#METABOLOMICS WORKBENCH tangshuo_20251029_193146 DATATRACK_ID:6609 STUDY_ID:ST004329 ANALYSIS_ID:AN007215 PROJECT_ID:PR002743 VERSION 1 CREATED_ON November 4, 2025, 10:55 am #PROJECT PR:PROJECT_TITLE Acute heat stress redirects coral carbon budgets: integrative evidence from PR:PROJECT_TITLE physiology and metabolomic PR:PROJECT_SUMMARY Marine heatwaves increasingly disrupt coral carbon budgets, yet how thermal PR:PROJECT_SUMMARY stress reshapes whole-holobiont carbon fixation across species remains PR:PROJECT_SUMMARY insufficiently resolved. Here, paired carbon-flux components—total PR:PROJECT_SUMMARY photosynthetic carbon fixation (TPCF) and total calcification-associated carbon PR:PROJECT_SUMMARY fixation (TCCF)—were quantified in three reef-building corals from the PR:PROJECT_SUMMARY northern South China Sea (Acropora hyacinthus, Pocillopora damicornis, Porites PR:PROJECT_SUMMARY lutea) under controlled acute warming and were integrated with photophysiology PR:PROJECT_SUMMARY and untargeted metabolomics. Across taxa, TPCF declined with temperature in PR:PROJECT_SUMMARY concert with reduced PSII efficiency, whereas TCCF decreased in all species and, PR:PROJECT_SUMMARY under severe heat, shifted to net dissolution in the branching A. hyacinthus and PR:PROJECT_SUMMARY P. damicornis; by contrast, the thick-tissued P. lutea retained a marginally PR:PROJECT_SUMMARY positive calcification-associated flux. Metabolomic enrichment indicated PR:PROJECT_SUMMARY heat-induced rewiring of central carbon metabolism away from growth toward PR:PROJECT_SUMMARY maintenance and repair, with constrained photosynthate preferentially routed to PR:PROJECT_SUMMARY nucleotide biosynthesis (purine/pyrimidine), translational supply PR:PROJECT_SUMMARY (aminoacyl-tRNA), and membrane-lipid remodeling. These coordinated reallocations PR:PROJECT_SUMMARY provide a mechanistic basis for divergent calcification outcomes—preservation PR:PROJECT_SUMMARY of minimal accretion in P. lutea versus rapid stalling of light-enhanced PR:PROJECT_SUMMARY calcification and skeletal dissolution in branching taxa. At the community PR:PROJECT_SUMMARY scale, the contrasted strategies imply that repeated heatwaves may favor more PR:PROJECT_SUMMARY stress-tolerant, thick-tissued assemblages and erode carbonate-budget capacity PR:PROJECT_SUMMARY in branching communities. Although the metabolomics employed here capture PR:PROJECT_SUMMARY holobiont-level signals and do not partition host versus Symbiodiniaceae PR:PROJECT_SUMMARY contributions, the paired-flux–omics framework establishes a process-level PR:PROJECT_SUMMARY link between photosynthate constraint, carbon-allocation decisions, and PR:PROJECT_SUMMARY calcification outcomes, yielding tractable indicators for forecasting functional PR:PROJECT_SUMMARY resilience and evaluating interventions under intensifying thermal extremes. PR:INSTITUTE Chinese Academy of Sciences PR:LAST_NAME Tang PR:FIRST_NAME Shuo PR:ADDRESS Guangzhou, Guangdong, China PR:EMAIL tangshuo23@mails.ucas.ac.cn PR:PHONE 19875477513 #STUDY ST:STUDY_TITLE Acute heat stress redirects coral carbon budgets: integrative evidence from ST:STUDY_TITLE physiology and metabolomics ST:STUDY_SUMMARY Marine heatwaves increasingly disrupt coral carbon budgets, yet how thermal ST:STUDY_SUMMARY stress reshapes whole-holobiont carbon fixation across species remains ST:STUDY_SUMMARY insufficiently resolved. Here, paired carbon-flux components—total ST:STUDY_SUMMARY photosynthetic carbon fixation (TPCF) and total calcification-associated carbon ST:STUDY_SUMMARY fixation (TCCF)—were quantified in three reef-building corals from the ST:STUDY_SUMMARY northern South China Sea (Acropora hyacinthus, Pocillopora damicornis, Porites ST:STUDY_SUMMARY lutea) under controlled acute warming and were integrated with photophysiology ST:STUDY_SUMMARY and untargeted metabolomics. Across taxa, TPCF declined with temperature in ST:STUDY_SUMMARY concert with reduced PSII efficiency, whereas TCCF decreased in all species