Fundulus as a Model for Comparative Toxicology:

Increasing the ecological relevance of shared modes of action through the incorporation of phylogenetics and

species sensitivities

Mark A. DugoJackson State University

Jackson, MS, USA

NOAA-ECSC Webinar SeriesJanuary 21, 2015

AcknowledgementsDr. Paul B. Tchounwou (JSU, Major Professor)NOAA, ECSC, Grand Bay NERRChristina Morhman (GBNERR – ECSC Site Coordinator)Hilliard Lackey (JSU/ECSC Correspondent)William T. Slack (U.S. Army Corps of Engineers - MMNS)Brian R. Kreiser (USM)Steven T. Ross (USM, emeritus)Robert Cashner (UNO emeritus)Mark S. Peterson (GCRL)Mississippi Museum of Natural Science (State Wildlife Grant)

CORE FOCUS AREAS:

Ecosystem Characterization

Ecological Processes

Social and Economic Processes

Forecasting and Modeling

Policy and Decision Tools

Processes: Towards increased understanding of biological structure and function at the population level to better understand responses to anthropogenic stressors.

Forecasting: To better predict ecological responses to anthropogenic changes

Policy:

Molecular Cellular Individual Population Community

Ecological RelevanceTranslating causality between the mode of action (MOA) of a stressor response at the

biochemical level beyond the organism to the population or community level possibly to the ecosystem level

Mode of Action Ecological Relevance

Increasing Complexity Ecosystems

Testing uncertainty

H1 – Species will respond differently to the same stressor

Null Hypothesis – Individual taxa will respond similarly to the same stressor

Global Climate Change2014 Hottest Year on Record

“No challenge poses a greater threat to future generations than climate change”- President Barack Obama

2015 State of the Union Address

2015

Fundulus (heteroclitus)Mummichog

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics

Burnett KG, Bain LJ, Baldwin WS, Callard GV, Cohen S, Di Giulio RT, Evans DH, Gómez-ChiarriM, Hahn ME, Hoover CA, Karchner SI, Katoh F, Maclatchy DL, Marshall WS, Meyer JN, NacciDE, Oleksiak MF, Rees BB, Singer TD, Stegeman JJ, Towle DW, Van Veld PA, Vogelbein WK, Whitehead A, Winn RN, Crawford DL.

Comp Biochem Physiol Part D Genomics Proteomics. 2007 Dec;2(4):257-286.

Mummichog – Model of Toxicology, Pollution Tolerance, and Adaptation

Natural physiological adaptive traits:Temperature – Distribution; Atlantic seaboard, natural longitudinal thermal cline (Burnett et al. 2009)

Salinity – Tolerance from 0- 120 parts per thousand (Griffith 1974)

Adaptation and Tolerance in Polluted environmentsA model of adaptive evolution –mechanism> PAH toxicities> Aryl Hydrocarbon Receptor,

CYP1A1

(Reviewed in Burnett et al. 2009)

Levels of UncertaintyF. heteroclitus has proven to be an ideal single species model for the delineation of MOAs in the context of adaptation and tolerance to environmental toxicant (PCBs, PAHs, and HAHs at Superfund sites)

(Oleksiak et al. 2011, Wills et al. 2010, Williams and Oleksiak 2008, Hahn et al. 2004, Bello et al. 2001, Willet et al. 2001, and others)

Levels of uncertainty:Intraspecific variabilityInterspecific variability - other species in the same coastal habitats are much less tolerant.

Using the sentinel work on F. heteroclitus as a springboard, future work should consider expanding investigations to include members of the Fundulus clade

North American Freshwater, Estuarine and Marine Fishes

Distribution map of Fundulines (Parenti 1981)

Diversity within Fundulus

Fundulus+ 30 species

across 4 subgenera

> 12 species of Fundulusoccur in Mississippi waters

DNA based Phylogeny overlain with osmotolerance ranking:freshwater (red)brackish (green)marine (blue)

Parenti 1981

FundulusPhylogenetic signal of osmotolerance

Whitehead 2010

Jackson State University Environmental Science Graduate Studies in collaboration with the NOAA Environmental Cooperative Science Center (ECSC);Grand Bay National Estuarine Research Reserve (GBNERR)

Estuarine SpeciesSalinity range 0-20 ‰ (freq.,6-12 ‰, midland marshes driven by freshwater influx) (Lopez et al. 2011)

September, 2011-Removed From NOAA “Species of Concern”

Currently Under Review “Candidate Species”

Endangered Species Act under jurisdiction of the

United States Fish and Wildlife Service

Phylogenetic EcotoxicologyIntegrative Research

Examples of Eco-physiological Diversity within Fundulus

1) Diverse range of osmotolerance among Fundulus(classically known)

3) F. nottii species complex – 5 backwater species, including two floodplain dependent species*. Floodplains are stressful places to live. Ecological diversity among closely related species.

