Insect Biochemistry and Molecular Biology 34 (2004) 871–877 www.elsevier.com/locate/ibmb High-throughput detection of knockdown resistance in Myzus persicae using allelic discriminating quantitative PCR James A. Anstead a, , Martin S. Williamson b , Ioannis Eleftherianos a , Ian Denholm a a Department of Plant and Invertebrate Ecology, Rothamsted Research, Harpenden AL5 2JQ, UK b Department of Biological Chemistry, Rothamsted Research, Harpenden AL5 2JQ, UK Received 1 March 2004; received in revised form 27 May 2004; accepted 1 June 2004 Abstract The peach–potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimi- nation PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman 1 MGB probes. In combination with assays for elevated carboxylesterase levels and modified acet- ylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK. # 2004 Elsevier Ltd. All rights reserved. Keywords: Myzus persicae; Insecticide resistance; Real-time PCR; kdr 1. Introduction Myzus persicae (Sulzer) (Hemiptera: Aphididae), the peach–potato aphid, is an important pest of a number of agriculturally important crops, causing damage through feeding and virus transmission. Control is achieved primarily by the application of insecticides, often with multiple applications each year. This has led to the evolution of insecticide resistance conferred by three genetically independent mechanisms (Devonshire et al., 1998). The first of these to be characterised was the over-production of one of two closely related car- boxylesterases (E4 and FE4), that sequester and detox- ify certain insecticides, giving high resistance to organophosphates and most carbamates and also lim- ited resistance to pyrethroids (e.g. deltamethrin) (Devonshire and Moores, 1982). This increase in car- boxylesterase synthesis/production is caused by ampli- fication of the esterase structural genes (Field et al., 1988). Depending on the amount of carboxylesterase produced, individuals are classified into one of four somewhat arbitrary categories: S (susceptible), R 1 (moderately resistant), R 2 (highly resistant) or R 3 (extremely resistant) (Devonshire et al., 1986). Two types of target-site insensitivity conferring insec- ticide resistance have also been found in M. persicae. Individuals with modified acetylcholinesterase (MACE) show high levels of resistance to dimethyl carbamates such as pirimicarb and triazamate (Moores et al., 1994). The MACE phenotype has recently been shown to be associated with a single amino acid substitution (serine to phenylalanine, S431F) within the active site of the enzyme (Andrews et al., 2002; Nabeshima et al., 2003). M. persicae with overproduced carboxylesterases and MACE are now widespread in Europe, including the UK (Foster et al., 1998; Field and Foster, 2002; Mazzoni and Cravedi, 2002). Mutations in a voltage- gated sodium channel gene (homologous to the Drosophila para gene) conferring resistance to pyre- throids (e.g. deltamethrin) were first identified in the  Corresponding author. Tel.: +44-1582-763133x2353; fax: +44- 1582-760981. E-mail address: ansteadj@bbsrc.ac.uk (J.A. Anstead). 0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.06.002