#METABOLOMICS WORKBENCH tangshuo_20250706_225747 DATATRACK_ID:6138 STUDY_ID:ST004143 ANALYSIS_ID:AN006867 PROJECT_ID:PR002606 VERSION 1 CREATED_ON August 22, 2025, 1:11 pm #PROJECT PR:PROJECT_TITLE National Key R&D Program Project(2022YFC3103602, 2021YFF0502800) PR:PROJECT_SUMMARY Soft corals (order Alcyonacea), characterized by minimal or absent skeletal PR:PROJECT_SUMMARY structures, have received far less attention compared to reef-building hard PR:PROJECT_SUMMARY corals (order Scleractinia), despite their vulnerability to heat stress. This PR:PROJECT_SUMMARY study investigated the thermal response mechanisms of two symbiotic corals, PR:PROJECT_SUMMARY Galaxea fascicularis (stony coral) and Clavularia inflata (soft coral), which PR:PROJECT_SUMMARY could be found in the same water region and depth in the South China Sea. Using PR:PROJECT_SUMMARY controlled heat stress experiments, we analyzed their photophysiological and PR:PROJECT_SUMMARY metabolomic response. While G. fascicularis maintained stable net photosynthetic PR:PROJECT_SUMMARY rates (Pnet) and increased dark respiration (Rd), C. inflata showed a decline in PR:PROJECT_SUMMARY Rd, a slight Pnet reduction, and a symbiont loss. Metabolic changes in G. PR:PROJECT_SUMMARY fascicularis were primarily focused on energy production (e.g., D-mannose, PR:PROJECT_SUMMARY D-sorbitol, and Succinate) and amino acid metabolism (e.g., D-proline, Glutamic PR:PROJECT_SUMMARY acid, and Histidine). In contrast, C. inflata exhibited a broader range of PR:PROJECT_SUMMARY metabolic shifts, including Glucosamine-6-phosphate (involved in energy PR:PROJECT_SUMMARY pathways), D-proline, Histidine, Tryptophan, and Taurine (amino acid PR:PROJECT_SUMMARY metabolism), Purines and Pyrimidines (nucleotide metabolism), Carnosic acid, PR:PROJECT_SUMMARY Linolenic acid, and Retinene (antioxidants), as well as the dipeptides His-Ser PR:PROJECT_SUMMARY and Lys-Lys. These findings revealed that metabolic adjustments played a key PR:PROJECT_SUMMARY role in thermotolerance in stony coral, while soft coral exhibited a more PR:PROJECT_SUMMARY diverse response and incurred energetic costs in response to thermal stress. PR:PROJECT_SUMMARY This research provided insights into lineage-specific resilience mechanisms, PR:PROJECT_SUMMARY shedding light on why soft corals may be less competitive under heatwaves and PR:PROJECT_SUMMARY contributing to our understanding of shifts in coral reef community composition PR:PROJECT_SUMMARY driven by climate change. PR:INSTITUTE South China Sea Institute of Oceanology, Chinese Academy of Sciences PR:LAST_NAME Yuan PR:FIRST_NAME Xiangcheng PR:ADDRESS Xingang West Road, Haizhu District, Guangzhou, Guangdong, 510000, China PR:EMAIL xcyuan@scsio.ac.cn PR:PHONE 18078826651 PR:FUNDING_SOURCE National Key R&D Program Project (2022YFC3103602, 2021YFF0502800). Major Program PR:FUNDING_SOURCE of National Natural Science Foundation of China (42494882). Hainan Province Key PR:FUNDING_SOURCE R&D Program Project (ZDYF2023SHFZ131). The Key Special Project for Introduced PR:FUNDING_SOURCE Talents Team of Southern Marine Science and Engineering Guangdong Laboratory PR:FUNDING_SOURCE (Guangzhou) (GML2019ZD0404). Natural Resources Science and Technology Strategy PR:FUNDING_SOURCE Research Project (2023-ZL-66). #STUDY ST:STUDY_TITLE Lineage-Specific Thermal Tolerance Mechanisms in Stony and Soft Corals: Insights ST:STUDY_TITLE from Photophysiology and Metabolomics ST:STUDY_SUMMARY Soft corals (order Alcyonacea), characterized by minimal or absent skeletal ST:STUDY_SUMMARY structures, have received far less attention compared to reef-building hard ST:STUDY_SUMMARY corals (order Scleractinia), despite their vulnerability to heat stress. This ST:STUDY_SUMMARY study investigated the thermal response mechanisms of two symbiotic corals, ST:STUDY_SUMMARY Galaxea fascicularis (stony coral) and Clavularia inflata (soft coral), which ST:STUDY_SUMMARY could be found in the same water region and