and, ST:STUDY_SUMMARY under severe heat, shifted to net dissolution in the branching A. hyacinthus and ST:STUDY_SUMMARY P. damicornis; by contrast, the thick-tissued P. lutea retained a marginally ST:STUDY_SUMMARY positive calcification-associated flux. Metabolomic enrichment indicated ST:STUDY_SUMMARY heat-induced rewiring of central carbon metabolism away from growth toward ST:STUDY_SUMMARY maintenance and repair, with constrained photosynthate preferentially routed to ST:STUDY_SUMMARY nucleotide biosynthesis (purine/pyrimidine), translational supply ST:STUDY_SUMMARY (aminoacyl-tRNA), and membrane-lipid remodeling. These coordinated reallocations ST:STUDY_SUMMARY provide a mechanistic basis for divergent calcification outcomes—preservation ST:STUDY_SUMMARY of minimal accretion in P. lutea versus rapid stalling of light-enhanced ST:STUDY_SUMMARY calcification and skeletal dissolution in branching taxa. At the community ST:STUDY_SUMMARY scale, the contrasted strategies imply that repeated heatwaves may favor more ST:STUDY_SUMMARY stress-tolerant, thick-tissued assemblages and erode carbonate-budget capacity ST:STUDY_SUMMARY in branching communities. Although the metabolomics employed here capture ST:STUDY_SUMMARY holobiont-level signals and do not partition host versus Symbiodiniaceae ST:STUDY_SUMMARY contributions, the paired-flux–omics framework establishes a process-level ST:STUDY_SUMMARY link between photosynthate constraint, carbon-allocation decisions, and ST:STUDY_SUMMARY calcification outcomes, yielding tractable indicators for forecasting functional ST:STUDY_SUMMARY resilience and evaluating interventions under intensifying thermal extremes. ST:INSTITUTE Chinese Academy of Sciences ST:LAST_NAME Tang ST:FIRST_NAME Shuo ST:ADDRESS Guangzhou, Guangdong, China ST:EMAIL tangshuo23@mails.ucas.ac.cn ST:PHONE 19875477513 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Acropora hyacinthus, Pocillopora damicornis, Porites lutea SU:TAXONOMY_ID 55974,46731,51062 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - QC-1 Sample source:holobiont | treatment:- Species=-; RAW_FILE_NAME(Raw file name)=QC-1.mzXML SUBJECT_SAMPLE_FACTORS - QC-2 Sample source:holobiont | treatment:- Species=-; RAW_FILE_NAME(Raw file name)=QC-2.mzXML SUBJECT_SAMPLE_FACTORS - QC-3 Sample source:holobiont | treatment:- Species=-; RAW_FILE_NAME(Raw file name)=QC-3.mzXML SUBJECT_SAMPLE_FACTORS - QC-4 Sample source:holobiont | treatment:- Species=-; RAW_FILE_NAME(Raw file name)=QC-4.mzXML SUBJECT_SAMPLE_FACTORS - AH27_1 Sample source:holobiont | treatment:control Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH27_1.mzXML SUBJECT_SAMPLE_FACTORS - AH27_2 Sample source:holobiont | treatment:control Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH27_2.mzXML SUBJECT_SAMPLE_FACTORS - AH27_3 Sample source:holobiont | treatment:control Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH27_3.mzXML SUBJECT_SAMPLE_FACTORS - AH27_4 Sample source:holobiont | treatment:control Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH27_4.mzXML SUBJECT_SAMPLE_FACTORS - AH27_5 Sample source:holobiont | treatment:control Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH27_5.mzXML SUBJECT_SAMPLE_FACTORS - AH27_6 Sample source:holobiont | treatment:control Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH27_6.mzXML SUBJECT_SAMPLE_FACTORS - AH31_1 Sample source:holobiont | treatment:moderate heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH31_1.mzXML SUBJECT_SAMPLE_FACTORS - AH31_2 Sample source:holobiont | treatment:moderate heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH31_2.mzXML SUBJECT_SAMPLE_FACTORS - AH31_3 Sample source:holobiont | treatment:moderate heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH31_3.mzXML SUBJECT_SAMPLE_FACTORS - AH31_4 Sample source:holobiont | treatment:moderate heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH31_4.mzXML SUBJECT_SAMPLE_FACTORS - AH31_5 Sample source:holobiont | treatment:moderate heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH31_5.mzXML SUBJECT_SAMPLE_FACTORS - AH31_6 Sample source:holobiont | treatment:moderate heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH31_6.mzXML SUBJECT_SAMPLE_FACTORS - AH34_1 Sample source:holobiont | treatment:severe heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH34_1.mzXML SUBJECT_SAMPLE_FACTORS - AH34_2 Sample source:holobiont | treatment:severe heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH34_2.mzXML SUBJECT_SAMPLE_FACTORS - AH34_3 Sample source:holobiont | treatment:severe heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH34_3.mzXML SUBJECT_SAMPLE_FACTORS - AH34_4 Sample source:holobiont | treatment:severe heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH34_4.mzXML SUBJECT_SAMPLE_FACTORS - AH34_5 Sample source:holobiont | treatment:severe heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH34_5.mzXML SUBJECT_SAMPLE_FACTORS - AH34_6 Sample source:holobiont | treatment:severe heat stress Species=Acropora hyacinthus; RAW_FILE_NAME(Raw file name)=AH34_6.mzXML SUBJECT_SAMPLE_FACTORS - PD27_1 Sample source:holobiont | treatment:control Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD27_1.mzXML SUBJECT_SAMPLE_FACTORS - PD27_2 Sample source:holobiont | treatment:control Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD27_2.mzXML SUBJECT_SAMPLE_FACTORS - PD27_3 Sample source:holobiont | treatment:control Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD27_3.mzXML SUBJECT_SAMPLE_FACTORS - PD27_4 Sample source:holobiont | treatment:control Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD27_4.mzXML SUBJECT_SAMPLE_FACTORS - PD27_5 Sample source:holobiont | treatment:control Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD27_5.mzXML SUBJECT_SAMPLE_FACTORS - PD27_6 Sample source:holobiont | treatment:control Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD27_6.mzXML SUBJECT_SAMPLE_FACTORS - PD31_1 Sample source:holobiont | treatment:moderate heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD31_1.mzXML SUBJECT_SAMPLE_FACTORS - PD31_2 Sample source:holobiont | treatment:moderate heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD31_2.mzXML SUBJECT_SAMPLE_FACTORS - PD31_3 Sample source:holobiont | treatment:moderate heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD31_3.mzXML SUBJECT_SAMPLE_FACTORS - PD31_4 Sample source:holobiont | treatment:moderate heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD31_4.mzXML SUBJECT_SAMPLE_FACTORS - PD31_5 Sample source:holobiont | treatment:moderate heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD31_5.mzXML SUBJECT_SAMPLE_FACTORS - PD31_6 Sample source:holobiont | treatment:moderate heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD31_6.mzXML SUBJECT_SAMPLE_FACTORS - PD34_1 Sample source:holobiont | treatment:severe heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD34_1.mzXML SUBJECT_SAMPLE_FACTORS - PD34_2 Sample source:holobiont | treatment:severe heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD34_2.mzXML SUBJECT_SAMPLE_FACTORS - PD34_3 Sample source:holobiont | treatment:severe heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD34_3.mzXML SUBJECT_SAMPLE_FACTORS - PD34_4 Sample source:holobiont | treatment:severe heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD34_4.mzXML SUBJECT_SAMPLE_FACTORS - PD34_5 Sample source:holobiont | treatment:severe heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD34_5.mzXML SUBJECT_SAMPLE_FACTORS - PD34_6 Sample source:holobiont | treatment:severe heat stress Species=Pocillopora damicornis; RAW_FILE_NAME(Raw file name)=PD34_6.mzXML SUBJECT_SAMPLE_FACTORS - PL27_1 Sample source:holobiont | treatment:control Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL27_1.mzXML SUBJECT_SAMPLE_FACTORS - PL27_2 Sample source:holobiont | treatment:control Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL27_2.mzXML SUBJECT_SAMPLE_FACTORS - PL27_3 Sample source:holobiont | treatment:control Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL27_3.mzXML