Delivery of dsRNA to improve RNAi efficiency by cell-penetrating disulfide polymer for spodoptera frugiperda control

doi:10.21203/rs.3.rs-5052291/v1

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Delivery of dsRNA to improve RNAi efficiency by cell-penetrating disulfide polymer for spodoptera frugiperda control

https://doi.org/10.21203/rs.3.rs-5052291/v1

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The application of RNA is regarded as innovative and sustainable, and it has shown excellent control efficiency against pest control in the world. However, the efficiency of RNAi in insects could be more inefficient and unreliable, and exploring a superb delivery system is considered a crucial factor in improving RNAi. The fall armyworm, Spodoptera frugiperda (FAW), is a worldwide agricultural pest and has caused enormous losses to global food production. Herein, a novel and effective method of conjugating double-stranded RNA (dsRNA) with cell-penetrating disulfide polymer (CPD) was reported to improve the stability and RNAi efficiency of the dsRNA. Chitin synthase B gene (CHSB) and methoprene-tolerant gene (Met)gene were used as the target genes, which are essential for the development and growth of FAM. The CPD was prepared using a two-step method to deliver the dsRNA. The synthesized CPD/dsRNA reached a size from 161.9 nm to 226.2 nm and protected dsRNA from nuclease degradation. The biological application of CPD in Sf9 cells and fall armyworm(FAW)indicated the low cytotoxicity and high cell viability of CPD. Moreover, the dsRNA loaded by CPD could enter the cell within 6 h and had excellent lysosomal escape function and gene transfection efficiency. The bioassays of FAW showed the relative expression levels of CHSB and Met genes were 48.14% and 37.60% of the control at 72 h, respectively. Meanwhile, the weight and body length of the larval decreased significantly on the sixth day, and the mortality rate of CPD/dsCHSB reached 30% on the 10th day. This CPD has been shown for the first time to have excellent delivery performance in insects and is expected to become a new and effective tool for pest control, marking a significant advancement in the field of pest management.

RNA interference

dsRNA

cell-penetrating disulfide polymer

delivery

fall armyworm

The fall armyworm(FAW) is polyphagous and destructive which has caused a serious threat to global economic losses, and developed resistance to chemical pesticides. Therefore, safe and effective initiatives for FAW control are imminent.
RNAi-based control is an effective and environmentally friendly strategy to control the pest.
A novel and facile-synthesized RNAi system based on the cell-penetrating disulfide polymer (CPD) delivery system and gene silencing was developed to interfere with the CHSB and Met of FAW.
The dsCHSB and dsMet based on the CPD delivery resulted in significant gene downregulation and growth inhibition of FAW through feeding of dsRNA produced by Escherichia coli.

Since double-stranded RNA-mediated RNA interference (RNAi) was found in the Caenorhabditis elegans, it has been applied as the reverse genetics tool to study gene function(Fire et al. 1998). It is recently used to knock down target genes in plants, insects, animals, and humans(Agrawal et al. 2003; Li et al. 2022). The dsRNA that targets specific genes in an organism is processed into small interfering RNAs (siRNAs) of 21–25 nucleotides by Dicer enzymes. Further, the siRNAs are recruited to the RNA-induced silencing complex (RISC), which locates the complementary messenger RNA (mRNA) of target mRNAs and induces sequence-specific cleavage of the target mRNA(Paroo et al. 2007). Because of the specificity of dsRNA, it has the advantages of precision, biosecurity, and environmental safety and has a wide range of application prospects(Prentice et al. 2019). Given this, it possesses great potential to effectively defend against insect pests in recent years.

Many RNAi-based products have been developed. In 2017, The United States Environmental Protection Agency (EPA) approved a new generation of insect-resistant transgenic corn SmartStax®PRO expressing the dsRNA of the Snf7 gene, which protects maize roots from the damage of western corn rootworms(Zotti et al. 2018). However, there are still concerns about foods containing genetically modified plants(Bailey-Serres et al. 2019), so developing the spray green RNAi insecticides containing dsRNA is a promising approach(Jain et al. 2022). However, two key issues must be addressed: mass production of dsRNA and improved delivery of dsRNA in pests. The yield of BL21(DE3) RNase III- expressing dsRNA is three times higher than that of the L4440-HT115-(DE3) system commonly used in laboratories, which meets the large-scale preparation of dsRNA and produces RNAi effects in many insects(Shukla et al. 2016; Zhongzheng et al. 2020). However, the control effect of RNAi in pests and diseases is highly correlated with insect species, the function of the target gene, the expression site, and the delivery mode of dsRNA. RNAi is highly efficient and systematic in Coleopteran insects(Christiaens et al. 2020). In contrast, RNAi is relatively inefficient and variable in Lepidopteran species(Huvenne et al. 2009; Scott et al. 2013). Some mechanisms, such as dsRNA degradation in the hemolymph and midgut lumen, reduced uptake by the cells, reduced induction of RNAi components upon exposure to dsRNA, and accumulation of dsRNA in endosomes, have been studied to be possible reasons for the difference in RNAi efficiency among insects(Cooper et al. 2019; Garbutt et al. 2013; Gurusamy et al. 2020). Nanomaterials emerging nucleic acid delivery carriers are playing an important role in improving the transfection efficiency, interference efficiency, and stability of nucleic acid pesticides. A few types of gene delivery systems, such as polymer nanoparticles(Lichtenberg et al. 2019), nanoliposomes(Castellanos et al. 2019; Lin et al. 2017), and inorganic nanoparticles, have been studied(Yu et al. 2023). They have been demonstrated varying degrees of ability of efficient protection and delivery of dsRNA in insects. Li et al. synthesized a hyperbranched spherical low-cost polymer (SPc) composed of repeating units with a large number of amine groups using inexpensive pentaerythritol as substrate in a simple reaction step, which could be used to prepare SPc/dsRNA (260.0 nm, + 4.0 mv) complexes with dsRNA support. The SPc/dsRNA was administered orally to the insect Agrotis ypsilon, which significantly down-regulated the expression of the key developmental gene V-ATPase in the third instar larvae and then inhibited the growth and development of Agrotis ypsilon(Li et al. 2019). Lin et al. used liposome-loaded dsRNA Germany the Blattella germanica tubulin gene (α-tubulin, Tub) to reduce the degradation of dsTub in the midgut effectively, and injected liposome/dsTub into Germany cockroaches to achieve up to 60% gene silencing, significantly increasing the mortality rate of Germany cockroach nymphs(Lin et al. 2017). In addition, Ramesh et al. complexed cationic polymers, protamine, and cationic liposomes with dsRNA to generate nanocarriers, down-regulating the expression of the IAP gene and achieving a nymph mortality rate of 55%(Dhandapani et al. 2022). Similarly, Yu et al. used a one-pot method to synthesize ZIF-8, a metal-organic frame (MOF) nano-delivery vehicle, to load dsRNA and insecticide and exhibit the RNAi efficiency of dsRNA against Nilaparvata lugens. The MOF could also significantly increase and improve the characteristics of dsRNA in rice that are easy to slip off(Yu et al. 2023). These studies indicate the potential of nanopesticides for agricultural insect management.

