#METABOLOMICS WORKBENCH tangshuo_20251029_193146 DATATRACK_ID:6609 STUDY_ID:ST004329 ANALYSIS_ID:AN007214 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 POSITIVE 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:- L-Lysine 742494.9412 398893.4165 360173.8502 674492.7045 6541404.489 3568195.926 293336661.5 292531055.6 180733344.1 160315264 120487294.9 70419174.07 1171193.377 147721710.2 96013376.66 146412531.2 9412078.526 3962739.626 221591.8211 466595.7783 4800396.13 264690.6978 174007.7297 3663879.517 228175736.5 420267535.9 96181873.88 262433042.6 194783905.8 197211229.6 86403618.54 2873936.378 1122798.659 204715987.8 139271335.1 79447388.66 137968046.6 128995979 7335539.323 363560.4881 436877.9298 27349.4266 581050712.1 220702782.4 714942474.7 73662405.23 120626160.1 606219689.4 161158463.8 98772954.06 133612651.7 7555271.917 58887320.77 3910879.218 93147467.78 84928144.47 88680282.3 87796214 L-Arginine 11014853.22 7359261.161 13829530.35 9346327.52 9782692.491 12368955.59 1275886423 1291891030 834188981 640674990.2 644794351.2 483572778.8 2243924.607 7729231.969 6139233.373 5971248.024 8756761.608 6684516.656 4868585.712 6362084.148 6345707.72 7509802.906 19272408.43 37117467.13 1093104269 1812989831 451803454 1177458308 756058580.7 924797029 2994431.94 4866592.743 7176364.376 6271306.51 3898665.836 5260843.743 15752225.58 30679953.25 61244404.38 20919694.25 10235353.19 8105769.341 2306616507 1181373019 2704463800 743875703 958188608.9 2287792812 5747899.031 5453130.423 6102602.088 12862657.14 6833369.801 12949302.33 301013699.1 277058662.2 281573535.9 283909971.1 L-Phenylalanine 104384717.2 102520139.5 35131377.94 91585165.44 170537658.1 100367575.7 8949009781 9537751795 3046604620 7622567287 3014607365 3747007348 3585371834 4239277273 5531315111 3238159747 1685182179 3487811820 63039261.27 100902735 94845135.37 58479444.48 114385691.9 340445246.5 10401344093 10981071914 5620930945 9061205992 7542466743 8726486560 1698479839 3274216565 3964855834 5745441257 3674666167 4156135267 316057931.8 1182055425 602856694.5 63942572.27 64143458.02 40875836.99 12430394310 9175198302 13091999681 10500328772 11126910031 11510378374 3672753047 1947294867 2716892217 49390893.84 1283986693 34773761.8 3766845398 3620432563 3748084667 3680065124 L-Tyrosine 19389963.38 16657449.61 16452965.36 13247072.93 15476813.41 12089554.13 2344202004 2850018401 381439720.5 2009029468 623649616.6 431454643.5 102255883.2 310892995.9 65261775.99 1203116309 553592188.7 1083294246 7232125.65 15797554.04 15224216.26 11208355.89 12874529.68 21673661.98 3042363974 821775297.7 912941534.9 1898483576 935642429.5 2377714779 474186435 97535190.35 58409742.88 639825948.8 670792040 56979480.89 68740144.58 226236840.9 108843545.8 12196448.4 15118257.59 8762818.246 2604545107 1251171270 2974295432 1708238686 1463193403 2441651620 912536029.5 438893622.8 46987889.19 19246448.31 647095678.8 21138752.36 616246132.2 587368975.7 607842128.3 428880148.3 Choline 70690200.05 106177819.3 150894876.2 146408011.2 81221378.4 57808313.47 146929416.1 155781035.9 247737380.3 183568551.8 248973720.7 295155912.1 6304640.762 4138827.201 3959084.398 4169462.561 2716842.191 5550416.545 197326707.7 186278853.1 168152633.5 83106424.48 39672192.7 18353147.4 218621594.8 195671090.6 280296496 281070487 185952789.1 190421047.9 16232698.06 8393497.234 8548097.242 10808809.48 10155216.79 6183933.669 16976429.55 12090728.17 3824209.746 61904719.2 262721422 140948874.8 413326654.5 452191992.3 270505483.6 544325551.6 552063309.7 1489125.819 7309586.637 24815286.29 5528892.156 10744579.35 21004440.75 18134163.87 154257356.3 216327478.1 243625244.4 239324679.9 L-Histidine 32573931.28 22198809.1 52618747.77 16815172.32 34684544.77 37109371.43 3449608.889 1688573.629 9659647.61 18700481.65 4118148.147 5763369.797 13261359.71 13242142.61 18684110.92 