Browsing by Author "Mysore, Keshava"

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    A Conserved Female-Specific Requirement for the GGT Gene in Mosquito Larvae Facilitates RNAi-Mediated Sex Separation in Multiple Species of Disease Vector Mosquitoes
    (MDPI, 2022-01-27) Mysore, Keshava; Sun, Longhua; Li, Ping; Roethele, Joseph B.; Misenti, Joi K.; Kosmach, John; Igiede, Jessica; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Although several emerging mosquito control technologies are dependent on mass releases of adult males, methods of sex-sorting that can be implemented globally have not yet been established. RNAi screens led to the discovery of siRNA, which targets gamma-glutamyl transpeptidase (GGT), a gene which is well conserved in multiple species of mosquitoes and located at the sex-determining M locus region in Aedes aegypti. Silencing the A. aegypti, Aedes albopictus, Anopheles gambiae, Culex pipiens, and Culex quinquefasciatus GGT genes resulted in female larval death, with no significant impact on male survival. Generation of yeast strains that permitted affordable expression and oral delivery of shRNA corresponding to mosquito GGT genes facilitated larval target gene silencing and generated significantly increased 5 males:1 female adult ratios in each species. Yeast targeting a conserved sequence in Culex GGT genes was incorporated into a larval mass-rearing diet, permitting the generation of fit adult male C. pipiens and C. quinquefasciatus, two species for which labor-intensive manual sex separation had previously been utilized. The results of this study indicate that female-specific yeast-based RNAi larvicides may facilitate global implementation of population-based control strategies that require releases of sterile or genetically modified adult males, and that yeast RNAi strategies can be utilized in various species of mosquitoes that have progressed to different stages of sex chromosome evolution.
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    A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes
    (MDPI, 2021-09-28) Mysore, Keshava; Sun, Longhua; 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; Medical and Molecular Genetics, School of Medicine
    Prevention of mosquito-borne infectious diseases will require new classes of environmentally safe insecticides and novel mosquito control technologies. Saccharomyces cerevisiae was engineered to express short hairpin RNA (shRNA) corresponding to mosquito Rbfox1 genes. The yeast induced target gene silencing, resulting in larval death that was observed in both laboratory and outdoor semi-field trials conducted on Aedes aegypti. High levels of mortality were also observed during simulated field trials in which adult females consumed yeast delivered through a sugar bait. Mortality correlated with defects in the mosquito brain, in which a role for Rbfox1 as a positive regulator of Notch signaling was identified. The larvicidal and adulticidal activities of the yeast were subsequently confirmed in trials conducted on Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus, yet the yeast had no impact on survival of select non-target arthropods. These studies indicate that yeast RNAi pesticides targeting Rbfox1 could be further developed as broad-based mosquito larvicides and adulticides for deployment in integrated biorational mosquito control programs. These findings also suggest that the species-specificity of attractive targeted sugar baits, a new paradigm for vector control, could potentially be enhanced through RNAi technology, and specifically through the use of yeast-based interfering RNA pesticides.
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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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    Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes
    (Springer Nature, 2023-12-15) Mysore, Keshava; Njoroge, Teresia M.; Stewart, Akilah T. M.; Winter, Nikhella; Hamid‑Adiamoh, Majidah; Sun, Longhua; Shui Feng, Rachel; James, Lester D.; Mohammed, Azad; Severson, David W.; Duman‑Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    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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    Characterization of an adulticidal and larvicidal interfering RNA pesticide that targets a conserved sequence in mosquito G protein-coupled dopamine 1 receptor genes
    (Elsevier, 2020) Hapairai, Limb K.; Mysore, Keshava; Sun, Longhua; Li, Ping; Wang, Chien-Wei; Scheel, Nicholas D.; Lesnik, Alexandra; Scheel, Max P.; Igiede, Jessica; Wei, Na; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    G protein-coupled receptors (GPCRs), key regulators of a variety of critical biological processes, are attractive targets for insecticide development. Given the importance of these receptors in many organisms, including humans, it is critical that novel pesticides directed against GPCRs are designed to be species-specific. Here, we present characterization of an interfering RNA pesticide (IRP) targeting the mosquito GPCR-encoding dopamine 1 receptor (dop1) genes. A small interfering RNA corresponding to dop1 was identified in a screen for IRPs that kill Aedes aegypti during both the adult and larval stages. The 25 bp sequence targeted by this IRP is conserved in the dop1 genes of multiple mosquito species, but not in non-target organisms, indicating that it could function as a biorational mosquito insecticide. Aedes aegypti adults treated through microinjection or attractive toxic sugar bait delivery of small interfering RNA corresponding to the target site exhibited severe neural and behavioral defects and high levels of adult mortality. Likewise, A. aegypti larval consumption of dried inactivated yeast tablets prepared from a Saccharomyces cerevisiae strain engineered to express short hairpin RNA corresponding to the dop1 target site resulted in severe neural defects and larval mortality. Aedes albopictus and Anopheles gambiae adult and larval mortality was also observed following treatment with dop1 IRPs, which were not toxic to non-target arthropods. The results of this investigation indicate that dop1 IRPs can be used for species-specific targeting of dop1 GPCRs and may represent a new biorational strategy for control of both adult and larval mosquitoes.
