Browsing by Author "Realey, Jacob S."

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    Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes
    (Springer Nature, 2019-05-22) Mysore, Keshava; Li, Ping; Wang, Chien-Wei; Hapairai, Limb K.; Scheel, Nicholas D.; Realey, Jacob S.; Sun, Longhua; Severson, David W.; Wei, Na; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    BACKGROUND: RNA interference (RNAi), which has facilitated functional characterization of mosquito neural development genes such as the axon guidance regulator semaphorin-1a (sema1a), could one day be applied as a new means of vector control. Saccharomyces cerevisiae (baker's yeast) may represent an effective interfering RNA expression system that could be used directly for delivery of RNA pesticides to mosquito larvae. Here we describe characterization of a yeast larvicide developed through bioengineering of S. cerevisiae to express a short hairpin RNA (shRNA) targeting a conserved site in mosquito sema1a genes. RESULTS: Experiments conducted on Aedes aegypti larvae demonstrated that the yeast larvicide effectively silences sema1a expression, generates severe neural defects, and induces high levels of larval mortality in laboratory, simulated-field, and semi-field experiments. The larvicide was also found to induce high levels of Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus mortality. CONCLUSIONS: The results of these studies indicate that use of yeast interfering RNA larvicides targeting mosquito sema1a genes may represent a new biorational tool for mosquito control.
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    Evaluation of large volume yeast interfering RNA lure-and-kill ovitraps for attraction and control of Aedes mosquitoes
    (Wiley, 2021-09) Hapairai, Limb K.; Mysore, Keshava; James, Lester D.; Scheel, Nicholas D.; Realey, Jacob S.; Sun, Longhua; Gerber, Laura E.; Feng, Rachel Shui; Romero-Severson, Ethan; Mohammed, Azad; Duman-Scheel, Molly; Severson, David W.; Medical and Molecular Genetics, School of Medicine
    Aedes mosquitoes (Diptera: Culicidae), principle vectors of several arboviruses, typically lay eggs in man-made water-filled containers located near human dwellings. Given the widespread emergence of insecticide resistance, stable and biofriendly alternatives for mosquito larviciding are needed. Laboratory studies have demonstrated that inactivated yeast interfering RNA tablets targeting key larval developmental genes can be used to facilitate effective larvicidal activity while also promoting selective gravid female oviposition behaviour. Here we examined the efficacy of transferring this technology toward development of lure-and-kill ovitraps targeting Aedes aegypti (L.) and Aedes albopictus (Skuse) female mosquitoes. Insectary, simulated field and semi-field experiments demonstrated that two mosquito-specific yeast interfering RNA pesticides induce high levels of mortality among larvae of both species in treated large volume containers. Small-scale field trials conducted in Trinidad, West Indies demonstrated that large volume ovitrap containers baited with inactivated yeast tablets lure significantly more gravid females than traps containing only water and were highly attractive to both A. aegypti and A. albopictus females. These studies indicate that development of biorational yeast interfering RNA-baited ovitraps may represent a new tool for control of Aedes mosquitoes, including deployment in existing lure-and-kill ovitrap technologies or traditional container larviciding programs.
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    Oral RNAi for Gene Silencing in Mosquitoes: From the Bench to the Field
    (Cold Spring Harbor Laboratory Press, 2022-07-12) Mysore, Keshava; Hapairai, Limb; Realey, Jacob S.; Sun, Longhua; Roethele, Joseph B.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    RNA interference (RNAi) has played a key role in the field of insect functional genomics, a discipline that has enhanced the study of developmental, evolutionary, physiological, and molecular biological phenomena in a wide variety of insects, including disease vector mosquitoes. Here we introduce a recently optimized RNAi procedure in which adult mosquitoes are fed with a colored sugar bait containing small interfering RNA (siRNA). This procedure effectively and economically leads to gene silencing, is technically straightforward, and has been successfully used to characterize a number of genes in adult mosquitoes. We also discuss how, in addition to laboratory applications, this oral RNAi procedure might one day be used in the field for controlling insect pests.
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    Preparation and Use of a Yeast shRNA Delivery System for Gene Silencing in Mosquito Larvae
    (Humana Press, 2019) Mysore, Keshava; Hapairai, Limb K.; Wei, Na; Realey, Jacob S.; Scheel, Nicholas D.; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the dengue and Zika virus vector Aedes aegypti and the primary African malaria vector Anopheles gambiae. RNA interference (RNAi) has facilitated gene silencing experiments in both of these disease vector mosquito species and could one day be applied as a new method of vector control. Here, we describe a procedure for the genetic engineering of Saccharomyces cerevisiae (baker’s yeast) that express short hairpin RNA (shRNA) corresponding to mosquito target genes of interest. Following cultivation, which facilitates inexpensive propagation of shRNA, the yeast is inactivated and prepared in a ready-to-use dry tablet formulation that is fed to mosquito larvae. Ingestion of the yeast tablets results in effective larval target gene silencing. This technically straightforward and affordable technique may be applicable to a wide variety of mosquito species and potentially to other arthropods that feed on yeast.
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    Sugar-Baited Delivery of Small Interfering RNA for Gene Silencing in Adult Mosquitoes
    (Cold Spring Harbor Laboratory, 2022-07-12) Mysore, Keshava; Hapairai, Limb; Realey, Jacob S.; Sun, Longhua; Roethele, Joseph B.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    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.
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    Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality
    (BioMed Central, 2017-11-13) Mysore, Keshava; Hapairai, Limb K.; Sun, Longhua; Harper, Elizabeth I.; Chen, Yingying; Eggleson, Kathleen K.; Realey, Jacob S.; Scheel, Nicholas D.; Severson, David W.; Wei, Na; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Background Although larviciding can reduce the number of outdoor biting malaria vector mosquitoes, which may help to prevent residual malaria transmission, the current larvicide repertoire is faced with great challenges to sustainability. The identification of new effective, economical, and biorational larvicides could facilitate maintenance and expansion of the practice of larviciding in integrated malaria vector mosquito control programmes. Interfering RNA molecules represent a novel class of larvicides with untapped potential for sustainable mosquito control. This investigation tested the hypothesis that short interfering RNA molecules can be used as mosquito larvicides. Results A small interfering RNA (siRNA) screen for larval lethal genes identified siRNAs corresponding to the Anopheles gambiae suppressor of actin (Sac1), leukocyte receptor complex member (lrc), and offtrack (otk) genes. Saccharomyces cerevisiae (baker’s yeast) was engineered to produce short hairpin RNAs (shRNAs) for silencing of these genes. Feeding larvae with the engineered yeasts resulted in silenced target gene expression, a severe loss of neural synapses in the larval brain, and high levels of larval mortality. The larvicidal activities of yeast interfering RNA larvicides were retained following heat inactivation and drying of the yeast into user-friendly tablet formulations that induced up to 100% larval mortality in laboratory trials. Conclusions Ready-to-use dried inactivated yeast interfering RNA larvicide tablets may someday be an effective and inexpensive addition to malaria mosquito control programmes and a valuable, biorational tool for addressing residual malaria transmission. Electronic supplementary material The online version of this article (10.1186/s12936-017-2112-5) contains supplementary material, which is available to authorized users.