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The development of molecular tools to monitor the physiological response of shellfish to ocean acidification<br />David Me...
Outline<br />Introduce Ruditapesphilippinarum<br />Background on ocean acidification and physiology<br />Experimental desi...
Manila ClamRuditapesphilippinarum<br /><ul><li>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).</li></ul> Photo: Taylor Shellfish<br />
Ocean Acidification<br />CO2<br />CO2<br />H2O<br />H2CO3<br />H+<br />+<br />HCO3<br />CO32-<br />CO2<br />pH<br />Aragon...
Ocean Acidification and Physiology<br />Decreases in aragonite and calcite saturation state inhibits the ability of calcif...
Experimental Design<br />400uatm<br />520uatm<br />1000uatm<br />3 pCO2 Levels<br />6 Replicates/pCO2 treatment<br />~30,0...
Experimental Conditions<br />pH<br />400ppm<br />520ppm<br />1000ppm<br />Day<br /><ul><li>pH was maintaned and constant l...
Are clams ok?<br />
OA and Oxidative Stress<br /><ul><li>Oxidative stress: Cellular damage (e.g. DNA, proteins) caused by reactive oxygen spec...
ROS: Compounds containing oxygen with unpaired electrons</li></ul>e.g. H2O2<br /><ul><li>Sources of ROS:	Cellular metaboli...
Breakdown of pollutants
Detoxification of ROS:	Reduced by enzymes</li></li></ul><li>OA and Oxidative Stress<br />Oxidate stress response proteins<...
Measuring physiological changes<br />At the genomic level….<br />qPCR<br />Sequence specific primers<br />Gene of Interest...
Quantify</li></li></ul><li>OA and Oxidative Stress<br />400ppm<br />Glutathione Peroxidase<br />Thioredoxin<br />520ppm<br...
Previous studies measuring GPx gene expression noted a significant decrease under high pCO2 conditions</li></li></ul><li>F...
Ocean Acidification and Physiology<br />Decreases in aragonite and calcite saturation state inhibits the ability of calcif...
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The development of molecular tools to monitor the physiological response of shellfish to ocean acidification

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The development of molecular tools to monitor the physiological response of shellfish to ocean acidification

