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The shortage of commercially available and reliable laboratory spraying equipment for testing different preparations can be a major obstacle to achieve field-comparable results in the laboratory conditions. RNA interference is natural biological process which, when used for plant protection, can be designed method combining sustainability and minimal environmental side effects. Spraying of dsRNA is a field-relevant method that should ensure consistency and repeatability if conducted in laboratory. We built a portable spray device for laboratory use and tested its suitability for dsRNA application. For that, we carried out bioassay on three plant species with different leaf surface textures. DsRNA were detected in all samples 3 days post-treatment indicating its suitability for dsRNA delivery. We built a portable spray device for laboratory use and tested its suitability for dsRNA application. For that, we carried out:•Bioassay on three plant species with different leaf surface textures. DsRNA were detected in all samples 3 days post-treatment indicating its suitability for dsRNA delivery.

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Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

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Globodera pallida, an obligate sedentary endoparasite, is a major economic pest that causes substantial potato yield losses. This research aimed to study the effects of gene silencing of three FMRFamide-like peptides (FLPs) genes to reduce G. pallida infestation on potato plants by using kaolinite nanoclay as a carrier to deliver dsRNAs via drenching. A dsRNA dosage of 2.0 mg/ml silenced flp-32c by 89.5%, flp-32p by 94.6%, and flp-2 by 94.3%. J2s incubated for 5 and 10 h showed no phenotypic changes. However, J2s of G. pallida efficiently uptake dsRNA of all targeted genes after 15 h of incubation. On the other hand, J2s that had been kept for 24 h had a rigid and straight appearance. Under fluorescence microscopy, all dsRNA-treated nematodes showed fluorescein isothiocyanate (FITC) signals in the mouth, nervous system, and digestive system. The untreated population of J2s did not show any FITC signals and was mobile as usual. The drenching of potato cultivar Kufri Jyoti with the dsRNA-kaolinite formulations induced deformation and premature death of J2s, compared with untreated J2s that entered J3 or J4 stages. This study validates that the nanocarrier-delivered RNAi system could be employed effectively to manage G. pallida infestations.

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BACKGROUND: Chemical insecticides are an important tool to control damaging pest infestations. However, lack of species specificity, the rise of resistance and the demand for biological alternatives with improved ecotoxicity profiles means that chemicals with new modes of action are required. RNA interference (RNAi)-based strategies using double-stranded RNA (dsRNA) as a species-specific bio-insecticide offer an exquisite solution that addresses these issues. Many species, such as the fruit pest Drosophila suzukii, do not exhibit RNAi when dsRNA is orally administered due to degradation by gut nucleases and slow cellular uptake pathways. Thus, delivery vehicles that protect and deliver dsRNA are highly desirable. RESULTS: In this work, we demonstrate the complexation of D. suzukii-specific dsRNA for degradation of vha26 mRNA with bespoke diblock copolymers. We study the ex vivo protection of dsRNA against enzymatic degradation by gut enzymes, which demonstrates the efficiency of this system. Flow cytometry then investigates the cellular uptake of Cy3-labelled dsRNA, showing a 10-fold increase in the mean fluorescence intensity of cells treated with polyplexes. The polymer/dsRNA polyplexes induced a significant 87% decrease in the odds of survival of D. suzukii larvae following oral feeding only when formed with a diblock copolymer containing a long neutral block length (1:2 cationic block/neutral block). However, there was no toxicity when fed to the closely related Drosophila melanogaster. CONCLUSION: We provide evidence that dsRNA complexation with diblock copolymers is a promising strategy for RNAi-based species-specific pest control, but optimisation of polymer composition is essential for RNAi success. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Nanocarriers are widely used for efficient delivery of different cargo into mammalian cells; however, delivery into plant cells remains a challenging issue due to physical and mechanical barriers such as the cuticle and cell wall. Here, we discuss recent progress on biodegradable and biosafe nanomaterials that were demonstrated to be applicable to the delivery of nucleic acids into plant cells. This review covers studies the object of which is the plant cell and the cargo for the nanocarrier is either DNA or RNA. The following nanoplatforms that could be potentially used for nucleic acid foliar delivery via spraying are discussed: mesoporous silica nanoparticles, layered double hydroxides (nanoclay), carbon-based materials (carbon dots and single-walled nanotubes), chitosan and, finally, cell-penetrating peptides (CPPs). Hybrid nanomaterials, for example, chitosan- or CPP-functionalized carbon nanotubes, are taken into account. The selected nanocarriers are analyzed according to the following aspects: biosafety, adjustability for the particular cargo and task (e.g., organelle targeting), penetration efficiency and ability to protect nucleic acid from environmental and cellular factors (pH, UV, nucleases, etc.) and to mediate the gradual and timely release of cargo. In addition, we discuss the method of application, experimental system and approaches that are used to assess the efficiency of the tested formulation in the overviewed studies. This review presents recent progress in developing the most promising nanoparticle-based materials that are applicable to both laboratory experiments and field applications.

