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Cost of tolerance: physiological consequences of evolved resistance to inhabit a polluted environment in teleost fish Fundulus heteroclitus Nishad Jayasundara, Pani W. Fernando, Joshua S. Osterberg, Kristina M Cammen, Thomas F. Schultz, and Richard T. Di Giulio Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01913 • Publication Date (Web): 06 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017
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Environmental Science & Technology
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Cost of tolerance: physiological consequences of evolved resistance to inhabit a polluted
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environment in teleost fish Fundulus heteroclitus.
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Nishad Jayasundara1,2,*, Pani W. Fernando3, Joshua S. Osterberg4, Kristina M.
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Cammen2,4, Thomas F. Schultz4, and Richard T. Di Giulio2
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School of Marine Sciences, University of Maine, Orono, ME, USA.
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Nicholas School of the Environment, Duke University, Durham, NC, USA.
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Department of Mathematics and Information Technology, University of Leoben,
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Leoben, Austria.
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USA.
Duke Marine Lab, Nicholas School of the Environment, Duke University, Beaufort, NC,
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Corresponding author :
[email protected] 16 17
Key words: Adaptation, fitness costs, polycyclic aromatic hydrocarbons, pollution,
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metabolic rate, thermal plasticity
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Abstract
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Anthropogenic stressors, including pollutants, are key evolutionary drivers. It is
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hypothesized that rapid evolution to anthropogenic changes may alter fundamental
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physiological processes (e.g., energy metabolism), compromising an organism’s capacity
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to respond to additional stressors. The Elizabeth River (ER) Superfund site represents a
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“natural-experiment” to explore this hypothesis in several subpopulations of Atlantic
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killifish that have evolved a gradation of resistance to a ubiquitous pollutant—polycyclic
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aromatic hydrocarbons (PAH). We examined bioenergetic shifts and associated
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consequences in PAH-resistant killifish by integrating genomic, physiological, and
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modeling approaches. Population genomics data revealed that genomic regions encoding
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bioenergetic processes are under selection in PAH-adapted fish from the most
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contaminated ER site and ex vivo studies confirmed altered mitochondrial function in
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these fish. Further analyses extending to differentially PAH-resistant subpopulations
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showed organismal level bioenergetic shifts in ER fish that are associated with increased
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cost of living, decreased performance, and altered metabolic response to temperature
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stress—an indication of reduced thermal plasticity. A movement model predicted a
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higher energetic cost for PAH-resistant subpopulations when seeking an optimum habitat.
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Collectively, we demonstrate that pollution adaption and inhabiting contaminated
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environments may result in physiological shifts leading to compromised organismal
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capacity to respond to additional stressors.
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Introduction
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Anthropogenic activities have contributed to rapid environmental change, altering the
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trajectory and rate of biological evolution.1 Adaptive changes to abiotic stressors are
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likely to alter physiological and biochemical processes to maintain homeostasis, but may
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have tradeoffs. Notably, abiotic stressors often affect energy metabolism via effects on
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reaction rates and macromolecular structures, and due to the increased energy demand to
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mount an adequate response. Thus, stress responses over evolutionary time may pose a
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selection pressure on organismal metabolic phenotype, potentially altering the capacity of
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organisms to respond to additional stressors.2 Here, we examined this hypothesis in
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subpopulations of the estuarine fish, Atlantic killifish (Fundulus heteroclitus), inhabiting
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the Elizabeth River (ER), Virginia, USA that have evolved resistance to a highly toxic
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class of ubiquitous pollutants, polycyclic aromatic hydrocarbons (PAHs).3
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Evolutionary adaptation to anthropogenic contaminants is a key element of biological
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pollution response. Pollution adaptation has occurred across taxa4-6 including several
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teleost species such as Atlantic killifish that show evolved resistance to complex
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hydrocarbon compounds and mixtures.3, 7-10 Despite a number of studies examining the
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mechanisms of resistance in these fish, potential tradeoffs, including the capacity to
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mount an adequate response to additional stressors, are poorly understood.7, 11 In fact,
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consequences of contemporary evolutionary shifts in physiological processes across
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vertebrates are largely unknown, but remain an important consideration in determining
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“winners” and “losers” of global environmental change.12
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Killifish are remarkably eurytolerant, withstanding fluctuations in temperature, salinity,
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and oxygen in estuaries along the North American Atlantic Coast.13 This eurytolerance to
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physical stressors and evolved resistance to PAHs, enables the use of ER killifish as a
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unique model to investigate tradeoffs of pollution adaptation. Several sites of the ER are
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differentially PAH contaminated and the degree of resistance to developmental toxicity
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of PAHs in fish inhabiting these sites directly corresponds to the level of contaminants
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(Figure 1)3, 14, and are genetically different compared to fish from clean sites.8, 15, 16
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Therefore, these differentially resistant ER subpopulations provide a “natural
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experiment” to investigate consequences of evolved resistance and inhabiting a polluted
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environment, and the impact of adaptive or ancillary shifts in physiological and
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biochemical processes on capacity of organisms to maintain optimum performance in a
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multi-stressor context.
