposure to the acclimation temperature again significantly increased courtship duration courtship song was unchanged. These results illustrate a balance between costs and benefits of short-term acclimation on survival and reproductive components of fitness and demonstrate the short-term acclimation environment can have a pronounced effect on reproductive success.
More detail: Everman ER et al. 2018. PLoS One PMCID: PMC5965859; DOI: 10.1371/journal.pone.0197822
Overwintering in Drosophila suzukii
In temperate regions, seasonal and diurnal temperature variation presents novel challenges to small invasive ectotherms; however, phenotypic plasticity can facilitate survival and persistence. In collaboration with members of the Morgan and Ragland Labs at Kansas State University, I tested the role of developmental acclimation, adult long-term acclimation, and photoperiod on the induction of overwintering phenotypes including reproductive diapause and adult survival in a low genetic diversity population of Drosophila suzukii cultured from a recently established population in Topeka, Kansas (USA). We found that both temperature and photoperiod resulted in reduced ovary size and level of development relative to control females. Reduction in ovary development was observed in response to each acclimation temperature, with adult long-term acclimation at 11°C resulting in the largest reduction in ovary development and size. Additionally, reduction in ovary development was observed at warmer temperatures relative to previous reports of the induction of diapause in populations sampled in the northern USA and southern Canada. We also found evidence suggesting that D. suzukii is capable of short-term hardening, contrary to previous reports. Our study highlights the central role of phenotypic plasticity in response to winter-like laboratory conditions and provides an essential geographic comparison to previously published assessments of diapause and short-term hardening survival response for D. suzukii collected in southern Canada.
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More detail: Everman ER et al. 2018. Environmental Entomology. DOI: 10.1093/ee/nvy074
Population Genetics and Invasion of Eleutherodactylus coqui
As an undergraduate, I investigated the role of human-facilitation in the spread of the Coqui frog on Hawai'i Island where it is considered a nuisance due to its loud call and an ecological threat due to its voracious appetite for invertebrates. This project was an awesome introduction to the world of research and is the reason I decided to pursue research as a career. You can read all about my experiences as an undergraduate student here. I used microsatellites to infer genetic relatedness of populations distributed around the road network of Hawai'i Island and ultimately determined that populations were likely established through jump dispersal, where humans had transferred the frogs inadvertently (or on purpose) around the island. This research suggests that the frog has relatively low dispersal rates when unaided by humans, and highlights the importance of early detection and eradication of populations to help control the spread of this species. Read more here!
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More detail: Everman ER and Klawinski PD. 2013. Journal of Biogeography. DOI: https://doi.org/10.1111/jbi.12146
