Environmental Physiology (FISH441) group presentation of research project

1. ACIDIFICATION STRESS
RESPONSE IN PACIFIC
OYSTER
DAN BASCOM
ASHLEY TONGE
LEN TRAN
MORGAN TUFF

2. BACKGROUND
Adult Pacific oysters (Crassostrea gigas), photo
credit: NOAA/Taylor Seafoods

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

4. Experimental Design
CO2 (Praxair)
~pH = 7.24 ~pH = 5.24

5. Experimental Design
18x 2n
15x 3n

6. Experimental Design
12x 2n 6x 2n
10x 3n 5x 3n
~pH = 7.24 ~pH = 5.24

7. Experimental Design
24 hours
~pH = 5.24
Sample
6x 2n
5x 3n

8. Experimental Design
6x 2n
5x 3n
120 hours
Dry
~pH = 7.24
12x 2n 6x 2n
10x 3n 5x 3n
~pH = 7.24
Wet

9. Experimental Design
6x 2n
5x 3n
24 hours
Sample
6x 2n
5x 3n
~pH = 7.24

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

13. HSP-70 GENE EXPRESSION
• 3n
supposedly
more
resistant
• Lower in
Hsp70 in Dry

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
•UNSUPPRTED
•Hypothesis 2: There is a difference in metallothionein gene expression in
response to ocean acidification between C. gigas ploidy
•UNSUPPRTED

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