Roberts Insight

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Roberts Insight

  1. 1. changes in the environment and changes in expression: insight from oysters Steven Roberts University of Washington School of Aquatic and Fishery Sciences
  2. 2. research program overview environmental stressors shellfish
  3. 3. research program overview environmental stressors } chemotaxis binding protease cells oxidative burst antimicrobial hemolymph shellfish serum
  4. 4. research program overview pathogens environmental carbon dioxide stressors microbes mechanical stress microbes microbes stress response transcriptome proteome shellfish epigenome*
  5. 5. rationale comparative biology
  6. 6. rationale aquaculture
  7. 7. rationale environmental sciences
  8. 8. today pathogens environmental carbon dioxide stressors mechanical stress anthropogenic activities stress response transcriptome proteome oysters epigenome*
  9. 9. outline pathogens 1. Hemocyte gene carbon dioxide discovery mechanical stress anthropogenic activities 2. Multiple stressors 3. Characterizing natural populations
  10. 10. hemocyte (plated) cDNA library Prior to washing After washing
  11. 11. ESTs modified from Roberts et al 2009
  12. 12. vibrio exposure Roberts et al 2009
  13. 13. interleukin 17 •cytokine •large number of cytokines found in vertebrates are not found in invertebrates •interleukin 17 is not similar to other interleukins •vertebrates- interleukin expressed in activated memory T cells
  14. 14. interleukin 17 Roberts et al 2008
  15. 15. interleukin 17 Roberts et al 2008
  16. 16. interleukin 17 Roberts et al 2008
  17. 17. summary •interleukin 17 could be an early cytokine present before divergence of vertebrates and invertebrates •expression analysis indicates it is a rapid response signaling molecule •complete signaling pathway and presence of other molecules is not known in the oyster
  18. 18. outline pathogens 1. Hemocyte gene carbon dioxide discovery mechanical stress anthropogenic activities 2. Multiple stressors
  19. 19. multiple stressors
  20. 20. ocean acidification Sabine et al 2004
  21. 21. ocean acidification
  22. 22. ocean acidification
  23. 23. mechanical control control mechanical CO2 960 ppm control 24 hours 5 min 40 min
  24. 24. mechanical stress
  25. 25. mechanical control control mechanical CO2 960 ppm control 24 hours 5 min
  26. 26. microbial community Horner-Devine
  27. 27. summary •multiple environmental stressors could contribute to increase stressor susceptibility •data suggests stress response could be at capacity and not able to properly respond to secondary stressor •organisms will likely adapt to chronic changes •not clear how global change will affect normal physiological processes
  28. 28. outline pathogens 1. Hemocyte gene carbon dioxide discovery mechanical stress anthropogenic activities 2. Multiple stressors 3. Characterizing natural populations
  29. 29. urban, agriculture, water fowl, marinas, seals low population, low fecal coliform
  30. 30. Transcriptomics 16 million ~40 bp HQ reads 16 million ~40 bp HQ reads
  31. 31. 32 million reads v 17 million matched Sigenae consensuses 29 thousand features Upregulated features | min 10 unique hits & 2 fold increase 1329 1316 22 specific 25 specific
  32. 32. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
  33. 33. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
  34. 34. Upregulation (4-fold) low population, low fecal coliform urban, agriculture, water fowl, marinas, seals
  35. 35. RNAseq vs quantitative PCR
  36. 36. low population, qPCR low fecal coliform urban, agriculture, water fowl, marinas, seals steroid 17-alpha-hydroxylase
  37. 37. x fold = qPCR complement C1q steroid 17-alpha-hydroxylase serine protease inhibitor TNF-related protein 4 2 fold 3 fold 4 fold gonadotropin-releasing calmodulin-like metalloproteinase inhibitor 3 hormone II receptor specific 13 fold 3 fold
  38. 38. gene discovery
  39. 39. summary (biology) •limited categorical differences •selection and adaptation will play a significant role in physiological response •other normal physiological processes are occurring (out of sync)
  40. 40. summary (technical) •promising technology •issues include; genome plasticity, multiple isoforms, lack of genome •analysis is not trivial
  41. 41. What is the functional role of DNA methylation in shellfish? How do environmental conditions impact the epigenome?
  42. 42. background global methylation no methylation (except CpG islands) mosiac pattern - ~40-60% methylation
  43. 43. 5-methylcytosine and DNA repair cytosine uracil deamination 5-methylcytosine thymine Over time.. loss of methylated CpGs
  44. 44. oysters in  silico   analysis   of  ~30k  gene   clusters
  45. 45. biological process • DNA methylation is an important transcriptional control mechanism
  46. 46. environmental effects sites
  47. 47. epigenetics: implications and direction •Epigenetic variation will redefine our concept of “genetic” diversity •Novel mechanism impacted by environmental change •long-term effects ? •Candidate process that could explain other phenomenon
  48. 48. conclusions & directions • comparative and evolutionary aspects can provide valuable insight into more complex systems • with a better understanding of mechanisms, populations that are better able to respond to stressors can be identified. This information will be applicable for conservation, aquaculture, and predicting ecosystem change. • characterizing natural populations to better understand biology and the environment will continue to be complex, however deep sequencing will prove to be a valuable tool
  49. 49. acknowledgements Yannick Gueguen (Ifremer) Julien de Lorgeril (Ifremer) Frederick Goetz (WATER Institute) Giles Goetz (WATER Institute) Samuel White (UW) Rachel Thompson (UW - student) Claire Horner-Devine (UW) Mackenzie Gavery (UW - student) funding Joth Davis (Taylor Shellfish) USDA-NRAC Dustin Lennon (UW) NOAA SK Program Paul Sampson (UW) UW-SAFS

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