Keshava Mysore’s research while affiliated with Université Notre Dame d'Haïti and other places

Spotted wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), are invasive vinegar flies of East Asian origin that are an increasingly global threat to the small fruit industry. It is essential that new classes of eco-friendly insecticides and cost-effective strategies for SWD control are developed. Here, we describe the preparation of a strain of RNA interference (RNAi) Saccharomyces cerevisiae expressing shRNA that specifically targets the SWD RNA-binding Fox protein 1 (Rbfox1) gene. The yeast effectively silences the SWD Rbfox1 gene, resulting in significant loss of fly neural activity. Laboratory trials demonstrated that the RNAi yeast can be mixed with soda, which functions as SWD attractive targeted sugar bait (ATSB) that can be delivered in a soda bottle feeder. The ATSB, mixed with yeast that was heat-killed prior to suspension in the ATSB, resulted in 92 ± 1% mortality of SWD flies that consumed it, yet had no impact on non-target dipterans. Rbfox.687 yeast delivered in ATSB feeders may one day be a useful component of integrated SWD control programs.

Background: Drosophila suzukii, or spotted-wing drosophila (SWD), (Diptera: Drosophilidae), are invasive vinegar flies of East Asian origin that have wreaked havoc on the small fruit and berry industry. In locations where SWD are well established, weekly chemical insecticide applications are necessary, resulting in increased economic costs, unwanted environmental impacts ensuing from loss of non-targeted organisms, and the eventual emergence of populations that are resistant to these insecticides. It is therefore critical that new classes of biorational pesticides and cost-effective technologies for controlling SWD are identified. Results: Here, we used the attractive properties of Saccharomyces cerevisiae, bakers yeast, which was designed to express an RNA interference (RNAi) pesticide that specically targets the SWD Shaker (Sh) gene, to lure and kill flies that feed on the yeast, which was delivered in a feeder as a component of an attractive targeted sugar bait (ATSB). The yeast, which was heat killed prior to preparation of the ATSB, silenced the Sh gene, resulting in severe neural defects and fly mortality in laboratory trials. The RNAi yeast was successfully fed to the flies in an easily assembled soda bottle feeder that continuously rewetted the yeast with soda, which lured and killed the flies in simulated field trials. Despite this toxicity observed in SWD, consumption of the yeast had no impact on the survival of other dipteran insects. Conclusion: This promising ATSB technology, which was prepared with a new class of RNAi yeast insecticides, could one day be an effective component in integrated SWD control programs.

Large household water storage containers are among the most productive habitats for Aedes aegypti (Linnaeus, 1762), the primary mosquito vector for dengue and other arboviral pathogens. Increasing concerns for insecticide resistance and larvicide safety are limiting the successful treatment of large household water storage containers, which are among the most productive habitats for Aedes juveniles. The recent development of species-specific RNAi-based yeast larvicides could help overcome these problems, particularly if shelf stable ready-to-use formulations with significant residual activity in water can be developed. Here we examine the hypothesis that development of a shelf-stable controlled-release RNAi yeast formulation can facilitate lasting control of A. aegypti juveniles in large water storage containers. In this study, a dried inactivated yeast was incorporated into a biodegradable matrix containing a mixture of polylactic acid, a preservative, and UV protectants. The formulation was prepared using food-grade level components to prevent toxicity to humans or other organisms. Both floating and sinking versions of the tablets were prepared for treatment of various sized water containers, including household water storage tank-sized containers. The tablets passed accelerated storage tests of shelf life stability and demonstrated up to six months residual activity in water. The yeast performed well in both small and large containers, including water barrels containing 20-1000 larvae each, and in outdoor barrel trials. Future studies will include the evaluation of the yeast larvicide in larger operational field trials that will further assess the potential for incorporating this new technology into integrated mosquito control programs worldwide.

Large household water storage containers are among the most productive habitats for Aedes aegypti , the primary mosquito vector for dengue and other arboviral pathogens. Increasing concerns for insecticide resistance and larvicide safety are limiting the successful treatment of large household water storage containers, which are among the most productive habitats for Aedes juveniles. The recent development of species-specific RNAi-based yeast larvicides could help overcome these problems, particularly if shelf stable ready-to-use formulations with significant residual activity in water can be developed. Here we examine the hypothesis that development of a shelf-stable controlled-release RNAi yeast formulation can facilitate lasting control of A. aegypti juveniles in large water storage containers. In this study, a dried inactivated yeast was incorporated into a biodegradable matrix containing a mixture of polylactic acid, a preservative, and UV protectants. The formulation was prepared using food-grade level components to prevent toxicity to humans or other organisms. Both floating and sinking versions of the tablets were prepared for treatment of various sized water containers, including household water storage tank-sized containers. The tablets passed accelerated storage tests of shelf life stability and demonstrated up to six months residual activity in water. The yeast performed well in both small and large containers, including water barrels containing 20-1000 larvae each, and in outdoor barrel trials. Future studies will include the evaluation of the yeast larvicide in larger operational field trials that will further assess the potential for incorporating this new technology into integrated mosquito control programs worldwide.

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.

Simple Summary Mosquito-borne infectious diseases threaten millions of people worldwide, and it is critical that we identify new methods for controlling these insects. We recently developed a new class of mosquito insecticides that consists of yeasts which produce interfering RNA that is custom-designed to turn off mosquito genes yet does not hurt other organisms. Here, we test a newly optimized robust yeast strain which is suitable for industry-scale yeast production. Following heat inactivation and drying, the yeast was shipped to St. Augustine, Trinidad and Bangkok, Thailand, where it was evaluated in outdoor studies performed in tropical settings. The yeast effectively killed a variety of different types of mosquito larvae that died following yeast consumption. It could also be supplied to adult mosquitoes in a sugar bait solution, resulting in high levels of mosquito death. The yeast killed mosquitoes more quickly in outdoor experiments than in laboratory trials. These promising results indicate that yeast insecticides could one day be used globally as a new means of eco-friendly mosquito control and disease prevention. Abstract Eco-friendly new mosquito control innovations are critical for the ongoing success of global mosquito control programs. In this study, Sh.463_56.10R, a robust RNA interference (RNAi) yeast insecticide strain that is suitable for scaled fermentation, was evaluated under semi-field conditions. Inactivated and dried Sh.463_56.10R yeast induced significant mortality of field strain Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus larvae in semi-field larvicide trials conducted outdoors in St. Augustine, Trinidad, where 100% of the larvae were dead within 24 h. The yeast was also stably suspended in commercial bait and deployed as an active ingredient in miniature attractive targeted sugar bait (ATSB) station sachets. The yeast ATSB induced high levels of Aedes and Culex mosquito morbidity in semi-field trials conducted in Trinidad, West Indies, as well as in Bangkok, Thailand, in which the consumption of the yeast resulted in adult female mosquito death within 48 h, faster than what was observed in laboratory trials. These findings support the pursuit of large-scale field trials to further evaluate the Sh.463_56.10R insecticide, a member of a promising new class of species-specific RNAi insecticides that could help combat insecticide resistance and support effective mosquito control programs worldwide.

Several emerging mosquito control technologies require mass releases of adult male mosquitoes. Previous studies resulted in the generation of a laboratory female-specific larvicidal yeast strain targeting the GGT gene, which facilitated the laboratory sex separation of male Culex quinquefasciatus mosquitoes. Global deployment of this yeast-based sex-separation technology requires engineering second generation yeast strains which can be used in industrial-scale fermentations to support global mosquito control programs. In this study, the RNA-guided Cas-CLOVER system was used in combination with piggyBac transposase to generate robust Saccharomyces cerevisiae strains with multiple integrated copies of the insecticidal GGT shRNA expression cassette. Top expressing Cas-CLOVER strains killed Culex quinquefasciatus female larvae which consumed the yeast, facilitating male sex separation. Scaled fermentation resulted in kilogram-scale production of the yeast, which can be heat-killed and dried for global deployment to mosquito mass-rearing facilities.

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention.

RNA interference (RNAi), an innate regulatory mechanism that is conserved across many eukaryotic species, has been harnessed for experimental gene silencing in many organisms, including mosquitoes. This protocol describes an optimized method for inducing RNAi in adult Aedes aegypti and Anopheles gambiae mosquitoes that involves feeding them a red-colored sugar bait containing small interfering RNA (siRNA). This oral delivery method is less physically disruptive than delivery by subcutaneous injection, and the use of siRNAs (in contrast to long dsRNAs) for RNAi enables the design of molecules that target conserved sites so that gene function can be studied in multiple species. After feeding, the behavioral and morbidity phenotypes that result from the suppression of target gene expression can then be analyzed.