SUBJECT_SAMPLE_FACTORS - PL27_4 Sample source:holobiont | treatment:control Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL27_4.mzXML SUBJECT_SAMPLE_FACTORS - PL27_5 Sample source:holobiont | treatment:control Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL27_5.mzXML SUBJECT_SAMPLE_FACTORS - PL27_6 Sample source:holobiont | treatment:control Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL27_6.mzXML SUBJECT_SAMPLE_FACTORS - PL31_1 Sample source:holobiont | treatment:moderate heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL31_1.mzXML SUBJECT_SAMPLE_FACTORS - PL31_2 Sample source:holobiont | treatment:moderate heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL31_2.mzXML SUBJECT_SAMPLE_FACTORS - PL31_3 Sample source:holobiont | treatment:moderate heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL31_3.mzXML SUBJECT_SAMPLE_FACTORS - PL31_4 Sample source:holobiont | treatment:moderate heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL31_4.mzXML SUBJECT_SAMPLE_FACTORS - PL31_5 Sample source:holobiont | treatment:moderate heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL31_5.mzXML SUBJECT_SAMPLE_FACTORS - PL31_6 Sample source:holobiont | treatment:moderate heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL31_6.mzXML SUBJECT_SAMPLE_FACTORS - PL34_1 Sample source:holobiont | treatment:severe heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL34_1.mzXML SUBJECT_SAMPLE_FACTORS - PL34_2 Sample source:holobiont | treatment:severe heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL34_2.mzXML SUBJECT_SAMPLE_FACTORS - PL34_3 Sample source:holobiont | treatment:severe heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL34_3.mzXML SUBJECT_SAMPLE_FACTORS - PL34_4 Sample source:holobiont | treatment:severe heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL34_4.mzXML SUBJECT_SAMPLE_FACTORS - PL34_5 Sample source:holobiont | treatment:severe heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL34_5.mzXML SUBJECT_SAMPLE_FACTORS - PL34_6 Sample source:holobiont | treatment:severe heat stress Species=Porites lutea; RAW_FILE_NAME(Raw file name)=PL34_6.mzXML #COLLECTION CO:COLLECTION_SUMMARY Coral samples for this study were collected in September 2024 from three CO:COLLECTION_SUMMARY reef-building species: Acropora hyacinthus, Pocillopora damicornis, and Porites CO:COLLECTION_SUMMARY lutea, sourced from the Luhuitou Fringing Reef in Sanya Bay, Hainan, China CO:COLLECTION_SUMMARY (18°12′N, 109°28′E). The corals were harvested from approximately 3 meters CO:COLLECTION_SUMMARY depth in the reef ecosystem. Three colonies were obtained for each species, with CO:COLLECTION_SUMMARY each colony fragmented into 2-cm nubbins, resulting in a total of 36 nubbins per CO:COLLECTION_SUMMARY species. These nubbins were carefully selected from non-marginal, visibly CO:COLLECTION_SUMMARY healthy portions of the colonies. Each nubbin was fixed to a numbered ceramic CO:COLLECTION_SUMMARY plug using cyanoacrylate gel and allowed to acclimate for 7 days in the CO:COLLECTION_SUMMARY station’s indoor coral aquaculture system. This acclimatization was conducted CO:COLLECTION_SUMMARY to minimize stress caused by fragmentation and handling, replicating natural CO:COLLECTION_SUMMARY environmental conditions as closely as possible, including temperature control, CO:COLLECTION_SUMMARY light intensity, and water flow. Upon completion of the acclimation period, the CO:COLLECTION_SUMMARY nubbins were randomly assigned to one of three temperature treatment groups CO:COLLECTION_SUMMARY (control, moderate heat, severe heat) and placed in separate 12-L tanks under CO:COLLECTION_SUMMARY controlled conditions. The formal experiment, spanning from September 24 to CO:COLLECTION_SUMMARY September 30, 2024, exposed the coral nubbins to varying temperatures: 27°C CO:COLLECTION_SUMMARY (control), 31°C (moderate heat), and 34°C (severe heat). The water temperature CO:COLLECTION_SUMMARY was monitored and adjusted to reflect the target