Dataset: Acropora cervicornis protein measurements: nutrient- and disease-exposed from samples collected at Mote Marine Laboratory in situ nursery from June to July 2022

Proteins were extracted over ice using a Paansche airbrush with coral extraction buffer (50 mmol tris buffer, pH 7.8, with 0.05 mmol dithiothreitol). Tissues were then homogenized using a VWR 200 tissue homogenizer with a medium saw tooth generator for 60 seconds on ice. Samples were then left on ice for 10 minutes. From the resulting extract, 1 mL was reserved for melanin analysis. The remaining volume was centrifuged for 5 minutes at 4°C and 3500 RPM in an Eppendorf centrifuge 5810R. The resulting supernatant, or coral extract, was split into two ~2 mL aliquots mL which were frozen and stored at -80°C. 

Total host protein (total HP) in each sample was determined using Bradford reagent standardized to BSA. These concentrations were used to standardize all biochemical assays conducted on the samples. 

All colorimetric assays were run in triplicate on 96-well plates using a Synergy H1 Hybrid Multi-Mode microplate reader and Gen5 software. Samples were stored at -80°C and thawed immediately prior to processing.

To measure peroxidase (POX), 20 μL of sample were diluted with 20 μL 10 mM phosphate buffer, pH 6.0. Then 25 μL of 25 mM guaiacol in 10 mM phosphate buffer, pH 6.0, was added to each well. The reaction was initiated with 20 μL of 20 mM hydrogen peroxide and optical density was measured every 34 seconds for 15 minutes at 470 nm. Results were calculated as change in absorbance per minute, normalized according to mg of protein.
To measure prophenoloxidase (PPO), 20 μL of sample were diluted with 20 μL 50 mM phosphate buffer, pH 7.0. Next, samples were incubated for 30 minutes in 25 μL of trypsin (0.1 mg/mL). Just prior to the assay, 30 μL of 10 mM L-1,3- dihydroxphenylalanine (L-dopa) was added to each sample. Absorbance was then read every minute for 20 minutes at 490 nm at 26C. Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.
To measure phenoloxidase (PO), 20 μL of sample were diluted with 20 μL 50 mM phosphate buffer, pH 7.0. Next, samples were incubated for 30 minutes in 25 μL of molecular grade water. Just prior to the assay, 30 μL of 10 mM L-1,3- dihydroxphenylalanine (L-dopa) was added to each sample. Absorbance was then read every minute for 20 minutes at 490 nm at 26°C. Results were calculated as change in absorbance per minute at the steepest point of the curve, normalized according to mg of protein.

A Superoxide Dismutase (SOD) Activity Assay Kit from Sigma Aldrich was used to measure superoxide dismutase activity following manufacturer's protocols. Briefly, 20 μL of coral extract was incubated with WST dye and 20 μL xanthine oxidase for 30 minutes at 25°C. A standard curve was performed using the provided SOD enzyme. Percent inhibition of absorbance was then measured at 450 nm by comparing the absorbance of the samples to that of the control wells. Activity is reported as superoxide dismutase activity standardized by mg protein.