Transcriptomic analysis provides insights into molecular mechanisms of thermal physiology.

Physiological trait variation underlies health, responses to global climate change, and ecological performance. Yet, most physiological traits are complex, and we have little understanding of the genes and genomic architectures that define their variation. To provide insight into the genetic architecture of physiological processes, we related physiological traits to heart and brain mRNA expression using a weighted gene co-expression network analysis. mRNA expression was used to explain variation in six physiological traits (whole animal metabolism (WAM), critical thermal maximum (CTmax), and four substrate specific cardiac metabolic rates (CaM)) under 12 °C and 28 °C acclimation conditions. Notably, the physiological trait variations among the three geographically close (within 15 km) and genetically similar F. heteroclitus populations are similar to those found among 77 aquatic species spanning 15-20° of latitude (~ 2,000 km). These large physiological trait variations among genetically similar individuals provide a powerful approach to determine the relationship between mRNA expression and heritable fitness related traits unconfounded by interspecific differences. Expression patterns explained up to 82% of metabolic trait variation and were enriched for multiple signaling pathways known to impact metabolic and thermal tolerance (e.g., AMPK, PPAR, mTOR, FoxO, and MAPK) but also contained several unexpected pathways (e.g., apoptosis, cellular senescence), suggesting that physiological trait variation is affected by many diverse genes.

Feeling the heat: variation in thermal sensitivity within and among populations.

Physiology defines individual responses to global climate change and species distributions across environments. Physiological responses are driven by temperature on three time scales: acute, acclimatory and evolutionary. Acutely, passive temperature effects often dictate an expected 2-fold increase in metabolic processes for every 10°C change in temperature (Q10). Yet, these acute responses often are mitigated through acclimation within an individual or evolutionary adaptation within populations over time. Natural selection can influence both responses and often reduces interindividual variation towards an optimum. However, this interindividual physiological variation is not well characterized. Here, we quantified responses to a 16°C temperature difference in six physiological traits across nine thermally distinct Fundulus heteroclitus populations. These traits included whole-animal metabolism (WAM), critical thermal maximum (CTmax) and substrate-specific cardiac metabolism measured in approximately 350 individuals. These traits exhibited high variation among both individuals and populations. Thermal sensitivity (Q10) was determined, specifically as the acclimated Q10, in which individuals were both acclimated and assayed at each temperature. The interindividual variation in Q10 was unexpectedly large: ranging from 0.6 to 5.4 for WAM. Thus, with a 16°C difference, metabolic rates were unchanged in some individuals, while in others they were 15-fold higher. Furthermore, a significant portion of variation was related to habitat temperature. Warmer populations had a significantly lower Q10 for WAM and CTmax after acclimation. These data suggest that individual variation in thermal sensitivity reflects different physiological strategies to respond to temperature variation, providing many different adaptive responses to changing environments.

Evolved genetic and phenotypic differences due to mitochondrial-nuclear interactions.

The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only pathway with both nuclear and mitochondrial encoded proteins. The importance of the interactions between these two genomes has recently received more attention because of their potential evolutionary effects and how they may affect human health and disease. In many different organisms, healthy nuclear and mitochondrial genome hybrids between species or among distant populations within a species affect fitness and OxPhos functions. However, what is less understood is whether these interactions impact individuals within a single natural population. The significance of this impact depends on the strength of selection for mito-nuclear interactions. We examined whether mito-nuclear interactions alter allele frequencies for ~11,000 nuclear SNPs within a single, natural Fundulus heteroclitus population containing two divergent mitochondrial haplotypes (mt-haplotypes). Between the two mt-haplotypes, there are significant nuclear allele frequency differences for 349 SNPs with a p-value of 1% (236 with 10% FDR). Unlike the rest of the genome, these 349 outlier SNPs form two groups associated with each mt-haplotype, with a minority of individuals having mixed ancestry. We use this mixed ancestry in combination with mt-haplotype as a polygenic factor to explain a significant fraction of the individual OxPhos variation. These data suggest that mito-nuclear interactions affect cardiac OxPhos function. The 349 outlier SNPs occur in genes involved in regulating metabolic processes but are not directly associated with the 79 nuclear OxPhos proteins. Therefore, we postulate that the evolution of mito-nuclear interactions affects OxPhos function by acting upstream of OxPhos.

