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Published in: Malaria Journal 1/2017

Open Access 01-12-2017 | Research

Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality

Authors: Keshava Mysore, Limb K. Hapairai, Longhua Sun, Elizabeth I. Harper, Yingying Chen, Kathleen K. Eggleson, Jacob S. Realey, Nicholas D. Scheel, David W. Severson, Na Wei, Molly Duman-Scheel

Published in: Malaria Journal | Issue 1/2017

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Abstract

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.
Appendix
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Literature
1.
go back to reference WHO. World malaria report 2016. Geneva: World Health Organization; 2016. WHO. World malaria report 2016. Geneva: World Health Organization; 2016.
2.
go back to reference WHO. Larval source management: a supplementary measure for malaria vector control: an operational manual. Geneva: World Health Organization; 2013. WHO. Larval source management: a supplementary measure for malaria vector control: an operational manual. Geneva: World Health Organization; 2013.
4.
go back to reference Afrane YA, Mweresa NG, Wanjala CL, Gilbreath Iii TM, Zhou G, Lee MC, Githeko AK, Yan G. Evaluation of long-lasting microbial larvicide for malaria vector control in Kenya. Malar J. 2016;15:577.CrossRefPubMedPubMedCentral Afrane YA, Mweresa NG, Wanjala CL, Gilbreath Iii TM, Zhou G, Lee MC, Githeko AK, Yan G. Evaluation of long-lasting microbial larvicide for malaria vector control in Kenya. Malar J. 2016;15:577.CrossRefPubMedPubMedCentral
5.
go back to reference Yu N, Christiaens O, Liu J, Niu J, Cappelle K, Caccia S, Huvenne H, Smagghe G. Delivery of dsRNA for RNAi in insects: an overview and future directions. Insect Sci. 2013;20:4–14.CrossRefPubMed Yu N, Christiaens O, Liu J, Niu J, Cappelle K, Caccia S, Huvenne H, Smagghe G. Delivery of dsRNA for RNAi in insects: an overview and future directions. Insect Sci. 2013;20:4–14.CrossRefPubMed
6.
go back to reference Zhang H, Li HC, Miao XX. Feasibility, limitation and possible solutions of RNAi-based technology for insect pest control. Insect Sci. 2013;20:15–30.CrossRefPubMed Zhang H, Li HC, Miao XX. Feasibility, limitation and possible solutions of RNAi-based technology for insect pest control. Insect Sci. 2013;20:15–30.CrossRefPubMed
9.
go back to reference Moffat J, Reiling JH, Sabatini DM. Off-target effects associated with long dsRNAs in Drosophila RNAi screens. Trends Pharmacol Sci. 2007;28:149–51.CrossRefPubMed Moffat J, Reiling JH, Sabatini DM. Off-target effects associated with long dsRNAs in Drosophila RNAi screens. Trends Pharmacol Sci. 2007;28:149–51.CrossRefPubMed
10.
go back to reference Mysore K, Flannery EM, Tomchaney M, Severson DW, Duman-Scheel M. Disruption of Aedes aegypti olfactory system development through chitosan/siRNA nanoparticle targeting of semaphorin-1a. PLoS Negl Trop Dis. 2013;7(5):e2215.CrossRefPubMedPubMedCentral Mysore K, Flannery EM, Tomchaney M, Severson DW, Duman-Scheel M. Disruption of Aedes aegypti olfactory system development through chitosan/siRNA nanoparticle targeting of semaphorin-1a. PLoS Negl Trop Dis. 2013;7(5):e2215.CrossRefPubMedPubMedCentral
11.
go back to reference Mysore K, Andrews E, Li P, Duman-Scheel M. Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti. BMC Dev Biol. 2014;14:9.CrossRefPubMedPubMedCentral Mysore K, Andrews E, Li P, Duman-Scheel M. Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti. BMC Dev Biol. 2014;14:9.CrossRefPubMedPubMedCentral
12.
go back to reference Mysore K, Flannery E, Leming MT, Tomchaney M, Shi L, Sun L, O’Tousa JE, et al. Role of semaphorin-1a in the developing visual system of the disease vector mosquito Aedes aegypti. Dev Dyn. 2014;243:1457–69.CrossRefPubMedPubMedCentral Mysore K, Flannery E, Leming MT, Tomchaney M, Shi L, Sun L, O’Tousa JE, et al. Role of semaphorin-1a in the developing visual system of the disease vector mosquito Aedes aegypti. Dev Dyn. 2014;243:1457–69.CrossRefPubMedPubMedCentral
