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
¤ 1Watson et al. 2009. Early larval development of the Sydney rock oyster Saccostrea glomerata under near-future predictions of CO2-driven ocean acification. Journal of Shellfish Research. 28
(3)): 431-437.
¤ 2 Gaylord et al. 2011 Functional impacts of ocean acidification in an ecologically critical foundation species. J Exp Biol. 214: 2586-2594.
¤ 3 Parker et al. 2010. Comparing the effect of elevated pCO2 and temperature on the fertilization and early development of 2 species of oyster. Marine Biology. 157(11): 2435-2452.
¤ 4 Findlay et al. 2009. Post-larval development of 2 intertidal barnacles at elevated CO2 and temperature. Mar Biol. 157: 725-735.
¤ 5 Todham & Hofmann 2009 Transcriptomic response of sea urchin larvae Strongylocentrotus purpuratus to CO2-driven seawater acidification. J Exp Biol. 212: 2579-2594.
¤ 6 Stumpp et all. 2011. CO2 induced seawater acidification impacts sea urchin larval development II: Gene expression patterns in pluteus larvae. Comparative Biochemistry and Physiology – Part
A. 160(3): 320-330.
¤ 7 Wong et al. 2011. Response of larval barnacle proteome to CO2-driven seawater acidification. Comparative Biochemistry and Physiology – Part D. 6(3): 310-321.
¤ 8 Stumpp et al. 2011. CO2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay.
Comparative Biochemistry and Physiology – Part A. 160(3): 331-340.
¤ 9 Bechmann et al 2011. Effects of ocean aciidification on early life stages of shrimp (Pandalus borealis) and mussel (Mytilus edulis). J Toxicol Environ Health. 74(7-9): 424-438.
¤ 10 Martin et al. 2011. Early development andmolecular plasticity in the Mediterranean sea urchin Paracentrotus lividus exposed to CO2-driven aciidification. J Exp Biol. 214(8): 1357-1368.
¤ 11 DuPont et al. 2010. Near future ocean acidification increases growth of lecithotrophic larvae and juveniles of the sea star Crossaster papposus. J Exp Biol Part B. 314B(5): 382-389.
¤ 12 Kurihara et al. 2007. Effects of increased seawater pCO2 on early development of the oyster C.rassostrea gigas. Aquat Biol. 1:91-98.
¤ 13 Dupont et al. 2008. Near-future level of CO2-driven ocean acidification radically affects larval survival and development in the brittlestar Ophiothrix fragilis. Mar Ecol Prog Ser. 373: 285-294.
¤ 14 O’Donnell et al. 2009. Predicted impact of ocean acidification on marine invertebrate larvae: elevated CO2 alters response to thermal stress in sea urchin larvae. 156(3): 439-446.