DNA Methylation of Crassostrea gigas in Response to Ocean

AcidificationBy Andy Davison, Selina Cho, and

Ying-Ying Lin

Why do we care about Pacific oysters?

Why do we care about ocean acidification?

Why do we care about epigenetics?

ObjectivesQuantify the physiological

response of diploid and triploid oysters exposed to various acidified conditions

Implications of varying levels of DNA methylation

Methods

Restriction Enzyme Digestion

Methylated Cytosine Dot Blot

Gel Electrophoresis

Results

Triploid and diploid oyster mortality for control and high CO2

treatments

Restriction Enzyme Digestions PCR

amp- gigasin 2

All samples show elicit bands (~1140bp)

Row 2 sample 4- empty row

Row 2 sample 17- same band patterns

Hsp70

Smaller, fainter bands under only MspI samples

Bands appeared for both MspI and HpaII samples

Quantitative PCR control samples showed expression levels with a median of 1.3e-07hpaII digested samples- median of 2.5e-08

mspI digested samples- median 1.5e-08

Methylated Cytosine Dot Blot

Samples with 400ng of DNA showed methylation more frequently than 200ng samples

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

All samples of triploid, low pH (400ng) showed methylation

ConclusionsWhat went wrong?

◦Dot blot◦Restriction enzymes

Diploids and Triploids are the same

Phenotypic Plasticity-stress response

Thank you