#METABOLOMICS WORKBENCH Andressa_Stuart_20211112_035200 DATATRACK_ID:2926 STUDY_ID:ST001990 ANALYSIS_ID:AN003244 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Metabolomics of the interaction between a consortium of entomopathogenic fungi PR:PROJECT_TITLE and their target insect: mechanisms of attack and survival PR:PROJECT_TYPE Untargeted Metabolomics PR:PROJECT_SUMMARY One of the most concerning pests that attack strawberries in Brazil is PR:PROJECT_SUMMARY Duponchelia fovealis, a non-native moth with no registered control methods to PR:PROJECT_SUMMARY date. Our group recently observed that a fungal consortium formed by two strains PR:PROJECT_SUMMARY of Beauveria bassiana increased the mortality of D. fovealis more than PR:PROJECT_SUMMARY inoculation with each strain on its own. However, the molecular interaction PR:PROJECT_SUMMARY between the fungal consortium and the caterpillars is unknown, raising several PR:PROJECT_SUMMARY questions about the enhanced pest control observed. Furthermore, concerns over PR:PROJECT_SUMMARY the emergency of resistance and the selection for resistance to chemical and PR:PROJECT_SUMMARY biological products that are constantly applied in agriculture highlight the PR:PROJECT_SUMMARY need for careful examination of novel pest control methods. Thus, in this work, PR:PROJECT_SUMMARY we sought to pioneer the evaluation of the molecular interaction between a PR:PROJECT_SUMMARY fungal consortium of B. bassiana and D. fovealis caterpillars. We aimed to PR:PROJECT_SUMMARY understand the biocontrol process involved in this interaction and the defense PR:PROJECT_SUMMARY system of the caterpillar. Therefore, seven days after D. fovealis caterpillars PR:PROJECT_SUMMARY were inoculated with the B. bassiana consortium, the dead and surviving PR:PROJECT_SUMMARY caterpillars were analyzed using GC-MS and LC-MS/MS. PR:INSTITUTE Universidade Federal do Paraná PR:DEPARTMENT Patologia Básica PR:LABORATORY Laboratório de Microbiologia e Biologia Molecular PR:LAST_NAME Katiski da Costa Stuart PR:FIRST_NAME Andressa PR:ADDRESS Av. Cel. Francisco Heráclito dos Santos, 100, Curitiba, Paraná, 81530-000, PR:ADDRESS Brazil PR:EMAIL andressa.katiski@gmail.com PR:PHONE 5541991922779 PR:PROJECT_COMMENTS Previous studies by our research group showed the increased potential for PR:PROJECT_COMMENTS biocontrol on the insect pest Duponchelia fovealis when two strains of Beauveria PR:PROJECT_COMMENTS bassiana were used together as a fungal consortium. In this work we sought to PR:PROJECT_COMMENTS identify the metabolites involved in the interaction between the consortium PR:PROJECT_COMMENTS formed by different B. bassiana strains and its target insect, D. fovealis. We PR:PROJECT_COMMENTS identify the metabolites using non-targeted metabolomics, applying gas and PR:PROJECT_COMMENTS liquid chromatography coupled to mass spectrometers (GC-MS and LC-MS/MS). These PR:PROJECT_COMMENTS analyses aimed to elucidate the molecular mechanisms involved in the biocontrol PR:PROJECT_COMMENTS effect of the fungal consortium on D. fovealis and to examine possible PR:PROJECT_COMMENTS explanations for the survival of some caterpillars due to a potential resistance PR:PROJECT_COMMENTS mechanism. Dead and surviving caterpillars were analyzed separately. PR:DOI http://dx.doi.org/10.21228/M8B39F PR:CONTRIBUTORS Jason Lee Furuie, Thais Regiani Cataldi, Rodrigo Makowiecky Stuart, Maria PR:CONTRIBUTORS Aparecida Cassilha Zawadneak, Carlos Alberto Labate, Ida Chapaval Pimentel #STUDY ST:STUDY_TITLE Metabolomics of the interaction between a consortium of entomopathogenic fungi ST:STUDY_TITLE and their target insect: mechanisms of attack and survival ST:STUDY_TYPE Untargeted Metabolomics ST:STUDY_SUMMARY One of the most concerning pests that attack strawberries in Brazil is