13.
go back to reference Mysore K, Sun L, Tomchaney M, Sullivan G, Adams H, Piscoya AS, et al. siRNA-mediated silencing of doublesex during female development of the dengue vector mosquito Aedes aegypti. PLoS Negl Trop Dis. 2015;9:e0004213.CrossRefPubMedPubMedCentral Mysore K, Sun L, Tomchaney M, Sullivan G, Adams H, Piscoya AS, et al. siRNA-mediated silencing of doublesex during female development of the dengue vector mosquito Aedes aegypti. PLoS Negl Trop Dis. 2015;9:e0004213.CrossRefPubMedPubMedCentral
14.
go back to reference Tomchaney M, Mysore K, Sun L, Li P, Emrich SJ, Severson DW, et al. Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain. Biol Sex Differ. 2014;5:10.CrossRefPubMedPubMedCentral Tomchaney M, Mysore K, Sun L, Li P, Emrich SJ, Severson DW, et al. Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain. Biol Sex Differ. 2014;5:10.CrossRefPubMedPubMedCentral
15.
go back to reference Clemons A, Mori A, Haugen M, Severson DW, Duman-Scheel M. Culturing and egg collection of Aedes aegypti. Cold Spring Harb Protoc. 2010;pdb prot5507. Clemons A, Mori A, Haugen M, Severson DW, Duman-Scheel M. Culturing and egg collection of Aedes aegypti. Cold Spring Harb Protoc. 2010;pdb prot5507.
16.
go back to reference St Pierre SE, Ponting L, Stefancsik R, McQuilton P, FlyBase Consortium. FlyBase 102-advanced approaches to interrogating FlyBase. Nucleic Acids Res. 2014;42:D780–8.CrossRefPubMed St Pierre SE, Ponting L, Stefancsik R, McQuilton P, FlyBase Consortium. FlyBase 102-advanced approaches to interrogating FlyBase. Nucleic Acids Res. 2014;42:D780–8.CrossRefPubMed
17.
go back to reference Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.CrossRefPubMed Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.CrossRefPubMed
18.
go back to reference Blitzer EJ, Vyazunova I, Lan Q. Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti. Insect Mol Biol. 2005;14:301–7.CrossRefPubMed Blitzer EJ, Vyazunova I, Lan Q. Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti. Insect Mol Biol. 2005;14:301–7.CrossRefPubMed
19.
go back to reference Clemons A, Haugen M, Le C, Mori A, Tomchaney M, Severson DW, Duman-Scheel M. siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development. PLoS ONE. 2011;6:e16730.CrossRefPubMedPubMedCentral Clemons A, Haugen M, Le C, Mori A, Tomchaney M, Severson DW, Duman-Scheel M. siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development. PLoS ONE. 2011;6:e16730.CrossRefPubMedPubMedCentral
20.
go back to reference Singh AD, Wong S, Ryan CP, Whyard S. Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control. J Insect Sci. 2013;13:69.CrossRefPubMedPubMedCentral Singh AD, Wong S, Ryan CP, Whyard S. Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control. J Insect Sci. 2013;13:69.CrossRefPubMedPubMedCentral
21.
go back to reference WHO. Guidelines for laboratory and field testing of mosquito larvicides. Geneva: World Health Organization; 2005. WHO. Guidelines for laboratory and field testing of mosquito larvicides. Geneva: World Health Organization; 2005.
22.
go back to reference Fisher RA. Statistical methods for research workers. Edinburgh: Oliver and Boyd; 1925. Fisher RA. Statistical methods for research workers. Edinburgh: Oliver and Boyd; 1925.
23.
go back to reference Whyard S, Erdelyan CN, Partridge AL, Singh AD, Beebe NW, Capina R. Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs. Parasit Vectors. 2015;8:96.CrossRefPubMedPubMedCentral Whyard S, Erdelyan CN, Partridge AL, Singh AD, Beebe NW, Capina R. Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs. Parasit Vectors. 2015;8:96.CrossRefPubMedPubMedCentral
24.
go back to reference Mumberg D, Muller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156:119–22.CrossRefPubMed Mumberg D, Muller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156:119–22.CrossRefPubMed
