DOI: 10.1126/science.1078052, 96 (2002);298 Science

et al.Janet HemingwayAn Overview of Insecticide Resistance

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An Overview of Insecticide ResistanceJanet Hemingway,1 Linda Field,2 John Vontas3

Insecticide resistance poses a serious threat to current malaria controlefforts. The Anopheles gambiae genome will enable identification of newresistance genes and will provide new molecular targets for the design ofmore effective insecticides.

The introduction of DDT for control of themosquito vector of malaria in the late 1940s,and the early eradication of malaria from theperiphery of its transmission range by residualhouse spraying with this insecticide, led directlyto the malaria eradication campaign of the1960s backed by the World Health Organisa-tion. At the end of the 1960s, the concept oferadication was formally dropped in favor ofsustainable control, largely because insecticideresistance was being selected for among themosquito species that transmit malaria. Therehas recently been a resurgence in antimalarialactivities with the Roll Back Malaria initiativeand Global Fund for Health, which supportextensive use of pyrethroid-impregnated bednets for mosquitocontrol campaigns in Africaand other malaria-endemic regions. It is notclear how much the current large-scale pyre-throid resistance of mosquitoes in West Africawill affect these efforts, and what will replacethe pyrethroid-treated nets if selection of mul-tiresistance mechanisms results in widespreadfailure of this strategy (1). Genomics will playan increasingly important part in the develop-ment of new malaria control tools (Fig. 1).Comparing the genomes of the malaria vectorAnopheles gambiae and of the fruit fly Dro-sophila melanogaster will not only yield newhormonal, neuronal, and regulatory molec-ular targets for the development of newclasses of insecticides, but will also allowus to attack existing insecticide resistanceand to boost the life-span of currently avail-able insecticides.

The resistant phenotype—an insect that sur-vives a dose of insecticide that would normallyhave killed it—is relatively easy to monitorwith direct insecticide bioassays. However, inmany cases the actual molecular mechanismsresponsible for the resistant phenotypes are stillunknown. The availability of the A. gambiaegenome will allow us to determine the exactmolecular changes that have resulted in resis-tant phenotypes. For example, up-regulation ofone or more members of the cytochrome P450gene families can produce broad-spectrum in-secticide resistance. Currently, the mechanismsthat regulate insecticide resistance are poorly

understood. Drosophila may not be a goodmodel in this respect because it is not an insectpest, and so is not subjected directly to large-scale insecticide control programs. In contrast,A. gambiae has multiple resistance mechanismsthat have been field-selected in both East andWest Africa through exposure to DDT andpyrethroids (2, 3).

Research into insecticide resistance is obvi-ously ripe for the move from the static genomemap to the functional genomics approach,which will allow us to understand the evolutionof resistance in these complex organismsthrough modulation of gene expression. Mate-rial from East Africa has already been subjectedto standard genetic quantitative trait loci (QTL)mapping, which has defined a polytene chro-mosome region within which the regulator ofP450 gene expression must be encoded (4).The availability of the A. gambiae genomesequence now allows us to use new molecularmicrosatellite markers from the sequence tonarrow down this control region to a few kilo-bases of DNA. Open reading frames can thenbe identified and candidate genes analyzed forfunction with recently developed anophelinetransformation technology. Regulatory genescontrolling the expression of glutathionetransferases (enzyme families that are impor-

tant for protecting insect cells from insecti-cides) will be similarly defined from QTLsthat are already mapped to the A. gambiaepolytene chromosomes.

In Culex mosquitoes and aphids, elevatedesterases confer organophosphate resistancethrough gene amplification, with multiplecopies of DNA amplicons of about 30 kbbeing integrated stably into the insect ge-nome, either contiguously or, in the case ofaphids, sometimes disparately (5, 6). Theresistant phenotype results from a complextissue-specific interplay of differential up-regulation of these amplified genes and in thecase of aphids involves changes in DNAmethylation (7). The sequenced A. gambiaegenome is from an insecticide-susceptiblestrain, and there are obvious orthologs for theamplified Culex esterases. To date, there is noevidence of esterase gene amplification–based resistance in any Anopheles species.However, as new resistant strains of A.gambiae are investigated, amplificationsmay well be found, and analysis of thegenome sequence surrounding the ampli-cons will allow us to elucidate the size ofthe units and may shed light on the ampli-fication mechanisms.

The availability of the complete A. gam-biae genome sequence should stimulate arapid shift in research aimed at improving themanagement of insecticide resistance. If theproblem of resistance is subdivided into threestages—detection, monitoring, and manage-

1Liverpool School of Tropical Medicine, Liverpool L35QA, UK. 2Rothamsted, Harpenden, Herts AL5 2JQ,UK. 3Institute of Molecular Biology and Biotechnolo-gy, Crete.

Fig. 1. Potential steps in moving from A. gambiae genomics to improved, insecticide-basedstrategies for controlling transmission of malaria.

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ment—then the benefits of this genomesequence become obvious. The genomesequence will enable access to the major regu-latory genes involved in resistance, particu-larly if orthologous regulators control meta-bolically based resistance in insects generally.For example, management of resistance inpractice currently involves basic rotationsand mixtures or mosaics of different insecti-cides. Access to insect-specific metabolicenzyme regulators will provide a target for“add-ons” to current insecticides, whichshould expand their natural life-span byblocking common resistance pathways whileleaving mammalian toxicity unaffected.

