Dataset: brooded coral larvae 2 - respiration_photosyth_mortality
Deployment: lab_Edmunds_NMMBA

March 2010:
A Ruthenium-based optode (FOXY-R, 1.58 diameter, Ocean Optics) connected to a spectrophotometer (USB2000, Ocean Optics) and interfaced with a computer running Ocean Optics software (OOISensor, version 1.00.08) was used to measure the respiration of the larvae. The optode was 2-point calibrated using a zero solution (0.01 M Na2B4O7*10H2O saturated with Na2SO3) and 100% air saturation using water-saturated air at the treatment temperature. To measure respiration, 6 larvae were removed from the treatment containers 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 in each vial (Edmunds et al. 2011). Respiration measurements were completed after the 24-h incubation period. Larvae were dark-adapted prior to measurements so that respiration would not be stimulated by light (Edmunds and Davies 1988). Initial O2 concentration in the seawater filling the vials was determined before the vials were sealed, and vials without larvae were 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 (±0.1C, Hipoint, models LC-06 and LC-10). Incubation times were selected to ensure that O2 concentrations remained[75%. On completion of the incubations, vials were removed from the chillers, gently inverted to mix the seawater, and analyzed for O2 saturation. O2 saturation was converted to concentration using gas tables [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 converted to nmol O2 min-1 larvae-1, after adjusting for control O2 fluxes.

Larval photophysiology was assessed using pulse amplitude modulated (PAM) fluorometry to measure the maximum photochemical efficiency of open reaction centers of photosystem II (RCIIs) following a period of dark adaptation (i.e., Fv/Fm) of their Symbiodinium. Changes in Fv/Fm can detect damage to the photosynthetic apparatus, with declines under elevated temperature indicating damage to PSII (Jones et al. 1998; Bhagooli and Hidaka 2003). These measurements were conducted after the 24-h incubation period, with larvae being dark-adapted during the final 2 h of the incubation. After this period of darkness, Fv/Fm was measured using a diving PAM (Walz, GmbH) fitted with an 8-mm diameter probe and standardized for measuring intensity (setting: 10) and gain (setting: 10). Fluorescence was measured by loading 8 larvae into a drop of seawater on the tip of the probe, with these manipulations completed under weak red illumination. Two groups of 8 larvae were measured for Fv/Fm from each tank, and the average value in each tank was used for statistical analysis.

To assess the number of larvae dying in the treatments, at the conclusion of the incubations, tubs were removed from the treatments and the number of swimming larvae and settled recruits (with tissue) recorded. Due to the rapid breakdown of dead larvae (Yakovleva et al. 2009), larvae that could not be accounted for were assumed dead. Mortality was expressed as a percentage of the number of larvae added at the start of the experiment.

March 2012:
The second experiment was designed to be virtually identical to the first (n = 8 tanks, 2 tanks treatment-1), although the volume of the incubation tanks was increased to 120 L, and the sample size (number of replicate tubs containing larvae in each tank) was doubled with the objective of increasing statistical power and testing for tank effects for the dependent biological variables. Experimental difficulties affecting a single tank made it problematic to include the tank in the statistical analyses, and therefore, replicates were pooled between tanks in each treatment combination. Larvae were sampled from each replicate tub, and their response to the treatment conditions was assessed using dark respiration, photophysiology (Fv/Fm), and mortality, and each dependent variable was measured as described above.