#METABOLOMICS WORKBENCH tangshuo_20250706_225747 DATATRACK_ID:6138 STUDY_ID:ST004143 ANALYSIS_ID:AN006868 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 NEGATIVE 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:- Thymol-beta-D-glucoside 1610992.09 1711590.344 2761249.488 4686396.496 4209093.572 5268486.231 576315.9066 2303892.266 2753324.812 988899.6265 1154897.562 509192.6519 1844165.004 1815247.487 1839522.428 288705332 294718465.9 286661056.7 306937131.5 258846104.6 254955410.3 177736959.7 168012247 168012247 580667.2403 168012247 207600.9801 454206663.5 453658997.2 447942246.9 454245780.8 428056338.5 447908682.3 Ostruthin 616677.9597 755386.5437 1062394.07 1852622.289 1568072.364 2038379.616 262574.153 728727.8401 803739.838 398994.1123 455951.3139 212629.4056 714844.2107 705213.2714 705209.1917 1164842.146 1348522.782 803383.6306 1175434.642 1141325.142 1124098.622 559002.0867 576020.7359 558568.3911 1196307.156 1253649.313 761509.5321 1148556.226 1095037.266 1081737.834 1265682.771 1152263.462 1143927.217 D-proline 51459.74027 14661.12891 66259.51736 72609.84102 77598.02736 110825.7891 78090.2432 176181.0491 159074.7144 132111.0972 126156.8692 63773.52429 71992.74789 71996.361 70722.96075 1465319.429 1599063.987 1407795.003 1665919.94 1718950.512 1940810.194 1175209.858 1500983.427 1984767.008 2071926.617 2581064.571 2583846.002 1723102.113 1894912.094 1720471.975 1824387.965 2570999.909 1663835.675 Myo-inositol 121276.6399 182560.3813 151624.332 133870.36 230612.5293 158859.1315 247061.9516 681386.8524 538222.1412 401065.4959 494633.194 198443.3053 263895.9699 263005.6708 263897.0458 1931824.394 1847285.604 1652096.189 2223043.314 1851189.451 900540.9825 1417755.33 1092254.186 1555355.899 1307223.446 1368394.131 1282603.575 1835926.767 1892966.295 1898424.418 1836485.939 1891602.521 1761446.837 Ginsenoside F1 126181.1056 81197.89771 162686.5593 177232.6161 115098.2395 224489.7617 32901.98135 55828.51145 199633.5113 62271.38203 36760.24257 41147.03163 98318.1892 98351.1993 95758.71802 598224.7058 657364.4572 822262.3078 902181.0975 889325.5093 656247.5596 847326.7691 521349.0117 910400.3711 770900.1277 339565.8678 598588.3268 526824.8389 552959.2833 646499.6921 538814.9647 561739.532 416249.2426 Histidine 22177.31015 64104.19948 70365.81762 75322.36544 68391.5603 91418.10009 123186.776 105801.9045 207926.5996 132732.2256 115639.7257 45172.63263 41508.78832 44166.01349 41602.77186 1625660.108 2653453.067 3862855.386 1292391.834 2554947.161 3202038.262 4324897.486 3338240.163 907964.9009 2692741.072 2571278.967 3694283.861 3161620.651 3499465.925 3188715.783 3434816.14 2783044.382 3177851.567 Ganoderic acid H 67416.99117 58899.32921 45828.54266 51841.25696 29523.84247 63756.31929 25322.46034 55026.2216 29722.76012 24854.94771 44536.599 19361.43429 35222.12487 35216.10444 33837.58887 983856.884 1484514.896 998747.5131 1183012.93 1274318.106 1237258.405 555710.8432 614227.7308 997366.6236 544062.9886 677718.9153 388953.3136 841137.0613 812151.1911 891265.0053 866179.2492 866428.8442 849140.1002 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name RT m/z HMDB-ID Thymol-beta-D-glucoside 0.478 311.16832 -- Ostruthin 0.482 297.15242 -- D-proline 5.064 114.05512 HMDB0003411 Myo-inositol 6.382 179.05533 HMDB0000211 Ginsenoside F1 0.458 637.44787 HMDB0039555 Histidine 6.664 154.06105 HMDB0000177 Ganoderic acid H 2.613 553.26495 HMDB0035987 METABOLITES_END #END