Timmins schiffman afs 2011

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Timmins schiffman afs 2011

  1. 1. Gene Expression as an Indicatorof Environmental Stress in thePacific Oyster, Crassostrea gigasEmma Timmins-Schiffman University of WashingtonSteven Roberts SAFSMackenzie Gavery AFS, Conservation Genetics and Genomics in Fisheries September 8, 2011
  2. 2. Outline¤  Climate change in the Pacific Northwest¤  Oyster mortalities in hatcheries¤  Transcriptomic tools for monitoring environmental change ¤  Targeted gene approach ¤  Whole transcriptome (NGS) approach¤  Applications of transcriptomics
  3. 3. Climate Change in the PNW IPCC (2007) Projected Estimates of Atmospheric CO2 ¤  Ocean acidification ¤  As CO2 dissolves in 800 seawater, pH goes Estimate of atmospheric CO2 (ppm) down. 700 Kilometers along transect 600 500 400 300 1750 1800 1850 1900 1950 2000 2050 2100Feely et al. 2010 Year
  4. 4. WA Dept. of Ecology 2007 Commercial/Industry High Population Density
  5. 5. Hatchery mortalities¤  Larval mortalities have been linked to acidic, upwelled waters and disease Elston et al. 2008
  6. 6. Transcriptomic Tools for Conservation ¤  What are transcriptomics? ¤  Measurement of physiological response ¤  Range/plasticity of response ¤  Plasticity of population response linked to underlying genetic diversity Adaptation?Population Environmental Bottleneck? Change Acclimatization? Mortality?
  7. 7. Transcriptomics and Conservation¤  How do these host of environmental changes affect all life stages of Pacific oyster?¤  How can we learn about population conservation and response to environmental change through use of transcriptomic tools?
  8. 8. Transcriptomic Tools for Conservation ¤  2 methods ¤  Targeted gene approach (bottom up) ¤  Global transcriptome/next-generation sequencing (top down)Which genes/pathways might change? What response do we see?
  9. 9. Targeted Gene Approach¤  Oyster larvae exposed to 3 levels of pCO2 for 4 days – ambient (400 µatm), and 2 elevated (700 and 1000 µatm)¤  Variables measured: water chemistry, size, calcification, developmental stage, and gene expression¤  Genes chosen: ¤  Molecular chaperone – hsp70 ¤  Oxidative stress – SOD, GPx, Prx6
  10. 10. Oyster Morphometrics: Results Hinge Length by Treatment and Day Shell Height by Treatment and Day 80 70 70 60Hinge Length (!m) Shell Height (!m) 60 50 40 50 30 Day 1 Day 3 40 Day 1 Day 3 400 700 1000 400 700 1000 D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000 400 700 1000 400 700 1000 D1 400 D1 700 D1 1000 D3 400 D3 700 D3 1000 Day and pCO2 (!atm) Day and pCO2 (!atm) Treatment (µatm) Treatment (µatm)
  11. 11. Hsp70 Stress ResponseSTRESS Protein damage/ unfolding Hsp70Chaperones bind to proteins to either repair or remove
  12. 12. Heat Shock Protein 70 25Fold Over Minimum Expression 20 15 10 5 400 700 1000 Treatment (!atm)
  13. 13. Oxidative Stress Genes Stress Response STRESS •  Increase metabolism •  Kill pathogens Prx6 ROSSOD GPx
  14. 14. Fold Over Minimum Expression 0.00 0.05 0.10 0.15 0.20 400 700Treatment (!atm) Superoxide Dismutase 1000
  15. 15. Fold Over Minimum Expression 0.0e+00 5.0e+24 1.0e+25 1.5e+25 400 700Treatment (!atm) Glutathione Peroxidase 1000 Fold Over Minimum Expression 0e+00 1e+22 2e+22 3e+22 4e+22 5e+22 6e+22 400 700Treatment (!atm) Peroxiredoxin 6 1000
  16. 16. Targeted Gene Summary¤  Changes in pCO2 affect larval size – what underlying physiological mechanisms are affected?¤  Oxidative stress is an important response to ocean acidification¤  Acidification may cause larval oysters to increase production of ROS (metabolism?), necessitating up- regulation of oxidative stress genes
  17. 17. Whole Transcriptome Approach¤  2 sites: “pristine” and high human impact¤  Oysters collected cDNA pooled transcriptomes sequenced using SOLiD
  18. 18. v 32 million reads 17 million matched Publicly available contigs29 thousand contigsUpregulated contigs| min 10 unique hits & 2 fold increase 1329 1316 22 specific 25 specific
  19. 19. p-value cellular component movement 0.04 cell recognition cell projection organization cell adhesion 8E-15semantic space Y regulation of multicellular organismal processes negative regulation of cellular component organization cell regulation of biological communication quality hormone metabolism maintenance of location in cell immune maintenance of response location developmental localization of maturation cellular developmental cell process semantic space X
  20. 20. Whole Transcriptome: Results PCA of Oyster Gene Expression at 15 Genes 1PC2 (30%) 0 MDR49 HMGP CATL GSTO1 CALL RETST CP17A GNRR2 GSTA TIMP3.BB SPI DPGN TIMP3.DH -1 CIQT4 DEF1 Big Beef Creek Drayton Harbor -2 -1 0 1 2 PC1 (31%)
  21. 21. Whole Transcriptome Summary¤  A variety of novel processes involved in environmental response were discovered (cell adhesion)¤  Global picture of how the environment at the 2 sites is affecting the oyster physiological response
  22. 22. Conclusions¤  Both the targeted gene and the whole transcriptome approaches provide valuable tools for understanding oysters’ plastic response to environmental change.¤  Gene expression analyses should be considered important management tools for populations that may be experiencing environmental stress.
  23. 23. AcknowledgementsEmily Carrington•Matt George•Jeff Hard•MichelleHerko•Laura Newcomb•Ken Sebens•Jim & LisaSeeb•Richard Strathmann•Adam Summers•BillieSwalla•Sam WhiteNOAA Saltonstall-Kennedy grant

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