Synergistic control of Spodoptera frugiperda based on nanoparticle-mediated dsRNA and insecticide

Synergistic control of Spodoptera frugiperda based on nanoparticle-mediated dsRNA and insecticide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic control of Spodoptera frugiperda based on nanoparticle-mediated dsRNA and insecticide Suman Zong, Hao Hong, Jing Zhao, Liubin Xiao, Keyan Zhu-Salzman, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6184980/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract RNAi is currently the most promising gene silencing-based biotechnology for pest control. However, double-stranded RNA (dsRNA) is easily degraded in the environment, resulting in poor stability and ineffectiveness. In this study, we designed a nanoparticle chitosan-polyethylene glycol-carboxyl (CS-PEG-COOH), which can spontaneously assemble with dsRNA to form a dsRNA/CS-PEG-COOH complex. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) revealed the complex size being 543.66 nm. CS-PEG-COOH was able to prevent dsRNA from being degraded by midgut fluid or RNase A, thereby significantly improving the dsRNA stability under various environmental conditions. CS-PEG-COOH gradually released dsRNA at pH > 8. Cy3 fluorescent labeling confirmed that CS-PEG-COOH significantly enhanced the delivery of dsRNA to insects, and was water-washing resistant on plants. Subsequently, ds VGSC fragments were expressed in E. coli using selected VGSC fragments. Their silencing efficiency was significantly enhance by CS-PEG-COOH. Feeding Spodoptera frugiperda the nanocomposite in combination with metaflumizone caused down-regulation of genes related to cuticle synthesis, cell metabolism, and drug metabolism. Metaflumizone/ds VGSC /CS-PEG-COOH significantly enhanced pest control effect by inhibiting insect growth and development. Spodoptera frugiperda RNAi metaflumizone Nanocarrier Pest management Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Spodoptera frugiperda is an important pest that attacks corn and various other crops of the Gramineae family. Notorious for its wide host range, strong migration ability, and high reproduction rate, it causes serious damage to global agriculture [ 1 – 3 ]. Long-term and widespread use of chemical pesticides can lead to pest resistance [ 4 , 5 ] and cause adverse effects on the environment and human health. There is a urgent need for effective and safe alternative control methods [ 6 ]. The research and development of nucleic acid pesticides based on RNA interference (RNAi) technology has provided a promising direction for agricultural pest control [ 7 ]. This specific degradation of homologous mRNA induced by exogenous double-stranded RNA (dsRNA) inhibits the expression of insect target genes, thereby impairing the growth and reproduction of pests [ 8 – 10 ]. Due to its specificity and environmental safety, RNAi has become a new pest management strategy [ 11 ]. Despite its success in some coleopterans, RNAi is less efficient in Lepidopteran insects [ 12 – 15 ]. Difficulty may be due to poor dsRNA/siRNA delivery, degradation of dsRNA by dsRNA nucleases (dsRNases), and lack of RNAi machinery [ 16 – 18 ]. Injecting dsRNA synthesized in vitro into insects is the most commonly used in gene function research because it is effective and easy to handle dsRNA [ 19 – 20 ]. Currently, this method has been applied to a variety of insects, such as the cotton bollworm and migratory locust [ 21 – 23 ]. However, it also has disadvantages such as high cost and needing professional skills and equipment [ 24 – 26 ]. In addition, dsRNA injection can cause mechanical damage to the insect’s body, resulting in high mortality, thus unsuitable for field applications [ 27 , 28 ]. On the other hand, oral administration of dsRNA has been successful in many insects, such as world moths, skullcaps, and aphids [ 29 – 31 ]. The feeding method, simple to operate without causing mechanical damage to insects, is more suitable for field pest control. dsRNA is a high molecular weight, hydrophilic, and negatively charged molecule that cannot easily pass through the cell membrane to achieve gene silencing in the cytoplasm [ 32 ]. Therefore, it is necessary to develop safe and effective dsRNA carrier systems to protect dsRNA from nuclease degradation and enhance its transport to the cytoplasm for processing and RNA interference. In recent years, nanoparticle-mediated dsRNA delivery has emerged as a viable option [ 33 ]. Nanoparticles can combine with dsRNA through electrostatic interactions, hydrogen bonds, van der Waals forces, protecting dsRNA from being degraded by various enzymes in the digestive system. Nanoparticle/dsRNA complexes can activate clathrin-mediated endocytosis and promote dsRNA endosome release, thereby improving RNAi efficiency [ 34 ]. Over the past several years, many nanoparticles, such as chitosan, liposomes, cationic dendrimers, and quantum dots, have been used to deliver dsRNA for RNAi in insects. Among them, natural polymer chitosan (CS) with properties of low toxicity, good biocompatibility, and good biodegradability, forms stable complexes with dsRNA through electrostatic interactions [ 35 , 36 ]. Gurusamy et al. found that chitosan helps dsRNA escape from endosomes and improves RNAi efficiency in fruit cells and tissues [ 37 ]. Polyethylene glycol (PEG) is a hydrophilic macromolecular compound widely used in the biomedical field, and has beneficial properties such as biocompatibility, non-toxicity, and hydrophilicity. Interestingly, PEG can significantly improve the physical properties of CS, by stabilizing siRNA/nanocomposites, and extending the residence time of siRNA in the body [ 38 ]. The carboxyl group can react with the amino group to form a stable amide bond, or it can react with the hydroxyl group to form an ester bond. Polyethylene glycol can be chemically linked to other hydrophobic polymer materials such as chitosan through carboxyl modification to form amphiphilic polymer materials. At the same time, the carboxyl group, as an acidic group, can be used as a pH-responsive element in nanomaterials and can be protonated under alkaline conditions, thus affecting the drug loading efficiency of nanoparticles [ 39 , 40 ]. Appropriate target genes are also key to the successful application of RNAi-based pest management strategies [ 41 ]. VGSC is a complete transmembrane protein that exists in neurons, muscle cells, endocrine cells, and ovarian cells. It participates in the initiation and propagation of excitable cell action potentials and changes the propagation dynamics of nerve impulses [ 42 ]. Sodium ion channels are targets of a series of insecticides such as metaflumizone, indoxacarb and pyrethroids. Metaflumizone exerts stomach poisoning effects on target pests. Poisoned insects show symptoms of acute neurological poisoning, such as pseudoparalysis, paralysis, and the ability to move and be accompanied by violent tremors after being stimulated by external stimuli. With its widespread use around the world, insects in many places have developed varying degrees of resistance and cross-resistance to metaflumizone. Field populations of rice leaf rollers in Changsha and Nanning were 8.4 and 26.2 times resistant to metaflumizone, respectively [ 43 ]. Roditakis et al. reported a 17-fold cross-resistance to metaflumizone in Greek tomato field populations [ 44 ]. Therefore, synergism between RNAi and metaflumizone is expected to achieve a higher control effect. In this study, we prepared a new nanocarrier material CS-PEG-COOH and demonstrated that it can form a complex with dsRNA. We found that CS-PEG-COOH can release dsRNA at pH = 8. Further, we verified the stability and delivery efficiency of CS-PEG-COOH-loaded dsRNA in S. frugiperda and corn leaves. Subsequently, we selected VGSC as the target gene, synthesized dsRNA using the in vitro transcription system as well as the E. coli expression method, prepared the ds VGSC /CS-PEG-COOH complex, and used the feeding method to study the effects of dsRNAs synthesized by different methods on S. frugiperda RNAi efficiency. In addition, the compound was treated with metaflumizone to determine the mortality and development of S. frugiperda , providing a reference for combining efficient RNAi technology with traditional pesticides. Finally, transcriptome analysis shed some light on the synergistic effect of dsRNA/CS-PEG-COOH and metaflumizone. Materials and methods Insect rearing The susceptible strain of S. frugiperda was provided by the insectarium of the Institute of Plant Protection, Jiangsu Academy of Agricultural Science (China) and reared under temperature of 25 ± 1℃, relative humidity of 70 ± 10%, and photoperiod of L:D = 16h:8 h, supplemented 10% honey water in the adult stage. Synthesis of CS-PEG-COOH Methoxy polyethylene glycol carboxyl group (mPEG-COOH, 97%, MW 5000), chitosan (CS, degree of deacetylation ≥ 95%, MW 50,000–60,000), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 98.5%) and N-hydroxysuccinimide (NHS, 98%) were purchased from Macklin (Shanghai, China). Synthesis of CS-PEG-COOH had three steps: First, mPEG-COOH (0.1 g), EDC (20 mg) and NHS (13 mg) were added to a brown glass bottle containing 20 ml of deionized water and stirred at room temperature to activate the reaction for 2 h. Then the chitosan (0.2 g) was added and stirred continuously at room temperature for 48 h. Mixture was then centrifuged at 3000 r/min and the pH value of the upper clear solution was adjusted to 12. Finally, the solution was dialyzed with deionized water at least three times and freezedried to obtain CS-PEG-COOH samples. Expression of dsRNA For in vitro transcription of dsRNA, total RNA was extracted from the S. frugiperda larvae with AG RNAex Pro reagent (Accurate Biology, Hunan, China). The cDNA was synthesized using MMLV reverse transcriptase (Promega, Madison, WI) with an oligo (dT) 18 primer, at 42 ℃ (90 min) and 95 ℃ (10 min). All primers were synthesized by Sangon Biotech Co. Ltd (China), and listed in Table S1 . The target gene fragments ( VGSC : GenBank accession No. OR334597) were amplified using the 2 × Exp Taq Master Mix (Accurate Biology, Hunan, China). The amplified sequences were cloned into the pMD19T-vector (TaKaRa) and transformed into DH5α competent cells (Tsingke, Beijing, China). Plasmids extracted from E. coli were used as the templates for dsRNA synthesis using the T7 RiboMAX Express RNAi System (Promega, USA). The green fluorescent protein gene ( GFP ) was used as the control, and fragments were amplified from GFP plasmids stored in the laboratory using specific primers (Table S1 ). For bacterially expressed dsRNA, the dsRNAs were selected to express in plasmid- E. coli HT115 (DE3) (Tsingke, Beijing) system. The primers of dsRNA (L4440- VGSC , Table S1 ) were designed by DNAMAN 8.0 and CE Design V1.04 software (Vazyme, Nanjing, China), and the primers contained Pst I and Nhe I restriction sites of L4440 vector (Tiandz, Beijing, China). The fragments of the target gene were amplified using the 2 × Exp Taq Master Mix (Accurate Biology, Hunan), and the amplified sequences were cloned into L4440 vector digested with Pst I (Takara, Beijing) and Nhe I (Takara, Beijing) by ABclonal MultiF Seamless Assembly Mix (ABclonal, Wuhan, China) to generate L4440- VGSC . L4440-dsRNA expression plasmid vectors, L4440- VGSC , were then transformed into E. coli HT115 (DE3) cells for dsRNA expression. Single clone of HT115 (DE3) was cultured for 14 h with shaking in LB liquid media with 100 mg/L ampicillin (Amp) and 12.5 mg/L tetracycline (Tet) followed by shaking at 37 ℃, 200 rpm. When the OD 600 value reached 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM) was added and incubated for 5 h under the same conditions. Five ml of HT115 cell cultures was centrifuged at 12000 rpm for 5 min For total RNA extraction using AG RNAex Pro reagent (Accurate Biology, Hunan). The expressed dsRNAs were purified by MEGAclear Kit (Thermo