I'm Back!

It has been awhile. A long long while. I know, I know. My New Year's resolution was to starting writing regularly on the blog again. Better late than never.

My only excuse for my excessively long absence is that I have been writing. So, I'll take a little me-moment to catch you up on what I've been up to:

Fun with Fundulus: The Evolution of Pollution Resistance in Killifish

(credit to Evan D'Alessandro, Rosenstiel School of Marine and Atmospheric Science)

In honor of Mr. Charlie Darwin’s birthday I thought I would read an evolution paper. Put that together with the turn my career has taken into ecotoxicology (and the associated steep learning curve), I was steered towards a study about adapting to pollution.

Let me start by introducing you to today’s study organism: The mummichog (Fundulus heteroclitus) is a species of non-migratory killifish found along the Atlantic coast of North America. They can be found in the brackish waters of tidal creeks, saltwater marshes, and estuaries. These fish are remarkably hardy, adaptable, and easy to study. Throughout the decades, a great deal of knowledge has been gathered about their life history, genetics, behavior, and endocrinology. They have also been used to study embryological processes and responses to chemicals and toxins. The mummichog’s adaptability to varying temperature, salinity, and oxygen along with their ability to survive in highly polluted areas has made them a popular subject in toxicology.

We know that animal populations adapt to environmental stressors through genetic and epigenetic (heritable changes in gene activity that are not caused by changes in the DNA sequence) changes. Changes that, in turn, affect gene expression and/or protein function. In this way, toxic chemicals can drive selection. A big part of the field of toxicology is understanding the molecular basis of these changes as natural populations adapt to altered environments. The mummichog’s ability to live in grossly contaminated waters has been used to better test and understand the molecular mechanisms by which natural populations adapt to long-term, multi-generational exposure to pollution.

Toxicologists, like geneticists, seem to love acronyms. And once you start reading chemical names, you know why. So let’s get some chemical terminology out of the way first. There are several chemicals that are under the umbrella of “dioxin-like compounds” (DLCs) which are by-products of various industrial processes and are all highly toxic. Aromatic hydrocarbons such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polycyclic aromatic hydrocarbons (PAHs) all cause toxicity similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and DLCs. This toxicity manifests as interference in embryonic development, reproductive problems, immune impairment, and other not-so-pretty consequences.

A new study in BMC Evolutionary Biology takes a look at the Fundulus of New Bedford Harbor, Massachusetts. This 18,000 acre estuary and seaport is one of the EPA’s Superfund sites. It is highly contaminated with PCBs and heavy metals. Through their various stages of development and into adulthood, the killifish of these waters are less sensitive and/or resistant to the effects of the toxins. The researchers took a candidate gene approach to investigate the molecular basis of this adaptation to DLCs. To do this, they went to New Bedford Harbor and other polluted sites to collect fish. They also collected from reference sites where the PCB sensitivities of the killifish have been characterized and measured. Their methods included a lot of genetic work that, for the sake of space and sanity, I’m not going to detail. Let’s just skip along to the results shall we?

The researchers found a repeated evolution of resistance to DLCs in widely separated populations of Fundulus along the east coast of the U.S. There is strong evidence that this adaptation involves altered sensitivity of the aryl hydrocarbon receptor (AHR). Genes encoding proteins in the (AHR)-dependent signaling pathway are a master regulator of responses to many of the most toxic DLCs. The AHR is a ligand-activated transcription factor that exhibits high affinity for DLCs, regulates the expression of a large set of genes in response to DLC exposure, and is required for TCDD or PCB toxicity in fish (and mammals too). Two paralogs (or clades) of the AHR pathway have been identified in mummichog, AHR1 and AHR2, as well as an AHR repressor (AHRR). The loci (gene locations) for these three genes were found to contain a large number of polymorphisms, many of which encoded changes in amino acids. Perhaps most interesting was AHR2, the predominant form expressed in many fishes which has 951 amino acids whose variants lead to 26 different forms of the protein. The genetic diversity at these the three loci was not significantly different between contaminated and reference sites except in the case of AHR2. This paralog had significant FST values (the fixation index that measures population differentiation due to genetic structure) and showed very low nucleotide variability (0.1%).

So what's happening here? When polluted sites are compared to reference sites there is similar genetic diversity. However, when you look at specific nucleotides you see a story start to emerge. AHR1, AHR2, and AHRR are resistance genes that mediate toxic effects, and populations of killifish exhibit strong genetic structure at all three of these loci. The selection observed at AHR1 and AHR2, specifically the latter, at the highly polluted New Bedford Harbor site suggests an adaptation to the PCBs present there. AHR2 seems to be one of the genes, possibly the major one, involved in this resistance and may be one of the recurring targets for selection during local adaptation to DLCs. This adaptation allows the mummichog to survive in a really polluted environment. These results are consistent with several lines of evidence from similar studies both in the field and the lab.

Witnessing, quantifying, and mapping these mechanisms greatly advances our understanding of the consequences of environmental toxins. Overall, this is a very interesting example of adaptation in an ever changing environment.


