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