#METABOLOMICS WORKBENCH yash_thsti_20250620_223850 DATATRACK_ID:6070 STUDY_ID:ST004001 ANALYSIS_ID:AN006598 PROJECT_ID:PR002506 VERSION 1 CREATED_ON June 21, 2025, 5:16 am #PROJECT PR:PROJECT_TITLE Metabolic and Lipidomic Trade-offs in Helicoverpa armigera: Dynamics Under Plant PR:PROJECT_TITLE Protease Inhibitor-Induced Stress PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY Plant protease inhibitors retard the growth and development of insects by PR:PROJECT_SUMMARY inhibiting their digestive proteases. In response, insects try to adapt to these PR:PROJECT_SUMMARY plant defensive molecules by modulating their protease expression. However, PR:PROJECT_SUMMARY their survival mechanisms might not be limited only to digestive plasticity. To PR:PROJECT_SUMMARY explore this, we performed a comprehensive lipidomics and metabolomics analysis PR:PROJECT_SUMMARY in Helicoverpa armigera fed with a recombinant Capsicum annuum protease PR:PROJECT_SUMMARY inhibitor (rCanPI-7) having unique four inhibitory repeat domains with potent PR:PROJECT_SUMMARY activity against insect trypsins and chymotrypsins. These results revealed that PR:PROJECT_SUMMARY H. armigera employs a dynamic and multifaceted physiological response to dietary PR:PROJECT_SUMMARY stress induced by rCanPI. Upon ingestion of rCanPI-7, down regulation of PR:PROJECT_SUMMARY glycolysis and TCA cycle indicated a decrease in primary energy metabolism while PR:PROJECT_SUMMARY oxidative stress was evident from the depletion of reduced glutathione, PR:PROJECT_SUMMARY peroxidation of membrane lipids, and accumulation of ceramides which are the PR:PROJECT_SUMMARY hallmarks of mitochondrial dysfunction. Investigation of the dynamics in the PR:PROJECT_SUMMARY turnover of different molecules hints that H. armigera activated multiple PR:PROJECT_SUMMARY compensatory strategies such as mobilizing triglycerides and amino acid PR:PROJECT_SUMMARY catabolism as an alternative source of energy, upregulation of antioxidants, PR:PROJECT_SUMMARY membrane remodeling, activation of apoptosis, and shifts in neuromodulatory PR:PROJECT_SUMMARY metabolites linked to cognitive adaptation. Collectively, these findings point PR:PROJECT_SUMMARY to a tightly regulated physiological tug-of-war in H. armigera, where the PR:PROJECT_SUMMARY damaging impact of rCanPI-induced oxidative and nutritional stress is PR:PROJECT_SUMMARY counteracted by a suite of compensatory metabolic, structural, and PR:PROJECT_SUMMARY neuromodulatory adjustments. To our knowledge, this is the first report of PR:PROJECT_SUMMARY lipidomic profiling in H. armigera, providing novel insights into its PR:PROJECT_SUMMARY biochemical resilience and identifying potential metabolic vulnerabilities for PR:PROJECT_SUMMARY enhancing biopesticide strategies. PR:INSTITUTE Translational health science and technology institute PR:DEPARTMENT NCD PR:LABORATORY Biomarker lab PR:LAST_NAME Kumar PR:FIRST_NAME Yashwant PR:ADDRESS NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India PR:EMAIL y.kumar@thsti.res.in PR:PHONE 01292876796 #STUDY ST:STUDY_TITLE Metabolic and Lipidomic Trade-offs in Helicoverpa armigera: Dynamics Under Plant ST:STUDY_TITLE Protease Inhibitor-Induced Stress ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY Plant protease inhibitors retard the growth and development of insects by ST:STUDY_SUMMARY inhibiting their digestive proteases. In response, insects try to adapt to these ST:STUDY_SUMMARY plant defensive molecules by modulating their protease expression. However, ST:STUDY_SUMMARY their survival mechanisms might not be limited only to digestive plasticity. To ST:STUDY_SUMMARY explore this, we performed a comprehensive lipidomics and metabolomics analysis ST:STUDY_SUMMARY in Helicoverpa armigera fed with a recombinant Capsicum annuum protease ST:STUDY_SUMMARY inhibitor (rCanPI-7) having unique four inhibitory repeat domains with potent ST:STUDY_SUMMARY activity against insect trypsins and chymotrypsins. These results revealed that ST:STUDY_SUMMARY H. armigera employs a dynamic and multifaceted physiological response to dietary ST:STUDY_SUMMARY stress induced by rCanPI. Upon ingestion of rCanPI-7, down regulation of ST:STUDY_SUMMARY glycolysis