Herein, we synthesized a cell-penetrating disulfide polymer (CPD), exhibiting great potential for nucleic acid molecule delivery(Wan et al. 2023). Compared to other bio-delivered macromolecules, CPD combines several characteristics. First, it contains dynamically reversible disulfide bonds, allowing the CPD to be taken up into the cells via the thiol-mediated pathway due to the abundant thiol expression on the cell surface. Thus, CPD could avoid endosomal capture(Du et al. 2018; Niu et al. 2021). Further, when CPD is taken up by cells, the disulfide bonds of the CPD backbone are rapidly biodegradable in the intracellular reductive glutathione (GSH), and the loaded drug by the CPD is released(Nehate et al. 2019). Therefore, the degradation and GSH-responsive drug release of CPD could effectively reduce the toxicity of polymers in cells as well(Nehate, Nayal, and Koul 2019).

The fall armyworm, Spodoptera frugiperda (FAW), One of the lepidopteran insects, could spread and multiply rapidly, which has caused great worldwide crop losses(Makale et al. 2022; Miraldo et al. 2016; Pogue 2002). At the same time, chemical pesticides could also have serious effects on the environment and non-target organisms, which greatly limits the application of pesticides(Jin et al. 2021; Wang et al. 2021). In this study, we wanted to enhance pest control in an RNAi-based way by improving the stability of dsRNA and the efficiency of RNAi delivery. Chitin synthase B gene (CHSB) and methoprene-tolerant gene (Met) gene of FAW were selected as the target genes, which have been shown to be critical for the growth and development of FAW(Saha et al. 2016; Zhang et al. 2012; Zhu et al. 2019). A diagram illustrating the preparation and delivery protocol for CPD to deliver the dsRNA is shown in Fig. 1. Firstly, we used the BL21(DE3) RNase III- system to express a dsRNA fragment with 500 bp in length and synthesized the cell-penetrating disulfide polymer, which combined to form stable nanoparticles, effectively protected the dsRNA from being degraded by nucleases, and studied the cellular effect of CPD/dsRNA on Sf9 cell culture, confirming the cytotoxicity and transfection ability of CPD on Sf9 cells. Finally, the impact of CPD/dsRNA on FAW was determined. CPD delivery dsRNA was applied to insects for the first time in this research, aiming to combine RNAi technology and nanotechnology to manage pests and provide a promising delivery method for crop protection.

Materials

Lipoic acid, 1,1'-carbonyldiimidazole (CDI), L-Arginine methyl ester dihydrochloride, N, N-diisopropylethylamine, Triethanolamine (TEOA), and 2-Iodoacetamide were obtained from Aladdin Biochemical Reagents Co., Ltd. (Shang Hai, China). Dimethylformamide (DMF), Dichloromethane (DCM), and Ethyl ether (Et₂O) Reagent Co., Ltd. All chemicals were of analytical grade and used without any further purification. The ReverTra Ace qPCR RT Master Mix with gDNA remover kit and SYBR qPCR Mix were purchased from TOYOBO (Japan), all Restriction enzymes, T4 ligase, Grace’s insect medium, Fetal bovine serum (FBS), and penicillin − streptomycin were purchased from Invitrogen (Carlsbad, CA, USA), All primers were synthesized at Qingke Biotechnology Co., Ltd (Bei Jing, China).

Insects and cell culture

The Sf9 cells, originating from ovarian tissue of female fall armyworm pupae, were cultured in Grace’s insect medium with 10% fetal bovine serum (FBS) and 1% penicillin − streptomycin at 27 ± 1°C.

FAW eggs were obtained from Shanghai Tahoe Chemical Co., Ltd (Shang Hai, China). They were maintained on corn seedlings in a climate-controlled incubator at 27 ± 1°C with a photoperiod of 16:8 (light: dark). Larvae were fed an artificial diet.

Construction of plasmid vectors for expression of dsRNA and extraction of dsRNA

The protocol of construction of plasmid vectors of dsRNA and the isolation of RNA from Escherichia coli was adapted in the published research(Murphy et al. 2016; Posiri et al. 2013). In brief, the total RNA was extracted from four third-instar nymphs of FAW by using the RNAiso Plus solution. Further, cDNA was synthesized by RT-PCR using the ReverTra Ace qPCR RT Master Mix with gDNA remover kit. Two Fragments, VG-L and VG-R for generating dsRNA, were amplified with primers from cDNA with primers shown in the Table S1, respectively, using the following program: 30 s at 94°C, 30 s at 55°C, and 10 s at 72°C for 30 cycles. The VG-L contained a stem–loop structure was digested with Bam HI–Eco RI and digested with Eco RI–Xho I to generate the complementary VG-R. Products were purified by Omega Gel Extraction Kit and were cloned into the pET-28a digested with Bam HI–Xho I together. Then, pET28a was transformed into BL21(DE3) RNase III- which previously knocked out the rnc gene encoding endoribonuclease. After cells were cultured at 37°C overnight, a single colony was verified to be correct and cultured in 5 ml of LB liquid medium with kanamycin (50 mg/mL) overnight and diluted into the 50 ml shake flask. 1 mM IPTG was added and cultured for 4–5 h at 37°C when the OD₆₀₀ value reached 0.6. The cell culture medium was treated at 80°C for 20 minutes, then centrifuged at 12,000 rpm for 3 minutes. 75% absolute ethanol mixed with phosphate-buffered saline (PBS) was added and centrifuged at 8000 rpm for 4 minutes. 250 µL of 150 mM NaCl solution was added to resuspend the cells, and the cell culture was centrifuged at 12 000 rpm for 2 min to obtain the coarsely extracted dsRNA. RNase-free DNase (5 µL) and RNase A solution (2 µL) were added to 250 µL of the dsRNA solution for 30 min to remove from DNA and single-stranded RNA. The solution was purified with magnetic beads according to the manufacturer’s programs and then quantified using a NanoDrop 2000 spectrophotometer.

