Medical and Veterinary Entomology (1997) 11, 290-296

Interactions of human malaria parasites, Plasmodium vivax and P. falciparum, with the midgut of Anopheles mosquitoes MANTHRI S . R A M A S A M Y , RANJITH K U L A S E K E R A , ISHANI C . W A N N I A R AC H C H I , K . A L AG A R AT N A M S R I KR I S H N A R A J and RAN J A N RAM A S AM Y Division of Life Sciences, Institute of Fundamental Studies, Kandy, Sri Lanka

Abstract. Present understanding of the development of sexual stages of the human malaria parasites Plasmodium vivax and Pfalciparum in the Anopheles vector is reviewed, with particular reference to the role of the mosquito midgut in establishing an infection. The sexual stages of the parasite, the gametocytes, are formed in human erythrocytes. The changes in temperature and pH encountered by the gametocyte induce gametogenesis in the lumen of the midgut. Macromolecules derived from mosquito tissue and second messenger pathways regulate events leading to fertilization. In An.tessellatus the movement of the ookinete from the lumen to the midgut epithelium is linked to the release of trypsin in the midgut and the peritrophic matrix is not a firm barrier to this movement, The passage of the Rvivax ookinete through the peritrophic matrix may take place before the latter is fully formed. The late ookinete development in Pfalciparurn requires chitinase to facilitate penetration of the peritrophic matrix. Recognition sites for the ookinetes are present on the midgut epithelial cells. N-acetyl glucosamine residues in the oligosaccharide side chains of An.tessellarus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition sites for Pvivax and Pfalciparum ookinetes. It is possible that ookinetes penetrating epithelial cells produce stress in the vector. Mosquito molecules may be involved in oocyst development in the basal lamina, and encapsulation of the parasite occurs in vectors that are refractory to the parasite. Detailed knowledge of vector-parasite interactions, particularly in the midgut and the identification of critical mosquito molecules offers prospects for manipulating the vector for the control of malaria.

Key words. Anopheles, Plasmodium vivax, Pfalciparurn, midgut glycoproteins, trypsin, gametocytes, ookinetes.

Introduction

Anopheles mosquitoes (Diptera: Culicidae) are vectors of Plasmodium vivax Grassi and Pfalciparum Welch (Haemosporodida: Plasmodiidae), the two most prevalent species of human malaria parasites. Malaria infection is initiated in humans when a female Anopheles mosquito injects Plasmodium sporozoites while obtaining a bloodmeal. Sporozoites develop

Editorial footnote: This refereed review was first presented verbally at a symposium on ‘Arthropod-Host-Pathogen Interaction’ at the XX International Congress of Entomology, Florence, August 1996.

Correspondence: Dr Manthri S. Ramasamy, Division of Life Sciences, Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka.

and undergo schizogony in the liver and subsequently within erythrocytes. Sexual stages, the gametocytes, are occasionally formed in erythrocytes where they mature but do not develop further until taken up in the bloodmeal of a vector Anopheles mosquito.

The asexual and sexual stages of Pvivar and Pfalciparum and their anopheline vectors are targets in malaria control prog- rammes. Malaria parasites have increasingly developed resistance to commonly used drugs such as chloroquine, sulphonamides and dihydrofolate reductase inhibitors. The development of anti- parasite vaccines has become therefore a part of the strategy to eradicate malaria (Collins & Paskewitz, 1995). Vaccines based on surface antigens of gametocytes, zygotes and ookinetes that block the transmission of Plasmodium to the vector and termed

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transmission-blocking vaccines are envisaged to be able to prevent the transmission of viable sexual stages of the malaria parasite from humans to the vector (Kaslow, 1990). Immunogenicity and efficacy studies in experimental animals with Rfalciparum transmission blocking antigens have been carried out (Ban et al., 199 I). Conventional approaches to vector control depend on the use of chemical insecticides and biological control agents. A different approach is based on developing immunity to mosquito midgut antigens (Ramasamy & Ramasamy, 1990b), a method analogous to a procedure that has achieved considerable success with ticks (Willadsen et al., 1989). The genetic manipulation of vector populations to control malaria and other vector-borne diseases is also being considered (Carlson et al., 1995). The essential concept in this process is to express stably genes that are able to prevent or disrupt malaria development in the mosquito. Understanding the mechanisms by which the parasite interacts with mosquito tissues after the passage of the gametocytes into the mosquito midgut is important for identifying such genes.