13654126.55 9158333.207 18464795.02 43253027.05 31396123.54 43630545.29 24428914.3 31388559.42 42149394.79 7386700.89 14600803.74 2087266.266 5160168.031 4128327.566 1405207.325 10718171.58 11436686.07 22630608.33 15020255.49 14259379.2 24392275.34 7702781.603 6642794.307 7381824.525 15859733.8 42908331.04 25905505.93 81966802.26 41884163.07 69742258.92 23388480.79 45091517.17 109290537.8 16116020.02 8371472.233 15352160.97 5815012.541 12733091.35 8896880.691 11744445.23 10957003.26 9750324.524 10482123.46 (S)-MALATE 4586056.264 5397860.823 6840512.912 5815363.122 4742230.732 5902739.667 4990312.16 4125596.576 4280928.651 4906621.322 5871516.374 6272976.933 5407372.397 8387915.588 5313268.39 6732229.618 8387248.384 5205764 5472763.919 4517805.326 5792182.212 5294618.801 4543729.626 4606252.877 3611044.436 2691931.834 4195391.25 2978089.717 2936419.358 3148072.62 8343597.907 5077432.646 3984685.596 4098814.288 7661649.924 3498509.699 28460692.1 28380458.07 9933787.354 4040556.742 4296193.489 3804582.991 5145999.177 5017276.898 3744904.44 9283605.362 9298529.901 5485022.788 4041596.971 5429106.842 5937735.682 9292629.232 2827279.761 5811746.536 6825806.186 4912857.874 4961252.127 3648300.529 Agmatine 1433167634 1146299917 1015150534 1354957516 1651402089 1726044811 22414210.39 9945640.93 44616726.31 2943754.675 209095008.7 215222125.8 1325777565 1381983340 1138488320 2169182219 1619115737 1665551157 1331235116 1119350361 933363140.8 1186354988 981713730.4 826670334.3 1291609.49 1215342.262 20878342.15 2056248.77 1542643.929 4498143.865 1032523688 1301175359 947322004.6 957713117.1 1344309282 771842191.1 121850607.5 67808905.03 24922806.33 1310017423 1397931468 919935006.7 4332409.778 12548524.98 2285008.04 11886316.76 36273444.7 7854872.148 847748590.7 844879190.4 1350090348 2550192757 620707360.1 1203619440 1059236362 992341874.5 940508723.7 992535766 cotinine-glucuronide 310203.6758 468428.8988 632066.71 445654.8744 232658.98 700729.0894 5724733.084 173591.4787 221937.4348 465547.2667 129110816.8 410047.5162 3449527.624 1285234.72 49986.32322 120306.1772 114650.8829 1441899.942 175823.2202 265109902.7 690908.5283 89515.75632 1021304.661 679823.0405 809907.5025 101539.5184 854549.3094 1092187.847 214645.3187 2988756.227 3514956.956 1816836.062 50472.70339 334574.6537 159797.5983 141513.866 7110850.992 950009.9085 1548861.79 356389.1846 376885.1892 555450.0453 1553645.896 725650.0633 801326.8868 2243995.785 18046031.14 55027864.37 358849.016 475696.6569 767900.366 1100283.87 59308.91013 181318.4511 5622754.015 5279397.821 5839869.074 3230576.984 Gluconolactone 2713322.43 3461763.532 3037303.737 1769286.275 4083357.743 4260251.857 258495.2905 682298.4685 7610116.35 3125600.38 4505413.793 4394815.361 18194590.77 26856546.17 29645123.79 26666968.78 36608112.67 32969342.28 3256631.916 5116891.592 4130344.238 5642996.202 6735554.641 505210.929 2156497.226 8176804.97 3402748.288 1892947.718 1977428.133 1807784.024 29790625.9 20666337.78 26909541.54 14625572.18 14858462.9 18182041.41 11568788.56 10713818.63 2789520.558 6470392.674 5128692.074 4090688.281 2641852.002 2089519.282 4237831.199 2272603.852 2827842.441 1477624.589 18212262.89 32902871.3 33216240.33 25412881.9 52280621.96 44897984.32 9039338.544 8199586.483 8115637.658 9253676.082 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z  HMDB ID KEGG ID L-Lysine 1.258 147.11284 HMDB0000182 C00047 L-Arginine 1.352 175.11896 HMDB0000517 C00062 L-Phenylalanine 4.896 166.08628 HMDB0000159 C00079 L-Tyrosine 2.436 182.08127 HMDB0000158 C00082 Choline 10.192 104.10695 HMDB0000097 C00114 L-Histidine 1.31 156.07675 HMDB0000177 C00135 (S)-MALATE 1.192 152.0556 HMDB0000156 C00149 Agmatine 1.307 131.12919 HMDB0001432 C00179 cotinine-glucuronide 6.016 353.13504 HMDB0001013 C00190 Gluconolactone 1.492 179.05496 HMDB0000150 C00198 METABOLITES_END #END