Testing Insecticidal Activity of Novel Chemically Synthesized siRNA against Plutella xylostella under Laboratory and Field Conditions

Background Over the last 60 years, synthetic chemical pesticides have served as a main tactic in the field of crop protection, but their availability is now declining as a result of the development of insect resistance. Therefore, alternative pest management agents are needed. However, the demonstration of RNAi gene silencing in insects and its successful usage in disrupting the expression of vital genes opened a door to the development of a variety of novel, environmentally sound approaches for insect pest management. Methodology/Principal Findings Six small interfering RNAs (siRNAs) were chemically synthesized and modified according to the cDNA sequence of P. xylostella acetylcholine esterase genes AChE1 and AChE2. All of them were formulated and used in insecticide activity screening against P. xylostella. Bioassay data suggested that Si-ace1_003 and Si-ace2_001 at a concentration of 3 µg cm−2 displayed the best insecticidal activity with 73.7% and 89.0%, mortality, respectively. Additional bioassays were used to obtain the acute lethal concentrations of LC50 and LC90 for Si-ace2_001, which were 53.66 µg/ml and 759.71 µg/ml, respectively. Quantitative Real-time PCR was used to confirm silencing and detected that the transcript levels of P. xylostella AChE2 (PxAChE2) were reduced by 5.7-fold compared to the control group. Consequently, AChE activity was also reduced by 1.7-fold. Finally, effects of the siRNAs on treated plants of Brassica oleracea and Brassica alboglabra were investigated with different siRNA doses. Our results showed that Si-ace2_001 had no negative effects on plant morphology, color and growth of vein under our experimental conditions. Conclusions The most important finding of this study is the discovery that chemically synthesized and modified siRNA corresponding to P. xylostella AChE genes cause significant mortality of the insect both under laboratory and field conditions, which provides a novel strategy to control P. xylostella and to develop bio-pesticides based on the RNA interference technology.

[1]  G. Cao,et al.  BmTGIF, a Bombyx mori Homolog of Drosophila DmTGIF, Regulates Progression of Spermatogenesis , 2012, PloS one.

[2]  Anthony M. Shelton,et al.  Biology, Ecology, and Management of the Diamondback Moth , 1993 .

[3]  Maneesh Kumar,et al.  Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA affects larval growth and its life cycle. , 2009, Journal of insect physiology.

[4]  Y. Koh,et al.  Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.). , 2007, Biochemical and biophysical research communications.

[5]  Y. Pang,et al.  Genome Organization, Phylogenies, Expression Patterns, and Three-Dimensional Protein Models of Two Acetylcholinesterase Genes from the Red Flour Beetle , 2012, PloS one.

[6]  Michel Raymond,et al.  A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non–homologous to the ace gene Drosophila , 2002, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[7]  R. A. Dybas,et al.  Avermectins, a novel class of compounds: implications for use in arthropod pest control. , 1991, Annual review of entomology.

[8]  S. Brimijoin,et al.  Selective and Irreversible Inhibitors of Aphid Acetylcholinesterases: Steps Toward Human-Safe Insecticides , 2009, PloS one.

[9]  Ronald D. Flannagan,et al.  Characterizing the Mechanism of Action of Double-Stranded RNA Activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte) , 2012, PloS one.

[10]  J. Thacker,et al.  An Introduction to Arthropod Pest Control , 2002 .

[11]  J. Doudna,et al.  A three-dimensional view of the molecular machinery of RNA interference , 2009, Nature.

[12]  D. Onstad,et al.  Effect of Insecticides and Plutella xylostella (Lepidoptera: Plutellidae) Genotype on a Predator and Parasitoid and Implications for the Evolution of Insecticide Resistance , 2012, Journal of economic entomology.

[13]  Wen-Jer Wu,et al.  Alterations of the acetylcholinesterase enzyme in the oriental fruit fly Bactrocera dorsalis are correlated with resistance to the organophosphate insecticide fenitrothion. , 2008, Insect biochemistry and molecular biology.

[14]  Gregor Bucher,et al.  Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium , 2008, Genome Biology.

[15]  P. Batterham,et al.  The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina. , 2001, Insect biochemistry and molecular biology.

[16]  Isabel Gómez,et al.  RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. , 2011, Journal of insect physiology.

[17]  A. Worden.  Toxicological methods. , 1974, Toxicology.

[18]  Michael J Furlong,et al.  Diamondback moth ecology and management: problems, progress, and prospects. , 2013, Annual review of entomology.

[19]  J. D. da Silva,et al.  Insecticide resistance in populations of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), from the state of Pernambuco, Brazil. , 2011, Neotropical entomology.

[20]  K. B. Temeyer,et al.  Identification and characterization of a cDNA encoding the acetylcholinesterase of Haematobia irritans (L.) (Diptera: Muscidae) , 2007, DNA sequence : the journal of DNA sequencing and mapping.

[21]  A. Shelton,et al.  Current control methods for diamondback moth and other brassica insect pests and the prospects for improved management with lepidopteran-resistant Bt vegetable brassicas in Asia and Africa , 2010 .

[22]  Zan Zhang,et al.  Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects , 2012, Journal of insect science.

[23]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[24]  Y. Tomoyasu,et al.  Larval RNAi in Tribolium (Coleoptera) for analyzing adult development , 2004, Development Genes and Evolution.