Population genomics of rapid evolution in natural populations: polygenic selection in response to power station thermal effluents.

BACKGROUND: Examples of rapid evolution are common in nature but difficult to account for with the standard population genetic model of adaptation. Instead, selection from the standing genetic variation permits rapid adaptation via soft sweeps or polygenic adaptation. Empirical evidence of this process in nature is currently limited but accumulating. RESULTS: We provide genome-wide analyses of rapid evolution in Fundulus heteroclitus populations subjected to recently elevated temperatures due to coastal power station thermal effluents using 5449 SNPs across two effluent-affected and four reference populations. Bayesian and multivariate analyses of population genomic structure reveal a substantial portion of genetic variation that is most parsimoniously explained by selection at the site of thermal effluents. An FST outlier approach in conjunction with additional conservative requirements identify significant allele frequency differentiation that exceeds neutral expectations among exposed and closely related reference populations. Genomic variation patterns near these candidate loci reveal that individuals living near thermal effluents have rapidly evolved from the standing genetic variation through small allele frequency changes at many loci in a pattern consistent with polygenic selection on the standing genetic variation. CONCLUSIONS: While the ultimate trajectory of selection in these populations is unknown and we survey only a minority of genomic loci, our findings suggest that polygenic models of adaptation may play important roles in large, natural populations experiencing recent selection due to environmental changes that cause broad physiological impacts.

Evolutionary Toxicogenomics of the Striped Killifish (Fundulus majalis) in the New Bedford Harbor (Massachusetts, USA).

In this paper, we used a Genotyping-by-Sequencing (GBS) approach to find and genotype more than 4000 genome-wide SNPs (Single Nucleotide Polymorphisms) from striped killifish exposed to a variety of polychlorinated biphenyls (PCBs) and other aromatic pollutants in New Bedford Harbor (NBH, Massachusetts, USA). The aims of this study were to identify the genetic consequences of exposure to aquatic pollutants and detect genes that may be under selection. Low genetic diversity (HE and π) was found in the site exposed to the highest pollution level, but the pattern of genetic diversity did not match the pollution levels. Extensive connectivity was detected among sampling sites, which suggests that balanced gene flow may explain the lack of genetic variation in response to pollution levels. Tests for selection identified 539 candidate outliers, but many of the candidate outliers were not shared among tests. Differences among test results likely reflect different test assumptions and the complex pollutant mixture. Potentially, selectively important loci are associated with 151 SNPs, and enrichment analysis suggests a likely involvement of these genes with pollutants that occur in NBH. This result suggests that selective processes at genes targeted by pollutants may be occurring, even at a small geographical scale, and may allow the local striped killifish to resist the high pollution levels.

Acclimation and acute temperature effects on population differences in oxidative phosphorylation.

Temperature changes affect metabolism on acute, acclamatory, and evolutionary time scales. To better understand temperature's affect on metabolism at these different time scales, we quantified cardiac oxidative phosphorylation (OxPhos) in three Fundulus taxa acclimated to 12 and 28°C and measured at three acute temperatures (12, 20, and 28°C). The Fundulus taxa (northern Maine and southern Georgia F. heteroclitus, and a sister taxa, F. grandis) were used to identify evolved changes in OxPhos. Cardiac OxPhos metabolism was quantified by measuring six traits: state 3 (ADP and substrate-dependent mitochondrial respiration); E state (uncoupled mitochondrial activity); complex I, II, and IV activities; and LEAK ratio. Acute temperature affected all OxPhos traits. Acclimation only significantly affected state 3 and LEAK ratio. Populations were significantly different for state 3. In addition to direct effects, there were significant interactions between acclimation and population for complex I and between population and acute temperature for state 3. Further analyses suggest that acclimation alters the acute temperature response for state 3, E state, and complexes I and II: at the low acclimation temperature, the acute response was dampened at low assay temperatures, and at the high acclimation temperature, the acute response was dampened at high assay temperatures. Closer examination of the data also suggests that differences in state 3 respiration and complex I activity between populations were greatest between fish acclimated to low temperatures when assayed at high temperatures, suggesting that differences between the populations become more apparent at the edges of their thermal range.