Fisher Scientific, USA), and the concentration and integrity of the dsRNAs were assessed using the NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis. Gel retardation test of CS-PEG-COOH-loaded dsRNA To analyze the binding capacity of CS-PEG-COOH with dsRNA at various mass ratios, CS-PEG-COOH was dissolved in 0.1 M sodium acetate buffer (pH 6.5), and in vitro transcribed dsGFP (200 ng) was incubated with CS-PEG-COOH (200 ng) at the mass ratios of 1 : 0, 0 : 1, 3 : 1, 2 : 1, 1 :1, 1 : 2, 1 : 3, 1 : 4 and 1 : 5. E. coli HT115 expressing L4440-ds GFP (100 ng) was incubated with CS-PEG-COOH (100 ng) at the mass ratios of 1 : 0, 0 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 1 : 3, 1 : 4 and 1 : 5, respectively. Each mixture was incubated at room temperature for 5 min, and then the agarose gel electrophoresis images were analyzed using Image J software. Complete loading of dsRNA onto CS-PEG-COOH was confirmed when the dsRNA/CS-PEG-COOH complex remained in the well and failed to migrate through a 1% agarose gel. Isothermal titration calorimetry (ITC) assays To determine the interaction of purified ds GFP with CS-PEG-COOH, 2.50 mg ds GFP was titrated with 40 mg CS-PEG-COOH using the Nano ITC (TA Instruments Waters, USA). The heat of interaction during each injection were calculated by the integration of each titration peak via NanoAnalyze TM software (USA). The test was carried out at 25°C, and ΔG was calculated using the formula ΔG = ΔH − TΔS. Characterization of dsRNA/CS-PEG-COOH complex For the preparation of dsRNA/CS-PEG-COOH complex, in vitro transcribed dsRNA was mixed with CS-PEG-COOH solution in a mass ratio of 1 : 3 and incubated for 5 min. The morphology of ds GFP /CS-PEG-COOH composite was determined using transmission electron microscopy (TEM) and atomic force microscopy (AFM). For TEM observation, a droplet of ds GFP /CS-PEG-COOH complex was immobilized on copper microgrid and then observed and imaged under TEM (HRTEM, JEOL F200, Japan). According to the method described by Zhang et al [ 45 ]. AFM (Bruker Dimension Icon, Germany) was used to examine the ds GFP /CS-PEG-COOH complex by using a tapping mode with a high aspect ratio tip. The size and zeta potentials of ds GFP , CS-PEG-COOH and ds GFP /CS-PEG-COOH complex were measured using dynamic light scattering by a Zetasizer Nano ZSE (Malvern, UK) at 25°C. Each treatment included three independent samples. Stability of ds GFP /CS-PEG-COOH complex In order to study the stability of dsGFP/CS-PEG-COOH complex in S. frugiperda midgut fluid, 3rd instar larvae were dissected in pre-cooled PBS to obtain the midgut. The sample was homogenized for 30 s, centrifuged at 4℃ for 20 min, and the supernatant was stored at -80℃ before use. The protein concentration in midgut fluid was detected using the Biuret method for protein content determination kit (Solarbio, Beijing, China). Different concentrations of midgut fluid were mixed with 200 ng ds GFP . The mixture was incubated for 1 h at room temperature and subsequently analyzed by 1% agarose gel electrophoresis. To study the stability of the ds GFP /CS-PEG-COOH complex, we treated the ds GFP /CS-PEG-COOH complex with a concentration capable of degrading the midgut content of 200 ng ds GFP , and analyzed it by 1% agarose gel. Similar to the above steps, midgut fluid was replaced with RNaseA (Rromege, USA) to study the stability of ds GFP /CS-PEG-COOH complex after RNase A treatment. Because the pH value of the intestine can affect the stability of nanomaterials and the degradation rate of dsRNA [ 46 – 47 ], the ds GFP /CS-PEG-COOH complex (200 ng) was exposed to various pH values ranging from 1 to 11 for 30 min, and then analyzed by 1% agarose gel electrophoresis. The relative band density was determined using ImageJ (National Institutes of Health, USA) from three independent samples. To determine the stability of the ds GFP /CS-PEG-COOH complex at various temperatures, ds GFP /CS-PEG-COOH complex (200 ng) was incubated at -20, 4, 25, 37 and 50°C for 30 min, and the stability of ds GFP was determined similarly as above. Each treatment included three independent samples. Delivery efficiency of dsRNA/CS-PEG-COOH complex in S. frugiperda In vitro transcribed fluorescent ds GFP detectable under a fluorescence microscope (Leica, Germany), was synthesized using the Cy3 labeling mix (Shanghai Plant Science Biotechnology Co., Ltd., China). Then delivery efficiency of dsRNA/CS-PEG-COOH complex was examined in S. frugiperda via oral feeding. The solutions of Cy3-ds GFP /CS-PEG-COOH complex (Cy3-ds GFP : 100 ng/µL), Cy3-ds GFP and CS-PEG-COOH were added into artificial diet, respectively. Then the 3rd instar larvae were transferred to the artificial diet for feeding. At 6 h and 24 h after treatment, the middle part of the larvae was cut into 10 µm sections transversely to prepare frozen sections. Fluorescent images of cross-sections of the larval midgut were captured using a fluorescence microscope. Fluorescence intensity of all samples from three independent samples was quantified using ImageJ software. Adhesion of CS-PEG-COOH loaded dsRNA to plants Solutions (200 µL) of fluorescent Cy3-ds GFP /CS-PEG-COOH complex (Cy3-ds GFP : 200 ng/µL), Cy3-ds GFP and CS-PEG-COOH were applied to the surface of corn leaves, respectively. The treated corn leaves were washed with PBS and fluorescent photos of the leaf surfaces were taken before and after cleaning. dsRNA fragment screening and RNAi efficiency analysis in S. frugiperda Three fragments of VGSC (6261 bp, Genbank: accession No. OR334597), named VGSC 1 (486 bp), VGSC 2 (499 bp), VGSC 3 (496 bp), in S. frugiperda were selected for dsRNA synthesis using the T7 RiboMAX expression (Promega, USA). Three kinds of dsRNAs (8000ng) were injected into the third instar larvae respectively, RNA were extracted on day 1 and day 3 after injection to examine the RNAi efficiency, and the experiment was repeated four times. The housekeeping gene GAPDH (Gene ID: 118271716) was used as a reference gene for quantification. Primers for qRT-PCR are listed in Table S2. SYBR Green I (Vazyme, Nanjing) was used for qRT‐PCR on a LightCycler®480 Real‐Time PCR detection system. The relative expression level of VGSC 1, VGSC 2 and VGSC 3 were obtained by the 2 −ΔΔCt method [ 48 ]. The two dsRNA was incubated with CS-PEG-COOH at the mass ratio to prepare dsRNA/CS-PEG-COOH complex (dsRNA concentration was 4 µg/µL). These complexes were applied on artificial diet cubes (3 mm × 3 mm) and fed to 3rd instar larvae. After consumption of the treated diet, larvae were given fresh artificial diet (1 × 1 cm). The RNA samples were extracted at 1 day, 2 day and 3 day after the oral feeding to perform Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) to examine the RNAi efficiency, and the experiment was replicated for four times. Primers for qRT-PCR are listed in Table S2. SYBR Green I (Vazyme, Nanjing) was used for qRT‐PCR on a LightCycler®480 Real‐Time PCR detection system. Synergism between ds VGSC / CS-PEG-COOH complex and metaflumizone Acetone was used to prepare working liquid for different chemicals, followed by serial dilutions in water to achieve five concentrations. Artificial feed (0.2 g) was soaked in the liquid for 10 seconds, then air dried and placed into a 24-well insect culture plate for later use. To test larvae, the 3nd instar larvae were introduced into the 24-well plate, one larva per well. LC 50 values were calculated after 3 days of treatment. Mortality was determined on days 3 and 7 and photos were taken on day 7. RNA-seq analysis To evaluate synergistic effect, metaflumizone and metaflumizone/ds VGSC /CS-PEG-COOH complex were applied on artificial diet cubes (3 mm × 3 mm) and fed to 3rd instar larvae of S. frugiperda . Total RNA was extracted on day 3 after the oral feeding using the AG RNAex Pro reagent (Accurate Biology, Hunan). Each treatment included three independent samples. The transcript libraries were constructed via Illumina sequencing platform. Clean reads were processed by trimming adaptor sequences and low-quality reads (Q ≤ 5) [ 49 ]. The resulting clean reads were assembled using Trinity software [ 50 ]. TopHat2 was used to achieve the sequence alignment with the reference genome, and obtain the localization information of reads on the reference genome [ 51 ]. The expression level of each transcript was presented by FPKM value. DESeq2 was applied for differential expression analysis between transcripts, and |log 2 (fold change)| ≥ 1.0 and padj ≤ 0.05 were screen conditions [ 52 ]. BLASTX was used to annotate single genes in gene databases. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out for gene enrichment and functional annotation. The expression levels of target genes were assessed using qRT-PCR. All primer sequences used for qRT-PCR are listed in Table S2. SYBR Green I (Vazyme, Nanjing) was used for qRT-PCR on a LightCycler®480 Real-Time PCR detection system. Statistical analysis. The significant differences in biological parameters were analyzed using statistical software SPSS 27.0 (SPSS Inc., USA), with p < 0.05 indicating statistical significance. All results are expressed as means ± standard errors. Results and discussion Self-assembly and characterization of ds GFP /CS-PEG-COOH complex Incubation of ds GFP with CS-PEG-COOH at room temperature for 5 min could achieve the self-assembly of ds GFP /CS-PEG-COOH complex (Fig. 1 a). The in vitro synthesized ds GFP showed complete loading at dsRNA/CS-PEG-COOH mass ratio of 1 : 3 (Fig. 1 b), and the bacterially expressed ds GFP showed complete loading of all bands including higher molecular weight RNA at the mass ratio of 1 : 4 (Fig. 1 c). In addition, we also used the ITC method to determine the interaction between dsRNA and CS-PEG-COOH, which is a high-precision method for determining the binding affinity between nanoparticles and biomolecules through changes in heat [ 53 ]. As shown in Fig. 1 d, e, the binding constant (Ka) of 4.219 × 10 5 M − 1 indicated that there was an effective interaction between CS-PEG-COOH and ds GFP . The Gibbs free energy ΔG is negative (-1656.39 kJ/mol), indicating an automatic reaction between the CS-PEG-COOH and dsRNA. The positive enthalpy (ΔH) indicates that the complexation between the CS-PEG-COOH and dsRNA is mainly due to non-covalent intermolecular interactions such as hydrogen bonding and hydrophobic interactions [ 54 – 55 ]. TEM (Fig. 1 d-f) and AFM (Fig. 1 g-i) showed linearity of ds GFP , and typical spherical shape of CS-PEG-COOH. The ds GFP was adsorbed on the surface of CS-PEG-COOH to form the dsGFP/CS-PEG-COOH complex. At 25℃, the particle sizes of ds GFP , CS-PEG-COOH and ds GFP /CS-PEG-COOH complex were 201.24, 38.12 and 543.66 nm, and their zeta potentials were − 13.10, 6.44 and − 21.66 mV, respectively (Table 1 ). The results showed that the CS-PEG-COOH had a strong positive surface charge that could adsorb negatively charged dsRNA. Table 1 Particle sizes and Zeta potentials of ds GFP , CS-PEG-COOH and dsGFP/CS-PEG-COOH complex. Sample Particle size (nm) Zeta potential (mV) dsGFP 201.24 + 3.77 -13.10 + 3.22 CS-PEG-COOH 38.12 + 5.60 6.44 + 1.90 dsGFP/CS-PEG-COOH complex 543.66 + 19.55 -21.66 + 2.05 Mean ± SE. Protection of dsRNA by CS-PEG-COOH When incubated with 50 ng of midgut fluid or 10 ng of RNase A solution (Fig. 2 a-d), dsGFP alone could be rapidly degraded by midgut fluid or RNase A (Fig. 2 a,c), compared to untreated dsGFP that migrated to the middle of the gel. Since dsRNA was tightly bound to CS-PEG-COOH, the midgut fluid-treated dsRNA/CS-PEG-COOH complex was immobilized in the well (Fig. 2 b). RNase A-treated dsRNA/CS-PEG-COOH complex was also immobilized in the well (Fig. 2 d). This indicates that CS-PEG-COOH effectively protected dsRNA from being degraded by RNase and midgut fluid degradation activities. In addition, CS-PEG-COOH was able to slow down the release of dsRNA in the midgut complex to improve the effectiveness of dsRNA interference. The