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RNA interference (RNAi) is recognized as a new and environmentally friendly pest control strategy due to its high specificity. However, the RNAi efficiency is relatively low in many sucking insect pests, such as Apolygus lucorum. Therefore, there is an urgent need to develop new and effective ways of dsRNA delivery. Bacterially expressed or T7 synthesized dsRNA targeting a G Protein-Coupled Receptor Kinase 2 gene was mixed with chitosan in a 1:2 ratio by mass. The size of the chitosan/dsRNA nanoparticles was 69 ± 12 nm, and the TEM and AFM images showed typical spherical or ellipsoidal structures. The chitosan nanoparticles protected the dsRNA from nuclease activity, and pH and temperature-dependent degradation, and the fluorescently-tagged nanoparticles were found to be stable on the surface of green bean plants (48 h) (Phaseolus vulgaris) and were absorbed by midgut epithelial cells and transported to hemolymph. Once fed to the A. lucorum nymph, chitosan/dsRNA could effectively inhibit the expression of the G protein-coupled receptor kinase 2 gene (70%), and led to significantly increase mortality (50%), reduced weight (26.54%) and a prolonged developmental period (8.04%). The feeding-based and chitosan-mediated dsRNA delivery method could be a new strategy for A. lucorum management, providing an effective tool for gene silencing of piercing-sucking insects.

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BACKGROUND: Recently, RNA interference (RNAi)-based biopesticide, a species-specific pest control alternative, has been deregulated and commercialized in the US and Canada. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is a major pest for rosaceous plants, which has been controlled primarily by synthetic pesticides. To address the emerging resistance issues in A. viennensis, we initiated a project to develop RNAi-based biopesticides. RESULTS: In this study, we (i) developed a dietary RNAi system for A. viennensis using leaf disc, (ii) assessed the suitability of multiple control genes to distinguish sequence-specific silencing from non-specific effects within this RNAi system, and (iii) screened for the target gene candidates. As a result, ß-Glucuronidase (GUS), an enzyme derived from E. coli and a broadly used reporter for plants is the appropriate control for A. viennensis RNAi, while green fluorescent protein (GFP), is not suitable due to its significantly higher mortality than the other controls. For target gene screening, suppression was confirmed for all the candidates, including two housekeeping genes (Vacuolar-type H + -ATPase subunit A (V-ATPase A) and Glyceraldehyde 3-phosphate dehydrogenase, (GAPDH)), and three genes associated with development (ATP-dependent RNA Helicase DDX3Y (Belle), CREB-binding protein (CBP), and Farnesoic acid O-methyltransferase (FaMet)). Knocking down of V-ATPase A resulted in the highest mortality (~ 90%) and reduced fecundity (over 90%) than other candidates. As for the genes associated with development, suppression of Belle and CBP, led to approximately 65% mortality, as well as 86% and 40% reduction in fecundity, respectively. Silencing of FaMet, however, had negligible biological impacts on A. viennensis. CONCLUSION: The combined efforts not only establish an effective dsRNA delivery method, but also provide potential target genes for RNAi-based biopesticides against A. viennensis, a devastating invasive pest for fruit trees and woody ornamental plants throughout Asia and Europe. © 2023 Society of Chemical Industry.

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Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

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BACKGROUND: RNA interference (RNAi) technology has been considered as a promising pest control strategy due to its species selectivity. One of the popular RNAs is exogenous double strand RNA (dsRNA). However, dsRNA is easily degraded by nucleases and is difficult to pass through the insect body walls, and these factors restrict the application of RNAi technology in pest management. Here, the brown planthopper (BPH, Nilaparvata lugens), a major hemipteran pest of rice in Asia countries was used as a model insect, and a dsRNA topical delivery system was constructed. RESULTS: The carrier part of the delivery system was composed of rosin-modified polyethylene glycol and chitosan, termed ROPE@C. When the N/P ratio was greater than 1:1.25, the dsRNA/ROPE@C complex encouraged full binding of the dsRNA. The gel electrophoresis results showed that ROPE@C improved the stability of dsRNA in the presence of nucleases in gut and lumen contents for at least 6 h and in the temperature range from 4 °C to 37 °C. The dsNlCHSA/ROPE@C/alkyl polyglycoside (APG) nano-formulation directly penetrated the body walls reaching hemocoel within 6 h, and consequently, the relative expression of chitin synthetase A (CHSA) in BPH was reduced by 54.3% and the mortality rate was 65.8%. CONCLUSION: We developed an appropriate delivery method for dsRNA application in BPH, which is helpful for a large-scale application of RNAi pesticides. © 2022 Society of Chemical Industry.

