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Roberts GRC
- 1. Shellfish as indicators of environmental change Steven Roberts School of Aquatic and Fishery Sciences University of Washington
- 3. Oceans and Human Health practical omic approaches ? medium to large scale characterization of transcripts, proteins, community members (metagenome), metabolites, epigenetic differences.... one health inter-relationships among human, animal, and environmental health and seeks to enhance communication, cooperation, and collaboration in integrating these areas for the health and well-being of all species
- 4. Oceans and Human Health
- 5. Oceans and Human Health immune response pathogen physiology bioindicator human pathogens
- 6. Oceans and Human Health immune response pathogen physiology bioindicator
- 7. Oceans and Human Health immune response pathogen physiology bioindicator
- 8. Oceans and Human Health bioindicator
- 9. Oceans and Human Health immune response pathogen physiology bioindicator
- 10. research program overview environmental stressors shellfish
- 11. research program overview pathogens environmental carbon dioxide stressors microbes mechanical stress microbes microbes stress response transcriptome proteome shellfish epigenome*
- 12. rationale comparative biology
- 14. rationale environmental sciences
- 16. Oceans and Human Health immune response pathogen physiology bioindicator
- 17. Oceans and Human Health immune response How does environmental change impact physiology (populations)?
- 18. Oceans and Human Health immune response How does environmental change impact physiology (populations)? How will changes in immune response impact pathogen dynamics?
- 19. Oceans and Human Health immune response Pacific Oysters - Vibrio - Oyster Herpes Virus
- 20. Oceans and Human Health immune response Pacific Oysters - Vibrio - Sanger - Oyster Herpes Virus - HTS How advances in technology are changing science
- 21. hemocyte (plated) cDNA library Prior to washing After washing
- 22. ESTs modified from Roberts et al 2009
- 23. vibrio exposure Roberts et al 2009
- 24. 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
- 25. interleukin 17 Roberts et al 2008
- 26. summary An omic approach that has a two step process •gene discovery & expression analysis
- 27. Oceans and Human Health immune response Pacific Oysters - Vibrio - Sanger - Oyster Herpes Virus - HTS How advances in technology are changing science
- 28. high throughput sequencing (HTS)
- 29. ABI SOLiD
- 30. high throughput sequencing (HTS)
- 31. Oyster Larvae & Oyster Herpes Virus Burge and Friedman (unpublished)
- 32. Oyster Larvae Exposures Active Virus Days Burge and Friedman (unpublished)
- 33. Transcriptomics 16 million ~40 bp HQ reads 16 million ~40 bp HQ reads Oyster Herpes Virus
- 34. Transcriptomics
- 35. Downregulated immune response genes
- 36. Oceans and Human Health immune response pathogen physiology bioindicator human pathogens
- 37. Transcriptomics
- 38. Transcriptomics pathogen physiology
- 39. bonus Ostreid herpesvirus 1 - complete genome coverage
- 40. Genome (strain variation / epidemiology) bonus Gene Expression (physiology / virulence) Ostreid herpesvirus 1 - complete genome coverage
- 41. Ostreid herpesvirus 1 - complete genome coverage
- 42. Ostreid herpesvirus 1 - complete genome ORF104 ORF107 ORF113
- 43. Ostreid herpesvirus 1 - complete genome ORF104 ORF107 ORF113
- 44. ORF107
- 45. coverage
- 46. ORF80 ORF90
- 47. ORF80 ORF90 membrane protein
- 48. summary HTS offers a non biased approach for characterization physiological responses in non- model host-pathogen systems
- 49. Oceans and Human Health immune response pathogen physiology bioindicator
- 51. Ocean Acidification and Emerging Diseases Acidification will compromise shell structure in many shellfish There is little known about how other physiological processes will be impacted Using genomic approaches to determine how multiple stressors impact host-pathogen system
- 52. Ocean Acidification and Emerging Diseases Pacific Oyster Vibrio tubiashii
- 53. How do changes in the environment influence Vibrio tubiashii physiology? tool development
- 54. Proteomics* Protein Extraction 2D gels ID differences
- 55. V. tubiashii and host presence oysters control
- 58. V. tubiashii and host presence oysters control
- 59. pI MW 3.7 29k 4.5 49k 4.8 26k
- 60. pI MW 3.7 29k 4.5 49k MW 4.8 26k
- 61. low oxygen conditions Chart 6 biosynthesis response to endogenous stimulus transport ion transport signal transduction Control cell communication transcription protein metabolism cell cycle carbohydrate metabolism protein biosynthesis cell organization and biogenesis response to external stimulus amino acid and derivative metabolism morphogenesis protein modification generation of precursor metabolites and energy death response to abiotic stimulus Low Oxygen cell death cell differentiation cell homeostasis nucleic acid metabolism secondary metabolism catabolism lipid metabolism DNA metabolism response to stress 0 0.025 0.050 0.075 0.100
