Roberts GRC
description
Transcript of Roberts GRC
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Shellfish as indicators of environmental change
Steven Roberts School of Aquatic and Fishery SciencesUniversity of Washington
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Oceans and Human Health
one health
omic approaches
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
medium to large scale characterization of transcripts, proteins, community members (metagenome), metabolites, epigenetic differences....
practical?
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Oceans and Human Health
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
human pathogens
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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Oceans and Human Health
bioindicator
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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research program overview
environmental stressors
shellfish
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research program overview
environmental stressors
shellfish
pathogenscarbon dioxidemechanical stress
stress response transcriptome proteome epigenome*
microbesmicrobes
microbes
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rationale
comparativebiology
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rationale
aquaculture
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rationale
environmentalsciences
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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Oceans and Human Health
immune response
How does environmental change impact physiology (populations)?
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Oceans and Human Health
immune response
How does environmental change impact physiology (populations)?
How will changes in immune response impact pathogen dynamics?
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Oceans and Human Health
immune response
Pacific Oysters- Vibrio - Oyster Herpes Virus
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Oceans and Human Health
immune response
Pacific Oysters- Vibrio - Sanger - Oyster Herpes Virus - HTS
How advances in technology are changing science
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hemocyte (plated) cDNA library
Prior to washing After washing
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ESTs
modified from Roberts et al 2009
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vibrio exposure
Roberts et al 2009
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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
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interleukin 17
Roberts et al 2008
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summary
An omic approach that has a two step process
•gene discovery & expression analysis
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Oceans and Human Health
immune response
Pacific Oysters- Vibrio - Sanger - Oyster Herpes Virus - HTS
How advances in technology are changing science
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high throughput sequencing (HTS)
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ABI SOLiD
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high throughput sequencing (HTS)
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Oyster Larvae & Oyster Herpes Virus
Burge and Friedman (unpublished)
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Oyster Larvae Exposures
Burge and Friedman (unpublished)
Days
Act
ive
Viru
s
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Transcriptomics
16 million~40 bp
HQ reads
16 million~40 bp
HQ reads
Oyster Herpes Virus
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Transcriptomics
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Downregulated immune response genes
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
human pathogens
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Transcriptomics
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Transcriptomics
pathogenphysiology
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bonus
Ostreid herpesvirus 1 - complete genome
cove
rage
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bonus
Ostreid herpesvirus 1 - complete genome
cove
rage
Genome (strain variation / epidemiology)
Gene Expression (physiology / virulence)
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Ostreid herpesvirus 1 - complete genome
cove
rage
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Ostreid herpesvirus 1 - complete genome
ORF104 ORF107 ORF113
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Ostreid herpesvirus 1 - complete genome
ORF104 ORF107 ORF113
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ORF107
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cove
rage
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ORF80 ORF90
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ORF80 ORF90
membrane protein
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summary
HTS offers a non biased approach for characterization physiological responses in non-model host-pathogen systems
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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ocean acidification
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Ocean Acidification and Emerging Diseases
Acidification will compromiseshell structure in many shellfish
There is little known abouthow other physiological processes will be impacted
Using genomic approachesto determine how multiplestressors impact host-pathogen system
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Ocean Acidification and Emerging Diseases
Pacific Oyster
Vibrio tubiashii
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How do changes in the environment influence Vibrio tubiashii physiology?
tool development
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Proteomics*
Protein Extraction
2D gels
ID differences
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V. tubiashii and host presence
oysters
control
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oysters
control
V. tubiashii and host presence
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pI MW3.7 29k4.5 49k4.8 26k
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pI MW3.7 29k4.5 49k4.8 26k M
W
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biosynthesis response to endogenous stimulus
transport ion transport
signal transduction 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 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
Chart 6
Low Oxygen
Control
low oxygen conditions
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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
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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Oceans and Human Health
bioindicator
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shellfish as bioindicators
• sessile
• continuously filter water
• robust
• ubiquitous
• contaminantsaccumulate
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Physiological Response ofOysters in Puget Sound
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PROPS
- gene expression
- response to secondary stressor
- DNA methylation
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PROPS
- gene expression
- DNA methylation
- response to secondary stressor
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mechanical stress
subject oysters to stress then measure noradrenaline levels
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urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
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Transcriptomics
16 million~40 bp
HQ reads
16 million~40 bp
HQ reads
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v32 million reads
17 million matched Sigenae consensuses
29 thousand features
Upregulated features | min 10 unique hits & 2 fold increase
132922 specific
131625 specific
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urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
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urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
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urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
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RNAseq vs quantitative PCR
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qPCR
steroid 17-alpha-hydroxylase
low
pop
ulat
ion,
lo
w fe
cal c
olifo
rm
urba
n, a
gric
ultu
re,
wat
er fo
wl,
mar
inas
, sea
ls
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qPCR
steroid 17-alpha-hydroxylase
calmodulin-like
serine protease inhibitor complement C1q
TNF-related protein 4
metalloproteinase inhibitor 3gonadotropin-releasing
hormone II receptor
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summary
•HTS offers non-biased, one-stop characterization
•provides ability to identify organismal stressorswithout prior knowledge of contaminant
•natural variation in physiology needsand genetics needs to be taken intoconsideration
practical?
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Oceans and Human Health
bioindicator
pathogenphysiology
immune response
transcriptomeepigenome
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epigenetics
DNA Methylation
Histone Modification
Small InterferingRNA
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•controls normal developmental processes
•implicated in human diseases including cancer
•possible means for adaptation of changing environmental condition - heritable for several generations
epigenetics
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environmental epigenomics
- agouti viable yellow (Avy)- no methylation = yellow- methylation = normal
- maternal BPA exposure influences offspring- DNA hypomethylation
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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
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Oceans and Human Health
bioindicator
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What is the functional role of DNA methylation in shellfish?
How do environmental conditions impact the epigenome?
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background
no methylationglobal methylation
(except CpG islands)
mosiac pattern - ~40-60% methylation
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oysters
insilicoanalysis
of~30kgeneclusters
Gavery and Roberts (submitted)
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5-methylcytosine and DNA repair
Over time..loss of methylated
CpGs
cytosine
5-methylcytosine
deamination
thymine
uracil
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oysters
insilicoanalysis
of~30kgeneclusters
Gavery and Roberts (submitted)
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biological process
Gavery and Roberts (submitted)
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environmental effects
sites
Gavery, unpublished
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current direction
Bi-sulfiteSequencing
Me-DIParray
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Me-DIP comparative hybridization array
M. Settles
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Me-DIP comparative hybridization array
M. Settles
Practical
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bisulfite sequencing
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bisulfite sequencing
Bormann Chung CA, Boyd VL, McKernan KJ, Fu Y, Monighetti C, et al. (2010) Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding. PLoS ONE 5(2): e9320. doi:10.1371/journal.pone.0009320
- Less Practical? - Comprehensive
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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
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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
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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)
Mackenzie Gavery (UW)Joth Davis (Taylor Shellfish)Dustin Lennon (UW)Paul Sampson (UW)
fundingUSDA-NRACNOAA SK ProgramWashington SeaGrantUW-SAFS