depth in the South China Sea. Using ST:STUDY_SUMMARY controlled heat stress experiments, we analyzed their photophysiological and ST:STUDY_SUMMARY metabolomic response. While G. fascicularis maintained stable net photosynthetic ST:STUDY_SUMMARY rates (Pnet) and increased dark respiration (Rd), C. inflata showed a decline in ST:STUDY_SUMMARY Rd, a slight Pnet reduction, and a symbiont loss. Metabolic changes in G. ST:STUDY_SUMMARY fascicularis were primarily focused on energy production (e.g., D-mannose, ST:STUDY_SUMMARY D-sorbitol, and Succinate) and amino acid metabolism (e.g., D-proline, Glutamic ST:STUDY_SUMMARY acid, and Histidine). In contrast, C. inflata exhibited a broader range of ST:STUDY_SUMMARY metabolic shifts, including Glucosamine-6-phosphate (involved in energy ST:STUDY_SUMMARY pathways), D-proline, Histidine, Tryptophan, and Taurine (amino acid ST:STUDY_SUMMARY metabolism), Purines and Pyrimidines (nucleotide metabolism), Carnosic acid, ST:STUDY_SUMMARY Linolenic acid, and Retinene (antioxidants), as well as the dipeptides His-Ser ST:STUDY_SUMMARY and Lys-Lys. These findings revealed that metabolic adjustments played a key ST:STUDY_SUMMARY role in thermotolerance in stony coral, while soft coral exhibited a more ST:STUDY_SUMMARY diverse response and incurred energetic costs in response to thermal stress. ST:STUDY_SUMMARY This research provided insights into lineage-specific resilience mechanisms, ST:STUDY_SUMMARY shedding light on why soft corals may be less competitive under heatwaves and ST:STUDY_SUMMARY contributing to our understanding of shifts in coral reef community composition ST:STUDY_SUMMARY driven by climate change. ST:INSTITUTE South China Sea Institute of Oceanology, Chinese Academy of Sciences ST:LAST_NAME Yuan ST:FIRST_NAME Xiangcheng ST:ADDRESS Xingang West Road, Haizhu District, Guangzhou, Guangdong, 510000, China ST:EMAIL xcyuan@scsio.ac.cn ST:PHONE 18078826651 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Galaxea fascicularis, Clavularia inflata SU:TAXONOMY_ID 46745, 360968 #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:- RAW_FILE_NAME(Raw file name)=QC-1.raw SUBJECT_SAMPLE_FACTORS - QC-2 Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-2.raw SUBJECT_SAMPLE_FACTORS - QC-3 Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-3.raw SUBJECT_SAMPLE_FACTORS - QC-4 Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-4.raw SUBJECT_SAMPLE_FACTORS - QC-5 Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-5.raw SUBJECT_SAMPLE_FACTORS - QC-6 Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-6.raw SUBJECT_SAMPLE_FACTORS - RT0-1 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=RT0-1.raw SUBJECT_SAMPLE_FACTORS - RT0-2 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=RT0-2.raw SUBJECT_SAMPLE_FACTORS - RT0-3 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=RT0-3.raw SUBJECT_SAMPLE_FACTORS - RT0-4 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=RT0-4.raw SUBJECT_SAMPLE_FACTORS - RT0-5 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=RT0-5.raw SUBJECT_SAMPLE_FACTORS - RT0-6 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=RT0-6.raw SUBJECT_SAMPLE_FACTORS - RT1-1 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=RT1-1.raw SUBJECT_SAMPLE_FACTORS - RT1-2 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=RT1-2.raw SUBJECT_SAMPLE_FACTORS - RT1-3 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=RT1-3.raw SUBJECT_SAMPLE_FACTORS - RT1-4 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=RT1-4.raw SUBJECT_SAMPLE_FACTORS - RT1-5 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=RT1-5.raw SUBJECT_SAMPLE_FACTORS - RT1-6 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=RT1-6.raw SUBJECT_SAMPLE_FACTORS - QC-1_Discosoma Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-1_POS.wiff and QC-1_NEG.wiff SUBJECT_SAMPLE_FACTORS - QC-2_Discosoma Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-2_POS.wiff and QC-2_NEG.wiff