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The Biology of Malarial Parasite in the Mosquito - A Review
Amauri Braga Simonetti
Departamento de Microbiologia, Instituto de Biociencias,
Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite
500, 90050-170 Porto Alegre, RS, Brasil
Received 20 December 1995
Code Number: OC96099 Sizes of Files: Text: 126K Graphics: line drawings (gif) - 15.4K [TABLES AND FIGURES AT END OF TEXT] The purpose of this review is to summarize the biology of Plasmodium in the mosquito including recent data to contribute to better understanding of the developmental interaction between mosquito and malarial parasite. The entire sporogonic cycle is discussed taking into consideration different parasite/vector interactions and factors affecting parasite development to the mosquito.
Key words: malaria - mosquito - sporogonic cycle - cell biology The control and prevention of malaria has been pursued for a long time. Although campaigns against malaria were initially successful in some areas, the emergence of resistance of the parasite to drugs and of the mosquito vector to insecticides, combined with the difficulties in implementing and maintaining effective control schemes have led to a resurgence of the disease in many parts of the world (Wernsdorfer 1991, Schapira et al. 1993, Roush 1993). Much of the current work on malaria focuses on immunological aspects of the disease and there has been remarkable progress in the identification of a variety of parasite antigens in different stages of the parasite development, some of which may be included in a future vaccine (Nussenzweig & Nussenzweig 1989, Mitchel 1989, Targett 1989, Nussenzweig 1990, Hommel 1991, Good 1991a). However, experimental and field studies have shown the complexity of the immune response to parasites, indicating that an efficient malaria vaccine is difficult to achieve (Cattani 1989, Good 1991b, 1992, Philips 1992). Therefore, for control of the disease greater understanding of the biological mechanisms involved in host/parasite/vector interactions is essential. In recent years an increasing number of studies have concentrated on the mosquito stages of the parasite development. Comprehensive reviews on biological, ultrastructural, biochemical, molecular and immunological aspects of the parasite and the vector can be obtained in the literature (Sinden 1978, 1983, 1984, Carter & Graves 1988, Carter et al. 1988, Alano & Carter 1990, Billingsley 1990, Crampton et al. 1990, 1994, Alano 1991, Brey 1991, Coluzzi 1992, Kaslow et al. 1994a, Sinden et al. 1996). This review foccuses on the cell biology of malarial parasites at different stages of its development in the mosquito and also includes factors that may affect the parasite infectivity. The sporogonic cycle Some erythrocytic parasites differentiate into sexual forms called gametocytes. Mature and functional gametocytes ingested by an appropriate species of mosquito in a bloodmeal are stimulated to transform into the stages which establish the parasites in their vector (Garnham 1966). Under the influence of changes in the mosquito midgut environment the gametocytes become extracellular within 8-15 min of ingestion. After emergence from the red blood cell (exflagellation) the male gametes fertilize the female gametes within 60 min of ingestion of blood. The fertilized macrogamete (zygote) differentiates into a single motile ookinete over the next 10- 25 hr, which migrates from the bloodmeal through the midgut wall to form an oocyst underneath the basal lamina of the midgut. Each oocyst produces many thousands of invasive sporozoites over a period of 7-12 days. The sporozoites escape from the oocyst and then invade the salivary glands, here they stay for possibly very long periods until injected into another vertebrate host when the next bloodmeal is taken (Sinden 1984, Carter & Graves 1988). A diagram of the sporogonic cycle in the mosquito is shown in Figure. Development of gametocytes (gametocytogenesis) As with other members of the order Haemosporina, gametocytes of Plasmodium can arise from merozoites from pre- erythrocytic parasites. Nevertheless, during natural infections, most gametocytes arise from merozoites of blood- stage origin (Alano & Carter 1990). Two alternative possibilities by which malaria parasites could become committed to either sexual or asexual development have been proposed (Inselburg 1983, Mons 1985, 1986, Bruce et al. 1990): first, the merozoite is not committed at the time of invasion of a red blood cell. During early growth (as a ring form), the parasite is suceptible to factors that will commit it to either sexual or asexual development. Second, during growth of an asexual parasite, environmental factors influence it so that at maturity the schizont produces merozoites that are precommitted to form asexual parasites or committed to form gametocytes upon subsequent invasion of a red blood cell. A number of studies have attempted to identify factors that may influence the differentiation and production of sexual stage parasites including cAMP, presence of antimalarial drugs and other environmental factors associated to the host such as the presence of serum undetermined components (reviewed by Sinden 1983, Mons 1986, Carter & Graves 1988, Dearsly et al. 1990). In a recent report (Schneweis et al. 1991) it was claimed that the production of infective gametocytes in vitro can be enhanced by products of haemolysis but the nature of the erythrocytic factor was not identified. Gametocyte development takes longer than asexual schizogony, e.g. 26 hr as opposed to 22.5 hr in P. berghei (Mons et al. 1985) or in the more extreme case of P. falciparum 8-12 days as opposed to 48 hr (Sinden 1983). The proportion of parasites that develop into gametocytes varies greatly during the course of natural infections even at its peak but is very low in relation to the total parasitaemia (Smalley et al. 1981). The growth and differentiation of gametocytes of P. falciparum has been divided into five stages (I to V) covering about eight days from merozoite invasion to mature gametocyte, each stage being distinguished by successive changes in the organization of the cell (Hawking et al. 1971). Little is known about commitment of gametocytes to be male or female. The fact that both female and male can be produced by haploid blood-stage parasite, the sex of a gametocyte does not result from chromosomal differences between both types of cell but their development must be due to selective gene expression. In general, the number of female gametocytes predominates