The vector could undergo unfavourable pathological changes caused by damage to the midgut, competition for nutrients between the vector and the growing oocysts, etc., as reported in the artificial vector-parasite combination of An.stephensi Liston and the rodent malaria, Rberghei Vincke (Maier et al., 1987). However, the mortality of An.gambiae Giles, a natural vector of Rfalciparum, was not influenced by the parasite (Robert et al., 1990). Similarly, An.ressellatus Theobald did not show an increase in mortality due to infections with either Rfalciparum or R v i v a (our unpublished observation). Therefore, in nature, the relationship between the developing malaria parasite and the vector could be one that does not disadvantage the vector. Manipulation of genes responsible for interactions between Plasmodium and Anopheles aims to target the development of the parasite in the vector, without significantly affecting the fitness of the vector (Collins & Paskewitz, 1995). In this respect the genes confemng refractoriness of Anopheles sp. to Rvivar and Rfalciparum infections are particularly relevant (Collins er al., 1986).

This paper examines current knowledge on vector-midgut interactions in the relationship between Anopheles and Plasmodium with special emphasis on An. tessellatus, a natural vector of Rvivax and Rfalciparum in Sri Lanka (Mendis et al., 1990).

Parasite development In the mldgut

The environmental changes encountered by Plasmodium game- tocytes during their passage from man to the mosquito midgut lumen trigger the development of gametocytes. The gametocytes respond to these changes by simultaneously initiating macro- and microgametogenesis, during which both female and male parasites escape from the red blood cells, facilitating gamete dispersal and fertilization (Sinden et al., 1996), the processes being completed in approximately 20min (Alano & Carter, 1990). However, other workers have reported that fertilization may take longer (Sinden et al., 1996). The morphological and biochemical changes encountered in male and female gametocytes are discussed by Sinden et al. (1996). The further

development of the male gametocytes involves the liberation of flagellated motile microgametes from the microgametocyte, a process known as exflagellation. In passing from man to mosquito, the gametocytes encounter a reduced temperature (from 37°C to below 30"C), which probably acts as the major stimulus for the induction of gametogenesis. Studies by Sinden & Croll (1975) and Sinden (1983) have demonstrated a relationship between fall in temperature and exflagellation of Plasmodium, in v i m . At temperatures above 30"C, cultured Rfalciparum gametocytes remain quiescent, but lowering the temperature results in the gametocytes becoming extracellular and in exflagellation of the microgametocyte (Ogwan'g et al., 1993a). However, a drop in temperature is not the sole inducer of the exflagellation process. The change in pH, from pH 7.4 in humans to more alkaline condition, pH 8.0, of the mosquito midgut, may be an additional stimulus for induction of gametogenesis. Nijhout & Carter (1978) have demonstrated that in vitm the presence of bicarbonate and an elevated pH brings about the emergence of gametes in Rgullinaceurn Brumpt, an avian malaria parasite. In Rberghei, microgametogenesis consists of a temperature- dependent DNA synthesis and developmental events leading to exflagellation which was further dependent of an elevated pH, induced by Na+M+ exchange (Kawamoto et al., 1991). Exflagellation is also stimulated in vitm by macromolecules derived from mosquito tissues. For example, a dialysable small molecular-weight substance from the midgut of the mosquito which was neither species or sex specific and termed the exflagellation factor, induced exflagellation in Rgallinaceum (Nijhout, 1979). This exflagellation factor may probably be a co-inducer of gametogenesis within the lumen of the mosquito midgut in natural Plasmodium infections (Nijhout, 1979; Alano & Carter, 1990). The exflagellation of cultured Rfalciparum microgametocytes was also influenced by midgut homogenates from six species of anopheline mosquitoes in a species- independent manner (Mendis et al., 1994). Intracellular second messenger pathways, e.g. the inositol phosphate/calcium- calmodulin pathway (Kawamoto et al., 1990, 1993; Ogwan'g et al., 1993b) and protein kinases (Kawamoto et al., 1993) have also been reported to regulate microgametogenesis.

