Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site.

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

A multidimensional density dependent matrix population model for assessing risk of stressors to fish populations.

Modeling exposure and recovery of fish and wildlife populations after stressor mitigation serves as a basis for evaluating remediation success. Herein, we develop a novel multidimensional density dependent matrix population model that analyzes both size-structure and age class-structure simultaneously. This modeling approach emphasizes application in conjunction with field monitoring efforts (e.g., through effects-based monitoring programs) and/or laboratory analysis to link effects due to stressors to outcomes in populations. We applied the model to investigate Atlantic killifish (Fundulus heteroclitus) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin with effects on fertility and survival rates. The Atlantic killifish is an important and well-studied model organism for understanding the effects of pollutants and other stressors in estuarine and marine ecosystems. For each exposure concentration, the corresponding plots of total population size, population size structure, and age structure over time were generated. The present study serves as an example of how a multidimensional matrix population model can integrate effects across the life cycle, provide a linkage between endpoints observed in the individual and ecological risk to the population as a whole, and project outcomes for multiple generations.

Genetic basis for rapidly evolved tolerance in the wild: adaptation to toxic pollutants by an estuarine fish species.

Atlantic killifish (Fundulus heteroclitus) residing in some urban and industrialized estuaries of the US eastern seaboard demonstrate recently evolved and extreme tolerance to toxic aryl hydrocarbon pollutants, characterized as dioxin-like compounds (DLCs). Here, we provide an unusually comprehensive accounting (69%) through quantitative trait locus (QTL) analysis of the genetic basis for DLC tolerance in killifish inhabiting an urban estuary contaminated with PCB congeners, the most toxic of which are DLCs. Consistent with mechanistic knowledge of DLC toxicity in fish and other vertebrates, the aryl hydrocarbon receptor (ahr2) region accounts for 17% of trait variation; however, QTL on independent linkage groups and their interactions have even greater explanatory power (44%). QTL interpreted within the context of recently available Fundulus genomic resources and shared synteny among fish species suggest adaptation via interacting components of a complex stress response network. Some QTL were also enriched in other killifish populations characterized as DLC-tolerant and residing in distant urban estuaries contaminated with unique mixtures of pollutants. Together, our results suggest that DLC tolerance in killifish represents an emerging example of parallel contemporary evolution that has been driven by intense human-mediated selection on natural populations.

Integrating Monitoring and Genetic Methods To Infer Historical Risks of PCBs and DDE to Common and Roseate Terns Nesting Near the New Bedford Harbor Superfund Site (Massachusetts, USA).

Common and roseate terns are migratory piscivorous seabirds with major breeding colonies within feeding range of the polychlorinated biphenyl (PCB)-contaminated New Bedford Harbor (NBH, MA, USA) Superfund site. Our longitudinal study shows that before PCB discharges into NBH ceased (late 1970s), tern eggs had very high but variable PCB concentrations. However, egg concentrations of PCBs as well as DDE (1,1-bis(p-chlorophenyl)-2,2-dichloroethene), the degradation product of the ubiquitous global contaminant DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane), have since declined. Rate constants for temporal decline of PCB congeners in tern eggs varied inversely with log10KOW (n-octanol-water partition coefficient), shifting egg congener patterns away from those characterizing NBH sediment. To estimate the toxic effects on tern eggs of PCB dioxin-like congener (DLC) exposures, we extrapolated published laboratory data on common terns to roseate terns by characterizing genetic and functional similarities in species aryl hydrocarbon receptors (AHRs), which mediate DLC sensitivity. Our assessment of contaminant risks suggests that terns breeding near NBH were exposed historically to toxic levels of PCBs and DDE; however, acute effects on tern egg development have become less likely since the 1970s. Our approach demonstrates how comparative genetics at target loci can effectively increase the range of inference for chemical risk assessments from tested to untested and untestable species.

Targeted approach to identify genetic loci associated with evolved dioxin tolerance in Atlantic killifish (Fundulus heteroclitus).