Gene by environmental interactions affecting oxidative phosphorylation and thermal sensitivity.

The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only metabolic pathway with proteins encoded by both nuclear and mitochondrial genomes. In studies examining mitonuclear interactions among distant populations within a species or across species, the interactions between these two genomes can affect metabolism, growth, and fitness, depending on the environment. However, there is little data on whether these interactions impact natural populations within a single species. In an admixed Fundulus heteroclitus population with northern and southern mitochondrial haplotypes, there are significant differences in allele frequencies associated with mitochondrial haplotype. In this study, we investigate how mitochondrial haplotype and any associated nuclear differences affect six OxPhos parameters within a population. The data demonstrate significant OxPhos functional differences between the two mitochondrial genotypes. These differences are most apparent when individuals are acclimated to high temperatures with the southern mitochondrial genotype having a large acute response and the northern mitochondrial genotype having little, if any acute response. Furthermore, acute temperature effects and the relative contribution of Complex I and II depend on acclimation temperature: when individuals are acclimated to 12°C, the relative contribution of Complex I increases with higher acute temperatures, whereas at 28°C acclimation, the relative contribution of Complex I is unaffected by acute temperature change. These data demonstrate a complex gene by environmental interaction affecting the OxPhos pathway.

Phenotypic plasticity in gene expression contributes to divergence of locally adapted populations of Fundulus heteroclitus.

We examine the interaction between phenotypic plasticity and evolutionary adaptation using muscle gene expression levels among populations of the fish Fundulus heteroclitus acclimated to three temperatures. Our analysis reveals shared patterns of phenotypic plasticity due to thermal acclimation as well as non-neutral patterns of variation among populations adapted to different thermal environments. For the majority of significant differences in gene expression levels, phenotypic plasticity and adaptation operate on different suites of genes. The subset of genes that demonstrate both adaptive differences and phenotypic plasticity, however, exhibit countergradient variation of expression. Thus, expression differences among populations counteract environmental effects, reducing the phenotypic differentiation between populations. Finally, gene-by-environment interactions among genes with non-neutral patterns of expression suggest that the penetrance of adaptive variation depends on the environmental conditions experienced by the individual.

Natural variation in cardiac metabolism and gene expression in Fundulus heteroclitus.

Individual variation in gene expression is important for evolutionary adaptation and susceptibility to diseases and pathologies. In this study, we address the functional importance of this variation by comparing cardiac metabolism to patterns of mRNA expression using microarrays. There is extensive variation in both cardiac metabolism and the expression of metabolic genes among individuals of the teleost fish Fundulus heteroclitus from natural outbred populations raised in a common environment: metabolism differed among individuals by a factor of more than 2, and expression levels of 94% of genes were significantly different (P < 0.01) between individuals in a population. This unexpectedly high variation in metabolic gene expression explains much of the variation in metabolism, suggesting that it is biologically relevant. The patterns of gene expression that are most important in explaining cardiac metabolism differ between groups of individuals. Apparently, the variation in metabolism seems to be related to different patterns of gene expression in the different groups of individuals. The magnitude of differences in gene expression in these groups is not important; large changes in expression have no greater predictive value than small changes. These data suggest that variation in physiological performance is related to the subtle variation in gene expression and that this relationship differs among individuals.

Genomic and physiological responses to strong selective pressure during late organogenesis: few gene expression changes found despite striking morphological differences.