over the number of male, but this predominance may vary at different times between cloned infections (Cornelissen 1988, Schall 1989). Gametogenesis Mature and functional gametocytes ingested by the mosquito in a bloodmeal are stimulated by the midgut environment to transform into gametes. Studies indicate that various triggers induce gametocytes to undergo differentiation. Microgameto- genesis in vitro is, optimally, dependent upon a rise in pH (Nijhout & Carter 1978), a rise in pCO2 and bicarbonate levels (Carter & Nijhout 1977, Nijhout & Carter 1978), a fall in temperature of a few degrees below that of the vertebrate host (Sinden & Croll, 1975) or a very potent factor termed mosquito exflagellation factor (MEF). The latter is a small heat stable molecule from the mosquito head and gut which stimulates gametogenesis via a bicarbonate- and pH-independent mechanism (Nijhout 1979). Recently, Kawamoto et al. (1991) showed in vitro that induction of exflagellation of P. berghei is triggered by a rise in the intracellular pH (pHi) which is mediated by Ca^++ and cGMP regulation. pHi can be modulated by alkaline media and is controlled by a complex series of interdependent ion pumps and channels controlling Na^+, K^+, Cl^- and HCO3^- transport between the parasite and the environment. Other influential factors described include cAMP analogues and inhibitors of phosphodiesterase (Martin et al. 1978). The duration of microgametogenesis is both temperature and species dependent, e.g. at 20-22 C it may take 7-15 min for P. falciparum in vitro, although exflagellation may be detected after shorter periods in the fluid excreted by feeding Anopheles (Sinden 1983). There is no evidence that exflagellation is influenced by factors released by digestion of the blood meal since digestion normally begins several hours later (Graf et al. 1986). Microgamete formation involves three mitotic divisions with a rapid assembly of eight axonemes on the single microtubule organizing centre that divides and passes to the spindle poles. This division simultaneously segregates the genome and the axoneme so that each of the eight emergent gametes receives a single axoneme and haploid genome, both being connected to a common microtubule organizing centre. After exflagellation the microgametes, normally bearing a single axoneme, a single condensed nucleus and a single kinetosome with its sphere and granule at the distal end, are torn from the microgametocyte surface and rapidly move away into the blood meal (Sinden & Croll 1975). Macrogametogenesis at the morphological level involves little more than escape from the host cell (Sinden 1984). At the cellular level there is de novo synthesis of the proteins which are expressed on the surface of macrogamete (Kumar & Carter 1984). It was recently identified a gametocyte specific protein of P. falciparum called Pf11-1 and there is some evidence that this protein might be involved in the emergence of gametes from the infected erythrocyte (Scherf et al. 1992, 1993). Interactions between the vertebrate host and the parasite do not cease when the blood meal is taken. Following induction of gametogenesis the parasite is liberated into the blood meal. It has been shown that the gametes are susceptible to the phagocytes in the host's blood (Rutledge et al. 1969, Sinden & Smaley 1976) and that they are susceptible to activation of the components of the complement pathway (Grotendorst et al. 1986, 1987). Zygote formation (fertilization) and ookinete development Fertilization is rapid usually occurring within 1 hr of gamete formation, the plasmalemma of the two gametes fuse and the microgamete axoneme and nucleus enter the macrogamete cytoplasm (Sinden 1983). During zygote development, structural changes occur and its transformation to ookinete is in part determined by the gradual assembly of the apical complex including the collar and pollar rings, rhoptries and micronemes (Canning & Sinden 1973, Davies 1974, Sinden 1978). Intensive protein synthesis also begins in the fertilized macrogamete and continues in the zygote and developing ookinete as reported for different Plasmodium species (Kaushal et al. 1983a, Kaushal & Carter 1984, Kumar & Carter 1985, Vermeulen et al. 1985a, 1986, Sinden et al. 1987). Included in this repertoir of proteins are many of the targets of proposed transmission blocking immunity. It has been reported that An. gambiae can concentrate the bloodmeal by a factor of 1.2 to 1.8 reducing the production of ookinetes when compared to An. freeborni which can not concentrate (Sinden et al. 1996). The mature ookinete is a motile cell that varies from 7 to 18 mm in length and 2 to 4 mm in diameter (Sinden, 1978). Locomotion of the ookinete has been described in different Plasmodium species as a linear or snake-like gliding motion (Freyvogel 1966). Studying P. berghei ookinetes in primary mosquito cell cultures, Speer et al. (1974) described spiral waves on the surface of some ookinetes, especially in the anterior half of the body, which might be involved in ookinete locomotion. Some factors could influence the development, survival and infectivity of the parasite during its residence in the midgut lumen. Eyles (1952) has showed that the parasite development ceases at the ookinete stage unless a macromolecular (non- dialyzable) component is present in the blood meal. Studying the influence of red blood cells on the ability of P. gallinaceum zygotes fertilized in vitro to infect Aedes aegypti Rosenberg et al. (1984) found a linear relationship between erythrocyte density and the number of oocysts up to a 50% hematocrit. Furthermore, they deduced that there are one or more nondialyzable substances (erythrocytic factors) contained in normal erythrocytes, and released by mosquito digestion, that are essential for ookinete invasion of the gut epithelium. When they added trypsin inhibitor to the bloodmeal there was an inhibition of midgut penetration by ookinetes. Recent studies have shown that when mosquitoes are fed with cultured P. falciparum (Ponnudurai et al. 1989) and P. berghei (Sinden 1989) gametocytes, upon dilution with fresh red cells, more oocysts result at initial (low) dilutions whereas further dilution reduces oocyst counts. The involvement of blood factors and/or its digestive products in infectivity has been studied in different parasite-vector models. Using a selected line of An. stephensi, Feldmann and Ponnudurai (1989) found mature P. falciparum ookinetes in the midgut lumen of refractory mosquitoes but no further penetration of the gut epithelium was observed. The reasons