25.
go back to reference Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 1999;285:901–6.CrossRefPubMed Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 1999;285:901–6.CrossRefPubMed
26.
go back to reference Patel NH. In situ hybridization to whole mount Drosophila embryos. In: Krieg PA, editor. A laboratory guide to RNA: isolation, analysis, and synthesis. New York: Wiley-Liss; 1996. p. 357–70. Patel NH. In situ hybridization to whole mount Drosophila embryos. In: Krieg PA, editor. A laboratory guide to RNA: isolation, analysis, and synthesis. New York: Wiley-Liss; 1996. p. 357–70.
29.
go back to reference Mysore K, Flister S, Muller P, Rodrigues V, Reichert H. Brain development in the yellow fever mosquito Aedes aegypti: a comparative immunocytochemical analysis using cross-reacting antibodies from Drosophila melanogaster. Dev Genes Evol. 2011;221:281–96.CrossRefPubMed Mysore K, Flister S, Muller P, Rodrigues V, Reichert H. Brain development in the yellow fever mosquito Aedes aegypti: a comparative immunocytochemical analysis using cross-reacting antibodies from Drosophila melanogaster. Dev Genes Evol. 2011;221:281–96.CrossRefPubMed
30.
go back to reference Klagges BR, Heimbeck G, Godenschwege TA, Hofbauer A, Pflugfelder GO, Reifegerste R, et al. Invertebrate synapsins: a single gene codes for several isoforms in Drosophila. J Neurosci. 1996;16:3154–65.PubMed Klagges BR, Heimbeck G, Godenschwege TA, Hofbauer A, Pflugfelder GO, Reifegerste R, et al. Invertebrate synapsins: a single gene codes for several isoforms in Drosophila. J Neurosci. 1996;16:3154–65.PubMed
31.
go back to reference Lee S, Kim S, Nahm M, Kim E, Kim TI, Yoon JH, Lee S. The phosphoinositide phosphatase Sac1 is required for midline axon guidance. Mol Cells. 2011;32:477–82.CrossRefPubMedPubMedCentral Lee S, Kim S, Nahm M, Kim E, Kim TI, Yoon JH, Lee S. The phosphoinositide phosphatase Sac1 is required for midline axon guidance. Mol Cells. 2011;32:477–82.CrossRefPubMedPubMedCentral
32.
go back to reference Pulido D, Campuzano S, Koda T, Modolell J, Barbacid M. Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J. 1992;11:391–404.PubMedPubMedCentral Pulido D, Campuzano S, Koda T, Modolell J, Barbacid M. Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J. 1992;11:391–404.PubMedPubMedCentral
33.
go back to reference Winberg ML, Tamagnone L, Bai J, Comoglio PM, Montell D, Goodman CS. The transmembrane protein Off-track associates with Plexins and functions downstream of Semaphorin signaling during axon guidance. Neuron. 2001;32:53–62.CrossRefPubMed Winberg ML, Tamagnone L, Bai J, Comoglio PM, Montell D, Goodman CS. The transmembrane protein Off-track associates with Plexins and functions downstream of Semaphorin signaling during axon guidance. Neuron. 2001;32:53–62.CrossRefPubMed
34.
go back to reference Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 2007;448:151–6.CrossRefPubMed Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 2007;448:151–6.CrossRefPubMed
35.
go back to reference Jankovics F, Henn L, Bujna A, Vilmos P, Kiss N, Erdelyi M. A functional genomic screen combined with time-lapse microscopy uncovers a novel set of genes involved in dorsal closure of Drosophila embryos. PLoS ONE. 2011;6:e22229.CrossRefPubMedPubMedCentral Jankovics F, Henn L, Bujna A, Vilmos P, Kiss N, Erdelyi M. A functional genomic screen combined with time-lapse microscopy uncovers a novel set of genes involved in dorsal closure of Drosophila embryos. PLoS ONE. 2011;6:e22229.CrossRefPubMedPubMedCentral
36.
go back to reference Kucherenko MM, Pantoja M, Yatsenko AS, Shcherbata HR, Fischer KA, Maksymiv DV, et al. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan–dystrophin complex. PLoS ONE. 2008;3:e2418.CrossRefPubMedPubMedCentral Kucherenko MM, Pantoja M, Yatsenko AS, Shcherbata HR, Fischer KA, Maksymiv DV, et al. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan–dystrophin complex. PLoS ONE. 2008;3:e2418.CrossRefPubMedPubMedCentral