[25]  W. Grant,et al.  The development, regulation and use of biopesticides for integrated pest management , 2011, Philosophical Transactions of the Royal Society B: Biological Sciences.

[26]  K. Boo,et al.  Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée). , 2006, Biochimica et biophysica acta.

[27]  A. Shelton,et al.  Monitoring of Diamondback Moth (Lepidoptera: Plutellidae) Resistance to Spinosad, Indoxacarb, and Emamectin Benzoate , 2006, Journal of economic entomology.

[28]  Horacio Samaniego,et al.  Insecticide Resistance Mechanisms in the Green Peach Aphid Myzus persicae (Hemiptera: Aphididae) I: A Transcriptomic Survey , 2012, PloS one.

[29]  Y. Pang,et al.  Cholinergic and non-cholinergic functions of two acetylcholinesterase genes revealed by gene-silencing in Tribolium castaneum , 2012, Scientific Reports.

[30]  J. Whangbo,et al.  Environmental RNA interference. , 2008, Trends in genetics : TIG.

[31]  M. King,et al.  An immobilized nanoparticle-based platform for efficient gene knockdown of targeted cells in the circulation , 2009, Gene Therapy.

[32]  P. Marrone.  Microbial Pesticides and Natural Products as Alternatives , 1999 .

[33]  Y. Won,et al.  Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. , 2007, Journal of controlled release : official journal of the Controlled Release Society.

[34]  G. Smagghe,et al.  Insecticidal and fungicidal activity of new synthesized chitosan derivatives. , 2005, Pest management science.

[35]  T. Ho,et al.  Assessment of Chitosan-Affected Metabolic Response by Peroxisome Proliferator-Activated Receptor Bioluminescent Imaging-Guided Transcriptomic Analysis , 2012, PloS one.

[36]  W. Hunter,et al.  RNAi: future in insect management. , 2013, Journal of invertebrate pathology.

[37]  Jianzhen Zhang,et al.  Chitosan/double‐stranded RNA nanoparticle‐mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae) , 2010, Insect molecular biology.

[38]  Zhaojun Han,et al.  Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae). , 2009, Biochemical and biophysical research communications.

[39]  A. Fire.  Gene silencing by double-stranded RNA (Nobel Lecture). , 2007, Angewandte Chemie.

[40]  T. Tan,et al.  Insecticidal and Fungicidal Activities of Chitosan and Oligo-chitosan , 2003 .

[41]  Salwa A. Khalaf,et al.  Production and Characterization of Fungal Chitosan Under Solid-State Fermentation Conditions , 2008 .

[42]  K. Courtney,et al.  A new and rapid colorimetric determination of acetylcholinesterase activity. , 1961, Biochemical pharmacology.

[43]  Hongyu Zhang,et al.  RNA Interference of Four Genes in Adult Bactrocera dorsalis by Feeding Their dsRNAs , 2011, PloS one.

[44]  L. Gong,et al.  Cloning and characterization of three chemosensory proteins from Spodoptera exigua and effects of gene silencing on female survival and reproduction , 2012, Bulletin of Entomological Research.

[45]  K. Mathiopoulos,et al.  Genomic structure, organization and localization of the acetylcholinesterase locus of the olive fruit fly, Bactrocera oleae , 2012, Bulletin of Entomological Research.

[46]  Q. Boese,et al.  siRNA as a tool for streamlining functional genomic studies , 2003 .

[47]  J. Diaz‐ruiz,et al.  RNAi of ace1 and ace2 in Blattella germanica reveals their differential contribution to acetylcholinesterase activity and sensitivity to insecticides. , 2009, Insect biochemistry and molecular biology.

[48]  Lijuan Zhou,et al.  Monitoring resistance of field populations of diamondback moth Plutella xylostella L. (Lepidoptera: Yponomeutidae) to five insecticides in South China: A ten-year case study , 2011 .

[49]  P Taylor,et al.  The cholinesterases: from genes to proteins. , 1994, Annual review of pharmacology and toxicology.

[50]  J. Linden,et al.  Organic disease control elicitors. , 2000 .

[51]  Guy Smagghe,et al.  Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. , 2010, Journal of insect physiology.

[52]  B. Falk,et al.  Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli , 2011, PloS one.

[53]  F. Zeng,et al.  Feeding-Based RNA Intereference of a Gap Gene Is Lethal to the Pea Aphid, Acyrthosiphon pisum , 2012, PloS one.

[54]  W. S. Abbott,et al.  A method of computing the effectiveness of an insecticide. 1925. , 1925, Journal of the American Mosquito Control Association.

[55]  Hai-Sheng Qi,et al.  Improvement of Pest Resistance in Transgenic Tobacco Plants Expressing dsRNA of an Insect-Associated Gene EcR , 2012, PloS one.

[56]  Guiling He,et al.  RNA interference of ace1 and ace2 in Chilo suppressalis reveals their different contributions to motor ability and larval growth , 2011, Insect molecular biology.

[57]  Liang Gong,et al.  Silencing of Rieske iron-sulfur protein using chemically synthesised siRNA as a potential biopesticide against Plutella xylostella. , 2011, Pest management science.

[58]  Liping Sun,et al.  Chitosan kills bacteria through cell membrane damage. , 2004, International journal of food microbiology.

[59]  D. Heckel,et al.  Widespread pyrethroid resistance in Australian diamondback moth, Plutella xylostella (L.), is related to multiple mutations in the para sodium channel gene , 2011, Bulletin of Entomological Research.