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.
Elemental analysis
Extrapallial fluid, seawater, and shell samples 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 leached in the same manner as those for shell samples. The samples were acidified with ultra-pure nitric acid (Fisher TraceMetal Grade Nitric Acid UN2031). Shell samples were also acidified with ultra-pure nitric acid for analysis.
Extrapallial fluid, seawater, and shell samples were analyzed for a suite of 57 elements (including Ca) by ActLabs, Ontario, Canada. In addition to the common suite of elements, liquid samples were analyzed for Si and shell samples were analyzed for S, P, Au, B, and Re. Liquid samples were analyzed using the ActLabs ICPMS method. Shell samples were analyzed using the ActLabs ICPMS Ultratrace 4 method:
https://actlabs.com/geochemistry/exploration-geochemistry/4-acid-near-total-digestion/
This dataset represents molar ratio data that were processed using R (v. 4.0.3; R Core Team 2020) using the graphical interface RStudio (v. 1.0.1073) and then converted to molar ratios to calcium. For each sample type (EPF, seawater, shell), values were identified as outliers if the E/Ca ratio deviated by more than 103 from the mean E/Ca ratio for that element. This approach to identifying outliers removes those caused by sampling or equipment error but is more conservative than Tukey's method to identify and remove outliers.