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  • 1. The  Physiological  Response   of  Crassostrea  gigas  to  CO2-­‐‑ Induced  Ocean   Acidification Emma Timmins-Schiffman Carolyn Friedman Steven Roberts University of Washington School of Aquatic and Fishery Sciences Seattle, WA
  • 2. Plan  of  Talk •  Background: Ocean acidification and marine bivalves•  Aim of experiment•  Methods•  Results (proteomics)•  Conclusion 10 8 Oyster Shell Weight (g) 6 4 400 ppm 600 ppm 2 800 ppm 1000 ppm 1200 ppm 1400 ppm 0 0 2 4 6 8 10 12 Oyster Buoyant Weight (g)
  • 3. Ocean  Acidification  and   the  Environment •  Rising pCO2 in the atmosphere equilibrates with ocean surface water, reducing the pH
  • 4. Ocean  Acidification  and   the  Environment •  Rising pCO2 in the atmosphere equilibrates with ocean surface water, reducing the pH•  Current and projected changes in pCO2 are unprecedented Zeebe  2011
  • 5. Ocean  Acidification  and   Bivalves •  How do changes in oceanic pCO2 and/or pH affect the physiology of bivalves? o  C. gigas larvae show a developmental delay (Timmins-Schiffman et al., in press Marine Biology) o  Alterations to shell growth/maintenance o  Changes in metabolic rate o  Changes in responses to other stressors Lannig  et  al.  2010
  • 6. Experimental  Objective Assess the effects of ocean acidification on Crassostrea gigas at the molecular and whole organism level
  • 7. Methods •  Juvenile C. gigas were exposed to one of 6 pCO2 treatments for 1 month
  • 8. 427 µatm638 µatm810 µatm991 µatm1182 µatm2847 µatm
  • 9. 427 µatm 2012638 µatm810 µatm991 µatm1182 µatm2847 µatm
  • 10. 427 µatm 2012638 µatm810 µatm991 µatm 21001182 µatm2847 µatm
  • 11. Experimental  Design pCO2 (µatm) 400 600 800 1000 1200 2800t0: shell weight 1 month exposureNo additional Mechanicalstress stress Heat shock •  2 sublethal temperatures: •  Shell weight 42 & 43°C •  Gill tissue: •  1 lethal •  Transcriptomics temperature: •  Proteomics 44°C •  Shell structure/strength •  Whole body: lipids? •  Histology
  • 12. Experimental  Design pCO2 (µatm) 400 600 800 1000 1200 2800t0: shell weight 1 month exposureNo additional Mechanicalstress stress Heat shock •  2 sublethal temperatures: •  Shell weight 42 & 43°C •  Gill tissue: •  Transcriptomics •  1 lethal •  Proteomics temperature: •  Shell structure/strength 44°C •  Whole body: lipids? •  Histology
  • 13. Methods •  Shotgun proteomics on LC MS/MS (LTQ-Orbitrap)•  4 oysters from 4 treatments•  Assigned spectra to database•  Comparative expression by spectral counting 2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1 2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1 T: Average spectrum MS2 1240.28 (5584-5952) T: Average spectrum MS2 1240.28 (5584-5952) 2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1 1344.0381 1344.0381 100 1221.0632 T: ITMS + c NSI d Full ms2 1820.34@cid35.00 [490.00-2000.00] 100 1221.0632 95 95 1661.2975 90 100 90 85 85 95 80 80 75 1178.4297 1178.4297 75 90 70 70 85 65 65 60 60 Relative Abundance Relative Abundance 80 55 1039.4359 1039.4359 55 50 75 50 45 45 70 40 40 1424.5980 1424.5980 35 2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1 35 65 1490.5931 T: ITMS + c NSI 30 Full ms2 1820.34@cid35.00 [490.00-2000.00] d 1490.5931 30 60 25 25 1661.2975Relative Abundance 1912.4304 1912.4304 20 100 20 869.9010 1607.7299 1607.7299 55 545.8882 823.7305 545.8882 869.9010 15 15 823.7305 479.2288 95 479.2288 