Larval respiration was measured in a confined respirometer use a Ruthenium-based optrode (FOXY-R, 1.58 diameter, Ocean Optics) connected to a spectrophotometer (USB2000, Ocean Optics) and interfaced with a computer running the manufacturers software (OOISensor, version 1.00.08). The optrode was calibrated using a zero solution (0.01 M Na2B4O7·10H2O supersaturated with Na2SO3) and 100% air saturation usingwater-saturated air at the treatment temperature. Tomeasure respiration, 6 larvae were removed from the tubs in the treatment tanks and placed into 2-mL glass Wheaton vials filled with filtered seawater from the same treatment tank and sealed with Parafilm. A study conducted concurrently with the present analysis demonstrated that respiration of P. damicornis larvae in identical vials could be measured accurately with 5 larvae vial-1 (Edmunds et al., 2011).
Initial O2 concentration in the seawater was determined before the vials were sealed, and vials without larvae used as controls. Larvae in the sealed vials were incubated at their temperature treatments for 1.5-2 h in the dark using water baths set to the temperature of the respective treatments (±0.1 °C, Hipoint, models LC-06 and LC-10); vials were carefully moved throughout the incubations to ensure the seawater remained mixed. Incubation timeswere selected to ensure O2 concentrations remained >75% saturation. On completion of the incubations, vials were removed from the water baths, gently inverted to mix the seawater, and analyzed for O2 saturation. O2 saturation was converted to concentration using tabulated values of O2 solubility (N. Ramsing and J. Gundersen at http://www.unisense.com [based on Garcia and Gordon, 1992]) and the temperature and salinity of the seawater, and the change in O2 concentration used to calculate O2 flux after adjusting for controls. The respiration rates are expressed as nmol O2 mg protein-1 min-1.
Protein content was determined spectrophotometrically using the Bio-Rad Coomassie Blue assay in the microtiter plate protocol (BioRad Life Sciences Research, CA). At each sampling, 1 group of 8 larvae from each tub within each tank (i.e., 2 replicates tank-1) was frozen in liquid nitrogen and stored. Proteins were solubilized in 0.1 M NaOH, aided by sonication (10% amplitude for 15 s using a Branson Digital Sonifier S-250D, USA) and warming at 50 °C for 5 h. The extract was neutralized with 1 M HCl and processed in triplicate with the addition of the dye reagent. Following 30 min incubation, absorbances were measured at 595 nm using a plate reading spectrophotometer (Biotek Synergy H4 Hybrid Reader, USA), and converted to protein using a calibration prepared from bovine serum albumin. Protein content was expressed as µg larva-1.
The 'ambient' and 'high' pCO2 levels: 49.4 Pa versus 86.2 Pa
The 'ambient' and 'high' temperatures: 24.00 °C [ambient] versus 30.49 °C
Data also available from PANGAEA: doi:10.1594/PANGAEA.823582
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