3. 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.comhealth
10. METHODS
qPCR-used to quantify expression of a specific DNA sequence using pre-
designed primers
SDS-PAGEWestern Blot-used to separate proteins by size then stain
specific proteins for quantification using antibodies
11. SDSPAGE/Western Blot
• Wells 2 and 3-high
pH/dry diploid
• Well 4-high pH/wet
diploid
• Wells 5 and 6-low pH
diploid
12. 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
14. HSP-70 GENE EXPRESSION
• Results not
significant
• Hypotheses for
low Hsp-70
• Increased
acidification Shut
down transcription
• Not stressed: Hsc70
15. 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.
16. Metallothionein - Results
Average MT Gene Expression 2n vs. 3n Crassostrea
Gigas in Control pH (7.24pH)
0.025
0.02
Relative Gene Expression
0.017457467
0.015
0.008987637
0.01
0.005
0
MT_2n MT_3n
Ploidy
t-Test: Two-Sample Assuming Unequal Variances
Variable 1 Variable 2
Mean 0.017457467 0.008987637
Variance 5.84378E-05 1.4464E-05
Observations 2 3
Hypothesized Mean Difference 0
df 1
t Stat 1.45170504
P(T<=t) one-tail 0.192004587
t Critical one-tail 6.313751514
P(T<=t) two-tail 0.384009175
t Critical two-tail 12.70620473
17. Metallothionein - Results
Average MT Gene Expression 2n vs. 3n
Crassostrea Gigas in Low pH (5.24 pH)
0.00006
0.00005
Relative Gene Expression
0.00004 4.20392E-05
0.00003
2.21536E-05
0.00002
0.00001
0
MT_2n MT_3n
Ploidy
Variable 1 Variable 2
Mean 4.20392E-05 2.21536E-05
Variance 1.04276E-09 1.97468E-10
Observations 3 4
Hypothesized Mean Difference 0
df 3
t Stat 0.998085905
P(T<=t) one-tail 0.195897224
t Critical one-tail 2.353363435
P(T<=t) two-tail 0.391794448
t Critical two-tail 3.182446305
18. Metallothionein - Results
MT Gene Expression in Control pH vs. Low pH for 2n and 3n
Crassostrea Gigas
0.018 0.017457467
0.016
0.014
Relative Gene Expression
0.012
0.01 0.008987637
0.008 Control pH
Low pH
0.006
0.004
0.002
4.20392E-05 2.21536E-05
0
MT_2n MT_3n
Ploidy
MT_3n
MT_2n
Variable 1 Variable 2
Variable 1 Variable 2
Mean 0.008987637 2.21536E-05
Mean 0.017457467 4.20392E-05
Variance 1.4464E-05 1.97468E-10
Variance 5.84378E-05 1.04276E-09
Observations 3 4
Observations 2 3
Hypothesized Mean Difference 0
Hypothesized Mean Difference 0
df 2
df 1
t Stat 4.083085249
t Stat 3.221806557
P(T<=t) one-tail 0.027536942
P(T<=t) one-tail 0.095797511
t Critical one-tail 2.91998558
t Critical one-tail 6.313751514
P(T<=t) two-tail 0.055073883
P(T<=t) two-tail 0.191595021
t Critical two-tail 4.30265273
t Critical two-tail 12.70620473
19. Results – What do they mean?
•Statistically no significant differences – p < 0.05 & t Stat < t Critical
•2n vs. 3n
•Control pH
t Stat 1.45170504
t Critical two-tail 12.70620473
P(T<=t) two-tail 0.384009175
•Low pH
t Stat 0.998085905
t Critical two-tail 3.182446305
P(T<=t) two-tail 0.391794448
•Low pH vs. High pH
MT_2n
MT_3n
t Stat 3.221806557
t Stat 4.083085249
t Critical two-tail 12.70620473 t Critical two-tail 4.30265273
P(T<=t) two-tail 0.191595021 P(T<=t) two-tail 0.055073883
20. 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
21. 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
22. What next?
• Time span
– Longer study
• Time for acclimatization
• Different pHs
– Closer to actual seawater
– Moderate acidic deviation from control
Hinweis der Redaktion
Pacific oyster (Crassostrea gigas)-native to Japan, introduced to the North American west coast and Puget Sound in early 1900’s for aquaculture. Now the most commonly farmed oyster in the world, hatcheries produce diploid and triploid varieties. Prefers habitats in the intertidal zone, during low tide, its shell closes for protection and to prevent water loss.