Summarized from: Keister, E.F., Gantt, S.E., Reich, H.G., Turnham, K.E., Bateman, T.G., LaJeunesse, T.C., Warner, M.E., Kemp, D.W. Similarities in biomass and energy reserves among coral colonies from contrasting reef environments. Scientific Reports (2023).DOI:10.1038/s41598-023-28289-6

Field sampling
For each species, coral colonies were sampled (n= 3 to 14 individuals) at depths of 5 to 10 meters for offshore colonies and 2 to 5 meters for nearshore colonies. The sampled colonies were spaced at least 10 meters apart. All colonies selected for sampling were of similar sizes, representative of typical sizes for each species, and fragments were taken from the top and center of the colonies. Prior to sample collection, the coral colonies displayed no visible signs of stress, and there were no reported thermal anomalies or bleaching events.

The sampled coral colonies were carefully transported back to the Palau International Coral Research Center (PICRC) in coolers filled with seawater. At PICRC, the coral samples were individually placed into Whirlpaks® and immediately frozen at a temperature of -40°C. The frozen samples were then transported to the University of Alabama at Birmingham (UAB) in the United States, where they were stored at -80°C until further processing.

Laboratory processing
While still frozen, the coral fragments were cut into approximately 4 cm^² pieces using a Torque Master Tile Saw (QEP) equipped with a diamond blade. All excess skeleton, boring sponges, and epibionts were carefully removed from the coral fragments. To determine the surface area of each fragment, 3D scanning was performed using a Capture Mini 3D scanner along with Geomagic® Controlx64™ software from 3DSystems. Subsequently, the coral fragments were lyophilized for 36 hours using a Labconco Freeze Dry System and weighed to determine their total dry mass.

The lyophilized coral fragments were individually pulverized into a fine, homogenized powder using a SPEX Sample Prep ball mill. This powder consisted of the complete coral holobiont, including the animal host, endosymbiotic dinoflagellate communities, and microbiome. The powdered samples were then partitioned for further analysis, including the examination of tissue biomass and energy reserves such as total lipids, soluble protein, and carbohydrates.

Tissue biomass
To assess tissue biomass, the dry powdered coral fragments were weighed, ranging from approximately 0.5 to 3.3 grams. These fragments were then subjected to combustion in a muffle furnace for a duration of 12 hours at a temperature of 500°C. This process allowed for the determination of the total organic content. The ash-free dry weight (AFDW) was calculated by subtracting the weight of the ash after combustion from the initial dry weight of the fragments. The ratio of AFDW to the total dry weight was utilized to calculate the overall AFDW, representing the total coral tissue biomass per surface area of the entire fragment.

Energy reserves
For the measurement of energy reserves, the sample sizes varied depending on the availability of samples. For some species, the offshore sampling yielded anywhere from 3 to 14 individuals, while the nearshore sampling had 6 to 8 individuals. Energy reserves were assessed alongside the tissue biomass as part of the study.

Lipid analyses were conducted to determine the total lipids and quantify different lipid classes present in the coral samples. Approximately 0.6 grams of lyophilized coral fragment powder were used for the lipid extraction process. Triplicate, independent lipid extractions were performed for each sample using a modified Folch method. In brief, a solvent system consisting of chloroform, methanol, and 0.88% NaCl in specific ratios (8:4:3) was used to extract the total lipids. The solvent volume was maintained at a 20:1 ratio relative to the dry sample weight. The lower chloroform phase was collected in pre-weighed glass tubes and dried under a continuous stream of nitrogen gas. The gravimetric determination of total lipid concentration was performed, and the values were converted to Joules (J) per unit of ash-free dry weight (AFDW) per gram. To achieve a concentration of 10 mg/mL, 100% chloroform was added to the dried lipid extract.

For the quantification of lipid classes, 1 μL of the lipid extracts was spotted in duplicate on individual silica Chromarods®. Thin-layer chromatography (TLC) was then conducted using a two-step solvent system. The first step involved a mixture of chloroform, methanol, and water (50:20:2 by volume) and allowed for the elution of phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol (PS-PI), phosphatidylcholine (PC), and lysophosphatidylcholine (LPC). The second step involved a mixture of hexane, ethyl ether, and formic acid (60:15:1.5 by volume) and allowed for the elution of wax ester (WAX), triacylglycerol (TAG), sterol (ST), and diacylglycerols (DAG). The Chromarods were dried at 100°C for 10 minutes before being analyzed using an Iatroscan MK 6S thin-layer flame ionization detector (TLC-FID) for identification and quantification of lipid classes. Calibration of the Iatroscan MK 6S was performed using compound classes in the concentration range of 0.1-10.0 mg/mL, including l-alpha-phosphatidyl-l-serine for PS-PI, l-alpha-phosphatidylethanolamine for PE, l-alpha-phosphatidylcholine for PC, l-alpha-lysophosphatidylcholine for LPC, palmityl palmitate for WAX, tripalmitin for TAG, cholesterol for ST, and dipalmitin for DAG.

All phospholipid lipid classes (PE, PS-PI, PC, LPC) were grouped together and analyzed as a single unit. The values for all lipid classes were presented in units of milligrams per unit of ash-free dry weight (AFDW) per gram.

For the analysis of carbohydrates and proteins, specific protocols were followed. To quantify soluble proteins, approximately 0.5 grams of lyophilized and crushed coral samples were used. Following the procedure described by McLachlan et al., the samples were placed in 15 mL tubes, and 1 mL of a diluted 1x solution of radioimmunoprecipitation (RIPA, Sigma-Aldrich) was added. The samples underwent three freeze-thaw cycles to lyse the cells and solubilize the proteins. After centrifugation for 20 minutes at 4122g at 4°C, 1 mL of the supernatant containing the solubilized protein was transferred to a 2 mL tube. A modified Bradford assay, using bovine serum albumin (BSA) as a standard, was then employed to quantify the soluble protein levels. All samples were analyzed in triplicate using a microplate reader (EPOCH 2, Agilent), measuring absorbance at 465 nm and 595 nm. The values for soluble proteins were converted to Joules per unit of ash-free dry weight (AFDW) per gram.

For the quantification of carbohydrates, around 0.3 grams of lyophilized and crushed coral samples were placed in a 2 mL tube with 1 mL of Milli-Q water. The samples were sonicated at 35% intensity for 2 minutes using a Fisher Scientific model CL-18 sonicator. After centrifugation for 10 minutes at 1000g, 1 mL of the supernatant containing total carbohydrates was collected. A modified DuBois method, described in Masuko et al., was employed to quantify carbohydrates, with glucose used to create a standard curve. All samples were run in triplicate on a microplate reader (EPOCH 2, Agilent), measuring absorbance at 485 nm and 750 nm for the determination of total carbohydrates. The carbohydrate values were then converted to Joules per unit of ash-free dry weight (AFDW) per gram.