Browsing by Author "Roethele, Joseph B."

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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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    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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    A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae
    (Springer Nature, 2021-05-20) Mysore, Keshava; Hapairai, Limb K.; Li, Ping; Roethele, Joseph B.; Sun, Longhua; Igiede, Jessica; Misenti, Joi K.; Duman‑Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.
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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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    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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    A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide
    (MDPI, 2021-11-02) Mysore, Keshava; Sun, Longhua; Hapairai, Limb K.; Wang, Chien-Wei; Igiede, Jessica; Roethele, Joseph B.; Scheel, Nicholas D.; Scheel, Max P.; Li, Ping; Wei, Na; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Concerns for widespread insecticide resistance and the unintended impacts of insecticides on nontarget organisms have generated a pressing need for mosquito control innovations. A yeast RNAi-based insecticide that targets a conserved site in mosquito Irx family genes, but which has not yet been identified in the genomes of nontarget organisms, was developed and characterized. Saccharomyces cerevisiae constructed to express short hairpin RNA (shRNA) matching the target site induced significant Aedes aegypti larval death in both lab trials and outdoor semi-field evaluations. The yeast also induced high levels of mortality in adult females, which readily consumed yeast incorporated into an attractive targeted sugar bait (ATSB) during simulated field trials. A conserved requirement for Irx function as a regulator of proneural gene expression was observed in the mosquito brain, suggesting a possible mode of action. The larvicidal and adulticidal properties of the yeast were also verified in Aedes albopictus, Anopheles gambiae, and Culexquinquefasciatus mosquitoes, but the yeast larvicide was not toxic to other nontarget arthropods. These results indicate that further development and evaluation of this technology as an ecofriendly control intervention is warranted, and that ATSBs, an emerging mosquito control paradigm, could potentially be enriched through the use of yeast-based RNAi technology.