The development of molecular tools to monitor the physiological response

of shellfish to ocean acidification

David Metzger1

Shallin Busch2

Michael Maher2

Paul McElhany2

Carolyn Friedman1

Steven Roberts1

1University of WashingtonSchool of Aquatic and Fishery Sciences

Seattle, WA

2NOAA’s Northwest Fishery Science CenterSeattle, WA

Outline1.Introduce Ruditapes philippinarum

2.Background on ocean acidification and physiology

3.Experimental design

4.Results

5.Future directions

Manila ClamRuditapes philippinarum

Photo: Taylor Shellfish

• Focus on larvae

• Economically important, locally farmed bivalve species

• Few studies on the impacts of ocean acidification on R. philippinarum.• OA results in increase in heavy metal uptake (Lopez

et al., 2010).

Ocean AcidificationCO2

CO2 H2O H2CO3

HCO3 CO32-

H++

Photo by David Mackwww.visualphotos.com

CO2 pH AragoniteCalcite

Ocean Acidification and Physiology

Decreases in aragonite and calcite saturation state inhibits the ability of calcifying organisms to properly form shells.

Changes in metabolism and energy allocationsMore energy put towards calcification and and less

allocated towards other vital physiological processes

Disrupts ion homeostasis

Changes in immune response

Generates reactive oxygen species (ROS) = increased cytological damage

Experimental Design

3 pCO2 Levels

6 Replicates/pCO2 treatment

~30,000 larvae/jar

Sampling schedule: Days in system = 1, 4, 7, 11, 14 days

400uatm 520uatm 1000uatm

Experimental Conditionsp

H

1 4 7 11 147.3

7.4

7.5

7.6

7.7

7.8

7.9

8

Day

pH was maintaned and constant levels for the duration of the experiment

1000ppm520ppm400ppm

Percent Survival +/- 95%ci

1 4 7 11 140.00%

40.00%

80.00%

120.00%

There was no significant difference in survival between treatments

Perc

en

t S

urv

ival

Day

1000ppm520ppm400ppm

Larval Size +/- 95%ciS

hell

Are

a (

μm

)

0 1 4 7 11 140

2000

4000

6000

8000

10000

12000

14000

16000

18000

DaysThere was no significant difference in shell area

between treatments

1000ppm520ppm400ppm

SummaryConducted an experiment in which pCO2

conditions were held at three constant levels.

Increased pCO2 levels up to 1000ppm had no effect on survival or size of 5 day old manila clams.

Increased pCO2 levels up to 1000ppm have no effect on the transcription of GPx or Thioredoxin.

Are clams ok?

OA and Oxidative Stress• Oxidative stress: Cellular damage (e.g. DNA,

proteins) caused by reactive oxygen species (ROS)

• ROS: Compounds containing oxygen with unpaired electrons

e.g. H2O2

• Sources of ROS: Cellular metabolismImmune responseCell signalingEnvironment• Natural• Breakdown of pollutants

• Detoxification of ROS: Reduced by enzymes

OA and Oxidative StressOxidate stress response proteins

1. Glutathione peroxidase (GPx):Converts hydrogen peroxide to water

2. ThioredoxinReduces oxidized forms of thioredoxin peroxidase

H2O2

GPxH2O

GSH GSSG

GR

H2O2

TRX peroxidaseH2O

TRX-SH2TRX(SH)2

TRX reductase

Measuring physiological changes

At the genomic level….

qPCRGene of Interest

Copies of target region

Sequence specific primers

• Identify specific genes

• Quantify

OA and Oxidative Stress

1 Day 1 Week0

100

200

300

400

500

600

Glutathione Peroxidase

1 Day 1 Week012345678

Thioredoxin

No significant difference in between treatments

Previous studies measuring GPx gene expression noted a significant decrease under high pCO2 conditions

Fold

Ch

an

ge

1000ppm

520ppm

400ppm

Future Plans: Next Generation Sequencing

qPCR: Candidate Gene

NGS: Sequence entire transcriptome

Gene of Interest

Copies of target region

Sequence specific primers

Total mRNA

millions of short sequences

Ocean Acidification and Physiology

Decreases in aragonite and calcite saturation state inhibits the ability of calcifying organisms to properly form shells.

Changes in metabolism and energy allocationsMore energy put towards calcification and and less

allocated towards other vital physiological processes

Disrupts ion homeostasis

Changes in immune response

Generates reactive oxygen species (ROS) = increased cytological damage

AcknowledgementsUniversity of WashingtonRoberts Lab:Sam WhiteSteven RobertsEmma Timmins-SchiffmanCaroline StorerMackenzie Gavery

Friedman Lab: Carolyn FriedmanBrent VadopalasLiza RayLisa CrossonElene DorfmeierSammi BrombackerRobyn Strenge

NOAA NWFSCShallin BuschPaul McElhanyMike MaherJason MillerSarah Norberg

Taylor ShellfishGreg JacobJoth Davis

FundingWashington Sea GrantSaltonstall-KennedyUniversity of Washington

Special Thanks!Student Sponsors

Chelsea Farms LLC

Chuckanut Shellfish

Little Skookum Shellfish Growers

NOAA

PCS-NSA

Rock Point Oyster Co.

Seattle Shellfish

Taylor Shellfish