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Your Environment. Your Health.

Killifish Hint at Genetic Basis for Human Toxicant Susceptibility

Killifish populations have adapted to survive and reproduce in polluted waters. Researchers have studied the evolutionary and genetic basis for this adaptation, discovering that it comes with a cost.

Killifish (Fundulus heteroclitus)

Killifish (Fundulus heteroclitus) appear to have evolved resistance to several harmful pollutants in the past 50-60 years
(Photo courtesy of Boston University SRP Center)

For more than two decades, the Duke University and Boston University Superfund Research Program (SRP) Centers have used these 2-3 inch long fish to understand the toxicity, mechanisms, and health effects of two groups of hazardous contaminants, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)

The Problem

PAHs and PCBs pollute air, water, and soil, and are linked to health problems in humans. The chemicals do not dissolve easily in water, but instead stick to solid particles and settle to the bottom of rivers, lakes, and estuaries.

To learn more about these chemicals, Duke and Boston University SRP researchers study contaminated sites on the east coast. The Atlantic Wood Industries Superfund site in Virginia, which closed in 1992, is contaminated with a mixture of chemicals, including PAHs from the manufacture of preservatives used to treat wood. Runoff from the site has contaminated the nearby Elizabeth River. The New Bedford Harbor Superfund site in Massachusetts is contaminated with PCBs from the manufacture of electrical equipment. Even though manufacture and use of these contaminants have stopped at these sites, the chemicals remain.

Boston University and Duke University SRP Centera ctivities, between 1995, when Bostons SRP Center began, to present when two centers began integrating data the SRP data supplement to better understand the mechanisms of resistance

SRP Solutions

Richard Di Giulio, Ph.D. and Mark Hahn, Ph.D.

Di Giulio (left) and Hahn (right) study how exposure to contaminants over generations leads to population-wide genetic changes, a field called evolutionary toxicology.
(Photo courtesy of Duke SRP Center and WHOI)

Evolutionary Toxicology Provides Insights into Adaptation

Richard Di Giulio, Ph.D., leads a Duke University SRP Center project focused on understanding the mechanisms and consequences of killifish adaptation to PAHs. His early studies explored how fish adapt over many generations and sought to understand whether adaptation was inherited through genes.

Mark Hahn, Ph.D., senior scientist at Woods Hole Oceanographic Institution (WHOI) in Massachusetts, leads a similar project at the Boston University SRP Center on the mechanisms and impacts of PCB resistance in the fish. Like Di Giulio, his studies focus on the genetic basis for adaptation.

Evolution and Cost of Adaptation

  • Discovered killifish resistance to pollutants is at least partly genetic.
  • Analyzed how adapting to contaminants leaves killifish more susceptible to other stressors.

Di Giulio examined whether resistance to PAHs was passed down through many generations. In an early study, his group compared fish from the polluted Elizabeth River with fish from non-polluted rivers and monitored their offspring for two generations. In the lab, Di Giulio’s team exposed the fish to contaminated sediments from the Elizabeth River. The two generations of offspring from the contaminated site were more resistant to PAHs and more likely to survive than those from the non-polluted site. Elizabeth River offspring were also less likely to have heart deformities, tail deformities, and other morphological issues resulting from PAH exposure than fish from a clean estuary in Virginia.

Elizabeth River map

The Elizabeth River site is shown in the rectangle at the bottom of the map, with the non-polluted King’s Creek located to the north.
(Photo courtesy of Duke SRP Center)

Interestingly, fish resistant to PAHs were more sensitive to natural stressors like phototoxicity and low oxygen. Phototoxicity, an increased sensitivity to sunlight due to the presence of a chemical, caused skin lesions in some adapted killifish. While killifish are known to tolerate a wide range of dissolved oxygen levels, adapted killifish were more sensitive to low levels of oxygen. The results point to an evolutionary tradeoff between adaptation and fitness.

More recently, Di Giulio and his team examined the role of early life exposure in killifish behavior . Non-adapted killifish exposed to contaminated water from the Elizabeth River as embryos showed altered locomotion and behavior later in life. Adapted killifish exposed as embryos behaved typically. However, exposure level played a role, as water with lower levels of contaminants did not affect behavior of non-adapted fish in the long-term.

According to the authors, because adapted killifish evolved with Elizabeth River sediment, the sediment might be protective. That is, it might keep fish healthy by regulating bacteria and parasite populations, and even influencing the gut microbiome.

Similar to the earlier work, the researchers uncovered physiological and survivorship costs of adaptation. For example, the researchers found that when raised in unpolluted water in the lab, adapted embryos faced higher mortality in the long-term.

Genome-Wide Association Studies and the Aryl Hydrocarbon Receptor

  • Identified genes involved in contaminant resistance.
  • Discovered genes in AHR pathways that likely play a role in resistance.
Map of New Bedford Harbor

New Bedford Harbor is showed in the yellow box on the map of Massachusetts.
(Photo courtesy of Map courtesy of Boston University SRP Center)

Hahn’s research focuses on the biochemical changes behind PCB resistance in killifish. He studies the aryl hydrocarbon receptor (AHR) signaling pathway, which regulates an animal’s biological response to chemicals. PCBs activate AHR proteins to alter gene expression and cell function.

Hahn and colleagues found that killifish have polymorphisms, or variations in DNA sequences for AHR. They identified 98 single-nucleotide polymorphisms within three different genes related to the AHR. That is, variations in the DNA sequence coding for amino acids that make up the AHR protein.

