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changes in the environment and changes in expression: insight from oysters
Steven RobertsUniversity of WashingtonSchool of Aquatic and Fishery Sciences
research program overview
environmental stressors
shellfish
research program overview
environmental stressors
shellfishhemolymph
cells
serum
}chemotaxisbindingproteaseoxidative burstantimicrobial
research program overview
environmental stressors
shellfish
pathogenscarbon dioxidemechanical stress
stress response transcriptome proteome epigenome*
microbesmicrobes
microbes
rationale
comparativebiology
rationale
aquaculture
rationale
environmentalsciences
today
environmental stressors
oysters
pathogenscarbon dioxidemechanical stressanthropogenic activities
stress response transcriptome proteome epigenome*
outline
pathogenscarbon dioxidemechanical stressanthropogenic activities
1. Hemocyte gene discovery
2. Multiple stressors
3. Characterizingnatural populations
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
interleukin 17
Roberts et al 2008
interleukin 17
Roberts et al 2008
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
outline
pathogenscarbon dioxidemechanical stressanthropogenic activities
1. Hemocyte gene discovery
2. Multiple stressors
multiple stressors
ocean acidification
Sabine et al 2004
ocean acidification
ocean acidification
24 hours 5 min 40 min
control
CO2
960 ppm
mechanical
control
mechanical
control
mechanical stress
24 hours 5 min
controlmechanical
control
mechanical
control
CO2
960 ppm
microbial community
Horner-Devine
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
outline
pathogenscarbon dioxidemechanical stressanthropogenic activities
1. Hemocyte gene discovery
2. Multiple stressors
3. Characterizingnatural populations
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
Upregulation (4-fold)
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
2 fold
3 fold13 fold
calmodulin-like
serine protease inhibitor complement C1q
TNF-related protein 4
3 fold
metalloproteinase inhibitor 3
specific
gonadotropin-releasing hormone II receptor
4 fold
x fold =
gene discovery
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)
summary (technical)
•promising technology
•issues include; genome plasticity, multiple isoforms, lack of genome
•analysis is not trivial
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
5-methylcytosine and DNA repair
Over time..loss of methylated
CpGs
cytosine
5-methylcytosine
deamination
thymine
uracil
oysters
in silico analysis
of ~30k gene clusters
biological process
• DNA methylation is an important transcriptional control mechanism
environmental effects
sites
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
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
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)Joth Davis (Taylor Shellfish)Dustin Lennon (UW)Paul Sampson (UW)
fundingUSDA-NRACNOAA SK ProgramUW-SAFS