Synthesis of M

Following a slightly adapted procedure(Bang et al. 2013), a solution of lipoic acid (2.06 g, 10 mmol) in anhydrous DMF (20 ml), CDI (1.62 g, 10 mmol) was added, and the mixture was stirred at 25℃ under N2 atmosphere for 2 h. A solution of L-arginine methyl ester dihydrochloride (1.31 g, 5.0 mmol) and N, N-Diisopropylethylamine (0.87 mL, 5.0 mmol) in anhydrous DMF (20 ml) was added, and the reaction mixture was continued to stir at 25℃ under N2 atmosphere for 3.5 h. Afterward, the reaction mixture was added dropwise into Et2O (100 ml). The mixture was centrifuged (3 min, 3000 rpm), and the supernatant was discarded. The remaining yellow oil is washed three times with a DCM/Et2O mixture (1:2, 5×10 ml). After drying the residue in vacuo, a pale yellow solid M (346 mg, Cl salt, 84%) was obtained. 1H NMR (400 MHz, Deuterium Oxide) δ 4.30 (dd, J = 9.2, 5.0 Hz, 1H), 3.63 (m, 4H), 3.10 (m, 2.9 Hz, 4H), 2.54–2.14 (m, 4H), 1.84 (dd, J = 14.8, 6.9 Hz, 2H), 1.72–1.63 (m, 2H), 1.58–1.51 (m, 4H), 1.38–1.27 (m, 2H).

Synthesis of cell-penetrating disulfide polymer (CPD)

M and initiator, the N-acetyl-L-cysteine methyl ester, were dissolved into the TEOA buffer solution (1.0 M, pH = 7), respectively. For polymerization, the M (100 mM) and initiator 4 (0.2 mM) are stirred vigorously at 25°C under an N₂ atmosphere in 20 ml TEOA buffer. After a fixed 30 min, the mixture was quenched with a terminator solution (40 mL, 250 mM) in water and purified by dialysis in water for 3 days, and then, the CPD was obtained. The structure of CPD was validated by ¹H NMR (400 MHz, D₂O), and the relative molecular weight was analyzed by gel permeation chromatography (GPC).

Gel retardation assay

The ability of CPD to condense dsRNA was evaluated through gel electrophoresis. The complexes were incubated at mass ratios of 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, and 8:1 by loading 0.25, 0.5, 1, 2, 4, 6, and 8 µg of CPD with 1 µg dsRNA for 30 min at room temperature. The mixture was estimated with 1.0% agarose gels made from 1×TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH = 8). The gel electrophoresis was carried out at 150 v for 20 min.

Particle size and zeta potential analysis.

The CPD was dissolved in water to form nanoparticles with the dsRNA. The particle size and zeta potential of the CPD and CPD/RNA complexes were analyzed in triplicate at 25°C with a Zetasizer Nano ZS. The appearance of the CPD/RNA complexes was observed by transmission electron microscopy (TEM).

dsRNA Protection Performance.

The protection of dsRNA from nuclease degradation was evaluated using RNA enzymes. Briefly, naked dsRNA solution (1600 ng) and CPD/dsRNA were incubated with 25 U of nucleic acid allozyme at 37°C for different periods (0, 2h) in triplicates. Following incubation, 100 mM NaBH4 was added instantly to released dsRNA, and 0 h of dsRNA was used as the control. The different dsRNA samples were extracted and subjected to 1% agarose gel containing Gel Blue stain, followed by quantitative analysis using Quantity One software.

Cell viability assay

The cells were transfected with different concentrations of nanoparticles to characterize cell viability, which was evaluated using the CCK-8 assay kit. Briefly, Sf9 cells are seeded into 96-well plates at a density of 5 × 10⁴ cells per well. After seeding for 2 h, the CPD and CPD/dsRNA complexes were transfected in 100 µl culture medium. The concentration of CPD and CPD/dsRNA complexes varied from 0 to 64 µg/ml, and the quantity of dsRNA was fixed at 100 ng per well. After incubation for 24 and 48 h, respectively, the medium was removed, and 100 µL fresh medium containing 10 µL of CCK8 solution was added into each well with PBS-washed medium for 1.5 h. The absorption of each well was measured at 450 nm with a microplate reader to calculate the OD values. The cell viability was calculated by the formula:

\(\:Cell\:viability=\frac{\left(A-{A}_{0}\right)}{({A}_{1}-{A}_{0})}\times\:100\) %

where A represents the absorbance of the cells treated with various complexes,

A₁ represents the absorbance of medium blanks, and A₀ is the absorbance of the cells without any treatment.

Plasmid construction and mRNA in vitro transcription

The plasmid PIB-V5-His (pDNA)and mRNA encoding Green Fluorescent Protein (GFP) were constructed to test the cell transfection efficiency on the Sf9 cell line. Cells were seeded in 24-well plates at a concentration of 1.5×10⁵ cells/well in 0.25 mL of SF 900 II medium and incubated overnight at 27°C. CPD was incubated with a pDNA and mRNA (500 ng) solution at a mass ratio of 6:1 for 30 min at room temperature to prepare the CPD/pDNA and CPD/mRNA complexes. Then, the complexes were added to the cell medium. The PEI/pDNA solution, according to the manufacturer’s protocol, was added. pDNA alone and CPD were set as the controls. After 48 h of transfection, the green fluorescence was used to reflect the delivery of CPD into insect cells by fluorescence microscopy.

