Mark`s Presentation - PDF - NOAA Environmental Cooperative


Mark`s Presentation - PDF - NOAA Environmental Cooperative
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. Dugo
Jackson State University
Jackson, MS, USA
NOAA-ECSC Webinar Series
January 21, 2015
Dr. Paul B. Tchounwou (JSU, Major Professor)
Christina 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)
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
Forecasting: To better predict ecological
responses to anthropogenic changes
Ecological Relevance
Translating 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
Testing uncertainty
H1 – Species will respond differently to the same
Null Hypothesis – Individual taxa will respond
similarly to the same stressor
Global Climate Change
2014 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
Fundulus (heteroclitus)
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-Chiarri
M, Hahn ME, Hoover CA, Karchner SI, Katoh F, Maclatchy DL, Marshall WS, Meyer JN, Nacci
DE, 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
(Burnett et al. 2009)
– Tolerance from 0- 120 parts per thousand
(Griffith 1974)
Adaptation and Tolerance in Polluted environments
A model of adaptive evolution –
mechanism> PAH toxicities>
Aryl Hydrocarbon Receptor,
(Reviewed in Burnett et al. 2009)
Levels of Uncertainty
F. 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 variability
Interspecific 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
Diversity within Fundulus
North American Freshwater,
Estuarine and Marine Fishes
+ 30 species
across 4 subgenera
> 12 species of Fundulus
occur in Mississippi waters
Distribution map of
Fundulines (Parenti 1981)
Phylogenetic signal of osmotolerance
DNA based Phylogeny overlain with
osmotolerance ranking:
freshwater (red)
Whitehead 2010
Parenti 1981
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 Species
Salinity range 0-20 ‰
(freq.,6-12 ‰, midland marshes driven by
freshwater influx) (Lopez et al. 2011)
September, 2011Removed From NOAA “Species of Concern”
Currently Under Review
“Candidate Species”
Endangered Species Act
under jurisdiction of the
United States Fish and Wildlife Service
Phylogenetic Ecotoxicology
Integrative Research
Examples of Eco-physiological Diversity within
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
Molecular Systematics – Fundulus notti species complex
Field Collections – 54 Independent Collections
Specimens – 3,523 Individuals
DNA Samples – 220 Individuals
DNA Sequences – 28 Individuals
Comparative Toxicology
RNA Samples
(Liver and Gills) – 378 Samples
Genes for Expression
Studies – CYP1A1 and FMO A
3-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 Methods
DNA sequencing
mitochondrial cyt b
1140 base pairs
Phylogenetic Analysis – PAUP*4.0b10
Maximum Parsimony
NJ, Kimura 2-parameter Distance Measures
Mr. Bayes 3.1
Bayesian Analysis - GTR+G model
2 independent runs, 4 Markov chains,
1,000,000 generations, sampled every 100
generation; 2,500 burn- in
Bootstrapping, 1,000 rounds resampling
Fundulus nottii species group
F. lineolatus
F. escambiae
F. nottii
F. dispar
F. blairae
Wiley 1977; Ghedotti and Grose 1997
Modified from: Wiley 1977; See also Wiley and Mayden 1985
F. notti species complex
Based on Molecular
F. dispar
F. lineolatus
F. blairae
F. notti
F. escambiae
Fundulus – Phylogenetic Ecotoxicology
Genbank +2010
BLAST search
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.
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)
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.
* Statistically significant (p<0.005); ** Statistically significant (p<0.05),
using Student’s t-test.
The magnitude of CYP1A1
up regulation is salinity
dependent, being more
pronounced in the higher salinity
treatment (20ppt).
FMO A mRNA Expression
FMO A mRNA Expression
down regulated in 3-M salinity
treatment groups.
10ppt salinity treatment down
regulation > than 20 ppt
treatment group.
Freshwater groups does not
appear to express FMO mRNA in
controls or 3-M treatment
This is the first toxicology study
to document FMO modulation in
Fundulus according to a toxicant
or salinity.
This 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
Sequence Data
Signaling Pathways
- Evolutionary History
Molecular Systematics
- Population Genetics
Metapopulation Dynamics
Phenotypic Plasticity
- Modes of Action
Phenotypic Plasticity
Cancer Model
Fundulus heteroclitus (Linnaeus, 1766)
Complete Genomic Sequencing Underway
Flavin-containing monooxygenases (FMOs)
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)
Eswaramoorthy al. 2006
Xenobiotic Induction of FMOs
Thioester 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).

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