Development of a soybean leaf disc assay for determining oral insecticidal activity in the lepidopteran agricultural pest Helicoverpa armigera.

Pest insects pose a heavy burden on global agricultural industries with small molecule insecticides being predominantly used for their control. Unwanted side effects and resistance development plagues most small molecule insecticides such as the neonicotinoids, which have been reported to be harmful to honeybees. Bioinsecticides like Bacillus thuringiensis (Bt) toxins can be used as environmentally-friendly alternatives. Arachnid venoms comprise another promising source of bioinsecticides, containing a multitude of selective and potent insecticidal toxins. Unfortunately, no standardised insect models are currently available to assess the suitability of insecticidal agents under laboratory conditions. Thus, we aimed to develop a laboratory model that closely mimics field conditions by employing a leaf disk assay (LDA) for oral application of insecticidal agents in a bioassay tray format. Neonate larvae of the cotton bollworm (Helicoverpa armigera) were fed with soybean (Glycine max) leaves that were treated with different insecticidal agents. We observed dose-dependent insecticidal effects for Bt toxin and the neonicotinoid insecticide imidacloprid, with imidacloprid exhibiting a faster response. Furthermore, we identified several insecticidal arachnid venoms that were active when co-applied with sub-lethal doses of Bt toxin. We propose the H. armigera LDA as a suitable tool for assessing the insecticidal effects of insecticidal agents against lepidopterans.

Foliar application of clay-delivered RNA interference for whitefly control.

Whitefly (Bemisia tabaci) is a phloem-feeding global agricultural pest belonging to the order Hemiptera. Foliar application of double-stranded RNA (dsRNA) represents an attractive avenue for pest control; however, limited uptake and phloem availability of the dsRNA has restricted the development of RNA interference (RNAi)-based biopesticides against sap-sucking insects. Following high-throughput single and combinational target gene identification for additive effects, we report here that foliar application of dsRNA loaded onto layered double hydroxide (LDH), termed BioClay, can effectively disrupt multiple whitefly developmental stages in planta. Adjuvants were shown to enhance uptake and movement of foliar-applied dsRNA to vascular bundles and into the whitefly. Notably, delivering the dsRNA as a BioClay spray instead of as naked dsRNA improved protection against immature insect stages, demonstrating the platform's potential to extend the benefits offered by RNA insecticides towards complete life cycle control of whitefly and potentially other pests.

Current scenario of RNAi-based hemipteran control.

RNA interference (RNAi) is an homology-dependent gene silencing mechanism that is a feasible and sustainable avenue for the management of hemipteran pests. Commercial implementation of RNAi-based control strategies is impeded by limited knowledge about the mechanism of double-stranded RNA (dsRNA) uptake, the function of core RNAi genes and systemic RNAi mechanisms in hemipteran insects. This review briefly summarizes recent progress in RNAi-based studies aimed to reduce insect populations, viral transmission and insecticide resistance focusing on hemipteran pests. This review explores RNAi-mediated management of hemipteran insects and offers potential solutions, including in silico approaches coupled with laboratory-based toxicity assays to circumvent potential off-target effects against beneficial organisms. We further explore ways to mitigate degradation of dsRNA in the environment and the insect such as stacking and formulation of dsRNA effectors. Finally, we conclude by considering nontransformative RNAi approaches, concatomerization of RNAi sequences and pyramiding RNAi with active constituents to reduce dsRNA production and application cost, and to improve broad-spectrum hemipteran pest control. © 2020 Society of Chemical Industry.

RNAi-Based Functional Genomics in Hemiptera.

