Mosquitoes and Strategies to Control Malaria

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1 Mosquitoes and Strategies to Control Malaria Abhinav Sinha Abstract Malaria kills millions of people every year, yet there has been little progress in controlling this disease. Moreover, the number of cases is rising, due to the emergence of drugresistant parasites and insecticide-resistant mosquitoes. For transmission to occur, the malaria parasite has to complete a complex developmental cycle in the mosquito. The mosquito is therefore a potential weak link in malaria transmission, generating mosquito populations that are refractory to the parasite. The mosquito is the obligatory vector for malaria transmission. After the mosquito ingests an infected blood meal, the parasite undergoes a complex developmental cycle, which includes mating of the parasite gametes and the transformation into three different forms: ookinete, oocyst and sporozoites and also the crossing of two different epithelia (mid-gut and salivary glands). This complex development within the mosquito and mechanisms of the mosquito s immune system implicated in killing parasites offers multiple potential targets to interfere with transmission. This can be explored by analyses of mosquito genes to define and characterize mosquito responses to components of Plasmodium-infected blood. A lot of research showed that the journey of the Plasmodium inside its vector, the Anopheles mosquito, and the multi-faceted interactions between the vector and the parasite offer many novel sites and directions from which the disease can be attacked. However, the interactions are not fully understood and many new facts are waiting to be unmasked. Completion of the Anopheles gambiae and Plasmodium falciparum genome mapping has opened the opportunity to apply high throughput methods to the analysis of gene function. The burst of information generated by these approaches and the use of molecular markers to investigate the biology of these interactions is broadening our understanding of this complex system. The ability to disrupt individual genes expressed in mosquito stages of the parasite has generated a wealth of information on the basic biology and survival strategies of the parasite, but many genes still remain to be characterized. A detailed analysis of the spatial and temporal expression of the effectors molecules mediating the interactions between the mosquito and the parasite will provide an integrated picture of this fascinating and complex journey. Each of these critical steps is a potential target to interfere with malaria transmission to humans. Introduction The impact of malaria to families and economies is simply appalling and cannot be ignored. In Africa, it kills over one million children under five every year. Wen Kilama, now Chair of the African Malaria Vaccine Testing Network, puts this in perspective: It is like loading up seven Boeing 747 airliners each day, then deliberately crashing them into Mount Kilimanjaro ( The vast majority of those who die are among the poorest of society (WHO, 1988). Malaria kills millions of people every year, yet there has been little progress in controlling this disease. Moreover, the number of cases is rising, due to the emergence of drug-resistant parasites and insecticide-resistant mosquitoes (Ashburner, 2002).

2 34 Proceeding of National Symposium on Tribal Health Hundreds of dedicated malariologists have devoted their careers to combat malaria. Yet, as Robert Desowitz so graphically tells us, the situation is now getting worse, not better (Desowitz, 1991). In the 1960s, for example, malaria had been all but eradicated in India and Sri Lanka; today there are some 20,000 deaths a year due to malaria in India alone (Sharma, 1999). Two of the currently implemented vector control strategies, indoor insecticide spraying and insecticide-treated bednets (ITNs) have proven effective in reducing transmission, especially among children. However, multiple insecticide resistances among the main mosquito vectors Anopheles gambiae and Anopheles funestus (George, 2005), and various complications in the introduction, distribution and proper use of ITNs indicate that these tools alone will be insufficient to reduce malaria transmission to a level that could eventually lead to eradication of the disease. Parasite control appears even more problematic: an effective antigen for vaccine production has remained elusive for many decades; drug resistance is increasing; and new, more effective drugs are unaffordable. On top of this, increasing poverty, bad hygiene conditions and devastating civil conflicts in many endemic countries worsen the situation. In Africa alone, over a quarter of a billion people are newly infected with malaria every year, of which over one million die. Thus new, effective control strategies are an urgent need (George, 2005). Critical Control Points in the Journey of Plasmodium inside the Mosquito 1. Entry of plasmodium into anopheles and its subsequent development : After the mosquito ingests an infected blood meal, the parasite undergoes a complex developmental cycle, which includes mating of the parasite gametes and the transformation into three different forms: ookinete, oocyst and sporozoites and also the crossing of two different epithelia (mid-gut and salivary glands) (Ghosh et al, 2000). This complex development within the mosquito offers multiple potential targets to interfere with transmission. In the mosquito, ookinetes must survive the harsh environment of a blood meal undergoing digestion and cross several barriers, including the epithelial cells, before reaching the mid-gut basal lamina. When they come in direct contact with the mosquito immune system, multiple complex interactions determine whether the parasite will be recognized and destroyed. The transit and development in the mosquito is a dangerous and difficult journey that results in substantial parasite losses. For every 1000 Plasmodium berghei gametocytes ingested, only two viable ookinetes will be generated, and only 2 20% of them will successfully invade the midgut and develop to mature oocysts (Alavi et al, 2003). Plasmodium can tolerate such great losses, because the population re-expands during the oocyst stage (Carolina Barillas-Mury, 2005). Strong circumstantial evidence suggests that invasion depends on recognition of mosquito surface molecules by Plasmodium proteins. Major obstacles are encountered in the mid-gut tissue, where most parasites are killed by the mosquito s immune system (Dong et al, 2006). It has already been demonstrated that mosquitoes genetically modified with a gene that interferes with parasite invasion of the mid-gut are impaired for parasite transmission (Ito et al, 2002). Moreover, when transgenic mosquitoes ingested a Plasmodium-infected blood meal, they had a distinct fitness advantage over their nontransgenic counterparts. These discoveries are important for devicing methods to spread the inserted gene through wild mosquito populations. Little information is available 34

3 Sinha 35 about genes that are expressed during parasite development in the mosquito. These genes are involved in protection of the mosquito from the parasite. 2. The mosquito s response to parasite invasion : In order to cope with the risk of infection, from the frequent and diverse microbial exposure, insects have developed several structural barriers and a multifaceted innate immune system comprising a variety of synergistic defence mechanisms. The first line defence against microbes is represented by the structural barriers, which include the hardened outer exoskeleton, the peritrophic matrix of the mid-gut and the chitinous linings of the trachea. The midgut epithelium, apart from being an immune competent organ, is also serving as a structural barrier for microbes and parasites. The peritrophic matrix is a chitinous sack that facilitates digestion and also protects the mid-gut epithelium from direct contact with the meal and a large proportion of the microbial mid-gut flora which can be boosted up to a fold after a blood meal in some haematophagous insects (Ito et al, 1996; Shao et al, 2001). Malaria parasites have developed a specific mechanism, utilizing a chitinase, to traverse the peritrophic matrix before invasion of the mosquito mid-gut epithelium (Shahabuddin et al, 1993). Pathogens that have made it through the insects structural barriers will encounter its innate immune system that is less complex than the adaptive immune system of vertebrates due to the absence of antibodies and B-cell memory. It is however, rapidly activated after challenge and has a certain degree of specificity to different microbial classes. It comprises a variety of effective mechanisms that ultimately can control infection. Insect innate immune responses involve both cellular and humoral defence mechanisms that are triggered by PRRs (pattern recognition receptors) capable of specific binding to PAMPs (pathogen associated molecular patterns) (Medzhitov et al, 2002). These PRRs can mediate microbial killing directly, through phagocytosis, or indirectly by the triggering of serine protease cascades that in turn can activate defence reactions such as melanotic encapsulation or initiate intracellular immune signalling pathways which regulate the transcription of antimicrobial peptide genes and other effective genes (Hoffmann et al, 1996; Hoffmann et al, 2002). Anopheline mosquitoes are strongly activating their immune system when the parasites are invading the epithelial tissues and subsequently when they migrate through their open circulatory system. These immune responses peak at the stages when the largest parasite losses occur and have been linked to parasite elimination (Dimopoulos et al, 1997; Dimopoulos et al, 1998; Richman et al, 1997; Luckhart et al, 1998; Lowenberger et al, 1999). A genetically selected refractory mosquito strain, L 3-5, can completely block Plasmodium development through a melanotic encapsulation mechanism that appears to involve components of the innate immune system (Collins et al, 1986). A detailed understanding of the mechanisms involved in Plasmodium killing and the determination of mosquito permissiveness to malaria infection can ultimately be used for the development of novel malaria control strategies (Dimopoulos et al, 1998). The existence of a high density genetic map, several immune competent cell lines, the complete genome sequence, expression profiling tools and transgenic tools, renders A. gambiae an important model organism for the study of the insect innate immune system and its interactions with a protozoan parasite (Zheng et al, 1997; Muller et al, 1999; Dimopoulos et al, 2000; Dimopoulos et al, 2000a; Dimopoulos et al, 2000b; Grossman et al, 2001; Blandin et al, 2002; Holt et al, 2002). What are the mechanisms of the mosquito s immune system implicated in killing parasites? This can be explored by analyses of mosquito genes to define and characterize mosquito responses to components of Plasmodium-infected blood. These analyses will 35

4 36 Proceeding of National Symposium on Tribal Health permit selection of key candidate defense genes for further functional characterization. Researchers at Malaria Research Center (JHMRI) have already identified a pattern recognition receptor family of genes that is likely to participate in Plasmodium killing, and several members of this gene family are now being molecularly characterized. They are also studying mosquito salivary gland physiology to better understand its function in blood feeding and transmission and to identify promoters that are suitable for expression of plasmodiocidal proteins in mosquitoes (Dimopoulos G). The mosquito responses against these pathogens are quite diverse, and the defense against the malaria parasite involved both common and species-specific components. Malaria-infected blood was sufficient to activate anti-plasmodium immune responses, even in the absence of mid-gut invasion. Through this mechanism, the mosquito can initiate its defense against Plasmodium prior to invasion of the gut. Mosquito genes that could negatively influence Plasmodium development were also capable of regulating the resistance to bacterial infection, but several of the antibacterial genes had no effect on Plasmodium; thus, the mosquito apparently utilizes its antibacterial defense systems against the malaria parasite (Dong et al, 2006). 3. Parasite-vector interactions : Vector and parasite interact at multiple stages and locations, and the nature and effectiveness of this reciprocal interaction determines the success of transmission. Many of the interactions engage the mosquito s innate immunity, a primitive but very effective defense system. In some cases, the mosquito kills the parasite, thus blocking the transmission cycle. However, not all interactions are antagonistic; some represent immune evasion. Pointing out that transgenic approach will no doubt prove powerful; Alexander Raikhel has targeted the mosquito s immune system to respond to parasite infections in two new ways. In one case, he describes a transgenic mosquito that produces an endogenous anti-microbial agent that destroys the parasite. In another example, Raikhel has altered the insect s immune system so that it becomes susceptible to infection after a blood meal (Phillips, 2003). Analysis of gene expression responses to infection can provide a wealth of information on how a variety of biological processes are affected in the mosquito. Infected blood alone stimulated the regulation of 13% of all mosquito genes. The specificity of these parasites vector interactions makes them attractive targets for those working to develop synthetic refractory mechanisms in mosquitoes. The development of anti-sporozoite effector genes is ongoing work. Combining these genes with others that target ookinetes and prevent oocyst formation should permit producing a multi-genic phenotype of no sporozoites in the salivary glands. Furthermore, the use of multiple effector genes may be necessary to prevent the selection of resistance to any one mechanism. This could prevent the breakdown of a control strategy based on a genetic approach. In addition, a balance among fitness effects, effectiveness of the molecule, ease of engineering of the phenotypes and mechanism for spreading the phenotypes through a population will dictate the practicality of any one strategy. Ultimately, saving lives will be the most important measure of these approaches (Phillips, 2003). 4. Strategies to interfere with Plasmodium development in the mosquito : The sequence of the A. gambiae genome revealed numerous potential components of the innate immune system, and it established that they evolve rapidly. The development of functional genomic methodologies has provided novel opportunities for understanding the immune system and the nature of its interactions with the parasite. In this context, 36

5 Sinha 37 identification of both Plasmodium antagonists and protectors in the mosquito represents a significant conceptual advance. In addition to providing fundamental understanding of primitive immune systems, studies of mosquito interactions with the parasite open unprecedented opportunities for novel interventions against malaria transmission. The generation of transgenic mosquitoes that resist malaria infection in the wild and the development of anti-malarial smart sprays capable of disrupting interactions that are protective of the parasite, or reinforcing others that are antagonistic, represent technical challenges but also immense opportunities for improvement of global health (Osta et al, 2004; Riehle et al, 2003). The ability to disrupt individual genes expressed in mosquito stages of the parasite has generated a wealth of information on the basic biology and survival strategies of the parasite, but many genes still remain to be characterized. A detailed analysis of the spatial and temporal expression of the effector molecules mediating the interactions between the mosquito and the parasite will provide an integrated picture of this fascinating and complex journey. Each of these critical steps is a potential target to interfere with malaria transmission to humans (Barillas-Mury et al, 2005). i. Transmission blocking vaccines: Transmission blocking vaccines consist of antibodies that are ingested by the mosquito with the blood meal and interfere with parasite development. Proteins expressed on the surface of gametes (e.g. Pfs47/48, Pfs230) and ookinetes (e.g. Pfs25 and Pfs28) have been tested for such vaccines (Carter, 2001; Healer et al, 1999; Duffy et al, 1997). Antibodies against these proteins bind to the parasite and presumably block ookinete invasion of the midgut epithelium. Various reports that polyclonal antibodies against mosquito midgut proteins interfere with Plasmodium oocyst formation have been published (Ramasamy et al, 1990; Srikrishnaraj et al, 1995; Lal et al, 1994; Lal et al, 2001), but in no case have the relevant antigens been identified (Jacobs-Lorena et al, 1995). It should be noted that transmissionblocking vaccines do not protect the immunized individual but act by preventing infection of people in the surrounding community. Thus, for ethical reasons and for increased effectiveness, transmission-blocking antigens will have to be incorporated into conventional vaccines that target the vertebrate stages of the parasite. ii. Paratransgenesis: Paratransgenesis, the genetic manipulation of commensal or symbiotic bacteria to alter the host s ability to transmit a pathogen, is an alternative means of preventing malaria transmission. Bacteria can be engineered to express and secrete peptides or proteins that block parasite invasion or kill the parasite in the midgut. This strategy has shown promise in controlling transmission of Trypanosoma cruzi by Rhodnius prolixus under laboratory conditions (Grossman et al, 2001). Furthermore, symbiotic bacteria in the tsetse fly have been isolated, transformed with a reporter gene, and reinserted into the fly (Ito et al, 2002). For this strategy to be used in malaria control, bacteria that can survive in the mosquito s midgut must be identified. Gram-positive and Gram-negative bacteria, including Escherichia, Alcaligenes, Pseudomonas, Serratia and Bacillus, have been identified in the midgut of wild anopheline adults (Demaio et al, 1996; Straif et al, 1998). These bacteria are easily cultured in the laboratory and may be suitable targets for genetic manipulation. Whether these bacteria are stable or transient residents of the midgut of adult mosquitoes remains to be determined. To successfully control malaria the refractory proteins or peptides expressed by the bacteria must act on the midgut stages of the malaria parasites, maintain their bioactivity in the mid-gut environment, and be expressed 37

6 38 Proceeding of National Symposium on Tribal Health in sufficient quantities. When An. stephensi mosquitoes were fed E. coli that express a fusion protein of ricin and a single-chain antibody against Pbs21 (a P. berghei ookinete surface protein), oocyst formation was inhibited by up to 95% (Yoshida et al, 2001). Other effector molecules, such as SM1 and PLA2, are also in consideration. The use of paratransgenesis for the control of malaria will require the development of methods to introduce genetically modified bacteria into field mosquitoes. Transgenesis is a novel tool with potential use for the control of vector-transmitted diseases (Grossman et al, 2001; Coates et al, 1998; Catteruccia et al, 2000; Moreira et al, 2000; Kokoza et al, 2000). Recently, Ito et al. (2002) and Moreira et al. (2002) demonstrated that a synthetic peptide (SM1) and the bee venom phospholipase A2, respectively, can interfere with Plasmodium development in mosquitoes (Ito et al, 2002; Gardner et al, 2002). While these results are encouraging, it is important to consider that the Plasmodium genome is known for its plasticity (George et al, 2004) and the possibility of the emergence of resistant parasite strains is a likely possibility. For this reason, it will be important to develop multiple effector genes that can interfere with parasite development in mosquitoes by independent mechanisms. The concept of genetic control of insect pests is not new and dates back over in the 1930s-40s, and successful field trials aiming to induce sterility in insect populations have taken place in the following years. In 1968, C. Curtis proposed the use of translocations to fix and drive through insect populations genes that are favorable to man and not deleterious to the insect. A means to introduce such genes in the insect germ line was found in 1982, when Drosophila melanogaster became the first insect ever to be stably transformed with the use of a transposable element (Rubin and Spradling, 1982). Some years later (1991), a joint meeting of the World Health Organization/ Special Program on Research and Training in Tropical Diseases (WHO/TDR) and the Mac Arthur Foundation established genetic modification of vector species as a desirable approach to fight the disease (Alphey et al., 2002). The research agenda for malaria control that arose from that meeting anticipated the development of tools for stable germ line transformation of anophelines by 2000, engineering of mosquitoes incapable of carrying malaria by 2005, and controlled experiments to test how to drive the engineered trait through wild populations by 2010 (James et al., 1999). Based on recent scientific achievements, the genetic strategy to deplete or incapacitate a disease-transmitting insect population became one of the Grand Challenges in Global Health that were announced in October 2003 (Varmus et al., 2003) (George, 2005). There has been considerable progress over the last decade towards developing the tools for creating a refractory mosquito. Accomplishments include germline transformation of several important mosquito vectors, the completed genomes of the mosquito Anopheles gambiae and the malaria parasite Plasmodium falciparum, and the identification of promoters and effector genes that confer resistance in the mosquito. These tools have provided researchers with the ability to engineer a refractory mosquito vector, butthere are fundamental gaps in our knowledge of how to transferthis technology safely and effectively into field populations (Michael, 2003). Therefore, control of parasite development in the mosquito has considerable promise as a new approach in the fight against malaria. The task is twofold - to engineer mosquitoes with a genetic trait that confers resistance to malaria or causes population suppression; and to drive the new trait through field populations (George, 2005). 38

7 Sinha 39 Researchers led by Marcelo Jacobs-Lorena of Case Western Reserve University in Cleveland, Ohio, showed that adding a synthetic gene for a peptide called SM1 into the related species A. stephensi almost completely disrupted the mosquito s ability to transmit Plasmodium berghei, which causes malaria in mice. The peptide resembles a receptor in the mosquito s gut and salivary glands that is thought to allow the parasite to burrow from one to the other. Flooding the mosquito s body with a similar molecule seems to confuse the parasite, preventing its progress. Meanwhile, Andrea Crisanti and his colleagues at Imperial College, London, are working on genetic manipulations that direct A. gambiae s immune system against P. falciparum. Once the best target is identified it would be a matter of months before we have a modified mosquito, Crisanti asserts. Other, more generic approaches are also being considered, such as genetic modifications that render the offspring of the engineered insects sterile (Anderson et al, 1999). a. Challenges with the genetic route: Even if a transgenic refractory mosquito can be produced, how can we ensure that it completely infiltrates wild populations? Would the genetic modification remain stable? And what are the public-health implications if the scheme is only a partial success? Creating genetically modified mosquitoes in the lab is just the start. How can they be made to spread their altered genes through wild A. gambiae populations? It s a tall order, but genetic elements that could fit the bill exist naturally in insect populations. These transposons, or jumping genes, copy themselves and move around the genome, being passed down the generations. Once they enter a population, they can spread rapidly through it (Anderson et al, 1999). Even if lab-reared strains are continually crossed with vigorous wild-type mosquitoes, the genetic modification itself may also reduce their fitness, as unpublished preliminary work indicates. There is a cost to mosquitoes of carrying a foreign gene, suggests Peter Billingsley, a molecular physiologist who studies malaria transmission at the University of Aberdeen, UK. On the positive side, the introduced genes would free the transgenic mosquitoes from the burden of carrying P. falciparum. There is some evidence that the parasite has a detrimental effect on A. gambiae s fitness (Ahmed et al, 2002; Jasinskiene et al, 1998), but scientists would want to conduct extensive experiments to see whether this would outweigh any fitness deficits before going ahead with field releases (Anderson et al, 1999). Population geneticists also warn that the refractoriness genes could become unhitched from their transposon and be left behind. Once relieved of its burden, the transposon will steam on ahead, says Chris Curtis, a medical entomologist at the London School of Hygiene and Tropical Medicine. And after a particular transposon has spread through a population, it can t be used again. Perhaps the biggest worry, however, is whether the modification would remain stable in the long term. It could sweep through the global population only to be selected out after a number of years. Alternatively, the malaria parasite might develop resistance to the modification just as it has to antimalarial drugs. Both scenarios have profound implications for public health. A temporary halt in malaria transmission could result in people losing all natural immunity, rendering the disease even more devastating once the parasite returns. If malaria were eliminated from Africa for a short period of time, the consequences could be terrible, says Andrew Spielman, a medical entomologist at Harvard University (Anderson et al, 1999). Other concerns are more speculative. As a transposon jumps around the A. gambiae genome, for example, it could disrupt other genes, with unpredictable results. 39