and TCA cycle indicated a decrease in primary energy metabolism while ST:STUDY_SUMMARY oxidative stress was evident from the depletion of reduced glutathione, ST:STUDY_SUMMARY peroxidation of membrane lipids, and accumulation of ceramides which are the ST:STUDY_SUMMARY hallmarks of mitochondrial dysfunction. Investigation of the dynamics in the ST:STUDY_SUMMARY turnover of different molecules hints that H. armigera activated multiple ST:STUDY_SUMMARY compensatory strategies such as mobilizing triglycerides and amino acid ST:STUDY_SUMMARY catabolism as an alternative source of energy, upregulation of antioxidants, ST:STUDY_SUMMARY membrane remodeling, activation of apoptosis, and shifts in neuromodulatory ST:STUDY_SUMMARY metabolites linked to cognitive adaptation. Collectively, these findings point ST:STUDY_SUMMARY to a tightly regulated physiological tug-of-war in H. armigera, where the ST:STUDY_SUMMARY damaging impact of rCanPI-induced oxidative and nutritional stress is ST:STUDY_SUMMARY counteracted by a suite of compensatory metabolic, structural, and ST:STUDY_SUMMARY neuromodulatory adjustments. To our knowledge, this is the first report of ST:STUDY_SUMMARY lipidomic profiling in H. armigera, providing novel insights into its ST:STUDY_SUMMARY biochemical resilience and identifying potential metabolic vulnerabilities for ST:STUDY_SUMMARY enhancing biopesticide strategies. ST:INSTITUTE Translational health science and technology institute ST:DEPARTMENT NCD ST:LABORATORY Biomarker lab ST:LAST_NAME Kumar ST:FIRST_NAME Yashwant ST:ADDRESS NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India ST:EMAIL y.kumar@thsti.res.in ST:PHONE +911292876796 #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Helicoverpa armigera ( 29058) SU:TAXONOMY_ID 29058 #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 - EC_1 Sample source:Early | Treatment:Control RAW_FILE_NAME(Raw file name)=EC_1.mzXML SUBJECT_SAMPLE_FACTORS - EC_2 Sample source:Early | Treatment:Control RAW_FILE_NAME(Raw file name)=EC_2.mzXML SUBJECT_SAMPLE_FACTORS - EC_3 Sample source:Early | Treatment:Control RAW_FILE_NAME(Raw file name)=EC_3.mzXML SUBJECT_SAMPLE_FACTORS - EI_1 Sample source:Early | Treatment:Fed RAW_FILE_NAME(Raw file name)=EI_1.mzXML SUBJECT_SAMPLE_FACTORS - EI_2 Sample source:Early | Treatment:Fed RAW_FILE_NAME(Raw file name)=EI_2.mzXML SUBJECT_SAMPLE_FACTORS - EI_3 Sample source:Early | Treatment:Fed RAW_FILE_NAME(Raw file name)=EI_3.mzXML SUBJECT_SAMPLE_FACTORS - LC_1 Sample source:Late | Treatment:Control RAW_FILE_NAME(Raw file name)=LC_1.mzXML SUBJECT_SAMPLE_FACTORS - LC_2 Sample source:Late | Treatment:Control RAW_FILE_NAME(Raw file name)=LC_2.mzXML SUBJECT_SAMPLE_FACTORS - LC_3 Sample source:Late | Treatment:Control RAW_FILE_NAME(Raw file name)=LC_3.mzXML SUBJECT_SAMPLE_FACTORS - LI_1 Sample source:Late | Treatment:Fed RAW_FILE_NAME(Raw file name)=LI_1.mzXML SUBJECT_SAMPLE_FACTORS - LI_2 Sample source:Late | Treatment:Fed RAW_FILE_NAME(Raw file name)=LI_2.mzXML SUBJECT_SAMPLE_FACTORS - LI_3 Sample source:Late | Treatment:Fed RAW_FILE_NAME(Raw file name)=LI_3.mzXML SUBJECT_SAMPLE_FACTORS - MC_1 Sample source:Mid | Treatment:Control RAW_FILE_NAME(Raw file name)=MC_1.mzXML SUBJECT_SAMPLE_FACTORS - MC_2 Sample source:Mid | Treatment:Control RAW_FILE_NAME(Raw file name)=MC_2.mzXML SUBJECT_SAMPLE_FACTORS - MC_3 Sample source:Mid | Treatment:Control RAW_FILE_NAME(Raw file name)=MC_3.mzXML SUBJECT_SAMPLE_FACTORS - MI_1 Sample source:Mid | Treatment:Fed RAW_FILE_NAME(Raw file name)=MI_1.mzXML SUBJECT_SAMPLE_FACTORS - MI_2 Sample source:Mid | Treatment:Fed RAW_FILE_NAME(Raw file name)=MI_2.mzXML SUBJECT_SAMPLE_FACTORS - MI_3 Sample source:Mid | Treatment:Fed RAW_FILE_NAME(Raw file name)=MI_3.mzXML #COLLECTION CO:COLLECTION_SUMMARY Three biological replicates of AD-fed and rCanPI-fed insects from early, mid, CO:COLLECTION_SUMMARY and late responses were used. Metabolites were extracted by adding 500 µl of CO:COLLECTION_SUMMARY 80% chilled methanol (MS-grade, Waters) to 25 mg of frozen and crushed tissue. CO:COLLECTION_SUMMARY The suspension was vortexed for 1 min and frozen at -80 °C for 10 min. The CO:COLLECTION_SUMMARY freeze-thaw cycle was repeated twice, followed by centrifugation