Internalization of CPD-conjugated CypHer-5E-labeled dsRNA

In the current work, CypHer5E-labeled dsRNA was labeled in red, Lyso-Tracker™ was employed to stain lysosomes in green, and the Hoechst33258 was used to label the nuclear in blue to explore the cellular uptake and lysosomal escape of CPD. 4×10⁵ Sf9 cells were exposed to the proper amounts of CypHer5E-labeled dsRNA-CPD in 1 ml of serum-free culture medium at 2 h,4 h, and 6 h, respectively, and the naked CypHer-5E-labeled dsRNA was used as a control. Then, the cells were stained by Lyso-Tracker™ and Hoechst 33258, respectively. The images were scanned by Nikon A1 confocal laser scanning microscopy.

Bioassays

The larvae of FAW were fed on an artificial diet for the duration of the assay; the second instars with the same body size were placed in the sterile Petri dish with a diameter of 5 cm for the later RNAi experiment. 10 instars were placed in each plate, and the dsRNA at a concentration of 1000 ng/µL and CPD were mixed to make the artificial diet, and then fully mixed with artificial food of about 1 cm³ and then evenly added to the Petri dish. The ones treated with CPD/dsGFP and CPD were used as the control group, and each experimental group had 5 replicates. There are 50 nymphs in total. The nymphs were put into the incubator at 26 ± 1°C and the photoperiod of 14:10 h. The food was changed every 2 days and was stopped after 6 days, and the normal food was changed back to continue feeding. Changes in body weight and body length in each treatment group are recorded on the sixth day, and insect mortality is recorded on the tenth day.

Interference Efficiency analyzed by qPCR

Total RNA was extracted from larvae using RNAiso Plus reagent, and cDNA was synthesized with one microgram of total RNA using a reverse transcription kit. The relative mRNA levels were performed by the 2^−ΔΔCt method(Livak et al. 2001), and the RpL10 was the reference gene. The primers are shown in Table S1. qPCR was performed with SYBR Green Supermix, and the reaction system contained 10 µL of SYBR qPCR Mix, 1 µL cDNA, 0.4 µl each of 10 µM forward and reverse primers and 8.2 µL of RNase-Free water in 20 µL final volume. The reaction conditions were followed by 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s settings.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 9 software (GraphPad Software Inc.). All statistics were shown as the mean ± standard deviation (SD). The Student’s t-test for two samples and one-way ANOVA with a Tukey’s post-hoc test for more than two groups were applied. P < 0.05 is considered statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001).

Synthesis and Verification of dsRNA in Vitro

The pET28a-BL21(DE3) RNase III-system was used to produce the dsRNA at a low cost and high-efficiency way, which expressed three times more than the widely used L4440-HT115. The CHSB and Met gene segments were cloned into the pET28a plasmid, and the constructed plasmids were transformed into BL21(DE3) RNase III- system to express the target dsRNA (Fig. 2A). The single T7 promoter activated RNA transcription to transcribe dsRNA with reverse complementary fragments that formed dsRNA. The crude dsRNA was obtained from the Escherichia coli using 75% ethanol (Fig. S1). The length of purified three dsRNA fragments is approximately 400 bp, and there were obvious main bands between 250 and 500 bp marker in length by agarose gel electrophoresis (Fig. 2B).

Preparation and characterization of CPD and CPD/dsRNA.

To enhance the delivery of dsRNA in insect cells and tissues, CPD was synthesized to load dsRNA. The polymer containing guanidyl groups could have electrostatic interaction with the phosphate group of nucleic acid and be conducive to transmembrane transport via the disulfide bonds. As shown in Scheme 1, First, the strained disulfide monomer M with guanidyl groups was prepared by a CDI-mediated coupling reaction between the carboxyl group of lipoic acid and the primary amines of L-arginine methyl ester dihydrochloride. The structural characterization of M was confirmed by ¹H NMR (Fig. 3A) and LC-MS (Fig. S2). N-acetyl-L-cysteine methyl ester was used as an initiator to carry out through the ring-opening polymerization of the monomer, and the polymerization progress was terminated by adding 2-iodoacetamide. CPD was obtained as a white powder after being freeze-dried to remove water. The successful structure of CPD was analyzed by ¹H NMR. As shown in Fig. S3, the number of monomeric repeat units of CPD was calculated to be 17 based on the integration of two peaks. The purified CPD was calculated the number-average molecular weight (Mn) as ca. 5975 Da based on the integration from the proton peaks of PEG as the standard by GPC, which displayed a unimodal in GPC traces and was eluted at relatively low elution times (Fig. 3B), indicating its polymeric nature. The low molecular weight distributions (Mw/Mn = 1.1) had shown the good dispersion of CPD (Table S2). Both ¹H NMR and GPC data indicated that a relatively pure polymer had been obtained by simple synthesis with a relatively low cost, which further meets the need for large-scale use in practice.

Further, the CPD was first tested for its ability to load dsRNA, and the physicochemical properties of the resulting complexes were also evaluated. As shown in Fig. 3C, the resulting lane brightness gradually weakened as the mass ratio of the complex was increased. When the mass ratio reached 6, the CPD/dsRNA could be loaded completely because the larger nanoparticles were stuck in the wells, resulting in no strip in the electrophoresis lane, so mass ratio 6 of the CPD/dsRNA was selected for further evaluation. The size and Zeta potential charge of complexes of CPD and CPD/dsRNA were further measured by dynamic light scattering (DLS) analysis. As shown in Fig. 3D and Fig. 3E, the CPD was positively charged (+ 38.9 mV) owing to the guanidyl groups in the chain and was bound to the negatively charged RNA. Thus, the zeta potential reduced to 27.5 mV. The size of CPD/RNA likewise increased from 169 nm to 220 nm due to binding dsRNA, tested by DLS. The microstructures of the CPD/RNA complexes were observed by TEM. The particle size was basically the same as that of DLS. These data indicated that CPD has successfully interacted with the dsRNA to form stable nanoparticles (Fig. 3F).

