Chitinase inhibition induces transcriptional dysregulation altering ecdysteroid-mediated control of Spodoptera frugiperda development

Summary

Chitinases and ecdysteroid hormones are vital for insect development. Crosstalk between chitin and ecdysteroid metabolism regulation is enigmatic. Here, we examined chitinase inhibition effect on Spodoptera frugiperda ecdysteroid metabolism. In vitro studies suggested that berberine inhibits S. frugiperda chitinase 5 (SfCht5). The Berberine feeding resulted in defective S. frugiperda development. Berberine-fed insects showed higher SfCht5 and Chitinase 7 expression and cumulative chitinase activity. Chitinase inhibition led to overexpression of chitinases, ecdysteroid biosynthesis, and responsive genes. SfCht5 silencing and overexpression resulted in ecdysone receptor deregulation. Transcription factors, like Broad Complex Z4, regulate the ecdysteroid metabolism and showed high expression upon berberine ingestion. Broad Complex Z4 binding in 5′ UTR of Ecdysone receptor, SfCht5, Chitinase 7, Phantom, Neverland, and other ecdysteroid biosynthesis genes might lead to their upregulation in berberine-fed insects. As a result, berberine-fed insects showed ecdysone overaccumulation. These findings underscore chitinase activity’s impact on ecdysone biosynthesis and its transcriptional crosstalk.

Graphical abstract

Highlights

• •Chitinase inhibition led to chitin and ecdysteroid metabolism deregulation
• •Chitinase silencing and overexpression resulted in ecdysone receptor deregulation
• •Ecdysteroid biosynthesis genes were upregulated on chitinase inhibition
• •Chitinase activity impacts on ecdysone biosynthesis and its transcriptional crosstalk

Classification Description: Biological sciences; Microbial metabolism

Introduction

Insect development is governed by an interplay of molecular regulators, among which chitinases and ecdysteroid hormones are pivotal contributors. Insect exoskeletal and peritrophic matrix has chitin as a major constituent. It protects insects from physical, mechanical, chemical, and infectious damage.^1,2,3 The insect exoskeleton is modified during development and maturation by shedding old and replacing it with a new cuticle. This suggests its indispensability during molting in the larval stages, pupation, and eclosion. Simultaneously, ecdysteroid hormones orchestrate these crucial stages in insect development. However, the crosstalk between these two essential components remains enigmatic, representing a critical knowledge gap in understanding insect development.

It is known that diverse isoforms of chitinases act during different life stages, physiological processes, and cellular responses. Chitin degradation is an integral part of exoskeleton remodeling. The hydrolysis of the β-1-4-glycosidic bond by endochitinase and β-N-acetylglucosaminidase (exochitinase) forms N-acetylglucosamine (NAG). NAG can be recycled and used as a monomer for chitin biosynthesis.² Apart from getting synthesized by recycling NAG, chitin can also be produced from trehalose and glycogen.⁴ Insect chitinases are classified into eight groups (groups I to VIII) based on sequence similarity and domain organization.⁵ They are known for their distinctive functions, such as group I chitinases are involved in adult ecdysis,⁶ while group II chitinases play a role in embryo hatching and molting.⁷ Group III chitinases are expressed during late developmental stages and are important for tissue differentiation, regulating abdominal contraction, and wing development.⁸ In contrast, group IV chitinases are specifically expressed in the gut during feeding stages (larva and moth) and aid peritrophic matrix-associated chitin turnover or digestion of dietary chitin.⁹ Group V comprises imaginal disc growth factors associated with cell proliferation and differentiation.¹⁰ However, the functional roles of the chitinases belonging to group VI, VII, and VIII in insects remain poorly characterized.² Chitinase silencing or its inhibition in several insects resulted in a defective peritrophic matrix, abortive molting, growth retardation, and developmental abnormalities. For example, dsRNA-mediated silencing of the group IV chitinase in Ostrinia nubilalis midgut resulted in increased chitin accumulation in the peritrophic matrix and a significant decrease in the larval mass.⁹ Likewise, dsRNA injection against chitinase in Tribolium castaneum caused severe defects during larva to larva, larva to pupa, and pupa to adult molting, depending on the dsRNA administration timing.⁷ In the case of Spodoptera frugiperda, chitinase silencing affects the molting and eclosion.^11,12 Moreover, natural molecules like shikonin, wogonin, quercetin, kaempferol, and berberine are known insect chitinase inhibitors. Shikonin and wogonin treatment caused larval growth retardation and increased mortality in S. frugiperda, O. furnacalis, and Mythimna separata.¹³ Berberine feeding has resulted in impaired growth and metamorphosis in O. furnacalis.¹⁴ Chitinase silencing and inhibition resulting in developmental defect is well known, yet effect of chitin degradation impairment on the developmental processes other than chitin remodeling is largely unexplored.

Insect molting and metamorphosis are under hormonal regulation. Known as the primary molting hormone 20-hydroxyecdysone (20E), titer significantly increases before molting. This may trigger the expression of molting-related genes such as the 20E receptor, Ecdysone receptor (EcR), Broad-Complex Zinc Finger 4 (BR-C Zs), and Ecdysone-induced protein 74 (E74).¹⁵ 20E also regulates chitin biosynthetic pathway genes such as Chitin synthase (CHS-A and CHS-B), Trehalase, and Glucose-6-P isomerase.^16,17 Moreover, chitinase expression increased after elevation of 20E levels, indicating chitinase is a potentially 20E-responsive gene.⁴ During metamorphosis in Bombyx mori, 20E increases the Chitinase 5 expression through Broad-Complex Zinc Finger 4 (Br-Z4)-mediated regulation. However, the detailed molecular interactions between chitinases and ecdysteroids through transcriptional regulations that underpin their concerted action remain insufficiently explored. This highlights the need to unravel chitin and ecdysteroid metabolism crosstalk under altered chitin degradation, at gene expression, protein activity, and metabolite levels.

Here, we sought to examine the molecular effects of the chitinase inhibitor, berberine, on the metabolism and physiology of S. frugiperda. The impact of chitinase inhibition on chitin metabolism and recycling was analyzed. Further, its effect on ecdysteroid biosynthesis and response is accounted. Also, the effect of chitinase inhibition, silencing, and overexpression on S. frugiperda molting-related genes and their transcriptional control was assessed. Our study aspires to contribute valuable insights into the intricate crosstalk between chitinases and ecdysteroid hormones upon chitinase inhibition, shedding light on insect development’s molecular intricacies and potentially uncovering unique targets for pest control strategies and agricultural applications.

