the development of molecular tools to monitor the physiological response of shellfish to ocean...
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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
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Outline1.Introduce Ruditapes philippinarum
2.Background on ocean acidification and physiology
3.Experimental design
4.Results
5.Future directions
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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).
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Ocean AcidificationCO2
CO2 H2O H2CO3
HCO3 CO32-
H++
Photo by David Mackwww.visualphotos.com
CO2 pH AragoniteCalcite
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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
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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
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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
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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
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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
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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.
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Are clams ok?
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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
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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
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Measuring physiological changes
At the genomic level….
qPCRGene of Interest
Copies of target region
Sequence specific primers
• Identify specific genes
• Quantify
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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
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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
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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
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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
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Special Thanks!Student Sponsors
Chelsea Farms LLC
Chuckanut Shellfish
Little Skookum Shellfish Growers
NOAA
PCS-NSA
Rock Point Oyster Co.
Seattle Shellfish
Taylor Shellfish