Response of the Pacific Oyster Crassostrea gigas to multiple stressors:

An integrated molecular approach

INTEGRATIVE ENVIRONMENTAL PHYSIOLOGY

David BermanSammi Brombacker

Dave MetzgerPaul Pratt

Jason TayagRoss Whippo

15 DECEMBER 2010

• Background

Environmental Stress, Physiology

• Study Organism

• General Methods

• Experimental Design

• Specific Methods and Results

• What does this all mean?D. Berman

Environmental Stressors

http://www.paranormalknowledge.com/wp-content/uploads/2010/01/water-pollution.jpg

http://www.solutions4pollution.org/pollution.jpg

http://www.onlinefast.org/gaia/sites/default/files/photos/shannon%20point%20-%20algae%20bloom.jpg

• Common aquatic stressors include temperature, pH, anoxia, pathogens and ‘pollution’

• May be ‘natural’ or anthropogenically induced

• Stressors act synergistically

• Organisms use a variety biological mechanisms to cope

Integrative Approach

R. Whippo

DNA RNA Protein

Physiological Response

Pacific Oyster Crassostrea gigas• Introduced species to Pacific Northwest

• Valuable shellfish industry

• Intertidal and Subtidal

• Model organism as bioindicator

Sessile

Hardy

Experimental Design

Collect Oysters from Big Beef Creek

11/11/10

http://winksite.mobi/xhtml/ms_fo_pg_v.cfm?fid=15611&id=13825&susid=25641&s=1&s2=1

Relatively pristine

Easy access

Cobble

n = 32

S. Roberts

Experimental Design

OYSTERS ACCLIMATED (4 DAYS) n = 32

11/12/10

Collect Oysters from Big Beef Creek

11/11/10

• 12 degrees Celsius

• 4 Oysters in 5L seawater

Experimental Design

OYSTERS ACCLIMATED (4 DAYS) n = 32

No treatment n = 16

Copper treatment

n = 16

11/16/10

11/12/10

Collect Oysters from Big Beef Creek

11/11/10

http://www.writeonnewjersey.com/tag/copper-penny/

• Copper Ubiquitous aquatic contaminant Immunosuppressive effects Bioaccumulates in oyster tissues CuSO4 at 130ppm

Experimental Design

OYSTERS ACCLIMATED (4 DAYS) n = 32

No treatment n = 16

Copper treatment

n = 16

Vibrio treatment

n = 8

Vibrio treatment

n = 8

11/16/10

11/18/10

11/12/10

Collect Oysters from Big Beef Creek

11/11/10

http://www.eol.org/pages/97174

Experimental Design

OYSTERS ACCLIMATED (4 DAYS) n = 32

No treatment n = 16

Copper treatment

n = 16

Vibrio treatment

n = 8

Vibrio treatment

n = 8

11/16/10

11/18/10

11/12/10

Collect Oysters from Big Beef Creek

11/11/10

http://www.eol.org/pages/97174

• Vibrio tubiashii Reemerging bacterial

pathogen of shellfish Linked to massive oyster

mortality events Correlated with increased

sea surface temperature Concentration 103 CFU/ml

Experimental Design

OYSTERS ACCLIMATED (4 DAYS) n = 32

No treatment n = 16

Copper treatment

n = 16

Vibrio treatment

n = 8

Vibrio treatment

n = 8

SAMPLE n = 32

11/16/10

11/18/10

11/12/10

11/19/20

Collect Oysters from Big Beef Creek

11/11/10

Sampling

Gill - for mRNA and Protein - Interface with aquatic

environment. High concentration of hemocytes.

D. Berman

D. Berman

S. Roberts

Mantle -DNA analysis. - Look for changes in methylation pattern

Vibrio

V. VulnificusCDC public health library

-Gram negative rod shapped bacteria

-Disease causing agent in cholera (V. cholerae), paralytic shellfish poisoning (V. parahaemolyticus), and more!

-Foodborne infections from eating undercooked seafood.

Vibrio tubiashii• Pathogen identified by Tubiash et al. in 1965 as

bacillary necrosis.• Haeda et al. later identified the causative bacteria as

V. tubiashii in 1984.

• Reemergence of V. tubiashii in shellfish hatcheries along the west coast of North America.

• Reports of 59% loss in production in 2007.

The Question…

• Increases in V. tubiashii outbreaks typically associated with warmer temperatures.

• Evidence of vibriosis causing depletion of wild oyster stocks.

• Added stress associated with pollutants may impact oyster health, but does it effect the physiology of V. tubiashii?