Stability evaluation of CPD/dsRNA using nucleases

As we all know, RNA is susceptible to degradation by a variety of enzymes and microorganisms in the environment, which is a potential challenge to use rare dsRNA directly for pest management strategies, especially in the complex environmental conditions in the field, so it is crucial to select a suitable vector improve the stability of dsRNA. CPD could closely combine bound to nucleic acid molecules for the delivery of nucleic acid drugs in a variety of studies. Nucleases that efficiently degrade dsRNA were used in this study to explore the protective performance of CPD on dsRNA. The CPD/dsRNA was treated with nucleases, the disulfide bond was rapidly reduced by adding NaBH4, and then the remaining dsRNA was released. As shown in Fig, the untreated RNA at 0 h, the control, showed the clear bands gel electrophoresis. Although the migration of bands was slightly faster than expected, it could result from secondary structures in the RNA molecule duo to the loop structure. In the dsRNA nuclease-treated groups, the rare dsRNA after 1 h and 2 h had no band were observed, which indicated that the dsRNA had been completely degraded. In the CPD/dsRNA treatment group, the clear band was still observed after 2 h (Fi. 4A). The concentration of dsRNA at 1 h was 68% of the 0 h control group, and 62% of the concentration of the 0 h control group was still maintained after 2 h after quantitative analysis (Fig. 4B). This illustrated that half of the RNA was still retained in the CPD treatment group after nuclease treatment. This may be due to the fact that CPD was tightly bound to RNA rather than fully embedded, so nucleases still degraded some of the RNA. Overall, CPD had excellent protective effect on RNA. Moreover, due to the high activity and concentration of nucleases used in the laboratory, and optimal conditions (37°C) of the maximum activity of nucleases, the degradation process of dsRNA was greatly accelerated, which was difficult to achieve under natural conditions. Therefore, the protective properties of the CPD we prepared against dsRNA could greatly meet the requirements of practical applications.

Cell cytotoxicity analysis

In addition to having a good nucleic acid loading rate, biosafety is also a vital feature of nucleic acid delivery vectors for in vivo application. We used the CCK-8 method to evaluate the cytotoxicity of CPD and CPD/RNA nanovector at different concentrations (RNA concentration fixed at 100 ng) in the study. As shown in Fig. 4B, the cell viability of CPD was greater than 80% at different working concentrations, and its cell viability at the highest concentration was significantly higher than that of the commercial transfection reagent PEI. After co-incubation with cells at various concentrations for 24 h and 48 h, the cell viability of CPD/dsRNA nanovector was greater than 80% (Fig. 4D), indicating that CPD has low cytotoxicity and good cytocompatibility. The reason is that the reaction substrates are biosafe substances, and the toxic regent was removed by dialysis.

Uptake of CPD/dsRNA nanopolymers by Sf9 cells

Studies have shown that CPD could deliver short oligonucleotides into mammalian cells, such as Hela and 293T cells(Hei et al. 2023; Zhou et al. 2019). Still, it has not been reported whether CPD could successfully be delivered in insect cells. Further, to investigate whether CPD can successfully deliver dsRNA into cells, CPD was incubated with Cypher-5E-labeled dsGFP at a mass ratio of 6:1 for 2 h, 4 h, and 6 h in the Sf9 cells, respectively, with naked dsRNA as the control. At 2 h, it could be observed that CPD/dsRNA had begun to enter the cell, but the fluorescence of Cy5 was weak, indicating that the proportion of CPD/dsRNA nanocomplexes entering the cell was low. It could be observed that the fluorescence of Cy5 was evenly dispersed in the cell at 4 h, indicating that most of the complexes had crossed the cell membrane barrier into the cell. At 6 h, the red fluorescence of Cy5 had been observed to be basically evenly dispersed into the cell (Fig. 5A). There was no attenuation of fluorescence, indicating that the CPD/dsRNA nanocomplexes had basically entered the cell at this time (Fig. 5B). Only weak fluorescence was observed after 6 h in the control, indicating that the bare dsRNA had difficulty entering Sf9 cells. In contrast, the CPD loading dsRNA could enter Sf9 cells successfully and be maintained in cells.

Endosomal escape analysis of CPD/dsRNA nanoparticles in Sf9 cells

After CPD/dsRNA enters the cell, it mainly enters the cell through the thiol-mediated pathway. At the same time, a few would undergo clathrin-mediated endosomal capture and be delivered to the endosome(Guo et al. 2021). Successful endosomal escape of nanocarriers was one of the key steps in the cytoplasmic delivery of drugs. To explore whether endosomal escape occurs for CPD/dsRNA that enter cells in an endosome-mediated manner, we labeled the dsRNA with Cy5 in red, the endosome with Lyso-Tracker™ in green, and the nucleus with the Hoechst33258 in blue. The results of the CPD/dsRNA complexes were incubated with Sf9 cells for 2 h and 6 h, respectively. The fluorescence staining results showed that the red fluorescence and green fluorescence overlapped with yellow fluorescence at 2 h. It was demonstrated that the nanocarrier entering the cell was captured by lysosomes, and some of the lysosomes escaped. The Pearson coefficient was 0.45. With the incubation time extended to 6 h, the red and green fluorescence was separated (Fig. 6A). The Pearson coefficient of 0.27 (Pearson's R value) was calculated to be lower than 0.5 (the minimum value indicating the relationship between the two images) and significantly lower than the Pearson coefficient value (0.45) at 2 h (Fig. 6B), suggesting that some of the nanocarriers had successfully escaped lysosomes and entered the cytoplasm. It may be since disulfide polymers could fuse with the endosomal membrane under acidic conditions, and after the anionic lipid bilayer on the cytoplasmic side of the lysosomal is rearranged into multiple layers, the membrane is unstable, and membrane fusion and pore formation will occur. The nano-system escapes from the endosomes and completes the release of dsRNA(Guo et al. 2021).