1851.9578 50 10 10 1851.9578 5 90 5 45 0 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z 2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1 85 400 600 800 1000 1200 1400 1600 1800 2000 40 T: Average spectrum MS2 1240.28 (5584-5952) m/z 1915.7020 1426.2880 1344.0381 80 35 100 1221.0632 95 75 30 90 85 70 25 80 65 20 1710.3446 75 1178.4297 15 70 60 Relative Abundance 979.8818 1102.1235 65 1255.5396 10 60 55 1349.2131 Relative Abundance 55 1039.4359 5 50 50 0 45 45 600 800 1000 1200 1400 1600 1800 2000 40 1424.5980 m/z 40 35 1490.5931 1915.7020 30 1426.2880 35 25 1912.4304 20 1607.7299 30 869.9010 15 545.8882 823.7305 479.2288 25 1851.9578 10 5 20 1710.3446 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z 15 979.8818 1102.1235 1255.5396 10 1349.2131 5 0 600 800 1000 1200 1400 1600 1800 2000 m/z
  • 14. Methods Gigasdatabase (Sigenae) contig numberPeptides deduced from spectra that match to this protein
  • 15. Shell  Weight 0.7 0.6 Elevated pCO2 hasGain in Shell Weight (g) a negative effect 0.5 on growth 0.4 0.3 0.2 0.1 0.0 400 2800 800 1200 600 1000 Treatment (µatm)
  • 16. Proteome •  Identified 897 unique proteins across all samples•  Proteins were annotated by 453 distinct GO categories
  • 17. Proteome cell  cycle  and   proliferation cell  organization   cell  adhesion and  biogenesis cell-­‐‑cell  stress  response transport signaling death protein   RNA  metabolism metabolism signal  transduction DNA  metabolism All proteins developmental   processes
  • 18. Proteome Entire proteome represented by GO terms
  • 19. Exposure  Comparison Decreased Expression High pCO2 Similar Expression(150 Unique Proteins) Level Elevated Expression High pCO2(130 Unique Proteins)
  • 20. Exposure  Comparison Proteinsexpressed in high pCO2 only
  • 21. Exposure  comparison •  Proteins expressed at high pCO2 are related to the physiological processes o  Metabolism o  Oxidative stress o  Transport (proton and calcium) o  Cellular stress o  Translation•  These processes imply that the oyster is maintaining a significant response to stress even after 1 month of exposure
  • 22. Exposure  Comparison 15 400 µatm + MS 400 µatm 10 5 PC 2 (20.5%) 0 -5 2800 µatm + MS -10 2800 µatm -15 -15 -10 -5 0 5 10 15 PC 1 (49.7%)
  • 23. Results •  MS approach expands on previous 2D gel analyses•  Shotgun sequencing allow for: 1.  Identification of more proteins 2.  Greater potential to detect more nuanced responses and protein interactions
  • 24. Conclusions •  We were able to identify hundreds of proteins in the C. gigas gill proteome•  Even after 1 month of exposure, oysters were maintaining an energetically costly stress response 10 8 Oyster Shell Weight (g) 6 4 400 ppm 600 ppm 2 800 ppm 1000 ppm 1200 ppm 1400 ppm 0 0 2 4 6 8 10 12 Oyster Buoyant Weight (g)
  • 25. Further  Work •  Evaluate physiological response to combined stressors•  Comparison of transcriptomic and proteomic responses•  Develop a more robust system for protein identifications
  • 26. Acknowledgements •  UWPR (Proteomics) o  Priska von Haller, Jimmy Eng, Tahmina Jahan•  UW SAFS and Biology o  Michael O’Donnell, Emily Carrington, Ken Sebens, Matt George o  Sam White, Mackenzie Gavery, Caroline Storer, Dave Metzger•  UW Medicinal Chemistry o  Dave Goodlett, Brook Nunn•  Oyster collection and care o  Sam Garson, Ronen Elad, Joth Davis, Jason Ragan, Dustin Johnson•  Funding o  Research: crowdfunding through RocketHub o  Travel: UW SAFS