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We provide a one-stop online platform, namely, dsRNAEngineer, to help users optimize dsRNA for RNAi-based pest control.dsRNAEngineer incorporates a range of pest and non-pest species to enable large-scale transcriptome-level analysis for dsRNA design.dsRNAEngineer contains four functionalities, namely, screen-target, on-target, off-target, and multiple-target functions, to design rational dsRNAs that sufficiently target pests but are safe for non-target organisms. Over the past two decades, many double-stranded (ds)-RNAs have been synthesized to silence target genes for exploration of gene functions in pests. Some of these dsRNAs are lethal to pests, leading to a new category of pesticides. The generation of these environmentally friendly pesticides requires precise in silico design of dsRNA molecules that target pests but not non-pest organisms. Current efforts in dsRNA design focus mainly on the analysis of the target gene sequence, lacking comprehensive analysis of all transcripts of the whole transcriptome per given species, causing low efficiency and imprecise dsRNA target exploration. To address these limitations, we created the dsRNAEngineer online platform (https://dsrna-engineer.cn), which allows comprehensive and rational dsRNA design, incorporating hundreds of pest and non-pest transcriptomes. Developed functionalities include screen-target (screen conserved genes for cotargets of various pest species), on-target, off-target, and multi-target to generate optimal dsRNA for precise pest control. [Display omitted] dsRNAEngineer incorporates hundreds of pest and non-pest transcriptomes to provide comprehensive dsRNA design for pest control. With the application of several RNA-based pesticides in the field, dsRNAEngineer illustrates a framework for designing pest-specific and biosafe dsRNA, which will greatly promote the development of RNAi biotechnology as a pest control strategy. Over the past two decades, RNAi biotechnology has been intensively investigated for pest control, and several products have been registered and applied in the field. However, current methods of double-stranded (ds)RNA design lack a comprehensive analysis of all transcripts at the transcriptome level for both pest and non-pest species. To solve these problems, our timely developed tool, dsRNAEngineer (https://dsrna-engineer.cn), which has four functionalities (screen-target, on-target, off-target, and multi-target functions) was developed to help users design rational dsRNAs that sufficiently target pests but are otherwise safe for non-target organisms, based on large-scale transcriptome-level analysis of both pest and non-pest species. With the emergence of RNA pesticides, we believe that dsRNAEngineer can be considered as a gold standard in dsRNA design to promote pest control based on RNAi biotechnology.

Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method.

In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi’s potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.

The drastic loss of biodiversity has alarmed the public and raised sociopolitical demand for chemical pesticide-free plant production, which is now treated by governments worldwide as a top priority. Given this global challenge, RNAi-based technologies are rapidly evolving as a promising substitute to conventional chemical pesticides. Primarily, genetically modified (GM) crops expressing double-stranded (ds)RNA-mediating gene silencing of foreign transcripts have been developed. However, since the cultivation of GM RNAi crops is viewed negatively in numerous countries, GM-free exogenous RNA spray applications attract tremendous scientific and political interest. The sudden rise in demand for pesticide alternatives has boosted research on sprayable RNA biopesticides, generating significant technological developments and advancing the potential for field applications in the near future. Here we review the latest advances that could pave the way for a quick lab-to-field transition for RNA sprays, which, as safe, selective, broadly applicable, and cost-effective biopesticides, represent an innovation in sustainable crop production. Given these latest advances, we further discuss technological limitations, knowledge gaps in the research, safety concerns and regulatory requirements that need to be considered and addressed before RNA sprays can become a reliable and realistic agricultural approach.

The spotted wing Drosophila ( Drosophila suzukii ) is an invasive and serious economic pest to small and stone fruits and its control is difficult. RNA interference (RNAi) or double-stranded RNA (dsRNA)-mediated gene silencing is rapidly becoming a widely used functional genomics tool in insects and holds great potential for insect pest control. This study investigates whether RNAi is functional in D. suzukii and whether oral delivery of dsRNA can elicit gene silencing and insecticidal activity. Firstly, microinjection of dsRNA targeting two essential genes ( alpha COP and shrb ) into the haemolymph of adult flies was performed, confirming that the RNAi system is functional and that gene silencing results in mortality. Secondly, dsRNA targeting alpha - COP and two extra essential genes, rpl13 and vha26 , was mixed with artificial diet and fed to the larval and adult stages of D. suzukii . With naked dsRNA, no clear silencing and mortality were scored. However, combining dsRNA with a transfection reagent led to a significant increase in gene silencing and insect mortality. The best results were obtained with ds-Vha26. The results are discussed in relation to future optimization of the production, formulation, combinations and delivery of dsRNA.

