Strategies for the production of dsRNA biocontrols as alternatives to chemical pesticides

Current crop pest control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. However, many insects, plant viruses, and fungi have no current means of control or have developed resistance against traditional pesticides. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. The successful commercialisation of dsRNA based biocontrols for effective pest management strategies requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. A number of methods exist for the production and delivery of dsRNA based biocontrols and here we review alternative methods currently employed and emerging new approaches for their production. Additionally, we highlight potential challenges that will need to be addressed prior to widespread adoption of dsRNA biocontrols as novel sustainable alternatives to traditional chemical pesticides

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC

Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA. We have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications. The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length. Moreover, further analysis under non-denaturing conditions revealed the presence of heterogeneous dsRNA species. IP-RP-HPLC fractionation and AFM analysis revealed that these alternative RNA species do not arise from different lengths of individual dsRNA molecules in the product, but represent misannealed RNA species that present as larger assemblies or multimeric forms of the RNA. These results for the first time provide direct structural insight into dsRNA produced both in vivo in bacterial systems and in vitro, highlighting the structural heterogeneity of RNA produced. These results are the first example of detailed characterisation of the different forms of dsRNA from two production systems and establish atomic force microscopy as an important tool for the characterisation of long dsRNA

Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: a potential new tool for improving RNA-based plant protection

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either are not currently controlled by artificial means or have developed resistance against chemical pesticides. Long dsRNAs are capable of inducing RNAi in insects and are emerging as novel, highly selective alternatives for sustainable insect management strategies. However, there are significant challenges associated with RNAi efficacy in insects. In this study, we synthesized a range of chemically modified long dsRNAs in an approach to improve nuclease resistance and RNAi efficacy in insects. Our results showed that dsRNAs containing phosphorothioate modifications demonstrated increased resistance to southern green stink bug saliva nucleases. Phosphorothioate-modified and 2′-fluoro-modified dsRNA also demonstrated increased resistance to degradation by soil nucleases and increased RNAi efficacy in Drosophila melanogaster cell cultures. In live insects, we found chemically modified long dsRNAs successfully resulted in mortality in both stink bug and corn rootworm. These results provide further mechanistic insight into the dependence of RNAi efficacy on nucleotide modifications in the sense or antisense strand of the dsRNA in insects and demonstrate for the first time that RNAi can successfully be triggered by chemically modified long dsRNAs in insect cells or live insects

Maize genetics Committee on Outreach, Diversity, Inclusion, and Education (CODIE) 2020-2021 update.

Virtual meeting

Effects of next-generation, dual-active-ingredient, long-lasting insecticidal net deployment on insecticide resistance in malaria vectors in Tanzania : an analysis of a 3-year, cluster-randomised controlled trial

Background Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. Methods For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). Findings Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. Interpretation Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies