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Keywords:

  • malaria;
  • Anopheles;
  • Plasmodium;
  • vector control;
  • genomics

Abstract

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Remarkable progress has been made towards a deeper understanding of mosquito biology since the completion of the Anopheles gambiae Giles genome project. Combined with the development of efficient transgenic technologies for genetic modification of major vector species and the availability of powerful molecular, genetic and bioinformatics tools, this is allowing the identification of genes involved in mosquito biological functions crucial to malaria transmission, ranging from host-seeking behaviour and innate immunity to insecticide resistance. Moreover, population genetic studies are beginning to elucidate the complex structure of vector populations. Finally, novel methods for malaria control are emerging that are based on the use of genetically modified mosquitoes either to interrupt the journey of the Plasmodium parasite within its insect host or to suppress those mosquito species that function as vectors for parasite transmission. Copyright © 2007 Society of Chemical Industry


1 INTRODUCTION

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Malaria represents a leading cause of morbidity and mortality worldwide, causing over 1 million deaths each year.1, 2 Almost 90% of these deaths occur in sub-Saharan Africa, especially among young children. In spite of intense efforts, the number of malaria cases is on the rise, and it has been predicted to double over the next two decades.3 The development of novel tools to improve our understanding of the biology of the mosquito vectors is providing a unique opportunity for targeted interventions to fight the disease.

The completion of the Anopheles gambiae Giles genome sequence4 is facilitating a series of projects aimed at addressing three major challenges:5 (1) to perform studies on field populations to analyse natural intra- and interspecific variations in the ability to transmit disease; (2) to expand our understanding of mosquito biology with a view to identifying new targets for vector control; (3) to gain genetic information aimed at developing strategies to introduce refractory traits in natural populations and to reduce the size of natural vector populations. This review will report on some of the most promising molecular and genetic advances made since the completion of the sequencing project towards achieving these goals.

2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Historically, malaria control measures attacking the mosquito vector have been, and still are, the most effective way of tackling the disease. Among the hundreds of anthropophilic Anopheles species, only about 40 are regarded as important for malaria transmission,6 suggesting that the ability of a vector to support parasite development depends on differences in genetics, ecology and behaviour between mosquito species.7, 8 Mosquito control strategies are often complicated by the presence of multiple vectors in the same area. For example, in Africa, as many as five different anopheline species can function as malaria vectors, either simultaneously or seasonally.9 The role of each in malaria transmission can vary according to the season and ecological factors, and the relative importance of each vector species has a large impact on malaria transmission patterns.10 Genomic tools are being utilized to study the complex population biology of malaria vectors and understand the basis of reproductive isolation, providing crucial information relevant to malaria control. With the completion of the A. gambiae genome sequence, the number of neutral genetic markers for estimating gene flow between populations has dramatically increased. Microsatellite markers have been widely used to study intra- and interspecific differences among species of the A. gambiae complex.11–13 This complex includes six different species, not all of which participate in malaria transmission. Anopheles gambiae sensu stricto is the most efficient vector species in the complex and comprises two molecular forms, M and S, which are postulated to have diverged very recently, possibly less than 10 000 years ago.14 The M form is restricted to West Africa, where the two forms (which do not seem to differ in their capability of supporting Plasmodium falciparum (Welch) infections) are sympatric. Although in the laboratory there are no apparent constraints to mating within these forms, and hybrids are viable and fertile, in nature there is significant restriction to gene flow between M and S individuals, as they interbreed at a low frequency (about 1.2%).15 In an attempt to elucidate the genetic basis of this reproductive isolation, genomic DNA from the M and S forms of A. gambiae was hybridized to Affymetrix GeneChip microarrays, and three major regions of difference were identified on the basis of the intensity of the hybridization signal detected.16 The analysis of these regions should help reveal the genes responsible for reproductive isolation and aid our understanding of the speciation processes in Anopheles mosquitoes, solving a conundrum that has long been puzzling population biologists, and providing crucial information for future malaria control programmes.

The striking intraspecific differences in the ability of anopheline mosquitoes to transmit malaria are also being investigated. A recent study mapped resistance to natural P. falciparum infections in a wild-type A. gambiae population from Mali to a small region of chromosome 2L, using microsatellites to analyse isofemale pedigrees.17 Notably, refractory traits were observed at relatively high frequencies, giving support to the view that mosquito vectors have an intrinsic ability to interfere with parasite development.18 The complexity and heterogeneity of malaria transmission in Africa is, however, not fully elucidated, and in some large geographical areas detailed information on the composition of the anopheline vectors is not available.