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    Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae
    (JOVE, 2015-03-25) Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W.; Zhu, Kun Yan; Duman-Scheel, Molly; Department of Medical and Molecular Genetics, IU School of Medicine
    Vector mosquitoes inflict more human suffering than any other organism-and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.
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    Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti
    (Springer Nature, 2014-02-19) Mysore, Keshava; Andrews, Emily; Li, Ping; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Background: Essentially nothing is known about the genetic regulation of olfactory system development in vector mosquitoes, which use olfactory cues to detect blood meal hosts. Studies in Drosophila melanogaster have identified a regulatory matrix of transcription factors that controls pupal/adult odorant receptor (OR) gene expression in olfactory receptor neurons (ORNs). However, it is unclear if transcription factors that function in the D. melanogaster regulatory matrix are required for OR expression in mosquitoes. Furthermore, the regulation of OR expression during development of the larval olfactory system, which is far less complex than that of pupae/adults, is not well understood in any insect, including D. melanogaster. Here, we examine the regulation of OR expression in the developing larval olfactory system of Aedes aegypti, the dengue vector mosquito. Results: A. aegypti bears orthologs of eight transcription factors that regulate OR expression in D. melanogaster pupae/adults. These transcription factors are expressed in A. aegypti larval antennal sensory neurons, and consensus binding sites for these transcription factors reside in the 5' flanking regions of A. aegypti OR genes. Consensus binding sites for Single-minded (Sim) are located adjacent to over half the A. aegypti OR genes, suggesting that this transcription factor functions as a major regulator of mosquito OR expression. To functionally test this hypothesis, chitosan/siRNA nanoparticles were used to target sim during larval olfactory development. These experiments demonstrated that Sim positively regulates expression of a large subset of OR genes, including orco, the obligate co-receptor in the assembly and function of heteromeric OR/Orco complexes. Decreased innervation of the antennal lobe was also noted in sim knockdown larvae. These OR expression and antennal lobe defects correlated with a larval odorant tracking behavioral defect. OR expression and antennal lobe defects were also observed in sim knockdown pupae. Conclusions: The results of this investigation indicate that Sim has multiple functions during larval and pupal olfactory system development in A. aegypti.
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    A conserved female-specific larval requirement for MtnB function facilitates sex separation in multiple species of disease vector mosquitoes
    (BMC, 2021-06-26) Mysore, Keshava; Sun, Longhua; Roethele, Joseph B.; Li, Ping; Igiede, Jessica; Misenti, Joi K.; Duman‑Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Background: Clusters of sex-specific loci are predicted to shape the boundaries of the M/m sex-determination locus of the dengue vector mosquito Aedes aegypti, but the identities of these genes are not known. Identification and characterization of these loci could promote a better understanding of mosquito sex chromosome evolution and lead to the elucidation of new strategies for male mosquito sex separation, a requirement for several emerging mosquito population control strategies that are dependent on the mass rearing and release of male mosquitoes. This investigation revealed that the methylthioribulose-1-phosphate dehydratase (MtnB) gene, which resides adjacent to the M/m locus and encodes an evolutionarily conserved component of the methionine salvage pathway, is required for survival of female larvae. Results: Larval consumption of Saccharomyces cerevisiae (yeast) strains engineered to express interfering RNA corresponding to MtnB resulted in target gene silencing and significant female death, yet had no impact on A. aegypti male survival or fitness. Integration of the yeast larvicides into mass culturing protocols permitted scaled production of fit adult male mosquitoes. Moreover, silencing MtnB orthologs in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus revealed a conserved female-specific larval requirement for MtnB among different species of mosquitoes. Conclusions: The results of this investigation, which may have important implications for the study of mosquito sex chromosome evolution, indicate that silencing MtnB can facilitate sex separation in multiple species of disease vector insects.
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    Correction: Mysore et al. A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes. Pathogens 2021, 10, 1251
    (MDPI, 2022-08-23) Mysore, Keshava; Sun, Longhua; 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; Medical and Molecular Genetics, School of Medicine
    In the original publication [1], there was a mistake in Figure 1 as published. The wrong graph was inadvertently included in panel 1f (dose–response curve). Additionally, the original image for the gel shown in panel 1a is now included in the Supplementary Materials.
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    Demonstration of RNAi Yeast Insecticide Activity in Semi-Field Larvicide and Attractive Targeted Sugar Bait Trials Conducted on Aedes and Culex Mosquitoes
    (MDPI, 2023-12-15) Stewart, Akilah T. M.; Mysore, Keshava; Njoroge, Teresia M.; Winter, Nikhella; Shui Feng, Rachel; Singh, Satish; James, Lester D.; Singkhaimuk, Preeraya; Sun, Longhua; Mohammed, Azad; Oxley, James D.; Duckham, Craig; Ponlawat, Alongkot; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    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.