Physiological effects of Cu(II) on Vt

• Measured mRNA expression of RseA.

• What’s RseA?- Negatively regulates activity of RpoE

RpoE

• Extracellular stress response (thermal and chemical).

• Induced in response to protein misfolding.

• Induced upon Cu(II) exposure in E. coli.

RpoE and Copper

• Influence of 2mM Copper on growth of rpoE mutants in E. coli

Wild-typerpoE deletion

rpoE deletion complemented in trans

Egler, M., Grosse, C., Grass, G., Nies, D. 2005. Role of the extracytoplasmic function protein family sigma factor RpoE in metal resistance of Escherichia coli. J. Bacteriol. 187(7). 2297-2307.

RpoE transcriptional regulation• RseA negative regulated rpoE function

Missiakas, D., Mayer, M.P., Lemaire, M., Georgopoulos C., Raina, S. (1997). Modulation of the Escherichia coli sE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins. Mol. Micro. 24(2), 355-371.

Methods

500ml2um filter

RseA expression in Vt.

-Reduced expression of RseA may be required for infection.-Vt may require increased activity of RpoE during infection, thus decreasing transcriptional activity of sigma E-In ability of Vt to decrease RseA expression in presence of Cu to appropriate “infection” levels may decrease pathogenicity.

Future Research

• No known effects of RpoE/RseA onpathogenicty of bacteria.

• Other genes associated with pathogenicity such as metalloprotease.

• Measure RpoE directly

DNA Methylation

• What is it?

• What can it tell us?

• Methylation in oysters

• Sample processing

• What did I find?

http://commons.wikimedia.org/wiki/File:DNA-fragment-3D-vdW.png

What is DNA Methylation?

• Methyl group attached to DNA at CpG site

• Found in vertebrates and invertebrates

• Influences expression of genes

• Normal part of organismal physiology - Epigenetics

• Heritable!

UNMETHYLATED

METHYLATED

DNA

DNAGATAGATACGATAGATA

GATAGATACGATAGATA

methyl group

Methylation In Oysters

• Relatively new line of inquiry

• Shown to have ‘mosaic methylation’

Housekeeping genes

Immune function

• Hsp70, Hsp25, Cytochrome P450, MEP1-like protein, 14-3-3 protein gamma (Gavery & Roberts 2010)

MOUSE SEASTAR FRUITFLY

Bird & Taggart 1980

UNMETHYLATED

METHYLATED

UNMETHYLATED

METHYLATED

MspIHpaII

Procedure• DNA extractions

From mantle tissue Nine individuals, three each

from control, copper, vibrio

• Enzyme digestions (CCGG)

UNMETHYLATED

METHYLATED

UNMETHYLATED

METHYLATED

MspIHpaII

Procedure• DNA extractions

From mantle tissue Nine individuals, three each

from control, copper, vibrio

• Enzyme digestions (CCGG)

CUT

CUT

CUT

Procedure

• qPCR run

• Hsp70 selected as gene of interest

Regularly methylated

Stress response – protein integrity, inhibits apoptosis

• Two runs produced consistent results

http://1ststagespringhill.org/blog/2008/03/learning-by-doing.html

Analysis

• qPCR data

• Critical threshold set manually

• ‘Close calls’ determined by 2-c(t) limit

Results

• Expect to see: + - +

• Two samples didn’t meet expectation

• Control sample unmethylated

• Copper sample did not amplify properly Extractions Digestions

+ - +

Conclusions

• Oysters can be unmethylated at Hsp70

• No clear pattern associated with stressors

• Need to look deeper into methylation patterns and stress

How often is Hsp70 unmethylated?

Do different stressors have different effects?

HSP 70 Gene Expression

photo: SGC

Why HSP 70?

• They act as molecular chaperonso  Protect proteins from unfoldingo  Mark denatured proteins

• Stress inducible

• Associated with a cellular response when exposed to heavy metals (Boutet et al., 2003 and Boutet et al., 2004).

photo: SGC

RNA Methods

1.RNA was extracted from Big Beef Creek C. gigas gill tissue using TRI Reagent®

2.Quantified using Nanodrop spectrophotometer3.50 μl of each RNA sample was DNased using Turbo

DNA-free™ Kit4.RNA templates were normalized to 2 μg/RT reaction5.All samples were reverse transcribed6.225 uL of nuclease free water was added for a final

dilution 

photo: bioweb.wku.edu

RNA Methods

• Primers were designed using NCBI Primer-Blasto HSP 70o Copper oxidase - Davido Metallothionein IV - Jason