(*revealed in current study)

Effort of Current Research

Field Collections – 54 Independent CollectionsSpecimens – 3,523 IndividualsDNA Samples – 220 IndividualsDNA Sequences – 28 Individuals

RNA Samples (Liver and Gills) – 378 Samples

Genes for Expression Studies – CYP1A1 and FMO A

Treatments3-Methylcholanthrene (3-M) (PAH), using a corn oil vehicle

IP Injected (30mg/kg) (Powers et al. 1998)

4 days exposure in freshwater, 10ppt or 20ppt

*Controls – Corn Oil IP Injection,(no 3-M)

Molecular Systematics – Fundulus notti species complex

Comparative Toxicology

Molecular Methods

DNA sequencingmitochondrial cyt b1140 base pairs

Phylogenetic Analysis – PAUP*4.0b10Maximum ParsimonyNJ, Kimura 2-parameter Distance Measures

Mr. Bayes 3.1Bayesian Analysis - GTR+G model2 independent runs, 4 Markov chains, 1,000,000 generations, sampled every 100 generation; 2,500 burn- in

Bootstrapping, 1,000 rounds resamplingTREEVIEW 1.40

F. lineolatus F. escambiae F. nottii F. dispar F. blairae

Fundulus nottii species group

Wiley 1977; Ghedotti and Grose 1997

Modified from: Wiley 1977; See also Wiley and Mayden 1985

F. notti species complex Based on Molecular

Phylogeny

F. dispar

F. blairae

F. lineolatus

F. escambiaeF. notti

Fundulus – Phylogenetic Ecotoxicology

Genbank + BLAST searchWhitehead 2010

FMOs and Osmoregulation

FMO oxidizes choline derived trimethylamine (TMA) to trimethylamine oxide (TMAO). TMAO has osmotic function in retaining water and minimizing salt intake.

FMO acts to reduce stress. (Trout, Medaka and flounder.)

FMO is upregulated in saline environments(El-Alfy et al. 2002, Schlenk et al. 1996, Rodriquez-Fuentes et al. 2008, Wang et

al. 2001).

*FMO’s have not been explored in Fundulus; however Fundulus is described as a model test organism (Burnett et al. 2007.)

Aims of Comparative Toxicology Study:1) Assess differential toxicological dose response among

closely related Fundulus species (i.e. Phylogenetic Toxicology), according to CYP and FMO activity levels.

Ha1: Species dependent toxicities will reflect signatures of ecological tolerance.

2) Assess differential toxicological dose response among estuarine Fundulids in relation to varied salinities, according to CYP and FMO activity levels.

Ha2: Salinity will alter toxicity on a species dependent basis (i.e. more sensitive species, more toxic)

3) Determine the utility of the FMO enzyme system in Fundulus species

Ha3: FMO activity will be present in Fundulus

CYP1A1 mRNA Expression

CYP1A1 mRNA Expression

CYP1A1 mRNA levels are globally up regulated following 3-M treatment and were more pronounced in the liver versus gills.

Interspecies variability of modulation is evident.

The magnitude of CYP1A1up regulation is salinity dependent, being more pronounced in the higher salinity treatment (20ppt).* Statistically significant (p<0.005); ** Statistically significant (p<0.05),

using Student’s t-test.

FMO A mRNA Expression

FMO A mRNA ExpressionFMO A mRNA is down regulated in 3-M salinity treatment groups.

10ppt salinity treatment down regulation > than 20 ppttreatment group.

Freshwater groups does not appear to express FMO mRNA in controls or 3-M treatment groups.

This is the first toxicology study to document FMO modulation in Fundulus according to a toxicant or salinity.

RelevanceThis study considers the genetic and ecological diversity among Fundulus sp. in terms of salinity preference and or “floodplain dependence” to assess interspecies differences of stress response.

This approach is specifically useful in the context of habitat loss and climate change. Anthropogenic activities are causing a decline in the abundance of quality salt marsh and floodplain habitats. Natural fluctuations within these habitats are further subject to pronounced environmental shifts due to climate change.

F. jenkinsi - longstanding NOAA species of concern, and recent candidate for listing under the Endangered Species Act.

F. dispar and F. blairae - herein verified as distinct species, have also been identified through field collections, as obligates to the floodplains of larger rivers. These species may have differential physiological tolerances relative to other members of the F. nottii clade, which may translate into variable sensitivity to xenobiotic insults among closely related species.

Thank You

Fundulus as a Genomic Model at Multiple Levels of Molecular

Comparison

Sequence Data- Evolutionary History

Molecular SystematicsPhylogeography

- Population Genetics Metapopulation Dynamics Phenotypic Plasticity

Signaling Pathways- Modes of Action

AdaptationPhenotypic PlasticityCancer Model

Fundulus heteroclitus (Linnaeus, 1766)Complete Genomic Sequencing Underway

Flavin-containing monooxygenases (FMOs)

Eswaramoorthy al. 2006

FMOs attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion.

(Eswaramoorthy al. 2006)

5 FMO Genes globally (Krueger, and Williams 2005, Schlenk 1998)

*Clinically popularized - Trimethylaminuria (TMAU), FMO3 mutation, inhibits metabolism of trimethylamine (TMA) into trimethylamine oxide (TMAO) (Treacy et al. 1998)

Xenobiotic Induction of FMOsThioester pesticides are the most commonly studied modulators of FMO catalytic function

(i.e. Aldicarb; a cholinesterase inhibitor ) (Rodriquez-Fuentes et al. 2008, El-Alfy et al. 2002, Wang et al. 2001, Schlenk 1998)

Most Recently: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene (BaP), and 3-Methylcholanthrene (3-M) have been shown to modulate FMO mRNA levels, however; protein levels and catalytic function are not always linear to mRNA induction.

Most interestingly, TCDD, BaP and 3-M modulation of FMO mRNA is dependent on the AhR receptor i.e. (Dioxin Receptor) (Celius et al. 2010, Celius et al. 2010)

*Several PAH’s have been identified to modulate both FMO and CYP; however there is variation among species and tissue types within species. Sex Dependent

(Celius et al 2008, Lewis et al. 2004, Novick et al. 2009, Zhou et al. 2001).