Methods for May Cohorts (from Waldbusser et al.)
The investigators measured the isotopic composition of carbon in larval shell, larval tissue, food, and holding tank water during growth and development of a commercial larval Crassostrea gigas cohort in May 2011. These measurements were coupled to bulk measures of larval biochemical composition to estimate energetic status and demands for the initial shell development.

Setting and Experimental Design
Crassostrea gigas larvae were raised according to commercial spawning protocols at Whiskey Creek Shellfish Hatchery in Netarts Bay, Oregon, as previously described in detail in Barton et al., 2012. Briefly, successfully fertilized Willapa Bay oyster larvae from several conditioned broodstock oysters were added to a 22 cubic meter tank an hour after fertilization at an estimated density of approximately 5 x 10⁷ individuals per cubic meter. Water was changed in culture tanks every 2-3 days during cohort development. The pH of fertilization water was 8.15 (measured with a YSI 30 on the NBS scale), and developing embryos were reared under a P_CO2 of 396 uatm and aragonite saturation state of 2.38. Samples for P_CO2 and TCO₂ of culture tanks were collected at each tank filling and analyzed following Bandstra et al. (2006) with an uncertainty of less than 5% and 0.2% for P_CO2 and TCO2, respectively. The rest of the carbonate system was calculated using standard dissociation constants as previously described Barton et al., 2012. Conditions in culture tanks at filling had an average P_CO2 (± 1 S.D.) of 364 (± 40) uatm aragonite saturation state with of 2.58 (± 0.33) at the culture temperature of 25 degrees C, and a salinity of 26.5 (± 2.29). Larvae were fed a diet of Isocrysis galbani cultured in a continuous culture system during the first week following fertilization and this was switched to a mixed algae diet consisting primarily of Chaetocerous gracilis, Thalassiorsira spp., and Isochrysis galbana cultured in batches. Estimated cell densities were 5-7 x 10¹⁰ cells per cubic meter.

During each tank change, all larvae were captured on an appropriately sized sieve and a primary sample of the entire cohort was randomly collected with a clean stainless steel spatula. The mass of each primary sample varied from ~0.5 to 1.0 g representing an estimated 2.8 x 10⁶ to 5.6 x 10⁶ two-day old larvae or 2.3 x 10⁵ to 4.6 x 10⁵ 19-day old larvae. All 400 million larvae in the cohort were reared collectively in one tank, so replicate primary samples would have been pseudo-replicates of larvae from the same tank.

The larval samples were quickly rinsed with deionized water, immediately frozen, and freeze dried within 2 weeks with a Labconco FreeZone 6 Freeze Drier. Sub-samples from each primary sample were then used for subsequent analyses. Approximately 100’s to 10’s of larvae for stable isotope analyses of shell carbonates from Day 2 and 19 samples were needed, and lipid extractions required roughly 10⁴ to 10⁵ larvae for Day 2 and 19, respectively. Analyses of stable isotopes and elemental analyses of tissue required approximately 10³ to 10⁴ larvae. Variable volumes (200-800 ml) of algal cultures were vacuum filtered onto pre-combusted onto glass fiber filters (GF/F 47mm, Whatman) to achieve a similar analytical mass for all analyses.

δ¹³C Measurements
All stable carbon isotope samples were analyzed in Oregon State University’s CEOAS Stable Isotope Laboratory and details for standard analyses may be found at http://stable-isotope.coas.oregonstate.edu/. All runs were calibrated and checked with NIST certified (NBS 19, NBS 20 UQ6) and in-house calibrated standards, with precision and accuracy noted below.