• 18s Ribosomal subunit was used as a reference

• qPCR reaction ingredients were 1x Immunomix master mix, 500 nM forward and reverse primers, and 0.2 mM Syto-13 dyeo 2 μl cDNA = templateo 2 μl sterile water = negative control

HSP 70 Results

• One Vt sample had no C(t) reading and the same well had a very low melting temperature

• A Cu sample had a low melt temperature

F= 1.29p = 0.30

• Vt in the absence of Cu lowers expression, but in the presence almost eliminates

• High p-value could be improved with additional replicates

HSP 70 Conclusion

• Contradictory reports for metal exposureo Demonstrate a decrease in expression (Moraga et

al., 2005)o Demonstrate an increase in expression in Mytilus edulis

(Sanders, 1988)• Metals (especially copper) have an inhibitory effect on the

HSP protein synthesis after 7 days (Moraga et al., 2005)• Using tissue exposed to the environment shows the most

successo gillo digestive tract - food

References

Boutet et al., 2003 I. Boutet, A. Tanguy, S. Rousseau, M. Auffret and D. Moraga, Molecular identification and expression of Heat Shock Cognate 70 (HSC70) and Heat Shock Protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas, Cell Stress Chaperones 8 (2003), p. 7685.

Boutet et al., 2004 I. Boutet, A. Tanguy and D. Moraga, Response of the Pacific oyster Crassostrea gigas to hydrocarbon contamination under experimental conditions, Gene 329 (2004), pp. 147–157.

Moraga, D., Meisterzheim, A., Tanguy-Royer, S., Boutet, I., Tanguy, A., Donval, A.  2005.. Stress response in Cu 2+ and Cd 2+ exposed oysters (Crassostrea gigas): An immunohistochemical approach. Comparative Biochemistry and Physiology, Part C. 141: 151-156.

Sanders, B.M., 1993. Stress proteins in aquatic organisms: an environmental perspective. Crit. Rev. Toxicol. 23, 29–75.

Metallothioneins (MT)

• Thiol (cysteine)-rich• Regulate useful metals in diet (Zn)• Bind heavy metals (Cu, Hg, Cd, etc.) and

transports out of cells• May be affected by bacteria (Paul-Pont et al.

2010)• MT-IV should increase transcription for gene

with an increase in Cu2+

MT structure varies

Metallothionein IV Results

• qPCR amplification• Vibrio tubiashii really seems to inhibit MT-IV

expression• Anova P=0.09 – More likely the result of too

few replicates. Vt means were really different.

Figure 1: Average MT-IV expression

Metallothionein IV

• Decrease in total MT protein with copper exposure (Geret et al. 2002)

• Being used up and cannot be replenished because transcription of the gene is inhibited.

• Oxidative stress uncontrolled• Oxidative stress leads to cancer, death• Cu accumulates, harmful for consumption

Copper Oxidase

What does it do?

• Is a defense mechanism of Oysters and many other organisms against copper which is extremely toxic (Grass 2001)

• Binds copper to itself to keep it from causing harm to the cells by interrupting cell processes

• Oysters can store this copper in their tissues• In high concentration turns their tissue green

Methods

• RNA was extracted and then Dnased to get rid of any carryover DNA

• Reverse Transcription to change RNA into cDNA

• Used qPCR amplification to test for gene expression

Copper Oxidase Expression in OystersCopper Oxidase Expression in Oysters

10

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

Copper Oxidase Expression in Oysters

Control Cu Vt Cu/Vt

Expr

essio

n Le

vel (

fold

/min

)

Detrimental Effects of Vibrio

10

50

100

150

200

250

Copper Oxidase Comparing Negative effects of vibrio

Control Vt Cu/Vt

Gene

Exp

ress

ion

(fold

/min

)

Things to Consider

• This data shows that Vibrio is inhibiting the transcription of copper-oxidase in some way

• This is extremely detrimental and leaves the oysters subject to copper poisoning

• Shows the importance of multiple stressor testing by showing clearly how one stressor can leave an organism defenseless to another

Western Blot

Methods

Gill Tissue Extraction SDS-PAGE

http://ycl.enicp.net/web/ycl/files/MiniVerticalElectrophoresisSystemEV3.jpg

Protein Extraction

Western Blot

Image Analysis

Data & Analysis

 Control Vt Cu Cu/Vt

Control -

0.1752

0.0021

0.0021

Vt0.175

2 -0.003

40.003

3

Cu0.002

10.003

4 -0.391

4

Cu/Vt0.002

10.003

30.391

4 -

Questions?