SUBJECT_SAMPLE_FACTORS - QC-3_Discosoma Sample source:holobiont | treatment:- RAW_FILE_NAME(Raw file name)=QC-3_POS.wiff and QC-3_NEG.wiff SUBJECT_SAMPLE_FACTORS - CK-1 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=CK-1_POS.wiff and CK-1_NEG.wiff SUBJECT_SAMPLE_FACTORS - CK-2 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=CK-2_POS.wiff and CK-2_NEG.wiff SUBJECT_SAMPLE_FACTORS - CK-3 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=CK-3_POS.wiff and CK-3_NEG.wiff SUBJECT_SAMPLE_FACTORS - CK-4 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=CK-4_POS.wiff and CK-4_NEG.wiff SUBJECT_SAMPLE_FACTORS - CK-5 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=CK-5_POS.wiff and CK-5_NEG.wiff SUBJECT_SAMPLE_FACTORS - CK-6 Sample source:holobiont | treatment:control RAW_FILE_NAME(Raw file name)=CK-6_POS.wiff and CK-3_POS.wiff SUBJECT_SAMPLE_FACTORS - jjsw-1 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=jjsw-1_POS.wiff and jjsw-1_NEG.wiff SUBJECT_SAMPLE_FACTORS - jjsw-2 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=jjsw-2_POS.wiff and jjsw-2_NEG.wiff SUBJECT_SAMPLE_FACTORS - jjsw-3 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=jjsw-3_POS.wiff and jjsw-3_NEG.wiff SUBJECT_SAMPLE_FACTORS - jjsw-4 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=jjsw-4_POS.wiff and jjsw-4_NEG.wiff SUBJECT_SAMPLE_FACTORS - jjsw-5 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=jjsw-5_POS.wiff and jjsw-5_NEG.wiff SUBJECT_SAMPLE_FACTORS - jjsw-6 Sample source:holobiont | treatment:heat stress RAW_FILE_NAME(Raw file name)=jjsw-6_POS.wiff and jjsw-6_NEG.wiff #COLLECTION CO:COLLECTION_SUMMARY Summary: Coral samples from the whole coral holobiont were collected for CO:COLLECTION_SUMMARY metabolomic analysis to investigate the physiological and thermal tolerance CO:COLLECTION_SUMMARY responses of Galaxea fascicularis and Cladiella inflata to acute heat stress. A CO:COLLECTION_SUMMARY total of three colonies per species were sampled, processed into 48 individual CO:COLLECTION_SUMMARY polyps, and subjected to controlled experimental conditions. Sample Source: CO:COLLECTION_SUMMARY Luhuitou Fringing Reef, Sanya Bay, Hainan, China, August 2023. Collection CO:COLLECTION_SUMMARY Method: Coral colonies were carefully collected by hand or appropriate tools CO:COLLECTION_SUMMARY from the reef, followed by separation into individual healthy polyps from CO:COLLECTION_SUMMARY non-marginal regions. Each polyp was affixed to a numbered ceramic base using CO:COLLECTION_SUMMARY animal glue (GEL-10, Aron Alpha, Japan) and allowed to recover for 7 days in an CO:COLLECTION_SUMMARY outdoor coral aquaculture system designed to replicate natural field conditions. CO:COLLECTION_SUMMARY Collection Site: Luhuitou Fringing Reef, Sanya Bay, Hainan, China. CO:SAMPLE_TYPE Holobiont #TREATMENT TR:TREATMENT_SUMMARY Coral fragments of Galaxea fascicularis (stony coral) and Clavularia inflata TR:TREATMENT_SUMMARY (soft coral) were subjected to an acute heat stress experiment. After a 3-day TR:TREATMENT_SUMMARY acclimation period at 27 °C, the temperature in the heat treatment group was TR:TREATMENT_SUMMARY gradually raised from 27 °C to 33 °C over 2 hours and then maintained at TR:TREATMENT_SUMMARY 33 °C for 72 hours. The control group was continuously kept at 27 °C TR:TREATMENT_SUMMARY throughout the experiment. Temperature was monitored every 15 minutes using a TR:TREATMENT_SUMMARY data logger to ensure precise thermal conditions. This setup simulated TR:TREATMENT_SUMMARY short-term heatwave events to evaluate the corals' thermal stress responses TR:TREATMENT_SUMMARY under controlled conditions. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY In this study, a standardized protocol was used to extract metabolites from SP:SAMPLEPREP_SUMMARY coral samples (Galaxea fascicularis and Clavularia inflata) for non-targeted SP:SAMPLEPREP_SUMMARY LC-MS/MS metabolomic analysis. Whole coral holobiont samples (including host SP:SAMPLEPREP_SUMMARY tissue and symbiotic algae) were flash-frozen in liquid nitrogen immediately SP:SAMPLEPREP_SUMMARY after collection and ground into a fine powder using a mortar and pestle. SP:SAMPLEPREP_SUMMARY Approximately 100 mg of each powdered sample was mixed with 1 mL of cold SP:SAMPLEPREP_SUMMARY methanol/acetonitrile/water (2:2:1, v/v/v). The homogenate was sonicated at low SP:SAMPLEPREP_SUMMARY temperature (30 minutes, twice) and centrifuged at 14,000 g for 20 minutes at SP:SAMPLEPREP_SUMMARY 4 °C. The resulting supernatant was collected and dried using a vacuum SP:SAMPLEPREP_SUMMARY concentrator. Prior to LC-MS analysis, the dried extracts were reconstituted in SP:SAMPLEPREP_SUMMARY 100 µL of acetonitrile/water (1:1, v/v). To ensure data quality and SP:SAMPLEPREP_SUMMARY instrument stability, pooled Quality Control (QC) samples were prepared by SP:SAMPLEPREP_SUMMARY mixing aliquots from all samples and were injected at regular intervals SP:SAMPLEPREP_SUMMARY throughout the analytical sequence. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) 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 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS TOF MS scan range: m/z 60–1000; Product ion scan range: m/z 25–1000; TOF MS MS:MS_COMMENTS scan accumulation time: 0.20 s/spectrum; Product ion scan accumulation time: MS:MS_COMMENTS 0.05 s/spectrum; Data acquired in IDA mode with high sensitivity; Collision MS:MS_COMMENTS energy: 35 ± 15 eV; Declustering potential: ±60 V. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples CK-1 CK-2 CK-3 CK-4 CK-5 CK-6 jjsw-1 jjsw-2 jjsw-3 jjsw-4 jjsw-5 jjsw-6 QC-1_Discosoma QC-2_Discosoma QC-3_Discosoma RT0-1 RT0-2 RT0-3 RT0-4 RT0-5 RT0-6 RT1-1 RT1-2 RT1-3 RT1-4 RT1-5 RT1-6 QC-1 QC-2 QC-3 QC-4 QC-5 QC-6 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:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- 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:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:heat stress Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Sample source:holobiont | treatment:- Phosphatidylethanolamine 990.0672746 748.8388682 1345.823384 443.5956596 1036.680874 1845.346976 1509.118219 1068.311235 926.7602031 1659.604153 1004.133631 1792.086841 1333.77726 1317.570406 1332.10132 852894.4916 96541.26268 163997.9001 619894.8779 176627.8879 178076.2462 615547.5583 465645.5438 780200.8998 658041.4421 519817.7226 584669.2499 338183.2001 629057.3719 571113.0375 206702.5624 580052.1719 588072.4821 Hypoxanthine 1193.444369 1743.12583 559.8322093 687.9376786 843.22876 609.1290462 846.3909909 2119.167691 2037.13156 802.6934738 598.1494522 523.6611887 888.2045784 781.9709838 783.6477017 106987.9326 698797.3307 122176.9364 216802.8852 635108.9511 147136.7431 411584.0876 713431.3681 706439.1741 511439.0289 701377.8488 559495.1619 732456.1418 806997.2555 786486.5447 765391.4671 765423.5862 788112.6826 Triethanolamine 5934.009536 11272.33718 9767.0234 11851.21677 21879.12291 12558.79212 6663.674534 20871.89398 17526.54011 10999.49496 17881.56281 7719.514438 12995.95848 12995.79927 13353.04416 25301283.79 25713422.66 27293988.25 21169754.05 24543949.77 29288829.11 28957802.12 21253668.81 23243506.55 26320670.96 26182038.14 28819882.15 26085427.8 23850316.76 26084717.14 24791356.16 24782479.64 27007873.16 4-imidazoleacrylic acid 882.2370976 746.6472578 1312.016536 1119.437829 831.8186901 787.0946162 913.6473028 888.0382698 584.6012518 705.7438896 948.9079551 1146.223344 1049.928348 912.9486421 1050.247617 2158589.362 2038952.147 2208364.416 2022801.464 1673648.546 2075907.234 2390430.927 2078091.446 2391565.974 2299078.958 1990498.92 2248955.87 2209804.484 2209497.889 2233956.598 2117143.598 2291762.889 2234346.206 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT m/z HMDB-ID Phosphatidylethanolamine 3.422 398.87205 -- Hypoxanthine 6.608 286.96582 -- Triethanolamine 5.098 325.11253 -- 4-imidazoleacrylic acid 6.6 395.85097 -- METABOLITES_END #END