{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001990","ANALYSIS_ID":"AN003244","VERSION":"1","CREATED_ON":"02-08-2024"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics of the interaction between a consortium of entomopathogenic fungi and their target insect: mechanisms of attack and survival","PROJECT_TYPE":"Untargeted Metabolomics","PROJECT_SUMMARY":"One of the most concerning pests that attack strawberries in Brazil is Duponchelia fovealis, a non-native moth with no registered control methods to date. Our group recently observed that a fungal consortium formed by two strains of Beauveria bassiana increased the mortality of D. fovealis more than inoculation with each strain on its own. However, the molecular interaction between the fungal consortium and the caterpillars is unknown, raising several questions about the enhanced pest control observed. Furthermore, concerns over the emergency of resistance and the selection for resistance to chemical and biological products that are constantly applied in agriculture highlight the need for careful examination of novel pest control methods. Thus, in this work, we sought to pioneer the evaluation of the molecular interaction between a fungal consortium of B. bassiana and D. fovealis caterpillars. We aimed to understand the biocontrol process involved in this interaction and the defense system of the caterpillar. Therefore, seven days after D. fovealis caterpillars were inoculated with the B. bassiana consortium, the dead and surviving caterpillars were analyzed using GC-MS and LC-MS/MS.","INSTITUTE":"Universidade Federal do Paraná","DEPARTMENT":"Patologia Básica","LABORATORY":"Laboratório de Microbiologia e Biologia Molecular","LAST_NAME":"Katiski da Costa Stuart","FIRST_NAME":"Andressa","ADDRESS":"Av. Cel. Francisco Heráclito dos Santos, 100, Curitiba, Paraná, 81530-000, Brazil","EMAIL":"andressa.katiski@gmail.com","PHONE":"5541991922779","PROJECT_COMMENTS":"Previous studies by our research group showed the increased potential for biocontrol on the insect pest Duponchelia fovealis when two strains of Beauveria bassiana were used together as a fungal consortium. In this work we sought to identify the metabolites involved in the interaction between the consortium formed by different B. bassiana strains and its target insect, D. fovealis. We identify the metabolites using non-targeted metabolomics, applying gas and liquid chromatography coupled to mass spectrometers (GC-MS and LC-MS/MS). These analyses aimed to elucidate the molecular mechanisms involved in the biocontrol effect of the fungal consortium on D. fovealis and to examine possible explanations for the survival of some caterpillars due to a potential resistance mechanism. Dead and surviving caterpillars were analyzed separately.","DOI":"http://dx.doi.org/10.21228/M8B39F","CONTRIBUTORS":"Jason Lee Furuie, Thais Regiani Cataldi, Rodrigo Makowiecky Stuart, Maria Aparecida Cassilha Zawadneak, Carlos Alberto Labate, Ida Chapaval Pimentel"},

"STUDY":{"STUDY_TITLE":"Metabolomics of the interaction between a consortium of entomopathogenic fungi and their target insect: mechanisms of attack and survival","STUDY_TYPE":"Untargeted Metabolomics","STUDY_SUMMARY":"One of the most concerning pests that attack strawberries in Brazil is Duponchelia fovealis, a non-native moth with no registered control methods to date. Our group recently observed that a fungal consortium formed by two strains of Beauveria bassiana increased the mortality of D. fovealis more than inoculation with each strain on its own. However, the molecular interaction between the fungal consortium and the caterpillars is unknown, raising several questions about the enhanced pest control observed. Furthermore, concerns over the emergency of resistance and the selection for resistance to chemical and biological products that are constantly applied in agriculture highlight the need for careful examination of novel pest control methods. Thus, in this work, we sought to pioneer the evaluation of the molecular interaction between a fungal consortium of B. bassiana and D. fovealis caterpillars. We aimed to understand the biocontrol process involved in this interaction and the defense system of the caterpillar. Therefore, seven days after D. fovealis caterpillars were inoculated with the B. bassiana consortium, the dead and surviving caterpillars were analyzed using GC-MS and LC-MS/MS.","INSTITUTE":"Universidade Federal do Paraná","DEPARTMENT":"Patologia Básica","LABORATORY":"Laboratório de Microbiologia e Biologia Molecular","LAST_NAME":"Katiski da Costa Stuart","FIRST_NAME":"Andressa","ADDRESS":"Av. Cel. Francisco Heráclito dos Santos, 100, Curitiba, Paraná, 81530-000, Brazil","EMAIL":"andressa.katiski@gmail.com","PHONE":"55 41 991922779","SUBMIT_DATE":"2021-11-12"},