8 40 Proceeding of National Symposium on Tribal Health It s extremely unlikely, but what if a transposon gave mosquitoes the ability to transmit other diseases? Changing the relationship between mosquito and malaria parasite also could have unforeseen ecological and evolutionary impacts. Could the parasite adapt quickly enough to use another vector to continue its deadly life cycle? If sterility genes were used to eliminate A. gambiae, would other species, which only occasionally bite humans, move in and replace them? Could these new mosquitoes transmit malaria? Given the catalogue of unknowns and the potential consequences if the plan were to go awry, experts say that an intensive, multi-stage research programme is needed, similar to the clinical trials of safety and efficacy carried out for experimental drugs. Laboratory studies would first assess the stability, fitness constraints and safety of various genetic modifications. Harmless and well-studied transgenes, such as that for jellyfish green fluorescent protein, could be used at first. Later on, experiments would move on to the genes that would be used for real, in a controlled environment that mimics the wild as closely as possible. A gigantic greenhouse in Kenya, dubbed the Malariasphere, could provide a suitable testing ground. Operated by the International Centre of Insect Physiology and Ecology in Nairobi, the Malariasphere contains mock-ups of huts, forest and breeding puddles for mosquitoes. It is currently being used to study the dynamics of A. gambiae populations and their ability to spread malaria. While such basic research is under way, researchers would need to investigate suitable sites for transgenic releases. Extensive baseline information on local populations of mosquitoes and the burden of malaria in the area would be needed for field workers to be able to tell if a later release of refractory mosquitoes is having the desired effect. Before giving the eventual goahead, however, authorities may want to see the principle demonstrated in the field under circumstances in which the stakes are not so high. Some argue that it would be best to begin on an island with no malaria, working with a mosquito that does not transmit the disease or bite humans. This way, the dynamics of an artificial gene moving through a population could be studied more safely (Anderson et al, 1999). One intriguing idea, championed by Edman, would be to use the geographically isolated Hawaiian Islands as a natural laboratory. iii. Genetically modified mosquitoes: Another promising approach is to genetically modify mosquitoes to express proteins or peptides that interfere with Plasmodium development. Methods to produce transgenic culicine (Catteruccia et al, 2000) and anopheline (Grossman et al, 2000; Burt, 2003) mosquitoes are now available. A review of the promoters to drive the transgenes and effector molecules whose products hinder parasite development are considered below. a. Promoters: An essential step in engineering mosquitoes with reduced vector competence is the identification of suitable promoters to drive the expression of antiparasitic genes. During its development in the mosquito, the parasite occupies three compartments: mid-gut lumen, hemocoel and salivary gland lumen. Thus, promoters that drive synthesis and secretion of proteins into these compartments need to be identified. In addition to spatial considerations, the time of protein synthesis relative to arrival of the parasite in each of these compartments needs to be considered. Control of transmission has the best chance of success if pre-sporozoite stages in the mid-gut lumen are targeted. Strong and ubiquitous promoters could also be used to drive the expression of effector genes. However, such promoters are not ideal because generalized expression may impose a fitness load on the mosquito and even promoters considered ubiquitous (e.g. actin) are not equally expressed in all cells. 40

9 Sinha 41 b. Effector genes: The quest for anti-parasite molecules has been directed towards identification of gene products that hinder transmission by either killing or interfering with parasite development. Although major advances have been accomplished in recent years, it is important that the search for new effector molecules and promoters continue for two reasons. First, considering how easily parasites acquire drug resistance, it is likely that parasites will be selected that can overcome the barrier imposed by the effector molecules. Secondly, maximum efficiency of blocking parasite development (ideally 100%) is important for the transgenic mosquito strategy to have a significant impact on disease transmission. Furthermore, while many of the tools for genetic modification of mosquitoes have