1. CHARACTERIZING THE RESPONSE OF THE PACIFIC OYSTER (Crassostrea gigas) TO ELEVATED CO2 AND DISEASE EXPOSURE
Emma Timmins-Schiffman
SAFS, Roberts Lab
UW Graduate Student Symposium
November 19, 2009

2. Purpose How does ocean acidification affect Pacific oyster physiology?
Genes give us insight into physiological pathways

3. Outline Background Proposed research plan
Ocean acidification
Effects on marine organisms
Proposed research plan
Pilot study and preliminary results

4. Outline Background Proposed research plan
Ocean acidification
Effects on marine organisms
Proposed research plan
Pilot study and preliminary results

5. Global Warming
Increased atmospheric CO2 = increased aqueous CO2 = lower oceanic pH

6. Acidification: effects on the oceanic environment
Water chemistry
CO2(aq) + H2O  H2CO3 (carbonic acid)  HCO3- (bicarbonate) + H+  CO3 -2 (carbonate) + 2H+
Metal bioavailability
Upwelling of “corrosive water” along west coast
Seasonal effects of wind and currents
[CO3 -2]
30%
2x pre-industrial CO2
Oceanic ability to absorb CO2
[H+]
60%

7. Outline Background Proposed research plan
Ocean acidification
Effects on marine organisms
Proposed research plan
Pilot study and preliminary results

8. Acidification: effects on marine organism calcification
Calcification: H+ interacts with carbonate so it is less available to form CaCO3
Prediction of ocean-wide shoaling of aragonite (CaCO3) undersaturated waters by 2100.
Highest CO2 concentrations are in near-shore waters.
Acidic waters are approaching near-shore habitats.

9. CaCO3 saturation state
<1.0 is associated with corrosive seawater (Fabry et al. 2008)

10. CO2 CO2 CO3-2 CO2 CO3-2 H+ CO2 CO3-2 CO3-2 H+ CO3-2 Anthropogenic CO2
CaCO3
CaCO3
CaCO3
CaCO3

11. More CO2 results in more H+ ions in the ocean.
CO2 + H20  H2CO3  HCO3- + H+  CO H+
CO2
More CO2 results in more H+ ions in the ocean.
H+ associates with CO3-2, taking this essential ion
away from calcifying organisms.
CO2
CO2
CO2
CO2
CO2
H2CO3
H2CO3
H2CO3 H+
Equilibrium chemical equation - will shift in order to balance when one compound is increased or decreased H+ H+
CO2
CO2
CO3-2
CO2 H+
CO3-2
H2CO3
CO2
CO2 X X X
CaCO3

12. Acidification: effects on marine organism physiology
pH also affects metabolic physiology
Energy allocated to dealing with one stress means less energy for other basic physiological functions.
Ecosystem-wide effects: biodiversity, trophic interactions
Internal acid-base balances.
Lower levels (phytoplankton, bivalves) compromised --> effects move up food chain

13. Oysters can be used as a model organism since the stress response is evolutionarily highly conserved.

14. Outline Background Proposed research plan
Ocean acidification
Effects on marine organisms
Proposed research plan
Pilot study and preliminary results

15. Overview of Proposed Research
Assess the interrelationship of temperature, pH, and disease on oyster physiology.
Which factors (in wild and hatchery settings) are specifically associated with poor larvae survival?

16. Proposed Research Methods
Samples
Hatchery larvae
Wild spawning events
In a controlled setting, expose oysters to varying conditions: pH, temperature, V. tubiashii presence/absence
Separately
Synergistically

17. Other Examples Xenobiotic stress in jelly fish (Schroth et al. 2005)
Metal load (copper) in mussels (Dondero et al. 2006)
Jellies: upregulation of genes that deal with oxidative stress proteins (antioxidants), general stress. Time dependency of response until very high loads of contaminant.
Mussels: upregulation of general stress genes, anitoxidant response, and basic cell repair

18. Proposed Research Objective
Characterize differentially-expressed genes in oysters exposed to changes in pH (CO2), temperature, and disease exposure.
(Develop panel of genes (~12) where expression is significantly affected
Microarray
Candidate gene (RT-qPCR)
Protein assays
Determine physiological pathways in which differentially-expressed genes are involved)

19. Outline Background Proposed research plan
Ocean acidification
Effects on marine organisms
Proposed research plan
Pilot study and preliminary results

20. Pilot Study: Methods Adult & juvenile Pacific oysters CO2 trial
Control (380 ppm)
Experimental (970 ppm)
V. tubiashii trial
Differential expression?

21. Pilot Study: Results Candidate gene approach SOD IL-17 AURKA CytP450
MDR
HIF
Hsp70
Prx6
Extract RNA (whole body juveniles)
Reverse-transcribe to cDNA
Real-time quantitative PCR to measure gene expression

22. Pilot Study: a closer look at Hypoxia-induced factor 1a
Not significant, but hint at possibilities for more trials. Energy allocation away from other stress pathways?

23. Pilot Study: Results Differential display
PCR cDNA with arbitrary primers - find expression difference then find genes

24. Next Steps…
Continue with differential display to find more expression differences.
Follow similar procedures for the disease-exposed oysters.
Continue candidate gene approach through more literature research and laboratory testing.

25. Questions? Thank you: Saltonstall-Kennedy Grant Program (NOAA)
Dr. Steven Roberts - UW, SAFS
Sam White - UW, SAFS
Rony Thi - UW, Forestry
Mackenzie Gavery - UW, SAFS
Dr. Tim Green - University of Queensland

26. References
Dondero, F., A. Dagnino, H. Jonsson, F. Capri, L. Gastaldi, A. Viarengo Assessing the occurrence of stress syndrome in mussls (Mytilus edulis) using a combined biomarker/gene expression approach. Aq. Tox. 78(Supp. 1): S13-S24.
Fabry, V., B. Seibel, R. Feely, J. Orr Impacts of ocean acidification on marine fauna and ecosystem processes. ICES J. Mar. Sci.65:
Feely,R., C. Sabine, J.M. Hernandez-Ayon, D. Ianson, B. Hales Evidence for Upwelling of Corrosive “Acidified” Water onto the Continental Shelf. Science. 320:
Orr, J. et al Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature. 437(29):
Sabine, C. et al The Oceanic Sink for Anthropogenic CO2. Science. 305:
Schroth, W., A. Ender, B. Schierwater Molecular biomarkers and adaptation to environmental stress in moon jelly (Aurelia spp.). Mar. Biotech. 7(5):
Pictures: Steven Roberts , (jellyfish), (drawn oyster), (mussel)