Seawater acidification and the general stress response of the Pacific oyster

Crassostrea gigas

Rachel ThompsonDecember 11, 2009

CO2 induced acidification

CO32-

Interaction with H+ makes it less available to calcifying organisms (Orr et al. 2005)

CaCO3

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

Acidification and cellular stress response

Todgham et al. 2009

Purple Sea Urchin Strongylocentrotus purpuratus

Biomineralization,Development

Cellular Stress Response

High CO2

Control

Climate change: Potential for multiple stressors

Temperature increase

Acidification

U.S. EPA

IPCC Projections

How will organisms be affected?

QuestionObjective

• How does acidification affect the general molecular stress response of the Pacific oyster?

• Hypothesis: Oysters living in seawater with high dissolved CO2 levels will have less energy available to implement a strong stress response to additional disturbances

Methods:Collection

Willapa Bay

Samish Bay

North Bay

Methods: Acclimation

48 oysters separated into 2 groups

Group 1: Normal Air

Group 2: CO2 (pH~7.2)

Maintained at constant temperature for 1 week

CO2

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

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

Stressed Non-stressedArbitrary Expression Value = 100.3012*Ct + 11.434

Results: Stress response to CO2?

Yes: Significant differences in expression of all 3 genes

Results: Mechanical stress response?

Yes: Significant differences in expression of HSP 70 and glutathione peroxidase

QuestionObjective

• How does acidification affect the general molecular stress response of the Pacific Oyster?

• Hypothesis: Oysters living in seawater with high

dissolved CO2 levels will have less energy available to implement a strong stress response to additional disturbances

Results: Does CO2 exposure affect the oyster’s response to mechanical stress?

Control Carbon Dioxide

Possibly…increases in expression levels in control oysters significant

ConclusionsHypothesis: Oysters living in seawater with high dissolved

CO2 levels will have less energy available to implement a strong stress response to additional disturbances

1. CO2 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?)

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

BONUS

ARISA (Automated ribosomal intergenic spacer analysis): Compare microbial communities between control and CO2 exposed oysters based on “DNA fingerprint”

Significant difference between control and CO2!

Horner-Devine lab

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

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.