- 62. summary • proteomic approaches offer global tool to directly examine functional responses to changes in environmental conditions (including those that contribute to virulence) • absence of a known genome makes protein identification challenging
- 63. Oceans and Human Health immune response pathogen physiology bioindicator
- 64. Oceans and Human Health bioindicator
- 65. shellfish as bioindicators • sessile • continuously filter water • robust • ubiquitous • contaminants accumulate
- 67. PROPS - gene expression - response to secondary stressor - DNA methylation
- 68. PROPS - gene expression - response to secondary stressor - DNA methylation
- 69. mechanical stress subject oysters to stress then measure noradrenaline levels
- 70. urban, agriculture, water fowl, marinas, seals low population, low fecal coliform
- 71. Transcriptomics 16 million ~40 bp HQ reads 16 million ~40 bp HQ reads
- 72. 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
- 73. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
- 74. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
- 75. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
- 76. RNAseq vs quantitative PCR
- 77. low population, qPCR low fecal coliform urban, agriculture, water fowl, marinas, seals steroid 17-alpha-hydroxylase
- 78. qPCR complement C1q steroid 17-alpha-hydroxylase serine protease inhibitor TNF-related protein 4 gonadotropin-releasing calmodulin-like metalloproteinase inhibitor 3 hormone II receptor
- 79. summary •HTS offers non-biased, one-stop characterization •provides ability to identify organismal stressors without prior knowledge of contaminant •natural variation in physiology needs and genetics needs to be taken into consideration practical?
- 80. Oceans and Human Health immune response pathogen physiology bioindicator transcriptome epigenome
- 81. epigenetics DNA Methylation Histone Small Interfering Modification RNA
- 82. epigenetics •controls normal developmental processes •implicated in human diseases including cancer •possible means for adaptation of changing environmental condition - heritable for several generations
- 83. environmental epigenomics - agouti viable yellow (Avy) - no methylation = yellow - methylation = normal - maternal BPA exposure influences offspring - DNA hypomethylation
- 84. environmental epigenomics • Rats treated with the estrogenic pesticide methoxychlor or the antiandrogenic fungicide vinclozolin during pregnancy • Male offspring that have decreased sperm capacity and fertility • Compromised fertility is passed through the adult male germ line for four generations Anway MD, Cupp AS, Uzumcu M, Skinner MK 2005 Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308:1466–1469
- 85. Oceans and Human Health bioindicator
- 86. What is the functional role of DNA methylation in shellfish? How do environmental conditions impact the epigenome?
- 87. background global methylation no methylation (except CpG islands) mosiac pattern - ~40-60% methylation
- 88. oysters in silico analysis of ~30k gene clusters Gavery and Roberts (submitted)
- 89. 5-methylcytosine and DNA repair cytosine uracil deamination 5-methylcytosine thymine Over time.. loss of methylated CpGs
- 90. oysters in silico analysis of ~30k gene clusters Gavery and Roberts (submitted)
- 91. biological process Gavery and Roberts (submitted)
- 92. environmental effects sites Gavery, unpublished
- 93. current direction Me-DIP array Bi-sulfite Sequencing
- 94. Me-DIP comparative hybridization array M. Settles
- 95. Me-DIP comparative hybridization array Practical M. Settles
- 97. bisulfite sequencing - Less Practical? Bormann Chung CA, Boyd VL, McKernan KJ, Fu Y, Monighetti C, et al. (2010) - Comprehensive Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding. PLoS ONE 5(2): e9320. doi:10.1371/journal.pone.0009320
- 98. summary • epigenetic processes are important mechanisms by which changing environmental conditions alter gene expression pattern • more research is needed to better understand mechanism and heritability in marine organisms
- 99. conclusions & directions • comparative and evolutionary aspects can provide valuable insight into predicting ecosystem changes • 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
- 100. acknowledgements Yannick Gueguen (Ifremer) Julien de Lorgeril (Ifremer) Frederick Goetz (WATER Institute) Giles Goetz (WATER Institute) Samuel White (UW) Colleen Burge (UW) Carolyn Friedman (UW) Tatyana Marushchak (UW) funding Mackenzie Gavery (UW) USDA-NRAC Joth Davis (Taylor Shellfish) NOAA SK Program Dustin Lennon (UW) Washington SeaGrant Paul Sampson (UW) UW-SAFS