at 15,000g for CO:COLLECTION_SUMMARY 10 min at 4 °C. The supernatant was collected in a separate tube, and 100 μl CO:COLLECTION_SUMMARY was dried using a speed vacuum at room temperature for 20 to 25 min. Samples CO:COLLECTION_SUMMARY were stored at -80 °C till further analysis. For sample injection, each sample CO:COLLECTION_SUMMARY was re-suspended in 25 μl of methanol-water mixture (3:17, methanol: water), CO:COLLECTION_SUMMARY vortexed briefly for 30 s, and centrifuged at 14,000 rpm for 10 min at 4 °C. CO:SAMPLE_TYPE Larvae #TREATMENT TR:TREATMENT_SUMMARY Experiment design and feeding assays were performed as per our previous study TR:TREATMENT_SUMMARY (Lomate et al., 2018). In brief, H. armigera larvae were maintained at optimal TR:TREATMENT_SUMMARY growth conditions in the laboratory with 27 ± 2°C, 60 ± 5% relative humidity TR:TREATMENT_SUMMARY and a photoperiod of 14 h light and 10 h dark. An artificial diet (AD) was TR:TREATMENT_SUMMARY prepared as per (Mahajan et al., 2013), and the PI diet was prepared by adding TR:TREATMENT_SUMMARY 150 μg of recombinant Capsicum annum protease inhibitor (rCanPI-7) to the TR:TREATMENT_SUMMARY artificial diet. Neonates were fed on artificial diet for 2 days, and then first TR:TREATMENT_SUMMARY instar larvae were transferred to the control artificial diet (AD-fed) and TR:TREATMENT_SUMMARY rCanPI-7 incorporated artificial diet (CanPI-fed) for 48 hours. Whole larvae TR:TREATMENT_SUMMARY were harvested at 0.5, 2, 6, 12, 24 and 48 h, each set containing 100 larvae. TR:TREATMENT_SUMMARY Pooled samples of 0.5, 2, and 6 h (early response), 12 and 24 h (mid response), TR:TREATMENT_SUMMARY and 48 h (late response) were studied using lipidomic and metabolomic studies. TR:TREATMENT_SUMMARY At each stage of bioassay, the harvested samples were snap frozen in liquid TR:TREATMENT_SUMMARY nitrogen and stored at -80°C until further use. Three biological replicates TR:TREATMENT_SUMMARY were used for both lipidomic and metabolomic study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Three biological replicates of AD-fed and rCanPI-fed insects from early, mid, SP:SAMPLEPREP_SUMMARY and late responses were used. Metabolites were extracted by adding 500 µl of SP:SAMPLEPREP_SUMMARY 80% chilled methanol (MS-grade, Waters) to 25 mg of frozen and crushed tissue. SP:SAMPLEPREP_SUMMARY The suspension was vortexed for 1 min and frozen at -80 °C for 10 min. The SP:SAMPLEPREP_SUMMARY freeze-thaw cycle was repeated twice, followed by centrifugation at 15,000g for SP:SAMPLEPREP_SUMMARY 10 min at 4 °C. The supernatant was collected in a separate tube, and 100 μl SP:SAMPLEPREP_SUMMARY was dried using a speed vacuum at room temperature for 20 to 25 min. Samples SP:SAMPLEPREP_SUMMARY were stored at -80 °C till further analysis. For sample injection, each sample SP:SAMPLEPREP_SUMMARY was re-suspended in 25 μl of methanol-water mixture (3:17, methanol: water), SP:SAMPLEPREP_SUMMARY vortexed briefly for 30 s, and centrifuged at 14,000 rpm for 10 min at 4 °C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY XBridge BEH Amide (Waters Corporation, Milford, MA, USA) column were used for CH:CHROMATOGRAPHY_SUMMARY reverse-phase and HILIC, respectively. For polar compound separation, solvent A CH:CHROMATOGRAPHY_SUMMARY consisted of 20 mM ammonium acetate in water (pH 9.0), while mobile phase B was CH:CHROMATOGRAPHY_SUMMARY 100% acetonitrile. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Waters XBridge BEH Amide (100 x 2.1mm,2.5um) CH:SOLVENT_A 20 mM ammonium acetate (pH-9.0) water CH:SOLVENT_B ACN CH:FLOW_GRADIENT 85% B and proceeds to 10% B over 14 minutes CH:FLOW_RATE 200 ul/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS An Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Waltham, MA, MS:MS_COMMENTS USA) equipped with a heated electrospray ionization (HESI) source was used for MS:MS_COMMENTS data acquisition following (Kumar et al., 2020). In brief, for MS1 acquisition, MS:MS_COMMENTS the mass resolution was set to 120,000, while MS2 was done at 30,000 with a mass MS:MS_COMMENTS range of 60 to 900 Da. MS:MS_RESULTS_FILE ST004001_AN006598_Results.txt UNITS:relative intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END