Plasmid and mRNA analysis of GFP in Sf9 cells

Further, to verify whether the synthesized CPD could deliver DNA and mRNA nucleic acids into cells and complete the correct translation of proteins. First, we incubated the plasmid coding GFP protein with CPD at a mass ratio of 1:6 and then transfected it into Sf9 cells. The commercially common PEI of 25 kDa and the pDNA were used as the positive control and the negative control, respectively. Only the CPD was transfected to eliminate the interference of the fluorescence background signal. As shown in Fig. 7A, the cells transfected with CPD/pDNA showed a significantly brighter green fluorescence compared with the commercial gene vector PEI, which only had a slight presence of GFP. In contrast, the fluorescence of the cells in the negative control group was feeble. In the meantime, Sf9 cells were transfected with GFP mRNA and CPD at a mass ratio of 1:6 as well, and the bare mRNA group was used as the control. The experimental group showed significant fluorescence after CPD transfection with mRNA, whereas the control group showed almost no fluorescent signal (Fig. 7B). These results indicate that CPD not only has excellent plasmid and RNA delivery ability in insect cells but also is higher than that of commercial PEI, indicating that the CPD was an effective gene vector for in vitro application.

CPD/dsRNA Improves RNAi in fall armyworm

Compared with traditional chemical pesticides for pest control, RNAi-based insecticide is highly specific to the target pest, which is considered environmentally friendly in agriculture. The above results suggest that the CPD might be suitable for in vivo DNA or RNA delivery. We ask whether CPD could deliver the dsRNA to achieve RNAi effectively for pest control. The CHSB gene encodes chitin synthase B in insects, which is closely related to chitin synthesis in insects, and the protein encoded by the Met gene is a receptor that regulates insect juvenile hormones, both of which are closely related to the growth and development of insects. After feeding diet mixed with 1000 ng/µL of CPD/dsCHSB and CPD/dsMet complexes, the morphological changes of larvae were analyzed on the sixth day, and the phenotypes of larvae fed with two CPD/dsRNA treatments were significantly different from the control group (CPD/dsGFP). The larvae fed CPD/dsCHSB developed only to third or fourth instar larvae on the sixth day, while control larvae fed CPD/dsGFP mostly developed to fourth instars (Fig. 8A). Larval size and weight declined significantly after feeding CPD/dsRNA compared to the control group. The average body length and body weight of the control group were 13.62 mm and 42.19 mg on the sixth day, and the average body length and body weight of the CPD/dsCHSB treatment group were 8.75 mm and 20.1 mg on the sixth day, which were only 64.2% and 47.6% of the control group. The average body length of the CPD/dsMet treatment group was 11.01 mm, and the average body weight was 27.61 mg, which was only 80.8% and 65.4% of that of the control group, respectively (Fig. 8).

RNA expression levels and survival of CPD/ds CHSB and CPD/dsMet

The total RNA of larvae feeding with CPD/dsCHSB and CPD/dsMet at 48 h and 72 h was extracted, respectively, and reverse transcribed into cDNA. The relative gene expression levels of the two genes at 48 h and 72 h were detected by RT-qPCR. The relative expression levels of CHSB and Met of larvae significantly declined at 48 h and 72 h, compared to the control group. The mRNA expression of the CHSB gene decreased to 53.76% and 48.14% at 48 h and 72 h, respectively (Fig. 9A). The mRNA expression of the Met gene decreased to 59.20% and 37.60% at 48 h and 72 h, respectively (Fig. 9B). The results of RT-qPCR indicated that the dsRNA delivered by CPD could successfully enter the cytoplasm of FAW, resulting in the downregulation of related genes, which might cause the phenomenon of delayed development and differences in body length and body weight in the larvae.

The survival rate of la at day 10 in the CPD/dsCHSB and CPD/dsMet experimental groups was statistically reduced. The results showed that compared with the survival rate of the control group (84%), the survival rate of the two target genes delivered by CPD into the cells could lead to a significant decrease in the survival rate of larvae. The survival rate of the CPD/dsCHSB treatment group was 70%, and the survival rate of the CPD/dsMet treatment group was 76% (Fig. 9D). The results showed that compared with the control group (9.33 days), the pupal stage of the CPD/dsCHSB experimental group was 10.6 days, which was significantly prolonged. In comparison, the pupal stage of the CPD/dsMet experimental group was 8.40 days, which was not substantially different from that of the control group (Fig. 9C). This may be due to the fact that the CHSB gene is related to chitinase synthesis, chitin is closely associated with the molting of adults, and the silencing of the CHSB gene in the early stage affects the pupation time of subsequent pupae. Although the down-regulation of CHSB and Met genes did not result in immediate nymph death, nor did there be a significant reduction in mortality in the CPD/dsMet experimental group, the down-regulation of related genes might cause the phenomenon of delayed development and differences in body length and weight in the larvae, and CHSB also affected long-term health traits (prolonged pupal stage).

In summary, we synthesized a simple and inexpensive cell-penetrating disulfide polymer (CPD) in the study. Two key genes, CHSB and Met, for the growth and development of fall armyworm were expressed by BL21(DE3) RNase III- system. The study preliminarily completed the efficient and low-cost preparation of CPD and significantly reduced the production cost of dsRNA. Using CPD combined with dsRNA, the synthetic nano-delivery system could effectively protect dsRNA from degradation, and the dsRNA delivered by CPD could not only complete cell uptake but also successfully undergo lysosomal escape. In addition, CPD has good cell transfection capacity in insect cells, while the vector also ensures low cytotoxicity. After CPD delivery, the relative gene expression level of mRNA in larvae was decreased in the FAW, and the growth was inhibited. The use of cell-penetrating disulfide polymers to deliver dsRNA, combining RNA interference technology with novel nano-delivery, provides a new approach and method for insecticide resistance management and a new direction for insecticide resistance management and sustainable agricultural production.

The authors declare no competing financial interest.

Author contribution

XW, WL,and YR conceived and designed research. XW, QX, DZ and MH conducted feld experiments and analysed data. XW wrote the initial draft. XY and YR led the project administration. All authors contributed to the conception, design, and research of this study. All authors read and approved the manuscript.

Acknowledgments

Prof. Zhiping Xu is acknowledged for providing insect experimental suggestions and engaging in insightful discussions.