pH of the insect midgut is one of the important factors for efficient delivery of dsRNA. Studies have shown that alkaline midgut pH of lepidopteran insectsleads to hydrolysis of dsRNA, causing low RNAi efficiency. To further test the stability of the complex, the dsGFP/CS-PEG-COOH complex was incubated under different pH gradients, and then the dissociated dsGFP was quantitatively analyzed (Fig. 2 e). At pH 8, the dsGFP/CS-PEG-COOH complex released part of the dsRNA, and when the pH increased from 9 to 11, the release of dsRNA increased significantly. This indicates that dsRNA can be released from the dsGFP/CS-PEG-COOH complex in an alkaline environment. In addition, we also tested the stability of dsGFP loaded with CS-PEG-COOH at different temperatures. The intensity of the electrophoresis bands did not change significantly, indicating that the dsGFP/CS-PEG-COOH complex can adapt well to temperature changes in the field (Fig. 2 f). CS-PEG-COOH improves dsRNA delivery efficiency in larvae and enhances dsRNA adhesion to plants The Cy3-ds GFP was employed to treat the S. exigua larvae through oral feeding to examine the delivery efficiency by fluorescence microscopy, followed by quantitative analysis of Cy3-ds GFP (Fig. 3 a,c). After oral feeding for 6 hours, clear Cy3-ds GFP signals were observed in the midgut of larvae treated with Cy3-ds GFP and Cy3-ds GFP /CS-PEG-COOH. The fluorescence intensity of larvae treated with Cy3-d sGFP /CS-PEG-COOH complex was 2.78 fold higher than that of Cy3-ds GFP . After 24 h feeding, clear Cy3-ds GFP signals were observed in the midgut of larvae treated with Cy3-ds GFP /CS-PEG-COOH complex, while no Cy3-ds GFP signals were seen in the larvae treated with Cy3-ds GFP alone, suggesting that CS-PEG-COOH improved dsRNA stability of in the gut. When fluorescent Cy3-dsGFP/CS-PEG-COOH was sprayed on corn leaves, adhesion of CS-PEG-COOH-loaded dsGFP to plants was detected. After rincing leaves with water, the fluorescence signal on corn treated with CS-PEG-COOH-loaded Cy3-dsGFP was much stronger than that treated with Cy3-dsGFP alone (Fig. 3 b). Quantitative results also confirmed this conclusion (Fig. 3 d). Similar to previous studies, these results support the use of chitosan for efficient delivery of dsRNA into plants. The greater stability and enhanced delivery of chitosan-loaded dsRNAs allows for better entry into plant cells and facilitates enhanced uptake in tobacco, legumes and other plants [ 56 – 57 ]. Efficient dsRNA fragment screening and synergistic effect with metaflumizone VGSC is an essential gene for potential conduction and growth and development of excited tissues, and it is involved in the initiation and propagation of excitable cellular action potentials [ 58 ]. Local injection of dsRNA of gene fragments synthesized in vitro.(Fig. 4 a,b). Results showed that, at 24h and 48h, ds VGSC1 significantly improved the interference efficiency of nymphs, therefore was adopted for subsequent experiments. Two methods were used to obtain dsRNA, one was bacterial expression, and the other was in vitro transcribed dsRNA. E. coli- expressed dsRNA (Fig. 4 c) was incubated with CS-PEG-COOH, and the biological activity of the complex was detected by a feeding method. Compared with ds GFP /CS-PEG-COOH, the dsRNAs/CS-PEG-COOH complex significantly reduced the expression level of the corresponding target genes, and its persistence was significantly higher than feeding dsRNAs alone (Fig. d). Since VGSC is the target of metaflumizone, we further studied the effect of co-treatment of dsRNA/ CS-PEG-COOH and metaflumizone on the growth and reproduction of S. frugiperda . The metaflumizone LC 50 value measured after 3 days was 6.244 mg/L (Table 2 ). The larval mortality results are shown in Fig. 4 e-f. The highest mortality rate of larvaes treated with metaflumizone/ds VGSC /CS-PEG-COOH complex within 3 days was 73.33%, while that of larvaes treated with ds VGSC /CS-PEG-COOH and metaflumizone separately were 6.00% and 58.33%, respectively. The highest mortality rate of nymphs treated with metaflumizone/ds VGSC /CS-PEG-COOH complex within 7 days was 93.40%, while the mortality rates of larvaes treated with ds VGSC /CS-PEG-COOH and metaflumizone alone were 10.30% and 70.00%. The development cycle of larvae is shown in Table 3 . There are differences in the larval lifespan between different treatments. The lifespan of S. frugiperda treated with metaflumizone/ds VGSC /CS-PEG-COOH complex was 67 days, significantly longer than that treated with metaflumizone (41 days) alone. Results of ds VGSC /CS-PEG-COOH were not different from the control, indicating that ds VGSC /CS-PEG-COOH and metaflumizone can significantly extend the development time of larvae and further weaken the growth ability and resistance of S. frugiperda , thus achieving the effect of increasing the efficiency and reducing the dosage of pesticides. Table 2 Toxicity of metaflumizone to 3rd instar larvae of S. frugiperda . Formulation Slope ± SE LC 50 /(mg/L) 95% Confidence limits Metaflumizone 0.466 ± 0.055 6.244 (5.515, 7.113) Table 3 The effect of different chemical treatments on development duration of S. frugiperda Formulation Duration of development /d 3th 4th 5th 6th Pupae Female Male Total duration Control 2.40 ± 0.24 c 2.80 ± 0.20 b 2.20 ± 0.20 b 2.20 ± 0.20 b 1.80 ± 0.44 b 10.20 ± 0.24 b 10.80 ± 0.20 b 37.20 ± 0.37 c dsVGSC/CS-PEG-COOH 2.40 ± 0.24 c 3.36 ± 0.60 b 2.80 ± 0.83 ab 2.20 ± 0.20 b 2.20 ± 0.44 ab 10.40 ± 0.20 b 10.60 ± 0.24 b 39.60 ± 0.40 c Metaflumizone 5.20 ± 0.48 b 4.40 ± 0.50 b 3.00 ± 0.31 ab 3.00 ± 0.10 a 2.60 ± 0.54 ab 10.80 ± 0.37 b 10.40 ± 0.24 b 47.20 ± 1.31 b Metaflumizone/dsVGSC/CS-PEG-COOH 9.40 ± 0.40 a 8.20 ± 0.58 a 3.80 ± 0.20 a 3.6 ± 0.24 a 2.80 ± 0.44 a 12.60 ± 0.24 a 12.80 ± 0.20 a 61.80 ± 0.73 a NOTE: Different lower letters above bars indicate highly significant differences between three treatments (p < 0.05, Tukey’s Honestly Significant Difference test). Synergistic mechanism between CS-PEG-COOH-loaded ds VGSC and metaflumizone To elucidate the synergistic mechanism between CS-PEG-COOH-loaded ds VGSC and metaflumizone, we extracted total RNA from surviving larvaes after 72 h of treatment for RNA-seq analysis. The results showed that the sample sequencing quality was high (Table S3) and so was the Pearson correlation coefficient (Fig. S1 ). Compared with metaflumizone alone, metaflumizone/ds VGSC /CS-PEG-COOH changed the expression of 5208 genes, including 2519 up-regulated genes and 2689 down-regulated genes (Fig. 5 a). DEGs are divided into different pathways such as tyrosine metabolism, lysosomes, glycolysis, biosynthesis of metabolites, drug metabolism, and endocytosis (Fig. 5 b). As shown in Fig. 5 c, after metaflumizone/ds VGSC /CS-PEG-COOH complex treatment, many stratum corneum structure-related genes were significantly down-regulated, such as aldo-keto reductase AKR2E4 , Ecdysone-induced protein 75B , endocuticle structural glycoprotein ABD-5 , In insects, ecdysteroids affect molting, and reproduction [ 59 ]. The ecdysteroid ecdysone was first isolated in 1954 [ 60 ]. and shown to be produced by the reduction of 3-dehydroedysone (3DE) by 3β-reductase [ 61 ]. which is then hydroxylated at position 20 to form the active steroid hormone 20-hydroxyecdysone. This 3β-reductase belongs to aldo-keto reductase (AKR) family 2, namely AKR2E4. This enzyme reduces 3DE to ecdysone in the presence of NADPH. The amount of AKR2E4 in silkworm hemolymph may vary with the hormonal activity of ecdysteroids [ 62 ]. The ecdysone-inducible gene E75 is the main target of the ecdysone receptor (EcR) and plays a key role in the molting process of arthropods: ecdysteroids may act through E75 to stimulate the degradation of epidermal chitin. We found that E75 protects the rhythm under stress conditions, indicating that steroid signaling plays a role in maintaining the circadian rhythm of Drosophila [ 63 ]. LmAbd-5 is mainly highly expressed in tissues of ectodermal origin. After silencing LmAbd-5, migratory locusts have no visible phenotype, but ultrastructural analysis found that it is involved in the formation of the inner epidermal lamellar structure of migratory locusts [ 64 ]. In the current study, many essential genes that promote cell metabolism were also significantly down-regulated, such as lysosomal alpha-mannosidase-like ( MAN2B1 ), Late endosomal/lysosomal adapter, MAPK and MTOR activator 1 ( LAMTOR1 ), Aryl hydrocarbon receptor nuclear translocator homolog tgo ( ARNT ). In animal cells, MAN2B1 not only affects the structure and physical and chemical properties of proteins, but also affects physiological processes such as cell adhesion, migration, growth, and differentiation [ 65 ]. LAMTOR1 can regulate receptor recycling through endosomes and the MAPK signaling pathway by recruiting some of its components to late endosomes. LAMTOR1 is involved in several processes, including cholesterol homeostasis as well as release and regulation of lysosomal uptake [ 66 ]. ARNT is a transcription factor that is reported to play a crucial role in regulating glycolysis, angiogenesis, and apoptosis [ 67 ]. In addition, there are down-regulation of genes related to drug metabolism, such as UDP-glycosyltransferase UGT5-like ( UGT2A1 ), glutathione S-transferase 1 ( GstD1 ), UDP-glycosyltransferase is also involved in regulating endogenous compounds, and they are important metabolic detoxification enzymes in insects, mediating the development of insect resistance. The expression levels of relevant genes were verified by qRT PCR, and the results were consistent with the transcriptome data (Fig. 5 d). Conclusion In summary, we proposed a novel and improved CS-PEG-COOH nanocarrier system for efficient RNAi delivery. In dsRNA/CS-PEG-COOH complex, CS-PEG-COOH significantly improves the stability and delivery efficiency of dsRNA, and protects it from RNase A degradation and the impact of the pH microenvironment. At the same time, the alkaline intestinal environment promotes some release of dsRNA. Cy3 fluorescent labeling confirmed that CS-PEG-COOH significantly enhanced the delivery of dsRNA to insects, and was water-washing resistant on plants. Through feeding, CS-PEG-COOH was shown to significantly improve the silencing effect of ds VGSC on S. frugiperda , increasing the pest control effectiveness and reducing the dosage of pesticides. Finally, RNA-seq analysis showed that the CS-PEG-COOH-loaded ds VGSC complex cooperated with metaflumizone to down-regulate genes related to cuticle biosynthesis, cell metabolism, and drug metabolism, thereby inhibiting the growth and development of insects. Declarations Acknowledgements Not applicable Author Contributions Suman Zong: Study design, Writing - Original draft, Investigation, Data curation. Hao Hong: Writing - Original draft, Investigation, Data curation. Jing Zhao: Study design, Writing - review and editing. Liubin Xiao: Writing - review and editing, Supervision. Keyan Zhu-Salzman: Writing - review and editing. Han Wu: Writing - review and editing, Supervision. Dejin Xu: Writing - review and editing, Supervision. Guangchun Xu: Writing -review and editing, Supervision. Linquan Ge: Conceptualization, Study design, Writing - review and editing, Supervision. Yongan Tan: Conceptualization, Study design, Writing - review and editing, Supervision. Funding This study was supported by Technology Innovation Fund [CX(22)2038], National Natural Science Foundation of China (32372631) and National Key Research and Development Program of the Ministry of Science and Technology (2022YFD1401800). Availability of date and materials The data sets used and/or analyzed in this study are available from the corresponding author upon reasonable request. Ethics and Consent to Publish declarations Not applicable Competing interests The authors declare no conflict of interest. References Goergen G, Kumar PL, Sankung SB, Togola A, Tamò M. First Report of Outbreaks of the Fall Armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a New Alien Invasive Pest in West and Central Africa. PLoS ONE. 2016;11(10):e0165632. Mendesil E, Tefera T, Blanco CA, Paula-Moraes SV, Huang F, Viteri DM, Hutchison WD. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6184980","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":426555956,"identity":"feef57db-5c70-41d5-9308-7d7cb3deb429","order_by":0,"name":"Suman Zong","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Suman","middleName":"","lastName":"Zong","suffix":""},{"id":426555958,"identity":"9a7745c2-5770-416a-9a68-8ca4bcee705c","order_by":1,"name":"Hao Hong","email":"","orcid":"","institution":"Nanjing University, Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Hong","suffix":""},{"id":426555959,"identity":"85ba8d49-9020-4b82-949c-4407e295fa1f","order_by":2,"name":"Jing Zhao","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhao","suffix":""},{"id":426555960,"identity":"4eb817e2-58f3-43d6-b042-b51fc08fca78","order_by":3,"name":"Liubin Xiao","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Liubin","middleName":"","lastName":"Xiao","suffix":""},{"id":426555962,"identity":"5036743e-5cc4-4484-b89f-c8ba323e6891","order_by":4,"name":"Keyan Zhu-Salzman","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Keyan","middleName":"","lastName":"Zhu-Salzman","suffix":""},{"id":426555963,"identity":"7a72cb37-ccc3-4bec-a441-3f24a2edb068","order_by":5,"name":"Han Wu","email":"","orcid":"","institution":"Jiangsu Provincial Agro-product Supervision \u0026 Testing Center","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Wu","suffix":""},{"id":426555966,"identity":"3fd8b17c-2801-4f5e-84b3-59acfb765a58","order_by":6,"name":"Dejin Xu","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dejin","middleName":"","lastName":"Xu","suffix":""},{"id":426555968,"identity":"afc243b9-ac8b-4e0a-8cea-01dbf89a01eb","order_by":7,"name":"Guangchun Xu","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guangchun","middleName":"","lastName":"Xu","suffix":""},{"id":426555969,"identity":"017d93dc-ced4-42c3-90b7-39198ae13ec0","order_by":8,"name":"Linquan Ge","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Linquan","middleName":"","lastName":"Ge","suffix":""},{"id":426555971,"identity":"ee84648f-1588-417b-a669-65bbdb8a8339","order_by":9,"name":"Yongan Tan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3PMQuCQBTA8WcHuly4nhT1FfwA0We5R9BWBC1CQSeGDhnNQR/CsTERcrG9UfeWNse6ppbUtqD7T+/g/R4cgEr1gzEg7kkOOtHcnDvLJkQTJ+AApuF5dp6dvyBWmPpWsSb1xApckbTL4dS+ou+g0MEMNrySdGgsEspHc0mueOwCyy5RJekxlIRg9CKZDjab1JB+IcnqRWbok3rSYZokCe7D2IdGxApRxIdxijvD9RjPzrT2LyxN4vw2WDzvG8W9dJY9M9hWE1mLvj3ox7X3tLLRmkqlUv1tD1V+Ua4cdQAoAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yongan","middleName":"","lastName":"Tan","suffix":""}],"badges":[],"createdAt":"2025-03-08 15:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6184980/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6184980/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78370943,"identity":"1b1f1229-e811-4311-9631-55f44d7d9f9f","added_by":"auto","created_at":"2025-03-12 14:02:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":606878,"visible":true,"origin":"","legend":"\u003cp\u003eSelf-assembly mechanism and characterization of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH compelx. \u003cstrong\u003ea\u003c/strong\u003eSchematic illustration of self-assembled ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex. \u003cstrong\u003eb \u003c/strong\u003eGel electrophoresis assay of ds\u003cem\u003eGFP\u003c/em\u003e retardation by CS-PEG-COOH. One μg ds\u003cem\u003eGFP\u003c/em\u003e was incubated with CS-PEG-COOH at various mass ratios, and the mixture (10 μL) was then analyzed. M: DNA marker. \u003cstrong\u003ec \u003c/strong\u003eGel electrophoresis assay of L4440-ds\u003cem\u003eGFP\u003c/em\u003e retardation by CS-PEG-COOH. One μg L4440-ds\u003cem\u003eGFP\u003c/em\u003ewas incubated with CS-PEG-COOH at various mass ratios, and the mixture (10 μL) was then analyzed. M: DNA marker. AFM images.\u003cstrong\u003e d and e\u003c/strong\u003e ITC titration of dsGFP into SPc solution. The heats of interaction during each injection were calculated by the integration of each titration peak. (\u003cstrong\u003ef-h\u003c/strong\u003e), TEM images (\u003cstrong\u003ei-k\u003c/strong\u003e) and particle size distributions (\u003cstrong\u003el-n\u003c/strong\u003e) of ds\u003cem\u003eGFP\u003c/em\u003e, CS-PEG-COOH and ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex.\u003c/p\u003e","description":"","filename":"Figure141.png","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/39563224b868d315e9bf8b69.png"},{"id":78370945,"identity":"837c2b83-5048-44c5-a6c2-729f2f03dad5","added_by":"auto","created_at":"2025-03-12 14:02:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175099,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced stability of CS-PEG-COOH-complexed ds\u003cem\u003eGFP\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e ds\u003cem\u003eGFP\u003c/em\u003edegradation by midgut content. The 200 ng of ds\u003cem\u003eGFP\u003c/em\u003e was incubated for 30min at 25℃ with different concentrations of midgut content. M: DNA maker. \u003cstrong\u003eb\u003c/strong\u003eBands of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex treated with or without 50 ng midgut content. The ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex was incubated with 50 ng midgut content at 37℃ for 30 min. All volume sample was analyzed by 1% agarose gel. \u003cstrong\u003ec\u003c/strong\u003eds\u003cem\u003eGFP\u003c/em\u003e degradation by RNase A. The 200 ng of ds\u003cem\u003eGFP\u003c/em\u003e was added with RNase A to prepare the reaction solution. M: DNA maker. \u003cstrong\u003ed\u003c/strong\u003e Bands of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex treated with or without RNase A. The ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex was incubated with RNase A (10 ng) at 37℃ for 30 min. All volume sample was analyzed by 1% agarose gel. \u003cstrong\u003ee\u003c/strong\u003e Stability of CS-PEG-COOH-loaded ds\u003cem\u003eGFP\u003c/em\u003eunder various pH environments. The ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex was incubated with pH 1-11 at room temperature for 15 min, respectively. The relative band density was determined using the ImageJ software from three independent samples. \u003cstrong\u003ef \u003c/strong\u003eStability of CS-PEG-COOH-loaded ds\u003cem\u003eGFP\u003c/em\u003eunder various temperature environments. The ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex was incubated at temperature (-20, 4, 25, 37 and 50℃) for 1 h, respectively. The relative band density was determined usingThe relative band density was determined using the ImageJ software from three independent samples.\u003c/p\u003e","description":"","filename":"Figure142.png","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/7aa50039890ecc531bafb74e.png"},{"id":78371975,"identity":"24b70b1e-a423-4d4c-944c-b035bbcf31f9","added_by":"auto","created_at":"2025-03-12 14:10:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639505,"visible":true,"origin":"","legend":"\u003cp\u003eCS-PEG-COOH improves dsRNA delivery efficiency in insects and adhesion to corn. \u003cstrong\u003ea\u003c/strong\u003e Fluorescence photographs of the nymphal intestine were taken through different treatments of larval feeding. \u003cstrong\u003eb\u003c/strong\u003e After spraying, wash and take fluorescence pictures of the leaf surface. \u003cstrong\u003ec\u003c/strong\u003eThe fluorescence intensity was determined using the ImageJ software from three independent samples of nymphal intestine. \u003cstrong\u003ed\u003c/strong\u003eThe fluorescence intensity was determined using the ImageJ software from three independent samples of corns. Asterisks above the bars represent statistical significance, respectively (Tukey’s HSD test, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure143.png","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/7e2cc8759e282116c649123c.png"},{"id":78371979,"identity":"6ff7c5ff-1acd-4340-9363-311f90dc1414","added_by":"auto","created_at":"2025-03-12 14:10:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":255280,"visible":true,"origin":"","legend":"\u003cp\u003edsRNA fragments screening of dsRNA/CS-PEG-COOH complexes and the synergistic control effect of dsRNA/CS-PEG-COOH and pesticides. \u003cstrong\u003ea \u003c/strong\u003eSchematic map of the locations of three dsRNAs in the mRNAs of \u003cem\u003eVGSC\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e Expression levels of \u003cem\u003eVGSC\u003c/em\u003eafter the RNAi. Asterisks above the bars represent statistical significance, respectively (Tukey’s HSD test, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003ec\u003c/strong\u003e dsRNA expressed in L4440 expression system. \u003cstrong\u003ed\u003c/strong\u003e Expression levels of \u003cem\u003eVGSC\u003c/em\u003e after feeding with different treatments. \u003cstrong\u003ee\u003c/strong\u003e Lethal effects of synergistic application of RNAi and Metaflumizone. Each treatment consisted of three replicates. \u003cstrong\u003ef \u003c/strong\u003eRepresentative photos of various treatments.\u003c/p\u003e","description":"","filename":"Figure144.png","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/6e5a96f5fa1cd4dd18186cb9.png"},{"id":78371974,"identity":"f337274e-7444-4c5e-b6cc-3c037f8f68bd","added_by":"auto","created_at":"2025-03-12 14:10:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144541,"visible":true,"origin":"","legend":"\u003cp\u003eComparative transcriptome analysis of\u003cem\u003e \u003c/em\u003eMetaflumizone and Metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH. \u003cstrong\u003ea\u003c/strong\u003e Analysis of differentially expressed genes (DEGs) with a volcano plot. Up- and down-regulated genes are represented by red and green dots, respectively. \u003cstrong\u003eb\u003c/strong\u003e KEGG enrichment analysis of DEGs. \u003cstrong\u003ec\u003c/strong\u003e Heat maps of cuticle-related, cell metabolism, and drug metabolism-related genes. \u003cstrong\u003ed \u003c/strong\u003eValidation of DEGs using qRT-PCR. The relative mRNA levels of target genes were normalized to the abundance of the \u003cem\u003eβ-action\u003c/em\u003e gene. Asterisk indicates significant difference according to independent \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure145.png","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/abe597cc1b8e81f69cdd105f.png"},{"id":78475375,"identity":"961dc175-df6f-417d-9ab0-32ccb663d418","added_by":"auto","created_at":"2025-03-13 16:46:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2892193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/3dd42673-b4f4-49ce-b9d8-644d7d6c3098.pdf"},{"id":78371976,"identity":"de837026-0732-4cae-b216-c9ec27ff8394","added_by":"auto","created_at":"2025-03-12 14:10:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":96761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditonal file\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Primers for dsRNA synthesis. \u003cstrong\u003eTable S2.\u003c/strong\u003e Primers for quantitative PCR (qRT-PCR). \u003cstrong\u003eTable S3. \u003c/strong\u003eSequencing quality and genome mapping for RNA-seq analysis. \u003cstrong\u003eFigure S1.\u003c/strong\u003e Pearson correlation between collected samples.\u003c/p\u003e","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6184980/v1/5bbf0c312169487a9631fdbe.