5. Objectives
Quantify the physiological response of
diploid and triploid oysters exposed to
various acidified conditions
Implications of varying levels of DNA
methylation
10. Results
Triploid and diploid oyster mortality for
control and high CO2 treatments
11. Restriction Enzyme Digestions
PCR
All samples show elicit
bands (~1140bp)
Row 2 sample 4- empty
row
Row 2 sample 17- same
band patterns
amp- gigasin 2
12. Hsp70
Bands
appeared for
both MspI and
HpaII samples
Smaller, fainter
bands under
only MspI
samples
13. Quantitative PCR control samples
showed
expression levels
with a median of
1.3e-07
hpaII digested
samples- median of
2.5e-08
mspI digested
samples- median
1.5e-08
14. Methylated Cytosine Dot Blot
Methylation most common in both triploid
and diploid oysters exposed to low pH
conditions
14 of the 22 samples from the low
pH treatment showed some level
of methylation
Four of the 22 samples from the
dry treatment showed signs of
methylation
two of the 18 samples from the
high pH, wet treatment
demonstrated methylation
Samples with 400ng of DNA showed
methylation more frequently than
200ng samples
All samples of triploid, low pH (400ng)
showed methylation
15. Conclusions
What went wrong?
◦ Dot blot
◦ Restriction enzymes
Diploids and Triploids are the same
Phenotypic Plasticity-stress response
global production of this species had expanded to 4.38 million tonnes, more than any other species of fish, molluscs or crustacea.
Pacific science association
The objective of this study was to quantify the physiological response of diploid and triploid oysters exposed to various acidified conditions by measuring stress related indicators, specifically DNA methylation, with various molecular techniques. The molecular techniques used were: methylated cytosine dot blots and restriction enzyme digestions amplified by polymerase chain reaction (PCR) and viewed on agarose gel after electrophoresis.
Restriction enzymes, methylated cytosine dot blot, PCR, qPCR gel electrophoresis
Bacterial enzymes that cut at specific recognition sequences. Methylation can prevent cutting (HpaII cannot cut methylated).
Confirming presence of methylated cytosine by antibody (probes) binding