The midgut epithelium of the mosquito, the first tissue barrier to be encountered by the sexual stages of Plasmodium, is a single layer of polarised cells composed of a larger number of digestive cells interspersed with endocrine and regenerative cells (Hecker, 1977; Houk & Hardy, 1982), which is separated from the haemocoel by a basal lamina. The apical (lumen) side of the digestive cells is folded into microvilli and these cells secrete enzymes into the lumen. The ingested bloodmeal is stored and digested in the posterior region of the midgut. In female Aedes aegypti more endocrine cells are present in the posterior region of the midgut compared to the anterior region (Brown et al., 1985). The large numbers of peptidergic endocrine cells associated with the mosquito midgut make it potentially the largest endocrine organ in the mosquito (Brown et al., 1985). It may be speculated that factors produced by the midgut endocrine cells may directly or indirectly through their effects on other cells of the midgut affect parasite invasion, the development of the gametes, the process of fertilization and ookinete motility. For example, the exflagellation factor may be released by endocrine cells into the lumen (Brown & Lea, 1989). The intake

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292 Manthri S . Ramasamy et al.

of a bloodmeal triggers several physiological events in Anopheles. These include the synthesis and release of the proteolytic enzymes trypsin, chymotrypsin and aminopeptidase (Billingsley & Hecker, 1991; Horler & Briegel, 1995). The peritrophic matrix is a lamellar structure that is formed around the bloodmeal, from secretions of the epithelial cells of the midgut. Dissections of the midgut of blood-fed An.tessellatus suggest that the peritrophic matrix first appears around 18 h after a bloodmeal, its development starting in the posterior region of the midgut. The peritrophic matrix persists until 30h post-blood feeding in An.tessellatus (our unpublished observation). Glycoproteins, proteoglycan and chitin are found in the peritrophic matrix (Peters, 1992). The glycoproteins of the peritrophic matrix have mainly high mannose N-linked oligosaccharides and few 0-linked or complex N-linked oligosaccharides in Ae.gambiae and Atwegypti (Moskalyk et al., 1995) and An.tessellatus (Ramasamy et al., 1997).

Following exflagellation, the extracellular macrogametocyte is fertilized by a motile microgamete within the bloodmeal. Meiotic division then takes place in the zygote, which changes its rounded shape, via a retort form, to a motile ellipsoid ookinete. Ookinetes then cross the peritrophic matrix and the midgut epithelium and lodge on the basal lamina on the haemocoel side of the midgut where they develop into oocysts. The formation of the ookinete and its passage through the midgut, the kinetics of protease release and the rapidity of bloodmeal digestion, peritrophic matrix formation and the release of other mosquito-derived molecules, probably all have a direct bearing on the establishment of the mosquito vector/ malaria parasite relationship as observed with An.tessellatus/ R vivax, An. tesse1latudR falciparum, An.gambiae/R falciparum, An. stephensi/Rfalciparum and Ae.aegyptilRgallinaceum.

Experimental modulation of Plasmodium infectivity in Anopheles sp.

Humoral responses to vector antigens can modulate the estab- lishment of Plasmodium infections in the vector. Ingestion of anti-mosquito antibodies raised to midgut tissues of An. farauti Laveran (Ramasamy & Ramasamy, 1990a) and An.stephensi (LA et al., 1994) reduced their susceptibility to infection with Pberghei. The prevalence of l? v i v a infections in An.tessel1atu.s was significantly reduced by the presence of rabbit IgG purified from An.tessellutus anti-midgut sera (Srikrishnaraj et al., 1995), and this transmission-blocking effect was complement indepen- dent. The reduction in susceptibility of An.tessellatus to Rvivax was due to the anti-midgut IgG exerting effects either on events leading to ookinete formation andor, possibly, the passage of the ookinete through the midgut epithelium. The anti-mosquito antibodies did not cause increased mortality in An.tessel1atu.s. but clearly showed the dependence of Rvivaxon mosquito-derived molecules to establish an infection in the midgut. Anti-midgut antibodies also prevented the formation of the peritrophic matrix in the posterior midgut of An.tessel1atu.s (Ramasamy et al., 1996a). Plasmodium v i v a and Rfalciparum oocysts are found predominantly in the posterior region of the An.tessellarus midgut. The IgG-mediated reduction in Rvivax infection in An.tessellatus (Srikrishnaraj et al., 1995), observed in conjunction

with the absence of a peritrophic matrix in the posterior region, suggests that the peritrophic matrix of An.tessellatus may not be a physical barrier to establishing a Rvivax infection in this mosquito. The presence or absence of peritrophic matrix in An.srephensi did not influence Pberghei infectivity (Billingsley & Rudin, 1992). It therefore appears that the peritrophic matrix does not constitute a firm barrier for the motile ookinete, and the development of Plasmodium in Anopheles.

The synthesis and release of the proteolytic enzymes trypsin, chymotrypsin and aminopeptidase are triggered by the intake of a bloodmeal in Anopheles sp. mosquitoes (Billingsley & Hecker, 1991; H6rler & Briegel, 1995; Chege et al., 1996; Ramasamy et al., 1996b). In Ae.aegypti, two groups of trypsin forms are produced following a bloodmeal, i.e. an early trypsin, which is a signal for activations of the late trypsin, which in turn plays a major role in digestion of the bloodmeal (Felix et al., 199 1 ; Barillas-Murray & Wells, 1993). Digestive proteinases from blood-fed Ae.aegypti have been shown to damage the ookinetes of Rgallinaceum (Gass & Yeates, 1979). The high attrition rate during the development of Plasmodium sexual stages in the Anopheles midgut (Sluiters et al., 1986) may be attributed partly to midgut proteolytic damage, particularly by trypsin. In Aagarnbiae, the rate of digestion of a bloodmeal in individual mosquitoes inversely correlates with the number of Rfalciparum oocysts that develop in the midgut (Meuwissen & Ponnudurai, 1988). The kinetics of appearance of trypsin and aminopeptidase do not, alone, regulate the early stage of Rfalciparum development as shown in An.freeborni Aitken, Angambiae and Aaalbimanus Wiedmann, which exhibit excellent, good, and poor susceptibility respectively to the parasite (Chege et al., 1996). The differences seen in the rates and amounts of trypsin and aminopeptidase in the midguts of these three Anopheles species could not account for the differences in their susceptibility to Rfalciparum. However, studies by Feldman et al. (1990) suggest that elevated aminopeptidase activity may be a factor which limits Pfalciparum development in a refractory strain of An.stephensi. From the view of the mosquito, the release of trypsin brings about digestion of the bloodmeal and, consequently, the synthesis of yolk proteins in the fat body and the development of eggs (Hagedorn, 1985). Mosquito;derived molecules within the midgut that are required for the formation of zygotes and ookinetes from gametocytes may be susceptible to damage by trypsin, chymotrypsin or aminopeptidase. Trypsin may also destroy components of the alternative pathway of com- plement activation that are deleterious to zygotes (Grotendorst & Carter, 1987) or trypsin may be required to activate a Rfalciparum ookinete chitinase (Huber et al., 1991) that is required for penetration of the chitinaceous peritrophic membrane (Shahabuddin et al., 1993).

The development of Rgallinaceum in Ae.aegypti and Rfalciparum in An.freeborni was blocked by inhibitors of chitinase (Shahabuddin et al., 1993). Specific trypsin inhibitors (Shahabuddin et al., 1995) and murine anti-trypsin antibodies blocked the infectivity Rgallinaceum to Ae. aegypti, and this effect was reversed by exogenous chitinase (Shahabuddin er al., 1996). Trypsin inhibitors in a bloodmeal delayed the onset of maximal trypsin activity in An.tessellafus, and the presence of protease inhibitors in the infective bloodmeal increased the proportion of An.tessellatus that became infected with Rvivax (Ramasamy

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Vector midgut and malaria parasites 293

