Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism

Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4′-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains.

[1]  M. Sharakhova,et al.  An Integrated Linkage, Chromosome, and Genome Map for the Yellow Fever Mosquito Aedes aegypti , 2013, PLoS neglected tropical diseases.

[2]  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.

[3]  A. Lynd,et al.  A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: functional characterisation and signatures of selection. , 2012, Insect biochemistry and molecular biology.

[4]  D. Gubler,et al.  Dengue/dengue hemorrhagic fever: the emergence of a global health problem. , 1995, Emerging infectious diseases.

[5]  Lee Ching Ng,et al.  The 2005 dengue epidemic in Singapore: epidemiology, prevention and control. , 2008, Annals of the Academy of Medicine, Singapore.

[6]  David Weetman,et al.  Does kdr genotype predict insecticide-resistance phenotype in mosquitoes? , 2009, Trends in parasitology.

[7]  J. Oakeshott,et al.  Biochemical Genetics and Genomics of Insect Esterases , 2005, Reference Module in Life Sciences.

[8]  J. Morgan,et al.  Field-Caught Permethrin-Resistant Anopheles gambiae Overexpress CYP6P3, a P450 That Metabolises Pyrethroids , 2008, PLoS genetics.

[9]  J. Casida,et al.  Metabolism of trans- and cis-permethrin, trans- and cis-cypermethrin, and decamethrin by microsomal enzymes. , 1979, Journal of agricultural and food chemistry.

[10]  M. Goddard,et al.  Copy Number Variation and Transposable Elements Feature in Recent, Ongoing Adaptation at the Cyp6g1 Locus , 2010, PLoS genetics.

[11]  M. Takagi,et al.  Widespread Distribution of a Newly Found Point Mutation in Voltage-Gated Sodium Channel in Pyrethroid-Resistant Aedes aegypti Populations in Vietnam , 2009, PLoS neglected tropical diseases.

[12]  J. Lewis,et al.  Probit Analysis (3rd ed). , 1972 .

[13]  J. Morgan,et al.  Pyrethroid and DDT cross‐resistance in Aedes aegypti is correlated with novel mutations in the voltage‐gated sodium channel gene , 2003, Medical and veterinary entomology.

[14]  L. Field,et al.  Insecticide resistance in the aphid Myzus persicae (Sulzer): chromosome location and epigenetic effects on esterase gene expression in clonal lineages , 2003 .

[15]  Taichi Q. Itoh,et al.  Point mutations in domain II of the voltage-gated sodium channel gene in deltamethrin-resistant Aedes aegypti (Diptera: Culicidae) , 2010 .

[16]  Janet Hemingway,et al.  The molecular basis of insecticide resistance in mosquitoes. , 2004, Insect biochemistry and molecular biology.

[17]  L. Field Methylation and expression of amplified esterase genes in the aphid Myzus persicae (Sulzer). , 2000, The Biochemical journal.

[18]  G. Georghiou,et al.  Mechanisms responsible for high levels of permethrin resistance in the house fly , 1986 .

[19]  L. Ng,et al.  Pyrethroid Resistance in Aedes aegypti Larvae (Diptera: Culicidae) from Singapore , 2014, Journal of medical entomology.

[20]  J. Hemingway,et al.  Genomic analysis of detoxification genes in the mosquito Aedes aegypti. , 2008, Insect biochemistry and molecular biology.

[21]  F. Winteringham,et al.  Absorption and metabolism of [14C] pyrethroids by the adult housefly, Musca domestica L., in vivo. , 1955, The Biochemical journal.

[22]  J Hemingway,et al.  Insecticide resistance in insect vectors of human disease. , 2000, Annual review of entomology.

[23]  A. Hobbs,et al.  A Piperonyl Butoxide Synergizable Resistance to Permethrin in Helicoverpa armigera Which Is Not Due to Increased Detoxification by Cytochrome P450 , 1993 .

[24]  Y. Nomura,et al.  A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids. , 2011, Insect biochemistry and molecular biology.

[25]  John Vontas,et al.  Insecticide resistance in the major dengue vectors Aedes albopictus and Aedes aegypti , 2012 .

