From the waters of Kāne‘ohe Bay, HI, four colonies of each coral species M. capitata and P. acuta were identified and collected under SAP 2019-60. Each of the four colonies for each species was fragmented into 30 pieces at the Hawaiʻi Institute of Marine Biology, located on Moku o Loʻe in Kāne‘ohe Bay, HI, and hot-glued to labeled plugs. (21N 157 W ; depth 1m).

Metabolite extraction and analysis conducted at Rutgers University.

Coral sampling: Sampling of the nubbins began after the onset of color score divergence between the treatment groups. Three sampling points were selected on 22 May 2019 (T1), 3 June 2019 (T3), and 7 June 2019 (T5) on the basis of reaching maximum treatment temperature, where the coral color score began to diverge by treatment and where the coral color score differences were maximized between treatments within species within the experimental time frame. Coral nubbins were selected for sampling randomly using a random number generator, and color scores were recorded for each nubbin after sampling. Corals were sampled at ~14:30 at each time point by removing them from their treatment, only touching the plastic bases and inserting them into sterile Whirl-Paks that were immediately submerged in liquid nitrogen and transferred to −80°C until metabolite extraction.

Metabolite extraction from coral nubbins: Metabolites were extracted using a protocol optimized for water-soluble polar metabolite analysis on LC-MS. The extraction buffer was a solution of 40:40:20 (methanol:acetonitrile:water) (v/v/v) + 0.1 M formic acid. The extraction buffer was stored at −20°C before usage. Immediately preceding the metabolite extraction, 1 ml of extraction buffer was added to a 2-ml glass Dounce homogenizer that had chilled on ice. Pieces of the −80°C preserved nubbins were then clipped, weighed, and added to the cold extraction buffer in the Dounce and left to incubate for 5 min. The pestle of the Dounce was then used to homogenize the coral tissue until there was a visible accumulation of coral skeleton at the bottom of the Dounce and the homogenate was visibly pigmented. An additional 500-μl aliquot of cold 40:40:20 + 0.1M formic acid extraction buffer was then used to rinse down the sides of the Dounce and pestle. The total 1.5-ml volume was then strained through a sterile 100-μm cell strainer into a 50-ml receptacle. There was a visible amount of skeleton collected in the strainer. The rest of the homogenate was then transferred to a 1.5-ml Eppendorf tube, vortexed for 10 s, and then centrifuged for 10 min at 16,000g at 4°C. After centrifugation, there was a pellet at the bottom of the tube. A final 500-μl aliquot of the homogenate was then pipetted to a second clean Eppendorf tube, to which 44 μl of 15% NH4HCO3 was added to neutralize the acid in the buffer. This was the final extract and was ready to be loaded to instrument vials for analysis.

Cultivation of Aiptasia: The animals were held at 27°C under a 12-hour light/12-hour dark cycle at 25 μmol photons m−2 s−1. All individuals were from the clonal population CC7, which naturally contains the algal symbionts of the Symbiodiniaceae clade A species Symbiodinium linuchae (Sunagawa et al. 2009). CC7 animals were made aposymbiotic as previously described (Xiang et al. 2013). This resulted in the strain CC7-Apo. CC7-Apo animals subsequently exposed to algae of the clonal axenic strain SSB01 (Symbiodiniaceae clade B species Breviolum minutum) (Baumgarten et al. 2015) and cultured under standard conditions. After these anemone strains were created, each strain was continued and propagated though asexual reproduction in long-term culture for >1 year before experimentation. By using long-term, stable anemone cultures, we intended to minimize the effects of the stress on the animals required to generate the CC7-Apo and CC7-SSB01 anemones that were used for metabolomic analysis. Aiptasia were aliquoted individually to the 1.5-ml Eppendorf tubes in 500 μl of artificial seawater, flash-frozen, and stored at −80°C until processing at Rutgers University.

Metabolite extraction from Aiptasia: Metabolites were extracted using a protocol optimized for water-soluble polar metabolite analysis on LCMS. The extraction buffer used was a solution of 40:40:20 (methanol:acetonitrile:water) (v/v/v) + 0.1 M formic acid and was stored at −20°C before usage. CC7-Apo and CC7-SSB01 animals were removed from the artificial seawater storage, and intragroup animals were pooled to produce six replicates, each having an input weight of 25 mg in a 1.5-ml Eppendorf tube. Replicates were kept on dry ice before extraction. A total of 500 μl of extraction buffer was added; then, samples were vortexed for 10 s and transferred to crushed ice to incubate for 10 min. Samples were then centrifuged for 10 min, and the supernatant was transferred to a 1.5-ml Eppendorf tube; the procedure was repeated for a second round of extraction. A total of 1 ml of supernatant was then centrifuged, and a final 500-μl aliquot of the homogenate was pipetted to a clean Eppendorf tube, to which 44 μl of 15% NH4HCO3 was added to neutralize the acid in the buffer. This was the final extract and was ready to be loaded to instrument vials for analysis.

UHPLC conditions: The HILIC separation was performed on a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA) with XBridge BEH Amide column (150 mm by 2.1 mm, 2.5-μm particle size; Waters, Milford, MA) using a gradient of solvent A [95%:5% H2O:acetonitrile with 20 mM acetic acid and 40 mM ammonium hydroxide (pH 9.4)] and solvent B [20%:80% H2O:acetonitrile with 20 mM acetic acid and 40 mM ammonium hydroxide (pH 9.4)]. The gradient was 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B. The flow rate was 300 μl/min. The injection volume was 5 μl, and the column temperature was 25°C. The autosampler temperature was set to 4°C, and the injection volume was 5 μl. Full-scan MS: The full-scan MS analysis was performed on a Thermo Fisher Scientific Q Exactive Plus with a HESI source, which was set to a spray voltage of −2.7 kV under negative mode and 3.5 kV under positive mode. The sheath, auxiliary, and sweep gas flow rates were 40, 10, and 2 (arbitrary unit), respectively. The capillary temperature was set to 300°C, and aux gas heater was 360°C. The S-lens radio frequency (RF) level was 45. The m/z scan range was set to 72 to 1000 m/z under both positive and negative ionization mode. The automatic gain control (AGC) target was set to 3e6, and the maximum injection time (IT) was 200 ms. The resolution was set to 70,000.

Parallel reaction monitoring MS: The MS2 spectra generation was performed on a Thermo Fisher Scientific Q Exactive Plus with a HESI source, which was set to a spray voltage of −2.7 kV under negative mode and 3.5 kV under positive mode. The sheath, auxiliary, and sweep gas flow rates were 40, 10, and 2 (arbitrary unit), respectively. The capillary temperature was set to 300°C, and aux gas heater was 360°C. The S-lens RF level was 45. The m/z scan ranges were specified for the four dipeptides and monitored for the full 22-min run time. The AGC target was set to 2 × 105, and the maximum IT was 100 ms. The resolution was set to 17,500. The isolation window was set to 2.0 m/z. Collision energy was set to a stepwise 30, 50, and 80 normalized collision energy (NCE). Our results suggest that a single MS2 spectrum may contain irrelevant m/z signals and the pseudospectrum, generated by correlating multiple MS2 spectra to the MS1-extracted ion chromatogram, is an effective approach to “clean” the MS2 spectrum. The pseudospectra were generated using a modified version of COVINA (Su et al. 2020).

Synthesis of standards: The dipeptide and tripeptide standards were synthesized and purified to 95% purity by GenScript USA (Piscataway, NJ). All standards were shipped at 25°C and stored at −20°C until they were prepared for analysis on the mass spectrometer. Parallel reaction monitoring was used to generate the MS2 spectra for comparison with samples. A total of 12 pmol of each standard was used in the analysis.