Results

Berberine inhibits chitinase activity by blocking active-site access

Phylogenetic analysis revealed that S. frugiperda chitinase 5 (SfCht5) belongs to group I chitinases, which are expressed in all insect developmental stages and vital for molting (Figure 1A). In silico interaction, studies predicted that berberine (Figure 1B; Table S1) binds in the SfCht5 active site (Figure 1C) and forms mainly Pi-Pi stacked and Pi-Pi T-shaped interaction with lining aromatic residues like W106, Y271, F308, and W371 (Figure 1D). Next, we expressed SfCht5 in E. coli and purified recombinant protein was assayed for activity inhibition by berberine (Figures 1E and 1F). With an IC₅₀, ∼158 μM, berberine showed concentration-dependent inhibition of recombinant SfCht5. Further, berberine at ∼160 μM concentration inhibited 85% of S. frugiperda endochitinase activity with IC₅₀ ∼ 6.8 μM (Figure S1). These results indicate that berberine can inhibit SfCht activity in vitro and ex vivo and can interfere with chitin metabolism.

Figure 1.

In vitro interaction and biochemical characterization of SfCht5 inhibition by BER

(A) Phylogenetic analysis of SfCht5 with differentially expressed chitinases from BER-fed insect transcriptome (represented with the diamond shape) and chitinases from other insects. Phylogenetic analysis was done using the maximum likelihood method with 1,000 bootstrap value.

(B) Chemical structure of BER.

(C) In silico docking pose of BER with SfCht5 represented in surface representation using PyMOL molecular visualization tool.

(D) Three-dimensional map of intermolecular interaction between the BER and binding pocket of SfCht5, where R371 forms polar contact with OH group of BER and F308 is involved in Pi-interaction with central aromatic ring.

(E) Purified recombinant SfCht5 was checked on 12% SDS-PAGE.

(F) Inhibition kinetics of SfCht5 activity with BER showed competitive inhibition with IC₅₀ of 137 μM (Data are represented as mean ± SEM).

Berberine ingestion impedes S. frugiperda growth and development by affecting chitin metabolism

We examined the effect of berberine feeding on insect growth and development. S. frugiperda second instar larvae fed on an artificial diet (AD) with different berberine concentrations (100, 500, and 1,000 ppm) showed significantly reduced larval mass. Compared to the control group fed on AD, 22^nd day post diet (DPD), we observed 42%, 53%, and 80% mortality in 100, 500, and 1,000 ppm berberine-fed larvae, respectively (Figure 2A). On the 6^th DPD, larvae fed on 100, 500, and 1,000 ppm berberine had around 15%, 54%, and 69% less larval mass, respectively (Figures 2B and 2C). In summary, berberine ingestion resulted in increased mortality and developmental delay due to reduced larval mass. The chitin content in berberine-fed larvae was relatively high compared to the control (Figure 2D). Further, at 6^th DPD, the berberine-fed larvae exhibited increased endochitinase activity, with fluorescence intensities of ∼15,201 RFU, while the control larvae exhibited ∼3,480 RFU (Figure 2E). Despite high endochitinase activity, high chitin content in berberine-fed larvae could be due to inhibition of overexpressed endochitinases by berberine. Berberine feeding also impacted pupa stage, resulting in decreased mass and delayed and aberrant pupal development (Figures 2F, S2A, and S2B). On 14^th DPD, 100%, 57%, 39%, and 9% pupation rates were observed in control, 100, 500, and 1,000 ppm berberine-fed insects, respectively (Figures S2B and S2C). Further, the moth’s emergence has reduced significantly in pupa originating from berberine-fed larvae; out of total pupas, 83%, 44%, 15%, and 12% emerged as viable moths in control, 100, 500, and 1,000 ppm berberine-fed insects. The berberine feeding has also affected moths’ development. The moths originated from 100 ppm berberine-fed larvae had pupal skin attached to the thorax. Feeding at high berberine concentration, moths showed a lower emergence rate and abnormal development (Figure S2D). Importantly, berberine is stable in an insect gut environment (Figure 2G). These findings indicate that berberine interferes with the growth, development, and survival of S. frugiperda by inhibiting chitinase activity.

Figure 2.

BER hinders the growth and development of Spodoptera frugiperda

(A) Comparative percent mortalities versus time of larvae fed on artificial diet (Control) and artificial diet with different concentrations of BER (100, 500, and 1,000 ppm).

(B) The average size of larvae photographed on 6^th day post diet (DPD).

(C) Comparative mass gain of S. frugiperda larvae fed on control and BER-containing diet (100,500, and 1,000 ppm) (n = 16).

(D) Chitin content assessment in larvae fed with control and BER (500 ppm) diet at 6^th DPD.

(E) Residual chitinase activity in larvae fed with control and BER (500 ppm) diet at 6^th DPD.

(F) Developmental delay at 12^th DPD in larvae fed with BER.

(G) Stability of BER was assessed in the frass of the larvae fed with and without BER (500 ppm) at 6^th DPD. Asterisks indicate significant changes compared to control as calculated by Student’s unpaired t test (Data are represented as mean ± SEM; ∗p value <0.05; ∗∗p value <0.01; ∗∗∗p value <0.001, ∗∗∗∗p-value <0.0001).

Chitinase inhibition significantly altered chitin remodeling and its hormonal control in S. frugiperda

To study the effects of berberine feeding on gene expression and, eventually, on the metabolism, we performed high-throughput transcriptome sequencing of larvae fed on AD and 500 ppm berberine containing AD on 6^th DPD. Transcriptome analysis showed that the expression of 3,855 genes were significantly altered, out of which 897 genes were upregulated (log₂ fold change ≥1.5 and p value ≤0.05) and 2,958 genes were downregulated (log₂ fold change ≤ −0.5 and p value ≤0.05) upon berberine feeding compared with control larvae at 6^th DPD (Figure S3A). Gene ontology enrichment analysis mapped DEGs to vital physiological processes such as cuticle development, electron transport chain, and ATP synthesis (Figures S3B–S3D).