Killifish from the polluted New Bedford Harbor had different genetic variations than killifish from non-polluted sites. The researchers hypothesized that the variants in adapted fish may create a nonfunctional AHR protein. With the AHR pathway turned off, PCBs can no longer activate the protein to alter the gene expression and function of cells that typically leads to toxicity. According the researchers, AHR is likely an important genetic target driving PCB resistance.

Subsequent to these findings, Hahn collaborated with WHOI biologist Sibel Karchner, Ph.D., University of California, Davis researchers Andrew Whitehead, Ph.D. and Noah Reid, Ph.D., to sequence the genomes of almost 400 killifish. The genome wide study, which involved four groups of fish from polluted sites and four groups of fish from non-polluted sites, examined which parts of the genome were under selection. The results led the researchers back to the AHR pathway.

The team found genetic hotspots, or places in the genome that appeared to undergo natural selection in the pollution-resistant fish. The strongest hotspots appeared in all four groups of fish from polluted sites and included genes involved in the AHR signaling pathway. These findings strengthen the link between contaminant adaptation and genetic variation in AHR, according to the researchers.

In a similar study, Di Giulio’s group sequenced adapted and non-adapted killifish DNA from the Elizabeth River and a non-polluted site. They identified eight places in the genome with unique variations in the adapted population. Like Hahn, Di Giulio’s lab identified genetic adaptation related to the AHR pathway, suggesting reduced AHR activation in response to PAHs protects against toxicity.

Biological Resilience and Resistance in New Bedford Harbor

By studying killifish, researchers can learn more about chemical toxicity in humans. Both killifish and humans are vertebrates, and both utilize AHR pathways. Previous studies in mice, birds, and some fish show that variation in the AHR protein itself can control sensitivity to dioxin-like chemicals such as PCBs. Genetic variation in AHR in humans exists, but it does not fully account for differences in chemical susceptibility or resilience. In their studies, Hahn and Di Giulio found that other genes encoding proteins in the AHR pathway may contribute to variations in sensitivity, and the same could be true for humans.

Research Translation and Community Engagement

Not only do the Atlantic Wood and New Bedford Harbor Superfund sites provide a living laboratory for research, they also provide opportunities for connecting with communities and taking part in cleanup activities.

The Duke SRP Center works alongside federal and state agencies and nonprofit organizations to restore the Elizabeth River. The river is undergoing extensive restoration of its habitats and wetlands. Emphasis is placed on increasing public awareness of evolution and preventing future inputs of contaminants.

The Boston University SRP Center’s Community Engagement and Research Translation Core helps community organizations understand and address exposures to chemicals in the New Bedford Harbor. One of these community organizations, Hands Across the River (HARC), requested the Center’s assistance in understanding health concerns and strengthening their cleanup initiatives. The Center has also helped connect HARC with legal advice and other expertise to support cleanup efforts.

Future Directions: Integrating and Sharing Data

Hahn and Di Giulio joined forces and received an SRP data supplement to collaborate on integrating their genomic data to better understand mechanisms of susceptibility and their inheritance.

With the supplement, the researchers will integrate the Hahn team’s whole-genome data for 384 killifish with Di Giulio’s sequenced genome samples from 270 fish. Initial analyses of data from both projects identified variation in the AHR signaling pathway as a common feature associated with pollution resistance. They will also integrate datasets showing genetic variation in rodent and human models.

The team is making data available on the open-source genome browser, JBrowse. In the long-term, they hope that creating archives of publicly accessible data will allow researchers to link killifish data with animal and human data to look for similar genes across species, which could help further explain genetic susceptibility to hazardous substances.

SRP encourages data sharing among its grant recipients to accelerate new discoveries, stimulate new collaborations, and increase scientific transparency and rigor. SRP data supplements emphasize FAIR Guiding Principles for Scientific Data Management and Stewardship, meaning that data should be findable, available, interoperable, and reusable.


Carvan M. 2007. Roundtable discussion: Fish models in toxicology. Zebrafish 4(1): 9-20. doi: 10.1089/zeb.2006.9998

Brown DR, Bailey JM, Oliveri AN, Levin ED, Di Giulio RT. 2016. Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish. Neurotoxicol Teratol 53:55-63. PMID: 26548404

Di Giulio RT & Clark BW. 2015. The Elizabeth River Story: A Case Study in Evolutionary Toxicology. J Toxicol Environ Health B Crit Review 18(6): 259-298. PMID: 26505693

Meyer JN, Di Giulio RT. 2003. Heritable Adaptation and Fitness Costs in Killifish (Fundulus heteroclitus) Inhabiting a Polluted Estaury. Ecological Society of America 13(2). 490-503. doi: 10.1890/1051-0761(2003)013[0490:HAAFCI]2.0.CO;2

Osterberg JS, Cammen KM, Schultz TF, Clark BW, Di Giulio RT (2018) Genome-wide scan reveals signatures of selection related to pollution adaptation in non-model estuarine Atlantic killifish (Fundulus heteroclitus). Aquat Toxicol 200: 73-82. PMID: 29727773

Reid NM, Proestou DA, Clark BW, Warren WC, Colbourne JK, Shaw JR, Karchner SI, Hahn ME, Nacci D, Oleksiak MF, Crawford DL, Whitehead A (2016) The genomic landscape of rapid repeated evolutionary adaptation to toxic pollution in wild fish. Science 354: 1305-1308. PMID: 27940876

Reitzel AM, Karchner SI, Franks DG, Evans BR, Nacci DE, Champlin D, Vieira VM, Hahn ME. 2014. Genetic variation at aryl hydrocarbon receptor (AHR) loci in populations of Atlantic killifish (Fundulus heteroclitus) inhabiting polluted and reference habitats. BMC Evol Biol 14:6. doi: 10.1186/1471-2148-14-6

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