1. The Physiological Response
of Crassostrea gigas to CO2-­‐‑
Induced Ocean
Acidification
Emma Timmins-Schiffman
Carolyn Friedman
Steven Roberts
University of Washington
School of Aquatic and Fishery Sciences
Seattle, WA

2. Plan of Talk
• Background: Ocean acidification and marine
bivalves
• Aim of experiment
• Methods
• Results (proteomics)
• Conclusion
10
8
Oyster Shell Weight (g)
6
4
400 ppm
600 ppm
2
800 ppm
1000 ppm
1200 ppm
1400 ppm
0
0 2 4 6 8 10 12
Oyster Buoyant Weight (g)

3. Ocean Acidification and
the Environment
• Rising pCO2 in the atmosphere equilibrates with
ocean surface water, reducing the pH

4. Ocean Acidification and
the Environment
• Rising pCO2 in the atmosphere equilibrates with
ocean surface water, reducing the pH
• Current and projected changes in pCO2 are
unprecedented
Zeebe 2011

5. Ocean Acidification and
Bivalves
• How do changes in oceanic pCO2 and/or pH
affect the physiology of bivalves?
o C. gigas larvae show a developmental delay (Timmins-Schiffman et al., in press
Marine Biology)
o Alterations to shell growth/maintenance
o Changes in metabolic rate
o Changes in responses to other stressors
Lannig et al. 2010

6. Experimental Objective
Assess the effects of ocean acidification on
Crassostrea gigas at the molecular and
whole organism level

7. Methods
• Juvenile C. gigas were
exposed to one of 6 pCO2
treatments for 1 month

8. 427 µatm
638 µatm
810 µatm
991 µatm
1182 µatm
2847 µatm

9. 427 µatm 2012
638 µatm
810 µatm
991 µatm
1182 µatm
2847 µatm

10. 427 µatm 2012
638 µatm
810 µatm
991 µatm 2100
1182 µatm
2847 µatm

11. Experimental Design
pCO2 (µatm)
400 600 800 1000 1200 2800
t0: shell weight
1 month exposure
No additional Mechanical
stress stress Heat shock
• 2 sublethal
temperatures:
• Shell weight 42 & 43°C
• Gill tissue: • 1 lethal
• Transcriptomics temperature:
• Proteomics 44°C
• Shell structure/strength
• Whole body: lipids?
• Histology

12. Experimental Design
pCO2 (µatm)
400 600 800 1000 1200 2800
t0: shell weight
1 month exposure
No additional Mechanical
stress stress Heat shock
• 2 sublethal
temperatures:
• Shell weight 42 & 43°C
• Gill tissue:
• Transcriptomics
• 1 lethal
• Proteomics temperature:
• Shell structure/strength 44°C
• Whole body: lipids?
• Histology

13. Methods
• Shotgun proteomics on LC MS/MS (LTQ-Orbitrap)
• 4 oysters from 4 treatments
• Assigned spectra to database
• Comparative expression by spectral counting
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1 2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1
T: Average spectrum MS2 1240.28 (5584-5952) T: Average spectrum MS2 1240.28 (5584-5952)
2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1 1344.0381 1344.0381
100 1221.0632
T: ITMS + c NSI d Full ms2 1820.34@cid35.00 [490.00-2000.00] 100 1221.0632
95 95
1661.2975 90
100 90
85 85
95 80 80
75 1178.4297 1178.4297
75
90
70 70
85 65 65
60 60
Relative Abundance
Relative Abundance
80 55 1039.4359 1039.4359
55
50
75 50
45 45
70 40 40
1424.5980 1424.5980
35 2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1
35
65 1490.5931
T: ITMS + c NSI 30 Full ms2 1820.34@cid35.00 [490.00-2000.00]
d 1490.5931
30
60 25 25
1661.2975
Relative Abundance
1912.4304 1912.4304
20 100 20
869.9010 1607.7299 1607.7299
55 545.8882 823.7305 545.8882
869.9010
15 15 823.7305
479.2288 95 479.2288
1851.9578
50 10 10 1851.9578
5 90 5
45 0 0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1
85 400 600 800 1000 1200 1400 1600 1800 2000
40 T: Average spectrum MS2 1240.28 (5584-5952)
m/z
1915.7020
1426.2880 1344.0381 80
35 100 1221.0632
95 75
30 90
85 70
25
80
65
20 1710.3446 75 1178.4297
15
70 60
Relative Abundance
979.8818
1102.1235 65
1255.5396
10 60
55
1349.2131
Relative Abundance
55 1039.4359
5 50
50
0 45 45
600 800 1000 1200 1400 1600 1800 2000 40
1424.5980
m/z 40
35
1490.5931 1915.7020
30
1426.2880
35
25
1912.4304
20
1607.7299
30
869.9010
15 545.8882 823.7305
479.2288 25
1851.9578
10
5
20 1710.3446
0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
15 979.8818
1102.1235
1255.5396
10 1349.2131
5
0
600 800 1000 1200 1400 1600 1800 2000
m/z