"SUBJECT":{"SUBJECT_TYPE":"Insect","SUBJECT_SPECIES":"Duponchelia fovealis"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"CatS_1",
"Factors":{"Factor":"Control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_2",
"Factors":{"Factor":"Control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_3",
"Factors":{"Factor":"Control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_4",
"Factors":{"Factor":"Control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_5",
"Factors":{"Factor":"Control"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov2_1",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov2_2",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov2_3",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov2_4",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana"},
"Additional sample data":{"RAW_FILE_NAME":"CatM_Bov2_4","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"CatM_Bov2_5",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov2 strain of Beauveria bassiana"},
"Additional sample data":{"RAW_FILE_NAME":"CatM_Bov2_5","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"CatM_Bov3_1",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov3_2",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov3_3",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Bov3_4",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana"},
"Additional sample data":{"RAW_FILE_NAME":"CatM_Bov3_4","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"CatM_Bov3_5",
"Factors":{"Factor":"Duponchelia fovealis killed by the Bov3 strain of Beauveria bassiana"},
"Additional sample data":{"RAW_FILE_NAME":"CatM_Bov3_5","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"CatM_Cons_1",
"Factors":{"Factor":"Duponchelia fovealis killed by the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Cons_2",
"Factors":{"Factor":"Duponchelia fovealis killed by the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Cons_3",
"Factors":{"Factor":"Duponchelia fovealis killed by the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatM_Cons_4",
"Factors":{"Factor":"Duponchelia fovealis killed by the consortium"},
"Additional sample data":{"RAW_FILE_NAME":"CatM_Cons_4","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"CatM_Cons_5",
"Factors":{"Factor":"Duponchelia fovealis killed by the consortium"},
"Additional sample data":{"RAW_FILE_NAME":"CatM_Cons_5","RAW_FILE_NAME":"-","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"CatS_Bov2_1",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov2_2",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov2_3",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov2_4",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov2_5",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 2 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov3_1",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov3_2",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov3_3",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov3_4",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Bov3_5",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the Bov 3 strain of Beauveria bassiana"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Cons_1",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Cons_2",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Cons_3",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Cons_4",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the consortium"},
"Additional sample data":{"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 ID":"-",
"Sample ID":"CatS_Cons_5",
"Factors":{"Factor":"Duponchelia fovealis that survived the application of the consortium"},
