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Shellfish as indicators of environmental change
Steven Roberts
School of Aquatic and Fishery Sciences
University of Washing...
Oceans and Human Health


                                                           practical
omic approaches            ...
Oceans and Human Health
Oceans and Human Health


    immune
   response
                           pathogen
                           physiology...
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
Oceans and Human Health




      bioindicator
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
research program overview


  environmental
    stressors




                            shellfish
research program overview

                              pathogens
  environmental               carbon dioxide
    stress...
rationale

            comparative
              biology
rationale




aquaculture
rationale




            environmental
              sciences
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
Oceans and Human Health


    immune
   response




   How does environmental change impact physiology (populations)?
Oceans and Human Health


    immune
   response




   How does environmental change impact physiology (populations)?

  ...
Oceans and Human Health


    immune
   response


                Pacific Oysters
                 - Vibrio
              ...
Oceans and Human Health


    immune
   response


                  Pacific Oysters
                   - Vibrio - Sanger
 ...
hemocyte (plated) cDNA library

       Prior to washing          After washing
ESTs




       modified from Roberts et al 2009
vibrio exposure




                  Roberts et al 2009
interleukin 17

•cytokine

•large number of cytokines found in vertebrates are not
 found in invertebrates

•interleukin 1...
interleukin 17




                 Roberts et al 2008
summary



An omic approach that has a two step process

 •gene discovery & expression analysis
Oceans and Human Health


    immune
   response


                  Pacific Oysters
                   - Vibrio - Sanger
 ...
high throughput sequencing (HTS)
ABI SOLiD
high throughput sequencing (HTS)
Oyster Larvae & Oyster Herpes Virus




                               Burge and Friedman (unpublished)
Oyster Larvae Exposures
Active Virus




                    Days

                           Burge and Friedman (unpublis...
Transcriptomics



                                   16 million
                                     ~40 bp
             ...
Transcriptomics
Downregulated immune response genes
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
Transcriptomics
Transcriptomics



                  pathogen
                  physiology
bonus


                   Ostreid herpesvirus 1 - complete genome
coverage
Genome (strain variation / epidemiology)
           bonus        Gene Expression (physiology / virulence)


              ...
Ostreid herpesvirus 1 - complete genome
coverage
Ostreid herpesvirus 1 - complete genome




ORF104        ORF107           ORF113
Ostreid herpesvirus 1 - complete genome




ORF104        ORF107           ORF113
ORF107
coverage
ORF80   ORF90
ORF80          ORF90

membrane protein
summary




HTS offers a non biased approach for
characterization physiological responses in non-
model host-pathogen syst...
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
ocean acidification
Ocean Acidification and Emerging Diseases


Acidification will compromise
shell structure in many shellfish

There is little ...
Ocean Acidification and Emerging Diseases



              Pacific Oyster




                    Vibrio tubiashii
How do changes in the environment
influence Vibrio tubiashii physiology?




          tool development
Proteomics*

Protein Extraction

                     2D gels


                               ID differences
V. tubiashii and host presence


       oysters




       control
V. tubiashii and host presence


       oysters




       control
pI   MW
3.7   29k
4.5   49k
4.8   26k
pI   MW
3.7   29k
4.5   49k
            MW




4.8   26k
low oxygen conditions                                              Chart 6

                                      biosynth...
summary


• proteomic approaches offer global tool to directly examine
  functional responses to changes in environmental ...
Oceans and Human Health


    immune
   response
                          pathogen
                          physiology

...
Oceans and Human Health




      bioindicator
shellfish as bioindicators

• sessile

• continuously
  filter water

• robust

• ubiquitous

• contaminants
  accumulate
Physiological
Response of
Oysters in
Puget
Sound
PROPS


- gene expression


- response to
secondary stressor


- DNA methylation
PROPS


- gene expression


- response to
secondary stressor


- DNA methylation
mechanical stress




subject oysters to
stress then
measure
noradrenaline
levels
urban, agriculture, water
  fowl, marinas, seals




low population, low fecal
       coliform
Transcriptomics


                  16 million
                    ~40 bp
                   HQ reads




                ...
32 million reads
v

                                            17 million matched
                                       ...
low population, low fecal   urban, agriculture, water
       coliform               fowl, marinas, seals
low population, low fecal   urban, agriculture, water
       coliform               fowl, marinas, seals
low population, low fecal   urban, agriculture, water
       coliform               fowl, marinas, seals
RNAseq vs quantitative PCR
low population,                                     qPCR
low fecal coliform


urban, agriculture,
   water fowl,
  marinas...
qPCR
                                   complement C1q
steroid 17-alpha-hydroxylase                                   seri...
summary

•HTS offers non-biased, one-stop characterization

•provides ability to identify organismal stressors
 without pr...
Oceans and Human Health


    immune
   response
                                 pathogen
                               ...
epigenetics




                        DNA Methylation




           Histone        Small Interfering
          Modificat...
epigenetics


 •controls normal developmental processes

 •implicated in human diseases including cancer

 •possible means...
environmental epigenomics


                       - agouti viable yellow (Avy)
                       - no methylation = ...
environmental epigenomics

• Rats treated with the estrogenic pesticide methoxychlor
 or the antiandrogenic fungicide vinc...
Oceans and Human Health




      bioindicator
What is the functional role of DNA methylation in shellfish?

How do environmental conditions impact the epigenome?
background




 global methylation
                                      no methylation
     (except CpG islands)




    ...
oysters




    in
silico

    analysis

 of
~30k
gene

    clusters




                 Gavery and Roberts (submitted)
5-methylcytosine and DNA repair



cytosine                 uracil


           deamination


5-methylcytosine         thy...
oysters




    in
silico

    analysis

 of
~30k
gene

    clusters




                 Gavery and Roberts (submitted)
biological process




                     Gavery and Roberts (submitted)
environmental effects




                        sites
                                Gavery, unpublished
current direction




Me-DIP
 array
   Bi-sulfite
  Sequencing
Me-DIP comparative hybridization array




                                         M. Settles
Me-DIP comparative hybridization array

                                         Practical




                           ...
bisulfite sequencing
bisulfite sequencing




- Less Practical?     Bormann Chung CA, Boyd VL, McKernan KJ, Fu Y, Monighetti C, et al. (2010)

-...
summary

• epigenetic processes are important mechanisms by
 which changing environmental conditions alter gene
 expressio...
conclusions & directions

• comparative and evolutionary aspects can provide valuable
 insight into predicting ecosystem c...
acknowledgements

Yannick Gueguen (Ifremer)
Julien de Lorgeril (Ifremer)
Frederick Goetz (WATER Institute)
Giles Goetz (WA...
Roberts GRC
Roberts GRC
Roberts GRC
Roberts GRC
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Transcript of "Roberts GRC"