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To this end, a comparative physiological approach and an analysis of an existing
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population genomic dataset was taken to examine bioenergetic consequences of PAH-
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resistance and inhabiting a chronically polluted environment in ER fish,14 relative to a
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clean reference subpopulation, Kings Creek (KC) (Figure 1). We focused on energy
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metabolic processes since they may be under significant selection pressure due to (i)
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increased ATP demand for PAH detoxification and to maintain cellular homeostasis; (ii)
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effects of PAHs on mitochondrial integrity17 as well as cardiac development and
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function18-20 potentially altering oxygen and nutrient circulation; and (iii) presumed role
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of the aryl hydrocarbon receptor, a key protein involved in PAH metabolism, as a
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mitochondrial regulator.21
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First, we examined a restriction site-associated DNA (RAD) genomic dataset comparing
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fish from a highly contaminated site (Atlantic Wood (AW) Superfund site; Fig 1) with
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Mains Creek (MC, very low PAH site in the ER) and KC to test if genes associated with
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energy metabolism are under selection. We then examined cardiac mitochondrial
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function utilizing an ex vivo oxygen consumption rate (OCR) assay in AW and KC fish.
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Considering the role of cardiac bioenergetics in defining thermal limits of teleosts,22-24 we
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evaluated cardiac mitochondrial function at 24°C and 34°C as an index for thermal
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plasticity. Subsequently, we extended our analysis to include four ER subpopulations
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(Figure 1) and characterized shifts in organismal aerobic metabolism at different
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temperatures and also measured thermal tolerance. To characterize potential ecological
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significance of altered bioenergetics, we measured swimming performance and the costs
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of finding an optimum environment based on a novel probabilistic movement model.
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Collectively, the current study provides a comprehensive analysis of bioenergetic
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consequences and potential ecological significances of rapid evolution to anthropogenic
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contaminants in inhabiting a chronically polluted environment in a vertebrate.
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Materials and methods
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Fish collection and care. Fish were collected from the reference (KC) and ER sites
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(AW-highly contaminated; Money Point (MP)-contaminated and under remediation;
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Jones Creek (JC)-moderately contaminated; MC-low levels of contaminants) (Figure 1,
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SI). MP remediation started in 2009 and fish were collected from a fully remediated
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section. Upon capture, fish were maintained in static glass tanks (30 cm × 30 cm × 75
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cm, at 24–25°C, 14:10 h light-dark cycle, and13–15 ppt artificial seawater (ASW; Instant
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Ocean, Foster & Smith, Rhinelander, WI, USA) and were used within 2-3 months of
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acclimation. Fish were fed ad libitum a mix of Cyclop-eeze (Argent Chemical
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Laboratories, Inc., WA, USA) and Zeigler’s Adult Zebrafish Complete Diet (Zeigler
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Bros., Inc., Gardners, PA, USA). Water changes were conducted once a week. Fish were
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carefully chosen to represent a similar size range; weights and lengths are in Table S1.
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Only males were chosen, but gender identification was difficult at times in small
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juveniles. We specifically focused on wild-caught lab acclimated fish, as opposed to lab
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reared F1 individuals, to characterize the metabolic phenotype of each subpopulation that
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is most representative of their physiological function at each ER site. Detailed methods
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for each experiment are available in SI.
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RAD analysis. A set of genomic scaffolds that were previously characterized as
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exhibiting genetic differentiation across multiple comparisons of fish from polluted and
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unpolluted sites (Osterberg and colleagues, unpublished data) were queried for genes
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related to energy metabolism and maintaining mitochondrial function and integrity based
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on user defined categories determined by Gene Ontology information and previous
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literature (Table S2). Data were drawn from a double digest RAD sequencing25, 26
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analysis of 83,144 loci and 12,071 single nucleotide polymorphisms (SNPs) that
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compared genome-wide variation among AW, KC, and MC fish (n=32 per population)
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(SI). RAD loci with smoothed pairwise FST values27 that were significantly greater than
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the average across the genome (P