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Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

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Specific gene silencing by RNA interference (RNAi) involving exogenous double stranded RNA (dsRNA) delivery has potential in Helicoverpa armigera control, a resistant insect pest. Here, ionotropically synthesized cationic chitosan nanoparticles (CNPs, 95 nm size, +36 mV charge) showed efficient dsRNA loading (95 %) and effective protection from insect gut nucleases and pH degradation. The CNPs were tagged with fluorescence and found to be stable on leaf surface (24 h) and were internalized by columnar insect gut cells. A single dose of CNPs:dsRNA complex (containing 0.1 µg dsRNA) ingested by H. armigera larvae via artificial/leaf feed effectively silenced lipase and chitinase target genes (2-2.7 fold downregulation) and suppressed their respective enzyme activities (2-5.3 fold). RNAi caused reduced pupation (5-fold) and impaired moth emergence. RNAi effects correlated significantly with 100% insect mortality (PCA 0.97-0.99). Furthermore, specific dsRNA did not affect non-target insects Spodoptera litura and Drosophila melanogaster. Developed CNPs:dsRNA complexes towards RNAi targets can serve as a safe, targeted insecticide for sustainable crop protection.

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In this work, we obtained carbon dots from glucose or saccharose as the nucleation source and passivated them with branched polyethylenimines for developing dsRNA nanocomposites. The CDs were fully characterized using hydrodynamic analyses, transmission electron microscopy, X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The ζ potential determined that the CDs had positive charges, good electrophoretic mobility and conductivity, and were suitable for obtaining dsRNA nanocomposites. DsRNA naked or coated with the CDs were delivered to leaves of cucumber plants by spraying. Quantitation of the dsRNA that entered the leaves showed that when coated with the CDs, 50-fold more dsRNA was detected than when naked dsRNA. Moreover, specific siRNAs derived from the sprayed dsRNAs were 13 times more abundant when the dsRNA was coated with the CDs. Systemic dsRNAs were determined in distal leaves and showed a dramatic increase in concentration when delivered as a nanocomposite. Similarly, systemic siRNAs were significantly more abundant in distal leaves when spraying with the CD-dsRNA nanocomposite. Furthermore, FITC-labeled dsRNA was shown to accumulate in the apoplast and increase its entry into the plant when coated with CDs. These results indicate that CDs obtained by hydrothermal synthesis are suitable for dsRNA foliar delivery in RNAi plant applications.

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RNA interference (RNAi) is an essential approach for studying gene function and has been considered as a promising strategy for pest control. However, RNAi method has not been conducted in Woolly apple aphid (Eriosoma lanigerum Hausmann), one of the most damaging apple pests in the world. In the study, we investigated the efficacy of RNAi of V-ATPase subunit D (ATPD), an efficacious target for RNAi in other insects, in E. lanigerum by a transdermal double-stranded RNA (dsRNA) delivery system with nanocarriers. Our results showed although topical application of dsATPD in E. lanigerum for 24 h produced 40.5% gene silencing, the additional help of nanocarriers extremely improved the interference efficiency with 98.5% gene silencing. Moreover, a 55.75% mortality was observed 5 days after topical application of nanocarriers and dsATPD, relative to the control (topical application of nanocarriers and double-stranded green fluorescent protein [dsGFP]). The nanocarrier-based transdermal dsRNA delivery system will promote the development of functional analysis of vital genes and also provide a potential target for RNAi-based management of E. lanigerum.

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The application of the RNA interference (RNAi) mechanism promotes the development of novel approaches toward sustainable crop protection. Compared with traditional double-stranded (ds)RNA delivery systems, nanoparticles offer great advantages in delivering dsRNA to improve RNAi efficiency, thus promoting the development and practice of RNAi-based pest management strategies. Here, we described a transdermal dsRNA delivery system with a nanosized star polycation, and presented a method to improve RNAi efficiency to increase the control effect against aphids. Insect gene functional analysis and pest management can be achieved by this method.

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RNA interference (RNAi) is a natural mechanism of gene regulation, highly conserved in eukaryotes. Since the elucidation of the gene silencing mechanism, RNAi became an important tool used in insect reverse genetics. The demonstration of effective target-gene silencing by ingestion of double-stranded RNA (dsRNA) produced by transgenic plants indicated the RNAi potential to be used in insect pest management, particularly in agriculture. However, the efficiency of gene silencing by RNAi in insects may vary according to the target taxa, and lepidopteran species have been shown to be quite recalcitrant to RNAi. Developing transgenic plants is a time-consuming and labor-intensive process, so alternative oral delivery systems are required to develop and optimize RNAi settings, such as selecting an efficient target gene, and dsRNA design, length, and stability, among other features. We have developed delivery systems to evaluate dsRNAs to silence genes from two important lepidopteran crop pests of tomato (Solanum lycopersicum) and sugarcane (Saccharum × officinarum): Tuta absoluta (Meyrick), the South American Tomato Pinworm, and Diatraea saccharalis (Fabricius), the Sugarcane Borer, respectively. The protocol described here can be used in similar species and includes (a) direct oral delivery by droplets containing dsRNA; (b) oral delivery by tomato leaflets that absorbed dsRNA solution; (c) delivery by Escherichia coli expressing dsRNA; and (d) delivery by transgenic plants expressing dsRNA.