ST:STUDY_SUMMARY Duponchelia fovealis, a non-native moth with no registered control methods to ST:STUDY_SUMMARY date. Our group recently observed that a fungal consortium formed by two strains ST:STUDY_SUMMARY of Beauveria bassiana increased the mortality of D. fovealis more than ST:STUDY_SUMMARY inoculation with each strain on its own. However, the molecular interaction ST:STUDY_SUMMARY between the fungal consortium and the caterpillars is unknown, raising several ST:STUDY_SUMMARY questions about the enhanced pest control observed. Furthermore, concerns over ST:STUDY_SUMMARY the emergency of resistance and the selection for resistance to chemical and ST:STUDY_SUMMARY biological products that are constantly applied in agriculture highlight the ST:STUDY_SUMMARY need for careful examination of novel pest control methods. Thus, in this work, ST:STUDY_SUMMARY we sought to pioneer the evaluation of the molecular interaction between a ST:STUDY_SUMMARY fungal consortium of B. bassiana and D. fovealis caterpillars. We aimed to ST:STUDY_SUMMARY understand the biocontrol process involved in this interaction and the defense ST:STUDY_SUMMARY system of the caterpillar. Therefore, seven days after D. fovealis caterpillars ST:STUDY_SUMMARY were inoculated with the B. bassiana consortium, the dead and surviving ST:STUDY_SUMMARY caterpillars were analyzed using GC-MS and LC-MS/MS. ST:INSTITUTE Universidade Federal do Paraná ST:DEPARTMENT Patologia Básica ST:LABORATORY Laboratório de Microbiologia e Biologia Molecular ST:LAST_NAME Katiski da Costa Stuart ST:FIRST_NAME Andressa ST:ADDRESS Av. Cel. Francisco Heráclito dos Santos, 100, Curitiba, Paraná, 81530-000, ST:ADDRESS Brazil ST:EMAIL andressa.katiski@gmail.com ST:PHONE 55 41 991922779 ST:SUBMIT_DATE 2021-11-12 #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Duponchelia fovealis #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - CatS_1 Factor:Control RAW_FILE_NAME=CatS_1; RAW_FILE_NAME=CatS_1_R001 CatS_1_R002 CatS_1_R003.; RAW_FILE_NAME=CatS_1_R001_NEG CatS_1_R002_NEG CatS_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_2 Factor:Control RAW_FILE_NAME=CatS_2; RAW_FILE_NAME=CatS_2_R001 CatS_2_R002 CatS_2_R003.; RAW_FILE_NAME=CatS_2_R001_NEG CatS_2_R002_NEG CatS_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_3 Factor:Control RAW_FILE_NAME=CatS_3; RAW_FILE_NAME=CatS_3_R001 CatS_3_R002 CatS_3_R003.; RAW_FILE_NAME=CatS_3_R001_NEG CatS_3_R002_NEG CatS_3_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_4 Factor:Control RAW_FILE_NAME=CatS_4; RAW_FILE_NAME=CatS_4_R001 CatS_4_R002 CatS_4_R003.; RAW_FILE_NAME=CatS_4_R001_NEG CatS_4_R002_NEG CatS_4_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_5 Factor:Control RAW_FILE_NAME=CatS_5; RAW_FILE_NAME=CatS_5_R001 CatS_5_R002 CatS_5_R003.; RAW_FILE_NAME=CatS_5_R001_NEG CatS_5_R002_NEG CatS_5_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov2_1 Factor:Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov2_1; RAW_FILE_NAME=CatM_Bov2_1_R001 CatM_Bov2_1_R002 CatM_Bov2_1_R003 CatM_Bov2_1_R004.; RAW_FILE_NAME=CatM_Bov2_1_R001_NEG CatM_Bov2_1_R002_NEG CatM_Bov2_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov2_2 Factor:Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov2_2; RAW_FILE_NAME=CatM_Bov2_2_R001 CatM_Bov2_2_R002 CatM_Bov2_2_R003 CatM_Bov2_2_R004.; RAW_FILE_NAME=CatM_Bov2_2_R001_NEG CatM_Bov2_2_R002_NEG CatM_Bov2_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov2_3 Factor:Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov2_3; RAW_FILE_NAME=CatM_Bov2_3_R001 CatM_Bov2_3_R002 CatM_Bov2_3_R003 CatM_Bov2_3_R004.; RAW_FILE_NAME=CatM_Bov2_3_R001_NEG CatM_Bov2_3_R002_NEG CatM_Bov2_3_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov2_4 Factor:Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov2_4; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatM_Bov2_5 