A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes

Mysore, Keshava; Sun, Lili; Hapairai, Limb K.; Wang, Chien-Wei; Roethele, Joseph B.; Igiede, Jessica; Scheel, Max P.; Scheel, Nicholas D.; Li, Ping; Wei, Na; Severson, David W.; Duman-Scheel, Molly

doi:10.3390/pathogens10101251

Cited by 12 publications

(21 citation statements)

References 69 publications

(127 reference statements)

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“…In vertebrates, Rbfox1 is necessary for proper neuronal migration and axonal growth ( Hamada et al, 2016 ), is induced by stress ( Amir-Zilberstein et al, 2012 ), and modulates the splicing of genes involved in axonal depolarization ( Lee et al, 2009 ; Gehman et al, 2011 ). Rbfox1 was recently shown to regulate sensory neuron specification in Drosophila ( Shukla et al, 2017 ) and brain development in the mosquito Aedes aegypti ( Mysore et al, 2021 ), suggesting its function in neuronal development is conserved. In vertebrate muscle, Rbfox1 binding sites are enriched around developmentally regulated, alternatively spliced exons in the heart ( Kalsotra et al, 2008 ), and Rbfox1 regulates alternative splicing of structural proteins as well as proteins in the calcium signaling pathway in skeletal muscle ( Pedrotti et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

et al. 2022

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Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila. Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type–specific gene and splice isoform expression, notably loss of an indirect flight muscle–specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3′-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type–specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type–specific splicing and expression dynamics of identity genes and structural proteins.

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Section: Introductionmentioning

confidence: 99%

Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

et al. 2022

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“…The adulticidal activity of 5-HTR.426 yeast was therefore assessed. This was initially pursued using a previously described lab-based sucrose feeder system 43 , and then in conjunction with a miniature bait station sachet system designed to mimic the commercial bait stations that are presently being evaluated in the phase 3 clinical trials 54 .…”

Section: Resultsmentioning

confidence: 99%

“…Based on previous functional characterizations of 5-HTRs in A. aegypti and A. gambiae 10 , 38 – 40 , it was hypothesized that silencing of 5-HTR1 in multiple disease vector mosquito species would result in significant larval and adult mortality. Recently, we have characterized and developed several species-specific interfering RNA pesticides for larval and adult mosquito control through RNAi-mediated gene silencing using modified baker’s yeast as the delivery system 19 , 41 – 43 . In this study, we developed and characterized a yeast RNAi pesticide targeting 5-HTR1 genes in larvae and adult A. aegypti , A .…”

Section: Introductionmentioning

confidence: 99%

Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes

Mysore,

Njoroge,

Stewart

et al. 2023

Sci Rep

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G protein-coupled receptors (GPCRs), which regulate numerous intracellular signaling cascades that mediate many essential physiological processes, are attractive yet underexploited insecticide targets. RNA interference (RNAi) technology could facilitate the custom design of environmentally safe pesticides that target GPCRs in select target pests yet are not toxic to non-target species. This study investigates the hypothesis that an RNAi yeast insecticide designed to silence mosquito serotonin receptor 1 (5-HTR1) genes can kill mosquitoes without harming non-target arthropods. 5-HTR.426, a Saccharomyces cerevisiae strain that expresses an shRNA targeting a site specifically conserved in mosquito 5-HTR1 genes, was generated. The yeast can be heat-inactivated and delivered to mosquito larvae as ready-to-use tablets or to adult mosquitoes using attractive targeted sugar baits (ATSBs). The results of laboratory and outdoor semi-field trials demonstrated that consumption of 5-HTR.426 yeast results in highly significant mortality rates in Aedes, Anopheles, and Culex mosquito larvae and adults. Yeast consumption resulted in significant 5-HTR1 silencing and severe neural defects in the mosquito brain but was not found to be toxic to non-target arthropods. These results indicate that RNAi insecticide technology can facilitate selective targeting of GPCRs in intended pests without impacting GPCR activity in non-targeted organisms. In future studies, scaled production of yeast expressing the 5-HTR.426 RNAi insecticide could facilitate field trials to further evaluate this promising new mosquito control intervention.

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“…In Anopheles gambiae , dsRNA in bacterial lysate mixed with glucose solution was used as a sugar source for feeding adult mosquitoes [ 4 , 6 ]. Mysore and co-authors [ 7 ] have also employed genetically engineered yeast that produced short hairpin RNAs (shRNAs) to suppress Rbfox1 genes in larvae and adult Aedes albopictus , Anopheles gambiae , and Culex quinquefasciatus via feeding, causing high larval and adult mortality.…”

Section: Introductionmentioning

confidence: 99%

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development

Naumenko,

Fritz

2024

Insects

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Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle.

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A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes

Cited by 12 publications

References 69 publications

Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

Rbfox1 is required for myofibril development and maintaining fiber type–specific isoform expression in Drosophila muscles

Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development

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