BACKGROUND: The most toxic aromatic hydrocarbon pollutants are categorized as dioxin-like compounds (DLCs) to which extreme tolerance has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly aromatic hydrocarbon-contaminated urban/industrialized estuaries of the US Atlantic coast. Multiple tolerant and neighboring sensitive killifish populations were compared with the expectation that genetic loci associated with DLC tolerance would be revealed. RESULTS: Since the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, single nucleotide polymorphisms (SNPs) from 42 genes associated with the AHR pathway were identified to serve as targeted markers. Wild fish (N = 36/37) from four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Similar to other killifish population genetic analyses, strong genetic differentiation among populations was detected, consistent with isolation by distance models. When DLC-sensitive populations were pooled and compared to pooled DLC-tolerant populations, multi-locus analyses did not distinguish the two groups. However, pairwise comparisons of nearby tolerant and sensitive populations revealed high differentiation among sensitive and tolerant populations at these specific loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP1A and 3A30), and the NADH dehydrogenase subunits. In addition, significant shifts in minor allele frequency were observed at AHR2 and CYP1A loci across most sensitive/tolerant pairs, but only AHR2 exhibited shifts in the same direction across all pairs. CONCLUSIONS: The observed differences in allelic composition at the AHR2 and CYP1A SNP loci were identified as significant among paired sensitive/tolerant populations of Atlantic killifish with multiple statistical tests. The genetic patterns reported here lend support to the argument that AHR2 and CYP1A play a role in the adaptive response to extreme DLC contamination. Additional functional assays are required to isolate the exact mechanism of DLC tolerance.

Genetic variation at aryl hydrocarbon receptor (AHR) loci in populations of Atlantic killifish (Fundulus heteroclitus) inhabiting polluted and reference habitats.

BACKGROUND: The non-migratory killifish Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other "dioxin-like compounds" (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci (AHR1, AHR2, AHRR) in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor (MA, USA), a PCB Superfund site, and nearby reference sites. RESULTS: We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant FST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus. CONCLUSION: Together, these data suggest that F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.

Independently evolved pollution resistance in four killifish populations is largely explained by few variants of large effect.

The genetic architecture of phenotypic traits can affect the mode and tempo of trait evolution. Human-altered environments can impose strong natural selection, where successful evolutionary adaptation requires swift and large phenotypic shifts. In these scenarios, theory predicts that adaptation is due to a few adaptive variants of large effect, but empirical studies that have revealed the genetic architecture of rapidly evolved phenotypes are rare, especially for populations inhabiting polluted environments. Fundulus killifish have repeatedly evolved adaptive resistance to extreme pollution in urban estuaries. Prior studies, including genome scans for signatures of natural selection, have revealed some of the genes and pathways important for evolved pollution resistance, and provide context for the genotype-phenotype association studies reported here. We created multiple quantitative trait locus (QTL) mapping families using progenitors from four different resistant populations, and using RAD-seq genetically mapped variation in sensitivity (developmental perturbations) following embryonic exposure to a model toxicant PCB-126. We found that one to two large-effect QTL loci accounted for resistance to PCB-mediated developmental toxicity. QTLs harbored candidate genes that govern the regulation of aryl hydrocarbon receptor (AHR) signaling. One QTL locus was shared across all populations and another was shared across three populations. One QTL locus showed strong signatures of recent natural selection in the corresponding wild population but another QTL locus did not. Some candidate genes for PCB resistance inferred from genome scans in wild populations were identified as QTL, but some key candidate genes were not. We conclude that rapidly evolved resistance to the developmental defects normally caused by PCB-126 is governed by few genes of large effect. However, other aspects of resistance beyond developmental phenotypes may be governed by additional loci, such that comprehensive resistance to PCB-126, and to the mixtures of chemicals that distinguish urban estuaries more broadly, may be more genetically complex.

Connecting Suborganismal Data to Bioenergetic Processes: Killifish Embryos Exposed to a Dioxin-Like Compound.

A core challenge for ecological risk assessment is to integrate molecular responses into a chain of causality to organismal or population-level outcomes. Bioenergetic theory may be a useful approach for integrating suborganismal responses to predict organismal responses that influence population dynamics. We describe a novel application of dynamic energy budget (DEB) theory in the context of a toxicity framework (adverse outcome pathways [AOPs]) to make quantitative predictions of chemical exposures to individuals, starting from suborganismal data. We use early-life stage exposure of Fundulus heteroclitus to dioxin-like chemicals (DLCs) and connect AOP key events to DEB processes through "damage" that is produced at a rate proportional to the internal toxicant concentration. We use transcriptomic data of fish embryos exposed to DLCs to translate molecular indicators of damage into changes in DEB parameters (damage increases somatic maintenance costs) and DEB models to predict sublethal and lethal effects on young fish. By changing a small subset of model parameters, we predict the evolved tolerance to DLCs in some wild F. heteroclitus populations, a data set not used in model parameterization. The differences in model parameters point to reduced sensitivity and altered damage repair dynamics as contributing to this evolved resistance. Our methodology has potential extrapolation to untested chemicals of ecological concern. Environ Toxicol Chem 2023;42:2040-2053. © 2023 Oak Ridge National Laboratory and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Independently evolved pollution resistance in four killifish populations is largely explained by few variants of large effect.