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic control of Spodoptera frugiperda based on nanoparticle-mediated dsRNA and insecticide","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e is an important pest that attacks corn and various other crops of the Gramineae family. Notorious for its wide host range, strong migration ability, and high reproduction rate, it causes serious damage to global agriculture [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Long-term and widespread use of chemical pesticides can lead to pest resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and cause adverse effects on the environment and human health. There is a urgent need for effective and safe alternative control methods [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The research and development of nucleic acid pesticides based on RNA interference (RNAi) technology has provided a promising direction for agricultural pest control [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This specific degradation of homologous mRNA induced by exogenous double-stranded RNA (dsRNA) inhibits the expression of insect target genes, thereby impairing the growth and reproduction of pests [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Due to its specificity and environmental safety, RNAi has become a new pest management strategy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite its success in some coleopterans, RNAi is less efficient in Lepidopteran insects [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Difficulty may be due to poor dsRNA/siRNA delivery, degradation of dsRNA by dsRNA nucleases (dsRNases), and lack of RNAi machinery [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Injecting dsRNA synthesized \u003cem\u003ein vitro\u003c/em\u003e into insects is the most commonly used in gene function research because it is effective and easy to handle dsRNA [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Currently, this method has been applied to a variety of insects, such as the cotton bollworm and migratory locust [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, it also has disadvantages such as high cost and needing professional skills and equipment [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, dsRNA injection can cause mechanical damage to the insect\u0026rsquo;s body, resulting in high mortality, thus unsuitable for field applications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. On the other hand, oral administration of dsRNA has been successful in many insects, such as world moths, skullcaps, and aphids [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The feeding method, simple to operate without causing mechanical damage to insects, is more suitable for field pest control. dsRNA is a high molecular weight, hydrophilic, and negatively charged molecule that cannot easily pass through the cell membrane to achieve gene silencing in the cytoplasm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, it is necessary to develop safe and effective dsRNA carrier systems to protect dsRNA from nuclease degradation and enhance its transport to the cytoplasm for processing and RNA interference.\u003c/p\u003e \u003cp\u003eIn recent years, nanoparticle-mediated dsRNA delivery has emerged as a viable option [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Nanoparticles can combine with dsRNA through electrostatic interactions, hydrogen bonds, van der Waals forces, protecting dsRNA from being degraded by various enzymes in the digestive system. Nanoparticle/dsRNA complexes can activate clathrin-mediated endocytosis and promote dsRNA endosome release, thereby improving RNAi efficiency [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Over the past several years, many nanoparticles, such as chitosan, liposomes, cationic dendrimers, and quantum dots, have been used to deliver dsRNA for RNAi in insects. Among them, natural polymer chitosan (CS) with properties of low toxicity, good biocompatibility, and good biodegradability, forms stable complexes with dsRNA through electrostatic interactions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Gurusamy et al. found that chitosan helps dsRNA escape from endosomes and improves RNAi efficiency in fruit cells and tissues [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Polyethylene glycol (PEG) is a hydrophilic macromolecular compound widely used in the biomedical field, and has beneficial properties such as biocompatibility, non-toxicity, and hydrophilicity. Interestingly, PEG can significantly improve the physical properties of CS, by stabilizing siRNA/nanocomposites, and extending the residence time of siRNA in the body [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The carboxyl group can react with the amino group to form a stable amide bond, or it can react with the hydroxyl group to form an ester bond. Polyethylene glycol can be chemically linked to other hydrophobic polymer materials such as chitosan through carboxyl modification to form amphiphilic polymer materials. At the same time, the carboxyl group, as an acidic group, can be used as a pH-responsive element in nanomaterials and can be protonated under alkaline conditions, thus affecting the drug loading efficiency of nanoparticles [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAppropriate target genes are also key to the successful application of RNAi-based pest management strategies [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. VGSC is a complete transmembrane protein that exists in neurons, muscle cells, endocrine cells, and ovarian cells. It participates in the initiation and propagation of excitable cell action potentials and changes the propagation dynamics of nerve impulses [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Sodium ion channels are targets of a series of insecticides such as metaflumizone, indoxacarb and pyrethroids. Metaflumizone exerts stomach poisoning effects on target pests. Poisoned insects show symptoms of acute neurological poisoning, such as pseudoparalysis, paralysis, and the ability to move and be accompanied by violent tremors after being stimulated by external stimuli. With its widespread use around the world, insects in many places have developed varying degrees of resistance and cross-resistance to metaflumizone. Field populations of rice leaf rollers in Changsha and Nanning were 8.4 and 26.2 times resistant to metaflumizone, respectively [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Roditakis et al. reported a 17-fold cross-resistance to metaflumizone in Greek tomato field populations [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, synergism between RNAi and metaflumizone is expected to achieve a higher control effect.\u003c/p\u003e \u003cp\u003eIn this study, we prepared a new nanocarrier material CS-PEG-COOH and demonstrated that it can form a complex with dsRNA. We found that CS-PEG-COOH can release dsRNA at pH\u0026thinsp;=\u0026thinsp;8. Further, we verified the stability and delivery efficiency of CS-PEG-COOH-loaded dsRNA in \u003cem\u003eS. frugiperda\u003c/em\u003e and corn leaves. Subsequently, we selected \u003cem\u003eVGSC\u003c/em\u003e as the target gene, synthesized dsRNA using the \u003cem\u003ein vitro\u003c/em\u003e transcription system as well as the \u003cem\u003eE. coli\u003c/em\u003e expression method, prepared the ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH complex, and used the feeding method to study the effects of dsRNAs synthesized by different methods on \u003cem\u003eS. frugiperda\u003c/em\u003e RNAi efficiency. In addition, the compound was treated with metaflumizone to determine the mortality and development of \u003cem\u003eS. frugiperda\u003c/em\u003e, providing a reference for combining efficient RNAi technology with traditional pesticides. Finally, transcriptome analysis shed some light on the synergistic effect of dsRNA/CS-PEG-COOH and metaflumizone.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsect rearing\u003c/h2\u003e \u003cp\u003eThe susceptible strain of \u003cem\u003eS. frugiperda\u003c/em\u003e was provided by the insectarium of the Institute of Plant Protection, Jiangsu Academy of Agricultural Science (China) and reared under temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃, relative humidity of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, and photoperiod of L:D\u0026thinsp;=\u0026thinsp;16h:8 h, supplemented 10% honey water in the adult stage.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis of CS-PEG-COOH\u003c/h3\u003e\n\u003cp\u003eMethoxy polyethylene glycol carboxyl group (mPEG-COOH, 97%, MW 5000), chitosan (CS, degree of deacetylation\u0026thinsp;\u0026ge;\u0026thinsp;95%, MW 50,000\u0026ndash;60,000), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 98.5%) and N-hydroxysuccinimide (NHS, 98%) were purchased from Macklin (Shanghai, China).\u003c/p\u003e \u003cp\u003eSynthesis of CS-PEG-COOH had three steps: First, mPEG-COOH (0.1 g), EDC (20 mg) and NHS (13 mg) were added to a brown glass bottle containing 20 ml of deionized water and stirred at room temperature to activate the reaction for 2 h. Then the chitosan (0.2 g) was added and stirred continuously at room temperature for 48 h. Mixture was then centrifuged at 3000 r/min and the pH value of the upper clear solution was adjusted to 12. Finally, the solution was dialyzed with deionized water at least three times and freezedried to obtain CS-PEG-COOH samples.\u003c/p\u003e\n\u003ch3\u003eExpression of dsRNA\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e transcription of dsRNA, total RNA was extracted from the \u003cem\u003eS. frugiperda\u003c/em\u003e larvae with AG RNAex Pro reagent (Accurate Biology, Hunan, China). The cDNA was synthesized using MMLV reverse transcriptase (Promega, Madison, WI) with an oligo (dT)\u003csub\u003e18\u003c/sub\u003e primer, at 42 ℃ (90 min) and 95 ℃ (10 min). All primers were synthesized by Sangon Biotech Co. Ltd (China), and listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The target gene fragments (\u003cem\u003eVGSC\u003c/em\u003e: GenBank accession No. OR334597) were amplified using the 2 \u003cem\u003e\u0026times; Exp Taq\u003c/em\u003e Master Mix (Accurate Biology, Hunan, China). The amplified sequences were cloned into the pMD19T-vector (TaKaRa) and transformed into DH5α competent cells (Tsingke, Beijing, China). Plasmids extracted from \u003cem\u003eE. coli\u003c/em\u003e were used as the templates for dsRNA synthesis using the T7 RiboMAX Express RNAi System (Promega, USA). The \u003cem\u003egreen fluorescent protein\u003c/em\u003e gene (\u003cem\u003eGFP\u003c/em\u003e) was used as the control, and fragments were amplified from \u003cem\u003eGFP\u003c/em\u003e plasmids stored in the laboratory using specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor bacterially expressed dsRNA, the dsRNAs were selected to express in plasmid-\u003cem\u003eE. coli\u003c/em\u003e HT115 (DE3) (Tsingke, Beijing) system. The primers of dsRNA (L4440-\u003cem\u003eVGSC\u003c/em\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed by DNAMAN 8.0 and CE Design V1.04 software (Vazyme, Nanjing, China), and the primers contained \u003cem\u003ePst\u003c/em\u003e I and \u003cem\u003eNhe\u003c/em\u003e I restriction sites of L4440 vector (Tiandz, Beijing, China). The fragments of the target gene were amplified using the 2 \u003cem\u003e\u0026times; Exp Taq\u003c/em\u003e Master Mix (Accurate Biology, Hunan), and the amplified sequences were cloned into L4440 vector digested with \u003cem\u003ePst\u003c/em\u003e I (Takara, Beijing) and \u003cem\u003eNhe\u003c/em\u003e I (Takara, Beijing) by ABclonal MultiF Seamless Assembly Mix (ABclonal, Wuhan, China) to generate L4440-\u003cem\u003eVGSC\u003c/em\u003e. L4440-dsRNA expression plasmid vectors, L4440-\u003cem\u003eVGSC\u003c/em\u003e, were then transformed into \u003cem\u003eE. coli\u003c/em\u003e HT115 (DE3) cells for dsRNA expression. Single clone of HT115 (DE3) was cultured for 14 h with shaking in LB liquid media with 100 mg/L ampicillin (Amp) and 12.5 mg/L tetracycline (Tet) followed by shaking at 37 ℃, 200 rpm. When the OD\u003csub\u003e600\u003c/sub\u003e value reached 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM) was added and incubated for 5 h under the same conditions. Five ml of HT115 cell cultures was centrifuged at 12000 rpm for 5 min For total RNA extraction using AG RNAex Pro reagent (Accurate Biology, Hunan). The expressed dsRNAs were purified by MEGAclear Kit (Thermo Fisher Scientific, USA), and the concentration and integrity of the dsRNAs were assessed using the NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003eGel retardation test of CS-PEG-COOH-loaded dsRNA\u003c/h3\u003e\n\u003cp\u003eTo analyze the binding capacity of CS-PEG-COOH with dsRNA at various mass ratios, CS-PEG-COOH was dissolved in 0.1 M sodium acetate buffer (pH 6.5), and \u003cem\u003ein vitro\u003c/em\u003e transcribed dsGFP (200 ng) was incubated with CS-PEG-COOH (200 ng) at the mass ratios of 1 : 0, 0 : 1, 3 : 1, 2 : 1, 1 :1, 1 : 2, 1 : 3, 1 : 4 and 1 : 5. \u003cem\u003eE. coli\u003c/em\u003e HT115 expressing L4440-ds\u003cem\u003eGFP\u003c/em\u003e (100 ng) was incubated with CS-PEG-COOH (100 ng) at the mass ratios of 1 : 0, 0 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 1 : 3, 1 : 4 and 1 : 5, respectively. Each mixture was incubated at room temperature for 5 min, and then the agarose gel electrophoresis images were analyzed using Image J software. Complete loading of dsRNA onto CS-PEG-COOH was confirmed when the dsRNA/CS-PEG-COOH complex remained in the well and failed to migrate through a 1% agarose gel.\u003c/p\u003e\n\u003ch3\u003eIsothermal titration calorimetry (ITC) assays\u003c/h3\u003e\n\u003cp\u003eTo determine the interaction of purified ds\u003cem\u003eGFP\u003c/em\u003e with CS-PEG-COOH, 2.50 mg ds\u003cem\u003eGFP\u003c/em\u003e was titrated with 40 mg CS-PEG-COOH using the Nano ITC (TA Instruments Waters, USA). The heat of interaction during each injection were calculated by the integration of each titration peak via NanoAnalyze TM software (USA). The test was carried out at 25\u0026deg;C, and ΔG was calculated using the formula ΔG\u0026thinsp;=\u0026thinsp;ΔH\u0026thinsp;\u0026minus;\u0026thinsp;TΔS.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of dsRNA/CS-PEG-COOH complex\u003c/h2\u003e \u003cp\u003eFor the preparation of dsRNA/CS-PEG-COOH complex, \u003cem\u003ein vitro\u003c/em\u003e transcribed dsRNA was mixed with CS-PEG-COOH solution in a mass ratio of 1 : 3 and incubated for 5 min. The morphology of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH composite was determined using transmission electron microscopy (TEM) and atomic force microscopy (AFM). For TEM observation, a droplet of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex was immobilized on copper microgrid and then observed and imaged under TEM (HRTEM, JEOL F200, Japan). According to the method described by Zhang et al [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. AFM (Bruker Dimension Icon, Germany) was used to examine the ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex by using a tapping mode with a high aspect ratio tip. The size and zeta potentials of ds\u003cem\u003eGFP\u003c/em\u003e, CS-PEG-COOH and ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex were measured using dynamic light scattering by a Zetasizer Nano ZSE (Malvern, UK) at 25\u0026deg;C. Each treatment included three independent samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStability of ds\u003c/b\u003e \u003cb\u003eGFP\u003c/b\u003e \u003cb\u003e/CS-PEG-COOH complex\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to study the stability of dsGFP/CS-PEG-COOH complex in \u003cem\u003eS. frugiperda\u003c/em\u003e midgut fluid, 3rd instar larvae were dissected in pre-cooled PBS to obtain the midgut. The sample was homogenized for 30 s, centrifuged at 4℃ for 20 min, and the supernatant was stored at -80℃ before use. The protein concentration in midgut fluid was detected using the Biuret method for protein content determination kit (Solarbio, Beijing, China). Different concentrations of midgut fluid were mixed with 200 ng ds\u003cem\u003eGFP\u003c/em\u003e. The mixture was incubated for 1 h at room temperature and subsequently analyzed by 1% agarose gel electrophoresis. To study the stability of the ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex, we treated the ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex with a concentration capable of degrading the midgut content of 200 ng ds\u003cem\u003eGFP\u003c/em\u003e, and analyzed it by 1% agarose gel. Similar to the above steps, midgut fluid was replaced with RNaseA (Rromege, USA) to study the stability of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex after RNase A treatment. Because the pH value of the intestine can affect the stability of nanomaterials and the degradation rate of dsRNA [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], the ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex (200 ng) was exposed to various pH values ranging from 1 to 11 for 30 min, and then analyzed by 1% agarose gel electrophoresis. The relative band density was determined using ImageJ (National Institutes of Health, USA) from three independent samples. To determine the stability of the ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex at various temperatures, ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex (200 ng) was incubated at -20, 4, 25, 37 and 50\u0026deg;C for 30 min, and the stability of ds\u003cem\u003eGFP\u003c/em\u003e was determined similarly as above. Each treatment included three independent samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDelivery efficiency of dsRNA/CS-PEG-COOH complex in\u003c/b\u003e \u003cb\u003eS. frugiperda\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e transcribed fluorescent ds\u003cem\u003eGFP\u003c/em\u003e detectable under a fluorescence microscope (Leica, Germany), was synthesized using the Cy3 labeling mix (Shanghai Plant Science Biotechnology Co., Ltd., China). Then delivery efficiency of dsRNA/CS-PEG-COOH complex was examined in \u003cem\u003eS. frugiperda\u003c/em\u003e via oral feeding. The solutions of Cy3-ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex (Cy3-ds\u003cem\u003eGFP\u003c/em\u003e: 100 ng/\u0026micro;L), Cy3-ds\u003cem\u003eGFP\u003c/em\u003e and CS-PEG-COOH were added into artificial diet, respectively. Then the 3rd instar larvae were transferred to the artificial diet for feeding. At 6 h and 24 h after treatment, the middle part of the larvae was cut into 10 \u0026micro;m sections transversely to prepare frozen sections. Fluorescent images of cross-sections of the larval midgut were captured using a fluorescence microscope. Fluorescence intensity of all samples from three independent samples was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdhesion of CS-PEG-COOH loaded dsRNA to plants\u003c/h3\u003e\n\u003cp\u003eSolutions (200 \u0026micro;L) of fluorescent Cy3-ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex (Cy3-ds\u003cem\u003eGFP\u003c/em\u003e: 200 ng/\u0026micro;L), Cy3-ds\u003cem\u003eGFP\u003c/em\u003e and CS-PEG-COOH were applied to the surface of corn leaves, respectively. The treated corn leaves were washed with PBS and fluorescent photos of the leaf surfaces were taken before and after cleaning.\u003c/p\u003e \u003cp\u003e \u003cb\u003edsRNA fragment screening and RNAi efficiency analysis in\u003c/b\u003e \u003cb\u003eS. frugiperda\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThree fragments of \u003cem\u003eVGSC\u003c/em\u003e (6261 bp, Genbank: accession No. OR334597), named \u003cem\u003eVGSC\u003c/em\u003e1 (486 bp), \u003cem\u003eVGSC\u003c/em\u003e2 (499 bp), \u003cem\u003eVGSC\u003c/em\u003e3 (496 bp), in \u003cem\u003eS. frugiperda\u003c/em\u003e were selected for dsRNA synthesis using the T7 RiboMAX expression (Promega, USA).\u003c/p\u003e \u003cp\u003eThree kinds of dsRNAs (8000ng) were injected into the third instar larvae respectively, RNA were extracted on day 1 and day 3 after injection to examine the RNAi efficiency, and the experiment was repeated four times. The housekeeping gene \u003cem\u003eGAPDH\u003c/em\u003e (Gene ID: 118271716) was used as a reference gene for quantification. Primers for qRT-PCR are listed in Table S2. SYBR Green I (Vazyme, Nanjing) was used for qRT‐PCR on a LightCycler\u0026reg;480 Real‐Time PCR detection system. The relative expression level of \u003cem\u003eVGSC\u003c/em\u003e1, \u003cem\u003eVGSC\u003c/em\u003e2 and \u003cem\u003eVGSC\u003c/em\u003e3 were obtained by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe two dsRNA was incubated with CS-PEG-COOH at the mass ratio to prepare dsRNA/CS-PEG-COOH complex (dsRNA concentration was 4 \u0026micro;g/\u0026micro;L). These complexes were applied on artificial diet cubes (3 mm \u0026times; 3 mm) and fed to 3rd instar larvae. After consumption of the treated diet, larvae were given fresh artificial diet (1 \u0026times; 1 cm). The RNA samples were extracted at 1 day, 2 day and 3 day after the oral feeding to perform Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) to examine the RNAi efficiency, and the experiment was replicated for four times. Primers for qRT-PCR are listed in Table S2. SYBR Green I (Vazyme, Nanjing) was used for qRT‐PCR on a LightCycler\u0026reg;480 Real‐Time PCR detection system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynergism between ds\u003c/b\u003e \u003cb\u003eVGSC\u003c/b\u003e \u003cb\u003e/ CS-PEG-COOH complex and metaflumizone\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAcetone was used to prepare working liquid for different chemicals, followed by serial dilutions in water to achieve five concentrations. Artificial feed (0.2 g) was soaked in the liquid for 10 seconds, then air dried and placed into a 24-well insect culture plate for later use. To test larvae, the 3nd instar larvae were introduced into the 24-well plate, one larva per well. LC\u003csub\u003e50\u003c/sub\u003e values were calculated after 3 days of treatment. Mortality was determined on days 3 and 7 and photos were taken on day 7.\u003c/p\u003e\n\u003ch3\u003eRNA-seq analysis\u003c/h3\u003e\n\u003cp\u003eTo evaluate synergistic effect, metaflumizone and metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH complex were applied on artificial diet cubes (3 mm \u0026times; 3 mm) and fed to 3rd instar larvae of \u003cem\u003eS. frugiperda\u003c/em\u003e. Total RNA was extracted on day 3 after the oral feeding using the AG RNAex Pro reagent (Accurate Biology, Hunan). Each treatment included three independent samples. The transcript libraries were constructed via Illumina sequencing platform. Clean reads were processed by trimming adaptor sequences and low-quality reads (Q\u0026thinsp;\u0026le;\u0026thinsp;5) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The resulting clean reads were assembled using Trinity software [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. TopHat2 was used to achieve the sequence alignment with the reference genome, and obtain the localization information of reads on the reference genome [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The expression level of each transcript was presented by FPKM value. DESeq2 was applied for differential expression analysis between transcripts, and |log\u003csub\u003e2\u003c/sub\u003e(fold change)| \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e 1.0 and padj\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;0.05 were screen conditions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. BLASTX was used to annotate single genes in gene databases. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out for gene enrichment and functional annotation. The expression levels of target genes were assessed using qRT-PCR. All primer sequences used for qRT-PCR are listed in Table S2. SYBR Green I (Vazyme, Nanjing) was used for qRT-PCR on a LightCycler\u0026reg;480 Real-Time PCR detection system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e The significant differences in biological parameters were analyzed using statistical software SPSS 27.0 (SPSS Inc., USA), with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistical significance. All results are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eSelf-assembly and characterization of ds\u003c/b\u003e \u003cb\u003eGFP\u003c/b\u003e \u003cb\u003e/CS-PEG-COOH complex\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIncubation of ds\u003cem\u003eGFP\u003c/em\u003e with CS-PEG-COOH at room temperature for 5 min could achieve the self-assembly of ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The \u003cem\u003ein vitro\u003c/em\u003e synthesized ds\u003cem\u003eGFP\u003c/em\u003e showed complete loading at dsRNA/CS-PEG-COOH mass ratio of 1 : 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), and the bacterially expressed ds\u003cem\u003eGFP\u003c/em\u003e showed complete loading of all bands including higher molecular weight RNA at the mass ratio of 1 : 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In addition, we also used the ITC method to determine the interaction between dsRNA and CS-PEG-COOH, which is a high-precision method for determining the binding affinity between nanoparticles and biomolecules through changes in heat [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e, the binding constant (Ka) of 4.219 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicated that there was an effective interaction between CS-PEG-COOH and ds\u003cem\u003eGFP\u003c/em\u003e. The Gibbs free energy ΔG is negative (-1656.39 kJ/mol), indicating an automatic reaction between the CS-PEG-COOH and dsRNA. The positive enthalpy (ΔH) indicates that the complexation between the CS-PEG-COOH and dsRNA is mainly due to non-covalent intermolecular interactions such as hydrogen bonding and hydrophobic interactions [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f) and AFM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i) showed linearity of ds\u003cem\u003eGFP\u003c/em\u003e, and typical spherical shape of CS-PEG-COOH. The ds\u003cem\u003eGFP\u003c/em\u003e was adsorbed on the surface of CS-PEG-COOH to form the dsGFP/CS-PEG-COOH complex. At 25℃, the particle sizes of ds\u003cem\u003eGFP\u003c/em\u003e, CS-PEG-COOH and ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH complex were 201.24, 38.12 and 543.66 nm, and their zeta potentials were \u0026minus;\u0026thinsp;13.10, 6.44 and \u0026minus;\u0026thinsp;21.66 mV, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results showed that the CS-PEG-COOH had a strong positive surface charge that could adsorb negatively charged dsRNA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticle sizes and Zeta potentials of ds\u003cem\u003eGFP\u003c/em\u003e, CS-PEG-COOH and dsGFP/CS-PEG-COOH complex.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"+\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"+\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edsGFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e201.24\u0026thinsp;+\u0026thinsp;3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c3\"\u003e \u003cp\u003e-13.10\u0026thinsp;+\u0026thinsp;3.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCS-PEG-COOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e38.12\u0026thinsp;+\u0026thinsp;5.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c3\"\u003e \u003cp\u003e6.44\u0026thinsp;+\u0026thinsp;1.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edsGFP/CS-PEG-COOH complex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e543.66\u0026thinsp;+\u0026thinsp;19.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c3\"\u003e \u003cp\u003e-21.66\u0026thinsp;+\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtection of dsRNA by CS-PEG-COOH\u003c/h2\u003e \u003cp\u003eWhen incubated with 50 ng of midgut fluid or 10 ng of RNase A solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-d), dsGFP alone could be rapidly degraded by midgut fluid or RNase A (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c), compared to untreated dsGFP that migrated to the middle of the gel. Since dsRNA was tightly bound to CS-PEG-COOH, the midgut fluid-treated dsRNA/CS-PEG-COOH complex was immobilized in the well (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). RNase A-treated dsRNA/CS-PEG-COOH complex was also immobilized in the well (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). This indicates that CS-PEG-COOH effectively protected dsRNA from being degraded by RNase and midgut fluid degradation activities. In addition, CS-PEG-COOH was able to slow down the release of dsRNA in the midgut complex to improve the effectiveness of dsRNA interference. The pH of the insect midgut is one of the important factors for efficient delivery of dsRNA. Studies have shown that alkaline midgut pH of lepidopteran insectsleads to hydrolysis of dsRNA, causing low RNAi efficiency. To further test the stability of the complex, the dsGFP/CS-PEG-COOH complex was incubated under different pH gradients, and then the dissociated dsGFP was quantitatively analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). At pH 8, the dsGFP/CS-PEG-COOH complex released part of the dsRNA, and when the pH increased from 9 to 11, the release of dsRNA increased significantly. This indicates that dsRNA can be released from the dsGFP/CS-PEG-COOH complex in an alkaline environment. In addition, we also tested the stability of dsGFP loaded with CS-PEG-COOH at different temperatures. The intensity of the electrophoresis bands did not change significantly, indicating that the dsGFP/CS-PEG-COOH complex can adapt well to temperature changes in the field (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCS-PEG-COOH improves dsRNA delivery efficiency in larvae and enhances dsRNA adhesion to plants\u003c/h2\u003e \u003cp\u003eThe Cy3-ds\u003cem\u003eGFP\u003c/em\u003e was employed to treat the \u003cem\u003eS. exigua\u003c/em\u003e larvae through oral feeding to examine the delivery efficiency by fluorescence microscopy, followed by quantitative analysis of Cy3-ds\u003cem\u003eGFP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,c). After oral feeding for 6 hours, clear Cy3-ds\u003cem\u003eGFP\u003c/em\u003e signals were observed in the midgut of larvae treated with Cy3-ds\u003cem\u003eGFP\u003c/em\u003e and Cy3-ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH. The fluorescence intensity of larvae treated with Cy3-d\u003cem\u003esGFP\u003c/em\u003e/CS-PEG-COOH complex was 2.78 fold higher than that of Cy3-ds\u003cem\u003eGFP\u003c/em\u003e. After 24 h feeding, clear Cy3-ds\u003cem\u003eGFP\u003c/em\u003e signals were observed in the midgut of larvae treated with Cy3-ds\u003cem\u003eGFP\u003c/em\u003e /CS-PEG-COOH complex, while no Cy3-ds\u003cem\u003eGFP\u003c/em\u003e signals were seen in the larvae treated with Cy3-ds\u003cem\u003eGFP\u003c/em\u003e alone, suggesting that CS-PEG-COOH improved dsRNA stability of in the gut. When fluorescent Cy3-dsGFP/CS-PEG-COOH was sprayed on corn leaves, adhesion of CS-PEG-COOH-loaded dsGFP to plants was detected. After rincing leaves with water, the fluorescence signal on corn treated with CS-PEG-COOH-loaded Cy3-dsGFP was much stronger than that treated with Cy3-dsGFP alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Quantitative results also confirmed this conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Similar to previous studies, these results support the use of chitosan for efficient delivery of dsRNA into plants. The greater stability and enhanced delivery of chitosan-loaded dsRNAs allows for better entry into plant cells and facilitates enhanced uptake in tobacco, legumes and other plants [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEfficient dsRNA fragment screening and synergistic effect with metaflumizone\u003c/h2\u003e \u003cp\u003e \u003cem\u003eVGSC\u003c/em\u003e is an essential gene for potential conduction and growth and development of excited tissues, and it is involved in the initiation and propagation of excitable cellular action potentials [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Local injection of dsRNA of gene fragments synthesized in vitro.(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Results showed that, at 24h and 48h, ds\u003cem\u003eVGSC1\u003c/em\u003e significantly improved the interference efficiency of nymphs, therefore was adopted for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwo methods were used to obtain dsRNA, one was bacterial expression, and the other was in vitro transcribed dsRNA. \u003cem\u003eE. coli-\u003c/em\u003eexpressed dsRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) was incubated with CS-PEG-COOH, and the biological activity of the complex was detected by a feeding method. Compared with ds\u003cem\u003eGFP\u003c/em\u003e/CS-PEG-COOH, the dsRNAs/CS-PEG-COOH complex significantly reduced the expression level of the corresponding target genes, and its persistence was significantly higher than feeding dsRNAs alone (Fig. d). Since VGSC is the target of metaflumizone, we further studied the effect of co-treatment of dsRNA/ CS-PEG-COOH and metaflumizone on the growth and reproduction of \u003cem\u003eS. frugiperda\u003c/em\u003e. The metaflumizone LC\u003csub\u003e50\u003c/sub\u003e value measured after 3 days was 6.244 mg/L (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The larval mortality results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-f. The highest mortality rate of larvaes treated with metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH complex within 3 days was 73.33%, while that of larvaes treated with ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH and metaflumizone separately were 6.00% and 58.33%, respectively. The highest mortality rate of nymphs treated with metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH complex within 7 days was 93.40%, while the mortality rates of larvaes treated with ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH and metaflumizone alone were 10.30% and 70.00%. The development cycle of larvae is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. There are differences in the larval lifespan between different treatments. The lifespan of \u003cem\u003eS. frugiperda\u003c/em\u003e treated with metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH complex was 67 days, significantly longer than that treated with metaflumizone (41 days) alone. Results of ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH were not different from the control, indicating that ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH and metaflumizone can significantly extend the development time of larvae and further weaken the growth ability and resistance of \u003cem\u003eS. frugiperda\u003c/em\u003e, thus achieving the effect of increasing the efficiency and reducing the dosage of pesticides.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eToxicity of metaflumizone to 3rd instar larvae of \u003cem\u003eS. frugiperda\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSlope\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e/(mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% Confidence limits\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetaflumizone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.466\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(5.515, 7.113)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cbr\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of different chemical treatments on development duration of \u003cem\u003eS. frugiperda\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eDuration of development /d\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3th\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4th\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5th\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6th\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePupae\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTotal duration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.44 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e37.