Water samples were analyzed by continuous mass flow spectrometry on a GasBench-Delta V system. The run had a precision of 0.02‰ and accuracy better than 0.06‰. Larval shell carbonate δ¹³C was measured by reacting the freeze-dried larvae with a viscous ~105% H₃PO₄solution in a Finnigan MAT Kiel III at 70 degrees C for 5 minutes after which the isotopic composition of the evolved and purified CO₂ was measured on a dual inlet mass spectrometer (MAT 252 IRMS). Each run had a precision and accuracy of 0.02‰ and 0.01 ‰, respectively. Repeatability of larval shell samples was within 0.02‰. To obtain measures of tissue δ¹³C composition of tissues, freeze-dried larvae acid fumed with concentrated HCl to eliminate the shell carbonate signal. The remaining sample material was then analyzed by flash combustion (>1000 degree C) using a Carlo Erba NA 1500 elemental analyzer interfaced by continuous-flow mass spectrometer (Delta-Plus XL). Repeatability of organic larval samples was 0.21‰. Algae samples, collected as noted above, were analyzed in the same way as larval tissue however without the acid fuming. Measurements of organic δ¹³C composition were made with a precision and accuracy of 0.17‰ and 0.12‰, respectively.

Larval Biochemical and Size Analyses
Fractions of organic tissue and shell (% of dry weight) were determined by first measuring %C on bulk freeze-dried larval samples and then decalcified samples (via HCl fuming) on a Carlo Erba NA1500 elemental analyzer. The difference between whole and decalcified larvae and the molar proportion of C in calcium carbonate were used to estimate the % calcium carbonate by weight, with the remainder being organic matter. These values for % organic matter were correlated to (r² = 0.89) loss on ignition (LOI) measurements, with LOI consistently over estimating % organic matter. Total lipid % was measured by extracting freeze-dried larvae with a methylene chloride: methanol solution (3:1) using a Dionex ASE 200 Accelerated Solvent Extractor and weighing an aliquot of the isolated total extractable lipid (TEL) fraction dissolved in CS₂ gravimetrically. Larval size was evaluated on 100-150 larvae per sample, taken as a separate sub-sample during tank water changes and evaluated on digital micrographs using standard image analysis techniques (ImageJ 1.44p). A standard mass at length model for C. gigas larvae (Bocheneck et al., 2001) was used to calculate a total individual mass. Measured proportional composition data were then applied to the mass at length estimate to compute per larva weights of shell, bulk organic, and lipid content.

Methods for August Cohorts (from Brunner et al.)
The investigators sampled multiple commercial cohorts of Pacific oyster larvae, Crassostrea gigas, at the Whiskey Creek Shellfish Hatchery (WCH), Netarts, Oregon, U.S.A. where carbonate chemistry within the hatchery is set by the ambient Netarts Bay conditions, but food availability (ad libitum) and temperature (25 degrees C) are controlled to optimal levels. The large (~400 million larvae) cohorts provided sufficient larvae to measure bulk biochemistry parameters such as organic content and composition while the sequential tank incubations allowed us to fully constrain the possible isotopic sources for incorporation into shell and tissue at discrete intervals during larval life (e.g., 0-2 days, 2-5 days).

Setting and Experimental Design
The investigators followed three cohorts of approximately 400 million Crassostrea gigas larvae from fertilization through metamorphosis: one cohort in May 2011, and two in August 2011. WCH’s spawning protocols are described in detail by Barton et al. (2012), and by Waldbusser et al. (2013). Briefly, larvae are maintained in static 22 cubic meter tanks, which have their water changed every 2-3 days, and are fed daily. The water for each tank change is pumped from Netarts Bay — a marine-dominated estuary on the northern Oregon coast (45.403N, 123.944W) which is flushed nearly every tidal cycle with water from the adjacent North Pacific Ocean (Whiting & McIntire 1985). With roughly two-thirds of the 9.41 square-kilometeraerial extent covered in Zostrera spp. beds, and the remaining tidal flats supporting microphytobenthos production, there is a diurnal PCO₂ cycle that reflects the daily cycle of photosynthesis and respiration in Spring and Summer such that the highest PCO₂ values are seen in the morning after a night of respiration and the lowest PCO₂ values are seen in the afternoon (Waldbusser & Salisbury 2014). Thus, the saturation state at the hatchery intake pipe is variable on hourly, weekly and seasonal timescales (Barton et al. 2012, Waldbusser & Salisbury 2014). To exaggerate the naturally-occurring PCO₂ conditions of the upwelling season in August, water was pumped in the morning. May 2011 was a period of high freshwater input into Netarts Bay and thus the water started with a lower salinity (~26) and thus alkalinity than usual. For the May cohort, water for each tank-fill was pumped in the afternoon, when PCO₂ was relatively low. All water is heated to 25 degree C before being pumped into the tanks.