14. Methods
Gigasdatabase (Sigenae) contig number
Peptides deduced from spectra that match to this protein

15. Shell Weight
0.7
0.6
Elevated pCO2 has
Gain in Shell Weight (g)
a negative effect
0.5
on growth
0.4
0.3
0.2
0.1
0.0
400 2800 800 1200 600 1000
Treatment (µatm)

16. Proteome
• Identified 897 unique proteins across all samples
• Proteins were annotated by 453 distinct GO
categories

17. Proteome
cell cycle and
proliferation
cell organization
cell adhesion
and biogenesis
cell-­‐‑cell
stress response
transport
signaling
death
protein
RNA metabolism
metabolism
signal
transduction
DNA metabolism
All proteins
developmental
processes

18. Proteome
Entire
proteome
represented
by GO terms

19. Exposure Comparison
Decreased Expression
High pCO2 Similar Expression
(150 Unique Proteins) Level
Elevated Expression
High pCO2
(130 Unique Proteins)

20. Exposure Comparison
Proteins
expressed in
high pCO2
only

21. Exposure comparison
• Proteins expressed at high pCO2 are related to the
physiological processes
o Metabolism
o Oxidative stress
o Transport (proton and calcium)
o Cellular stress
o Translation
• These processes imply that the oyster is maintaining
a significant response to stress even after 1 month of
exposure

22. Exposure Comparison
15 400 µatm + MS
400 µatm
10
5
PC 2 (20.5%)
0
-5
2800 µatm + MS
-10
2800 µatm
-15
-15 -10 -5 0 5 10 15
PC 1 (49.7%)

23. Results
• MS approach expands on previous 2D gel analyses
• Shotgun sequencing allow for:
1. Identification of more proteins
2. Greater potential to detect more nuanced responses and protein
interactions

24. Conclusions
• We were able to identify hundreds of proteins in the
C. gigas gill proteome
• Even after 1 month of exposure, oysters were
maintaining an energetically costly stress response
10
8
Oyster Shell Weight (g)
6
4
400 ppm
600 ppm
2
800 ppm
1000 ppm
1200 ppm
1400 ppm
0
0 2 4 6 8 10 12
Oyster Buoyant Weight (g)

25. Further Work
• Evaluate physiological response to combined
stressors
• Comparison of transcriptomic and proteomic
responses
• Develop a more robust system for protein
identifications

26. Acknowledgements
• UWPR (Proteomics)
o Priska von Haller, Jimmy Eng, Tahmina Jahan
• UW SAFS and Biology
o Michael O’Donnell, Emily Carrington, Ken Sebens, Matt
George
o Sam White, Mackenzie Gavery, Caroline Storer, Dave Metzger
• UW Medicinal Chemistry
o Dave Goodlett, Brook Nunn
• Oyster collection and care
o Sam Garson, Ronen Elad, Joth Davis, Jason Ragan, Dustin
Johnson
• Funding
o Research: crowdfunding through RocketHub
o Travel: UW SAFS