  1. 1. The development of molecular tools to monitor the physiological response of shellfish to ocean acidification<br />David Metzger1<br />Shallin Busch2<br />Michael Maher2<br />Paul McElhany2<br />Carolyn Friedman1<br />Steven Roberts1<br />1University of Washington<br />School of Aquatic and Fishery Sciences<br />Seattle, WA<br />2NOAA’s Northwest Fishery Science Center<br />Seattle, WA<br />
  2. 2. Outline<br />Introduce Ruditapesphilippinarum<br />Background on ocean acidification and physiology<br />Experimental design<br />Results<br />Future directions<br />
  3. 3. Manila ClamRuditapesphilippinarum<br /><ul><li>Focus on larvae
  4. 4. Economically important, locally farmed bivalve species
  5. 5. Few studies on the impacts of ocean acidification on R. philippinarum.
  6. 6. OA results in increase in heavy metal uptake (Lopez et al., 2010).</li></ul> Photo: Taylor Shellfish<br />
  7. 7. Ocean Acidification<br />CO2<br />CO2<br />H2O<br />H2CO3<br />H+<br />+<br />HCO3<br />CO32-<br />CO2<br />pH<br />Aragonite<br />Calcite<br />Photo by David Mack<br />www.visualphotos.com<br />
  8. 8. Ocean Acidification and Physiology<br />Decreases in aragonite and calcite saturation state inhibits the ability of calcifying organisms to properly form shells.<br />Changes in metabolism and energy allocations<br />More energy put towards calcification and and less allocated towards other vital physiological processes<br />Disrupts ion homeostasis<br />Changes in immune response<br />Generates reactive oxygen species (ROS) = increased cytological damage<br />
  9. 9. Experimental Design<br />400uatm<br />520uatm<br />1000uatm<br />3 pCO2 Levels<br />6 Replicates/pCO2 treatment<br />~30,000 larvae/jar<br />Sampling schedule: Days in system = 1, 4, 7, 11, 14 days <br />
  10. 10. Experimental Conditions<br />pH<br />400ppm<br />520ppm<br />1000ppm<br />Day<br /><ul><li>pH was maintaned and constant levels for the duration of the experiment</li></li></ul><li>Percent Survival +/- 95%ci<br />400ppm<br />520ppm<br />1000ppm<br />Percent Survival<br />Day<br /><ul><li>There was no significant difference in survival between treatments</li></li></ul><li>Larval Size +/- 95%ci<br />Shell Area (μm)<br />400ppm<br />520ppm<br />1000ppm<br />Days<br /><ul><li>There was no significant difference in shell area between treatments</li></li></ul><li>Summary<br />Conducted an experiment in which pCO2 conditions were held at three constant levels.<br />Increased pCO2 levels up to 1000ppm had no effect on survival or size of 5 day old manila clams.<br />Increased pCO2 levels up to 1000ppm have no effect on the transcription of GPx or Thioredoxin.<br />
  11. 11. Are clams ok?<br />
  12. 12. OA and Oxidative Stress<br /><ul><li>Oxidative stress: Cellular damage (e.g. DNA, proteins) caused by reactive oxygen species (ROS)
  13. 13. ROS: Compounds containing oxygen with unpaired electrons</li></ul>e.g. H2O2<br /><ul><li>Sources of ROS: Cellular metabolism</li></ul>Immune response<br />Cell signaling<br />Environment<br /><ul><li>Natural
  14. 14. Breakdown of pollutants
  15. 15. Detoxification of ROS: Reduced by enzymes</li></li></ul><li>OA and Oxidative Stress<br />Oxidate stress response proteins<br />Glutathione peroxidase (GPx):<br /> Converts hydrogen peroxide to water<br />2. Thioredoxin<br />Reduces oxidized forms of thioredoxin peroxidase<br />GPx<br />H2O2<br />H2O<br />GSH<br />GSSG<br />GR<br />TRX peroxidase<br />H2O2<br />H2O<br />TRX(SH)2<br />TRX-SH2<br />TRX reductase<br />
  16. 16. Measuring physiological changes<br />At the genomic level….<br />qPCR<br />Sequence specific primers<br />Gene of Interest<br />Copies of target region<br /><ul><li>Identify specific genes
  17. 17. Quantify</li></li></ul><li>OA and Oxidative Stress<br />400ppm<br />Glutathione Peroxidase<br />Thioredoxin<br />520ppm<br />1000ppm<br />Fold Change<br /><ul><li>No significant difference in between treatments
  18. 18. Previous studies measuring GPx gene expression noted a significant decrease under high pCO2 conditions</li></li></ul><li>Future Plans: Next Generation Sequencing<br />qPCR: Candidate Gene<br />NGS: Sequence entire transcriptome<br />Sequence specific primers<br />Gene of Interest<br />Copies of target region<br />Total mRNA<br />millions of short sequences<br />
  19. 19. Ocean Acidification and Physiology<br />Decreases in aragonite and calcite saturation state inhibits the ability of calcifying organisms to properly form shells.<br />Changes in metabolism and energy allocations<br />More energy put towards calcification and and less allocated towards other vital physiological processes<br />Disrupts ion homeostasis<br />Changes in immune response<br />Generates reactive oxygen species (ROS) = increased cytological damage<br />
  20. 20. Acknowledgements<br />University of Washington<br />Roberts Lab:<br />Sam White<br />Steven Roberts<br />Emma Timmins-Schiffman<br />Caroline Storer<br />Mackenzie Gavery<br />Friedman Lab: <br />Carolyn Friedman<br />Brent Vadopalas<br />Liza Ray<br />Lisa Crosson<br />EleneDorfmeier<br />SammiBrombacker<br />Robyn Strenge<br />NOAA NWFSC<br />Shallin Busch<br />Paul McElhany<br />Mike Maher<br />Jason Miller<br />Sarah Norberg<br />Taylor Shellfish<br />Greg Jacob<br />Joth Davis<br />Funding<br />Washington Sea Grant<br />Saltonstall-Kennedy<br />University of Washington<br />
  21. 21. Special Thanks!<br />Student Sponsors<br />Chelsea Farms LLC<br />Chuckanut Shellfish<br />Little Skookum Shellfish Growers<br />NOAA<br />PCS-NSA<br />Rock Point Oyster Co.<br />Seattle Shellfish<br />Taylor Shellfish<br />

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