Adam M. Reitzel, Sibel I. Karchner, Diana G. Franks, Brad R. Evans, Diane Nacci, Denise Champlin, Verónica M. Vieira, & Mark E. Hahn (2014). Genetic variation at aryl hydrocarbon receptor (AHR) loci in populations of Atlantic killifish (Fundulus heteroclitus) inhabiting polluted and reference habitats BMC Evolutionary Biology, 14 (1) DOI: 10.1186/1471-2148-14-6


Read more about this study in the Woods Hole Oceanographic Institute's New Release "Solving An Evolutionary Puzzle New Bedford Harbor Pollution Prompts PCB-Resistance in Atlantic Killifish"
(also the source of the above image)

EPA's New Bedford Harbor Superfund Site page

And you can learn more about how the mummichog became a model organism here:
Atz, J. W. 1986. Fundulus heteroclitus in the Laboratory: A History. Amer. Zool. 26(1): 111-120. DOI: 10.1093/icb/26.1.111 (LINK)

Heavy Metals in Fish: Toxicity and Tolerance

Today I found an interesting paper that fits right in to my new job in the field of aquatic ecotoxicology. As the name suggests, this field is a combination of ecology and toxicology that deals with the nature, effects, and interactions of harmful substances in the environment. In my case, it is aquatic, freshwater systems in particular. The paper I came across looks at the effects of metal contamination and tolerance in freshwater fish.

Metal contamination is something that occurs worldwide. A number of industrial metals (particularly copper, cadmium and nickel) have been well studied in freshwater systems. These studies have used gradients in contamination to demonstrate correlations between chronic metal exposure and physiological changes that occur as a result of the toxicity. These changes can include alterations in various metabolic processes as well as impaired growth and reproduction. This study focuses on how wild brown trout (Salmo trutta) respond when exposed to a water-borne mixture of metals.

The researchers looked at the brown trout that inhabit the River Hayle in Southwest England. Historically, this area has been mined, peaking during the 1800s. The drainage from these mining operations contaminates the river with a mixture of metals, and the middle region of the River Hayle is known to have extremely high metal concentrations. So high that few fish or invertebrates are able to live there. However, brown trout migrate between the upper and lower sections, including this area. Trout found in the lower regions of the river have been shown to have acute metal toxicity, including total zinc, copper and iron which average 639, 42 and 200 ug/L respectively. Despite these high levels, the fish are able to sustain a population with no evidence of reduced genetic diversity. The aim of this study is to figure out how this tolerance of metals is possible.

To answer this question, the researchers used an integrative approach, combining genomics with the analysis of metal accumulation in tissues. They collected embryo and adult fish from the metal-polluted River Hayle and their control, the River Teign. In the adults, the researchers sampled portions of gill, gut, kidney and liver tissues and processed them to measure the concentrations of seven metals: copper (Cu). lead (Pb), zinc (Zn), arsenic (As), cadmium (Cd), iron (Fe), and nickel (Ni). Since there is relatively little gene sequence information on brown trout, they then had to sequence, assemble and annotate transcriptome (the set of all RNA molecules [mRNA, rRNA, tRNA, and non-coding RNA]). I think you'll thank me for not going in to how they do that (if you are interested in these methods, the paper lays them out nicely), but suffice it to say it is laborious but informative. Then they performed a functional analysis for differentially expressed genes from each tissue.

When the researchers compared the metal concentrations they found all seven metals to be significantly higher in the Hayle trout than the Teign trout. Across all metals the fold change was highest in the gills (62.6-fold change) followed by the liver (33.7-fold change) then the kidney (18.5-fold change). They found no significant differences in the gut. This suggests that the gills are the primary uptake route for these metals. That makes sense considering the large surface area in direct contact with the water and the abundance of uptake carriers and transporters for these metals. After the metals are taken in by the gills, they are transported in the bloodstream to the rest of the body, accumulating in the liver and kidney. As these organs are responsible for processing, detoxification, storage and excretion it is easy to see why accumulation might happen here.

In both rivers, zinc was the most abundant metal in the gill, gut, and kidney, while copper was found to be highest in the liver. They also found zinc and copper to be the ones that increased to the greatest extent in the gills, liver and kidney. That's logical when you consider that these two metals were the ones elevated to the greatest extent between the two rivers (60- and 40-fold, respectively). They also found evidence that may link the uptake, storage and metabolism of iron, cadmium, and arsenic.

In order to identify potential mechanisms of toxicity and/or tolerance to these metals, gene expression patterns for the four selected tissues were examined in fish from both rivers. A total of 998 transcripts were differentially exposed in at least one tissue. You should expect the activity of the components involved in the body's metal homeostasis system (that ensures an adequate supply of essential [trace] metals) to change with increased metal exposure. And indeed, the researchers found at least one MT (glutathione and metallothioneins; act as buffers for metal ions entering cells and have an affinity for most metals), particularly metallothionein b, to be the most strongly up-regulated genes in the Hayle trout. This suggests that the trout's metal tolerance mechanism may be as a result of the sequestration of metals by MT. And although zinc and copper were found to be in the highest concentrations in tissues, only the zinc transporter gene was differentially expressed (down-regulated in the kidney). However, they did find changes in iron-metabolism related genes.  Since metals also disrupt the balance of ions in the body causing oxidative damage, the researchers also looked at the ion homeostasis system. They found differential expression of enzymes and a number of other genes encoding proteins that are important in maintaining ion balance.

All of this put together gives some interesting mechanisms of metal toxicity, demonstrating that these fish have developed strategies for dealing with the pollution in their environment.


Uren Webster, T. M., Bury, N.R., van Aerle, R., & Santos, E.M. (2013). Global transcriptome profiling reveals molecular mechanisms of metal tolerance in a chronically exposed wild population of brown trout Environmental Science & Technology DOI: 10.1021/es401380p


(image via Biopix)