1. The Effects of Ocean Acidification on
Pacific Oyster Larval Development
and Physiology
Emma Timmins-Schiffman
Steven Roberts
Carolyn Friedman
Michael O’Donnell
University of Washington
PCSGA
Salem, OR, September, 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
pH
7.5
400 !atm
700 !atm
1000 !atm
7.0
Time

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
Dissolution
threshold
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

12. Proportion Larvae at Pre-Hatching Stage at 6hpf
1.0
400 !atm
700 !atm
1000 !atm
0.8
Proportion at Pre-Hatching
Significantly
0.6
fewer larvae are
in an advanced
0.4
developmental
stage at higher
pCO2 (lower pH)
0.2
0.0
400 700 1000
Treatment

13. Larval Calcification: Methods
¤ Double polarization of light
¤ Qualify larval calcification

14. Larval Calcification at 24h
More larvae
1.0
400 !atm
700 !atm have started
1000 !atm calcification
0.8
at higher
Proportion Calcified
pCO2
0.6
0.4
0.2
0.0
400 700 1000
Treatment

15. Larval Calcification at 72h
Fewer larvae
1.0
are fully
calcified in
the highest
0.8
pCO2
Proportion Calcified
(lowest pH)
0.6
treatment
0.4
400 !atm
0.2
700 !atm
1000 !atm
0.0
400 700 1000
Treatment

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

17. 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)
Larvae are smaller at higher pCO2 at 3
days post-fertilization

18. Growth Rate by Treatment
15
Hinge
Height
Shell height
Growth Rate/Day (!m)
10
growth rate is
slower at the
highest pCO2
5
0
400 700 1000
Treatment (!atm)

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

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

21. Heat Shock Protein 70
25
Fold Over Minimum Expression
Increased
20
expression
of hsp70
with
15
increased
pCO2 could
indicate
10
cellular
stress
5
400 700 1000
Treatment (!atm)

22. Oxidative Stress Genes
Stress Response
STRESS • Increase metabolism
• Kill pathogens
ROS
Prx6 SOD

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

24. 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

25. Oxidative Stress Genes
Greater expression of SOD and Prx6 may
indicate increased oxidative stress during
exposure to ocean acidification

26. Conclusions
¤ pCO2 of 700 and 1000 µatm caused decreased growth
and calcification in C .gigas larvae through 72 hpf
¤ There is evidence of physiological stress
¤ Significant for exposure to other stressors
¤ Significant for continued growth, development, and survival

27. Thank you
Emily Carrington•Matt George•Michelle Herko•Laura
Newcomb•Ken Sebens•Richard Strathmann•Adam
Summers•Billie Swalla•Brent Vadopalas
Chelsea Farms LLC
Little Skookum Shellfish Growers
Rock Point Oyster Co.
Seattle Shellfish
Taylor Shellfish
NOAA Aquaculture
NSA, Pacific Coast Section

28. References
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