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
Separation of DNA sizes by electrical field
FISH441: Oysters, acidification and methylation
DNA Methylation of Crassostrea gigas in Response to Ocean AcidificationBy Andy Davison, Selina Cho, and Ying- Ying Lin
Results Triploid and diploid oyster mortality for control and high CO2 treatments
Restriction Enzyme DigestionsPCR All samples show elicit bands (~1140bp) Row 2 sample 4- empty row Row 2 sample 17- same band patterns amp- gigasin 2
Hsp70 Bands appeared for both MspI and HpaII samples Smaller, fainter bands under only MspI samples
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
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
Conclusions What went wrong? ◦ Dot blot ◦ Restriction enzymes Diploids and Triploids are the same Phenotypic Plasticity-stress response