for this limited development in non- compatible mosquitoes could be related to digestive function since early initiation of hemoglobin degradation and higher aminopeptidase activity have been described in refractory strains of An. stephensi (Feldmann et al. 1990). It has also been shown that P. gallinaceum develops up to the ookinete stage in the non-compatible mosquito An. stephensi, this development occurring over the same time period and with the same success as in the compatible vector Ae. aegypti. However, P. gallinaceum ookinetes did not escape from the midgut lumen in An. stephensi mosquitoes (Rudin et al. 1991). Possible mechanism inhibiting parasite development involves damage of the parasite by digestive enzymes present in the vector. Trypsin and aminopeptidases are the major proteolytic enzymes involved in blood digestion by female mosquitoes, and are produced by the midgut cells in direct response to blood feeding (Briegel & Lea 1975, Graf & Briegel 1982, Billingsley 1990, Billingsley & Hecker 1991). P. gallinaceum ookinetes 0-10 hr old (i.e. zygote to ookinete transition) were shown to be susceptible to mosquito enzymes in double feeding experiments (Gass 1977) and in vitro damage was observed to cultured ookinetes by proteinases from Ae. aegypti (Gass & Yeates, 1979). However, results found by Shahabuddin et al. (1993) using the same parasite/vector system suggest that the parasite secretes an inactive or partially active chitinase that is activated by a mosquito- produced serine protease. In a recent study Chege et al. (1996) examined the effect of digestive enzymes on the kinetics of P. falciparum ookinete development and oocyst infection rates in An. albimanus, An. freeborni and An. gambiae. Their data indicated that proteolytic enzymes alone do not limit the early stages of sporogonic development in these vector species of Anopheles. Peritrophic layer The peritrophic layer (PL) of the insect midgut forms a cylindrical sheet separating the midgut contents from the single cell-layered midgut endothelium. It is secreted within hours of the blood meal at different rates depending on the mosquito species (Billingsley 1990). Apical secretion granules are present in the midgut cells of Anopheles species, which are released into the periphery of the posterior midgut lumen during the feeding process in response to stretching of the gut wall. The vesicle contents coalesce and then condense to form a compacted PL between 8 and 24 hr (Hecker 1977, Berner et al. 1983). In culicine mosquitoes the PL is formed de novo by the posterior midgut cells in the proventriculus. In A. aegypti, formation starts immediately after the blood meal, but about 12 hr later it is a mature structure (Perrone & Spielman, 1988). The role of the mosquito PL as a barrier to ookinete invasion of the gut wall and pathogenic effects of Plasmodium species upon the vector are controversial. In ultrastructural studies on the interaction of P. falciparum ookinetes with the midgut epithelium of An. stephensi Meis & Ponnudurai (1987) frequentely found parasites trapped in the membrane. They also observed that if the PL was dissected out 36 hr after the blood meal many ookinetes were attached to its external surface. In the same study it was mentioned that the ookinete was capable of penetrating the newly formed, but not the thickened and hardened PL 36 hr after the feeding. A failure to cross this barrier or retarded penetration might increase the length of exposure of ookinetes to mosquito trypsin to which it is known to be sensitive. In contrast, using P. berghei-infected An. atroparvus mosquitoes, Sluiters et al. (1986) concluded that the PL would not function as physical barrier against migrating ookinetes which can pass through fenestrations. Billingsley and Rudin (1992) observed that infectivity of An. stephensi by P. berghei, measured by oocyst counts, was unnaffected by the presence or absence of the PL. However, in A. aegypti infected with P. gallinaceum its presence serves to reduce rather than prevent infection (Ponnudurai et al. 1988, Billingsley & Rudin 1992). These observations suggested to the authors that in compatible vector-parasite combinations, the PL acts as a limiting, rather than absolute barrier to the penetrating ookinete while in incompatible combinations the PL appears to be an absolute barrier to ookinete penetration. To reach the midgut epithelium the ookinetes must first cross the partially or fully formed PL. The PL may act as the recognition site for the penetrating ookinete via lectin- mediated mechanisms. The occurrence of sugar residues which could be involved in vector recognition by the parasite has been demonstrated in the PL of An. stephensi and Ae. aegypti (Berner et al. 1983). Rudin and Hecker (1989) showed the presence of binding sites for different lectins in midguts of P. berghei-infected An. stephensi, with high specificity for N-acetyl-D-galactosamine (GalNAc), and P. gallinaceum-infected Ae. aegypti, with high specificity for N-acetyl-D-glucosamine (GlcNAc). The authors concluded that it seems likely that lectin-binding phenomena play a role in the orientation of the parasites on their way out of the midgut lumen and that the PM and/or glycocalyx may be crucial structures for the penetration of the gut epithelium by the ookinete. This does not exclude the possibility that ookinetes penetrate the PL by an enzymatic process (see below). Ultrastructural observations on Ae. aegypti infected with P. gallinaceum showing an electron dense amorphous material in front of the parasite were consistent with a blockade of parasites within the PL (Sieber et al. 1991). The ookinetes appeared to disrupt the layers of the PL, suggesting an enzymatic mechanism for penetration. These observations were further investigated by Huber et al. (1991) who, using the same vector/parasite system, also suggested a possible role for GlcNAc in the binding of the ookinete to the PL, and demonstrated the presence of chitin in the PL. They observed that mature ookinetes transiently secreted a soluble chitinase, thought to be responsible for the digestion of the PL. Recently, Shahabuddin et al. (1993) reported an inhibition of P. gallinaceum chitinase and a transmission blocking activity of a chitinase inhibitor, allosamidin, on the sporogonic development of P. gallinaceum and P. falciparum in the respective vectors Ae. aegypti and An. freeborni. However, enzymatic mechanisms of penetration may differ in other vector/parasite systems since the PL of An. stephensi appears not to contain chitin (Berner et al. 1983).
Penetration of midgut epithelium Ookinetes found in the epithelial cell layer between 24 and 48 hr post-infection have successfully evaded the obstacles presented by the midgut lumen; however, they still might fail to develop at the normal site of development (Sinden 1984, Ponnudurai et al. 1988). It has been shown that the midgut wall is negatively charged (Houk et al. 1986), but there is no evidence of electrical charge interactions between ookinetes and epithelial cells. It has been a matter for discussion as to whether the ookinete follows an intra- or intercellular route to reach the ultimate site of development and encystment in the outer wall of the midgut epithelium. Intercellular movement of P. gallinaceum ookinetes was first described by Stohler (1957), but Mehlhorn et al. (1980) have found the same parasite in an intracellular position. Recently, Torii et al. (1992a) observed P. gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Ae. aegypti, which they interpret as that they first enter into the epithelium, then exit into the intercellular space and move to the basal lamina. Garnham et al. (1962) showed that the ookinete of P. cynomolgi bastianelli enters the epithelial cell by liquifying the cell membrane. Davies (1974) again postulated intercellular movement by P. berghei nigeriensis ookinetes. Although describing the ookinete of P. berghei in an intracellular position, as did Garnham et al. (1969), Canning and Sinden (1973) stated that the parasite might also migrate by an intercellular route. More recently, P. yoelii nigeriensis ookinetes have been described to take an intracellular route to the external wall of the midgut (Maier et al. 1987). However, when the same parasite was used to infect An. omorii, an intercellular route was mostly undertaken, although the intracellular occurrence was also observed (Syaffruddin et al. 1991). Meis and Ponnudurai (1987) presented evidence that P. falciparum ookinetes migrate between the epithelial midgut cells. Using a specific monoclonal antibody they also observed a track in the PL, which is related to the shedding of a 25 kD surface protein (Pfs25) during movement. The authors suggested that this protein may bind to receptors on the epithelial cells prior to an intercellular invasion, since it is reported to have epidermal growth factor (EGF)-like domains (Kaslow et al. 1988). It was recently shown that Pfs25 persits in the oocyst wall during parasite development in the mosquito (Lensen et al. 1993). The same group studied the migration of P. falciparum and P. berghei ookinetes through the midgut epithelium in An. stephensi by using ruthenium red staining (Meis et al. 1989). The results of previous studies were confirmed: P. falciparum ookinetes penetrated by intercellular route, but the rodent parasite P. berghei appeared to take an intracellular position, confirming that both mechanisms occur and are species-dependent. In the case of P. berghei a protein of 21 kD (Pbs21) present on the surface of the ookinete (Sinden et al. 1987) could play a role during the intracellular invasion of the midgut epithelium of An. stephensi mosquitoes. It was demonstrated in the midgut of P. berghei infected mosquitoes that expression of Pbs21 was predominantly localized on the ookinete surface one day after the infectious blood meal and thereafter expression declined to a minimum on days 2 and 3, the time of onset of oocyst development (Simonetti et al. 1993). The mode of penetration by ookinetes can perhaps be related to damage of the epithelial lining of the midgut. Intercellular migration may not damage cell membranes, and increased mortality does not occur in P. falciparum-infected An. stephensi and An. gambiae during this period, even with very heavy parasite loads (Meis & Ponnudurai, 1987). Similar observations have been reported in P. gallinaceum-infected Ae. aegypti (Freier & Friedman, 1987). They observed similar mortality rates in infected and uninfected batches of mosquitoes. However, when ookinetes use an intracellular route, as described previously, increased damage to midgut cells might occur, resulting in higher mosquito mortality. This is probably mediated by invasion of the hemolymph by opportunistic gram-negative bacteria and/or microsporidia (Maier et al. 1987, Seitz et al. 1987). Ultimately the ookinete penetrates the basement membrane, but fails to pass through the basal lamina of the midgut adjacent to hemocoele (Sinden 1984). Whether this is due to the inability of the ookinete to penetrate the basal lamina, or to the specific recognition of the lamina and consequent shut- down of the incisive process is not known. Interactions of parasites with vector extracellular matrix proteins (ECM) cannot be discounted (Kaslow et al. 1994b). Oocyst development
Oocyst development is predominantly extracellular (Duncan et al. 1960, Garnham et al. 1969, Howells & Davies 1971, Sinden 1975), but occasionally occurs within the midgut epithelial cell (Vanderberg et al. 1967, Bafort 1971, Beaudoin et al. 1974). The ookinete usually comes to rest beneath the basal lamina 18-24 hr after the infective blood meal (Sinden 1978). It rapidly rounds up between 18 and 72 hr after feeding and the apical complex is resorbed into the oocyst cytoplasm (Garnham et al. 1969). There is some evidence suggesting a significant role of the basal lamina in the development of the ookinete (Kaslow et al. 1994b). It was found that in vitro- cultured ookinetes injected directly into the hemolymph form clusters of oocysts adherent to the basal lamina throughout the hemocoele. Furthermore, binding of ookinetes to artificial surfaces, such as plastic, is enhanced at least 10- fold by addition of various components of basal lamina such as matrigel, collagen IV, and laminin (Warburg & Miller, 1992). The young oocyst is enveloped by a thick plasmalemma that is covered on the hemocoelomic surface by a fibrous basal lamina. Oocysts from the second day onwards are also covered by an amorphous capsule which becomes reduced in thickness at maturity (Vanderberg et al. 1967, Aikawa 1971, Strome & Beaudoin 1974, Sinden 1975). Despite the usual growth of the oocyst under the basal lamina of the midgut, oocyst development is not site specific. Weathersby (1952, 1954, 1960) has shown by injection of gametocytes directly into the hemocoele of susceptible mosquitoes that oocysts would develop to maturity if attached to other parts of the body than the stomach or even if they were floating freely in the hemocoel fluid. In his experiments he used different parasite-host combinations and concluded that the site of oocyst development is probably not a critical factor in the maturation process. Furthermore, the factors that are responsible for the death of parasite in refractory lines are not confined to the stomach wall. These results were supported by those reported by Ball and Chao (1957, 1960, 1961, 1976) who showed that oocysts of P. relictum may develop in vitro away from the intact stomach of the mosquito. The overall results of this series of studies by Ball and Chao demonstrated in vitro development of all stages from ookinetes to fully infective sporozoites without attachment of oocysts to the midgut. However, it was not possible to obtain complete sporogonic development in a single preparation. Rosenberg and Koontz (1984) injected cultured P. gallinaceum zygotes into the hemocoele of Ae. aegypti mosquitoes and observed development of ectopic oocysts in approximately 50% of the mosquitoes, with sporozoites being found in the salivary glands. These observations suggest that oocyst metabolism is not dependent upon direct transfer of nutrients from the midgut epithelium. Occasional intracellular oocyst development has been reported for P. berghei in An. stephensi and An. quadrimaculatus (Vanderberg et al. 1967). The same localization was described in P. vinckei by Bafort (1971) who concluded that both mechanical pressure and physiological mechanisms play a role in the movement of oocysts to the hemocoelomic surface. Studying the sporogonic development of P. berghei in An. stephensi, Beaudoin et al. (1974) found oocysts developing ectopically within the midgut epithelium following normal infection, eventually emptying their sporozoite content into the tissue itself or the midgut lumen. In addition, they observed no morphological and structural abnormalities in the luminal parasites which displayed good viability. In contrast to P. berghei, no ectopic development was seen in P. falciparum-infected An. stephensi mosquitoes (Meis et al. 1992b), confirming previous results observed with P. falciparum in naturally infected An. gambiae (Sinden & Strong 1978). Recent reports described an enhancement of oocyst development in vitro for P. berghei (Syaffruddin et al. 1992), P. gallinaceum (Warburg & Miller, 1992) and P. falciparum (Warburg & Schneider, 1993), when insect cell lines were added into the culture medium. From these observations it appears that nutritional or other regulatory requirements of the developing parasite can be met without a direct contact with midgut epithelium or haemolymph. The question of how the oocyst is supplied with nutritive material is an intriguing one. Little information is available on the uptake and source of nutrients for oocyst development. It is assumed that in vivo the source of nutrients is the hemolymph. Mack and Vanderberg (1978) analyzed hemolymph of An. stephensi collected from uninfected and P. berghei-infected mosquitoes at different stages of the parasite development. It was found that four days after the blood meal the osmotic pressure and the specific gravity were lower in infected mosquitoes compared with uninfected ones. The difference, however, was attributed to indirect effects of the quality of the ingested blood meal. These studies were complemented by analysis of the concentration of free amino acids in the hemolymph collected in similar conditions with results showing significantly lower concentrations in infected mosquitoes with decreases in valine and histidine, and a total loss of detectable methionine suggesting it is incorporated (Mack et al. 1979). This difference could be due to the utilization of some of these amino acids by the developing oocyst as suggested by Ball and Chao (1976) who analyzed the uptake of amino acids by P. relictum oocysts in vitro, comparing growth of uninfected and infected guts of Culex tarsalis in Grace's insect culture medium. They found significant decreases in the concentration of certain amino acids including arginine, asparagine, proline and histidine, and less marked decrease in concentrations of others like methionine, valine, leucine and isoleucine. From these studies it appears that the reduced amount of free aminoacids in the hemolymph is due to oocyst metabolism. Autoradiographic studies with P. gallinaceum in Ae. aegypti mosquitoes indicated that ^3H-leucine is uniformly incorporated throught the oocyst within 15 min of injection into hemocoele (Vanderberg et al. 1967).
Sporogony With increasing maturation the oocyst undergoes considerable cytoplasmic subdivision. Initially the plasmalemma forms invaginations and clefts that penetrate even deeper into the cytoplasm, thus subdividing the cell (Vanderberg et al. 1967, Terzakis 1971, Posthuma et al. 1988). In a transmission electron microscopy study of P. falciparum oocysts it was suggested that cleft formation was due to dilation of endoplasmic reticulum (Sinden & Strong 1978). Using immunogold labelling technique during sporogonic stage of the same parasite, Posthuma et al. (1988) considered the latter explanation unlikely. With increasing activity the cytoplasmic clefts become extended and the expanding vacuolar space more pronounced leading to the sporoblast formation. Along the clefts sporozoites are formed by a budding process at the surface of the limiting membrane (Vanderberg et al. 1967, Howells & Davies 1971, Canning & Sinden 1973, Sinden & Strong 1978). As the sporozoite continues to bud off, a nucleus and various cytoplasmic components are passed into it from the sporoblast. The membranes of the developing sporozoite pellicle are formed and other organelles like microtubules and rhoptries become discernible (Vanderberg et al. 1967, Sinden & Garnham 1973). When sporogony is completed (about 10-12 days after the infective feed), the oocyst is filled with sporozoites and one or more residual bodies (Sinden 1984). Estimations of number of sporozoites per oocyst have ranged widely. Garnham (1966) reported that the number in a single P. vivax oocyst varies from 1,000 to 10,000. Pringle (1965) estimated that a single oocyst of P. falciparum contains nearly 10,000 sporozoites. Studies carried out with mosquitoes fed on infected volunteers from Thailand showed a mean count of approximately 3,700 sporozoites per oocyst for P. vivax and 3,400 for P. falciparum, whereas for P. cynomolgi-infected mosquitoes a single oocyst contained about 7,500 (Rosenberg & Rungsiwongse 1991). Dependent upon species, the mature sporozoite varies from 9 to 16.5 mm in length and from 0.4 to 2.7 mm in diameter; aberrant forms have been described up to 40 mm long (Sinden 1978). So far, studies on synthesis and expression of proteins during sporogonic development have foccused on a polypeptide called the circumsporozoite protein (CSP) found on the mature sporozoite. Observations on the origin of CSP and its distribution through the mosquito stage were reported by several authors. It is now well established that these proteins are synthesized in maturing oocysts of different Plasmodium species from 6-7 days after the infective blood meal, before sporozoites are visible (Nagasawa et al. 1987, 1988, Posthuma et al. 1988, Hamilton et al. 1988, Boulanger et al. 1988, Torii et al. 1992b, Meis et al. 1992a). At this stage, CSP is present on the plasmalemma and at various sites within the cytoplasm and endoplasmic reticulum of the sporoblast. When the sporozoites bud from the sporoblast they are already covered with CSP which is also found in salivary gland sporozoites (Yoshida et al. 1981, Santoro et al. 1983, Tsuji et al. 1992). Humoral encapsulation of oocysts, which in malaria infected mosquitoes is known as Ross' black spores, has been described in P. berghei nigeriensis and P. vivax and seems to occur mainly in older oocysts which have begun to produce sporozoites (Sinden & Garnham, 1973). This phenomenon was studied in a selected line of An. gambiae that encapsulates different Plasmodium species (Collins et al. 1986). The authors demonstrated that refractoriness is manifested by melanization of the ookinete after its passage through the midgut epithelium. Paskewitz et al. (1989) localized phenoloxidase activity in the basal lamina of the epithelial cells of both encapsulating and susceptible mosquitoes prior to blood feeding. However, after an infective blood meal, this activity was still observed close to invading ookinetes in refractory mosquitos but it was reduced or absent in susceptible mosquitoes. When the non-compatible vector An. gambiae was fed with ookinetes of P. gallinaceum, invasion of the midgut epithelium by the ookinetes occurred but oocysts were infrequentely formed. Using the same system Vernick et al. (1989) and Vernick and Collins (1989) tried to elucidate mechanisms involved in vector-parasite incompatibility by injecting in vitro- cultured ookinetes into the hemocoel of mosquitoes and monitoring parasite development using specific rRNA probes. As no differences were found between susceptible and refractory mosquitoes the authors suggested that the specific lytic factor(s) in the refractory line are intracellular. The sporozoite and salivary gland invasion
The motile sporozoites emerge into the hemocoele through holes from an area of weakness in the oocyst wall. Holes are possibly produced by a combination of the muscular action of the gut wall and the activity of the sporozoites (Sinden 1978, Meis et al. 1992b). Within the hemocoel the sporozoites are distributed throughout the mosquito and can initially be found in many parts of the insect, even in the maxillary palps; within a day or two of their release from oocysts the sporozoites invade the salivary glands where they accumulate and remain until delivery (Vaughan et al. 1992). Thus, sporozoites do not adhere to most tissues, except for the salivary glands and rarely the midgut wall, hemocytes or thoracic muscles (Vanderberg 1974, Sinden 1975, 1978, Golenda et al. 1990, Vaughan et al. 1992). The latter observation, especially in heavily infected mosquitoes, could be associated with an impairment of flight activity in malaria-infected An. stephensi vectors demonstrated by some authors (Rowland & Boersma 1988). It has been estimated that in mosquitoes fed on individuals with naturally acquired P. vivax about 850 sporozoites per oocyst reached the glands (Sattabongkot et al. 1991) which follows, by calculation, that approximately 23 % of all P. vivax sporozoites released into the hemocoel subsequentely reach the salivary glands (Rosenberg & Rungsiwongse 1991). Vaughan et al. (1992), using regression analysis, calculated the approximately 650 salivary gland sporozoites were produced per oocyst and reported that virtually all oocyst infections produced salivary gland infections in An. gambiae infected with P. falciparum by membrane feeding. The same group has found a similar number by studying the sporogonic development of cultured P. falciparum in six species of laboratory-reared Anopheles mosquitoes (Vaughan et al. 1994). This is in contrast to observations on wild-caught An. gambiae from Burkino-Faso (Lombardi et al. 1987) and western Kenya (Beier et al. 1990) where sporozoites failed to enter the salivary glands in 43% and 10% of infected mosquitoes, respectively. However, when sporozoites from P. gallinaceum oocysts were injected into Ae. aegypti female mosquitoes only 6.5 to 10.4% of inoculated sporozoites invaded the salivary glands. Interestingly, injected salivary gland sporozoites did not reinvade the glands (Touray et al. 1992). Recently, a laboratory study on An. tesselatus mosquitoes infected with different isolates of P. vivax and P. falciparum from patients living in Sri Lanka showed that approximately 15% of mosquito batches in which oocysts developed failed to produce salivary gland sporozoites (Gamage-Mendis et al. 1993). This discrepancy between naturally and laboratory-infected mosquitoes could be attributed to different environmental conditions or mixed mosquito populations. It could be expected sporozoites would elicit a humoral response in the mosquito by activation of the prophenoloxidase cascade and as a result be killed by melanization. However, sporozoites in the hemocoele are rarely seen to be melanized (Brey 1991). Sporozoites might be protected from mosquito defense reactions against non-self' by antigen sharing. This has been demonstrated as a potential mechanism for avoidance of mosquito defence reactions by microfilariae of Brugia pahangi (Maier et al. 1987). Immunoreactivity to CSP was observed on the midgut wall of mosquitoes infected with P. falciparum (Boulanger et al. 1988) and P. yoelii (Beaudoin et al. 1990). The latter authors also detected reactivity on uninfected midguts and suggest the presence of a common determinant between the parasite and the mosquito. The migration of sporozoites from oocysts to salivary glands could be active (in contact with basal lamina), passive (in suspension) or both. If active, the parasite would use a gliding motility to move across the basal lamina and reach the gland. Vanderberg (1974) described circular gliding and attached waving locomotion and movement in sporozoites of different species when bovine serum albumin was added to Medium 199. Sporozoites that move over a substratum in vitro leave behind trails of CS proteins (Stewart & Vanderberg 1988, 1991, 1992). Soluble CSP was shown to be present throughout the hemocoel (Robert et al. 1988). Beier et al. (1992a) concluded that the release of CSP by sporozoites is a normal but complex mechanism that they interpreted to be associated with sporozoite survival in the host, is not site- specific and it would be regulated in response to background levels of soluble CSP in the environment (negative feedback mechanism). This idea is supported by previous results showing that the parasites cease to synthesize CSP during their journey through the hemolymph but shedding still occurs (Posthuma et al. 1988, 1989). There is no evidence yet for chemotaxis but the congregation of sporozoites in the vicinity of the glands before invasion may support, according to some workers, a tactile mechanism (Golenda et al. 1990, Meis et al. 1992b). Several polysacharides have been identified that may orient protozoa towards or away from a stimulus (Van Houten 1988). The selective invasion of mosquito salivary glands by malarial parasites is not well understood. Specific recognition of the glands was shown in P. knowlesi. Oocysts developed normally on the gut of An. freeborni but sporozoites were never found in salivary glands. When glands from the susceptible vector An. dirus were implanted into the abdomen, they did become infected (Rosenberg 1985). These experiments demonstrated that the salivary glands themselves determined specificity. Although invasion of salivary glands seems to require specific recognition, the mechanism by which sporozoites recognize, attach to, and penetrate the glands remains to be determined. Sporozoites preferentially invade the medial lobe and the distal portions of the lateral lobes of the salivary glands where the salivary duct is not chitinous in Anopheles species (Sterling et al. 1973, James & Rossignol 1991, Ponnudurai et al. 1991). Hence, the occurrence of a specific receptor-mediated invasion by sporozoites is plausible. Perrone et al. (1986) used lectins to characterize carbohydrate moieties on the basal lamina of Ae. aegypti salivary glands. As the median and distal lateral lobes bound a common lectin, RCA 120, whose substrate is b-D- galactose, the authors suggested that sugars that bind this lectin serve as candidate residues to which sporozoites may attach. In contrast, the sporozoite coat binds some lectins with very low efficiency (Schulman et al. 1980, Turner & Gregson 1982). However, in a recent scanning electron microscopic study Meis et al. (1992b) localized P. falciparum sporozoites in proximal and distal parts but were unable to identify any specific regions on the glands where sporozoites penetrate. These authors also showed sites where the sporozoites have pierced the basal lamina, which probably explains the presence of CSP on the basal lamina induced by shedding during penetration (Posthuma et al. 1989) and the presence of immunoreactive spots of 1-2 mm on the surface of infected glands (Golenda et al. 1990). Recently, Touray et al. (1994) developed an in vivo salivary gland invasion assay and have found that anti-salivary gland antibodies, sulfated glycosaminoglycans and some lectins, particularly Suc-WGA, block invasion of sporozoites. Although the mechanism of blocking is not yet known, those lectins that blocked invasion bound to salivary glands but did not bind to sporozoites. Beier et al. (1992a) and Beier (1993) proposed