UNCORRECTED PROOF Insect Molecular Biology (2006) © 2006 The Royal Entomological Society 1 I M B 6 4 7 Operator: Wang Jingjing Dispatch: 29.03.06 PE: Rodney Tonge Journal Name Manuscript No. Proofreader: Liang Lifen No. of Pages: 7 Copy-editor: Fernando Alonso Blackwell Publishing Ltd Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility G. J. Lycett*†¶, L. A. McLaughlin‡¶, H. Ranson§, J. Hemingway§, F. C. Kafatos*, T. G. Loukeris† and M. J. I. Paine‡ * European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg D-69117, Germany; † Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Vassilika Vouton, 711 10 Heraklion, Crete, Greece; ‡ Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK; and § Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK Abstract We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae , important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides. Keywords: Introduction The mosquito Anopheles gambiae is the most important vector of the human malaria parasite Plasmodium falciparum . Mosquito control programmes, aimed at reducing the impact of the disease, are being seriously undermined by the emergence of resistance to pyrethroid insecticides (Ranson et al ., 2002). Previous genetic and biochemical evidence have shown the involvement of the cytochrome P450 gene family in insecticide resistance through increased metabolism (Feyereisen, 1999; Ranson et al ., 2002), although the exact mechanisms are unknown. Recent genome sequencing has revealed the A. gambiae genome carries over 100 P450 genes and a single gene for the P450 redox partner NADPH cytochrome P450 oxidoreductase (CPR) (Holt et al ., 2002). Cytochromes P450 are a superfamily of haem-containing mono-oxygenases involved in a host of critical biochemical pathways through the metabolism of a wide range of xenobiotic and endogenous compounds. All P450 mono- oxygenation reactions occurring in the endoplasmic reticulum require electrons supplied by CPR, a diflavin enzyme that contains flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors that shuttle electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) through a series of redox- coupled reactions to P450 (Paine et al ., 2004). As well as cytochromes P450, CPR is capable of reducing other electron acceptors, including cytochrome b 5 (Schenkman & Jansson, 1999) and squalene hypoxidase, which are both involved in sterol biosynthesis (Ono et al ., 1977) and haem oxygenase (Wang & de Montellano, 2003), involved in cellular haem homeostasis (Maines, 1997). To date little is known about the in vivo localization and precise role of the P450 mono-oxygenase complex in mosquitoes. Early work in other insects has shown that cytochrome P450s, cytochrome b 5 , and CPR are abundant in crude extracts derived from proximal intestine, Malpighian tubules, and fat bodies in the insecticide resistant (LPR) strain of house-fly (Scott & Lee, 1993), and therefore potentially implicating these tissues in insecticide metabolism. doi: 10.1111/j.1365-2586.2006.00647.x Received 02 December 2005; accepted after revision 30 January 2006. Correspondence: Dr Mark J. I. Paine, Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. Tel.: +44(0)1382 496420; fax: +44(0)1382 669993; e-mail: m.j.paine@dundee.ac.uk ¶These authors contributed equally to this work. 1