Insecticide resistance can result from directchanges to the proteins that normally bind to theinsecticides. For example, mutations in sodiumchannels (the target of DDT and pyrethroids)and in acetylcholinesterase (AChE; the target oforganophosphates and carbamates) have beenwell documented in many insect species includ-ing, in the case of sodium channels, mosquitoes(3, 8). The Anopheles genome will provideinformation on the target site genes, facilitatingcloning and mutagenesis studies to determinethe precise nature of the mutations and to aid inpredicting interactions between insect proteinsand insecticides. In the longer term, this couldlead to new insecticidal molecular targets. Thisapproach may be especially important forAChE as there is increasing evidence for mul-tiple AChE genes from the Anopheles and Dro-sophila genome databases (9). Two AChEgenes are apparent in the A. gambiae genome as

detailed by Ranson et al. in this issue (10), andto date no resistance-linked mutations havebeen identified in mosquitoes predominantly instudies on the sex-linked AChE gene. TheAnopheles genome in conjunction with that ofDrosophila also provides sequences of nicotin-ic acetylcholine receptor subunits, which willfacilitate their cloning from other insect species.This receptor is the target of an important newgroup of agrochemicals, but until now studiesof insect receptors have relied on coexpressionof insect genes with a vertebrate subunit (11).

Many instances of resistance, resulting froma change in a single regulator gene, may triggercomplex cascades of expression of unrelatedgenes. The presence of the full genome se-quence will prompt a move away from thereductionist approach that has dominated thelast two decades of resistance research. Such anapproach has tended to result in a lack of ap-preciation for how the large physiologicalchanges that often accompany resistance caninfluence other characteristics of the insect vec-tor. For example, many of the large scale-upregulations of enzyme families that accompanyinsecticide resistance result in profound chang-es in oxidative stress levels in the cells wherethese enzymes are expressed. These are oftenthe identical tissues in which parasites or virus-es reside during transmission by insect vectorsfrom one human to another.

Microarray and cell biology approaches arebeing used to define mechanisms in A. gambiaethat enable this insect vector to be refractory toinfection by malaria and other parasites. Avail-

able data already strongly indicate that an en-hanced ability of the refractory insects to toler-ate oxidative stress is integral to their ability toresist parasite infection (12). The convergenceof these two lines of resistance research will begreatly facilitated by our ability to look at reg-ulation patterns in different phenotype combi-nations across the whole genome. There arealready indications that these are not mutuallyexclusive systems; for example, filarial para-sites fail to develop in highly insecticide-resis-tant Culex mosquitoes (13).

Overall, the A. gambiae genome providesus with exciting opportunities to move fromknowledge and understanding of how resis-tance genes work to the practical applicationof that knowledge in the field. This willfundamentally improve the control of malariaand other important vector-borne diseasesand will contribute to the wider studies ofresistance in agricultural pests.

References1. C. F. Curtis et al., Philos. Tran. R. Soc London Ser. B.353, 1769 (1998).

2. H. Ranson et al., Insect Mol. Biol. 9, 499 (2000).3. F. Chandre et al., Bull. WHO 77, 230 (1999).4. H. Ranson et al., Insect Mol. Biol. 11, 409 (2002).5. R. L. Blackman et al., Heredity 82, 80 (1999).6. M. G. Paton et al., Biochem. J. 346, 17 (2000).7. L. M. Field, Biochem. J. 349, 863 (2000).8. H. Ranson et al., Insect Mol. Biol. 9, 491 (2000).9. M. Grauso et al., FEBS Lett. 424, 279 (1998).10. H. Ranson et al., Science 298, 179 (2002).11. Y Huang et al., Neurosci. Lett. 284, 116 (2000).12. G. Dimopoulos et al., Proc. Natl. Acad. Sci. U.S.A. 99,

8814 (2002).13. L. McCarroll et al., Nature 407, 961 (2000).

V I E W P O I N T

The Anopheles Genome and ComparativeInsect Genomics

Thomas C. Kaufman,1 David W. Severson,2 Gene E. Robinson3

The Anopheles gambiae genome sequence, coupled with the Drosophilamelanogaster genome sequence, provides a better understanding of theinsects, a group that contains our friends, foes, and competitors.

The Phylum Arthropoda is the most species-rich and morphologically diverse animalgroup on the planet. Since their appearance inthe Early Cambrian and their subsequent ra-diation, arthropods have come to inhabit anddominate the vast majority of ecological hab-itats. From the many different arthropodgroups that existed in the Early Cambrian,

only four have survived to the present: theChelicerata, Myriapoda, Crustacea, and In-secta. Members of these four groups plagueus, transmit diseases, benefit us, and feed us.The genome sequence of the African malariavector, the mosquito Anopheles gambiae, re-ported on page 129 of this issue (1), coupledwith the Drosophila melanogaster genomesequence (2), provides us with new insightsinto the genetic makeup of two members ofthe Insecta, arguably the dominant group ofarthropods.

The genome sequences of A. gambiae andPlasmodium falciparum, the malaria parasiteit transmits (3, 4), will yield fresh insights

into parasite and vector biology that will leadto more efficient disease control strategies. Anew approach to vector-borne disease controlbased on the genetic manipulation of themosquito has already received considerableattention (5, 6). The A. gambiae genomesequence will accelerate efforts to identifymolecules that can inhibit parasite develop-ment in the vector and subsequently preventtransmission to humans. Stable germlinetransformation has been demonstrated forseveral vector mosquitoes (7–9). This is en-couraging news given that transgenicanopheline mosquitoes engineered to expressan anti-Plasmodium molecule turn out to beinefficient vectors for disease transmission inthe laboratory (10).

Of the �3500 mosquito species, molecu-lar information exists for only a small num-

1Department of Biology, Indiana University, Bloom-ington, IN 47405, USA. 2Center for Tropical DiseaseResearch and Training, Department of Biological Sci-ences, University of Notre Dame, IN 46556, USA.3Department of Entomology and Neuroscience Pro-gram, University of Illinois, 505 South Goodwin Ave-nue, Urbana, IL 61801, USA.

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