been developed, an extensive gap exists in our ability to transfer this technology to the field for the control of malaria. c. Challenges facing a successful field release : l The fitness cost of refractoriness - To maximize the likelihood of successfully introducing refractory genes into a wild mosquito population, transgenes should impose minimal fitness load. l Developing an effective drive mechanism - Two general strategies can be considered for introducing transgenic mosquitoes in the field: population replacement or a genetic drive mechanism. Population replacement, or inundatory release, requires a significant reduction of the resident mosquito population (for instance, with insecticides), followed by the release of large numbers of refractory mosquitoes to fill the vacated biological niche. This strategy is promising as a research tool and as a field test to assess the effectiveness of the transgenic mosquito approach for interrupting malaria transmission. However, this strategy cannot be considered for large-scale control purposes, because it is not possible to produce sufficient numbers of mosquitoes to achieve population replacement on a country- or continent-wide level. An efficient genetic drive mechanism is helpful because a manageable number of genetically modified mosquitoes can replace the wild population, even if the effector gene(s) imposes some fitness cost. A second possible drive mechanism is the use of so-called selfish genes. These genes drive themselves through a population by using the host cell DNA repair machinery (Thomas et al, 2000). A third potential drive mechanism to introduce an effector gene into a vector population involves the use of the symbiotic bacterium Wolbachia, which exerts its drive mechanism primarily through cytoplasmic incompatibility. Regardless of the driving mechanism employed, it will be essential that the effector gene (that interferes with Plasmodium development) be tightly linked to the driving element. A dissociation of the two (for instance, by recombination) will cause the driving gene to continue to spread through the population alone while the effector gene is lost. l Mass production of transgenic insects and genetic sexing mechanisms: Transgenic-based methods to reduce or eradicate vector populations, such as the release of insects carrying a dominant lethal (RIDL, Sinden, 2002) show promise for some species. However, their use as a malaria control program in Africa would be difficult to implement due to reproductively incompatible subspecies and migration of mosquitoes among villages. Even if successful, this approach would leave an ecological vacuum that another malaria vector could quickly fill. Therefore, replacement of wild 41

10 42 Proceeding of National Symposium on Tribal Health populations with transgenic mosquitoes carrying refractory genes instead of population suppression or eradication methods would be more appropriate. l Avoiding resistance to the refractory genes: Parasites facing a refractory mosquito population would be under strong selective pressure, similar to the one posed by antimalarials, and thus resistance may develop. Engineering a mosquito with multiple refractory genes that target different aspects of parasite development could minimize resistance to the refractory genes. Major advances in recent years, including successful germline transformation and characterization of promoters, are allowing researchers to test putative refractory genes. One important task for the near future is the identification of additional effector genes, and this will be greatly facilitated by the availability of the A.gambiae and P.falciparum genome sequences. This knowledge can be used to engineer a mosquito that inhibits or kills the malaria parasite during multiple developmental stages. With this ideal mosquito on the horizon, the most important task is to begin laying the groundwork for its introduction into the wild. References Africa Fighting Malaria [ Ahmed AM,.Baggott SL, Maingon R., Hurd. H The costs of mounting an immune response are reflected in the reproductive fitness of the mosquito Anopheles gambiae. Oikos. Vol.97(3). pp Alavi Y, Arai M, Mendoza J, Tufet-Bayona M, Sinha R, Fowler K, et al The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by Anopheles stephensi, Anopheles gambiae and Aedes aegypti. Int J Parasitol. Vol.33. pp Anderson RA, Knols BJG, Koella JC Plasmodium falciparum sporozoites increase feeding-associated mortality of their mosquito hosts Anopheles gambiae. Parasitology. Vol.120. pp Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC, Levashina EA Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the defensin gene. EMBO Report. Vol.3. pp Burt A Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc. R. Soc. Lond. B. Vol pp Carter R Transmission blocking malaria vaccines. Vaccine. Vol.19. pp Catteruccia F, Nolan T, Loukeris TG, Blass C, Savakis C, Kafatos FC, Crisanti A Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature. Vol pp Clarke T Mosquitoes minus malaria. Nature. Vol.419. pp Coates CJ, Jasinskiene N, Miyashiro L, James AA Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. USA. Vol.95. pp

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