BACKGROUND: Adaptations to a new environment, such as a polluted one, often involve large modifications of the existing phenotypes. Changes in gene expression and regulation during critical developmental stages may explain these phenotypic changes. Embryos from a population of the teleost fish, Fundulus heteroclitus, inhabiting a clean estuary do not survive when exposed to sediment extract from a site highly contaminated with polycyclic aromatic hydrocarbons (PAHs) while embryos derived from a population inhabiting a PAH polluted estuary are remarkably resistant to the polluted sediment extract. We exposed embryos from these two populations to surrogate model PAHs and analyzed changes in gene expression, morphology, and cardiac physiology in order to better understand sensitivity and adaptive resistance mechanisms mediating PAH exposure during development. RESULTS: The synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (ß-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (α-naphthoflavone), caused significant developmental delays, impaired cardiac function, severe morphological alterations and failure to hatch, leading to the deaths of reference embryos; resistant embryos were mostly unaffected. Unexpectedly, patterns of gene expression among normal and moderately deformed embryos were similar, and only severely deformed embryos showed a contrasting pattern of gene expression. Given the drastic morphological differences between reference and resistant embryos, a surprisingly low percentage of genes, 2.24% of 6,754 analyzed, show statistically significant differences in transcript levels during late organogenesis between the two embryo populations. CONCLUSIONS: Our study demonstrates important contrasts in responses between reference and resistant natural embryo populations to synergistic effects of surrogate model PAHs that may be important in adaptive mechanisms mediating PAH effects during fish embryo development. These results suggest that statistically significant changes in gene expression of relatively few genes contribute to the phenotypic changes and large morphological differences exhibited by reference and resistant populations upon exposure to PAH pollutants. By correlating cardiac physiology and morphology with changes in gene expression patterns of reference and resistant embryos, we provide additional evidence for acquired resistance among embryos whose parents live at heavily contaminated sites.

Variation in gene expression within and among natural populations.

Evolution may depend more strongly on variation in gene expression than on differences between variant forms of proteins. Regions of DNA that affect gene expression are highly variable, containing 0.6% polymorphic sites. These naturally occurring polymorphic nucleotides can alter in vivo transcription rates. Thus, one might expect substantial variation in gene expression between individuals. But the natural variation in mRNA expression for a large number of genes has not been measured. Here we report microarray studies addressing the variation in gene expression within and between natural populations of teleost fish of the genus Fundulus. We observed statistically significant differences in expression between individuals within the same population for approximately 18% of 907 genes. Expression typically differed by a factor of 1.5, and often more than 2.0. Differences between populations increased the variation. Much of the variation between populations was a positive function of the variation within populations and thus is most parsimoniously described as random. Some genes showed unexpected patterns of expression--changes unrelated to evolutionary distance. These data suggest that substantial natural variation exists in gene expression and that this quantitative variation is important in evolution.

Trans-Acting Genotypes Associated with mRNA Expression Affect Metabolic and Thermal Tolerance Traits.

Evolutionary processes driving physiological trait variation depend on the underlying genomic mechanisms. Evolution of these mechanisms depends on the genetic complexity (involving many genes) and how gene expression impacting the traits is converted to phenotype. Yet, genomic mechanisms that impact physiological traits are diverse and context dependent (e.g., vary by environment and tissues), making them difficult to discern. We examine the relationships between genotype, mRNA expression, and physiological traits to discern the genetic complexity and whether the gene expression affecting the physiological traits is primarily cis- or trans-acting. We use low-coverage whole genome sequencing and heart- or brain-specific mRNA expression to identify polymorphisms directly associated with physiological traits and expressed quantitative trait loci (eQTL) indirectly associated with variation in six temperature specific physiological traits (standard metabolic rate, thermal tolerance, and four substrate specific cardiac metabolic rates). Focusing on a select set of mRNAs belonging to co-expression modules that explain up to 82% of temperature specific traits, we identified hundreds of significant eQTL for mRNA whose expression affects physiological traits. Surprisingly, most eQTL (97.4% for heart and 96.7% for brain) were trans-acting. This could be due to higher effect size of trans- versus cis-acting eQTL for mRNAs that are central to co-expression modules. That is, we may have enhanced the identification of trans-acting factors by looking for single nucleotide polymorphisms associated with mRNAs in co-expression modules that broadly influence gene expression patterns. Overall, these data indicate that the genomic mechanism driving physiological variation across environments is driven by trans-acting heart- or brain-specific mRNA expression.

Convergence or redundancy: alternative views about the evolutionary genomics of character displacement.

