e timmins schiffman_physiomar12
TRANSCRIPT
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
Plan of Talk • Background: Ocean acidification and marine
bivalves • Aim of experiment • Methods • Results (proteomics) • Conclusion
0 2 4 6 8 10 120
24
68
10
Oyster Buoyant Weight (g)
Oys
ter S
hell
Wei
ght (
g)
400 ppm600 ppm800 ppm1000 ppm1200 ppm1400 ppm
Ocean Acidification and the Environment
• Rising pCO2 in the atmosphere equilibrates with ocean surface water, reducing the pH
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
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
Experimental Objective
Assess the effects of ocean acidification on Crassostrea gigas at the molecular and
whole organism level
Methods • Juvenile C. gigas were
exposed to one of 6 pCO2 treatments for 1 month
2847 µatm
638 µatm
1182 µatm
427 µatm
810 µatm 991 µatm
2847 µatm
638 µatm
1182 µatm
427 µatm
810 µatm 991 µatm
2012
2847 µatm
638 µatm
1182 µatm
427 µatm
810 µatm 991 µatm
2012
2100
Experimental Design pCO2 (µatm)
400 600 800 1000 1200 2800
1 month exposure
t0: shell weight
No additional stress
Mechanical stress Heat shock
• 2 sublethal temperatures: 42 & 43°C
• 1 lethal temperature: 44°C
• Shell weight • Gill tissue:
• Transcriptomics • Proteomics
• Shell structure/strength • Whole body: lipids? • Histology
Experimental Design pCO2 (µatm)
400 600 800 1000 1200 2800
1 month exposure
t0: shell weight
No additional stress
Mechanical stress Heat shock
• 2 sublethal temperatures: 42 & 43°C
• 1 lethal temperature: 44°C
• Shell weight • Gill tissue:
• Transcriptomics
• Proteomics • Shell structure/strength • Whole body: lipids? • Histology
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 #7891 RT: 69.73 AV: 1 NL: 3.19E1T: ITMS + c NSI d Full ms2 [email protected] [490.00-2000.00]
600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1661.2975
1915.70201426.2880
1710.3446
979.8818 1102.1235 1255.53961349.2131
2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1T: ITMS + c NSI d Full ms2 [email protected] [490.00-2000.00]
600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1661.2975
1915.70201426.2880
1710.3446
979.8818 1102.1235 1255.53961349.2131
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1T: Average spectrum MS2 1240.28 (5584-5952)
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1344.03811221.0632
1178.4297
1039.4359
1424.5980
1490.5931
1912.4304
1607.7299869.9010545.8882 823.7305
479.22881851.9578
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1T: Average spectrum MS2 1240.28 (5584-5952)
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1344.03811221.0632
1178.4297
1039.4359
1424.5980
1490.5931
1912.4304
1607.7299869.9010545.8882 823.7305
479.22881851.9578
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1T: Average spectrum MS2 1240.28 (5584-5952)
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1344.03811221.0632
1178.4297
1039.4359
1424.5980
1490.5931
1912.4304
1607.7299869.9010545.8882 823.7305
479.22881851.9578
Methods Gigasdatabase (Sigenae) contig number
Peptides deduced from spectra that match to this protein
400 2800 800 1200 600 1000
Treatment (µatm)
Gai
n in
She
ll W
eigh
t (g)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Shell Weight
Elevated pCO2 has a negative effect
on growth
Proteome • Identified 897 unique proteins across all samples • Proteins were annotated by 453 distinct GO
categories
cell adhesion cell cycle and proliferation cell organization
and biogenesis
cell-‐‑cell signaling
death
developmental processes
DNA metabolism
protein metabolism RNA metabolism
signal transduction
stress response transport
Proteome
All proteins
Proteome
Entire proteome
represented by GO terms
Exposure Comparison
Similar Expression Level
Elevated Expression High pCO2
(130 Unique Proteins)
Decreased Expression High pCO2
(150 Unique Proteins)
Exposure Comparison
Proteins expressed in high pCO2
only
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
-15 -10 -5 0 5 10 15
-15
-10
-50
510
15
PC 1 (49.7%)
PC
2 (2
0.5%
)
Exposure Comparison
2800 µatm
2800 µatm + MS
400 µatm 400 µatm + MS
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
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
0 2 4 6 8 10 120
24
68
10
Oyster Buoyant Weight (g)
Oys
ter S
hell
Wei
ght (
g)
400 ppm600 ppm800 ppm1000 ppm1200 ppm1400 ppm
Further Work • Evaluate physiological response to combined
stressors • Comparison of transcriptomic and proteomic
responses • Develop a more robust system for protein
identifications
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