3.1 Vectorial capacity
Anopheles gambiae mosquitoes are the world's most important malaria vector, and their efficiency for Plasmodium transmission depends on many factors, such as their remarkable preference for humans as host for blood-feeding, their high susceptibility to parasite infection and their longevity. The genetic basis regulating mosquito behaviours that are key to the ability of the mosquito to transmit disease are being extensively studied. Olfaction plays a crucial role in shaping behaviours such as host seeking and feeding, which determine the vectorial capacities of different mosquito species, and recent evidence suggests that in some cases it may also be involved in mating choices.19 The determination of the A. gambiae genome sequence has enabled the key components of the mosquito olfactory system to be identified. A total of 276 G protein-coupled receptors (GPCRs), which include 79 candidate odourant receptors (AgORs) and 76 candidate gustatory receptors (GPRgrs), have been identified.20 Comparative genomics showed that the AgOR family is rapidly expanding compared with the odorant receptors of Drosophila.20 Subsequent studies utilizing the genome data currently being generated for several vector and non-vector insects are helping to elucidate the role of the AgORs in shaping species-specific chemosensory processes that are likely to have evolved in the extremely anthropophilic A. gambiae mosquitoes. Experimental evidence based on transgenic expression of two candidate AgORs into targeted olfactory receptor neurons of Drosophila melanogaster Meig. has already confirmed the role of specific ORs in olfactory signalling in A. gambiae.21
The full catalogue of putative odourant-binding proteins (OBPs) has also been identified in A. gambiae. These proteins are the most abundantly expressed in olfactory tissues,22–24 and they have been postulated to either act as odourant carriers and/or mediate the catalytic removal of odourants from the lymph. As a whole, the gene families implicated in olfactory processes are regarded as promising novel targets for the design of novel mosquito attractants and/or repellents, and for the development of other pharmacological applications for mosquito control.
3.2 Vector–parasite interactions
The A. gambiae immune system has also been the focus of an impressive series of studies that have led to the identification of various mosquito molecules regulating parasite development. Comparative genomics have demonstrated large similarities between the immune signalling pathways of Drosophila and Anopheles,25 and in general have shown that only around 10% of the putative Anopheles proteins have no detectable homologues in any other genome that has been sequenced.26 The availability of genetic manipulation of Plasmodium parasites27 and of genetic and molecular tools such as microsatellite markers and microarray platforms, combined with the use of RNA interference (RNAi) in mosquitoes,28 is helping to unravel the genetic basis regulating Anopheles susceptibility or refractoriness to Plasmodium infection. One of the most promising studies looked at natural variation in the field using microsatellite markers to analyse alleles naturally associated with refractoriness to P. falciparum infections in a field A. gambiae population.17 This study identified a series of genomic loci implicated in protecting mosquitoes from parasite infections. A more detailed analysis of these loci identified one gene, Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1), as playing a major role in natural refractoriness.
Reverse genetic approaches have also been utilized for the identification of a battery of agonists and antagonists to Plasmodium development utilizing the model P. berghei murine parasites.29, 30 RNAi is applied to Anopheles mosquitoes by means of direct injection of in vitro synthesized dsRNA molecules into the thorax of adult mosquitoes.28 Two potentially immune genes, the complement-like thioester-containing protein TEP1 and the leucine-rich immune protein LRIM1, have been implicated as novel antagonists to Plasmodium development, causing parasite death in the midgut.29, 30 In contrast, two A. gambiae C-type lectins, CTL4 and CTLMA2, the RNAi-induced knock-out of which resulted in complete and partial melanization of the ookinetes respectively, have been shown to act as agonists of Plasmodium by protecting the ookinete from melanization in the mosquito midgut.30
The serine protease inhibitor genes (serpins), which function as negative regulators of the prophenoloxidase (PPO) activation pathway that invokes the melanization of malaria parasites, have also recently been shown to be involved in parasite development within the mosquito midgut.31 RNAi-mediated knockdown of SRPN2 caused a 97% reduction in parasite prevalence through increased ookinete lysis and melanization.31 Knockdown of SRPN6 in A. stephensi mosquitoes instead induced a substantial increase in parasite numbers, whereas its depletion in A. gambiae delayed progression of parasite lysis.32 CLIPB14 and CLIPB15, which share structural similarity to proteases involved in PPO activation in other insects, have also been implicated in parasite killing.33 How these immune genes are mutually coordinated or excluded in response to Plasmodium infection has not yet been clarified, although some evidence seems to indicate that NF-κB-like transcription factors present in the Anopheles cells play a role in their regulation.34, 35