Agrawal N, Dasaradhi P V N, Mohmmed A, Malhotra P, Bhatnagar Raj K, and Mukherjee Sunil K (2003) RNA Interference: Biology, Mechanism, and Applications. Microbiol. Mol. Biol. Rev 67: 657-685. https://doi.org/10.1128/mmbr.67.4.657-685.2003
Bailey-Serres J, Parker J E, Ainsworth E A, Oldroyd G E D, and Schroeder J I (2019) Genetic strategies for improving crop yields. Nature 575: 109-118. https://doi.org/10.1038/s41586-019-1679-0
Bang E-K, Gasparini G, Molinard G, Roux A, Sakai N, and Matile S (2013) Substrate-Initiated Synthesis of Cell-Penetrating Poly(disulfide)s. J. Am. Chem. Soc 135: 2088-2091. https://doi.org/10.1021/ja311961k
Castellanos N L, Smagghe G, Sharma R, Oliveira E E, and Christiaens O (2019) Liposome encapsulation and EDTA formulation of dsRNA targeting essential genes increase oral RNAi-caused mortality in the Neotropical stink bug Euschistus heros. Pest Manag Sci 75: 537-548. https://doi.org/https://doi.org/10.1002/ps.5167
Christiaens O, Whyard S, Vélez A M, and Smagghe G (2020) Double-Stranded RNA Technology to Control Insect Pests: Current Status and Challenges. Front Plant Sci 11:451. https://doi.org/10.3389/fpls.2020.00451.
Cooper A M W, Silver K, Zhang J, Park Y, and Zhu K Y (2019) Molecular mechanisms influencing efficiency of RNA interference in insects. Pest Manage. Sci. 75: 18-28. https://doi.org/https://doi.org/10.1002/ps.5126
Dhandapani R K, Gurusamy D, and Palli S R (2022) Protamine–Lipid–dsRNA Nanoparticles Improve RNAi Efficiency in the Fall Armyworm, Spodoptera frugiperda. J. Agric. Food Chem 70: 6634-6643. https://doi.org/10.1021/acs.jafc.2c00901
Du S, Liew S S, Li L, and Yao S Q (2018) Bypassing Endocytosis: Direct Cytosolic Delivery of Proteins. J. Am. Chem. Soc 140: 15986-15996. https://doi.org/10.1021/jacs.8b06584
Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, and Mello C C (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811. https://doi.org/10.1038/35888
Garbutt J S, Bellés X, Richards E H, and Reynolds S E (2013) Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica. J. Insect Physiol 59: 171-178. https://doi.org/https://doi.org/10.1016/j.jinsphys.2012.05.013
Guo J, Wan T, Li B, Pan Q, Xin H, Qiu Y, and Ping Y (2021) Rational Design of Poly(disulfide)s as a Universal Platform for Delivery of CRISPR-Cas9 Machineries toward Therapeutic Genome Editing. ACS Central Science 7: 990-1000. https://doi.org/10.1021/acscentsci.0c01648
Gurusamy D, Mogilicherla K, Shukla J N, and Palli S R (2020) Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda. Arch. Insect Biochem. Physiol 104: e21678. https://doi.org/https://doi.org/10.1002/arch.21678
Hei M-W, Zhan Y-R, Chen P, Zhao R-M, Tian X-L, Yu X-Q, and Zhang J (2023) Lipoic Acid-Based Poly(disulfide)s as Versatile Biomolecule Delivery Vectors and the Application in Tumor Immunotherapy. Mol. Pharm 20 (6):3210-3222. https://doi.org/10.1021/acs.molpharmaceut.3c00231
Huvenne H, and Smagghe G (2009) Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review. J. Insect Physiol 56: 227-235. https://doi.org/10.1016/j.jinsphys.2009.10.004
Jain R G, Fletcher S J, Manzie N, Robinson K E, Li P, Lu E, Brosnan C A, Xu Z P, and Mitter N (2022) Foliar application of clay-delivered RNA interference for whitefly control. Nature Plants 8: 535-548. https://doi.org/10.1038/s41477-022-01152-8
Jin R, Wang Y, He B, Zhang Y, Cai T, Wan H, Jin B R, and Li J (2021) Activator protein-1 mediated overexpression in the clothianidin resistance of (Stål). Pest Manage. Sci 77: 4476-4482. https://doi.org/https://doi.org/10.1002/ps.6482
Li J, Qian J, Xu Y, Yan S, Shen J, and Yin M (2019) A Facile-Synthesized Star Polycation Constructed as a Highly Efficient Gene Vector in Pest Management. ACS Sustainable Chemistry & Engineering 7: 6316-6322. https://doi.org/10.1021/acssuschemeng.9b00004
Li M, Ma Z, Peng M, Li L, Yin M, Yan S, and Shen J (2022) A gene and drug co-delivery application helps to solve the short life disadvantage of RNA drug. Nano Today 43: 101452. https://doi.org/https://doi.org/10.1016/j.nantod.2022.101452
Lichtenberg S S, Tsyusko O V, Palli S R, and Unrine J M (2019) Uptake and Bioactivity of Chitosan/Double-Stranded RNA Polyplex Nanoparticles in Caenorhabditis elegans. Environ. Sci. Technol 53: 3832-3840. https://doi.org/10.1021/acs.est.8b06560
Lin Y-H, Huang J-H, Liu Y, Belles X, and Lee H-J (2017) Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response. Pest Manage. Sci 73: 960-966. https://doi.org/https://doi.org/10.1002/ps.4407
Livak K J, and Schmittgen T D (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25: 402-408. https://doi.org/https://doi.org/10.1006/meth.2001.1262
Makale F, Mugambi I, Kansiime M K, Yuka I, Abang M, Lechina B S, Rampeba M, and Rwomushana I (2022) Fall armyworm in Botswana: impacts, farmer management practices and implications for sustainable pest management. Pest Manage. Sci 78: 1060-1070. https://doi.org/https://doi.org/10.1002/ps.6717
Miraldo L L, Bernardi O, Horikoshi R J, e Amaral F S A, Bernardi D, and Omoto C (2016) Functional dominance of different aged larvae of Bt-resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) on transgenic maize expressing Vip3Aa20 protein. Crop Protect 88: 65-71. https://doi.org/https://doi.org/10.1016/j.cropro.2016.06.004
Murphy K A, Tabuloc C A, Cervantes K R, and Chiu J C (2016) Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference. Sci. Rep 6: 22587. https://doi.org/10.1038/srep22587
Nehate C, Nayal A, and Koul V (2019) Redox Responsive Polymersomes for Enhanced Doxorubicin Delivery. ACS Biomater Sci Eng 5: 70-80. https://doi.org/10.1021/acsbiomaterials.8b00238
Niu B, Liao K, Zhou Y, Wen T, Quan G, Pan X, and Wu C (2021) Application of glutathione depletion in cancer therapy: Enhanced ROS-based therapy, ferroptosis, and chemotherapy. Biomaterials 277: 121110. https://doi.org/https://doi.org/10.1016/j.biomaterials.2021.121110
Paroo Z, Liu Q, and Wang X (2007) Biochemical mechanisms of the RNA-induced silencing complex. Cell Res. 17: 187-194. https://doi.org/10.1038/sj.cr.7310148
Pogue M G (2002) A world revision of the genus Spodoptera Guenée (Lepidoptera: Noctuidae).
Posiri P, Ongvarrasopone C, and Panyim S (2013) A simple one-step method for producing dsRNA from E. coli to inhibit shrimp virus replication. J. Virol. Methods 188: 64-69. https://doi.org/https://doi.org/10.1016/j.jviromet.2012.11.033
Prentice K, Smagghe G, Gheysen G, and Christiaens O (2019) Nuclease activity decreases the RNAi response in the sweetpotato weevil Cylas puncticollis. Insect Biochem. Mol. Biol 110: 80-89. https://doi.org/https://doi.org/10.1016/j.ibmb.2019.04.001
Saha T T, Shin S W, Dou W, Roy S, Zhao B, Hou Y, Wang X L, Zou Z, Girke T, and Raikhel A S (2016) Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression. Proc. Natl. Acad. Sci. U. S. A 113: E735-E743. https://doi.org/10.1073/pnas.1523838113
Scott J G, Michel K, Bartholomay L C, Siegfried B D, Hunter W B, Smagghe G, Zhu K Y, and Douglas A E (2013) Towards the elements of successful insect RNAi. J. Insect Physiol 59: 1212-1221. https://doi.org/https://doi.org/10.1016/j.jinsphys.2013.08.014
Shukla J N, Kalsi M, Sethi A, Narva K E, Fishilevich E, Singh S, Mogilicherla K, and Palli S R (2016) Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects. RNA Biol 13: 656-669. https://doi.org/10.1080/15476286.2016.1191728
Wan Y, Wang W, Lai Q, Wu M, and Feng S (2023) Advances in cell-penetrating poly(disulfide)s for intracellular delivery of therapeutics. Drug Discov. Today 28: 103668. https://doi.org/https://doi.org/10.1016/j.drudis.2023.103668
Wang Z-J, Liang C-R, Shang Z-Y, Yu Q-T, and Xue C-B (2021) Insecticide resistance and resistance mechanisms in the melon aphid, Aphis gossypii, in Shandong, China. Pestic. Biochem. Physiol 172: 104768. https://doi.org/https://doi.org/10.1016/j.pestbp.2020.104768
Yu C, Li J, Zhang Z, Zong M, Qin C, Mo Z, Sun D, Yang D, Zeng Q, Wang J, Ma K, Li J, Wan H, and He S (2023) Metal–Organic Framework-Based Insecticide and dsRNA Codelivery System for Insecticide Resistance Management. ACS Applied Materials & Interfaces 15: 48495-48505. https://doi.org/10.1021/acsami.3c09074
Zhang X, Zhang J, Park Y, and Zhu K Y (2012) Identification and characterization of two chitin synthase genes in African malaria mosquito, Anopheles gambiae. Insect Biochem. Mol. Biol 42: 674-682. https://doi.org/https://doi.org/10.1016/j.ibmb.2012.05.005
Zhongzheng M, Zhou H, Wei Y L, Yan S, and Shen J (2020) A novel plasmid–Escherichia coli system produces large batch dsRNAs for insect gene silencing. Pest Manage. Sci 76 (7):2505-2512. https://doi.org/10.1002/ps.5792
Zhou J, Sun L, Wang L, Liu Y, Li J, Li J, Li J, and Yang H (2019) Self-Assembled and Size-Controllable Oligonucleotide Nanospheres for Effective Antisense Gene Delivery through an Endocytosis-Independent Pathway. Angew. Chem. Int. Ed 58: 5236-5240. https://doi.org/https://doi.org/10.1002/anie.201813665
Zhu G H, Jiao Y Y, Chereddy S, Noh M Y, and Palli S R (2019) Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, <i>Aedes aegypti</i>. Proc. Natl. Acad. Sci. U. S. A 116: 21501-21507. https://doi.org/10.1073/pnas.1905729116
Zotti M, dos Santos E A, Cagliari D, Christiaens O, Taning C N T, and Smagghe G (2018) RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Manag Sci. 74: 1239-1250. https://doi.org/https://doi.org/10.1002/ps.4813

