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.

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.

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.
CONCLUSIONS: 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.

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.

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, β-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.
CONCLUSIONS: 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.

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.

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.

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.

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 armigera juvenile 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. armigera JHAMT 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.

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.