Impact of NPP feeding on the growth, development, and gene expression of Helicoverpa armigera and Spodoptera frugiperda.

(A) Schematic representation of NPP feeding assay with H. armigera larvae carried out for 10th days (n = 100, second-instar larvae, dpd= days post diet). Representation of insects on the 10th day and 15th day of NPP feeding bioassay, indicating delayed growth, pupation, reduced size, and no eclosion. (B) Gene expression analysis of trehalose metabolism genes using qRT-PCR. (C) Relative quantification of metabolites formed in trehalose metabolism. (D) Differential expression analysis of genes involved in trehalose metabolism and muscle development using NPP-fed insect transcriptomics data. Genes are clustered using hierarchical clustering. (E) Gene expression analysis of selected myogenic genes in control and treatment using qRT-PCR. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns= non-significant).

dsRNA mediated silencing of H. armigera HaTPS/TPP.

Schematic representation of dsRNA feeding assay carried out for 8th days (n = 60, second-instar larvae). (A) Silencing confirmation of HaTPS/TPP using qRT-PCR and percent ex-vivo residual activity of HaTPP. (B) Gene expression of HaTreh1 and HaTreh2 using qRT-PCR and percent ex-vivo residual activity of HaTreh. (C) Relative quantification of metabolite level of trehalose metabolism in HaTPS/TPP-silenced insects of H. armigera. (D) Phenotypic abnormalities associated with pupae and moths were observed upon dsHaTPP feeding. (E) Histological analysis of longitudinal sections (∼8um) fifth instar larvae of H. armigera upon feeding of EV and dsHaTPP. Scale bar=200nm. (F) Heatmap representing the expression pattern of genes involved in trehalose metabolism and muscle development upon HaTPS/TPP silencing. (G) Gene expression analysis of selected myogenic genes upon dsHaTPP feeding by qRT-PCR. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns= non-significant).

dsRNA mediated silencing of H. armigera Prm (n=60, Second instar larvae).

(A) Silencing confirmation of HaPrm using qRT-PCR. (B) Phenotypic abnormalities associated with pupae and moths were observed upon dsHaPrm feeding. (C) Histological analysis of longitudinal sections (∼8um) fifth instar larvae of H. armigera upon feeding of EV and dsHaPrm. Scale bar=200nm. (D) Heatmap represents the gene expression pattern involved in muscle development upon HaPrm silencing. (E) Gene expression analysis of myogenic genes upon HaPrm silencing using qRT-PCR. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns=non-significant).

Inhibition of trehalose metabolism disrupts energy homeostasis and affects the regulation of the cell cycle and transcription factor.

(A) Schematic of the flow of trehalose and glycolysis toward pyruvate. Metabolites that are significantly reduced upon HaTPS/TPP silencing compared to controls are shown in green and increased metabolite shown in red. (B) Heatmap represents the differential gene expression analysis of glycolytic genes upon HaTPS/TPP silencing. (C) Heatmap represents the differential gene expression analysis of cell cycle genes and energy-sensing genes upon HaTPS/TPP silencing. (D) Differential gene expression TFs upon silencing of HaTPS/TPP and HaPrm. (E) Validation of gene expression level of TFs by real-time PCR upon HaTPS/TPP and HaPrm silencing. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns=non-significant).

Functional gene regulatory network inference of the TPS/TPP silencing in H. armigera.

(A) Computational pipeline for RNA-seq data processing and network analysis. (B) KEGG pathways enriched upon TPS/TPP silencing, as identified by GSEA. Color scale is dependent upon adjusted P-value. The size of the circles is dependent on the number of enriched genes within each pathway. The X-axis shows the normalized enrichment scores (NES) of the different processes. (C) Gene regulatory subnetwork underlying the enriched motor protein KEGG gene set (motor protein - fcGRN). The network displays transcription factors with an E2F family transcription factor prominently featured at the centre, and their regulatory targets. Node size reflects the number of outgoing connections within the network such that TF with more number of connections will be highlighted. E2F family transcription factor is labelled in maroon, motor proteins are labelled in red and other proteins have a light-blue colour. (D) E2F family – enriched Gene set regulatory subnetwork. The source nodes are E2Fs and the target nodes are the genes belonging to 11 enriched gene sets. The network shows E2F transcription factor at the centre with regulatory connections to genes involved in various KEGG pathways. Nodes are color-coded according to their associated biological pathways as indicated in the legend.

Gene expression analysis and EMSA.

(A) Gene expression analysis of E2F and Dp upon HaTPS/TPP silencing. (B) Gene expression analysis of E2F and Dp upon HaPrm silencing. (C) Gene expression analysis of E2F and Dp upon feeding of 1000 ppm NPP to H. armigera larvae. (D) Binding of HaTPS/TPP promoter DNA with HaE2F transcription factor protein. Lane 1: HaTPS/TPP promoter DNA only (75 ng). Lane 2: HaE2F protein only 2 ug). Lane 3: DNA marker. Lane 4: HaTPS/TPP promoter DNA (75 ng) with HaE2F protein (0.5 ug). Lane 5: HaTPS/TPP promoter DNA (75 ng) with HaE2F protein (1 ug). Lane 6: HaTPS/TPP promoter DNA (75 ng) with HaE2F protein (2 ug). Lane 7: HaTPS/TPP promoter DNA (75 ng) with HaE2F protein (4 ug). Lane 8: HaTPS/TPP promoter DNA (75 ng) with HaE2F protein (8 ug). The gel shown in the upper panel was stained with SYBR Green EMSA stain. The gel shown in the lower panel is the same gel stained with SYPRO Ruby EMSA stain. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns=non-significant).

dsRNA mediated silencing of H. armigera HaDp to study the role of E2F in metabolism and muscle development (n=36, Second instar larvae).

(A) Silencing confirmation of HaE2F and HaDp using qRT-PCR. (B) Percent pupation rate calculated on the 18th day of assay. (C) Phenotypic abnormalities associated with moths were observed upon dsHaDp feeding. (D) Gene expression analysis of trehalose metabolic genes and myogenic genes by qRT-PCR

Exogenous feeding of 50 mM trehalose to the HaDp-silenced insects.

Schematic representation of rescue assay (n= 36, second instar larvae). (A) Insect body weight upon exogenous feeding of 50 mM trehalose to EV-fed insects. (B) Insect body weight upon exogenous feeding of 50 mM trehalose to dsHaDp-fed insects. (C) Percent pupation rate calculated 18th day of assay. (D) Normal adult moth emergence upon exogenous feeding of 50 mM trehalose to HaDp-silenced insects. (E) The gene expression level of TFs (HaE2F, HaDp), trehalose metabolic genes (HaTPS/TPP, HaTreh1), and myogenic genes (HaPrm, HaTm2) by real-time PCR upon exogenous feeding of 50 mM trehalose to HaDp-silenced insects. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns=non-significant).

Exogenous feeding of 50 mM trehalose leads to partial rescue of the HaTPS/TPP-silenced insects. Schematic representation of rescue assay (n= 60, second instar larvae). (A) Silencing confirmation and ex-vivo residual activity of HaTPP. (B) Gene expression analysis and residual activities of HaTPP and HaTreh compared between dsHaTPP and dsHaTPP plus 50 mM trehalose feeding. (C) Gene expression analysis of myogenic genes and selected TFs upon exogenous feeding of 50 mM trehalose to HaTPS/TPP-silenced insects. Asterisks indicate the significance level between control and treatment. Student’s unpaired t-test was used for calculating significance. Data are represented as mean ±SEM; (*p<0.05; **p value <0.01; *** p value <0.001; ****p value <0.0001; ns=non-significant).