Scheme 1 is available in the Supplementary Files section.

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Scheme 1. Synthesis route of the M and CPD.

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Delivery of dsRNA to improve RNAi efficiency by cell-penetrating disulfide polymer for spodoptera frugiperda control

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Version 1

Abstract

Figures

Key message

Introduction

Materials and methods

Materials

Insects and cell culture

Construction of plasmid vectors for expression of dsRNA and extraction of dsRNA

Synthesis of M

Synthesis of cell-penetrating disulfide polymer (CPD)

Gel retardation assay

Cell viability assay

Plasmid construction and mRNA in vitro transcription

Internalization of CPD-conjugated CypHer-5E-labeled dsRNA

Bioassays

Interference Efficiency analyzed by qPCR

Statistical Analysis

Results

Synthesis and Verification of dsRNA in Vitro

Stability evaluation of CPD/dsRNA using nucleases

Cell cytotoxicity analysis

Uptake of CPD/dsRNA nanopolymers by Sf9 cells

Endosomal escape analysis of CPD/dsRNA nanoparticles in Sf9 cells

Plasmid and mRNA analysis of GFP in Sf9 cells

CPD/dsRNA Improves RNAi in fall armyworm

RNA expression levels and survival of CPD/ds CHSB and CPD/dsMet

Conclusion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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Version 1