Interestingly, we found cuticle biosynthesis-related genes like Chitin synthase, Glutamine-fructose-6-P-aminotransferase, Phospho-N-acetyl-glucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, Chitin deacetylase isoforms, and Cuticle proteins were significantly overexpressed. The chitin recycling-related genes like chitinases also found to be dysregulated viz., chitinases 5 and chitinase 7 from group II and III, respectively, were upregulated (Figures 3A and 3B), whereas the chitinase 2 and chitinase EN03 belonging to group IV and V, respectively, were downregulated (Figure S4). Furthermore, genes involved in chitin biosynthesis from trehalose have significantly upregulated leading to glucosamine-6-phosphate overaccumulation (Figure 3B). On the contrary, β-N-acetyl-hexosaminidase involved in chitin depolymerization and SfCht2 from group IV, which has gut-specific expression during feeding stages, were downregulated (Figure 3A). This downregulation in β-N-acetyl-hexosaminidase resulted in a significant decrease in the N-acetylglucosamine-1-p level (Figure 3B). In addition, essential genes related to glycolysis, namely Pyruvate kinase, Aldolase, and Enolase, showed significant downregulation and Hexokinase and phosphoglucomutase showed increased expression (Figures 3A and 3B). While citrate cycle-related genes, like Citrate synthase and Malate dehydrogenase, were also downregulated. This resulted in reduced citrate and cis-aconitate levels (Figures 3A and 3B). Further, both the membrane-bound and soluble forms of trehalase are upregulated (Figures S5A and S5B), leading to a significant decrease in trehalose, while Trehalose transporter 1 is downregulated (Figure S6). On the contrary, glucose transporters, such as SWEET1, and glucose transporter type 1 were significantly upregulated in berberine-fed insects (Figure S6). Further, Fatty acid synthase and acetyl-CoA acetyltransferase, which are involved in fatty acid synthesis, also showed significant deregulation (Figures 3A and 3B).

Figure 3.

Chitinase inhibition affects chitin remodeling, energy metabolism, and endocrine control in S. frugiperda

(A) Heatmap for significant differentially expressed genes in larvae fed with control and BER (500 ppm) diet at 6^th DPD plotted using log₂FPKM values. Samples and genes are clustered using hierarchical clustering. Heatmap indicated overall overexpression of chitin metabolism, ecdysone metabolisms, and glycolysis-related genes in BER-fed insects. Whereas juvenile hormones metabolism and citrate cycle genes showed downregulation in BER-fed insects.

(B) Schematic representation of the chitin biosynthesis, recycling, and energy metabolism pathways in insects. The bar graph represents the candidate genes’ relative transcript levels (FPKM), and violin plots represent the candidate metabolites.

(C) Validation of selected genes with qRT-PCR. Asterisks indicate significant changes compared to the control as calculated by Student’s unpaired t test (Data are represented as mean ± SEM; ∗∗p value <0.0021; ∗∗∗ P value 0.0002; ∗∗∗∗p value <0.0001). (FPKM: fragments per kilobase of transcript per million mapped reads).

In response to chitinase inhibition, several endocrine system-related genes were found to be deregulated. Ecdysone oxidase, Ecdysone receptor, Eclosion hormone, Fushi tarazu (FTZ-F1), and Insulin Growth Factor (IGF)-binding protein were upregulated, whereas Juvenile Hormone (JH) Esterase, JH Epoxide hydrolase, JH-binding protein, and JH Repressible protein were downregulated (Figures 3A and S7). The overall transcriptome data were validated with quantitative real-time PCR (qRT-PCR) for the candidate genes (Figure 3C). Transcriptome and metabolomics data of berberine-fed insects suggested that chitin metabolism and endocrine control are significantly affected.

SfCht5 gene expression modulation impacts ecdysteroid biosynthesis and molting

Silencing of SfCht5 using dsRNA (Figure S8) resulted in around 70% reduction in Chitinase 5 expression on 6^th DPD (Figure 4A). The SfCht5 silencing exerted 15% mortality in insects; this indicates the indispensability of SfCht5 in insect’s overall survival and fitness (Figure 4B). No significant effect was observed in the larval weight of SfCht5-silenced group and most of them were able to pupate (Figure 4C). However, the eclosion of moths was hampered in the SfCht5-silenced insects, resulting in pharate moths’ emergence (Figure 4D). It has been observed that upon SfCht5 silencing, expression of Ecdysone receptor and Chitinase 7 is reduced significantly. Importantly, Chitin synthase 2 and Kruppel homolog showed upregulation while JH Esterase expression remained unaltered (Figure 4E).

Figure 4.

dsRNA-mediated silencing of S. frugiperda SfCht5

(A) Relative expression of SfCht5 in S. frugiperda larvae fed with control (diet with E. coli HT115 cells expressing L4440 Empty vector) and SfCht5 dsRNA (diet with E. coli HT115 cells expressing SfCht5 dsRNA) at 6^th DPD.

(B) Percent mortality of S. frugiperda larvae fed with control and SfCht5 dsRNA-containing diets.

(C) Relative body mass of S. frugiperda larvae fed on control and SfCht5 dsRNA-containing diet.

(D) Representative photo of S. frugiperda showing the effect of SfCht5 dsRNA on the moth eclosion.

(E) qRT-PCR-based relative expression analysis of selected genes in control and SfCht5 dsRNAs-fed larva at 6 DPD. Asterisks indicate significant changes compared to control as calculated by Student’s unpaired t test (Data are represented as mean ± SEM; ∗p value <0.05; ∗∗p value <0.01; ∗∗∗p value <0.001).

For overexpression of SfCht5, we mixed lipofectin with the SfCht5_pIBV5 plasmid and injected this mixture into S. frugiperda hemocoel (Figure 5A). The control insects were injected with EGFP_pIBV5 plasmid. qRT-PCR was used to measure SfCht5 mRNA 24 h post-injection. Three biological replicates (3 larvae in each) showed more than 4-fold increase in SfCht5 expression in SfCht5_pIBV5 plasmid-injected larvae as compared to the control (Figure 5B). Furthermore, total chitinase activity increased in SfCht5-overexpressed insects (Figure 5C). As an effect of SfCht5 overexpression, Phantom expression was significantly reduced, whereas, Ecdysone receptor showed increased expression (Figures 5D and 5E). This alteration in Phantom and Ecdysone receptor expression on SfCht5 overexpression supports the crosstalk of chitinase with ecdysteroid biosynthesis and response-related genes. Further, the SfCht5 overexpression also resulted in pupal deformities (Figure S9).

Figure 5.

Overexpression of SfCht5 in S. frugiperda

(A) Schematic representation of vector organization and strategy for overexpression by injection of pIBV5-SfCht5 plasmid mixed with lipofecting in 5^th instar S. frugiperda larvae (n = 6 in each treatment, in three biological replicate sets).

(B) Overexpression of SfCht5 in pIBV5-SfCht5 with lipofectin-injected larvae was confirmed with qRT-PCR analysis.

(C) SfCht5-overexpressed insects also showed an increased % chitinase activity.

(D and E) qRT-PCR analysis of the candidate genes from the ecdysone biosynthesis and signaling. Asterisks indicate significant changes compared to control as calculated by Student’s unpaired t test (Data are represented as mean ± SEM; ∗p value <0.05; ∗∗p value <0.01; ∗∗∗p value <0.001).

Transcription factors upregulated on chitinase inhibition result in overactivation of ecdysteroid biosynthesis and response

Transcript abundance of positive regulators of ecdysteroid biosynthesis has increased in berberine-ingested insects. Known transcription factors activating ecdysteroid biosynthesis and response such as SfFTZ-F1, SfBr-Z4, SfE75, and Ecdysone receptor showed increased expression in berberine-fed insects (Figure 6A). The high expression of candidate SfBr-Z4 in berberine-fed insects was validated by qRT-PCR (Figure S10). Transcription factor-binding site analysis in the 5′ UTR of SfCht5, Chitinase 7, Ecdysone receptor, Phantom, and Neverland showed the presence of SfBr-Z4-binding sites (Figure 6B). Further, electrophoretic mobility shift assays were performed to validate the in vitro interaction of the SfBr-Z4 protein (Figures S11A and S11B) with SfCht5 and SfEcR promoters. When incubated with the SfBr-Z4 protein, promoters of both SfCht5 and SfEcR showed retarded bands, suggesting the SfBr-Z4-promoter complex formation (Figures 6C and S11C). Phantom, Neverland, and other ecdysteroid biosynthesis-related genes showed increased transcript abundance in berberine-ingested insects (Figure 6D). Metabolite quantification showed overaccumulation of ecdysone in berberine-ingested insects (Figure 6E). Increased ecdysone levels were in accordance with the high expression of ecdysteroid biosynthesis-related genes. The overlapping function of high-expressing transcription factors such as SfBr-Z4 in the activation of chitinases and ecdysteroid biosynthesis-related genes could be a plausible mechanism by which ecdysteroid response is deregulated in berberine-ingested insects.

Figure 6.

Broad complex Z4 modulates the endocrine regulation of chitinase

(A) Digital gene expression analysis of transcription factors involved in chitinase and ecdysone biosynthesis regulation using BER-fed insects transcriptomics data. Heatmap is plotted using log₂FPKM values and clustered using hierarchical clustering, indicating that most transcription factors were upregulated. Br-Z4, a transcription factor common in ecdysteroid biosynthesis and chitinase expression, is overexpressed in BER-fed insects.

(B) Putative transcription factors binding sites in SfEcR, SfCht5, SfCht7, SfNvd, and SfPhm was predicted in +200 and −2,000 region from the transcription start site (TSS) using JASPER database. Br-Z4-binding sites were found to be abundant in putative promoter regions and common in candidate Chitinase genes and genes involved in ecdysone biosynthesis and its signaling.

(C) Binding of SfCht5 promoter DNA with Sf Br-Z4 transcription factor protein. Lane 1 and 5: SfCht5 promoter DNA only (75 ng). Lane 2 and 4: Sf Br-Zr protein only (1,500 ng). Lane 3: DNA markers. Lane 6–7: SfCht5 promoter DNA (75 ng) with Sf Br-Zr protein (1,500 ng). 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. The red star indicates the SfCht5 promoter: Sf Br-Z4 transcription factor complex, which is stained with both dyes.

(D) Transcript abundance of ecdysteroid biosynthesis-related genes found to be high in BER-fed insect transcriptome.

(E) Ecdysone levels were increased in S. frugiperda larvae fed with BER (500 ppm) as compared to control diet-fed larvae at 6 DPD.

Discussion

Group I chitinases are key for molting and metamorphosis. Interference in its activity or expression may lead to impairment in the molting process. Chitinase inhibition by natural molecules like shikonin, wogonin, quercetin, kaempferol, and berberine are known for insect developmental abnormalities. In the current study, S. frugiperda larvae fed on a berberine-containing diet showed increased mortality and reduced body mass due to antibiosis. Likewise, the pupation rate and pupal mass also decreased significantly on berberine ingestion. The larvae that survived upon berberine ingestion displayed delayed pupation. These results indicate that berberine potentially inhibits major chitinase isoforms and hampers chitin recycling and remodeling. It has been observed that berberine-fed insects show higher chitin content due to the inhibition of chitinase activity. Moreover, overexpression of chitinases from other groups may compensate for group I chitinase inhibition or silencing effect as cumulative chitinase activity can facilitate insect molting.⁷ Similarly, we observed increased chitinase activity, possibly due to compensatory overexpression of other chitinases.

Chitinase inhibition may hamper chitin recycling, activating chitin synthesis for molting and chitin remodeling. Chitinase inhibition results in increased chitin synthesis and higher expression of chitin synthase.¹⁸ Berberine-fed insects showed chitin synthase overexpression, which might be the putative reason for higher chitin content to facilitate chitin remodeling. Increased chitin synthesis was also reflected by the overaccumulation of the chitin precursor, glucosamine-6-phosphate. This glucosamine-6-phosphate overaccumulation might be due to the upregulation of trehalose degradation and amino sugar synthesis. Overexpression of Trehalase, Glutamine-fructose-6-P-aminotransferase, and UDP-N-acetylglucosamine pyrophosphorylase specifies that chitin synthesis is higher. Silencing of these genes in T. castaneum,¹⁹ Locusta migratoria,²⁰ Helicoverpa armigera,²¹ Spodoptera exigua,⁹ and Nilaparvata lugens²² resulted in chitin loss, increased mortality, and molting defects. Chitin deacetylation is essential for cuticular organization, and its disruption can lead to defective phenotypes.^23,24,25,26 Chitin deacetylase overexpression upon S. frugiperda chitinase inhibition predicts potential cuticle organization defect. Trehalose plays an essential role in insect chitin synthesis and molting. Silencing of chitinase leads to decreased levels of glucose, trehalose, and glycogen.^18,27 S. frugiperda chitinase inhibition leads to increased Trehalase expression and reduced trehalose levels, causing decreased expression of Trehalose transporter 1. In brief, increased trehalose hydrolysis upon chitinase inhibition may fulfill the glucose requirement for chitin synthesis. Hence, chitinase inhibition affects cuticular organization by dysregulation of chitin synthase, chitin deacetylase, and cuticular proteins expression.

Defective chitin metabolism leads to an imbalance in the use of glucose for glycolysis and citrate cycle.¹⁸ In the case of berberine-fed insects, these metabolic defects are reflected through the downregulation of Pyruvate kinase and Malate dehydrogenase. Moreover, reduced accumulation of fructose-6-P, citrate, and cis-aconitate corroborates with glycolysis and citrate cycle downregulation. This alteration in glucose flux and resultant glycolysis and citrate cycle inhibition may result in energy metabolism defect. Reduced energy and trehalose content may affect insects’ vitality, leading to decreased body mass.²⁸

Chitinase, Chitin synthase, and chitin deacetylase gene expression are under endocrine control and positively correlated with circulating 20E titers. 20E injection in B. mori and Manduca sexta larvae induces chitinase expression, while topical application of fenoxycarb (juvenile hormone mimic) suppresses chitinase expression.^29,30 In berberine-fed insects, genes related to ecdysteroid metabolism and response, like Ecdysone oxidase, Ecdysone receptor, Eclosion hormone, FTZ-F1, IGF-binding protein, and Kruppel homolog, were upregulated. While, JH degradation and suppression-related genes, such as JH Esterase, JH Epoxide hydrolase, JH-binding protein, and JH Repressible protein, were down-regulated. These observations indicate that chitinase inhibition causes upregulation of ecdysone-related genes as a feedback mechanism. High ecdysone levels in berberine-fed insects may activate the expression of late developmental genes like Titin, Yellow protein, HR 4, and Eclosion hormone. Several of these overexpressed late-developmental genes, like Ecdysone oxidase, Titin, and FTZ-F1, were found to be co-expressed with Chitinase 7. Furthermore, SfCht7 overexpression is one of the indicators of late larval stage induction in berberine-fed insects.^7,31 On the contrary, gut-specific Chitinase 2 isoform (SfCht2) was found to be downregulated. These changes in Chitinase 7 and Chitinase 2 expression patterns may represent the likely decline of feeding behavior and the emergence of the wandering stage.

To further dissect the effect of chitinase expression on ecdysone metabolism and response, we analyzed essential ecdysone-related genes in SfCht5-silenced tissue. On silencing of SfCht5, increased mortality and defective phenotypes were observed. Interestingly, the pupa to adult transition was defective, leading to pharate pupa formation or abnormal eclosion. Chitinase 7 showed reduced expression on SfCht5 silencing. While, Ecdysone receptor showed significant downregulation, suggesting deregulation of ecdysteroid response. Whereas other hormone regulation-related genes JH esterases and Kruppel homolog are unaltered. These results indicate that chitinase silencing may interfere with Ecdysone receptor gene expression. In vivo, overexpressed SfCht5 causes an increase in SfCht5 expression and cumulative chitinase activity. Likewise, Chitinase 5 overexpression also causes significant alterations in Ecdysone receptor and Phantom gene expression. Silencing and overexpression of SfCht5 resulted in the deregulation of ecdysteroid biosynthesis and response gene expression, suggesting their possible crosstalk with chitinase.

Transcription factors that positively regulate ecdysteroid biosynthesis are found to be upregulated in berberine-fed insects. In B. mori, BmCht5, was upregulated during larval-larval and larval-pupa transitions and induced by 20E.³² Promoter analysis of BmCht5 revealed the presence of BR-C Z4, BR-C Z2, and E74A cis-regulatory elements related to 20E response. Similarly, in the transcription factor-binding site analysis in 5′ UTR of SfCht5, Chitinase 7, Ecdysone receptor, Phantom, and Neverland, multiple Br-Z4-binding sites were predicted. This prediction suggests that Br-Z4 might be one of the transcription factors (TFs) involved in regulating chitinases and ecdysteroid biosynthesis-related genes. qRT-PCR analysis confirms that Br-Z4 is overexpressed in berberine-fed insects. Br-Z4 and other TFs overexpression might increase the expression of ecdysteroid biosynthesis-related genes, Phantom, Neverland, NPC cholesterol transporter, etc. Further, this overexpression of ecdysteroid biosynthesis genes increases overall ecdysone levels in the berberine-fed insects. This overaccumulation of ecdysone may cause endocrine disruption, resulting in developmental defects in berberine-fed insects.

Our study suggests that chitinase inhibition affects insect development in a multifactorial way. Chitinase inhibition undoubtedly alters chitin synthesis and remodeling; as a result, glucose flux and energy metabolisms are hampered. Collectively, this may cause delayed development and stunted growth. The insect might adjust ecdysone synthesis, metabolism, and responses to balance affected chitin metabolism. However, this metabolic rewiring and endocrine control adjustment resulted in severe developmental abnormalities and defects. This study highlights the dual potential of chitinase inhibitors as metamorphosis disruptors and hormonal deregulators for futuristic pesticide design.

Limitations of the study

This study paved the path for future investigations on the downstream effects of excess ecdysone induced by chitinase inhibition. It is essential to explore alteration in downstream signaling pathways and gene expression beyond the molting and metamorphosis. Further, examining the long-term consequences of an imbalance in chitin metabolism and its endocrine control on insect physiology, reproductive success, and overall fitness will be vital. Functional interaction and crosstalk of transcriptional regulators with chitin metabolism and the endocrine system need to be explored for an in-depth understanding of insect development.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
Escherichia coli BL21 (DE3)	Thermo Fisher Scientific Inc.	Cat#EC0114
Escherichia coli Shuffle T7	New England Biolabs	Cat#C3029J
Escherichia coli HT115 (DE3)	Laboratory of Dr. Sandhya Kaushik, TIFR, India	N/A
Escherichia coli Top10	Thermo Fisher Scientific Inc.	Cat#C404010
Chemicals, peptides, and recombinant proteins
9,10-Dimethoxy-5,6-dihydro-[1,3] dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium chloride dihydrate (Berberine)	BLDpharm	Cat#BD261206
Isopropyl ß-D-1-thiogalactopyranoside	Sisco Research Laboratories	Cat#67208
cOmplete™ His-Tag Purification Resin	Roche	Cat#5893682001
4-Methylumbelliferyl ß-D-N, N',N''-triacetylchitotrioside	Sigma-Aldrich	Cat#M5639
Calcofluor white stain	Sigma-Aldrich	Cat#18909
TRIzol reagent	Invitrogen	Cat#15596026
TB Green Premix Ex Taq II (Tli RNase H Plus)	TaKaRa	Cat#RR82WR
In-Fusion® HD Cloning Kit	TaKaRa	Cat#ST0344
Phusion High-Fidelity DNA Polymerase	New England Biolabs	Cat#M0530S
Lipofectin	Invitrogen	Cat#18292037
Critical commercial assays
NEBNext Ultra II RNA Library Prep Kit	New England Biolabs	Cat#E7775
Electrophoretic Mobility-Shift Assay (EMSA) Kit	Invitrogen	Cat#E33075
Deposited data
RNA-seq raw data	This study	BioProject ID: PRJNA974147
Experimental models: Organisms/strains
Spodoptera frugiperda	ICAR-National Bureau of Agricultural Insect Resources, Bengaluru	N/A
Oligonucleotides
See Table S2 for Primer sequences used	N/A	N/A
Recombinant DNA
Plasmid: pET28a-SfCht5	This paper	N/A
Plasmid: pET28b-SfBr-Z4	This paper	N/A
Plasmid: L4440-SfCht5-dsRNA	This paper	N/A
Plasmid: pIBV5-SfCht5	This paper	N/A
pIBV5-EGFP	This paper	N/A
Software and algorithms
Prism 8	GraphPad software	https://www.graphpad.com/
Robetta server	Kim at al.33	https://robetta.bakerlab.org/
AutoDock 4.0	AutoDock	https://autodock.scripps.edu/
Marvin sketch	N/A	http://www.chemaxon.com
PyMOL	Schrodinger, LLC.	https://pymol.org/
FastQC	Andrews34	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Fastp v0.12.4	Chen et al.35	https://github.com/OpenGene/fastp.
MultiQC	Ewels et al.36	https://multiqc.info/
bbmap's v38.18 bbduk algorithm	Bushnell37	https://www.osti.gov/biblio/1241166
STAR v2.7.10a	Dobin et al.38	https://github.com/alexdobin/STAR
featureCounts v2.0.0 software	Liao et al.39	http://subread.sourceforge.net
spearman rank correlation	Zar40	Correlation_matrix.xlsx
DESeq2 v1.34.1	Love et al.41	http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html
FunSet web server	Hale et al.42	http://funset.uno/
KOBAS server	Wu et al.43	http://kobas.cbi.pku.edu.cn
MS-DIAL software	Tsugawa wt al.44	http://prime.psc.riken.jp/Metabolomics_Software/MS-DIAL/index.html
TB tools-II v2.003	Chen et al.45
JASPAR database	Castro-Mondragon et al.46	https://jaspar.genereg.net/
Other
2x 150bp Novaseq 6000 sequencing	Illumina	https://www.lifecell.in/
MassBank of North America	N/A	http://mona.fiehnlab.ucdavis.edu
assembly AGI-APGP_CSIRO_Sfru_2.0; fall armyworm	N/A	https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_023101765.2/

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rakesh S. Joshi (rs.joshi@ncl.res.in).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •RNA sequencing data have been deposited into the National Center for Biotechnology Information, NIH, Sequence Read Archive as Bioproject (Submission ID: SUB13375930; BioProject ID: PRJNA974147).
• •This paper does not report the original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Spodoptera frugiperda diet and growth conditions

S. frugiperda eggs were procured from ICAR-National Bureau of Agricultural Insect Resources, Bengaluru, India. Upon egg hatching the larvae were reared on an artificial diet (AD) at 28 ± 1°C under a 14: 10 h (L:D) photoperiod and 70% relative humidity until they complete the life cycle. The 700ml artificial diet contains chickpea flour 110 gm, ascorbic acid 2.6 gm, methyl-p-hydroxy-benzoate 2 gm, yeast extract 20 gm, sorbic acid 0.5 gm, casein powder 10 gm, multivitamin mixture 1 gm, streptomycin sulphate 1 gm, cholesterol 0.1 gm, formaldehyde 1 ml, vitamin E 0.6 gm, and agar 10 gm. After one generation of laboratory conditions acclimatized, the second-generation larvae were used for feeding bioassays.

Method details

Molecular modeling and docking studies

The three-dimensional structure of SfCht5 was modeled using the Robetta server.³³ The best-predicted model was energy-minimized and prepared for docking analysis using AutoDock 4.0 tool. Structures of berberine and other known natural molecules which are chitinase inhibitors were retrieved from Pubchem and converted to three-dimension using Marvin sketch (http://www.chemaxon.com). Ligand structures were prepared for docking using AutoDock 4.0 tool as described earlier.⁴⁷ The docking grid was set around SfCht5 active site with dimension 24∗24∗24 A° and docking was performed using the AutoDock Vina tool.⁴⁸ The docked complex structure was visualized and analyzed using the PyMol visualization tool.

Escherichia coli expression and in vitro assay for chitinase inhibition by berberine

For recombinant expression coding sequence of S. frugiperda endochitinase (Uniprot ID: A0A8K1Z7B3) without signal peptide was cloned into the pET28a vector using EcoRI/XhoI restriction sites and confirmed by sequencing. Further, confirmed clone was transformed and expressed in Escherichia coli BL21 (DE3) cells. The secondary culture of BL21 (DE3) cells transformed with pET28-SfCht5 was incubated until optical density (O.D.) reached 0.6, and was induced with 1 mM of IPTG. The induced culture was incubated overnight for recombinant protein expression. The bacterial pellet was subjected to sonication and protein purification using a Ni-NTA agarose matrix (cOmplete™ His-Tag Purification Resin, Roche). Detailed procedure of overexpression and purification of SfCht5 was carried out as described earlier.⁴⁹ To check the in vitro endochitinase inhibition potential of BER, rSfCht5 was incubated with different concentrations of BER, and residual endochitinase activity was measured using the synthetic substrate. Percent inhibition of rSfCht5 activity at different BER concentrations were plotted to calculate IC₅₀ values.⁵⁰

Ex vivo S.frugiperda chitinases activity in the presence of BER

Crude enzyme extract of 5^th instar S. frugiperda larvae fed on an artificial diet was prepared as mentioned earlier.¹¹ Briefly, 500 mg finely powdered tissue of 5^th instar larvae was homogenized in 50 mM sodium phosphate buffer (pH 7) in 1:1 ratio (w/v) and kept at 4°C for 2 h. The suspension was centrifuged at 13,000 ˣ g, 4°C for 20 min and the resulting supernatant was used as a source of chitinase. Total endochitinases activity inhibition from S.frugiperda was carried out using 4-Methylumbelliferyl ß-D-N, N',N''-triacetylchitotrioside as substrate, endochinase cleaves the substrate to release fluorescent 4-Methylumbelliferone. A suitable amount of enzyme extract was pre-incubated with the different concentrations of berberine at 30°C for 20 min, and the residual enzyme activity was estimated as described above.⁵⁰

Feeding bioassay

S. frugiperda larvae were reared on an artificial diet (AD) at 28 ± 1°C under a 14: 10 h (L:D) photoperiod and 70% relative humidity. Feeding bioassays were conducted as described earlier.⁵¹ Briefly, the second instar S. frugiperda (n=52 larvae/treatment, collectively in three independent experiments) were fed on an artificial diet (Control) and artificial diet with 100, 500, and 1000 ppm berberine for 22 DPD or until they undergo pupation. Insect growth in terms of weight and survival on control and berberine-containing diet was monitored on alternate days for 21 DPD. On the 6^th DPD larvae from each group were harvested by freezing in liquid nitrogen and pulled together in a group of three and treated as one replicate. Such three replicates were further used for transcriptomic, biochemical, and metabolic studies from the control and 500 ppm berberine-fed larvae.

