Toxicity of a novel dsRNA-based insecticide to the Colorado potato beetle in laboratory and field trials.

BACKGROUND: The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) is one of the most notorious pests of the potato, Solanum tuberosum. Potato beetles are capable of developing resistance to various insecticides in relatively few generations. Novel and effective means of controlling Colorado potato beetle populations are constantly required to protect potato crops and prevent loss of yield. The knockdown of gene function through ribonucleic acid interference has been demonstrated in Colorado potato beetles, suggesting the use of this technology as a means of beetle management. A novel double-stranded RNA-based insecticide with the active ingredient, ledprona, has been tested in variable dose laboratory bioassays, followed by field studies. RESULTS: Exposure to ledprona resulted in both increased beetle mortality and decreased foliage consumption in all four instars and adult beetles. Effects decreased from earlier to later life stages. No ovicidal activity was detected. Onset of mortality was slower compared with existing chemical insecticides. Nevertheless, field applications of formulated ledprona to potato plots resulted in their protection comparable with that provided by spinosad and chlorantraniliprole. CONCLUSION: Based on the results of this study, formulated ledprona has attributes to become a useful tool in controlling Colorado potato beetle populations that is likely to be a good fit in integrated pest management protocols. © 2022 Society of Chemical Industry.

First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle (Leptinotarsa decemlineata).

Colorado potato beetle (CPB, Leptinotarsa decemlineata) is a major pest of potato and other solanaceous vegetables in the Northern Hemisphere. The insect feeds on leaves and can completely defoliate crops. Because of the repeated use of single insecticide classes without rotating active ingredients, many chemicals are no longer effective in controlling CPB. Ledprona is a sprayable double-stranded RNA biopesticide with a new mode of action that triggers the RNA interference pathway. Laboratory assays with second instar larvae fed Ledprona showed a dose-response where 25×10-6g/L of dsPSMB5 caused 90% mortality after 6days of initial exposure. We also showed that exposure to Ledprona for 6h caused larval mortality and decreased target messenger RNA (mRNA) expression. Decrease in PSMB5 protein levels was observed after 48h of larval exposure to Ledprona. Both PSMB5 mRNA and protein levels did not recover over time. Ledprona efficacy was demonstrated in a whole plant greenhouse trial and performed similarly to spinosad. Ledprona, currently pending registration at EPA, represents a new biopesticide class integrated pest management and insecticide resistance management programs directed against CPB.

Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops.

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

Safety Considerations for Humans and Other Vertebrates Regarding Agricultural Uses of Externally Applied RNA Molecules.

The potential of double-stranded RNAs (dsRNAs) for use as topical biopesticides in agriculture was recently discussed during an OECD (Organisation for Economic Co-operation and Development) Conference on RNA interference (RNAi)-based pesticides. Several topics were presented and these covered different aspects of RNAi technology, its application, and its potential effects on target and non-target organisms (including both mammals and non-mammals). This review presents information relating to RNAi mechanisms in vertebrates, the history of safe RNA consumption, the biological barriers that contribute to the safety of its consumption, and effects related to humans and other vertebrates as discussed during the conference. We also review literature related to vertebrates exposed to RNA molecules and further consider human health safety assessments of RNAi-based biopesticides. This includes possible routes of exposure other than the ingestion of potential residual material in food and water (such as dermal and inhalation exposures during application in the field), the implications of different types of formulations and RNA structures, and the possibility of non-specific effects such as the activation of the innate immune system or saturation of the RNAi machinery.

RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis.

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.

Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.

Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.

Development of RNAi method for screening candidate genes to control emerald ash borer, Agrilus planipennis.

The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed to siRNAs by EAB larvae within 72 h after ingestion. Also, feeding neonate larvae with IAP (inhibitor of apoptosis) or COP (COPI coatomer, ß subunit) dsRNA silenced their target genes and caused mortality. Both an increase in the concentration of dsRNA fed and sequential feeding of two different dsRNAs increased mortality. Here we provide evidence for successful RNAi in EAB, and demonstrate the development of a rapid and effective bioassay for oral delivery of dsRNA to screen additional genes.

RNA interference in the Asian Longhorned Beetle:Identification of Key RNAi Genes and Reference Genes for RT-qPCR.

Asian Longhorned Beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi) technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes including those coding for Dicer, Argonaute, and double-stranded RNA-binding proteins (dsRBP) as well as for proteins involved in dsRNA transport and the systemic RNAi. We also compared expression of six potential reference genes that could be used to normalize gene expression and selected gapdh and rpl32 as the most reliable genes among different tissues and stages of ALB. Injection of double-stranded RNA (dsRNA) targeting gene coding for inhibitor of apoptosis (IAP) into larvae and adults resulted in a significant knockdown of this gene and caused the death of 90% of the larvae and 100% of adults. No mortality of both larvae and adults injected with dsRNA targeting gene coding for green fluorescence protein (GFP, as a negative control) was observed. These data suggest that functional RNAi machinery exists in ALB and a potential RNAi-based method could be developed for controlling this insect.

Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera.

BACKGROUND: Diabrotica virgifera virgifera, western corn rootworm, is one of the most devastating species in North America. D. v. virgifera neonates crawl through the soil to locate the roots on which they feed. Carbon dioxide (CO2) is one of the important volatile cues that attract D. v. virgifera larvae to roots. RESULTS: In this study, we identified three putative D. v. virgifera gustatory receptor genes (Dvv_Gr1, Dvv_Gr2, and Dvv_Gr3). Phylogenetic analyses confirmed their orthologous relationships with known insect CO2 receptor genes from Drosophila, mosquitoes, and Tribolium. The phylogenetic reconstruction of insect CO2 receptor proteins and the gene expression profiles were analyzed. Quantitative analysis of gene expression indicated that the patterns of expression of these three candidate genes vary among larval tissues (i.e., head, integument, fat body, and midgut) and different development stages (i.e., egg, three larval stages, adult male and female). CONCLUSION: The Dvv_Gr2 gene exhibited highest expression in heads and neonates, suggesting its importance in allowing neonate larvae to orient to its host plant. Similar expression patterns across tissues and developmental stages for Dvv_Gr1 and Dvv_Gr3 suggest a potentially different role. Findings from this study will allow further exploration of the functional role of specific CO2 receptor proteins in D. v. virgifera.