Timmins schiffman wun 2011

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The Effects of 3 Levels of pCO 2 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

Transcript of Timmins schiffman wun 2011

Page 1: 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

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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

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Which physiological mechanisms are changing?

¤ Calcification

¤ Hydrogen ion balance across membranes

¤ Energy metabolism

¤ Timing of developmental processes

¤ Stress response

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How does ocean acidification affect development and physiology of Pacific oyster larvae (Crassostrea gigas)

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CO2-free air CO2 (canister)

Venturi injector

Treatment-equilibrated water

DuraFET pH probe

Honeywell Controller

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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

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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

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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)

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0 1 2 3

1600

1700

1800

1900

2000

Dissolved Inorganic Carbon

Day

DIC

(!m

ol/k

g)

400 !atm700 !atm1000 !atm

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0 1 2 3

01

23

4

Calcium Carbonate Saturation State

Day

Omega

400 !atm700 !atm1000 !atm

CalciteAragonite

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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

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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

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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

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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

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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

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Larval Calcification: Methods

¤ Double polarization of light

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

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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

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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

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Larval Size: Methods

¤ Size measured in 2 parameters – hinge length and shell height

¤ Measurements are from 24 and 72 hours post fertilization

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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)

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400 700 1000

05

1015

Growth Rate by Treatment

Treatment (!atm)

Gro

wth

Rat

e/D

ay (!

m)

HingeHeight

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Growth Rate

Prodissoconch I

Prodissoconch II

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Gene Expression

¤ 2 microcosms from each treatment at 96 hpf

¤ Oxidative stress genes (SOD, GPx, Prx6) and molecular chaperone (Hsp70)

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Hsp70

STRESS Stress Response

Protein damage/unfolding

Chaperones bind to proteins to either repair or remove

Hsp70

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400 700 1000

510

1520

25

Heat Shock Protein 70

Treatment (!atm)

Fold

Ove

r Min

imum

Exp

ress

ion

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Oxidative Stress Genes

STRESS Stress Response

•  Increase metabolism •  Kill pathogens

ROS Prx6

GPx SOD

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400 700 1000

0.00

0.05

0.10

0.15

0.20

Superoxide Dismutase

Treatment (!atm)

Expression

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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

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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

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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

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Thank you

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

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¤  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.

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¤  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.

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¤  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.