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.37 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003edsVGSC/CS-PEG-COOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.36\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.60 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.83 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.44 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.60\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e39.60\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.40 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetaflumizone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.48 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.50 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.00\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.31 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.00\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.10 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.60\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.54 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.37 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e47.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.31 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetaflumizone/dsVGSC/CS-PEG-COOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.40\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.40 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.58 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.44 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.60\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.20 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e61.80\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.73 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eNOTE: Different lower letters above bars indicate highly significant differences between three treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Tukey\u0026rsquo;s Honestly Significant Difference test).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003cb\u003eSynergistic mechanism between CS-PEG-COOH-loaded ds\u003c/b\u003e\u003cb\u003eVGSC\u003c/b\u003e \u003cb\u003eand metaflumizone\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003cp\u003eTo elucidate the synergistic mechanism between CS-PEG-COOH-loaded ds\u003cem\u003eVGSC\u003c/em\u003e and metaflumizone, we extracted total RNA from surviving larvaes after 72 h of treatment for RNA-seq analysis. The results showed that the sample sequencing quality was high (Table S3) and so was the Pearson correlation coefficient (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Compared with metaflumizone alone, metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH changed the expression of 5208 genes, including 2519 up-regulated genes and 2689 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). DEGs are divided into different pathways such as tyrosine metabolism, lysosomes, glycolysis, biosynthesis of metabolites, drug metabolism, and endocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, after metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH complex treatment, many stratum corneum structure-related genes were significantly down-regulated, such as \u003cem\u003ealdo-keto reductase AKR2E4\u003c/em\u003e, \u003cem\u003eEcdysone-induced protein 75B\u003c/em\u003e, \u003cem\u003eendocuticle structural glycoprotein ABD-5\u003c/em\u003e, In insects, ecdysteroids affect molting, and reproduction [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The ecdysteroid ecdysone was first isolated in 1954 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. and shown to be produced by the reduction of 3-dehydroedysone (3DE) by 3β-reductase [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. which is then hydroxylated at position 20 to form the active steroid hormone 20-hydroxyecdysone. This 3β-reductase belongs to aldo-keto reductase (AKR) family 2, namely AKR2E4. This enzyme reduces 3DE to ecdysone in the presence of NADPH. The amount of AKR2E4 in silkworm hemolymph may vary with the hormonal activity of ecdysteroids [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The ecdysone-inducible gene E75 is the main target of the ecdysone receptor (EcR) and plays a key role in the molting process of arthropods: ecdysteroids may act through E75 to stimulate the degradation of epidermal chitin. We found that E75 protects the rhythm under stress conditions, indicating that steroid signaling plays a role in maintaining the circadian rhythm of \u003cem\u003eDrosophila\u003c/em\u003e [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. LmAbd-5 is mainly highly expressed in tissues of ectodermal origin. After silencing LmAbd-5, migratory locusts have no visible phenotype, but ultrastructural analysis found that it is involved in the formation of the inner epidermal lamellar structure of migratory locusts [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In the current study, many essential genes that promote cell metabolism were also significantly down-regulated, such as \u003cem\u003elysosomal alpha-mannosidase-like\u003c/em\u003e (\u003cem\u003eMAN2B1\u003c/em\u003e), \u003cem\u003eLate endosomal/lysosomal adapter, MAPK and MTOR activator 1\u003c/em\u003e (\u003cem\u003eLAMTOR1\u003c/em\u003e), \u003cem\u003eAryl hydrocarbon receptor nuclear translocator homolog tgo\u003c/em\u003e (\u003cem\u003eARNT\u003c/em\u003e). In animal cells, MAN2B1 not only affects the structure and physical and chemical properties of proteins, but also affects physiological processes such as cell adhesion, migration, growth, and differentiation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. LAMTOR1 can regulate receptor recycling through endosomes and the MAPK signaling pathway by recruiting some of its components to late endosomes. LAMTOR1 is involved in several processes, including cholesterol homeostasis as well as release and regulation of lysosomal uptake [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. ARNT is a transcription factor that is reported to play a crucial role in regulating glycolysis, angiogenesis, and apoptosis [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In addition, there are down-regulation of genes related to drug metabolism, such as \u003cem\u003eUDP-glycosyltransferase UGT5-like\u003c/em\u003e (\u003cem\u003eUGT2A1\u003c/em\u003e), \u003cem\u003eglutathione S-transferase 1\u003c/em\u003e (\u003cem\u003eGstD1\u003c/em\u003e), UDP-glycosyltransferase is also involved in regulating endogenous compounds, and they are important metabolic detoxification enzymes in insects, mediating the development of insect resistance. The expression levels of relevant genes were verified by qRT PCR, and the results were consistent with the transcriptome data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we proposed a novel and improved CS-PEG-COOH nanocarrier system for efficient RNAi delivery. In dsRNA/CS-PEG-COOH complex, CS-PEG-COOH significantly improves the stability and delivery efficiency of dsRNA, and protects it from RNase A degradation and the impact of the pH microenvironment. At the same time, the alkaline intestinal environment promotes some release of dsRNA. Cy3 fluorescent labeling confirmed that CS-PEG-COOH significantly enhanced the delivery of dsRNA to insects, and was water-washing resistant on plants. Through feeding, CS-PEG-COOH was shown to significantly improve the silencing effect of ds\u003cem\u003eVGSC\u003c/em\u003e on \u003cem\u003eS. frugiperda\u003c/em\u003e, increasing the pest control effectiveness and reducing the dosage of pesticides. Finally, RNA-seq analysis showed that the CS-PEG-COOH-loaded ds\u003cem\u003eVGSC\u003c/em\u003e complex cooperated with metaflumizone to down-regulate genes related to cuticle biosynthesis, cell metabolism, and drug metabolism, thereby inhibiting the growth and development of insects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuman Zong:\u003c/strong\u003e Study design, Writing - Original draft, Investigation, Data curation. \u003cstrong\u003eHao Hong:\u003c/strong\u003e Writing - Original draft, Investigation, Data curation.\u003cstrong\u003e\u0026nbsp;Jing Zhao:\u003c/strong\u003e Study design, Writing - review and editing. \u003cstrong\u003eLiubin Xiao:\u003c/strong\u003e Writing - review and editing, Supervision. \u003cstrong\u003eKeyan Zhu-Salzman:\u0026nbsp;\u003c/strong\u003eWriting - review and editing. \u003cstrong\u003eHan Wu:\u003c/strong\u003e Writing - review and editing, Supervision. \u003cstrong\u003eDejin Xu:\u003c/strong\u003e Writing - review and editing, Supervision. \u003cstrong\u003eGuangchun Xu:\u0026nbsp;\u003c/strong\u003eWriting -review and editing, Supervision. \u003cstrong\u003eLinquan Ge:\u003c/strong\u003e Conceptualization, Study design, Writing - review and editing, Supervision. \u003cstrong\u003eYongan Tan:\u0026nbsp;\u003c/strong\u003eConceptualization, Study design, Writing - review and editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Technology Innovation Fund [CX(22)2038],\u0026nbsp;National Natural Science Foundation of China (32372631)\u0026nbsp;and National Key Research and Development Program of the Ministry of Science and Technology (2022YFD1401800).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of date and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoergen G, Kumar PL, Sankung SB, Togola A, Tam\u0026ograve; M. 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Oncol Lett. 2020;20(4):56.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spodoptera frugiperda, RNAi, metaflumizone, Nanocarrier, Pest management","lastPublishedDoi":"10.21203/rs.3.rs-6184980/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6184980/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNAi is currently the most promising gene silencing-based biotechnology for pest control. However, double-stranded RNA (dsRNA) is easily degraded in the environment, resulting in poor stability and ineffectiveness. In this study, we designed a nanoparticle chitosan-polyethylene glycol-carboxyl (CS-PEG-COOH), which can spontaneously assemble with dsRNA to form a dsRNA/CS-PEG-COOH complex. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) revealed the complex size being 543.66 nm. CS-PEG-COOH was able to prevent dsRNA from being degraded by midgut fluid or RNase A, thereby significantly improving the dsRNA stability under various environmental conditions. CS-PEG-COOH gradually released dsRNA at pH\u0026thinsp;\u0026gt;\u0026thinsp;8. Cy3 fluorescent labeling confirmed that CS-PEG-COOH significantly enhanced the delivery of dsRNA to insects, and was water-washing resistant on plants. Subsequently, ds\u003cem\u003eVGSC\u003c/em\u003e fragments were expressed in \u003cem\u003eE. coli\u003c/em\u003e using selected \u003cem\u003eVGSC\u003c/em\u003e fragments. Their silencing efficiency was significantly enhance by CS-PEG-COOH. Feeding \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e the nanocomposite in combination with metaflumizone caused down-regulation of genes related to cuticle synthesis, cell metabolism, and drug metabolism. Metaflumizone/ds\u003cem\u003eVGSC\u003c/em\u003e/CS-PEG-COOH significantly enhanced pest control effect by inhibiting insect growth and development.\u003c/p\u003e","manuscriptTitle":"Synergistic control of Spodoptera frugiperda based on nanoparticle-mediated dsRNA and insecticide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 14:02:44","doi":"10.21203/rs.3.rs-6184980/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd9c2664-9c28-4c26-bd06-5efc498dfb7f","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-13T16:38:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-12 14:02:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6184980","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6184980","identity":"rs-6184980","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}