et al., 1996b). The increase in infectivity was manifest both as prevalence of infection and an increase in the number of oocysts. In contrast, inhibition of trypsin activity reduced the prevalence of Rfalciparum infection in An.tessellatus. These opposite effects, observed on infection of An.tessellatus with R vivax and Rfalciparum, are probably due to specific differences in the biology of sexual stages within the midgut of the vector. One difference may be in the kinetics of zygote formation and ookinete migration through the midgut. The development of the Pjalciparum zygote to an ookinete occurs in about 24h after a bloodmeal (Meis et al., 1992). The maximal ookinete penetration activity occurs at around 22 h after a bloodmeal for An.atroparvus van Thiel infected with Rberghei (Sluiters et al., 1986) and between 15 and 18 h in An.omori infected with Ryoelli nigeriensis (Syafruddin et al., 1991). In An.aegypti infected with Rgallinaceum, the maximal penetration of the midgut by ookinetes takes place between 30 and 35 h after a bloodmeal (Torii et al., 1992) and in An.stephensi infected with R falciparum maximal ookinete penetration occurs between 32 and 36h after a bloodmeal (Meis & Ponnudurai, 1987). Therefore, in Ryoelli and Rberghei, ookinete formation, maturation and midgut penetration occur more rapidly than in Rfalciparum and Rgallinaceurn. Plasmodium v i v a ookinetes on the other hand have been detected in smears of the midgut of An.tessellatus at 12h after a bloodmeal, and a higher density of ookinetes was observed in the An.tessellatus midgut at 18h. No Rvivax ookinetes were seen in the midgut at 24-30h (our unpublished observations). Plasmodium falciparum is phylogenetically closer to the avian malaria parasite Rgallinaceum than to Pvivax (Waters et al., 1993; Escalante & Ayala, 1994). Therefore it is possible that Rfalciparum and Rgallinaceum differ from the two murine malaria parasites and Rvivax in having delayed ookinete formation. Trypsin levels in An.tessellatus are elevated between 12 and 2 1 h after a bloodmeal (Ramasamy et al., 1996b). Delaying the action of trypsin in the bloodmeal may therefore reduce the damage to zygotes and immature ookinetes and thus enhance Rvivax infectivity. However, the delay in the peak of trypsin activity in the midgut may have a deleterious effect on young Rfalciparum ookinetes, affecting their maturation. The passage of Pvivax ookinetes through the peritrophic matrix may take place well before the peritrophic matrix is fully formed and therefore be independent of the presence of a putative parasite chitinase. The production of chitinase by Rvivax ookinetes has not been documented. In contrast, due to the comparatively late ookinete development in Rfalciparum, the peritrophic matrix of An.tessellatus probably acts as a barrier to penetration (Meis & Ponnudurai, 1987) and the ookinete therefore requires chitinase to facilitate penetration (Shahabuddin etal., 1993,1995). Plasmodium falciparum ookinetes require trypsin to activate the prochitinase (Shahabuddin & Kaslow, 1994) and the presence of trypsin inhibitors may result in suboptimal activation of prochitinase.

After the peritrophic matrix, it is the midgut epithelium which constitutes a potential physical barrier to the motile ookinete. The penetration of P falciparum through the midgut of Amtephensi is believed to occur by an intercellular route between epithelial cells (Meis & Ponnudurai, 1987). Ookinetes of Rgallinaceum enter the midgut epithelial cells of Ae.aegypti intracellularly, then exit to the intercellular space between the

cells and move to the basal lamina (Toni et al., 1992). Recognition sites on the epithelial cells (either apical or lateral), used by the ookinetes are potential targets for disruption of development of Plasmodium in Anopheles. Ingested anti-mosquito midgut antibodies could reduce infectivity by blocking specific ligands on the midgut epithelium that are recognised by ookinetes during their passage across the midgut (Srikrishnaraj et al., 1995). A number of sugar residues have been demonstrated in the peritrophic matrix and microvilli of the midgut epithelial cells of An.stephensi and Ae. aegypti (Rudin & Hecker, 1989); N-acetyl galactosamine and N-acetylglucosamine in the midgut were proposed ligands for ookinete recognition during its passage across the peritrophic matrix and midgut epithelium (Rudin & Hecker, 1989). In an in vitro study using cultured Rberghei ookinetes, Billingsley (1994) was able to demonstrate the recog- nition of N-acetylglucosamine by the parasite. Glycoproteins of the midgut epithelium of non-bloodfed An.tessellatus have been partially characterised and both N-acetyl glucosamine and mannose residues are present (Ramasamy et al., 1997). N-acetyl glucosamine residues in the oligosaccharide side chains of An.tessellatus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition and orienting sites for Rvivax and Rfalciparum ookinetes since chitotriose or ingested antibodies to the glycoproteins reduce the infectivity of the two parasites (Ramasamy et al., 1997).

