Timmins schiffman wun 2011

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

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

0 1 2 3 4

6.0

6.5

7.0

7.5

8.0

8.5

pH

Day

pH

400 !atm700 !atm1000 !atm

Ran out of CO2

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

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

0 2 4 6 8

0500

1000

1500

2000

2500

3000

3500

Average Larval Density by Treatment

Day

Ave

rage

Den

sity

in 3

L400 !atm700 !atm1000 !atm

0 2 4 6 8

0500

1000

1500

2000

2500

3000

3500

Average Larval Density by Treatment

Day

Ave

rage

Den

sity

in 3

L400 !atm700 !atm1000 !atm

400 700 1000

Proportion Fertilized Eggs at 1 hpf

Treatment (!atm)

Pro

porti

on F

ertil

ized

0.0

0.2

0.4

0.6

0.8

1.0

400 700 1000

Proportion Larvae Hatched at 6hpf

Treatment ( !atm)

Pro

porti

on H

atch

ed0.0

0.2

0.4

0.6

0.8

1.0

Larval Calcification: Methods

¤ Double polarization of light

¤ Qualify larval calcification – uncalcified, partially calcified, fully calcified

400 700 1000

Proportion Larvae with Calcification at 24hpf

Treatment (!atm)

Pro

porti

on P

artia

lly C

alci

fied

0.0

0.2

0.4

0.6

0.8

1.0

400 700 1000

Proportion Larvae Fully Calcified at 72hpf

Treatment (!atm)

Pro

porti

on F

ully

Cal

cifie

d

0.0

0.2

0.4

0.6

0.8

1.0

1.2

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)

400 700 1000

05

1015

Growth Rate by Treatment

Treatment (!atm)

Gro

wth

Rat

e/D

ay (!

m)

HingeHeight

Growth Rate

Prodissoconch I

Prodissoconch II

Gene Expression

¤ 2 microcosms from each treatment at 96 hpf

¤ Oxidative stress genes (SOD, GPx, 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

25

Heat Shock Protein 70

Treatment (!atm)

Fold

Ove

r Min

imum

Exp

ress

ion

Oxidative Stress Genes

STRESS Stress Response

•  Increase metabolism •  Kill pathogens

ROS Prx6

GPx SOD

400 700 1000

0.00

0.05

0.10

0.15

0.20

Superoxide Dismutase

Treatment (!atm)

Expression

400 700 1000

0.0e+00

5.0e+24

1.0e+25

1.5e+25

Glutathione Peroxidase

Treatment (!atm)

Fold

Ove

r Min

imum

Exp

ress

ion

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

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

Thank you

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

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