[26]  S. Foster,et al.  Amplification of a Cytochrome P450 Gene Is Associated with Resistance to Neonicotinoid Insecticides in the Aphid Myzus persicae , 2010, PLoS genetics.

[27]  P. Batterham,et al.  A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital. , 2006, Insect biochemistry and molecular biology.

[28]  J. Hemingway,et al.  Quantitative Trait Loci Mapping of Genome Regions Controlling Permethrin Resistance in the Mosquito Aedes aegypti , 2008, Genetics.

[29]  S. Reynaud,et al.  Do pollutants affect insecticide-driven gene selection in mosquitoes? Experimental evidence from transcriptomics. , 2012, Aquatic toxicology.

[30]  S. Valles,et al.  Effects of the Synergists Piperonyl Butoxide and S,S,S-Tributyl Phosphorotrithioate on Propoxur Pharmacokinetics in Blattella germanica (Blattodea: Blattellidae) , 2001, Journal of economic entomology.

[31]  K. Goh,et al.  Dengue--a re-emerging infectious disease in Singapore. , 1997, Annals of the Academy of Medicine, Singapore.

[32]  B. F. Stone A formula for determining degree of dominance in cases of monofactorial inheritance of resistance to chemicals. , 1968, Bulletin of the World Health Organization.

[33]  P. Somboon,et al.  A novel F1552/C1552 point mutation in the Aedes aegypti voltage-gated sodium channel gene associated with permethrin resistance , 2010 .

[34]  May R Berenbaum,et al.  Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. , 2007, Annual review of entomology.

[35]  S. Kasai,et al.  Global spread and genetic variants of the two CYP9M10 haplotype forms associated with insecticide resistance in Culex quinquefasciatus Say , 2013, Heredity.

[36]  D. Gubler Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. , 2002, Trends in microbiology.

[37]  C. Qiao,et al.  The same esterase B1 haplotype is amplified in insecticide-resistant mosquitoes of the Culex pipiens complex from the Americas and China , 1995, Heredity.

[38]  T. Tomita,et al.  cDNA and deduced protein sequence of CYP6D1: the putative gene for a cytochrome P450 responsible for pyrethroid resistance in house fly. , 1995, Insect biochemistry and molecular biology.

[39]  D. Gubler,et al.  Dengue Prevention and 35 Years of Vector Control in Singapore , 2006, Emerging infectious diseases.

[40]  T. Shono Pyrethroid Resistance: Importance of the kdr-Type Mechanism , 1985 .

[41]  D. Gubler,et al.  Dengue and Dengue Hemorrhagic Fever , 1998, Clinical Microbiology Reviews.

[42]  R. ffrench-Constant,et al.  A Single P450 Allele Associated with Insecticide Resistance in Drosophila , 2002, Science.

[43]  J. Hemingway,et al.  Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in México , 2009, PLoS neglected tropical diseases.

[44]  Stone Bf A formula for determining degree of dominance in cases of monofactorial inheritance of resistance to chemicals. , 1968 .

[45]  Pauline Aw,et al.  Dengue Virus Surveillance for Early Warning, Singapore , 2010, Emerging infectious diseases.

[46]  S. Kasai,et al.  P450 monooxygenases are an important mechanism of permethrin resistance in Culex quinquefasciatus Say larvae , 1998 .

[47]  W. Muir,et al.  Genome‐wide analysis of phenobarbital‐inducible genes in Drosophila melanogaster , 2006, Insect molecular biology.

[48]  H. Ranson,et al.  Development and application of a simple colorimetric assay reveals widespread distribution of sodium channel mutations in Thai populations of Aedes aegypti. , 2008, Acta tropica.

[49]  Yuzhe Du,et al.  Diversity and Convergence of Sodium Channel Mutations Involved in Resistance to Pyrethroids. , 2013, Pesticide biochemistry and physiology.

[50]  D. Gubler,et al.  Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses , 2004, Nature Medicine.

[51]  J. Clark,et al.  Agrochemical resistance : extent, mechanism, and detection , 2001 .

[52]  E. R. Johnson,et al.  Quasi-synergism and penetration of insecticides. , 1972, Journal of Economic Entomology.

