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