Factor:Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov2_5; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatM_Bov3_1 Factor:Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov3_1; RAW_FILE_NAME=CatM_Bov3_1_R001 CatM_Bov3_1_R002 CatM_Bov3_1_R003 CatM_Bov3_1_R004.; RAW_FILE_NAME=CatM_Bov3_1_R001_NEG CatM_Bov3_1_R002_NEG CatM_Bov3_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov3_2 Factor:Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov3_2; RAW_FILE_NAME=CatM_Bov3_2_R001 CatM_Bov3_2_R002 CatM_Bov3_2_R003 CatM_Bov3_2_R004.; RAW_FILE_NAME=CatM_Bov3_2_R001_NEG CatM_Bov3_2_R002_NEG CatM_Bov3_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov3_3 Factor:Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov3_3; RAW_FILE_NAME=CatM_Bov3_3_R001 CatM_Bov3_3_R002 CatM_Bov3_3_R003 CatM_Bov3_3_R004.; RAW_FILE_NAME=CatM_Bov3_3_R001_NEG CatM_Bov3_3_R002_NEG CatM_Bov3_3_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Bov3_4 Factor:Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov3_4; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatM_Bov3_5 Factor:Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana RAW_FILE_NAME=CatM_Bov3_5; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatM_Cons_1 Factor:Duponchelia fovealis killed by the consortium RAW_FILE_NAME=CatM_Cons_1; RAW_FILE_NAME=CatM_Cons_1_R001 CatM_Cons_1_R002 CatM_Cons_1_R003 CatM_Cons_1_R004.; RAW_FILE_NAME=CatM_Cons_1_R001_NEG CatM_Cons_1_R002_NEG CatM_Cons_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Cons_2 Factor:Duponchelia fovealis killed by the consortium RAW_FILE_NAME=CatM_Cons_2; RAW_FILE_NAME=CatM_Cons_2_R001 CatM_Cons_2_R002 CatM_Cons_2_R003 CatM_Cons_2_R004.; RAW_FILE_NAME=CatM_Cons_2_R001_NEG CatM_Cons_2_R002_NEG CatM_Cons_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatM_Cons_3 Factor:Duponchelia fovealis killed by the consortium RAW_FILE_NAME=CatM_Cons_3; RAW_FILE_NAME=CatM_Cons_3_R001 CatM_Cons_3_R002 CatM_Cons_3_R003 CatM_Cons_3_R004.; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatM_Cons_4 Factor:Duponchelia fovealis killed by the consortium RAW_FILE_NAME=CatM_Cons_4; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatM_Cons_5 Factor:Duponchelia fovealis killed by the consortium RAW_FILE_NAME=CatM_Cons_5; RAW_FILE_NAME=-; RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - CatS_Bov2_1 Factor:Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov2_1; RAW_FILE_NAME=CatS_Bov2_1_R001 CatS_Bov2_1_R002 CatS_Bov2_1_R003.; RAW_FILE_NAME=CatS_Bov2_1_R001_NEG CatS_Bov2_1_R002_NEG CatS_Bov2_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov2_2 Factor:Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov2_2; RAW_FILE_NAME=CatS_Bov2_2_R001 CatS_Bov2_2_R002 CatS_Bov2_2_R003.; RAW_FILE_NAME=CatS_Bov2_2_R001_NEG CatS_Bov2_2_R002_NEG CatS_Bov2_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov2_3 Factor:Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov2_3; RAW_FILE_NAME=CatS_Bov2_3_R001 CatS_Bov2_3_R002 CatS_Bov2_3_R003.; RAW_FILE_NAME=CatS_Bov2_3_R001_NEG CatS_Bov2_3_R002_NEG CatS_Bov2_3_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov2_4 Factor:Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov2_4; RAW_FILE_NAME=CatS_Bov2_4_R001 CatS_Bov2_4_R002 CatS_Bov2_4_R003.; RAW_FILE_NAME=CatS_Bov2_4_R001_NEG CatS_Bov2_4_R002_NEG CatS_Bov2_4_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov2_5 Factor:Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov2_5; RAW_FILE_NAME=CatS_Bov2_5_R001 CatS_Bov2_5_R002 CatS_Bov2_5_R003.; RAW_FILE_NAME=CatS_Bov2_5_R001_NEG CatS_Bov2_5_R002_NEG CatS_Bov2_5_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov3_1 Factor:Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov3_1; RAW_FILE_NAME=CatS_Bov3_1_R001 CatS_Bov3_1_R002 