Ex vivo chitin measurement

Chitin content in larvae fed with control and 500 ppm berberine-containing diet was measured at 6^th DPD, by using Calcofluor white stain (Sigma-Aldrich).⁵² Aliquots of 50 mg crushed larval tissue was homogenized in 200 μl Milli-Q water. To estimate chitin, 100 μl Calcofluor white stain (1mg/ml) was mixed with 20 μl larval tissue suspension and incubated in the dark for 15 min, followed by centrifugation at 20,000 × g for 5 min. The pellet was resuspended and washed with Milli-Q water. The washed pellet was resuspended in 200 μl Milli-Q water and transferred to a black 96-well microplate (Corning). Fluorescence intensity was determined using a microplate reader (Glomax Promega) with excitation λ_ex 365 nm and emission at λ_em 433 nm. Fluorescent intensity is directly proportional to the chitin present to which, Calcofluor white stain binds.

S. frugiperda ex vivo endochitinase activity

S. frugiprda crude enzyme extracts were prepared by homogenizing the tissue of larvae fed on control and 500 ppm berberine-containing diet in 50 mM sodium phosphate buffer (pH 7) in 1:1 ratio (w/v) and kept at 4°C for 2 h. The suspension was centrifuged at 13,000 ˣ g, 4°C for 20 min and the resulting supernatant was used as a source of chitinase.⁵³ The endochitinase activity assay was performed according to Vidhate et al. (2019) with minor modifications.⁵⁴ The standard 100 μl reaction contains 40 μM 4-Methylumbelliferyl ß-D-N, N',N''-triacetylchitotrioside (Sigma-Aldrich), 50 mM sodium phosphate buffer (pH 7). The substrate and buffer were preincubated for 10 min at 30°C and then the reaction was started by adding 5 μl crude enzyme extract. After incubation for 30 min at 30°C, the reaction was stopped by addition of 100 μl of 1 M Glycine NaoH buffer (pH 10.6). The fluorescence was measured in a black 96-well microplate (Corning) using a microplate reader (Glomax Promega) with excitation λ_ex 365 nm and emission at λ_em 433 nm. The fluorescence is directly proportional to the endo-chitinase enzyme activity.

Transcriptome analysis of insect tissue

RNA-Seq libraries were prepared for control and 500 ppm BER-fed insects at 6^th DPD (n=3 for each treatment group with 3 insects pulled together in one biological replicate). Total RNA from the insect body was extracted by using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA-seq libraries were prepared by using NEBNext Ultra II RNA Library Prep Kit for Illumina. Indexed libraries were sequenced (2x 150bp Novaseq 6000 sequencing) at Lifecell International Pvt Ltd, Chennai. The raw data quality was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc)³⁴ and MultiQC.³⁶ The data was checked for base call quality distribution, % bases above Q30, %GC, and sequencing adapter contamination. The raw sequence reads were processed to remove adapter sequences, low-quality bases and read shorter than 50 bp using fastp v0.12.4 with default parameters.³⁵ From the quality trimmed data rRNA reads were removed using bbmap's v38.18 bbduk algorithm (https://www.osti.gov/biblio/1241166). The QC passed reads were aligned against indexed S. frugiperda (assembly AGI-APGP_CSIRO_Sfru_2.0; fall armyworm) reference genome using STAR v2.7.10a.³⁸ The gene level expression values were obtained from S. frugiperda (assembly AGI-APGP_CSIRO_Sfru_2.0; fall armyworm) annotation file read counts using featureCounts v2.0.0 software.³⁹ The expression similarity between the biological replicates was checked by spearman rank correlation (Correlation_matrix.xlsx) and Principal Components Analysis (PCA). For differential expression analysis, samples were grouped as Control and Treated. Differential expression analysis was performed using the DESeq2 v1.34.1 package after normalizing the read counts (variance stabilized normalized counts).⁴¹ Genes with less than 5 read in any of the samples were removed. Genes with log2 fold change ≥ 1.5, log₂ fold change ≤ -0.5, and p-value ≤ 0.05 were considered significant. The genes that showed significant differential expression were mapped on the Drosophila melanogaster genome and proceeded for Gene ontology (GO) and KEGG enrichment analysis in the FunSet web server (http://funset.uno/)⁴² and KOBAS server (http://kobas.cbi.pku.edu.cn/) respectively.⁴³ Raw RNA sequencing data is submitted to NCBI as Bioproject (Submission ID: SUB13375930; BioProject ID: PRJNA974147).

Quantitative Real-Time PCR (qRT-PCR) analysis

Expression analysis of selected genes was performed with three biological replicates for control and BER-fed larvae by qRT-PCR. Total RNA from the insect tissue was extracted by using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. DNase I (Sigma-Aldrich)-treated RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Gene-specific oligonucleotides were designed using Primer-BLAST software (NCBI). The qRT-PCR analysis using Takara TB Green Premix Ex Taq II (Tli RNase H Plus) was performed on 7500 Fast Real-Time PCR System (Applied Biosystems, Foster, CA, USA). The oligonucleotides used are listed in the Table S2. PCR reactions were performed as three independent replicates. Relative expression levels were estimated by calculating the 2^−ΔΔCt for each gene. The S. frugiperda elongation factor 1-alpha (SfEf1-alpha) was used as the internal control for mRNA abundance calculation.

