1. Seawater acidification and the general stress response of the Pacific oyster Crassostrea gigas Rachel Thompson December 11, 2009

2. CO 2 induced acidification CO 3 2- Interaction with H+ makes it less available to calcifying organisms (Orr et al. 2005) CaCO 3

3. Effects on C. gigas physiology Development and Morphology Decreased growth rate Increased larval mortality Shell deformation What is happening at the molecular level? Gazeau et al. 2007

4. Acidification and cellular stress response Todgham et al. 2009 Purple Sea Urchin Strongylocentrotus purpuratus Biomineralization, Development Cellular Stress Response High CO 2 Control

5. Climate change : Potential for multiple stressors Temperature increase Acidification U.S. EPA IPCC Projections How will organisms be affected?

6. Question Objective How does acidification affect the general molecular stress response of the Pacific oyster? Hypothesis : Oysters living in seawater with high dissolved CO 2 levels will have less energy available to implement a strong stress response to additional disturbances

7. Methods: Collection Willapa Bay Samish Bay North Bay

8. Methods: Acclimation 48 oysters separated into 2 groups Group 1: Normal Air Group 2: CO 2 (pH~7.2) Maintained at constant temperature for 1 week CO 2

9. Methods: Mechanical Stress Induces a “transient state of stress” (Lacoste et al. 2002) Used as surrogate for additional environmental stressor (e.g. heat, pathogen) Oysters experience mechanical stress during sorting and grading (aquaculture) 5 minutes 12 oysters from each acclimation group

10. Methods: Analysis of gene expression RNA isolated from gill tissue, reverse transcribed qPCR with primers for 3 stress response genes: -Heat shock protein 70 -Metallothionein IV -Glutathione peroxidase StressedNon-stressed Arbitrary Expression Value = 10 0.3012*Ct + 11.434

11. Results: Stress response to CO 2 ? Yes: Significant differences in expression of all 3 genes

12. Results: Mechanical stress response? Yes: Significant differences in expression of HSP 70 and glutathione peroxidase

13. Question Objective How does acidification affect the general molecular stress response of the Pacific Oyster? Hypothesis : Oysters living in seawater with high dissolved CO 2 levels will have less energy available to implement a strong stress response to additional disturbances

14. Results: Does CO 2 exposure affect the oyster’s response to mechanical stress? ControlCarbon Dioxide Possibly…increases in expression levels in control oysters significant

15. Conclusions Hypothesis: Oysters living in seawater with high dissolved CO 2 levels will have less energy available to implement a strong stress response to additional disturbances 1. CO 2 exposure results in up-regulation of HSP 70, MT IV and GPx 2. Mechanical stress generates an additional increase in expression, except for MT IV 3. The magnitude of the increase in expression tended to be larger for control oysters, as indicated by greater statistical significance (Evidence for hypothesis?)

16. Implications pH of ocean will continue to decrease Compounding effects from multiple stressors (acidification, temperature increase) Acidification will shift resources away from other physiological processes

17. BONUSBONUS ARISA (Automated ribosomal intergenic spacer analysis): Compare microbial communities between control and CO 2 exposed oysters based on “DNA fingerprint” Significant difference between control and CO 2 ! Horner-Devine lab

18. Future Work Different classes of genes Additional stress response genes Function of MT in relation to acidification? Not involved in other stress responses? Replace mechanical stress with environmental stress of interest

19. References Gazeau F, Quiblier C, Jansen JM, Galtuso JP, Middleburg JJ and Heip CHR. 2007. Impact of elevated CO2 on shellfish calcification. Geophysical Research Letters, 34(7). Lacoste A, Malham SK, Gelebart F, Cueff A and Poulet SA. 2002. Stress induced immune changes in the oyster Crassostrea gigas. Dev Comp Immunol, 26(1):1-9. Orr JC, Fabry VJ, Aumont O, Bopp L, Doney SC, Feely RA, Gnanadesikan A, Gruber N, Ishida A, Joos F, Key RM, Lindsay K, Maier-Reimer E, Matear R, Monfray P, Mouchet A, Najjar RG, Plattner GK, Rodgers KB, Sabine CL, Sarmiento JL, Schlitzer R, Slater RD, Totterdell IJ, Weirig MF, Yamanaka Y and Yool A. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature, 437(7059):681-6. Talmage SC and Gobler CJ. 2009. The effects of elevated carbon dioxide concentrations on the metamorphosis, size, and survival of larval hard clams (Mercenaria mercenaria), bay scallops (Argopecten irradians), and Eastern oysters (Crassostrea virginica). Limnology and Oceanography, 54(6): 2072-2080. Todgham AE and Hofmann GE. 2009. Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification. J Exp Biol, 212(Pt 16):2579-94.