Sample Collection and Maintenance
Three coral species, 1 octocoral and 2 scleractinians, were utilized in this experiment. The octocoral, Eunicea flexuosa, was collected from Wonderland Reef (24.558694, -81.503528) within the Florida Keys National Marine Sanctuary (FKNMS) under the state of Florida (FL) saltwater fishing permit (Permit number: I-H1R76333834 held by A.M. Reigel). Three axial branch tips were clipped from each of 10 healthy E. flexuosa colonies located at depths of ~5-8 meters (m). Branch tips were kept in seawater and immediately transported to the outdoor land-based nursery (Climate and Acidification Ocean Simulator (CAOS)) at Mote Marine Laboratory at Summerland Key, FL, USA where they were placed into a shaded, temperature- and pH-maintained flow-through tank and allowed to acclimate for ~24 hours. The two hard coral species, Acropora cervicornis and Orbicella faveolata were provided by Mote Marine Laboratory’s field (A. cervicornis; Coordinates: 24.562747, -81.400455) and land-based (O. faveolata) nurseries as permitted under the FKNMS-2015-163-A3. All hard coral fragments were placed in the same flow-through CAOS tanks as the E. Flexuosa samples and allowed to acclimate for ~24 hours.

To develop a representative sponge community for the Florida Keys reefs, we collected 5 individuals of each of 6 sponge species (Niphates digitalis, Verongula rigida, Aplysina fulva, Aplysina cauliformis, Xestospongia muta, Callyspongia aculeata) from Wonderland Reef under FL saltwater fishing permit (Permit #: I-H1R76333834 held by A.M. Reigel). Sponges were kept in seawater and immediately transported to the lab where they were placed in a shaded CAOS flow-through tank to acclimate for ~24 hours. Corals and sponges were not in the same CAOS tanks during the acclimation period.

Stable Isotope Pulse-Chase Incubations
Following 24 hours of acclimation time, sponges (n=2 per species) and coral fragments (n=4-5 per species) were removed from their respective acclimation tanks and immediately placed into labeled, sterile WhirlPak© bags and stored in the -20º Celsius (C) freezer (i.e., initial, or T₀, samples). The remaining sponges were moved from the acclimation tank to a 'pulse' tank for a 3-hour incubation. The 'pulse' tank was pre-filled with 84 liters of 0.22 micrometer (µm) filtered seawater spiked with isotopically labeled inorganic compounds in the following final concentrations: 0.1 grams per liter (g L⁻¹) sodium bicarbonate (NaH¹³CO₃), 0.01g L⁻¹ sodium nitrate (Na¹⁵NO₃), and 0.01g L⁻¹ ammonium chloride (¹⁵NH₄Cl). At the end of the 3-hour 'pulse', a subset of sponges ('pulse' samples) was destructively sampled following the same procedure detailed above. The remaining enriched sponges (n=5 per species) were transferred to five separate flow-through tanks, such that each tank contained one individual of each sponge species, for a 1-hour 'rinse' period to flush away any labeled compounds that were not incorporated into the sponge tissue. At the end of the rinse, two fragments from each coral species were placed into each of eight experimental tanks, five enriched sponge-containing tanks, and three no-sponge control tanks, for the 6-hr 'chase'. During the duration of the 'chase', the flow-through system was turned off and a submersible aquarium pump was added to each tank to maintain circulation and gas exchange. A single fragment from each coral species was destructively sampled from each experimental tank twice during the 'chase': at the half point (T₃) and at the end (T₆). All enriched sponges were also destructively sampled at the end of the 'chase'.

