ACIDIFICATION STRESS         RESPONSE IN PACIFIC      OYSTERDAN BASCOM ASHLEY TONGE   LEN TRAN     MORGAN TUFF
BACKGROUND             Adult Pacific oysters (Crassostrea gigas), photo             credit: NOAA/Taylor Seafoods
OBJECTIVESTo determine if acidification would generate a particular stress   response in Pacific oystersGene Expression•  ...
Experimental Design                             CO2 (Praxair)~pH = 7.24      ~pH = 5.24
Experimental Design18x 2n                  15x 3n
Experimental Design             12x 2n                6x 2n             10x 3n                5x 3n~pH = 7.24            ~...
Experimental Design                 24 hours~pH = 5.24                              Sample  6x 2n  5x 3n
Experimental Design                                               6x 2n                                               5x 3...
Experimental Design         6x 2n         5x 3n                     24 hours                                Sample        ...
METHODSqPCR-used to quantify expression of a specific DNA sequence using pre-   designed primersSDS-PAGEWestern Blot-used ...
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...
HSP-70 GENE EXPRESSION                         • 3n                           supposedly                           more   ...
HSP-70 GENE EXPRESSION                         • Results not                           significant                        ...
EFFECTS OF ACIDITY ON CALCIFICATION Internal acidification during low tide imitated by emersion for final 24-hour  period...
Metallothionein - Results                                   Average MT Gene Expression 2n vs. 3n Crassostrea              ...
Metallothionein - Results                                     Average MT Gene Expression 2n vs. 3n                        ...
Metallothionein - Results                                                   MT Gene Expression in Control pH vs. Low pH fo...
Results – What do they mean?•Statistically no significant differences – p < 0.05 & t Stat < t Critical     •2n vs. 3n     ...
Results – What do they mean?•Hypothesis 1: Increasing stressors like ocean acidification upregulatesmetallothionein gene e...
Whyyyyy??? =[• At pH 5.24, oysters died within 24 hours.  – Possible RNA degradation     • Misinterpreting gene expression...
What next?• Time span  – Longer study     • Time for acclimatization• Different pHs  – Closer to actual seawater  – Modera...
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FISH441: Oyster acidification: gene and protein expression

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FISH441: Oyster acidification: gene and protein expression

  1. 1. ACIDIFICATION STRESS RESPONSE IN PACIFIC OYSTERDAN BASCOM ASHLEY TONGE LEN TRAN MORGAN TUFF
  2. 2. BACKGROUND Adult Pacific oysters (Crassostrea gigas), photo credit: NOAA/Taylor Seafoods
  3. 3. OBJECTIVESTo determine if acidification would generate a particular stress response in Pacific oystersGene Expression• Len - metallothioneins• Ashley - gigasin2• Morgan - HSP70Protein Expression• Dan – HSP70 3D model of HSP70, photo credit usnon.comhealth
  4. 4. Experimental Design CO2 (Praxair)~pH = 7.24 ~pH = 5.24
  5. 5. Experimental Design18x 2n 15x 3n
  6. 6. Experimental Design 12x 2n 6x 2n 10x 3n 5x 3n~pH = 7.24 ~pH = 5.24
  7. 7. Experimental Design 24 hours~pH = 5.24 Sample 6x 2n 5x 3n
  8. 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. 9. Experimental Design 6x 2n 5x 3n 24 hours Sample 6x 2n 5x 3n~pH = 7.24
  10. 10. METHODSqPCR-used to quantify expression of a specific DNA sequence using pre- designed primersSDS-PAGEWestern Blot-used to separate proteins by size then stain specific proteins for quantification using antibodies
  11. 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. 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. 13. HSP-70 GENE EXPRESSION • 3n supposedly more resistant • Lower in Hsp70 in Dry
  14. 14. HSP-70 GENE EXPRESSION • Results not significant • Hypotheses for low Hsp-70 • Increased acidification Shut down transcription • Not stressed: Hsc70
  15. 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. 16. Metallothionein - Results Average MT Gene Expression 2n vs. 3n Crassostrea Gigas in Control pH (7.24pH) 0.025 0.02Relative 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. 17. Metallothionein - Results Average MT Gene Expression 2n vs. 3n Crassostrea Gigas in Low pH (5.24 pH) 0.00006 0.00005Relative 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. 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_3nMT_2n Variable 1 Variable 2 Variable 1 Variable 2 Mean 0.008987637 2.21536E-05Mean 0.017457467 4.20392E-05 Variance 1.4464E-05 1.97468E-10Variance 5.84378E-05 1.04276E-09 Observations 3 4Observations 2 3 Hypothesized Mean Difference 0Hypothesized Mean Difference 0 df 2df 1 t Stat 4.083085249t Stat 3.221806557 P(T<=t) one-tail 0.027536942P(T<=t) one-tail 0.095797511 t Critical one-tail 2.91998558t Critical one-tail 6.313751514 P(T<=t) two-tail 0.055073883P(T<=t) two-tail 0.191595021 t Critical two-tail 4.30265273t Critical two-tail 12.70620473
  19. 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. 20. Results – What do they mean?•Hypothesis 1: Increasing stressors like ocean acidification upregulatesmetallothionein gene expression •UNSUPPRTED•Hypothesis 2: There is a difference in metallothionein gene expression inresponse to ocean acidification between C. gigas ploidy •UNSUPPRTED
  21. 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. 22. What next?• Time span – Longer study • Time for acclimatization• Different pHs – Closer to actual seawater – Moderate acidic deviation from control

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