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Environmental Physiology (FISH441) group presentation of research project
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ACIDIFICATION STRESS RESPONSE IN PACIFIC OYSTER
D A N B A S C O M
A S H L E Y T O N G E
L E N T R A N
M O R G A N T U F F
BACKGROUND
Adult Pacific oysters (Crassostrea gigas), photo credit: NOAA/Taylor Seafoods
OBJECTIVES
To determine if acidification would generate a particular stress response in Pacific oysters
Gene Expression
• Len - metallothioneins
• Ashley - gigasin2
• Morgan - HSP70
Protein Expression
• Dan – HSP70
3D model of HSP70, photo credit usnon.com\health
Experimental Design
CO2 (Praxair)
~pH = 7.24 ~pH = 5.24
Experimental Design
18x 2n
15x 3n
Experimental Design
~pH = 7.24 ~pH = 5.24
12x 2n10x 3n
6x 2n5x 3n
Experimental Design
~pH = 5.24
6x 2n5x 3n
24 hours
Sample
Experimental Design
~pH = 7.24
12x 2n10x 3n
120 hours
6x 2n5x 3n
6x 2n5x 3n
~pH = 7.24
Dry
Wet
Experimental Design
24 hours
Sample
6x 2n5x 3n
6x 2n5x 3n
~pH = 7.24
METHODS
qPCR-used to quantify expression of a specific DNA sequence using pre-designed primers
SDS-PAGE\Western Blot-used to separate proteins by size then stain specific proteins for quantification using antibodies
SDSPAGE/Western Blot
• Wells 2 and 3-high pH/dry diploid
• Well 4-high pH/wet diploid
• Wells 5 and 6-low pH diploid
Results
• Ha: low pH treatments(wells 5 and 6) will have more HSP70 and thus appear brighter after staining– Reject
• Ho: high and low pH treatments will show equal levels of HSP70– Insufficient evidence to confirm
• HSPs 60-90 kilodaltons• No binding to HSP70 • Banding at <10 kilodaltons could be to homologous protein
fragments• Ubiquitin 8 kilodaltons, found in all cells
HSP-70 GENE EXPRESSION
• 3n supposedly more resistant
• Lower in Hsp70 in Dry
• Results not significant
• Hypotheses for low Hsp-70• Increased
acidification Shut down transcription• Not stressed:
Hsc70
HSP-70 GENE EXPRESSION
EFFECTS OF ACIDITY ON CALCIFICATION
Internal acidification during low tide imitated by emersion for final 24-hour period.
Limited or stopped Calcification and shell formation in order to maintain homeostasis in the stress of acidity.
Metallothionein - Results
MT_2n MT_3n0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0.0174574674447724
0.00898763676323815
Average MT Gene Expression 2n vs. 3n Crassostrea Gigas in Control pH (7.24pH)
Ploidy
Rela
tive
Gene
Exp
ress
ion
t-Test: Two-Sample Assuming Unequal Variances
Variable 1 Variable 2Mean 0.017457467 0.008987637Variance 5.84378E-05 1.4464E-05Observations 2 3Hypothesized Mean Difference 0df 1t Stat 1.45170504P(T<=t) one-tail 0.192004587t Critical one-tail 6.313751514P(T<=t) two-tail 0.384009175t Critical two-tail 12.70620473
Metallothionein - Results
MT_2n MT_3n0
0.000005
0.00001
0.000015
0.00002
0.000025
0.00003
0.000035
0.00004
0.0000454.20391606781662E-
05
2.21536177699932E-05
Average MT Gene Expression 2n vs. 3n Crassostrea Gigas in Low pH (5.24 pH)
Ploidy
Rela
tive
Gene
Exp
ress
ion
Variable 1 Variable 2Mean 4.20392E-05 2.21536E-05Variance 1.04276E-09 1.97468E-10Observations 3 4Hypothesized Mean Difference 0df 3t Stat 0.998085905P(T<=t) one-tail 0.195897224t Critical one-tail 2.353363435P(T<=t) two-tail 0.391794448t Critical two-tail 3.182446305
Metallothionein - Results
MT_2n Variable 1 Variable 2
Mean 0.017457467 4.20392E-05Variance 5.84378E-05 1.04276E-09Observations 2 3Hypothesized Mean Difference 0df 1t Stat 3.221806557P(T<=t) one-tail 0.095797511t Critical one-tail 6.313751514P(T<=t) two-tail 0.191595021t Critical two-tail 12.70620473
MT_3n Variable 1 Variable 2
Mean 0.008987637 2.21536E-05Variance 1.4464E-05 1.97468E-10Observations 3 4Hypothesized Mean Difference 0df 2t Stat 4.083085249P(T<=t) one-tail 0.027536942t Critical one-tail 2.91998558P(T<=t) two-tail 0.055073883t Critical two-tail 4.30265273
MT_2n MT_3n0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018 0.0174574674447724
0.00898763676323815
4.20391606781662E-05
2.21536177699932E-05
MT Gene Expression in Control pH vs. Low pH for 2n and 3n Crassostrea Gigas
Control pHLow pH
Ploidy
Rela
tive
Gene
Exp
ress
ion
Results – What do they mean?•Statistically no significant differences – p < 0.05 & t Stat < t Critical• 2n vs. 3n
• Control pH
• Low pH
• Low pH vs. High pH
t Stat 1.45170504
t Critical two-tail 12.70620473
P(T<=t) two-tail 0.384009175
t Stat 0.998085905
t Critical two-tail 3.182446305
P(T<=t) two-tail 0.391794448
MT_2n
t Stat 3.221806557
t Critical two-tail 12.70620473
P(T<=t) two-tail 0.191595021
MT_3n
t Stat 4.083085249
t Critical two-tail 4.30265273
P(T<=t) two-tail 0.055073883
Results – What do they mean?•Hypothesis 1: Increasing stressors like ocean acidification upregulates metallothionein gene expression
•UNSUPPRTED
•Hypothesis 2: There is a difference in metallothionein gene expression in response to ocean acidification between C. gigas ploidy
•UNSUPPRTED
Whyyyyy??? =[
• At pH 5.24, oysters died within 24 hours.– Possible RNA degradation• Misinterpreting gene expression results
• Metallothionein gene was expressed!– Sufficient level in terms of acidity• Minimal but sufficient for acidic conditions
What next?
• Time span– Longer study• Time for acclimatization
• Different pHs– Closer to actual seawater– Moderate acidic deviation from control
