Dataset: Oyster Mussel Carbonate Responses
Deployment: Waldbusser_HMSC

Methods (see SI in Waldbusser et al. 2015, Nature Climate Change for extended methods):
Water collection and stripping dissolved inorganic carbon:
For each experiment, 1 um filtered seawater was collected from Yaquina Bay, Oregon. The alkalinity was reduced by addition of trace metal grade HCl in near alkalinity equivalence, followed by bubbling with ambient air for 48 hours to strip (DIC) as CO2. The acidified, stripped seawater was then 0.22 um filtered, pasteurized, and stored at 2-5 degrees C. Prior to treatment manipulation, the seawater was bubbled with 0.2 um-filtered outside air until atmospheric conditions were achieved, then carbonate DIC and alkalinity values were determined for manipulations.

Experimental manipulation:
A 4x4 factorial experimental design was developed to target 16 total treatment combinations of PCO2 and War (saturation state with respect to aragonite) (Supplementary Table 1, Figure 1), with triplicate 500 ml biological oxygen demand (BOD) bottles per treatment. Two separate experiments were conducted with each species. DIC and alkalinity concentrations were calculated for each of the 16 target treatment combinations (PCO2 and War). Experimental treatments were created by gravimetric addition of mineral acids and bases to the decarbonated seawater in gas-impermeable bags customized with luer lock fittings. Aliquots of a concentrated, ambient-PCO2, solution of Na2CO3 and and NaHCO3 were added to adjust DIC to target treatment level followed by 0.1N HCl to adjust alkalinity. Immediately following chemical manipulation, the bags with treatment water were stored without head-space at 2-5 degrees C for up to several weeks before spawning broodstock. Antibiotics were added to BOD bottles (2 ppm chloramphenicol and 10 ppm ampicillin), which we found to have no negative effects on larvae or carbonate chemistry in prior trials. Controls were included to evaluate experimental manipulations and incubation conditions by hatching eggs in open culture containers, as well as by using stored seawater collected prior to decarbonation and not subjected to chemical manipulations described in this study. 

Carbonate chemistry measurements:
Carbonate chemistry samples were collected from the treatment water bags just prior to stocking larvae in BOD bottles, and also from each BOD bottle at the end of the incubation period. Carbonate chemistry samples were collected in 350 ml amber glass bottles with polyurethane-lined crimp-sealed metal caps and preserved by addition of 30 ml of saturated HgCl2. Analyses of PCO2 and DIC were carried out following the procedure of Bandstra et al. modified for discrete samples as in Hales et al. Gas and liquid standards that bracketed the experimental range (Supplementary Table 1) were employed to ensure accuracy.

Larval Rearing:
Broodstock for mussel (Mytilus galloprovincialis) and oyster (Crassostrea gigas) experiments were obtained from Carlsbad Aquafarm, Carlsbad, CA, or from selected stocks of the Molluscan Broodstock Program (MBP), Yaquina Bay, Oregon, respectively. Broodstock spawning was stimulated by rapid increase of 10 degrees C in ambient seawater temperature. Gametes were collected from at least two male and two female parents, and the eggs fertilized in ambient seawater. Developing embryos were added at a density of 10 larvae ml-1 to triplicate BOD bottles per treatment after visual verification of successful fertilization. Sealed BOD bottles were oriented on their side and incubated for 48 hours at culture temperature (18 degrees C for mussels and 22 degrees C and 25 degrees C for oyster trials 1 and 2, respectively). Larvae from each BOD bottle were concentrated after a filtered chemistry sample was collected, sampled in triplicate, and preserved in 10% formalin buffered to ~8.1-8.2. 

Larval shell development and size:
Larvae were examined microscopically to determine the proportion of normally and abnormally developed D-hinge (prodissoconch I) larvae as well as larval shell lengths. Normally developed larvae were characterized by a straight hinge, smooth curvature along the edge of the valve, and appearance of tissue within the translucent shells. Digital images were used to determined shell length (longest axis perpendicular to the hinge) of normally developed larvae only. Images were analyzed using ImageJ (V1.42).

Data analyses:
Proportion normal data were scaled to the un-manipulated, seawater control for each experiment by dividing treatment values by control values. We used a two-way analysis of variance (ANOVA), with PCO2 and War as the primary factors with experiment as a blocking factor. Proportion normal data were square-root arcsine transformed. Assumptions of normality and homoscedascity were checked and any violations were managed as noted. Initial data analyses found unequal variance across treatment groups in the transformed proportion normal data, and mean values per treatment were used to improve heteroscedascity as well as blocking by experiment. To evaluate pH effects on shell development we ran a series of regression analyses of transformed proportion normal regressed on pH, within each War treatment and experiment. We then used a Bonferroni correction for multiple tests of significance to reduce Type 1 error. Analyses were conducted with the SAS software suite (v9.3). Non-linear, least-squares regression in Sigma-Plot (v12.5) was used to fit functional responses of development (logistic) and shell length (power).

References:
Bandstra L, Hales B & Takahashi T. 2006. High-frequency measurements of total CO2: Method development and first oceanographic observations. Mar Chem 100(1-2): 24-38.

Hales B, Takahashi T & Bandstra L. 2005. Atmospheric CO2 uptake by a coastal upwelling system. Global Biogeochem Cycles 19(1).

Langdon CJ, Evans F, Jacobson D and Blouin, M. 2003. Yields of cultured Pacific oysters Crassostrea gigas Thunberg improved after one generation of selection. Aquaculture, 220:227-244.

American Society for Testing and Materials. 2004. Standard guide for conducting static   acute toxicity tests starting with embryos of four species of saltwater bivalve molluscs. E724-98. 22 pp.