An evolutionary debate contrasts the importance of genetic convergence versus genetic redundancy. In genetic convergence, the same adaptive trait evolves because of similar genetic changes. In genetic redundancy, the adaptive trait evolves using different genetic combinations, and populations might not share the same genetic changes. Here we address this debate by examining single nucleotide polymorphisms (SNPs) associated with the rapid evolution of character displacement in Anolis carolinensis populations inhabiting replicate islands with and without a competitor species (1Spp and 2Spp islands, respectively). We identify 215-outliers SNPs that have improbably large FST values, low nucleotide variation, greater linkage than expected and that are enriched for genes underlying animal movement. The pattern of SNP divergence between 1Spp and 2Spp populations supports both genetic convergence and genetic redundancy for character displacement. In support of genetic convergence: all 215-outliers SNPs are shared among at least three of the five 2Spp island populations, and 23% of outlier SNPS are shared among all five 2Spp island populations. In contrast, in support of genetic redundancy: many outlier SNPs only have meaningful allele frequency differences between 1Spp and 2Spp islands on a few 2Spp islands. That is, on at least one of the 2Spp islands, 77% of outlier SNPs have allele frequencies more similar to those on 1Spp islands than to those on 2Spp islands. Focusing on genetic convergence is scientifically rigorous because it relies on replication. Yet, this focus distracts from the possibility that there are multiple, redundant genetic solutions that enhance the rate and stability of adaptive change.

RNA-Seq reveals complex genetic response to Deepwater Horizon oil release in Fundulus grandis.

BACKGROUND: The release of oil resulting from the blowout of the Deepwater Horizon (DH) drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq) in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future. RESULTS: Our de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set. CONCLUSION: RNA-Seq may be successfully applied to feral and extremely polymorphic organisms that do not have an underlying genome sequence assembly to address timely environmental problems. Additionally, the observed changes in a large set of transcript expression levels are indicative of a complex response to the varied petroleum components to which the fish were exposed.

Ecologically and evolutionarily important SNPs identified in natural populations.

Evolution by natural selection acts on natural populations amidst migration, gene-by-environmental interactions, constraints, and tradeoffs, which affect the rate and frequency of adaptive change. We asked how many and how rapidly loci change in populations subject to severe, recent environmental changes. To address these questions, we used genomic approaches to identify randomly selected single nucleotide polymorphisms (SNPs) with evolutionarily significant patterns in three natural populations of Fundulus heteroclitus that inhabit and have adapted to highly polluted Superfund sites. Three statistical tests identified 1.4-2.5% of SNPs that were significantly different from the neutral model in each polluted population. These nonneutral patterns in populations adapted to highly polluted environments suggest that these loci or closely linked loci are evolving by natural selection. One SNP identified in all polluted populations using all tests is in the gene for the xenobiotic metabolizing enzyme, cytochrome P4501A (CYP1A), which has been identified previously as being refractory to induction in the three highly polluted populations. Extrapolating across the genome, these data suggest that rapid evolutionary change in natural populations can involve hundreds of loci, a few of which will be shared in independent events.

Cardiac physiology and metabolic gene expression during late organogenesis among F. heteroclitus embryo families from crosses between pollution-sensitive and -resistant parents.

BACKGROUND: The teleost fish Fundulus heteroclitus inhabit estuaries heavily polluted with persistent and bioaccumulative chemicals. While embryos of parents from polluted sites are remarkably resistant to toxic sediment and develop normally, embryos of parents from relatively clean estuaries, when treated with polluted sediment extracts, are developmentally delayed, displaying deformities characteristic of pollution-induced embryotoxicity. To gain insight into parental effects on sensitive and resistant phenotypes during late organogenesis, we established sensitive, resistant, and crossed embryo families using five female and five male parents from relatively clean and predominantly PAH-polluted estuaries each, measured heart rates, and quantified individual embryo expression of 179 metabolic genes. RESULTS: Pollution-induced embryotoxicity manifested as morphological deformities, significant developmental delays, and altered cardiac physiology was evident among sensitive embryos resulting from crosses between females and males from relatively clean estuaries. Significantly different heart rates among several geographically unrelated populations of sensitive, resistant, and crossed embryo families during late organogenesis and pre-hatching suggest site-specific adaptive cardiac physiology phenotypes relative to pollution exposure. Metabolic gene expression patterns (32 genes, 17.9%, at p < 0.05; 11 genes, 6.1%, at p < 0.01) among the embryo families indicate maternal pollutant deposition in the eggs and parental effects on gene expression and metabolic alterations. CONCLUSION: Heart rate differences among sensitive, resistant, and crossed embryos is a reliable phenotype for further explorations of adaptive mechanisms. While metabolic gene expression patterns among embryo families are suggestive of parental effects on several differentially expressed genes, a definitive adaptive signature and metabolic cost of resistant phenotypes is unclear and shows unexpected sensitive-resistant crossed embryo expression profiles. Our study highlights physiological and metabolic gene expression differences during a critical embryonic stage among pollution sensitive, resistant, and crossed embryo families, which may contribute to underlying resistance mechanisms observed in natural F. heteroclitus populations living in heavily contaminated estuaries.