37.
go back to reference Pazos Obregon F, Papalardo C, Castro S, Guerberoff G, Cantera R. Putative synaptic genes defined from a Drosophila whole body developmental transcriptome by a machine learning approach. BMC Genomics. 2015;16:694.CrossRefPubMedPubMedCentral Pazos Obregon F, Papalardo C, Castro S, Guerberoff G, Cantera R. Putative synaptic genes defined from a Drosophila whole body developmental transcriptome by a machine learning approach. BMC Genomics. 2015;16:694.CrossRefPubMedPubMedCentral
38.
go back to reference Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, et al. Control of coleopteran insect pests through RNA interference. Nat Biotechnol. 2007;25:1322–6.CrossRefPubMed Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, et al. Control of coleopteran insect pests through RNA interference. Nat Biotechnol. 2007;25:1322–6.CrossRefPubMed
39.
41.
go back to reference Whyard S, Singh AD, Wong S. Ingested double-stranded RNAs can act as species-specific insecticides. Insect Biochem Mol Biol. 2009;39:824–32.CrossRefPubMed Whyard S, Singh AD, Wong S. Ingested double-stranded RNAs can act as species-specific insecticides. Insect Biochem Mol Biol. 2009;39:824–32.CrossRefPubMed
42.
go back to reference Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov. 2010;9:57–67.CrossRefPubMed Jackson AL, Linsley PS. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov. 2010;9:57–67.CrossRefPubMed
44.
go back to reference Li H, Guan R, Guo H, Miao X. New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests. Plant Cell Environ. 2015;38:2277–85.CrossRefPubMed Li H, Guan R, Guo H, Miao X. New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests. Plant Cell Environ. 2015;38:2277–85.CrossRefPubMed
45.
go back to reference San Miguel K, Scott JG. The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide. Pest Manag Sci. 2016;72:801–9.CrossRefPubMed San Miguel K, Scott JG. The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide. Pest Manag Sci. 2016;72:801–9.CrossRefPubMed
46.
go back to reference Borovsky D, Nauwelaers S, Van Mileghem A, Meyvis Y, Laeremans A, Theunis C, et al. Control of mosquito larvae with TMOF and 60 kDa Cry4Aa expressed in Pichia pastoris. Pestycydy/Pesticides. 2011;1:5–15. Borovsky D, Nauwelaers S, Van Mileghem A, Meyvis Y, Laeremans A, Theunis C, et al. Control of mosquito larvae with TMOF and 60 kDa Cry4Aa expressed in Pichia pastoris. Pestycydy/Pesticides. 2011;1:5–15.
47.
go back to reference Van Ekert E, Powell CA, Shatters RG Jr, Borovsky D. Control of larval and egg development in Aedes aegypti with RNA interference against juvenile hormone acid methyl transferase. J Insect Physiol. 2014;70:143–50.CrossRefPubMed Van Ekert E, Powell CA, Shatters RG Jr, Borovsky D. Control of larval and egg development in Aedes aegypti with RNA interference against juvenile hormone acid methyl transferase. J Insect Physiol. 2014;70:143–50.CrossRefPubMed
48.
go back to reference Murphy KA, Tabuloc CA, Cervantes KR, Chiu JC. Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference. Sci Rep. 2016;6:22587.CrossRefPubMedPubMedCentral Murphy KA, Tabuloc CA, Cervantes KR, Chiu JC. Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference. Sci Rep. 2016;6:22587.CrossRefPubMedPubMedCentral
Metadata
Title
Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality
Authors
Keshava Mysore
Limb K. Hapairai
Longhua Sun
Elizabeth I. Harper
Yingying Chen
Kathleen K. Eggleson
Jacob S. Realey
Nicholas D. Scheel
David W. Severson
Na Wei
Molly Duman-Scheel
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Malaria Journal / Issue 1/2017
Electronic ISSN: 1475-2875
DOI
https://doi.org/10.1186/s12936-017-2112-5

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