"Additional sample data":{"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":{"COLLECTION_SUMMARY":"Groups of 10 third instar caterpillars were placed on strawberry leaves and sprayed with 1 mL of B. bassiana conidia-suspension: 2 x 107 conidia/mL in 0.85% saline solution with Tween 80® adhesive spreader added. Therefore, the treatments applied were: control caterpillars (consisting only of 0.85% saline solution and Tween 80®), caterpillars inoculated with Bov 3 strain of B. bassiana, caterpillars inoculated with Bov 2 strain of B. bassiana and caterpillars inoculated with the Bov3-Bov 2 consortium. On the seventh day, caterpillars that did not respond to the stimulus provided by the touch of a brush were considered dead. All caterpillars were identified as living or dead at the end of the seven days and were subsequently stored in a freezer at -80ºC.","COLLECTION_PROTOCOL_FILENAME":"Metabolite_Extraction","SAMPLE_TYPE":"Insect tissue","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"The treatments applied were: control caterpillars (consisting only of 0.85% saline solution and Tween 80®), caterpillars inoculated with Bov 3 strain of B. bassiana, caterpillars inoculated with Bov 2 strain of B. bassiana and caterpillars inoculated with the Bov3-Bov2 consortium.","TREATMENT_PROTOCOL_FILENAME":"Metabolite_Extraction","TREATMENT_DOSEVOLUME":"2 x 10^7 conidia/mL"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"After the direct contact bioassay, the caterpillars were arranged by treatment, then macerated in liquid nitrogen (N2). Extraction was performed with 200 mg of the macerate added to a 1 mL microtube (Eppendorf, Germany) previously treated with methanol. Following this, 125 μL of chloroform (CHCl3), 50 μL ultra-pure water (H2O), and 250 μL cold methanol (CH3OH) were added to the macerate. The microtubes were vigorously vortexed and placed in an ultrasonic bath (Odontobrás, Ribeirão-SP) at 20 Hz and approximately 4ºC for 10 minutes. Then, 50 μL of CHCl3 and 50 μL of H2O were added, and the tubes were vortexed again. The samples were centrifuged (Eppendorf, Germany) for 5 minutes at 14000 rpm and 4°C, and the supernatant was filtered on a Whatman® 0.22 µm filter (Merck, Germany) and transferred to a glass vial. The vial was taken to a lyophilizer (Thermo Fischer Scientific, MA) until the samples had completely dried. Finally, the lyophilized samples were resuspended in 200 µL of extraction solution and aliquoted for use in the GC-MS and LC-MS/MS.","SAMPLEPREP_PROTOCOL_FILENAME":"Metabolite_Extraction","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"METHODS_FILENAME":"Metabolomics_Methods","INSTRUMENT_NAME":"Waters Acquity UPLC","COLUMN_NAME":"Waters Acquity UPLC HSS (100 x 2.1mm, 1.7um)","COLUMN_TEMPERATURE":"35 ºC","FLOW_GRADIENT":"95% solvent A and 5% B. The gradient increased linearly to 75% A and 25% B over the next 6 min. The polarity was reversed to 25% A and 75% B for 6 min, and finally 5% A and 95% B for 1 min","FLOW_RATE":"0.5 mL·min-1","SOLVENT_A":"Water; formic acid","SOLVENT_B":"100% acetonitrile; formic acid.","CAPILLARY_VOLTAGE":"3 kV","CHROMATOGRAPHY_TYPE":"Reversed phase"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"metabolomics_methods.pdf"},

"MS":{"INSTRUMENT_NAME":"Waters Acquity UPLC","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","MS_COMMENTS":"Generated data were pre-processed using MassLynx 4.1 software (Waters Corporation, MA, USA) and then analyzed using MetaboAnalyst 4.0 online software. Fragmentation was performed under the same conditions as the ionization source, using collision energies between 15 and 50 eV. The search for metabolites was performed in the Human Metabolome Database (HMDB) using a mass tolerance of up to 0.1 Da and considering the adduct of [M-H]-. The structures of the molecules were imported and underwent in silico fragmentation using ACD/MS Structure ID software suite (ACD/labs, Toronto, Canada). The fragmentation profile of each molecule proposed by the program was then compared to the fragments generated by MS/MS to accept or reject the identification of metabolites according to similarity.","ION_MODE":"NEGATIVE","CAPILLARY_VOLTAGE":"3 kV","DRY_GAS_FLOW":"50 L/hr","SOURCE_TEMPERATURE":"150 ºC","DESOLVATION_GAS_FLOW":"550 L/hr.","MS_RESULTS_FILE":"ST001990_AN003244_Results.txt UNITS:Relative intensity Has m/z:Yes Has RT:Yes RT units:Minutes"}

}