Ceratitis capitata, known as the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A more sustainable and species-specific alternative strategy may be based on double-stranded RNA (dsRNA) delivery through feeding to disrupt essential functions in pest insects, which is poorly reported in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by specific nucleases in the intestinal lumen is among the major obstacles to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of the ATPase vital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness. In contrast, by simultaneously feeding dsRNA against the CcVha68-1, CcdsRNase1, and CcdsRNase2 genes, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest management.

RNA interference (RNAi)-based biopesticides are novel biologic products, developed using RNAi principles. They are engineered to target genes of agricultural diseases, insects, and weeds, interfering with their target gene expression so as to hinder their growth and alleviate their damaging effects on crops. RNAi-based biopesticides are broadly classified into resistant plant-based plant-incorporated protectants (PIPs) and non-plant-incorporated protectants. PIP RNAi-based biopesticides are novel biopesticides that combine the advantages of RNAi and resistant transgenic crops. Such RNAi-based biopesticides are developed through nuclear or plastid transformation to breed resistant plants, i.e., dsRNA-expressing transgenic plants. The dsRNA of target genes is expressed in the plant cell, with pest and disease control being achieved through plant-target organism interactions. Here, we review the action mechanism and strategies of RNAi for pest management, the development of RNAi-based transgenic plant, and the current status and advantages of deploying these products for pest control, as well as the future research directions and problems in production and commercialization. Overall, this study aims to elucidate the current development status of RNAi-based biopesticides and provide guidelines for future research.

Background/Objectives: The bird cherry-oat aphid, Rhopalosiphum padi, is a major global pest of cereal crops and exhibits wing polyphenism, producing both winged (dispersive) and wingless (reproductive) morphs. Methods: To identify potential RNAi targets that could specifically disrupt the migratory winged morph, we conducted a comparative transcriptomic analysis of adult aphids. Differentially expressed genes (DEGs) were identified, annotated for their functions, and analyzed for their involvement in metabolic pathways. Results: Significant differences were observed in 121 genes between morphs: 13 were upregulated in the winged morph, while 108 were downregulated. Most DEGs were enriched in lipid metabolism and circadian rhythm pathways, suggesting that wing polymorphism may be adaptively linked to energy resource allocation strategies. Conclusions: This study firstly reveals the adult-stage-specific regulatory roles of lipid metabolism and circadian rhythm pathways in wing polyphenism, identifying six candidate genes (BCORL1, AMP-L, Pfl, Lip3L, HLFL(X7), and HLFL(X4)) for RNAi-based biocontrol strategies targeting migratory morphs.

Selecting an appropriate target gene is critical to the success of oral RNA interference-based pest control. The selection of target genes has depended on their ability to induce lethality. However, the induction of lethality by oral RNAi is slow-acting, and crop damage can progress during this time. Here, we show that oral RNAi of the death - associated inhibitor of apoptosis protein 1 ( diap1 ), unlike two conventional target genes, induces acute feeding cessation in the solanaceous pest, Henosepilachna vigintioctopunctata within 24 h. We also found that our designed dsRNA has high specificity and does not cause death or reduce diap1 expression in two non-target insects. Our results suggest that diap1 is an appropriate target in the context of the rapid reduction of crop damage. We propose that the phenotype of acute feeding cessation should be used as a novel criterion for selecting target genes for RNAi-based pest control.

RNA interference (RNAi)-based pesticides are pest control agents that use RNAi mechanisms as the basis of their action. They are regarded as environmentally friendly and are a promising alternative to conventional chemical pesticides. The effective substance in RNAi-based pesticides is double-stranded RNA (dsRNA) designed to match the nucleotide sequence of a target essential gene of the pest of concern. When taken up by the pest, this exerts an RNAi effect and inhibits some vital biochemical/biological process in the pest. dsRNA products are also expected to be applied for the control of viral diseases in aquaculture by RNAi, especially in shrimp farming. A critical issue in the practical application of RNAi agents is that production of the dsRNA must be low-cost. Here, we review recent methods for microbial production of dsRNAs using representative microorganisms (Escherichia coli, Pseudomonas syringae, Corynebacterium glutamicum, Chlamydomonas reinhardtii, and others) as host strains. The characteristics of each dsRNA production system are discussed.