3 STUDIES ON ANOPHELES BIOLOGY

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

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

4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

4.1 Population replacement

Theoretically, natural disease-transmitting populations could be replaced with genetically modified anopheline mosquitoes44–46 refractory to malaria parasites, a control strategy known as population replacement. Various levels of refractoriness to Plasmodium transmission have been achieved in the laboratory using transgenic mosquitoes expressing a battery of effector genes.47–49 In particular, the expression of an artificial peptide SM1 (for salivary-gland- and midgut-binding peptide 1) strongly inhibited the crossing of the midgut epithelium by the P. berghei murine parasites in A. stephensi Liston mosquitoes.47 However, it has to be stressed that, so far, experiments on the efficacy of effector transgenes in blocking parasite development have focused on the P. berghei model and have had little success when tested against the human P. falciparum parasites. Moreover, these experiments have never achieved a total blockade of disease transmission, as would be required in a malaria control programme based on population replacement.50 Another parallel challenge is represented by the development of effective methods to spread these genes through natural populations. For this to happen, ‘refractoriness’ genes must be linked to a genetic drive, and several different drive mechanisms, including transposable elements, negative heterosis, wolbachia symbionts and, more recently, homing endonucleases, have been proposed and in some cases are being tested in the laboratory.51–56 Ensuring the spread of an effective refractory trait in entire field populations, however, represents a formidable task. Crucially, a tight link between the drive and the refractoriness gene(s) must be in place to avoid recombination and consequent spread and fixation of the ‘empty’ drive, which would occur especially if expression of the refractory gene(s) were associated with fitness costs. As mentioned above, the efficacy of the introduced genes in blocking malaria transmission must be nearly total to ensure a significant effect on malaria transmission and epidemiology.50 Moreover, the inevitable evolutionary response of the parasite could lead to the selection of immunosuppressive Plasmodium strains capable of evading mosquito defence mechanisms, thus reducing the effectiveness of a replacement programme. In spite of these challenges, population replacement remains an extremely attractive approach that holds the additional benefit of promoting exceptional research advances. However, even when meeting the technological challenges ahead, as will probably happen in the near future, this strategy will have to tackle the ethical and safety issues related to the release of transgenic individuals carrying effector genes that are actively spreading throughout field populations.57–59

4.2 Sterile insect technique

An alternative malaria control strategy based on the use of transgenic technologies relies on the suppression or eradication of mosquito species that are vectors of human malaria. This approach, known as the sterile insect technique (SIT), is based on the field release of mass numbers of sexually active but genetically sterile insects, normally males, over large areas.60, 61 As mating of the sterile males with field females produces no viable progeny, if releases are repeated over an adequate period of time they will result in the eradication of the local populations. SIT has been successfully applied to the eradication of the New World screwworm, Cochliomyia hominivorax Coq., from the southern states of the USA, Mexico and all of Central America,62–64 and to other insect pests of agricultural and medical importance.65, 66 In the case of anopheline mosquitoes, a series of releases have been performed with the aim of evaluating SIT-related parameters such as dispersal and mating capabilities of the released males, and in some cases of achieving suppression of local populations.67–69 These programmes, although mostly unsuccessful, with the exception of a sterile release of A. albimanus Weid. in El Salvador,67 have helped highlight a series of issues that will be crucial for the success of any future SIT programme. Two major problems are represented by the loss of male competitiveness owing to the sterilization procedure70 (irradiation or chemosterilization) and the difficulty in developing efficient genetic sexing mechanisms that are needed to ensure a release of male-only populations.71, 72 The latter represents an absolute requirement for malaria control programmes, as female mosquitoes, even when sterile, would still contribute to disease transmission. The use of transgenic technologies has been invoked to overcome these problems, thus improving the chances of success of SIT strategies.69 Indeed, research in this direction is showing promising results, and the generation of the first genetic sexing strain for Anopheles mosquitoes was recently accomplished in which efficient and automated separation of the two sexes could be achieved by the sperm-specific expression of an enhanced green fluorescent protein (EGFP) gene in male late third-instar larvae and throughout development.73 The fluorescent sperm could also be visualized on transfer to the female, providing an important tool for recapture studies and dispersal analysis in an eventual release programme. The sex determination pathway in A. gambiae is also being investigated, and the final double-switch gene in the somatic sex determination cascade, doublesex, and the male sex-determining gene fruitless have been recently identified and isolated.74, 75 The identification of developmentally regulated promoters, sex-specific splice mechanisms and genes essential for mosquito fertility that could be manipulated to achieve the development of efficient genetic sexing strains or to induce genetic sterility in males will be instrumental in the success of future SIT or similar strategies, such as RIDL (Release of Insects carrying a Dominant Lethal). In RIDL, a technique that has never been applied outside the laboratory but may hold some promises for pest control,76 the released males are fertile but carry a female-specific dominant lethal gene whose expression leads to the elimination of their female progeny.

It should be noted that these vector control methods are species-specific. While this feature represents an advantage in terms of possible repercussions on the ecosystem, as only the relevant species will be targeted, it somewhat limits their usefulness in malaria control programmes to areas where Plasmodium transmission is carried out by a single vector species, or by a combination of species all amenable to genetic manipulation.

5 CONCLUSIONS

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

The completion of the A. gambiae genome sequence4 is providing invaluable leads for functional studies. The genomes of other important mosquito species that are vectors of human disease, such as Aedes aegypti L.,77Culex quinquefasciatus Say and additional anopheline species (www.vectorbase.org) are being or will be sequenced in the near future. This will substantially enhance the power of the tools developed for A. gambiae mosquitoes, for instance the comprehensive microarray platforms that are already utilized to study gene expression in related anopheline species, and will allow comparative genomics of different disease vectors that will most likely reveal some exciting biology. These advances, combined with ongoing field studies of mosquito behaviour and ecology, afford an unprecedented opportunity to develop novel vector control strategies. Future studies will need to focus on the vector–parasite combinations actually found in the field, and high priority should be given to the most promising candidates showing notable effects against most, if not all, human Plasmodia. Malaria research at the beginning of the third millennium is experiencing a new renaissance, filled with expectations and hopes; it is essential that in the near future research efforts focus on developing means of translating this remarkable progress into reduction in malaria morbidity and mortality in the field, by strengthening or complementing existing methods of disease control.

REFERENCES

  1. Top of page
  2. Abstract
  3. 1 INTRODUCTION
  4. 2 MALARIA TRANSMISSION IN THE FIELD: A COMPLEX MATTER
  5. 3 STUDIES ON ANOPHELES BIOLOGY
  6. 4 STRATEGIES FOR REPLACING OR ERADICATING VECTOR POPULATIONS
  7. 5 CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES