Download - FISH441 Group Project (Oysters)
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?