Insect Molecular Biology (1998) 7(2), 107±120 A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens * C. A. Malcolm, 1 D. Bourguet, 2 { A. Ascolillo, 1 S. J. Rooker, 1 { C. F. Garvey, 1 L. M. C. Hall, 3 N. Pasteur 2 and M. Raymond 2 1 School of Biological Sciences, Queen Mary and West®eld College, University of London, London, UK, 2 Institut des Sciences de l'Evolution (UMR 5554), Laboratoire Ge  ne  tique et Environnement, Universite  Montpellier II (C.C. 065), Montpellier, France, and 3 Department of Medical Microbiology, London Hospital Medical College, University of London, London, UK Abstract An acetylcholinesterase (AChE) gene, Ace.x, showing 93% identity of deduced amino acid sequence to Anopheles stephensi Ace has been cloned from a Culex pipiens strain homozygous for insensitive AChE (iAChE) mediated insecticide resistance. DNA sequence of genomic DNA clones identi®ed exons 2±5. RFLP of six clones indicated four possible alleles. Linkage analysis located Ace.x to chromosome I, less than 0.8 centimorgans from the sex locus, whereas the locus conferring resistance was 2.0 centimorgans from plum-eye on chromosome II. Ace.1 coding for AChE1, which is associated with resistance, is therefore auto- somal. We propose that Ace.x is the recently postu- lated Ace.2 coding for the biochemically distinct AChE2, which is not associated with resistance. Keywords: acetylcholinesterase, sex-linked gene, Culex pipiens, insecticide resistance. Introduction AChE (EC 3.1.1.7) catalyses the hydrolysis of the neurotransmitter acetylcholine (ACh) and thus is a key enzyme at cholinergic synapses in the insect central nervous system (see Toutant, 1989). Its inhibition by organophosphates and carbamates causes a desensi- tization of the ACh receptor leading to a blockage of nerve impulse transmission (Eldefrawi, 1976; Elde- frawi et al. , 1982). Culex pipiens is a complex of species and/or geo- graphical, or physiological forms, with a worldwide distribution (Harbach et al. , 1985; Miller et al. , 1996). In tropical and subtropical regions they are major vectors of ®lariasis and arboviruses and in many temperate regions a serious biting nuisance. C. pipiens has been subject to extensive control measures using insecti- cides and has evolved multiple resistance mechan- isms. Important are two conferring resistance to organophosphates: one involves overproduction of esterases conferring an increased ability to detoxify the insecticide, the other is mediated by an acetylcho- linesterase (AChE) insensitive to insecticide inhibition (Bourguet et al ., 1996a; Raymond & Pasteur, 1996). Propoxur-insensitive AChE in C. pipiens was ®rst reported in a strain (MSE) selected from a resistant population collected in Southern France (Raymond et al ., 1985). Genetic analysis of this strain attributed most of the insecticide resistance to a single gene, which from the previous studies coded for an insensi- tive AChE (Raymond et al ., 1985, 1987). Insensitive AChEs have been detected in more than twenty-®ve insect species (Fournier & Mute ro, 1994). In order to identify the mutation(s) conferring insecticide insensitivity, genes coding AChE (Ace ) have been cloned and sequenced in some insect species includ- ing: Drosophila melanogaster (Hall & Spierer, 1986), Anopheles stephensi (Hall & Malcolm, 1991), Aedes aegypti (Anthony et al. , 1995), Leptinotarsa decem- lineata (Zhu & Clark, 1995) and Musca domestica (Williamson & Devonshire, pers. comm.). Although vertebrates have two cholinesterases, AChE and butyrylcholinesterase (BuChE, EC 3.1.1.8), *The results of this paper were determined separately by two labora- tories (1 + 2) and combined into a single paper for clarity. { Present address: Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver B.C., Canada V6T 1Z4. { Present address: Department of Molecular Genetics, Heart Science Centre, Hare®eld Hospital, Hill End Road, Hare®eld, Middlesex UB9 6JH, UK. Received 6 January 1997; accepted 27 August 1997. Correspondence: Dr C. A. Malcolm, School of Biological Sciences, Queen Mary and West®eld College, University of London, Mile End Road, London E1 4NS, UK. e-mail: c.a.malcolm@qmw.ac.uk. # 1998 Blackwell Science Ltd 107