conditions, with temperature CO:COLLECTION_SUMMARY data logged every 15 minutes. The experimental setup aimed to assess the CO:COLLECTION_SUMMARY physiological and metabolic responses of these coral species under heat stress CO:COLLECTION_SUMMARY conditions, integrating measurements of photosynthetic carbon fixation, CO:COLLECTION_SUMMARY calcification, and metabolomic shifts. This study focused on understanding CO:COLLECTION_SUMMARY species-specific adaptations to thermal stress by measuring changes in both CO:COLLECTION_SUMMARY metabolic fluxes and symbiotic carbon allocation. CO:SAMPLE_TYPE Coral holobiont #TREATMENT TR:TREATMENT_SUMMARY Treatment Summary: The experiment was conducted to assess the effects of acute TR:TREATMENT_SUMMARY heat stress on coral physiology and metabolism across three reef-building coral TR:TREATMENT_SUMMARY species: Acropora hyacinthus, Pocillopora damicornis, and Porites lutea. The TR:TREATMENT_SUMMARY coral nubbins were subjected to three distinct temperature treatments: control TR:TREATMENT_SUMMARY (27°C), moderate heat stress (31°C), and severe heat stress (34°C). TR:TREATMENT_SUMMARY Acclimation and Experimental Setup: Coral nubbins (36 per species) were TR:TREATMENT_SUMMARY acclimated for 7 days in the laboratory’s flow-through seawater system, TR:TREATMENT_SUMMARY mimicking natural conditions. The system utilized seawater drawn from 5 meters TR:TREATMENT_SUMMARY depth, filtered through a 0.45-μm cartridge and UV sterilized. After TR:TREATMENT_SUMMARY acclimation, nubbins were randomly assigned to three temperature treatment TR:TREATMENT_SUMMARY groups: Control (27°C): This represents the annual mean seawater temperature in TR:TREATMENT_SUMMARY Sanya. Moderate Heat Stress (31°C): Simulating moderate heat stress scenarios, TR:TREATMENT_SUMMARY this treatment represents a 4°C increase over the control. Severe Heat Stress TR:TREATMENT_SUMMARY (34°C): Simulating extreme heat stress, this treatment represents a 7°C TR:TREATMENT_SUMMARY increase over the control. Thermal Regimes: Temperature was ramped from the TR:TREATMENT_SUMMARY control temperature (27°C) to the moderate heat stress (31°C) and severe heat TR:TREATMENT_SUMMARY stress (34°C) conditions over a 6-hour period on the morning of September 24, TR:TREATMENT_SUMMARY 2024. These temperatures were maintained for the subsequent 7 days. During this TR:TREATMENT_SUMMARY period, water temperature was recorded every 15 minutes using HOBO data loggers. TR:TREATMENT_SUMMARY Light and Circulation Conditions: All tanks were illuminated with full-spectrum TR:TREATMENT_SUMMARY fluorescent lamps (Giesemann, Germany), providing light at approximately 120 TR:TREATMENT_SUMMARY μmol photons m⁻² s⁻¹ on a 12-hour light/dark cycle, simulating natural TR:TREATMENT_SUMMARY irradiance at 3 meters depth. Water circulation was maintained at ~350 L/h using TR:TREATMENT_SUMMARY submersible pumps (AT101S, Atman, Beijing, China). Sampling and Measurements: At TR:TREATMENT_SUMMARY the end of the experimental period (September 30, 2024), coral nubbins were TR:TREATMENT_SUMMARY assessed for their physiological responses to the thermal stress treatments, TR:TREATMENT_SUMMARY including net photosynthetic carbon fixation (NCP), respiratory carbon TR:TREATMENT_SUMMARY consumption (CR), and calcification (both light and dark calcification rates). TR:TREATMENT_SUMMARY These parameters were quantified using the total alkalinity anomaly method for TR:TREATMENT_SUMMARY calcification and dissolved inorganic carbon (DIC) changes for NCP and CR. TR:TREATMENT_SUMMARY Additionally, metabolomic profiling was conducted to capture the reprogramming TR:TREATMENT_SUMMARY of the coral holobiont's metabolic pathways under heat stress conditions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Biological material & design: Whole coral holobionts (host + Symbiodiniaceae) SP:SAMPLEPREP_SUMMARY from three scleractinians—Acropora hyacinthus (AH), Pocillopora damicornis SP:SAMPLEPREP_SUMMARY (PD), Porites lutea (PL)—after 