The genetic architecture of phenotypic traits can affect the mode and tempo of trait evolution. Human-altered environments can impose strong natural selection, where successful evolutionary adaptation requires swift and large phenotypic shifts. In these scenarios, theory predicts the influence of few adaptive variants of large effect, but empirical studies that have revealed the genetic architecture of rapidly evolved phenotypes are rare, especially for populations inhabiting polluted environments. Fundulus killifish have repeatedly evolved adaptive resistance to extreme pollution in urban estuaries. Prior studies, including genome scans for signatures of natural selection, have revealed some of the genes and pathways important for evolved pollution resistance, and provide context for the genotype-phenotype association studies reported here. We created multiple quantitative trait locus (QTL) mapping families using progenitors from four different resistant populations, and genetically mapped variation in sensitivity (developmental perturbations) following embryonic exposure to a model toxicant PCB-126. We found that a few large-effect QTL loci accounted for resistance to PCB-mediated developmental toxicity. QTLs harbored candidate genes that govern the regulation of aryl hydrocarbon receptor (AHR) signaling, where some (but not all) of these QTL loci were shared across all populations, and some (but not all) of these loci showed signatures of recent natural selection in the corresponding wild population. Some strong candidate genes for PCB resistance inferred from genome scans in wild populations were identified as QTL, but some key candidate genes were not. We conclude that rapidly evolved resistance to the developmental defects normally caused by PCB-126 is governed by few genes of large effect. However, other aspects of resistance beyond developmental phenotypes may be governed by additional loci, such that comprehensive resistance to PCB-126, and to the mixtures of chemicals that distinguish urban estuaries more broadly, may be more genetically complex.

Common mechanism underlies repeated evolution of extreme pollution tolerance.

Human alterations to the environment can exert strong evolutionary pressures, yet contemporary adaptation to human-mediated stressors is rarely documented in wildlife populations. A common-garden experimental design was coupled with comparative transcriptomics to discover evolved mechanisms enabling three populations of killifish resident in urban estuaries to survive normally lethal pollution exposure during development, and to test whether mechanisms are unique or common across populations. We show that killifish populations from these polluted sites have independently converged on a common adaptive mechanism, despite variation in contaminant profiles among sites. These populations are united by a similarly profound desensitization of aryl-hydrocarbon receptor-mediated transcriptional activation, which is associated with extreme tolerance to the lethal effects of toxic dioxin-like pollutants. The rapid, repeated, heritable and convergent nature of evolved tolerance suggests that ancestral killifish populations harboured genotypes that enabled adaptation to twentieth-century industrial pollutants.

MERGANSER: an empirical model to predict fish and loon mercury in New England lakes.

MERGANSER (MERcury Geo-spatial AssessmeNtS for the New England Region) is an empirical least-squares multiple regression model using mercury (Hg) deposition and readily obtainable lake and watershed features to predict fish (fillet) and common loon (blood) Hg in New England lakes. We modeled lakes larger than 8 ha (4404 lakes), using 3470 fish (12 species) and 253 loon Hg concentrations from 420 lakes. MERGANSER predictor variables included Hg deposition, watershed alkalinity, percent wetlands, percent forest canopy, percent agriculture, drainage area, population density, mean annual air temperature, and watershed slope. The model returns fish or loon Hg for user-entered species and fish length. MERGANSER explained 63% of the variance in fish and loon Hg concentrations. MERGANSER predicted that 32-cm smallmouth bass had a median Hg concentration of 0.53 µg g(-1) (root-mean-square error 0.27 µg g(-1)) and exceeded EPA's recommended fish Hg criterion of 0.3 µg g(-1) in 90% of New England lakes. Common loon had a median Hg concentration of 1.07 µg g(-1) and was in the moderate or higher risk category of >1 µg g(-1) Hg in 58% of New England lakes. MERGANSER can be applied to target fish advisories to specific unmonitored lakes, and for scenario evaluation, such as the effect of changes in Hg deposition, land use, or warmer climate on fish and loon mercury.

The effects of mitochondrial genotype on hypoxic survival and gene expression in a hybrid population of the killifish, Fundulus heteroclitus.

The physiological link between oxygen availability and mitochondrial function is well established. However, whether or not fitness variation is associated with mitochondrial genotypes in the field remains a contested topic in evolutionary biology. In this study, we draw on a population of the teleost fish, Fundulus heteroclitus, where functionally distinct subspecies hybridize, likely as a result of past glacial events. We had two specific aims: (i) to determine the effect of mtDNA genotype on survivorship of male and female fish under hypoxic stress and (ii) to determine the effect of hypoxic stress, sex and mtDNA genotype on gene expression. We found an unexpected and highly significant effect of sex on survivorship under hypoxic conditions, but no significant effect of mtDNA genotype. Gene expression analyses revealed hundreds of transcripts differentially regulated by sex and hypoxia. Mitochondrial transcripts and other predicted pathways were among those influenced by hypoxic stress, and a transcript corresponding to the mtDNA control region was the most highly suppressed transcript under the conditions of hypoxia. An RT-PCR experiment on the control region was consistent with microarray results. Effects of mtDNA sequence variation on genome expression were limited; however, a potentially important epistasis between mtDNA sequence and expression of a nuclear-encoded mitochondrial translation protein was discovered. Overall, these results confirm that mitochondrial regulation is a major component of hypoxia tolerance and further suggest that purifying selection has been the predominant selective force on mitochondrial genomes in these two subspecies.

Resistance to Cyp3a induction by polychlorinated biphenyls, including non-dioxin-like PCB153, in gills of killifish (Fundulus heteroclitus) from New Bedford Harbor.

Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.

Population genetic diversity and fitness in multiple environments.

BACKGROUND: When a large number of alleles are lost from a population, increases in individual homozygosity may reduce individual fitness through inbreeding depression. Modest losses of allelic diversity may also negatively impact long-term population viability by reducing the capacity of populations to adapt to altered environments. However, it is not clear how much genetic diversity within populations may be lost before populations are put at significant risk. Development of tools to evaluate this relationship would be a valuable contribution to conservation biology. To address these issues, we have created an experimental system that uses laboratory populations of an estuarine crustacean, Americamysis bahia with experimentally manipulated levels of genetic diversity. We created replicate cultures with five distinct levels of genetic diversity and monitored them for 16 weeks in both permissive (ambient seawater) and stressful conditions (diluted seawater). The relationship between molecular genetic diversity at presumptive neutral loci and population vulnerability was assessed by AFLP analysis. RESULTS: Populations with very low genetic diversity demonstrated reduced fitness relative to high diversity populations even under permissive conditions. Population performance decreased in the stressful environment for all levels of genetic diversity relative to performance in the permissive environment. Twenty percent of the lowest diversity populations went extinct before the end of the study in permissive conditions, whereas 73% of the low diversity lines went extinct in the stressful environment. All high genetic diversity populations persisted for the duration of the study, although population sizes and reproduction were reduced under stressful environmental conditions. Levels of fitness varied more among replicate low diversity populations than among replicate populations with high genetic diversity. There was a significant correlation between AFLP diversity and population fitness overall; however, AFLP markers performed poorly at detecting modest but consequential losses of genetic diversity. High diversity lines in the stressful environment showed some evidence of relative improvement as the experiment progressed while the low diversity lines did not. CONCLUSIONS: The combined effects of reduced average fitness and increased variability contributed to increased extinction rates for very low diversity populations. More modest losses of genetic diversity resulted in measurable decreases in population fitness; AFLP markers did not always detect these losses. However when AFLP markers indicated lost genetic diversity, these losses were associated with reduced population fitness.

Developing a High-Quality Linkage Map for the Atlantic Killifish Fundulus heteroclitus.