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Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

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Plant pathogenic fungi are the largest group of disease-causing agents on crop plants and represent a persistent and significant threat to agriculture worldwide. Conventional approaches based on the use of pesticides raise social concern for the impact on the environment and human health and alternative control methods are urgently needed. The rapid improvement and extensive implementation of RNA interference (RNAi) technology for various model and non-model organisms has provided the initial framework to adapt this post-transcriptional gene silencing technology for the management of fungal pathogens. Recent studies showed that the exogenous application of double-stranded RNA (dsRNA) molecules on plants targeting fungal growth and virulence-related genes provided disease attenuation of pathogens like Botrytis cinerea, Sclerotinia sclerotiorum and Fusarium graminearum in different hosts. Such results highlight that the exogenous RNAi holds great potential for RNAi-mediated plant pathogenic fungal disease control. Production of dsRNA can be possible by using either in-vitro or in-vivo synthesis. In this review, we describe exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Potential challenges that are faced while developing a RNAi strategy for fungal pathogens, such as off-target and epigenetic effects, with their possible solutions are also discussed.

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RNA interference (RNAi) targeting lethal genes in insects has great potential for sustainable crop protection. Compared with traditional double-stranded (ds)RNA delivery systems, nanoparticles such as chitosan, liposomes, and cationic dendrimers offer advantages in delivering dsRNA/small interfering (si)RNA to improve RNAi efficiency, thus promoting the development and practice of RNAi-based pest management strategies. Here, we illustrate the limitations of traditional dsRNA delivery systems, reveal the mechanism of nanoparticle-mediated RNAi, summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management, and finally address the prospects of nanoparticle-based RNA pesticides.

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Chickpea pod borer, Helicoverpa armigera, displays resistance to chemical insecticides and transgenics. The potential nontransformative RNAi approach of specific gene silencing by mRNA breakdown through exogenous double-stranded (dsRNA) delivery to Helicoverpa faces problems of degradation by nucleases and insect gut pH. We demonstrate that chitosan nanoparticles (CNPs) effectively mediate specific dsRNA delivery against Helicoverpa armigerajuvenile hormone methyltransferase (JHAMT) and acetylcholine esterase (ACHE) target genes. Ionotropically synthesized cationic CNPs (100 nm size, +32 mV charge) loaded dsRNA efficiently and protected it effectively from degradation by nucleases and insect gut pH. Tagging CNPs with Calcofluor fluorescence illustrated its efficient uptake in columnar insect gut cells. The potential of CNPs-mediated dsRNA delivery was elucidated with effective silencing of green fluorescent protein transformed Sf9 cells. Furthermore, CNPs-dsRNA complexes were stable for 5 d on leaf surfaces, and their ingestion with leaf effectively silenced H. armigeraJHAMT and ACHE genes to suppress related enzyme activities and caused 100% insect mortality. Further, in planta bioassay with CNPs-dsRNA spray confirmed the RNAi induced insect mortality. Moreover, CNPs-dsRNA fed nontarget insects Spodoptera litura and Drosophila melanogaster were unaffected, and no toxicity was observed for CNPs in cell line studies. Remarkably, only two low dose (0.028 g/ha) topical CNPs-ache-dsRNA sprays on chickpea displayed reduced pod damage with high yields on par with chemical control in the field, which was followed by CNPs-jhamt-dsRNA nanoformulation. These studies can pave the way for the development of topical application of CNPs-dsRNA spray as a safe, specific, innovative insecticide for sustainable crop protection.

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Climate change imposes a great threat to world food security and encourages insect pest proliferation and spreading. Because of these challenges, identifying novel biotechnology pest management and its applications is inevitable. RNA interference (RNAi) is a gene regulatory process used for the maintenance and regulation of host defences against invading viruses. Nevertheless, it is widely used for the analysis of gene function. In recent years, the potential use of RNA interference (RNAi) as a tool for manipulating crop traits, as well as an alternative for crop protection, has undergone outstanding developments. In this review, we describe some genes involved in insect dsRNA uptake and discuss the reasons for varying RNAi response in insect pests, emphasizing the presence of nucleases and double-stranded RNA binding protein. We explore recent breakthroughs in innovative dsRNA delivery for efficient and effective knockdown in insect pests. Conclusively, topical delivery of dsRNA combined with a nanoparticle complex holds great potential for RNAi-mediated pest control.

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