Besides immunity genes, other factors have been implicated in shaping vector–parasite interactions. Recently, a microarray analysis of over 9000 putative mosquito genes identified 650 genes that are transcriptionally regulated by ingestion of a blood meal infected with P. berghei parasites.36 These encompass various functional classes such as actin-cytoskeleton, microtubule organization and movement, anti-apoptotic and redox-related genes. The parallel RNAi analysis of 11 of these genes identified additional agonists and antagonists to Plasmodium development. In particular, silencing of the RFABG gene (a retinoid- and fatty-acid-binding glycoprotein, precursor of apolipophorin I and II, two major components of the insect lipid transporter)37 induced a 3.9-fold reduction in oocyst numbers, as well as a total, parasite-independent inhibition of egg development in ovaries of blood-fed mosquitoes.36
These studies show how targeting crucial mosquito genes can alter the vector–parasite relationship in laboratory settings. In principle this information could lead to the development of novel, Plasmodium-tailored strategies for intervention. These could be based, for instance, on the design of compounds exclusively targeting mosquito factors relevant to parasite development or on the development of transgenic mosquitoes overexpressing known parasite antagonists or temporally and spatially deprived of Plasmodium agonists, to be used in population replacement strategies, as shown later. However, a few reservations about the feasibility of these approaches for malaria control need to be raised. First of all, some evidence derived from work on SRPN2 and other genes indicates that laboratory studies based on the P. berghei mouse malaria model may in some instances not be a reliable predictor of the mosquito immune response against P. falciparum in the field38 (Michel K, private communication). In addition, it is clear that no single gene is sufficient on its own to prevent Plasmodium transmission completely, and hence multiple genes would have to be targeted to have a significant effect on parasite development. Importantly, as only a partial inhibition of protein expression by stable RNAi is generally achieved,39 complete blockade of transmission by this means is highly unlikely. Furthermore, targeting or overexpressing genes of the immune system would most likely have a detrimental effect on mosquito survival, as demonstrated in the case of SRPN2, the loss-of-function phenotype of which results in decreased mosquito survival.31 In general, in spite of the fascinating biology that they are revealing, the true potential of these approaches for malaria control still remains to be fully elucidated. Future studies will need to concentrate on the natural interactions occurring in the field between P. falciparum and its vectors, similar to those described by Riehle et al.,17 to identify genes showing unequivocal phenotypes impacting on the transmission of human malaria and to develop efficient ways to manipulate their function.
3.3 Investigating the mechanisms of insecticide resistance
The above methods hold promise for future novel methods to target Anopheles mosquitoes, but most of these strategies are many years from practical application. At present, and for the foreseeable future, the control of malaria vectors relies extensively on the use of indoor house spraying with residual insecticides and the use of insecticide-impregnated bednets. However, the lack of availability of licensed insecticides, coupled with the growing problem of insecticide resistance, is an area of concern for the sustainability of insecticide-based control programmes and is prompting studies on the identification of novel insecticidal targets and on the understanding of resistance mechanisms.
Genomic technologies are providing important clues to the genes responsible for insecticide resistance.40, 41 One approach to reduce the impact of insecticide resistance mechanisms is to block or delay the rate of metabolism of insecticides in the mosquitoes. To achieve this efficiently, it is necessary to identify the major enzymes responsible for insecticide detoxification. Microarrays have been used to identify specific members of the detoxification enzyme families whose expression is elevated in insecticide-resistant populations.40, 41 This has enabled a short list of candidate genes to be identified, and the enzymes are being functionally characterized to determine their role in insecticide metabolism. Among these is GTSE2, to date the only metabolic enzyme that has been unquestionably associated with insurgence of resistance in laboratory strains of DDT-resistant A. gambiae mosquitoes.42
The identification of genes implicated in resistance mechanisms will improve the tools available to assess the efficacy of insecticide-based malaria control strategies. Monitoring for the presence of insecticide resistance alleles in field populations provides an early warning mechanism for incipient resistance and also enables informed decisions to be made about the appropriate choice of alternative insecticides, should resistance have an operational effect on control. As evidence of this, molecular assays to detect mutations in the sodium channel gene, the target site of pyrethroid and DDT insecticides, are already routinely employed in many malaria control programmes.43