The document summarizes research on the effects of 3 levels of pCO2 (400, 700, and 1000 μatm) on the early development of the Pacific oyster (Crassostrea gigas). Key findings include: 1) Higher pCO2 levels delayed larval development, reduced calcification, and decreased larval size. 2) The proportion of fertilized eggs and hatched larvae was lower at higher pCO2 levels. 3) Calcification was reduced in larvae exposed to higher pCO2, with fewer larvae showing full calcification at 72 hours in the 700 and 1000 μatm treatments. 4) Growth rates over 24-72 hours, as measured by h

1. The Effects of 3 Levels of pCO2 on
Early Development of the Pacific
Oyster
Emma Timmins-Schiffman
Steven Roberts
Carolyn Friedman
Michael O’Donnell
University of Washington
Worldwide University Network
Friday Harbor Labs, August 30, 2011

2. How does OA affect larvae?
Effect of OA Organism Reference
Decreased shell Oyster, mussel, 1, 2, 3, 4, 9, 12
size, strength, barnacle, crab
calcification
Transcriptome/ Urchin 5, 6, 10
physiology
Protein Barnacle 7
Developmental Urchin, shrimp, 8, 9, 13
delay and change brittle star
in energy budget
Increased growth Sea star 11
rate
Abnormal Brittle star, urchin, 12, 2
morphology oyster
Response to other Urchin, barnacle, 14, 3
stressors crab

3. Which physiological mechanisms are
changing?
¤ Calcification
¤ Hydrogen ion balance across membranes
¤ Energy metabolism
¤ Timing of developmental processes
¤ Stress response

4. How does ocean acidification affect
development and physiology of Pacific oyster
larvae (Crassostrea gigas)

5. CO2-free air
CO2 (canister)
Honeywell Controller
Treatment-
equilibrated
water
DuraFET pH probe Venturi injector

6. Experimental Design
Equilibrate treatment water
Fertilization 1 hpf 6 hpf 24 hpf 72 hpf 96 hpf
Fix samples for
Sample for transcriptomics
developmental stage, size,
and calcification

7. pH
8.5
8.0
7.5
Ran out of CO2
pH
7.0
400 !atm
6.5
700 !atm
1000 !atm
6.0
0 1 2 3 4
Day

8. Relationship between TA and Salinity Total Alkalinity
2100
2030
400 !atm
700 !atm
2020
1000 !atm
2050
2010
TA (!mol/kg)
TA (!mol/kg)
2000
2000
1990
1950
1980
1970
1900
0 1 2 3
28.0 28.5 29.0
Day
Salinity (ppt)

9. Dissolved Inorganic Carbon
2000
1900
DIC (!mol/kg)
1800
1700
400 !atm
700 !atm
1000 !atm
1600
0 1 2 3
Day

10. Calcium Carbonate Saturation State
4
3
Omega
2
1
400 !atm Calcite
700 !atm Aragonite
1000 !atm
0
0 1 2 3
Day

11. Results: Larval Development, Growth,
and Calcification
¤ Larvae were fixed for later microscopy
¤ Developmental stage was assessed
¤ Growth was measured: hinge length, shell
height
¤ Calcification:
¤ double polarization of light
¤ SEM

12. Average Larval Density by Treatment
3500
400 !atm
700 !atm
3000
1000 !atm
Average Density in 3L
2500
2000
1500
1000
500
0
0 2 4 6 8
Day

13. Average Larval Density by Treatment
3500
400 !atm
700 !atm
3000
1000 !atm
Average Density in 3L
2500
2000
1500
1000
500
0
0 2 4 6 8
Day

14. Proportion Fertilized Eggs at 1 hpf
1.0
0.8
Proportion Fertilized
0.6
0.4
0.2
0.0
400 700 1000
Treatment (!atm)

15. Proportion Larvae Hatched at 6hpf
1.0
0.8
Proportion Hatched
0.6
0.4
0.2
0.0
400 700 1000
Treatment ( !atm)

16. Larval Calcification: Methods
¤ Double polarization of light
¤ Qualify larval calcification – uncalcified,
partially calcified, fully calcified

17. Proportion Larvae with Calcification at 24hpf
1.0
0.8
Proportion Partially Calcified
0.6
0.4
0.2
0.0
400 700 1000
Treatment (!atm)

18. Proportion Larvae Fully Calcified at 72hpf
1.2
1.0
Proportion Fully Calcified
0.8
0.6
0.4
0.2
0.0
400 700 1000
Treatment (!atm)

19. Larval Size: Methods
¤ Size measured in 2
parameters – hinge
length and shell
height
¤ Measurements are
from 24 and 72
hours post
fertilization

20. Hinge Length by Treatment and Day Shell Height by Treatment and Day
80
70
70
60
Hinge Length (!m)
Shell Height (!m)
50
60
40
50
30
40
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
Day and pCO2 (!atm)
Day and pCO2 (!atm)

21. Growth Rate by Treatment
15
Hinge
Growth Rate/Day (!m) Height
10
5
0
400 700 1000
Treatment (!atm)

22. Growth Rate
Prodissoconch II
Prodissoconch I

23. Gene Expression
¤ 2 microcosms from
each treatment at
96 hpf
¤ Oxidative stress
genes (SOD, GPx,
Prx6) and molecular
chaperone (Hsp70)

24. Hsp70
Stress Response
STRESS Protein damage/
unfolding
Hsp70
Chaperones bind to proteins to either repair or remove

25. Heat Shock Protein 70
25
Fold Over Minimum Expression
20
15
10
5
400 700 1000
Treatment (!atm)

26. Oxidative Stress Genes
Stress Response
STRESS • Increase metabolism
• Kill pathogens
Prx6
ROSSOD
GPx

27. Superoxide Dismutase
0.20
0.15
Expression
0.10
0.05
0.00
400 700 1000
Treatment (!atm)

28. Fold Over Minimum Expression
0.0e+00 5.0e+24 1.0e+25 1.5e+25
400
700
Treatment (!atm)
Glutathione Peroxidase
1000

29. Fold Over Minimum Expression
0e+00 1e+22 2e+22 3e+22 4e+22 5e+22 6e+22
400
700
Treatment (!atm)
Peroxiredoxin 6
1000

30. Conclusions
¤ pCO2 of 700 and 1000 µatm caused decreased growth in
C .gigas larvae at 96 hpf
¤ There is evidence of physiological stress
¤ Significant for exposure to other stressors
¤ Significant for continued growth, development, and survival

31. Thank you
Emily Carrington•Matt George•Michelle Herko•Laura
Newcomb•Ken Sebens•Richard Strathmann•Adam
Summers•Billie Swalla

32. References
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