Killifish (Fundulus heteroclitus) are widely distributed among different aquatic environments where they demonstrate an impressive range of highly-plastic and locally adaptive phenotypes. High-throughput sequencing has begun to unravel the mechanisms and evolutionary history of these interesting features by establishing relationships in the genotype-phenotype map. However, some genotype-phenotype analyses require a higher order of contiguity than what initial scaffolded (fragmented genome assembly where contigs have been assemble into scaffolds) genome assemblies can provide. Here, we used 5,685 high-quality RAD-Seq markers from a single mapping family to order 84% of the scaffolded genome assembly to 24 chromosomes. This serves to: 1) expand the killifish genomic toolkit, 2) estimate genome-wide recombination rates, and 3) compare genome synteny to humans and other fishes. After initially building our map, we found that the selection of thresholds for sequence data filtration highly impacted scaffold placement in the map. We outline each step of the approach that dramatically improved our map to help guide others toward more effective linkage mapping for genome assembly. Our final map supports strong conservation of genomic synteny among closely related fish species and reveals previously described chromosomal rearrangements between more distantly related clades. However, we also commonly found minor scaffold misorientations in F. heteroclitus and in other assemblies, suggesting that further mapping (such as optical mapping) is necessary for finer scale resolution of genome structure. Lastly, we discuss the problems that would be expected from misoriented/unplaced scaffolds and stress the importance of a quality mapped genome as a key feature for further investigating population and comparative genomic questions with F. heteroclitus and other taxa.

REGULATION OF REPRODUCTIVE PROCESSES WITH DYNAMIC ENERGY BUDGETS.

1. The simple bioenergetic models in the family of Dynamic Energy Budget (DEB) consist of a small number of state equations quantifying universal processes, such as feeding, maintenance, development, reproduction and growth. Linking these organismal level processes to underlying suborganismal mechanisms at the molecular, cellular and organ level constitutes a major challenge for predictive ecological risk assessments. 2. Motivated by the need for process-based models to evaluate the impact of endocrine disruptors on ecologically relevant endpoints, this paper develops and evaluates two general modeling modules describing demand-driven feedback mechanisms exerted by gonads on the allocation of resources to production of reproductive matter within the DEB modeling framework. 3. These modules describe iteroparous, semelparous and batch-mode reproductive strategies. The modules have a generic form with both positive and negative feedback components; species and sex specific attributes of endocrine regulation can be added without changing the core of the modules. 4. We demonstrate that these modules successfully describe time-resolved measurements of wet weight of body, ovaries and liver, egg diameter and plasma content of vitellogenin and estradiol in rainbow trout (Oncorynchus mykiss) by fitting these models to published and new data, which require the estimation of less than two parameters per data type. 5. We illustrate the general applicability of the concept of demand-driven allocation of resources to reproduction as worked out in this paper by evaluating one of the modules with data on growth and seed production of an annual plant, the common bean (Phaseolis vulgaris).

Characterization of the Fundulus heteroclitus embryo transcriptional response and development of a gene expression-based fingerprint of exposure for the alternative flame retardant, TBPH (bis (2-ethylhexyl)-tetrabromophthalate).

Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (n = 16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.

Altered lipid homeostasis in a PCB-resistant Atlantic killifish (Fundulus heteroclitus) population from New Bedford Harbor, MA, U.S.A.

Sentinel species such as the Atlantic killifish (Fundulus heteroclitus) living in urban waterways can be used as toxicological models to understand impacts of environmental metabolism disrupting compound (MDC) exposure on both wildlife and humans. Exposure to MDCs is associated with increased risk of metabolic syndrome, including impaired lipid and glucose homeostasis, adipogenesis, appetite control, and basal metabolism. MDCs are ubiquitous in the environment, including in aquatic environments. New Bedford Harbor (NBH), Massachusetts is polluted with polychlorinated biphenyls (PCBs), and, as we show for the first time, tin (Sn). PCBs and organotins are ligands for two receptor systems known to regulate lipid homeostasis, the aryl hydrocarbon receptor (AHR) and the peroxisome proliferator-activated receptors (PPARs), respectively. In the current study, we compared lipid homeostasis in laboratory-reared killifish from NBH (F2) and a reference location (Scorton Creek, Massachusetts; F1 and F2) to evaluate how adaptation to local conditions may influence responses to MDCs. Adult killifish from each population were exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126, dioxin-like), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153, non-dioxin-like), or tributyltin (TBT, a PPARγ ligand) by a single intraperitoneal injection and analyzed after 3 days. AHR activation was assessed by measuring cyp1a mRNA expression. Lipid homeostasis was evaluated phenotypically by measuring liver triglycerides and organosomatic indices, and at the molecular level by measuring the mRNA expression of pparg and ppara and a target gene for each receptor. Acute MDC exposure did not affect phenotypic outcomes. However, overall NBH killifish had higher liver triglycerides and adiposomatic indices than SC killifish. Both season and population were significant predictors of the lipid phenotype. Acute MDC exposure altered hepatic gene expression only in male killifish from SC. PCB126 exposure induced cyp1a and pparg, whereas PCB153 exposure induced ppara. TBT exposure did not induce ppar-dependent pathways. Comparison of lipid homeostasis in two killifish populations extends our understanding of how MDCs act on fish and provides a basis to infer adaptive benefits of these differences in the wild.