7-day exposure to 27°C (control), 31°C SP:SAMPLEPREP_SUMMARY (acute moderate), or 34°C (acute severe). Nominal n = 6 biological replicates SP:SAMPLEPREP_SUMMARY per species × temperature (total n = 54). Harvesting & snap-freezing: To avoid SP:SAMPLEPREP_SUMMARY handling artifacts associated with day-5 incubations, nubbins were snap-frozen SP:SAMPLEPREP_SUMMARY directly from treatment tanks at the end of day 7 (late afternoon; matched to SP:SAMPLEPREP_SUMMARY photoperiod). Pulverization: Tissues were powdered under liquid N₂ to a fine SP:SAMPLEPREP_SUMMARY cryopowder. Extraction: Weighed 100 mg cryopowder was extracted with 1.0 mL SP:SAMPLEPREP_SUMMARY MeOH/ACN/H₂O (2:2:1, v/v/v). Extracts were sonicated on ice (2 × 30 min) and SP:SAMPLEPREP_SUMMARY centrifuged (14,000 g, 20 min, 4°C); supernatants were collected. Dry-down & SP:SAMPLEPREP_SUMMARY reconstitution: Supernatants were dried under vacuum (SpeedVac) and SP:SAMPLEPREP_SUMMARY reconstituted in 100 µL ACN/H₂O (1:1, v/v), then clarified prior to SP:SAMPLEPREP_SUMMARY LC–MS/MS. Injection volume: 2 µL. Quality control & blanks: A pooled QC SP:SAMPLEPREP_SUMMARY sample was prepared by combining 10 µL from each extract and injected every SP:SAMPLEPREP_SUMMARY five injections; solvent blanks were interleaved. Run order: Within each SP:SAMPLEPREP_SUMMARY chromatography/ionization mode (HILIC/RPLC; ESI⁺/ESI⁻), samples were SP:SAMPLEPREP_SUMMARY randomized across species and temperatures. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1 mm, 1.7 µm) CH:SOLVENT_A 100% Water; 25 mM Ammonium acetate; 25 mM Ammonium hydroxide CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 0–0.5 min: 95% B 0.5–7.0 min: linear from 95% B to 65% B 7.0–8.0 min: CH:FLOW_GRADIENT linear from 65% B to 40% B 8.0–9.0 min: hold at 40% B 9.0–9.1 min: return to CH:FLOW_GRADIENT 95% B 9.1–12.0 min: re-equilibration at 95% B CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 25°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500+ QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS acquisition Comments: Comments: "Data acquired using an AB Sciex 6500+ QTrap MS:MS_COMMENTS Triple Quadrupole mass spectrometer in both positive and negative ESI modes. MS:MS_COMMENTS Positive mode for hydrophilic interaction chromatography (HILIC) and negative MS:MS_COMMENTS mode for reversed-phase chromatography (RPLC) as per the untargeted metabolomics MS:MS_COMMENTS protocol. The system was set to acquire data over the m/z range of 60–1,000 Da MS:MS_COMMENTS for TOF-MS scans and 25–1,000 Da for MS/MS scans in IDA mode." Data processing MS:MS_COMMENTS Comments: Comments: "LC-MS data were processed using the ProteoWizard MSConvert MS:MS_COMMENTS tool to convert raw data from wiff to mzXML format. The data were processed with MS:MS_COMMENTS XCMS using centWave (peak detection: 10 ppm, peak width: 10–60 sec) and CAMERA MS:MS_COMMENTS for isotope/adduct annotation. Features with >50% non-zero values in at least MS:MS_COMMENTS one group were retained for further analysis. QC data was incorporated to MS:MS_COMMENTS monitor instrument performance, and ComBat was applied after QC-RLSC for signal MS:MS_COMMENTS drift correction." Software/procedures used for feature assignments: Comments: MS:MS_COMMENTS "Feature identification was based on the comparison of the measured m/z values MS:MS_COMMENTS with an in-house database and public databases such as MassBank, METLIN, and MS:MS_COMMENTS MoNA. Theoretical fragmentation patterns were matched to assign putative MS:MS_COMMENTS identities. MSI Level 1 was reported for authentic standards, and MSI Level 2 MS:MS_COMMENTS for unconfirmed features. Data were normalized to the total ion count (TIC) for MS:MS_COMMENTS each sample." #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples AH27_1 AH27_2 AH27_3 AH27_4 AH27_5 AH27_6 PD27_1 PD27_2 PD27_3 PD27_4 PD27_5 PD27_6 PL27_1 PL27_2 PL27_3 PL27_4 PL27_5 PL27_6 AH31_1 AH31_2 AH31_3 AH31_4 AH31_5 AH31_6 PD31_1 PD31_2 PD31_3 PD31_4 PD31_5 PD31_6 PL31_1 PL31_2 