CatS_Bov3_1_R003.; RAW_FILE_NAME=CatS_Bov3_1_R001_NEG CatS_Bov3_1_R002_NEG CatS_Bov3_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov3_2 Factor:Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov3_2; RAW_FILE_NAME=CatS_Bov3_2_R001 CatS_Bov3_2_R002 CatS_Bov3_2_R003.; RAW_FILE_NAME=CatS_Bov3_2_R001_NEG CatS_Bov3_2_R002_NEG CatS_Bov3_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov3_3 Factor:Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov3_3; RAW_FILE_NAME=CatS_Bov3_3_R001 CatS_Bov3_3_R002 CatS_Bov3_3_R003.; RAW_FILE_NAME=CatS_Bov3_3_R001_NEG CatS_Bov3_3_R002_NEG CatS_Bov3_3_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov3_4 Factor:Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov3_4; RAW_FILE_NAME=CatS_Bov3_4_R001 CatS_Bov3_4_R002 CatS_Bov3_4_R003.; RAW_FILE_NAME=CatS_Bov3_4_R001_NEG CatS_Bov3_4_R002_NEG CatS_Bov3_4_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Bov3_5 Factor:Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana RAW_FILE_NAME=CatS_Bov3_5; RAW_FILE_NAME=CatS_Bov3_5_R001 CatS_Bov3_5_R002 CatS_Bov3_5_R003.; RAW_FILE_NAME=CatS_Bov3_5_R001_NEG CatS_Bov3_5_R002_NEG CatS_Bov3_5_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Cons_1 Factor:Duponchelia fovealis that survived the application of the consortium RAW_FILE_NAME=CatS_Cons_1; RAW_FILE_NAME=CatS_Cons_1_R001 CatS_Cons_1_R002 CatS_Cons_1_R003.; RAW_FILE_NAME=CatS_Cons_1_R001_NEG CatS_Cons_1_R002_NEG CatS_Cons_1_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Cons_2 Factor:Duponchelia fovealis that survived the application of the consortium RAW_FILE_NAME=CatS_Cons_2; RAW_FILE_NAME=CatS_Cons_2_R001 CatS_Cons_2_R002 CatS_Cons_2_R003.; RAW_FILE_NAME=CatS_Cons_2_R001_NEG CatS_Cons_2_R002_NEG CatS_Cons_2_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Cons_3 Factor:Duponchelia fovealis that survived the application of the consortium RAW_FILE_NAME=CatS_Cons_3; RAW_FILE_NAME=CatS_Cons_3_R001 CatS_Cons_3_R002 CatS_Cons_3_R003.; RAW_FILE_NAME=CatS_Cons_3_R001_NEG CatS_Cons_3_R002_NEG CatS_Cons_3_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Cons_4 Factor:Duponchelia fovealis that survived the application of the consortium RAW_FILE_NAME=-; RAW_FILE_NAME=CatS_Cons_4_R001 CatS_Cons_4_R002 CatS_Cons_4_R003.; RAW_FILE_NAME=CatS_Cons_4_R001_NEG CatS_Cons_4_R002_NEG CatS_Cons_4_R003_NEG. SUBJECT_SAMPLE_FACTORS - CatS_Cons_5 Factor:Duponchelia fovealis that survived the application of the consortium RAW_FILE_NAME=-; RAW_FILE_NAME=CatS_Cons_5_R001 CatS_Cons_5_R002 CatS_Cons_5_R003.; RAW_FILE_NAME=CatS_Cons_5_R001_NEG CatS_Cons_5_R002_NEG CatS_Cons_5_R003_NEG. #COLLECTION CO:COLLECTION_SUMMARY Groups of 10 third instar caterpillars were placed on strawberry leaves and CO:COLLECTION_SUMMARY sprayed with 1 mL of B. bassiana conidia-suspension: 2 x 107 conidia/mL in 0.85% CO:COLLECTION_SUMMARY saline solution with Tween 80® adhesive spreader added. Therefore, the CO:COLLECTION_SUMMARY treatments applied were: control caterpillars (consisting only of 0.85% saline CO:COLLECTION_SUMMARY solution and Tween 80®), caterpillars inoculated with Bov 3 strain of B. CO:COLLECTION_SUMMARY bassiana, caterpillars inoculated with Bov 2 strain of B. bassiana and CO:COLLECTION_SUMMARY caterpillars inoculated with the Bov3-Bov 2 consortium. On the seventh day, CO:COLLECTION_SUMMARY caterpillars that did not respond to the stimulus provided by the touch of a CO:COLLECTION_SUMMARY brush were considered dead. All caterpillars were identified as living or dead CO:COLLECTION_SUMMARY at the end of the seven days and were subsequently stored in a freezer at CO:COLLECTION_SUMMARY -80ºC. CO:COLLECTION_PROTOCOL_FILENAME Metabolite_Extraction CO:SAMPLE_TYPE Insect tissue CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The treatments applied were: control caterpillars (consisting only of 0.85% TR:TREATMENT_SUMMARY saline solution and Tween 80®), caterpillars inoculated with Bov 3 strain of B. TR:TREATMENT_SUMMARY bassiana, caterpillars inoculated with Bov 2 strain of B. bassiana and TR:TREATMENT_SUMMARY caterpillars inoculated with the Bov3-Bov2 consortium. TR:TREATMENT_PROTOCOL_FILENAME Metabolite_Extraction TR:TREATMENT_DOSEVOLUME 2 x 10^7 conidia/mL #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After the direct contact bioassay, the caterpillars were arranged by treatment, SP:SAMPLEPREP_SUMMARY then macerated in liquid nitrogen (N2). Extraction was performed with 200 mg of SP:SAMPLEPREP_SUMMARY the macerate added to a 1 mL microtube (Eppendorf, Germany) previously treated SP:SAMPLEPREP_SUMMARY with methanol. Following this, 125 μL of chloroform (CHCl3), 50 μL ultra-pure SP:SAMPLEPREP_SUMMARY water (H2O), and 250 μL cold methanol (CH3OH) were added to the macerate. The SP:SAMPLEPREP_SUMMARY microtubes were vigorously vortexed and placed in an ultrasonic bath SP:SAMPLEPREP_SUMMARY (Odontobrás, Ribeirão-SP) at 20 Hz and approximately 4ºC for 10 minutes. SP:SAMPLEPREP_SUMMARY Then, 50 μL of CHCl3 and 50 μL of H2O were added, and the tubes were vortexed SP:SAMPLEPREP_SUMMARY again. The samples were centrifuged (Eppendorf, Germany) for 5 minutes at 14000 SP:SAMPLEPREP_SUMMARY rpm and 4°C, and the supernatant was filtered on a Whatman® 0.22 µm filter SP:SAMPLEPREP_SUMMARY (Merck, Germany) and transferred to a glass vial. The vial was taken to a SP:SAMPLEPREP_SUMMARY lyophilizer (Thermo Fischer Scientific, MA) until the samples had completely SP:SAMPLEPREP_SUMMARY dried. Finally, the lyophilized samples were resuspended in 200 µL of SP:SAMPLEPREP_SUMMARY extraction solution and aliquoted for use in the GC-MS and LC-MS/MS. SP:SAMPLEPREP_PROTOCOL_FILENAME Metabolite_Extraction SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:METHODS_FILENAME Metabolomics_Methods CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Waters Acquity UPLC HSS (100 x 2.1mm, 1.7um) CH:COLUMN_TEMPERATURE 35 ºC CH:FLOW_GRADIENT 95% solvent A and 5% B. The gradient increased linearly to 75% A and 25% B over CH:FLOW_GRADIENT the next 6 min. The polarity was reversed to 25% A and 75% B for 6 min, and CH:FLOW_GRADIENT finally 5% A and 95% B for 1 min CH:FLOW_RATE 0.5 mL·min-1 CH:SOLVENT_A Water; formic acid CH:SOLVENT_B 100% acetonitrile; formic acid. CH:CAPILLARY_VOLTAGE 3 kV CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE metabolomics_methods.pdf #MS MS:INSTRUMENT_NAME Waters Acquity UPLC MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS Generated data were pre-processed using MassLynx 4.1 software (Waters MS:MS_COMMENTS Corporation, MA, USA) and then analyzed using MetaboAnalyst 4.0 online software. MS:MS_COMMENTS Fragmentation was performed under the same conditions as the ionization source, MS:MS_COMMENTS using collision energies between 15 and 50 eV. The search for metabolites was MS:MS_COMMENTS performed in the Human Metabolome Database (HMDB) using a mass tolerance of up MS:MS_COMMENTS to 0.1 Da and considering the adduct of [M-H]-. The structures of the molecules MS:MS_COMMENTS were imported and underwent in silico fragmentation using ACD/MS Structure ID MS:MS_COMMENTS software suite (ACD/labs, Toronto, Canada). The fragmentation profile of each MS:MS_COMMENTS molecule proposed by the program was then compared to the fragments generated by MS:MS_COMMENTS MS/MS to accept or reject the identification of metabolites according to MS:MS_COMMENTS similarity. MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 3 kV MS:DRY_GAS_FLOW 50 L/hr MS:SOURCE_TEMPERATURE 150 ºC MS:DESOLVATION_GAS_FLOW 550 L/hr. MS:MS_RESULTS_FILE ST001990_AN003244_Results.txt UNITS:Relative intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END