Coral and Zooxanthellae Separations
To prepare for downstream analyses the coral fractions, host and zooxanthellae, were manually separated. Scleractinian fragments were thawed and airbrushed with an aerosolized jet of 0.22 micrometer (µm) filtered seawater to physically separate the coral tissue and skeleton and suspend the coral tissue material into a homogenate. To separate host tissue from zooxanthellae cells, the homogenate was centrifuged at 2000g for 3-5 minutes. Centrifugation formed a pellet comprised of zooxanthellae cells and a homogenate of host material. The host homogenate was pipetted into a separate sterile 50 milliliter (ml) Falcon tube. The homogenate, zooxanthellae pellet, and skeletal fragments were frozen and transported to Appalachian State University where they were stored at -20 degrees Fahrenheit (F) until further processing. At Appalachian State, host homogenates and zooxanthellae pellets were thawed and checked for purity. Impure fractions were combined, homogenized with a tissue homogenizer (maximum speed for ~15 seconds) to physically separate zooxanthellae cells from host tissue, and centrifuged (3000 x g, 6 minutes) to pellet the zooxanthellae cells. Separated fractions were combined with original fractions each time and checked for purity under the microscope. The process was repeated until at least 80% purity was reached.

E. flexuosa fractions were separated using a different process. First, the frozen coral branches were lyophilized (Labconco™ FreeZone™ Bulk Tray Dryer) for 22-24 hours, until they were completely dry. Following lyophilization, the axial skeleton was removed and the tissue was ground up using a mortar and pestle (note: separate mortar and pestle sets were used for control and enriched samples). The ground tissue was weighed and then rehydrated in 10ml of MilliQ water in a sterile 15ml Falcon tube. Very quickly following rehydration, the sclerites (skeletal fragments) sank to the bottom of the tube and the remaining host homogenate was pipetted into a new tube taking care not to transfer the sclerites. The homogenate was homogenized using a tissue homogenizer for ~15 seconds at maximum speed and centrifuged at 4000g for 5 minutes to separate the fractions. The centrifugation step was repeated as necessary until the host homogenate and zooxanthellae pellets were pure. Following both octocoral and scleractinian fraction separations, 50 microliters (µl) of pure zooxanthellae from each sample was transferred to a cryovial with 50µl of 10% paraformaldehyde (PFA) to fix the cells and stored in the refrigerator for future zooxanthellae counts. The pure host homogenates and remaining zooxanthellae pellets were stored in the -20F freezer.

Stable Isotope Analysis
All sponge and zooxanthellae tissues and the octocoral host homogenate were lyophilized, weighed, and the dried material was homogenized with a mortar and pestle. Homogenized samples were decalcified via exposure to 12M HCL in desiccator chambers. Subsamples of ~1-2.5 milligrams (mg) of decalcified tissue from each sample were packed into silver cups. Scleractinian coral homogenates, made as previously detailed, contained filtered seawater, and if lyophilized, the salts could contaminate the host tissue. To remove the salts, host homogenates were passed through a sterile pre-weighed 1.6 µm GF/F filter that captured host material while seawater passed through and was discarded. The filters containing host material were frozen, dried overnight in a drying oven, weighed, decalcified as detailed above, and sent to the Marine Biological Laboratory (MBL) Stable Isotope Laboratory where they were also packed into silver cups.

Packed samples were analyzed at the MBL Stable Isotope Laboratory for δ¹⁵N and δ¹³C using a Europa 20-20 continuous-flow isotope ratio mass spectrometer interfaced with a Europa ANCA-SL elemental analyzer. The analytical precision based on replicate analyses of isotopically homogeneous international standards is +/- 0.1 ‰ for both δ¹⁵N and δ¹³C measurements.

Sampling and Analysis Dates
Following the 08-Dec-2020 sampling, all field-collected samples were acclimated in the aquarium system for 24 hours and the stable isotope 'pulse-chase' experiment was conducted over 11 hours on 09-Dec-2020. The host and zooxanthellae fractions were frozen and then the separations of the fractions from A. cervicornis and O. faveolata happened on 17-Dec-2020 and both fractions were frozen again. The other experimental steps occurred over several months in Spring 2021.

Known Issues/Problems
Four control (i.e., non-enriched) coral samples (Of_T6_7, AC_T3_3, AC_T6_1, and EF_T6_1) and their corresponding zooxanthellae have δ¹⁵N and δ¹³C values that suggest they may have been contaminated by the labeled isotopes at some point during the sample preparation process. These samples are found at the bottom of the data file and are identified as 'Control-Contaminated' in the Treatment column. These samples should be excluded or used with caution.

isotopes_raw_data.csv