Spawn and Larval Culture
On 18 May 2011 and again on 14 August 2011 at least 3 Willapa Bay females were strip-spawned and fertilized with sperm from at least one Willapa Bay male. The fertilized eggs in August were split into two different tanks ~ 1 hr after fertilization (a "buffered" and control, each starting with approximately 400 million larvae), and followed separately for the remainder of their larval period. The WCH buffer system treated incoming water with industrial grade soda ash (Na₂CO₃) to an aragonite saturation state (Ω_ar) equal to 4 during this experiment.

All larvae were fed a rotating mixture of algal cultures grown at WCH including Isochrysis sp., Chaetoceros gracilis, and Chaetoceros calcitrans. In the first 5-10 days the larvae were fed Isochrysis sp from a continuous culture bag system and these algae have a particularly low δ¹³C signal [around -45‰] which is due to the bubbling of culture water with compressed industrial grade CO₂. At each feeding, between 200 and 800 mL of algal feed cultures were filtered onto pre-combusted 47mm GF/F glass fiber filters (Whatman) and the algae retained on the filter dried at <45 degrees C for storage until analysis. The sample volume was determined to account for different densities of algal culture and achieve optimal amounts of organic material on each filter for analysis.

At each tank water change, the previous tank’s water was drained onto an appropriately sized sieve to capture all the larvae, and the larvae were then transferred into a newly-filled tank. A 0.5 – 2 g dry weight sub-sample (representing an estimated 3-10 x 10⁶ two-day old larvae or 2-9 x 10⁵ 19-day old larvae) of the cohort was collected from the sieve, briefly rinsed with DI water and immediately frozen. The samples were then freeze-dried within three weeks of collection (Labconco FreeZone 6 Freeze Drier). As all the larvae from each cohort were living in the same tank, taking multiple primary samples would have been pseudo-replication, and the investigators therefore assume that the sub-samples accurately represent the entire larval cohort. In May, after 5 days, the fastest growing larvae were selected by passing the cohort through an 80 um sieve, which caught about half of the larvae. This procedure for poorly performing cohorts commonly employed at WCH. The August cohorts were not sorted via this size-fraction screening as their general growth and fitness was better.

Isotopic and Compositional Measurements of Larvae and Algae
All stable carbon isotope samples were analyzed in Oregon State University’s College of Earth, Ocean, and Atmospheric Sciences Stable Isotope Laboratory. Detailed descriptions of these standard protocols can be found in Waldbusser et al. (2013) and at http://stable-isotope.coas.oregonstate.edu/. All runs were calibrated and checked with NIST certified and in-house calibrated standards. Precision and accuracy are noted for each analysis.

To measure larval tissue δ¹³C and δ¹⁵N, 6-10 mg freeze-dried larvae in Ag boats were acidified in a concentrated HCl atmosphere following the method of Hedges & Stern (1984). The samples were dried overnight, encapsulated in Sn boats, and loaded into a Costech Zero Blank Autosampler. Samples were flash combusted at >1020 degrees C using a Carlo Erba NA1500 Elemental Analyzer (Verardo et al. 1990) and the resulting gas was analyzed by continuous-flow mass spectrometry using a DeltaPlus XL isotope ratio mass spectrometer. Hole punches of filter (diameter ~7mm) with dried algae were analyzed via the same method as the larval tissue, without the acidification step. Repeatability of samples was 0.21‰ for δ¹³C, and 0.27‰ for δ¹⁵N.

