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

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