Mosquito factors affecting oocyst development

The ookinete, on reaching the haemocoel side of the epithelial cell, comes to rest on the basal lamina to develop into the next stage in the Plasmodium life cycle in the midgut, the oocyst. Here the oocyst assumes its round shape and develops sporozoites. The role the midgut basal lamina plays in recognition by the ookinete and its influence on the subsequent development of the oocyst are not documented. However, laminin and collagen type IV, which are major constituents of the basal lamina of Drosophila, have been identified in the basement membranes of Ae.aegypti and in Rgallinaceum oocyst capsules developing in mosquito midguts (Warburg, unpublished, cited in Warburg & Miller, 1992). Oocyst development Rgallinaceum and Rfalciparum has been demonstrated in vitm utilizing Dmelanogaster L2 cells and matrigel, a basement membrane- like extract containing laminin and collagen type IV, among other components (Warburg & Miller, 1992; Warburg & Schneider, 1993). The observations prompted the authors to suggest that extracellular matrix components may be important for oocyst development in the haemocoel.

The midgut epithelium of An.stephensi is damaged by ookinetes of Rberghei and Rberghei nigeriensis, during their intracellular penetration of the midgut epithelium (Canning & Sinden, 1973; Davies, 1974). Plasmodium berghei infections resulted in high mortality in Anfarauti (Ramasamy & Ramasamy, 1990a), the highest mortality occurring between 24 and 48 h after a bloodmeal. It is likely that damage to the gut caused by penetrating ookinetes is maximal during this time, provoking stress in the vector (Brey, 1994). Midgut epithelial cells show cytoplasmic changes around ookinetes even when there is no pathogenesis, so alterations may be responses to ookinete movement (Torii

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et af. , 1992). In Anopheles selected for refractoriness to infection with Plasmodium, the vector often reacts by the encapsulation of the oocysts in the midgut. The encapsulation reaction occurs early (between 16 and 24h after an infective bloodmeal) in An.gambiae (Collins et al., 1986). A strain of An.gambiae com- pletely refractory to Rcynomofgi shows varying refractioness to Rfalciparum, R v i v a and Rovafe , and refractioness is manifested by the ability of An.gambiae to recognize the live parasite and initiate the encapsulation process (Paskewitz et a f . , 1988; Collins & Paskewitz, 1995). Plasmodium gallinaceurn ookinetes are also susceptible to lysis within the midgut epithelium of An.gambiae and this lytic mechanism is regulated by a ‘recessive’ refractory gene (Vernick et al., 1995). The findings of Paskewitz et a f . (1988), Collins & Paskewitx ( 1 995) and Vernik et al. (1 995) can be logically extended to the selection of genetically refractory lines of the malaria vector which could then be used to replace susceptible vector populations in areas where malaria is prevalent.

Conclusions

The development of Plasmodium in the mosquito midgut is critically dependent on vector cells and molecules. Characteriz- ation of the relevant mosquito molecules is at a very early stage. Structural glycoproteins of the microvilli of midgut epithelial cells, peptides from the endocrine cells, and haemolymph components in particular need to be further investigated. Putative midgut epithelial receptors involved in parasite recognition are targets which are potentially useful for transmission-blocking vaccines. Mosquito trypsin has been proposed as a target for transmission blocking of Rfalciparum (Shahabuddin & Kaslow, 1994). In many parts of the world, including Sri Lanka, Pfalciparum and Rvivax have identical vectors (Ramasamy et al., 1992a, b) and since inhibiting trypsin has opposite effects on Pfalciparum and Pvivax (Ramasamy et af . , 1996b), trypsin is probably not a good target for use in situations where mixed infections of R v i v a and Pfalciparum occur in the same vector.

Methods for chromosomal integration and stable expression of foreign or modified genes in anophelines (Miller et al., 1987; Carlson e r a f . , 1995) are now being developed. Refractory strains of mosquitoes are also being produced by conventional selection and breeding (Collins et al., 1986). A better understanding of mosquito-parasite interactions at a molecular level therefore offers the prospect of objectively manipulating the vector for the control of malaria.

Acknowledgments

This research is currently supported by the Natural Resources Energy and Science Authority of Sri Lanka and the European Community.

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Accepted 26 March 1997

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