Bioaccumulation and effects of dietary exposure to the alternative flame retardant, bis(2-ethylhexyl) tetrabromophthalate (TBPH), in the Atlantic killifish, Fundulus heteroclitus.

Bis(2-ethylhexyl) tetrabromophthalate (TBPH), a high production volume flame retardant chemical used as a replacement for banned flame retardants, has been detected in media and human and wildlife tissues globally. We describe bioaccumulation and biological effects from dietary exposure of TBPH to an estuarine fish, Atlantic killifish, Fundulus heteroclitus. Briefly, adult fish were fed carrier control or chemically amended diets for 28 d, followed by 14 d of control diet feeding. Diets were amended with TBPH (TBPH_LO diet, 139 µg/g dry wt, or TBPH_HI diet, 4360 µg/g dry wt) or a polychlorinated biphenyl congener (PCB153 diet, 13 µg/g dry wt), which was included as a positive control for bioaccumulation. Although bioaccumulation of either chemical correlated with fish size, only a small proportion of the TBPH offered (<0.5% total TBPH) had bioaccumulated into TBPH-treated fish by 28 d. In contrast, 24.5% of the PCB153 offered was accounted for in 28-d PCB-treated fish. Although 28-d bioaccumulated concentrations of TBPH differed by sex and treatment, sexes did not differ in their rates of TBPH bioaccumulation, and the time to achieve 50% of 28 d concentration (T1/2 ) was estimated to be 13 d. Depuration rates of TBPH did not differ by sex or treatment, and the time after exposure to achieve T1/2 was estimated to be 22 d. Independent of treatment, male fish grew faster than female fish, but for both sexes reproductive condition (gonadal somatic index) declined unexpectedly over the experimental period. Across treatments, only the TBPH_LO treatment affected growth, reducing male but increasing female growth rates by small amounts relative to respective controls. In summary, our study used very high concentrations of dietary TBPH to contaminate fish tissues above the highest levels reported to date in wild biota, yet we observed few adverse biological effects. Environ Toxicol Chem 2018;37:2350-2360. © 2018 SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Stable genetic structure and connectivity in pollution-adapted and nearby pollution-sensitive populations of Fundulus heteroclitus.

Populations of the non-migratory estuarine fish Fundulus heteroclitus inhabiting the heavily polluted New Bedford Harbour (NBH) estuary have shown inherited tolerance to local pollutants introduced to their habitats in the past 100 years. Here we examine two questions: (i) Is there pollution-driven selection on the mitochondrial genome across a fine geographical scale? and (ii) What is the pattern of migration among sites spanning a strong pollution gradient? Whole mitochondrial genomes were analysed for 133 F. heteroclitus from seven nearby collection sites: four sites along the NBH pollution cline (approx. 5 km distance), which had pollution-adapted fish, as well as one site adjacent to the pollution cline and two relatively unpolluted sites about 30 km away, which had pollution-sensitive fish. Additionally, we used microsatellite analyses to quantify genetic variation over three F. heteroclitus generations in both pollution-adapted and sensitive individuals collected from two sites at two different time points (1999/2000 and 2007/2008). Our results show no evidence for a selective sweep of mtDNA in the polluted sites. Moreover, mtDNA analyses revealed that both pollution-adapted and sensitive populations harbour similar levels of genetic diversity. We observed a high level of non-synonymous mutations in the most polluted site. This is probably associated with a reduction in Ne and concomitant weakening of purifying selection, a demographic expansion following a pollution-related bottleneck or increased mutation rates. Our demographic analyses suggest that isolation by distance influences the distribution of mtDNA genetic variation between the pollution cline and the clean populations at broad spatial scales. At finer scales, population structure is patchy, and neither spatial distance, pollution concentration or pollution tolerance is a good predictor of mtDNA variation. Lastly, microsatellite analyses revealed stable population structure over the last decade.