PL31_3 PL31_4 PL31_5 PL31_6 AH34_1 AH34_2 AH34_3 AH34_4 AH34_5 AH34_6 PD34_1 PD34_2 PD34_3 PD34_4 PD34_5 PD34_6 PL34_1 PL34_2 PL34_3 PL34_4 PL34_5 PL34_6 QC-1 QC-2 QC-3 QC-4 Factors Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:control Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:moderate heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:severe heat stress Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Palmitoleic acid 451128516.6 417428245.3 479017424.3 669288931 551040433.6 436892302.2 2175179225 2021781976 1036842828 2352679626 1921410388 1914185838 720901254.3 756574724.9 615789563.5 638407414.1 963053567 836448496.8 417818768.2 426404307 629008812.8 995299563.3 360775215.3 481132192.3 2290551681 2190994098 2774241803 2020913377 2754998130 2462366159 901336106.4 792213418 650325822.5 955575984.7 1045975687 857396372.1 1063134324 2228939993 1327778512 817980756.2 450678443.6 470380312.8 1471615358 1200117039 1225778028 807133169.7 1187507422 482525911.7 1118827722 572081017.3 894534930.4 1127988136 653158876.4 888502767.5 972832188.8 1168030933 1119401367 1098164880 L-Aspartic acid 62419631.77 13278028.43 21472641.62 2359860.239 16223765.32 33966026.29 33058929.57 38935312.31 1263587.476 12466289.1 4591191.538 971080.128 120038943.4 141384974.2 192956082.7 114595646.5 167719274.9 104108467.9 37836513.25 21676740.73 21266837.65 4949994.567 22606520.21 323418.1473 10783334.02 222910621 2553004.067 14229671.9 10999888.38 7599531.681 103577629.7 143093377 113626372.1 143400056.7 179620358.1 126751464.8 2562657.639 738596.4817 3461543.521 11875424.5 29628706.53 44041976.93 56904516 3613471.953 120048410.3 3796343.711 2771764.355 18700114.16 32645762.63 93257817.44 118961200.9 79657926.7 14827478.08 139688143.7 58854475.68 32762552.58 65192113.78 58503445.33 L-Glutamic acid 563495253.9 260827550 344727354.2 48814810.8 236790837.7 399079982.1 223679105.1 240404828 7510565.633 66028679.57 29398216.48 5839053.786 504446583.9 547332074.5 628846835.8 460268256.3 509139987.3 382838052 390300734.3 262373696.1 331129056.6 110092582.6 426301750.3 11831070.13 77184951.34 780485295.3 17705173.79 94878353.93 84039010.94 54375539.17 544307969.4 608434319.8 426924397.6 659610069.9 537162398.7 502329286.9 2879634.172 1973019.415 29476394.13 169515290.5 491614034.1 530804985.5 363634250.7 23844775.25 681621733.3 21190223.4 13526647.38 143620001.4 285626096.2 399805333.3 430954422.6 330291074 114291921 183099912.1 318521338.8 305090436 321263592.6 309134011.7 L-Threonine 26888200.62 26442237.92 19160243.35 13282479.33 18014529.44 21069780.16 30860373.77 30533722.41 4833225.374 22998753.55 7161538.114 4443060.188 10893206.34 10271856.84 2719499.687 11765489.91 7836030.196 9309329.643 44140594.76 27767331.9 26923088.07 17500352.27 21269138.19 9495873.831 28811624.63 54965517.67 10036584.33 26857167.65 22871645.34 22404740.47 6675702.984 16422655.7 3194668.774 1777237.246 3325811.914 4118653.795 390967.0446 556626.7761 7670152.571 24355507.31 30958769.32 27842200.9 107585221.7 46153864.4 106059268.8 29078039.83 20295816.28 117617382.5 11498926.43 5090839.976 2574963.154 12861644.5 8599642.736 12459810.52 15026168.09 14260516.89 14002644.96 14091527.74 L-Tryptophan 84801152.91 145731364.7 63112017.16 230761175.2 139254079.5 117612689.1 306763425.9 275297935.5 20741605.54 335845977.1 79684743.25 65516892.99 795533036.2 867302160.6 1085252984 799054424.9 14260366.37 699294125.1 98022510.05 135136985.2 119458275 191577782.9 219093295.7 202226897.4 385285323.4 357172443.8 142765995.4 404058521.8 240091121.7 289171078.4 525872930.7 848780379.3 756883504.9 1004552542 756913350 840473059.7 66425295.5 184173463.5 155807086.5 145165601.6 182453984 334265488.2 696819823.1 601718502.8 637050531.8 915424928.7 612032428.3 1024163403 548923017.5 425252056.3 696015211.7 5747330.972 131185987.3 2752705.312 364672583.4 365712892 369447797.5 359049225.2 L-Asparagine 4515638.547 2425689.634 3188098.023 1658621.151 2987529.239 3833796.148 7314312.956 7274631.413 950729.3522 