RNA interference (RNAi) is a powerful approach for sequence-specific gene silencing, displaying tremendous potential for functional genomics studies in hemipteran insects. Exploiting RNAi allows the biological roles of critical genes to be defined and aids the development of RNAi-based biopesticides. In this review, we provide context to the rapidly expanding field of RNAi-based functional genomics studies in hemipteran insects. We highlight the most widely used RNAi delivery strategies, including microinjection, oral ingestion and topical application. Additionally, we discuss the key variables affecting RNAi efficacy in hemipteran insects, including insect life-stage, gene selection, the presence of nucleases, and the role of core RNAi machinery. In conclusion, we summarise the application of RNAi in functional genomics studies in Hemiptera, focusing on genes involved in reproduction, behaviour, metabolism, immunity and chemical resistance across 33 species belonging to 14 families.

Exogenous Application of RNAi-Inducing Double-Stranded RNA Inhibits Aphid-Mediated Transmission of a Plant Virus.

Plant viruses are difficult to control, and they decrease both the quality and yield of crops, thus threatening global food security. A new approach that uses topical application of double-stranded RNA (dsRNA) to induce antiviral RNA-interference has been shown to be effective at preventing virus infection in a range of plants following mechanical inoculation. In this study, topical application of dsRNA was effective against mechanical inoculation and aphid-mediated inoculation with the potyvirus bean common mosaic virus (BCMV). Topical application of dsRNAs targeting either the coding region of the potyviral nuclear inclusion b (NIb) protein (BCMVNIb-dsRNA) or the coat protein (CP) coding region (BCMVCP-dsRNA) protected Nicotiana benthamiana and cowpea (Vigna unguiculata) plants against mechanical inoculation with BCMV. BCMVCP-dsRNA was selected for subsequent aphid transmission experiments. BCMVCP-dsRNA was loaded onto layered double hydroxide nanoparticles to form BCMVCP-BioClay which is a more stable formulation for delivering dsRNA than naked dsRNA. BCMVCP-BioClay was shown to be successful in protecting plants against BCMV transmission by the aphid Myzus persicae. Spraying detached N. benthamiana leaves with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected the leaves from infection by BCMV. Importantly, spraying of intact N. benthamiana and cowpea plants with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected plants of both species from BCMV infection. This study demonstrates that topical application of dsRNA using BioClay protects plants from aphid-mediated virus transmission, which is an important first step toward developing practical application of this approach in crop protection.

Induction of virus resistance by exogenous application of double-stranded RNA.

Exogenous application of double-stranded RNA (dsRNA) for virus resistance in plants represents a very attractive alternative to virus resistant transgenic crops or pesticides targeting virus vectors. However, the instability of dsRNA sprayed onto plants is a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only 5 days. Innovative approaches, such as the use of nanoparticles as carriers of dsRNA for improved stability and sustained release, are emerging as key disruptive technologies. Knowledge is still limited about the mechanism of entry, transport and processing of exogenously applied dsRNA in plants. Cost of dsRNA and regulatory framework will be key influencers towards practical adoption of this technology.

Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses.

Topical application of pathogen-specific double-stranded RNA (dsRNA) for virus resistance in plants represents an attractive alternative to transgenic RNA interference (RNAi). However, the instability of naked dsRNA sprayed on plants has been a major challenge towards its practical application. We demonstrate that dsRNA can be loaded on designer, non-toxic, degradable, layered double hydroxide (LDH) clay nanosheets. Once loaded on LDH, the dsRNA does not wash off, shows sustained release and can be detected on sprayed leaves even 30 days after application. We provide evidence for the degradation of LDH, dsRNA uptake in plant cells and silencing of homologous RNA on topical application. Significantly, a single spray of dsRNA loaded on LDH (BioClay) afforded virus protection for at least 20 days when challenged on sprayed and newly emerged unsprayed leaves. This innovation translates nanotechnology developed for delivery of RNAi for human therapeutics to use in crop protection as an environmentally sustainable and easy to adopt topical spray.

Diet-delivered RNAi in Helicoverpa armigera--Progresses and challenges.

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.

Herpesvirus mutagenesis facilitated by infectious bacterial artificial chromosomes (iBACs).

A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction.

The essential and non-essential genes of Bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Random-insertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.