In this study, we examined the phenotypic and molecular responses in the extrapallial fluid in the adult eastern oyster (Crassostrea virginica) exposed to experimental ocean acidification (OA) over 80 days. The collection and culturing of C. virginica specimens are detailed in Downey-Wall, A.M., L.P. Cameron, B.M. Ford, E.M. McNally, Y.R. Venkataraman, S.B. Roberts, J.B. Ries, and K.E. Lotterhos. 2020. Ocean acidification induces subtle shifts in gene expression and DNA methylation in the mantle tissue of the Eastern oyster (Crassostrea virginica). Frontiers in Marine Science doi: 10.3389/fmars.2020.566419.
Extrapallial fluid (EPF) was extracted as described in Downey-Wall et al. (2020). Extrapallial fluid was extracted by inserting a sterile 5 milliliter (mL) syringe with a flexible 18-gauge polypropylene tip into the EPF cavity through the luer-lock port. The EPF was stored in 2 mL polypropylene microcentrifuge tubes with screw caps (Fisherbrand Catalog No. 02-682-558) and refrigerated at 6 degrees celsius until further analysis.
Since all tanks received water from the same header source, seawater from a subset of six tanks (2 treatment-1) was sampled for elemental analysis. Seawater samples were collected in 50-milliliter (mL) polypropylene centrifuge tubes outside of an oyster sampling timepoint near the halfway point of the experimental exposure (day 63).
Elemental analysis
Extrapallial fluid and seawater were analyzed for trace and minor elements by inductively coupled plasma mass spectrometry (ICPMS). Liquid samples (i.e., EPF, seawater) were diluted to less than 0.05 percent total dissolved solid content with ultra-pure deionized water in 15 mL polypropylene centrifuge tubes and acidified with ultra-pure nitric acid (Fisher TraceMetal Grade Nitric Acid UN2031).
Extrapallial fluid and seawater was analyzed for a suite of 57 elements (including Ca) by ActLabs, Ontario, Canada. Liquid samples were analyzed using the ActLabs ICPMS method.