that as sporozoites invade the salivary glands, the build-up of CSP is the signal for sporozoites to halt their active motility, and thus their release of CSP (down-regulation). The involvement of CSP and other proteins distinct from CSP such as PySSP2 (Charoenvit et al. 1987) and PfSSP2 (Robson et al. 1988, Rogers et al. 1992a,b) in binding of sporozoites to the basal lamina of mosquito salivary glands is speculative. As they share similarities in their structure they might be related to this process since it has been suggested region II of CSP is involved in hepatocyte invasion (Cerami et al. 1992a,b, Pancake et al. 1992). Rosenberg (1985) and King (1988) have suggested that sporozoites invade the salivary gland cells using a mechanism similar to invasion of erythrocytes by merozoites although there is no evidence of parasitophorous vacuole membrane formation observed in other stages of the parasite cycle (Sinden & Strong 1978, Meis & Verhave 1988). Membrane-limited vacuoles beneath the plasma membrane in An. stephensi distal salivary gland cells invaded by P. berghei have been described (Sterling et al. 1973). Penetration could also involve an unusual intercellular route as suggested by Golenda et al. (1990) who detected CSP in the region between cells of the median lobe of the gland. Posthuma et al. (1989) observed many sporozoites on the basal side of the cells, but also found trail-like CSP immunoreactivity at the lateral space between the cells. After penetration, sporozoites are present in bundles in the accini of gland cells in both proximal and distal areas. Most probably, the sporozoites which are present in proximal areas of the glands are unable to reach the draining duct because of the chitinized layer in that area whereas distally localized sporozoites reach the draining duct via the large unchitinized collecting tube (Meis et al. 1992b). This explanation would not be valid for Aedes species since the salivary ducts are lined with chitin and extend the full length of the glands (James & Rossignol 1991). Penetration by sporozoites could cause pathological vesiculation and cytoskeletal changes in salivary gland tissues as the infected cells are often deformed and swollen (Sterling et al. 1973, Maier et al. 1987). The efficiency of salivary gland invasion is poorly understood. It has been estimated that the median sporozoite load in the glands is less than 10,000 in colonized or wild Anopheles species (Shute 1945, Pringle 1966, Wirtz et al. 1987, Beier et al. 1991b, Sattabongkot et al. 1991) or slightly higher (Ponnudurai et al. 1991). The development of infectivity by the sporozoites appears to be asynchronous, in some cases taking place in the hemocoele, while in others not occuring until after they have invaded the salivary glands. Thus, it seems to be time-dependent rather than site-dependent (Vanderberg 1975, Daher & Krettli 1980). It was demonstrated in P. berghei-infected mice that populations of salivary gland sporozoites were more than 10,000 times as infective to the vertebrate host as populations of oocyst sporozoites from the same mosquitoes (Vanderberg 1975). Touray et al. (1992) found that as few as 10-50 salivary gland P. gallinaceum sporozoites were required to induce infection in chickens compared to 5,000 oocyst sporozoites. Sporozoite infectivity increases with time during the first week after the invasion of the salivary gland (Vanderberg 1975). Degeneration of sporozoites is not frequent in nature as observed by Barber (1936) who studied anophelines collected in Mediterranean areas. When degeneration occurred in a salivary gland or a lobe of a gland, in another gland or lobe the sporozoites were normal or in a different stage of degeneration. The number of sporozoites injected into the tissue or capillary of the vertebrate is very small compared to that found in the salivary glands. It has been estimated, by employing different methods, that each bite delivers fewer than 50 sporozoites with a tendency for most sporozoites to be ejected in the first droplets of saliva (Vanderberg 1977, Rosenberg et al. 1990, Ponnudurai et al. 1991, Beier et al. 1991a,b, Beier et al. 1992b, Li et al. 1992). Although a correlation between salivary gland sporozoite load and sporozoite inoculum has been reported (Rosenberg et al. 1990) ejection of sporozoites is probably a random process, more related to the architecture of the salivary gland duct system than to the number of sporozoites in this organ (Ponnudurai et al. 1991). Some studies reported that P. falciparum-infected mosquitoes deliver sporozoites in an unpredictable fashion, sometimes not at all (Ponnudurai et al. 1991), and others transmit inconsistently (Rickman et al. 1990). Clumping of sporozoites has been reported in infected salivary glands (Sterling et al. 1973) and clusters of sporozoites were detected after delivery when An. stephensi mosquitoes infected with P. falciparum were allowed to feed through fresh mouse skin (Ponnudurai et al. 1991). It has been observed that salivary glands are not depleted of sporozoites even in vectors that feed up to 15 times (Shute 1945), which allows infected mosquitoes to remain potentially infectious for life. The low sporozoite inoculum and the low entomological inoculation rates in natural conditions (Mendis et al. 1990a, Gordon et al. 1991) demonstrates the high efficiency with which injected sporozoites will develop malaria.
Factors affecting parasite development to the mosquito The process of infection of mosquitoes is exceedingly complex and regulated by a range of factors originating from the parasite, the vertebrate host and the mosquito vector, and from the interactions between all three (Sinden 1991). Many of these and other factors are known to affect fertilization and subsequent stages of parasite development, thus having great influence on transmission of the disease. To have an idea, when sporogonic development of cultured P. falciparum was evaluated in six species of Anopheles mosquitoes there was a total loss of approximately 31,600-fold in the parasite population from macrogametocyte to oocyst stage (Vaughan et al. 1994).