Sequencing Bait: Nuclear and Mitogenome Assembly of an Abundant Coastal Tropical and Subtropical Fish, Atherinomorus stipes.

Genetic data from nonmodel species can inform ecology and physiology, giving insight into a species' distribution and abundance as well as their responses to changing environments, all of which are important for species conservation and management. Moreover, reduced sequencing costs and improved long-read sequencing technology allows researchers to readily generate genomic resources for nonmodel species. Here, we apply Oxford Nanopore long-read sequencing and low-coverage (∼1x) whole genome short-read sequencing technology (Illumina) to assemble a genome and examine population genetics of an abundant tropical and subtropical fish, the hardhead silverside (Atherinomorus stipes). These fish are found in shallow coastal waters and are frequently included in ecological models because they serve as abundant prey for commercially and ecologically important species. Despite their importance in sub-tropical and tropical ecosystems, little is known about their population connectivity and genetic diversity. Our A. stipes genome assembly is about 1.2 Gb with comparable repetitive element content (∼47%), number of protein duplication events, and DNA methylation patterns to other teleost fish species. Among five sampled populations spanning 43 km of South Florida and the Florida Keys, we find little population structure suggesting high population connectivity.

Gene expression throughout a vertebrate's embryogenesis.

BACKGROUND: Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. RESULTS: Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. CONCLUSIONS: These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.

Transcriptomic assessment of resistance to effects of an aryl hydrocarbon receptor (AHR) agonist in embryos of Atlantic killifish (Fundulus heteroclitus) from a marine Superfund site.

BACKGROUND: Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) from F. heteroclitus populations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive). RESULTS: Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing). CONCLUSIONS: The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.

Evolutionary and functional analyses of cytochrome P4501A promoter polymorphisms in natural populations.

The functional importance of variable, transcriptional regulatory sequences within and among natural populations is largely unexplored. We analysed the cytochrome P4501A (CYP1A) promoter in three populations of the minnow, Fundulus heteroclitus, because two SNPs in the promoter and first intron of CYP1A are under selection in populations adapted to pollutants. To define the importance of these SNPs, 1630 bp of the CYP1A promoter and first intron and exon were sequenced in eight individuals from three populations: a population from a polluted environment resistant to some aromatic pollutants and two flanking reference populations. CYP1A is induced by many aromatic pollutants, but in populations adapted to pollutants, CYP1A has been shown to be refractory to induction. We were interested in understanding whether variation in the CYP1A promoter explains mechanism(s) of adaptation to these aromatic pollutants. The CYP1A promoter was extremely variable (an average of 9.3% of the promoter nucleotides varied among all populations) and exhibited no fixed differences between populations. As CYP1A is poorly inducible in adapted fish, we hypothesized that CYP1A promoter regions might vary functionally between populations. Unexpectedly, in vitro analysis showed significantly greater transcription from CYP1A promoters found in the population from the polluted environment relative to promoters found in both reference populations. Thus, despite extensive variation among populations and lack of fixed differences between populations, individuals from a polluted environment have significantly enhanced promoter activity. These data demonstrate that intraspecific variation, which provides the raw material for natural selection to act on, can occur while maintaining promoter function.