2863881.961 1651158.878 13667.30247 5196030.019 5768614.457 8093797.951 5319171.119 1137237.483 4509224.303 3618804.925 3073569.747 2775619.391 1777393.777 2756684.407 173090.8157 4185126.874 12740654.37 478907.5565 5386141.258 3973505.561 3308609.609 4803981.752 5596181.672 5376417.178 6656826.604 492722.8243 5342303.471 3605794.27 5692821.633 47701.38074 2762458.204 4506197.602 3587276.098 16317330.43 3293197.25 24089231.66 2586325.235 451667.3967 18124581.44 3435601.452 2808651.395 2346898.95 546349.0171 361722.7718 663281.593 3336391.395 3694307.277 3601017.262 3129098.166 Chenodeoxycholic acid 20110749.62 21533061.41 12593027.2 15768963.34 24226661.25 11064019.51 17393492.24 14604764.76 183558.7323 18733127.43 13450029 12268661.37 35888.34905 33209.19964 91889.51484 4076391.989 5471863.042 38506 32599279.08 20523297.65 21354700.75 19532913.59 29419031.24 15229197.9 18606614.21 16629791.46 6529984.43 22105707.63 16841912.98 10620642.09 84433.53548 23023.09349 72435.61246 30188.39465 41040.17297 61608.93035 4097446.17 627148.0112 422290.8597 24708120.53 30548469.26 40907314.59 28179579.1 25738630.01 37273686.2 59659297.87 37829623.66 30825008.41 254793.8753 199561.3602 55926.61942 39955.28157 167851.3469 224986.0845 10189461.65 11683741.16 11126515.27 11789487.48 Beta-Muricholic acid 604924.1395 667503.5204 182272.2458 403479.4724 382788.1962 132434.1826 1363645.752 1861881.311 776832.8049 2080091.003 1242901.793 1248592.203 1260228.226 742676.2322 1103486.123 609549.2769 668383.9483 805233.7033 492211.7106 753529.4697 788115.4971 650347.8838 503589.1194 945540.2769 2745313.371 4005346.42 1446049.747 2691386.726 2199495.858 1628805.526 813487.3464 1087686.703 1477273.265 9778996.24 563387.3645 2209856.609 10870594.07 3395186.461 8071278.61 1226668.178 826547.3228 629330.4386 2913915.966 4037249.723 3159127.522 3488719.354 2843029.632 3130872.535 1344455.732 1454775.675 1026322.484 1870836.869 2327655.593 1761354.195 2403801.465 1835831.034 2002400.929 1763073.494 homo-gamma-Linolenic acid 122886848.9 203056366.2 143615759.2 430239649.4 282599545.7 236281800.9 1795679644 1313671223 739634601.5 2363382009 1371720149 1483072270 475888355 415349176.6 406451452.1 444371605.1 822126560.4 544120932.6 324555048.6 255184715.4 295565564.3 597158958.6 327740543.3 148535800.5 1609833022 1239234034 2420714626 1330767050 1745509148 1602037162 841211471.2 893743555.8 690375131.7 849649305.2 1212039317 804798117 223848145.6 358602799.5 466882789 400991619.5 363981858.8 256765865.7 643477812.4 477708072.2 615644833.4 769909987.2 496096889.3 494627271.2 702547113.6 705542610.6 1125810681 763884838.9 257091437.3 613009969.2 989463536.3 953024226.4 964354666.8 844734803.8 Taurocholic acid 30840.77236 642873.4364 50685.97979 530558.7798 40043.72846 712588.9482 84135.12025 335574.9173 392629.0847 501907.4236 498217.106 170398.4196 99362.56718 1092600.214 188104.5723 154350.2953 133844.6382 171916.087 480594.9546 44918.32302 498496.8022 85220.88923 80691.61931 96304.0183 165308.449 80556.97254 433040.7459 531829.9623 165135.5685 86587.12355 672986.062 370264.1489 223013.3046 167444362.6 212735.8699 11333258.94 4224499.083 722928.8382 3355709.086 490120.0013 75847.43729 65309.54907 38695.31055 311863.2295 112137.6425 504121.5389 274418.502 225823.2408 114863.2457 611049.3009 107911.6344 122362.2176 757281.1347 1525729.24 3050041.835 3245693.504 3049662.474 1944944.084 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z  HMDB ID KEGG ID Palmitoleic acid 9.782 253.21728 HMDB0003229 C08362 L-Aspartic acid 1.432 132.03002 HMDB0000191 C00049 L-Glutamic acid 1.376 146.0457 HMDB0000148 C00025 L-Threonine 1.36 118.05083 HMDB0000167 C00188 L-Tryptophan 5.19 203.08254 HMDB0000929 C00078 L-Asparagine 1.383 113.03549 HMDB0000168 C00152 Chenodeoxycholic acid 7.787 391.28521 HMDB0000518 C02528 Beta-Muricholic acid 7.305 407.28042 HMDB0000415 C17726 homo-gamma-Linolenic acid 10.385 305.24865 HMDB0002925 C03242 Taurocholic acid 6.038 514.28418 HMDB0000036 C05122 METABOLITES_END #END