  1. 1. Shellfish as indicators of environmental change Steven Roberts School of Aquatic and Fishery Sciences University of Washington
  2. 2. 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
  3. 3. Oceans and Human Health
  4. 4. Oceans and Human Health immune response pathogen physiology bioindicator human pathogens
  5. 5. Oceans and Human Health immune response pathogen physiology bioindicator
  6. 6. Oceans and Human Health immune response pathogen physiology bioindicator
  7. 7. Oceans and Human Health bioindicator
  8. 8. Oceans and Human Health immune response pathogen physiology bioindicator
  9. 9. research program overview environmental stressors shellfish
  10. 10. research program overview pathogens environmental carbon dioxide stressors microbes mechanical stress microbes microbes stress response transcriptome proteome shellfish epigenome*
  11. 11. rationale comparative biology
  12. 12. rationale aquaculture
  13. 13. rationale environmental sciences
  14. 14. Oceans and Human Health immune response pathogen physiology bioindicator
  15. 15. Oceans and Human Health immune response How does environmental change impact physiology (populations)?
  16. 16. Oceans and Human Health immune response How does environmental change impact physiology (populations)? How will changes in immune response impact pathogen dynamics?
  17. 17. Oceans and Human Health immune response Pacific Oysters - Vibrio - Oyster Herpes Virus
  18. 18. Oceans and Human Health immune response Pacific Oysters - Vibrio - Sanger - Oyster Herpes Virus - HTS How advances in technology are changing science
  19. 19. hemocyte (plated) cDNA library Prior to washing After washing
  20. 20. ESTs modified from Roberts et al 2009
  21. 21. vibrio exposure Roberts et al 2009
  22. 22. 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
  23. 23. interleukin 17 Roberts et al 2008
  24. 24. summary An omic approach that has a two step process •gene discovery & expression analysis
  25. 25. Oceans and Human Health immune response Pacific Oysters - Vibrio - Sanger - Oyster Herpes Virus - HTS How advances in technology are changing science
  26. 26. high throughput sequencing (HTS)
  27. 27. ABI SOLiD
  28. 28. high throughput sequencing (HTS)
  29. 29. Oyster Larvae & Oyster Herpes Virus Burge and Friedman (unpublished)
  30. 30. Oyster Larvae Exposures Active Virus Days Burge and Friedman (unpublished)
  31. 31. Transcriptomics 16 million ~40 bp HQ reads 16 million ~40 bp HQ reads Oyster Herpes Virus
  32. 32. Transcriptomics
  33. 33. Downregulated immune response genes
  34. 34. Oceans and Human Health immune response pathogen physiology bioindicator human pathogens
  35. 35. Transcriptomics
  36. 36. Transcriptomics pathogen physiology
  37. 37. bonus Ostreid herpesvirus 1 - complete genome coverage
  38. 38. Genome (strain variation / epidemiology) bonus Gene Expression (physiology / virulence) Ostreid herpesvirus 1 - complete genome coverage
  39. 39. Ostreid herpesvirus 1 - complete genome coverage
  40. 40. Ostreid herpesvirus 1 - complete genome ORF104 ORF107 ORF113
  41. 41. Ostreid herpesvirus 1 - complete genome ORF104 ORF107 ORF113
  42. 42. ORF107
  43. 43. coverage
  44. 44. ORF80 ORF90
  45. 45. ORF80 ORF90 membrane protein
  46. 46. summary HTS offers a non biased approach for characterization physiological responses in non- model host-pathogen systems
  47. 47. Oceans and Human Health immune response pathogen physiology bioindicator
  48. 48. ocean acidification
  49. 49. 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
  50. 50. Ocean Acidification and Emerging Diseases Pacific Oyster Vibrio tubiashii
  51. 51. How do changes in the environment influence Vibrio tubiashii physiology? tool development
  52. 52. Proteomics* Protein Extraction 2D gels ID differences
  53. 53. V. tubiashii and host presence oysters control
  54. 54. V. tubiashii and host presence oysters control
  55. 55. pI MW 3.7 29k 4.5 49k 4.8 26k
  56. 56. pI MW 3.7 29k 4.5 49k MW 4.8 26k
  57. 57. 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
  58. 58. 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
  59. 59. Oceans and Human Health immune response pathogen physiology bioindicator
  60. 60. Oceans and Human Health bioindicator
  61. 61. shellfish as bioindicators • sessile • continuously filter water • robust • ubiquitous • contaminants accumulate
  62. 62. Physiological Response of Oysters in Puget Sound
  63. 63. PROPS - gene expression - response to secondary stressor - DNA methylation
  64. 64. PROPS - gene expression - response to secondary stressor - DNA methylation
  65. 65. mechanical stress subject oysters to stress then measure noradrenaline levels
  66. 66. urban, agriculture, water fowl, marinas, seals low population, low fecal coliform
  67. 67. Transcriptomics 16 million ~40 bp HQ reads 16 million ~40 bp HQ reads
  68. 68. 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
  69. 69. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
  70. 70. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
  71. 71. low population, low fecal urban, agriculture, water coliform fowl, marinas, seals
  72. 72. RNAseq vs quantitative PCR
  73. 73. low population, qPCR low fecal coliform urban, agriculture, water fowl, marinas, seals steroid 17-alpha-hydroxylase
  74. 74. qPCR complement C1q steroid 17-alpha-hydroxylase serine protease inhibitor TNF-related protein 4 gonadotropin-releasing calmodulin-like metalloproteinase inhibitor 3 hormone II receptor
  75. 75. 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?
  76. 76. Oceans and Human Health immune response pathogen physiology bioindicator transcriptome epigenome
  77. 77. epigenetics DNA Methylation Histone Small Interfering Modification RNA
  78. 78. epigenetics •controls normal developmental processes •implicated in human diseases including cancer •possible means for adaptation of changing environmental condition - heritable for several generations
  79. 79. environmental epigenomics - agouti viable yellow (Avy) - no methylation = yellow - methylation = normal - maternal BPA exposure influences offspring - DNA hypomethylation
  80. 80. 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
  81. 81. Oceans and Human Health bioindicator
  82. 82. What is the functional role of DNA methylation in shellfish? How do environmental conditions impact the epigenome?
  83. 83. background global methylation no methylation (except CpG islands) mosiac pattern - ~40-60% methylation
  84. 84. oysters in
silico
 analysis
 of
~30k
gene
 clusters Gavery and Roberts (submitted)
  85. 85. 5-methylcytosine and DNA repair cytosine uracil deamination 5-methylcytosine thymine Over time.. loss of methylated CpGs
  86. 86. oysters in
silico
 analysis
 of
~30k
gene
 clusters Gavery and Roberts (submitted)
  87. 87. biological process Gavery and Roberts (submitted)
  88. 88. environmental effects sites Gavery, unpublished
  89. 89. current direction Me-DIP array Bi-sulfite Sequencing
  90. 90. Me-DIP comparative hybridization array M. Settles
  91. 91. Me-DIP comparative hybridization array Practical M. Settles
  92. 92. bisulfite sequencing
  93. 93. 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
  94. 94. 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
  95. 95. 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
  96. 96. 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
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