Christopher K' Beachy and S' Randal Voss

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Development of an ecotoxicogenomic model using
salamander
Christopher K. Beachy and S. Randal
Voss Department of Biology, Minot State
University, Minot, ND 58707 and Department of
Biology, University of Kentucky, Lexington, KY
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Why salamanders and why axolotl?
Axolotl photos
(aside from the obvious)
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A phylogeny of model craniates
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Whats the problem?
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ENDOCRINE DISRUPTION
ATRAZINE
--Post application (Spring) runoff can be gt 20
ppb --typical surface and groundwater
concentrations at other times during other times
of the year, 0.001 to 1.0 ppb --3 ppb EPA
standard for drinking water
Males exposed to 0.1 ppb have lowered
testosterone, elevated estrogen, and intersex
gonads
Xenopus laevis Hayes et al, 2002, PNAS
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ENVIRONMENTAL CONTEXT COUNTS effects of
sublethal levels of carbaryl in a natural
context.
100
Hyla versicolor
Predator cues dont kill tadpoles
Bufo americanus
mortality
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Relyea, 2004
0
Carbaryl and predator cue
carbaryl
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Whatre we doing?
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What we will learn
Associated Null Hypotheses
Life history and morphological assays will not
differ between control and EDed salamanders
How endocrine disruption results in morphological
and life history effects
Evaluate dynamic expression patterns via GEP or
SAGE
There is no dynamic gene expression pattern
Patterns of gene expression will not differ
between control and EDed salamanders
Compare GEP between control and EDed salamanders
Correlate differences in GEPs with differences in
morphological expression in tissues and life
history features
There will be no association among ED-induced
gene expression patterns and tissue/life
history features
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ECOLOGY PART
Full-factorial MANOVA on
Maturation (gonad status and mass)
Metamorphic timing and size
Storage (fat body mass)
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MICROARRAY CONSTRUCTION AND ANALYSIS efficient,
first-pass method for identifying candidate genes
that may be important in metamorphosis and
endocrine disruption of this process
Identify genes up- and down-regulated
washed over
microarray (gene chip)
mRNA isolated from tissue
--during metamorphosis and between control and
EDed animals
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What we will learn
Associated Null Hypotheses
Life history and morphological assays will not
differ between control and EDed salamanders
How endocrine disruption results in morphological
and life history effects
Evaluate dynamic expression patterns via GEP or
SAGE
There is no dynamic gene expression pattern
Patterns of gene expression will not differ
between control and EDed salamanders
Compare GEP between control and EDed salamanders
Correlate differences in GEPs with differences in
morphological expression in tissues and life
history features
There will be no association among ED-induced
gene expression patterns and tissue/life
history features
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So, what do we have to do first?
Establish the genomic standard
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Our GeneChips 4800 ESTs --Housekeeping
genes --Replication --all that stuff
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0 hours (pre-TH application)
6 hours (post-TH treatment)
24 hours (post-TH treatment)
RBCs harvested at 3 developmental stages --each
stage represents premet, met, and postmet rbc
populations --comparison of 0 hour stages
provides standard GEP --comparison of 6 and 24
hour stages provides determination of
differential sensitivity
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GENOMICS PART
Gene expression profile/profiling (GEP)
Red blood cells harvested from three life history
stages
Time points
Pre-treatment
6 hours post TH treatment
24 hours post TH treatment
Replicate chips
3 X 3 X 6 54 chips
3 animals per chip 162 animals
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NEXT PART Compare GEPs between/among Atrazine-i
nduced salamanders Control animals
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The essential link back to nature
Comparisons with standard TH GEP profile and
ATR-affected GEP There is no assay that is
more sensitive than the organism. Beachy, this
meeting