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

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

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

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

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

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%

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

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.

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

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

More CO2 results in more H+ ions in the ocean. CO2 + H20  H2CO3  HCO3- + H+  CO3 -2 + 2H+ 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

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

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

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

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?

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

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

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)

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

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

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

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?

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

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.

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

References Dondero, F., A. Dagnino, H. Jonsson, F. Capri, L. Gastaldi, A. Viarengo. 2006. 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. 2008. Impacts of ocean acidification on marine fauna and ecosystem processes. ICES J. Mar. Sci.65: 414-432. Feely,R., C. Sabine, J.M. Hernandez-Ayon, D. Ianson, B. Hales. 2008. Evidence for Upwelling of Corrosive “Acidified” Water onto the Continental Shelf. Science. 320: 1490-1492. Orr, J. et al. 2005. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature. 437(29): 681-686. Sabine, C. et al. 2004. The Oceanic Sink for Anthropogenic CO2. Science. 305: 367-372. Schroth, W., A. Ender, B. Schierwater. 2005. Molecular biomarkers and adaptation to environmental stress in moon jelly (Aurelia spp.). Mar. Biotech. 7(5): 1436-2228. Pictures: Steven Roberts , http://www.funny-potato.com/jellyfish.html (jellyfish), http://jackpotresearch.wordpress.com/2009/03/ (drawn oyster), http://www.kdwp.state.ks.us/news (mussel)