1. Gene Expression as an Indicator
of Environmental Stress in the
Pacific Oyster, Crassostrea gigas
Emma Timmins-Schiffman University of Washington
Steven Roberts SAFS
Mackenzie Gavery
AFS, Conservation Genetics and Genomics in Fisheries
September 8, 2011

2. Outline
¤ Climate change in the Pacific Northwest
¤ Oyster mortalities in hatcheries
¤ Transcriptomic tools for monitoring
environmental change
¤ Targeted gene approach
¤ Whole transcriptome (NGS) approach
¤ Applications of transcriptomics

3. Climate Change in the PNW
IPCC (2007) Projected Estimates of Atmospheric CO2
¤ Ocean acidification
¤ As CO2 dissolves in
800
seawater, pH goes
Estimate of atmospheric CO2 (ppm)
down.
700
Kilometers along transect
600
500
400
300
1750 1800 1850 1900 1950 2000 2050 2100
Feely et al. 2010 Year

4. WA Dept. of Ecology 2007
Commercial/Industry
High Population
Density

5. Hatchery mortalities
¤ Larval mortalities have been linked
to acidic, upwelled waters and
disease
Elston et al. 2008

6. Transcriptomic Tools for Conservation
¤ What are transcriptomics?
¤ Measurement of physiological response
¤ Range/plasticity of response
¤ Plasticity of population response linked to underlying
genetic diversity
Adaptation?
Population Environmental Bottleneck?
Change
Acclimatization? Mortality?

7. Transcriptomics and Conservation
¤ How do these host of environmental changes affect all
life stages of Pacific oyster?
¤ How can we learn about population conservation and
response to environmental change through use of
transcriptomic tools?

8. Transcriptomic Tools for Conservation
¤ 2 methods
¤ Targeted gene approach (bottom up)
¤ Global transcriptome/next-generation sequencing (top
down)
Which genes/pathways might change? What response do we see?

9. Targeted Gene Approach
¤ Oyster larvae exposed to 3 levels of pCO2 for 4 days –
ambient (400 µatm), and 2 elevated (700 and 1000 µatm)
¤ Variables measured: water chemistry, size, calcification,
developmental stage, and gene expression
¤ Genes chosen:
¤ Molecular chaperone – hsp70
¤ Oxidative stress – SOD, GPx, Prx6

10. Oyster Morphometrics: Results
Hinge Length by Treatment and Day Shell Height by Treatment and Day
80
70
70
60
Hinge Length (!m)
Shell Height (!m)
60
50
40
50
30
Day 1 Day 3 40 Day 1 Day 3
400 700 1000 400 700 1000
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
400 700 1000 400 700 1000
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
Day and pCO2 (!atm) Day and pCO2 (!atm)
Treatment (µatm) Treatment (µatm)

11. Hsp70
Stress Response
STRESS Protein damage/
unfolding
Hsp70
Chaperones bind to proteins to either repair or remove

12. Heat Shock Protein 70
25
Fold Over Minimum Expression
20
15
10
5
400 700 1000
Treatment (!atm)

13. Oxidative Stress Genes
Stress Response
STRESS • Increase metabolism
• Kill pathogens
Prx6
ROSSOD
GPx

14. Fold Over Minimum Expression
0.00 0.05 0.10 0.15 0.20
400
700
Treatment (!atm)
Superoxide Dismutase
1000

15. Fold Over Minimum Expression
0.0e+00 5.0e+24 1.0e+25 1.5e+25
400
700
Treatment (!atm)
Glutathione Peroxidase
1000
Fold Over Minimum Expression
0e+00 1e+22 2e+22 3e+22 4e+22 5e+22 6e+22
400
700
Treatment (!atm)
Peroxiredoxin 6
1000

16. Targeted Gene Summary
¤ Changes in pCO2 affect larval size – what underlying
physiological mechanisms are affected?
¤ Oxidative stress is an important response to ocean
acidification
¤ Acidification may cause larval oysters to increase
production of ROS (metabolism?), necessitating up-
regulation of oxidative stress genes

17. Whole Transcriptome Approach
¤ 2 sites: “pristine” and high
human impact
¤ Oysters collected cDNA
pooled transcriptomes
sequenced using SOLiD

18. v 32 million reads
17 million
matched
Publicly available contigs
29 thousand contigs
Upregulated contigs| min 10 unique hits & 2 fold increase
1329
1316
22 specific
25 specific

19. p-value
cellular component movement 0.04
cell recognition
cell projection organization
cell adhesion 8E-15
semantic space Y
regulation of multicellular organismal
processes
negative regulation of cellular component
organization
cell
regulation of biological communication
quality
hormone metabolism
maintenance of location
in cell immune
maintenance of response
location
developmental
localization of maturation
cellular developmental
cell process
semantic space X

20. Whole Transcriptome: Results
PCA of Oyster Gene Expression at 15 Genes
1
PC2 (30%)
0
MDR49
HMGP
CATL
GSTO1
CALL
RETST
CP17A
GNRR2
GSTA TIMP3.BB
SPI
DPGN
TIMP3.DH
-1
CIQT4
DEF1
Big Beef Creek
Drayton Harbor
-2
-1 0 1 2
PC1 (31%)

21. Whole Transcriptome Summary
¤ A variety of novel processes involved in environmental
response were discovered (cell adhesion)
¤ Global picture of how the environment at the 2 sites is
affecting the oyster physiological response

22. Conclusions
¤ Both the targeted gene and the whole transcriptome
approaches provide valuable tools for understanding
oysters’ plastic response to environmental change.
¤ Gene expression analyses should be considered
important management tools for populations that may
be experiencing environmental stress.

23. Acknowledgements
Emily Carrington•Matt George•Jeff Hard•Michelle
Herko•Laura Newcomb•Ken Sebens•Jim & Lisa
Seeb•Richard Strathmann•Adam Summers•Billie
Swalla•Sam White
NOAA Saltonstall-Kennedy grant