Metabolite profiling of insect tissues

Each biological replicates had three insects pulled together (n=3) from control and 500 ppm BER-fed larve at 6^th DPD were used for metabolic extraction. Three biological replicates were used, around 100 mg of frozen larval tissue was homogenized in 80% methanol:water (v/v) with solid:liquid ratio kept 1:5 (w/v). The suspension was vortexed for 30 s, bath sonicated for 30 min at room temperature and centrifuged at 14,000g for 10 min. The extract was filtered through 0.22 μM filters. The Samples were analyzed using LC-HRMS (Q Exactive MA/MS). Separation of metabolites was performed on 150 mm χ 2.1 mm i.d. (internal diameter), 1.9 μM UPLC BEH C18 column (Sigma Aldrich). The mobile phase consisted of 0.1% formic acid in MS grade water (phase A) (Sigma-Aldrich) and 0.1% formic acid in acetonitrile (phase B). The flow rate was 0.3 ml min^-1, and the column temperature was kept at 40°C. The following liquid chromatography conditions were used for insect metabolite analysis: gradient started with 2% phase B over the first 0.3 min and phase B increased to 30% over the next 2 min, from 30% to 45% phase B was increased over the next 7 min and further increased to 98% over next 12 min, held at 98% phase B for 3 min. Then the column was equilibrated to initial conditions (98% phase A and 2% phase B) for the next 5 min.⁵⁵ MS-DIAL software (http://prime.psc.riken.jp/Metabolomics_Software/MS-DIAL/index.html) with LC–MS/MS spectral database of MassBank of North America (http://mona.fiehnlab.ucdavis.edu/) was utilized for peak extraction, baseline filtering, calibration, peak alignment, deconvolution, peak identification, and integration.⁴⁴

Vector construction and dsRNA preparation

For RNAi, a partial fragment of SfCht5 (476 bp) was cloned into L4440 vector between XhoI/NotI sites by using In-fusion cloning (Takara Bio) and clones were confirmed by sequencing. The resultant recombinant vectors (L4440-SfCht5-dsRNA) and L4440 empty vector were transformed into the E. coli HT115 (DE3) cells. To prepare the dsRNA expressing bacteria, a single colony of E. coli HT115 (DE3) containing recombinant vectors were grown overnight in liquid LB with 100ug/ml ampicillin and 12.5 μg/ml tetracycline at 37°C with shaking at 200 rpm. The overnight grown cultures were inoculated to the fresh LB (1:100 proportion) with appropriate antibiotics and grown until O.D. reached 0.6. The synthesis of dsRNAs' was induced with the addition of 1mM IPTG and these cultures were incubated for 4 hrs under similar conditions. Bacterial cells were pelleted down by centrifugation at 4000 rpm at 4°C. The dsRNA expression was confirmed by extracting total RNA from aliquots of induced E. coli HT115 cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions, and dsRNAs were visualized on 1.5 % agarose gel.⁵⁶

Feeding bioassay of SfChi-dsRNA expressing bacteria to the S. frugiperda

Bacterial cultures of dsRNA targeting SfCht5 and L4440 empty vector were used for feeding first instar S. frugiperda larvae. IPTG-induced 200ml bacterial cultures were centrifuged at 4000 rpm for 10 min at 4°C and resuspended in DEPC water. The artificial diet (AD) was prepared as per Chikate et al., 2016 and the bacterial cultures were added to AD at a final concentration of 1 O.D. Feeding bioassays were conducted on the first instar S. frugiperda (n=113 larvae/treatment, collectively in two independent experiments) by feeding on an artificial diet with L4440 empty vector bacterial culture (Control) and an artificial diet with bacterial culture expressing L4440-SfCht5-dsRNA.⁵⁶ Insect growth and survival on control and dsRNA-containing diet were monitored on an alternate day until pupation. On the 3^rd and 6^th DPD 18 larvae from each group were harvested by freezing in liquid nitrogen, pulled together in a group of six, and treated as one replicate. The efficacy of SfCht5 silencing was assessed by qRT-PCR analysis of selected genes as described earlier for BER feeding bioassay.

Overexpression of SfCht5 in S. frugiperda

For stable overexpression, SfCht5 and Enhanced green fluorescence protein (EGFP) were cloned into the pIB/V5-His-TOPO vector using TOPO cloning and transformed into E. coli Top10. The pIBV5-SfCht5 plasmid was mixed with lipofectin (1:1 v/v) and incubated at room temperature for 30 min. Fifth instar S. frugiperda were anesthetized on ice, and 2 μl of plasmid with lipofectin (∼150 ng) was injected into the abdomen (n = 28 in each treatment). The control larvae were injected with pIBV5-EGFP plasmid. The plasmid-injected larvae were fed on the artificial diet for 24 hr and harvested in liquid nitrogen (6 biological replicates, with three larvae each) for further analysis using qRT-PCR.

Transcription factor binding site prediction

Nucleotides in the region of -2200 to +200 of the start site of selected gene sequences were extracted. The putative promoter sequences and transcription start site were predicted using Berkley Drosophila Genome Project's Neural Network Promoter Prediction software (https://www.fruitfly.org/seq_tools/promoter.html), to predict a complete promoter sequence and a transcription start site. The transcription factor binding sites were predicted using the JASPAR database (https://jaspar.genereg.net/) for Insecta.⁴⁶

Electrophoretic Mobility Shift Assay

The binding of the Broad complex Z4 (Br-Z4) transcription factor protein to the promoters of the SfCht5 and SfECR was confirmed by the Electrophoretic Mobility Shift Assay (EMSA). The coding sequence of S. frugiperda Br-Z4 was cloned into the pET28a vector using NdeI/XhoI restriction sites. The sequence confirmed plasmid was expressed in Escherichia coli Shuffle T7 cells by IPTG induction. The recombinantly expressed Br-Z4 protein was purified by using a Ni-NTA agarose matrix. The promoters of the SfCht5 and SfECR were amplified from the S. frugiperda genomic DNA using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA) and purified by Wizard SV gel and PCR-clean-up system (Promega, Madison, WI). These promoters were confirmed with sequencing. The binding assay of the Br-Z4 protein with promoters was performed using an Electrophoretic mobility shift assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Briefly, 75 ng promoter DNA was incubated with the 1500 ng Br-Z4 protein at room temperature for 1 hr. Further these reactions were loaded into 1% agarose gel, which was run for 2 hr at 35 V and 4°C. Followed by electrophoresis the DNA was stained using SYBR® Green EMSA gel stain for 30 min and after washing with water the stained nucleic acids were visualized in 300 nm UV transillumination. Further proteins in the same gel were stained using SYPRO® Ruby EMSA (Thermo Fisher Scientific, Waltham, MA) protein gel stain with TCA for 3 hr and after destaining stained proteins were documented in 300 nm UV transillumination.

Quantification and statistical analysis

Statistical analysis and visualization were carried out using GraphPad Prism 8. Differences between control and BER containing diet fed insects were calculated by Student's unpaired t-test. Detailed information regarding the statistical methods employed for each experiment can be found in the figure legends of the corresponding figures.

Published: February 20, 2024