Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 36 (2006) 642–653 Acetylcholinesterase mutation in an insecticide-resistant population of the codling moth Cydia pomonella (L.) Stefano Cassanelli a,Ã , Maritza Reyes b , Magali Rault b , Gian Carlo Manicardi a , Benoıˆt Sauphanor b a Department of Agricultural Science, University of Modena and Reggio Emilia, Reggio Emilia, Via JFK Kennedy 17/19, RE 42100, Italy b UMR Ecologie des Inverte´bre´s, INRA Site Agroparc, 84 914 Avignon Cedex 09, France Received 21 December 2005; received in revised form 13 May 2006; accepted 15 May 2006 Abstract Two strains of Cydia pomonella (L.) (Lepidoptera: Tortricidae) were selected in the lab by exposure to increasing concentrations of diflubenzuron (Rdfb strain) or azinphos-methyl (Raz strain). Insecticide bioassays showed that the adults of the Rdfb strain exhibited a 2.6-fold and a 7.7-fold resistance ratio to azinphos-methyl and carbaryl, respectively compared to a susceptible strain (S) whereas the adults of the Raz strain exhibited a 6.7-fold resistance ratio to azinphos-methyl and a 130-fold resistance ratio to carbaryl. In the Raz strain, a target site resistance mechanism was suggested by the inhibition of acetylcholinesterase (AChE) activity. In fact the ki values did not discriminate the S and Rdfb strains, while the Raz strain exhibited a 1.7-fold and a 14-fold increase in ki value compared to the S strain for azinphos-methyl oxon and carbaryl, respectively. To verify this hypothesis, two cloned AChE cDNAs sequences (named cydpom-ace2 e cydpom-ace1) were compared between the susceptible and the resistant strains. No difference in the deduced amino acid sequence was found in cydpom-ace2 (orthologous to the Drosophila melanogaster AChE). In the putative cydpom-ace1 (paralogous to the Drosophila AChE), a single amino acid substitution F399V was exclusively present in the Raz strain. The F399 lined the active site of the enzyme and the F399V substitution likely could influence the accessibility of different types of inhibitors to the catalytic site of the insensitive cydpom-ace1. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cydia pomonella; Insensitive acetylcholinesterase; Insecticide resistance 1. Introduction The ability of insects to become resistant to insecticides is under dependence of target site insensitivity and of pharmacokinetic processes modifying the rate or the properties of the toxin delivered to the target site. In the codling moth, Cydia pomonella L., cross-resistances to different classes of insecticides have been related to enhanced detoxification (Sauphanor et al., 1997; Bouvier et al., 2002), whereas a kdr mutation through leucine to phenylalanine replacement at position 1014 of the voltage- dependent sodium channel gene has been identified in pyrethroid resistant populations (Brun-Barale et al., 2005). Organophosphorous (OP) insecticides are the most inten- sively used against C. pomonella worldwide and resistances to these compounds have frequently been recorded in this species (Welter et al., 1991; Bush et al., 1993; Knight et al., 1994; Sauphanor et al., 1998; Dunley and Welter, 2000; Reuveny and Cohen, 2004). OPs irreversibly inhibit acetylcholinesterase (AChE, EC 3.1.1.7) which is a key enzyme catalysing the hydrolysis of the neurotransmitter acetylcholine in the nervous system, thereby ending transmission of nerve impulses at cholinergic synapses. Modifications of this target protein resulting from intensive selection with OPs have been identified in several species (Mutero et al., 1994; Baxter and Barker, 1998; Chen et al., 2001; Weill et al., 2003; Anazawa et al., 2003; Li and Han, 2004). It has been shown that AChE resistance to insecticides is associated with seven amino acid replace- ments in four resistant strains of Drosophila melanogaster (Mutero et al., 1994) and three resistant strains of Musca ARTICLE IN PRESS www.elsevier.com/locate/ibmb 0965-1748/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2006.05.007 Ã Corresponding author. Tel.: +39 0522 522049; fax: +39 0522 522027. E-mail address: stcass@unimore.it (S. Cassanelli).