RNAi efficacy is enhanced by chronic dsRNA feeding in pollen beetle.
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ABSTRACT:
SUBMITTER: Willow J
PROVIDER: S-EPMC8024372 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9-16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3-4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).
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Project description:Spray-induced gene silencing (SIGS) is a potential strategy for agricultural pest management, whereby nucleotide sequence-specific double-stranded RNA (dsRNA) can be sprayed onto a crop; the desired effect being a consumption of dsRNA by the target pest, and subsequent gene silencing-induced mortality. Nucleotide sequence-specificity is the basis for dsRNA's perceived biosafety. A biosafe approach to pollen beetle (Brassicogethes aeneus) management in oilseed rape (Brassica napus) agroecosystems is needed. We examined the potential for SIGS in B. aeneus, via bud feeding, a field-relevant dsRNA exposure route. Oilseed rape buds were uniformly treated with dsRNA designed to target αCOP in B. aeneus. Our model control dsRNA (dsGFP) remained detectable on buds throughout the entire 3 d exposure period. When applied at 5 µg/µL, dsαCOP induced significant αCOP silencing 3 d after dietary exposure to buds treated with this dsαCOP concentration. We also observed a trend of increased αCOP silencing with increasing concentrations of dsαCOP at both 3 and 6 d. Furthermore, we observed a marginally significant and significant reduction in B. aeneus survival at 10 and 15 d, respectively. Our results suggest potential for developing a SIGS approach to B. aeneus management-though further experiments are needed to more fully understand this potential.
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Project description:Lepidoptera comprise some of the most devastating herbivorous pest insects worldwide. One of the most promising novel pest control strategies is exploiting the RNA interference (RNAi) mechanism to target essential genes for knockdown and incite toxic effects in the target species without harming other organisms in the ecosystem. However, many insects are refractory to oral RNAi, often due to rapid degradation of ingested dsRNA in their digestive system. This is the case for many lepidopteran insects, including the beet armyworm Spodoptera exigua, which is characterized by a very alkaline gut environment (pH > 9.0) and a strong intestinal nucleolytic activity. In this research, guanidine-containing polymers were developed to protect dsRNA against nucleolytic degradation, specifically in high pH environments. First, their ability to protect dsRNA against nucleolytic degradation in gut juice of the beet armyworm S. exigua was investigated ex vivo. Polymers with high guanidine content provided a strong protection against nucleolytic degradation at pH 11, protecting the dsRNA for up to 30 h. Next, cellular uptake of the dsRNA and the polyplexes in lepidopteran CF203 midgut cells was investigated by confocal microscopy, showing that the polymer also enhanced cellular uptake of the dsRNA. Finally, in vivo feeding RNAi bioassays demonstrated that using these guanidine-containing polymer nanoparticles led to an increased RNAi efficiency in S. exigua. Targeting the essential gene chitin synthase B, we observed that the mortality increased to 53% in the polymer-protected dsRNA treatment compared to only 16% with the naked dsRNA and found that polymer-protected dsRNA completely halted the development of the caterpillars. These results show that using guanylated polymers as a formulation strategy can prevent degradation of dsRNA in the alkaline and strongly nucleolytic gut of lepidopteran insects. Furthermore, the polymer also enhances cellular uptake in lepidopteran midgut cells. This new delivery strategy could be of great use in further fundamental research in lepidopterans, using RNAi as a research tool, and could lead to future applications for RNAi-based pest control of lepidopteran insects.
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Project description:RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults.
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Project description:RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.
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Project description:In this study, the use of dendrimer-coated carbon nanotubes (CNTs) as a delivery vehicle for dsRNA was assessed in Tribolium castaneum. Exposure to low dosages of polyamidoamine dendrimer carbon nanotubes (PAMAM-CNTs) did not affect T. castaneum larval mortality. Expression of key apoptotic factors, Dronc (Tc12580), Dredd (Tcn-like, Tc014026) and Buffy, (Tcinhib apop1), which can act as toxicity indicators, were not altered in T. castaneum larvae following injection of PAMAM-CNTs. The level of knockdown of two target genes, α-tubulin and mitochondrial RNA polymerase (mtpol), were significantly increased when larvae were injected with double-stranded RNA bound to CNTs (PAMAM-CNT-dsRNA), compared to those injected with target dsRNA alone. PAMAM-CNTs were visualised in cellular vacuoles and in the cell nucleus. Increase occurrence of a blistered wing phenotype was found in a subset of PAMAM-CNT-dsRNAαtub injected larvae, relative to the level seen in larvae injected with naked dsRNAαtub alone. These results suggest that the use of functionalised CNTs for dsRNA delivery could increase the efficacy of RNA interference in insect pest species.
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Project description:The limitations of conventional pesticides have raised the demand for innovative and sustainable solutions for plant protection. RNA Interference (RNAi) triggered by dsRNA has evolved as a promising strategy to control insects in a species-specific manner. In this context, we review the methods for mass production of dsRNA, the approaches of exogenous application of dsRNA in the field, and the fate of dsRNA after application. Additionally, we describe the opportunities and challenges of using nanoparticles as dsRNA carriers to control insects. Furthermore, we provide future directions to improve pest management efficiency by utilizing the synergistic effects of multiple target genes. Meanwhile, the establishment of a standardized framework for assessment and regulatory consensus is critical to the commercialization of RNA pesticides.
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Project description:BackgroundRNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.Methodology/principal findingsIn this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.Conclusions/significanceSimilar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.
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Project description:BACKGROUND:The field ecology of the pollen beetle Meligethes aeneus and its damaging effects on oilseed rape crops are well understood. However, the flight behaviour of M. aeneus, in particular the drivers for migratory movements across the landscape, is not well studied. We combined three established methodologies - suction traps, vertical-looking radar and high-altitude aerial netting - to demonstrate that M. aeneus flies at a range of altitudes at different points during its active season. RESULTS:By linking evidence of high-altitude mass migration with immigration of pollen beetles into oilseed rape fields, we were able to 'ground-truth' the results to characterise the seasonal movements of this pest across the landscape. CONCLUSION:We demonstrate that this novel combination of methodologies can advance our understanding of the population movements of pollen beetles and could provide an opportunity to develop predictive models to estimate the severity and timing of pest outbreaks. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Project description:RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.
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