Shellfish as indicators of environmental change
Steven Roberts School of Aquatic and Fishery SciencesUniversity of Washington
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?
Oceans and Human Health
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
human pathogens
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
Oceans and Human Health
bioindicator
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
research program overview
environmental stressors
shellfish
research program overview
environmental stressors
shellfish
pathogenscarbon dioxidemechanical stress
stress response transcriptome proteome epigenome*
microbesmicrobes
microbes
rationale
comparativebiology
rationale
aquaculture
rationale
environmentalsciences
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
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)?
How will changes in immune response impact pathogen dynamics?
Oceans and Human Health
immune response
Pacific Oysters- Vibrio - Oyster Herpes Virus
Oceans and Human Health
immune response
Pacific Oysters- Vibrio - Sanger - Oyster Herpes Virus - HTS
How advances in technology are changing science
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 17 is not similar to other interleukins
•vertebrates- interleukin expressed in activated memory T cells
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 - Oyster Herpes Virus - HTS
How advances in technology are changing science
high throughput sequencing (HTS)
ABI SOLiD
high throughput sequencing (HTS)
Oyster Larvae & Oyster Herpes Virus
Burge and Friedman (unpublished)
Oyster Larvae Exposures
Burge and Friedman (unpublished)
Days
Act
ive
Viru
s
Transcriptomics
16 million~40 bp
HQ reads
16 million~40 bp
HQ reads
Oyster Herpes Virus
Transcriptomics
Downregulated immune response genes
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
human pathogens
Transcriptomics
Transcriptomics
pathogenphysiology
bonus
Ostreid herpesvirus 1 - complete genome
cove
rage
bonus
Ostreid herpesvirus 1 - complete genome
cove
rage
Genome (strain variation / epidemiology)
Gene Expression (physiology / virulence)
Ostreid herpesvirus 1 - complete genome
cove
rage
Ostreid herpesvirus 1 - complete genome
ORF104 ORF107 ORF113
Ostreid herpesvirus 1 - complete genome
ORF104 ORF107 ORF113
ORF107
cove
rage
ORF80 ORF90
ORF80 ORF90
membrane protein
summary
HTS offers a non biased approach for characterization physiological responses in non-model host-pathogen systems
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
ocean acidification
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
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
oysters
control
V. tubiashii and host presence
pI MW3.7 29k4.5 49k4.8 26k
pI MW3.7 29k4.5 49k4.8 26k M
W
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
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
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
Oceans and Human Health
bioindicator
shellfish as bioindicators
• sessile
• continuously filter water
• robust
• ubiquitous
• contaminantsaccumulate
Physiological Response ofOysters in Puget Sound
PROPS
- gene expression
- response to secondary stressor
- DNA methylation
PROPS
- gene expression
- DNA methylation
- response to secondary stressor
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
16 million~40 bp
HQ reads
v32 million reads
17 million matched Sigenae consensuses
29 thousand features
Upregulated features | min 10 unique hits & 2 fold increase
132922 specific
131625 specific
urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
urban, agriculture, water fowl, marinas, seals
low population, low fecal coliform
RNAseq vs quantitative PCR
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
qPCR
steroid 17-alpha-hydroxylase
calmodulin-like
serine protease inhibitor complement C1q
TNF-related protein 4
metalloproteinase inhibitor 3gonadotropin-releasing
hormone II receptor
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?
Oceans and Human Health
bioindicator
pathogenphysiology
immune response
transcriptomeepigenome
epigenetics
DNA Methylation
Histone Modification
Small InterferingRNA
•controls normal developmental processes
•implicated in human diseases including cancer
•possible means for adaptation of changing environmental condition - heritable for several generations
epigenetics
environmental epigenomics
- agouti viable yellow (Avy)- no methylation = yellow- methylation = normal
- maternal BPA exposure influences offspring- DNA hypomethylation
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
Oceans and Human Health
bioindicator
What is the functional role of DNA methylation in shellfish?
How do environmental conditions impact the epigenome?
background
no methylationglobal methylation
(except CpG islands)
mosiac pattern - ~40-60% methylation
oysters
insilicoanalysis
of~30kgeneclusters
Gavery and Roberts (submitted)
5-methylcytosine and DNA repair
Over time..loss of methylated
CpGs
cytosine
5-methylcytosine
deamination
thymine
uracil
oysters
insilicoanalysis
of~30kgeneclusters
Gavery and Roberts (submitted)
biological process
Gavery and Roberts (submitted)
environmental effects
sites
Gavery, unpublished
current direction
Bi-sulfiteSequencing
Me-DIParray
Me-DIP comparative hybridization array
M. Settles
Me-DIP comparative hybridization array
M. Settles
Practical
bisulfite sequencing
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
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
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
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
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