Dataset: brooded coral larvae 1 - size_protein_symbionts_photosynth
Deployment: lab_Edmunds_NMMBA

Protein content of the larvae was determined spectrophotometrically using the microtiter plate protocol of the Bio-Rad protein assay with Coomassie brilliant blue dye (Bio-Rad, CA). At each sampling, four groups of larvae (40 group-1 in June and 10 group-1 in July) were frozen in liquid nitrogen and stored until processed. Proteins were solubilized by placing the larvae in 0.1 M NaOH, disrupting them with ultrasonic vibrations (Branson Digital Sonifier S-250D, USA), and warming at 50C for 5 h. The protein extract was neutralized with HCl, diluted where necessary, and processed in triplicate with the addition of the dye reagent on a microtiter plate. Following 30-min incubations, absorbances were measured at 595 nm using a plate reader (Biotek Synergy H4 Hybrid Reader, USA) and converted to protein using a calibration prepared from bovine serum albumin. Protein content was expressed as mg larva-1.

Maximum quantum yield (Fv/Fm) of Symbiodinium within the larvae: To assess the condition and number of Symbiodinium, larvae were processed for maximum photochemical efficiency of PSII (Fv/Fm) and Symbiodinium density. Fv/Fm was measured using pulse amplitude modulation fluorometry (PAM) with a Diving-PAM (Walz, GmbH) fitted with an 8-mm-diameter probe and operated at constant settings for measuring intensity and gain (both set at 10). Four replicates of 8 larvae were dark-adapted for 2 h and transferred under weak red light to the tip of the sensor where they were retained within a drop of water. Previous experiments demonstrated that fluorescent measures could be obtained reliably with C6 larvae. Larvae processed for Fv/Fm were removed from the tip of the sensor and used to evaluate size.

Larval size and Symbiodinium densities: To determine the size, larvae were placed individually on a microscope slide in a drop of seawater and photographed using a digital camera fitted to a dissecting microscope. Larval area (mm^2) was measured using ImageJ 1.42q software (Abramoff et al. 2004), and mean larval area (n = 8) from each tank at each time point was used for statistical analysis. Finally, these larvae were analyzed for Symbiodinium densities by preserving 4 replicates of 8 larvae in 10 % formalin, and later macerating them with a Teflon pestle. The Symbiodinium in the slurry were counted using a hemocytometer (4 replicate counts) and the algal population expressed as cells larva-1.