timmins schiffman pcsga 2011
TRANSCRIPT
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
How does OA affect larvae? Effect of OA Organism Reference
Decreased shell size, strength, calcification
Oyster, mussel, barnacle, crab
1, 2, 3, 4, 9, 12
Transcriptome/physiology
Urchin 5, 6, 10
Protein Barnacle 7
Developmental delay and change in energy budget
Urchin, shrimp, brittle star
8, 9, 13
Increased growth rate
Sea star 11
Abnormal morphology
Brittle star, urchin, oyster
12, 2
Response to other stressors
Urchin, barnacle, crab
14, 3
Which physiological mechanisms are changing?
¤ Calcification
¤ Hydrogen ion balance across membranes
¤ Energy metabolism
¤ Timing of developmental processes
¤ Stress response
How does ocean acidification affect development and physiology of Pacific oyster larvae (Crassostrea gigas)
CO2-free air CO2 (canister)
Venturi injector
Treatment-equilibrated water
DuraFET pH probe
Honeywell Controller
Experimental Design
Equilibrate treatment water
Fertilization 1 hpf 6 hpf 24 hpf 72 hpf 96 hpf
Fix samples for developmental stage, size, and calcification
Sample for transcriptomics
7.0
7.5
8.0
8.5
pH
Time
pH
400 !atm700 !atm1000 !atm
0 1 2 31900
1950
2000
2050
2100
Total Alkalinity
Day
TA (!
mol
/kg)
400 !atm700 !atm1000 !atm
28.0 28.5 29.0
1970
1980
1990
2000
2010
2020
2030
Relationship between TA and Salinity
Salinity (ppt)
TA (!
mol
/kg)
0 1 2 3
1600
1700
1800
1900
2000
Dissolved Inorganic Carbon
Day
DIC
(!m
ol/k
g)
400 !atm700 !atm1000 !atm
0 1 2 3
01
23
4
Calcium Carbonate Saturation State
Day
Omega
400 !atm700 !atm1000 !atm
CalciteAragonite
Dissolution threshold
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
Significantly fewer larvae are in an advanced developmental stage at higher pCO2 (lower pH)
400 700 1000
0.0
0.2
0.4
0.6
0.8
1.0
Proportion Larvae at Pre-Hatching Stage at 6hpf
Treatment
Pro
porti
on a
t Pre
-Hat
chin
g
400 !atm700 !atm1000 !atm
Larval Calcification: Methods
¤ Double polarization of light
¤ Qualify larval calcification
More larvae have started calcification at higher pCO2
400 700 1000
0.0
0.2
0.4
0.6
0.8
1.0
Larval Calcification at 24h
Treatment
Pro
porti
on C
alci
fied
400 !atm700 !atm1000 !atm
Fewer larvae are fully calcified in the highest pCO2 (lowest pH) treatment
400 700 1000
0.0
0.2
0.4
0.6
0.8
1.0
Larval Calcification at 72h
Treatment
Pro
porti
on C
alci
fied
400 !atm700 !atm1000 !atm
Larval Size: Methods
¤ Size measured in 2 parameters – hinge length and shell height
¤ Measurements are from 24 and 72 hours post fertilization
D1 400 D1 700 D1 1000 D3 400 D3 700 D3 100040
5060
7080
Shell Height by Treatment and Day
Day and pCO2 (!atm)
She
ll H
eigh
t (!m
)D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000
3040
5060
70
Hinge Length by Treatment and Day
Day and pCO2 (!atm)
Hin
ge L
engt
h (!
m)
Larvae are smaller at higher pCO2 at 3 days post-fertilization
400 700 1000
05
1015
Growth Rate by Treatment
Treatment (!atm)
Gro
wth
Rat
e/D
ay (!
m)
HingeHeight
Shell height growth rate is slower at the highest pCO2
Gene Expression
¤ 2 microcosms from each treatment at 96 hpf
¤ Oxidative stress genes (SOD,Prx6) and molecular chaperone (Hsp70)
Hsp70
STRESS Stress Response
Protein damage/unfolding
Chaperones bind to proteins to either repair or remove
Hsp70
400 700 1000
510
1520
25Heat Shock Protein 70
Treatment (!atm)
Fold
Ove
r Min
imum
Exp
ress
ion
Increased expression of hsp70 with increased pCO2 could indicate cellular stress
Oxidative Stress Genes
STRESS Stress Response
• Increase metabolism • Kill pathogens
ROS Prx6 SOD
400 700 1000
0.00
0.05
0.10
0.15
0.20
Superoxide Dismutase
Treatment (!atm)
Expression
400 700 1000
0e+00
1e+22
2e+22
3e+22
4e+22
5e+22
6e+22
Peroxiredoxin 6
Treatment (!atm)
Fold
Ove
r Min
imum
Exp
ress
ion
Oxidative Stress Genes
Greater expression of SOD and Prx6 may indicate increased oxidative stress during exposure to ocean acidification
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
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
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.