[53]  S. Kasai,et al.  Genomic structures of Cyp9m10 in pyrethroid resistant and susceptible strains of Culex quinquefasciatus. , 2010, Insect biochemistry and molecular biology.

[54]  M. Paine,et al.  Pinpointing P450s Associated with Pyrethroid Metabolism in the Dengue Vector, Aedes aegypti: Developing New Tools to Combat Insecticide Resistance , 2012, PLoS neglected tropical diseases.

[55]  H. Ranson,et al.  Pyrethroid resistance in Aedes aegypti from Grand Cayman. , 2010, The American journal of tropical medicine and hygiene.

[56]  Ian Denholm,et al.  Delayed cuticular penetration and enhanced metabolism of deltamethrin in pyrethroid-resistant strains of Helicoverpa armigera from China and Pakistan. , 2006, Pest management science.

[57]  Y. Nomura,et al.  Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel , 2013, Proceedings of the National Academy of Sciences.

[58]  J. Casida,et al.  Metabolism of permethrin isomers in American cockroach adults, house fly adults, and cabbage looper larvae , 1978 .

[59]  S. Dai,et al.  A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti. , 2009, Insect biochemistry and molecular biology.

[60]  衛介 舩城,et al.  ピレスロイド抵抗性イエバエにおけるフェンバレレートの in vitro および in vivo 代謝 , 1994 .

[61]  Evgeny M. Zdobnov,et al.  Genome Sequence of Aedes aegypti, a Major Arbovirus Vector , 2007, Science.

[62]  J. Bonnet,et al.  Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies) , 2009, BMC Genomics.

[63]  L. Harrington,et al.  Cytochrome P450 monooxygenase-mediated permethrin resistance confers limited and larval specific cross-resistance in the southern house mosquito, Culex pipiens quinquefasciatus , 2007 .

[64]  J. Hemingway,et al.  Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti , 2012, Insect molecular biology.

[65]  Shinji Kasai,et al.  Overexpression of cytochrome P450 genes in pyrethroid-resistant Culex quinquefasciatus. , 2010, Insect biochemistry and molecular biology.

[66]  R. Feyereisen,et al.  4.1 – Insect Cytochrome P450 , 2005 .

[67]  L. Ng,et al.  First detection of a putative knockdown resistance gene in major mosquito vector, Aedes albopictus. , 2011, Japanese journal of infectious diseases.

[68]  A. Devonshire,et al.  Metabolism of Esfenvalerate by Pyrethroid-Susceptible and -Resistant Australian Helicoverpa armigera (Lepidoptera: Noctuidae) , 1995 .

[69]  John Vontas,et al.  Gene Amplification, ABC Transporters and Cytochrome P450s: Unraveling the Molecular Basis of Pyrethroid Resistance in the Dengue Vector, Aedes aegypti , 2012, PLoS neglected tropical diseases.

[70]  S. Lee,et al.  An improved method for preparation, stabilization, and storage of house fly (Diptera: Muscidae) microsomes. , 1989, Journal of economic entomology.

[71]  M. Monastirioti,et al.  Transgenic expression of the Aedes aegypti CYP9J28 confers pyrethroid resistance in Drosophila melanogaster , 2012 .

[72]  S. Kasai,et al.  Cis-acting mutation and duplication: History of molecular evolution in a P450 haplotype responsible for insecticide resistance in Culex quinquefasciatus. , 2011, Insect biochemistry and molecular biology.

[73]  K. Dong,et al.  Mechanisms responsible for cypermethrin resistance in a strain of German cockroach, Blattella germanica , 2000 .

[74]  J. G. Scott,et al.  Cytochromes P450 and insecticide resistance. , 1999, Insect biochemistry and molecular biology.

[75]  J. Bergé,et al.  Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism. , 2004, European journal of biochemistry.

[76]  D. Bull,et al.  In vivo and in vitro fate of fenvalerate in house flies , 1990 .

[77]  J. Hemingway,et al.  A mutation in the voltage‐gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti , 2007, Insect molecular biology.

[78]  G. B. Craig,et al.  Genetic distortion of sex ratio in a mosquito, Aedes aegypti. , 1966, Genetics.