Corals sampled at six reefs within Kāne'ohe Bay, O'ahu, Hawai'i:

1.) USA: Hawaii HIMB: 21.436056, -157.786861
2.) USA: Hawaii Reef.11.13: 21.450806, -157.794944
3.) USA: Hawaii Reef.35.36: 21.473889, -157.833667
4.) USA: Hawaii Reef.18: 21.450806, -157.811139
5.) USA: Hawaii Lilipuna.Fringe: 21.429417, -157.791111
6.) USA: Hawaii Reef.42.43: 21.477194, -157.826889

Experiment conducted at the Hawai'i Institute of Marine Biology.

Photosynthesis and Respiration Rates: Prior to experimental exposures, 10 coral fragments (5 per species) were used to generate a photosynthesis-irradiance curve to determine saturating irradiance for assessing rates of photosynthesis. Fragments were exposed to 10 light levels: 0, 15, 30, 60, 91, 136, 227, 416, 529, and 756 µmol m-2 s-1 generated by two LED lights (Aqua Illumination Hydra FiftyTwo) hung above the incubation chambers (described below). Rates of oxygen consumption or evolution were extracted using curve fitting of a non-linear least squares fit for a non-rectangular hyperbola (NLLS; Marshall & Biscoe 1980, Heberling 2013) was used to identify PI curve characteristics of each species. This model is as follows: = фPAR+√(φPPFD + Pmax)2-4ΘφPAR Pmax2Θ-Rd . Theta was set at 0.6 for M. capitata and 0.64 for P. acuta. Photosynthesis-irradiance curves were performed four times throughout the experiment on fragments of both species in the HTHC treatment to determine that there were no significant changes in Ik that occured during bleaching, and thus no need to change light settings used to measure respiration and photosynthetic rates.

Fragments were placed in individual respiration chambers (~610mL), with individual temperature (Pt1000 temperature sensor, PreSens) and fiber-optic oxygen probes (Oxygen Dipping Probes DP-PSt7, accuracy = ± 0.05% O2, PreSens) connected to a 10-channel oxygen meter (OXY-10 ST, accuracy = ± 1.0 °C, resolution = 0.1 °C, PreSens), to evaluate photosynthesis under saturating light conditions, as determined by PI curves described above, and light enhanced dark respiration (LEDR; Edmunds and Davies 1988). The respirometry setup consisted of 10 chambers with stirbars. Samples were measured in a series of runs that consisted of eight fragments (n=4 per species and n=2 blank chambers) per run, and exposed to PAR irradiance of 590 ± 7.16 µmol photons m-2 s-1 for 15 minutes to assess photosynthetic rates. Immediately afterwards, these fragments were exposed to dark conditions (0 µmol photons m-2 s-1) for 20 minutes to assess LEDR. Following respirometry, fragments were immediately snap-frozen in liquid nitrogen and stored at -80°C.

Sample Collection and Endosymbiont Density: Coral tissue was removed from the coral skeletons by airbrushing (Iwata Eclipse HP-BCS) with ice-cold 1X Phosphate Buffer Saline (PBS) solution, separating the soft tissue from the calcium carbonate skeleton and the total volume was recorded. The tissue slurry was then homogenized for 15 seconds at high speed (PRO Scientific Bio-Gen PRO200 Homogenizer). Of the total tissue slurry, 500 µL was taken for endosymbiont density, 500 µL for host soluble protein and host total antioxidant capacity, 1 mL for chlorophyll concentration assays, and 5 mL for ash-free dry weight. The skeleton was rinsed in freshwater and soaked in 20% bleach for 24 hours and dried at room temperature for 24 hours. The single dip wax-dipping technique (Veal et al. 2010) was used to determine surface area of each dried fragment skeleton. Symbiodiniaeceae cell concentration was quantified with 6 technical replicate counts on a haemocytometer (Hausser Scientific Bright-Line Counting Chamber) after homogenizing the tissue slurry for 1 minute. The average count (cells mL-1) was standardized to homogenate volume and surface area to produce cell density values in cells cm-2.

Host soluble protein and total antioxidant capacity: To quantify host soluble protein and host total antioxidant capacity the host tissue was isolated from symbionts by centrifugation at 10,000g for 10 minutes at 4°C. Soluble host protein of the supernatant was measured using the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Waltham, Massachusetts, Catalog #23225) with two duplicates per sample measured by a 96 well microplate reader (BioTek Synergy HTX Multi-Mode Reader) at a wavelength of 562 nm. Resulting values were compared to a bovine serum albumin standard curve and standardized to homogenate volume and surface area to obtain host soluble protein values (mg cm-2). Host total antioxidant capacity was quantified in duplicates per sample with the Cell BioLabs OxiSelect Total Antioxidant Capacity (TAC) Assay Kit (Catalog #STA-360) manufacturer’s instructions and compared to a uric acid standard curve to obtain Copper Reducing Equivalents (CRE) in mmol L-1. CRE values were standardized to total soluble protein for final values in units of CRE μmol mg-1.

Chlorophyll concentration: To isolate symbiont cells for chlorophyll concentration measurements, a 1 mL aliquot of tissue homogenate was centrifuged at 13,000 g for 3 min and the supernatant was discarded. 1 mL of acetone was added to the symbiont pellet and incubated in the dark at 4°C for 24 hours. Samples and acetone blanks were read in duplicate (200 μL each) on a 96-well microplate reader at λ = 630 and 663 nanometers. Chlorophyll pigments a and c2 were calculated with the following dinoflagellate equations, respectively: 11.43E663 - 0.64E630 and 27.09E630 - 3.63E663 (Jeffrey and Humphrey 1975). Concentration values were multiplied by 0.584 to correct for the path length of the plate, and standardized by both surface area (μg cm-2) and cell density (μg cells-1).

Tissue Biomass: To quantify biomass as ash-free dry weight (AFDW), 5 mL of tissue slurry was centrifuged at 13,000 g for 3 minutes. 4 mL of the supernatant host tissue was removed and placed in pre-weighed burned aluminum pan and the symbiont pellet was resuspended in 1 mL PBS solution and added to separate pre-weighed burned aluminum pans. The samples were placed in a drying oven for 24 hours at 60°C (Thermo Scientific Heratherm General Protocol Oven, Catalog #51028112), weighed, and then placed in a muffle furnace for 4 hours at 500°C (Thermo Scientific Lindberg Blue M Muffle Furnace, Catalog #BF51728C-1). AFDW (mg cm-2) of the host and symbiont fractions were calculated as the post-drying oven weight (dry weight) - post-muffle furnace weight and final values were normalized to surface area. Symbiont to host (S:H) AFDW ratios were calculated by dividing the symbiont AFDW by the host AFDW.

heatwave_physiology.csv