#METABOLOMICS WORKBENCH yash_thsti_20250620_234052 DATATRACK_ID:6072 STUDY_ID:ST004011 ANALYSIS_ID:AN006616 PROJECT_ID:PR002506
VERSION             	1
CREATED_ON             	June 28, 2025, 9:27 pm
#PROJECT
PR:PROJECT_TITLE                 	Metabolic and Lipidomic Trade-offs in Helicoverpa armigera: Dynamics Under Plant
PR:PROJECT_TITLE                 	Protease Inhibitor-Induced Stress
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: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_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: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
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            	Experiment design and feeding assays were performed as per our previous study
CO:COLLECTION_SUMMARY            	(Lomate et al., 2018). In brief, H. armigera larvae were maintained at optimal
CO:COLLECTION_SUMMARY            	growth conditions in the laboratory with 27 ± 2°C, 60 ± 5% relative humidity
CO:COLLECTION_SUMMARY            	and a photoperiod of 14 h light and 10 h dark. An artificial diet (AD) was
CO:COLLECTION_SUMMARY            	prepared as per (Mahajan et al., 2013), and the PI diet was prepared by adding
CO:COLLECTION_SUMMARY            	150 μg of recombinant Capsicum annum protease inhibitor (rCanPI-7) to the
CO:COLLECTION_SUMMARY            	artificial diet. Neonates were fed on artificial diet for 2 days, and then first
CO:COLLECTION_SUMMARY            	instar larvae were transferred to the control artificial diet (AD-fed) and
CO:COLLECTION_SUMMARY            	rCanPI-7 incorporated artificial diet (CanPI-fed) for 48 hours. Whole larvae
CO:COLLECTION_SUMMARY            	were harvested at 0.5, 2, 6, 12, 24 and 48 h, each set containing 100 larvae.
CO:COLLECTION_SUMMARY            	Pooled samples of 0.5, 2, and 6 h (early response), 12 and 24 h (mid response),
CO:COLLECTION_SUMMARY            	and 48 h (late response) were studied using lipidomic and metabolomic studies.
CO:COLLECTION_SUMMARY            	At each stage of bioassay, the harvested samples were snap frozen in liquid
CO:COLLECTION_SUMMARY            	nitrogen and stored at -80°C until further use. Three biological replicates
CO:COLLECTION_SUMMARY            	were used for both lipidomic and metabolomic study.
CO:SAMPLE_TYPE                   	insect gut
#TREATMENT
TR:TREATMENT_SUMMARY             	NA
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Three biological replicates of AD-fed and rCanPI-fed insects of early, mid, and
SP:SAMPLEPREP_SUMMARY            	late response were used for lipid profiling. Total lipids were extracted using
SP:SAMPLEPREP_SUMMARY            	as (Matyash et al., 2008) with some modifications. Methanol (0.5 mL) was added
SP:SAMPLEPREP_SUMMARY            	to 25 mg of crushed tissue, followed by thorough vortexing for 30 s. Next, 1.25
SP:SAMPLEPREP_SUMMARY            	mL of methyl-tert-butyl ether was added, and the mixture was incubated for 1h on
SP:SAMPLEPREP_SUMMARY            	a shaker at room temperature (~25°C). Later, 0.3 mL MS-grade water was added to
SP:SAMPLEPREP_SUMMARY            	introduce phase separation, followed by incubation at 25°C for 10 min. Samples
SP:SAMPLEPREP_SUMMARY            	were then centrifuged at 400 rpm and 10°C for 5 min. The upper organic phase
SP:SAMPLEPREP_SUMMARY            	was collected and dried using a SpeedVac concentrator. Samples were stored at
SP:SAMPLEPREP_SUMMARY            	−80 °C till further use. Before running on LC-MS/MS, the extract was
SP:SAMPLEPREP_SUMMARY            	re-suspended in 100 μL of a 65:30:5 (acetonitrile: 2-propanol: water, v/v/v)
SP:SAMPLEPREP_SUMMARY            	solution.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	solvent A consisted of a 2:3 v/v ratio of water to acetonitrile, and solvent B
CH:CHROMATOGRAPHY_SUMMARY        	was a 9:1 v/v ratio of propanol to acetonitrile, both containing 10mM ammonium
CH:CHROMATOGRAPHY_SUMMARY        	formate and 0.1% formic acid. Temperature of the column was at 40°C and a
CH:CHROMATOGRAPHY_SUMMARY        	constant solvent flow rate of 0.3 mL/min was maintained.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Dionex Ultimate 3000 RS
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
CH:SOLVENT_A                     	40% Water/60% Acetonitrile; 10mM Ammonium formate; 0.1% Formic acid
CH:SOLVENT_B                     	90% Propanol/10% Acetonitrile; 10mM Ammonium formate; 0.1% Formic acid
CH:FLOW_GRADIENT                 	A gradient elution method was employed for an 18-min period, where solvent B
CH:FLOW_GRADIENT                 	increased from 30 to 97% over the first 0 to 12 min, followed by a 3 min hold,
CH:FLOW_GRADIENT                 	then, solvent B was returned to 30% from 15.2 to 18 min.
CH:FLOW_RATE                     	300 μL/min
CH:COLUMN_TEMPERATURE            	40°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Fusion Tribrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	A high-resolution mass spectrometer, Orbitrap Fusion (Thermo Fisher Scientific,
MS:MS_COMMENTS                   	Waltham, MA, USA), was used for data acquisition, equipped with a heated
MS:MS_COMMENTS                   	electrospray ionization source (ESI). The ESI sheath and auxiliary gases were
MS:MS_COMMENTS                   	set at 60 and 20, respectively, and both positive and negative spray voltages
MS:MS_COMMENTS                   	were operated at 3000 V. During the MS run, a resolution of 120,000 with an
MS:MS_COMMENTS                   	automatic gain control (AGC) target of 200,000 was utilized, covering a mass
MS:MS_COMMENTS                   	range from 250 to 1200. In the subsequent MS/MS runs, the resolution was
MS:MS_COMMENTS                   	adjusted to 30,000 with an AGC target of 50,000, and fragmentation was achieved
MS:MS_COMMENTS                   	using a collision energy of 27 ± 3 (normalized collision energy (NCE, %)).
MS:MS_RESULTS_FILE               	ST004011_AN006616_Results.txt	UNITS:relative intensity	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END