In order to measure larval shell carbonate δ¹³C, the investigators reacted 19-50 freeze-dried larvae with concentrated phosphoric acid at 70 degrees C for 5 minutes in a Finnigan Keil III device. The resulting CO₂ was isolated from other reaction products and measured on a MAT 252 mass spectrometer by dual inlet mass spectrometry. Accuracy was better than ±0.05‰, and repeatability was ±0.02 ‰.

Larval samples were also analyzed for organic and inorganic elemental composition before and after acidification as described above on a Carlo Erba NA-1500 elemental analyzer calibrated with at least 6 acetanilide standards following the methods of Verardo et al. (1990). Ash-free dry weight (AFDW) was determined by weight comparison before and after combustion at 490 degrees C for 4 hours.

Extractable Lipids, Length and SEM Imaging
At each tank change, an aliquot of larvae were fixed in 2.5% gluteraldehyde and 1% paraformaldehyde in a 0.1 M cacodylate buffer. Samples were later transferred to 50% ethanol and, imaged using an Olympus SZH10 dissecting microscope. 100 larval lengths per sampling point were measured (ImageJ 1.44p). Lengths were converted into weights using the dry-tissue weight to length relationship for larval C. gigas from Bochenek et al. (2001), accounting for our measured proportion shell (1-AFDW) at each time point, instead of the 75% shell assumption used by the model.

To measure extractable lipids, ~75 mg samples of freeze-dried larvae were extracted on a Dionex ASE 200 Accelerated Solvent Extractor for four cycles of 5 minutes at 1500 psi with 3:1 methylene chloride:methanol. The extracts were combined with 25 mL hexane and washed against 10 mL 50% saturated NaCl to remove hydrophilic impurities (proteins, carbohydrates). The aqueous phase was drained away and the lipid-containing fraction was dried under an N₂ gas stream. The lipid concentrate was then re-suspended in 500 uL CS₂and an aliquot (100 uL) moved to a tared nickle weighing boat, dried and re-weighed to get a gravimetric weight of lipid. This weight was compared to the original sample weight to find the fraction total lipid. As with the other bulk measurements, these values were normalized to the larval weights to estimate lipid (ng) per larvae. Additionally, it should be noted that the lipid measurements by Waldbusser et al. (2013) may be an over-estimate, due to the gravimetric extraction method used. The washed values presented here provide a better estimate of the true lipid content. Due to a sampling error, the investigators do not have a reliable washed lipid value for the 48 hour time point for the May cohort. Given the similarity between the washed and unwashed values in early life, the unwashed gravimetric lipid weight (as reported in Waldbusser et al. 2013) are used instead.

Carbonate chemistry
ater samples were collected at each tank filling prior to the addition of larvae or algae. For dissolved inorganic carbon (DIC) ¹³C analysis, aliquots of HgCl₂-fixed sample were pipetted into Labco glass vials sealed with rubber septa caps. The vials were cooled to 13 degrees C, allowed to equilibrate for 15 minutes, and purged with He for 5 minutes each. 85% phosphoric acid was then added to each vial via syringe. The samples were left to equilibrate for 10 hours and analyzed via continuous-flow mass spectrometry using a GasBench-DeltaV system. Each run was standardized using a combination of sodium bicarbonate (3 mM in solution) and calcium carbonate standards. These methods have a precision of 0.15 ‰ and repeatability better than 0.2 ‰.

Samples for PCO₂ and dissolved inorganic carbon (DIC) analysis were collected at each water change in 330 mL amber glass bottles, poisoned with HgCl₂ and analyzed following Bandstra et al. (2006) The uncertainty of these measurements is less than 2% and 0.2% for PCO₂ and DIC, respectively. The remaining carbonate system parameters were calculated using the carbonic acid dissociation constants of Millero (2010), the boric acid constants as defined by Dickson (1990), and the aragonite solubility as defined by Mucci (1983).