Ocean acidification induces subtle shifts in gene expression and DNA methylation in mantle tissue of the Eastern oyster (Crassostrea virginica)

Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in adult eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments (‘control’, 580 μatm; ‘moderate OA’, 1000 μatm; ‘high OA’, 2800 μatm) and sampled at 6 time points (24 hours - 80 days). We found that high OA initially induced an increase in the pH of the extrapallial fluid (pHEPF) relative to the external seawater that peaked at day 9, but then diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene express..., Collection and Experimental Conditions
Adult C. virginica were collected from three intertidal sites within Plum Island Sound, Massachusetts, USA (Site 1, 42.751636, -70.837023; Site 2, 42.725186, -70.855022; Site 3, 42.681764, -70.813498) in late April 2017. These sites are all within 8 km of each other and the oysters from these sites are not distinct genetic populations. 
In the lab, oysters were cleaned of epibionts and shell ports were installed over eight days while being maintained in 50-L flow-through tanks. To measure the carbonate chemistry of the EPF, a 2 mm hole was drilled into the right valve approximately 2 cm from the hinge to expose the EPF cavity, without damaging the underlying mantle tissue. The drilled hole was gently rinsed with filtered seawater and patted dry. The barbed end of a nylon luer-lock coupling (McMaster-Carr 51525K123) was trimmed to the length of the shell thickness, inserted into the hole and sealed in place using marine-safe cyanoacrylate (Starbond ..., General Notes
Note 1: For a complete step-by-step description of the RNA and DNA Methylation QC, mapping, and quantification pipelines see the manuscript github repository - `https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse`.
Note 2: Using scripts will require modifying data pathways. Statistical analyses use relative paths that correspond to the manuscript github repository - `https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse`.
Note 3: Raw sequence files corresponding to this study are located on NCBI with the BioProject ID - PRJNA594029. Reference files and annotations based on NCBI C. virginica genome files (002022765.2_C_virginica-3.0).
Note 4: Additional intermediate products can be found on the github repository - `https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse`.
Files
Seawater Chemistry

`/AE17_WaterChemistry _carbChem.csv` : Biweekly water samples (Total Carbon and Alkalinity) and carbonate chemistry calculati...