a) Host location and feeding behaviour - Of primary importance in malaria transmission is the proportion of mosquito feeds taken from humans and the proportion of these feeds taken from infected individuals. In nature a vector needs to survive longer than the sporogonic period after taking an infective blood meal; during this period the mosquito probably takes blood meals every 2-3 days, depending on its gonotrophic cycle and the availability of breeding sites (Ponnudurai et al. 1989). Parasitemic hosts tend to be sick and often less irritated by mosquito feeding. Additionally, the thrombocytopenia which is commonly associated with blood-borne parasitic diseases leads to facilitation of vessel location, resulting in increased feeding success by mosquitoes on parasitemic hosts (Rossignol et al. 1985). Salivary glands and saliva contain a whole range of components with pharmacological activities important for blood feeding success and subsequent bloodmeal processing, including anticoagulants, anti-inflammatory, vasodilatory and immunosuppressive compounds (Ribeiro et al. 1984, 1989, Ribeiro 1987, Titus & Ribeiro 1990, James & Rossignol 1991). Rossignol et al. (1984) concluded that the lesions caused by P. gallinaceum sporozoites in the salivary glands of Ae. aegypti result in reduced levels of salivary apyrase. Mosquitoes deficient in salivary apyrase experience difficulty in locating host blood and engorging; they therefore probe more often and may attempt to feed on several hosts in succession (Rossignol et al. 1984, 1986, Ribeiro et al. 1985). Li et al. (1992) demonstrated that probing time of P. berghei-infected An. stephensi is not affected by sporozoite invasion of salivary glands. Blood meal size may influence infectivity since it determines the number of gametocytes ingested and therefore subsequent infection. It has been shown that larger An. dirus females took larger bloodmeals by artificial feeding with cultured P. falciparum and developed significantly more oocysts (Kitthawee et al. 1990). In a field study, Lyimo and Koella (1992) reported that the proportion of An. gambiae mosquitoes infected with P. falciparum during a blood meal was independent of size but the number of oocysts harboured by infected mosquitoes increased with size of the mosquito.
b) Gametocyte carriers/transmission blocking immunity - Malarial infections induce host responses to both asexual and sexual stage malaria parasites that may modulate gametocyte infectivity. Great heterogeneity in infectiousness of different carriers has been noted, with apparently poor correlation between infectiousness and gametocyte density (Graves et al.1988). However, it remains unclear whether symptomatic and asymptomatic asexual infections differ in their ability to influence gametocyte infectivity. It has been reported that asymptomatic P. falciparum patients were more infectious than symptomatic patients (Boyd & Kitchen 1937, Jeffery & Eyles 1955, Muirhead-Thomson 1957, Carter & Graves 1988) while, in contrast, another report suggested that asymptomatic and symptomatic P. malariae patients were equally infective (Young & Burgess 1961). Specific and non-specific responses are believed to modulate parasite transmission. The induction of specific immunological responses to Plasmodium shows marked heterogeneity within human populations (Mendis et al. 1987, Graves et al. 1988, Targett 1990, Snow et al. 1993). The sexual stages can induce trasmission blocking immunity (TBI) and effective targets include antigens identified on the surface of the macro- and/or microgemetes ( pre-fertilization'antigens) as well as antigens present on the surface of the gamete, zygote and ookinete ( post-fertilization' antigens). Antibodies to pre-fertilization' antigens of P. falciparum such as Pfs230, Pfs48/50 and Pfs16 have been detected in humans (Graves et al. 1988, Premawansa et al. 1994, Hogh et al. 1994). A significant association between lacking of infectivity of P. falciparum gametocyte carriers and recognition of epitope IIa on Pfs48/50 by antibodies in their sera has been observed (Graves et al. 1992). Naturally acquired TBI to P. vivax sexual stage antigens has also been demonstrated (Mendis et al. 1987, 1990b, Mendis & Carter 1991, Ranawaka et al. 1988, Goonewardene et al. 1990, Gamage- Mendis et al. 1992) and appears to play a role on transmission of the disease (de Zoysa et al. 1988). In P. vivax malaria antibodies, at low concentrations, can also have a transmission-enhancing effect on infectivity of malarial parasite to mosquitoes (Mendis et al. 1987, Peiris et al. 1988, Naotunne et al. 1990, Gamage-Mendis et al. 1992). A recent report described TBI in P. vivax malaria when antibodies raised against a peptide blocked parasite development in the mosquito An. tesselatus (Snewin et al. 1995). As shown in a variety of Plasmodium species, within the mosquito vector antibody to the pre-fertilization' antigens may prevent fertilization by any of four mechanisms: (1) the agglutination of macro/microgametes limiting their mobility; (2) antibody coating of macro/microgamentes inhibiting cell-cell recognition; (3) opsonization in the bloodmeal or (4) complement dependent/independent lysis (Gwadz 1976, Carter & Chen 1976, Carter et al. 1979, 1985, 1990, Kaushal et al. 1983a,b, Rener et al. 1983, Harte et al. 1985a, Vermeulen et al. 1985b, Grotendorst et al. 1986, Grotendorst & Carter 1987, Quakyi et al. 1987, Peiris et al. 1988, Premawansa et al. 1990). Cellular responses are also involved in TBI and may have some influence on parasite infectivity (Harte et al. 1985b, Mendis et al. 1990b, Riley & Greenwood 1990, Goonewardene et al. 1990). Immunity to `post-fertilization' antigens such as Pfs25, Pbs21, Pgs25 and Prs25 also plays an important role on transmission of the disease by suppressing parasite infectivity at different stages of its development in the mosquito as demonstrated by many workers in different Plasmodium species (Grotendorst et al. 1984, Vermeulen et al. 1985a, Sinden et al. 1987, Winger et al. 1988, Fries et al. 1989, Carter & Kaushal 1984, Carter et al. 1989a,b, Kaslow et al. 1991, 1992, 1994b, Foo et al. 1991, Sieber et al. 1991, Tirawanchai et al. 1991, Duffy et al. 1993, Paton et al. 1993, Ranawaka et al. 1993, 1994). The mechanisms of blockade could be the same four described above and/or the antibody may act by damaging the parasite surface coat (Ponnudurai et al. 1987). Non-specific responses to the asexual stages are believed to modulate parasite trasmission (Naotunne et al. 1991, Kwiatkowski 1992). Numerous non-specific factors may correlate with changes in gametocyte infectivity. Acute phase reactants like C-reactive protein (CRP), which are non-specific indicators of inflammatory activity, are elevated in patients with P. falciparum malaria (Ree 1971, Naik & Voller 1984, Chagnon et al. 1992). Some cytokines such as interferon (IFN-g), tumor necrosis factor (TNF-a) and interleukin 6 (IL- 6) are elevated in sera fom patients with P. falciparum and P. vivax malaria (Grau et al. 1989, Kern et al. 1989, Kwiatkowski et al. 1990, Mendis et al. 1990c, Karunaweera et al. 1992). Both IFN-g and TNF-a appear to cause a transient but marked drop in the infectivity of gametocytes to mosquitoes due to the intraerythrocytic killing of parasites (Naotunne et al. 1991, Karunaweera et al. 1992). However, a recent study has shown no elevation in blood levels of cytokines IL-2, IL-6, TNF-a and IFN-g nor reactive nitrogen intermediates (Hogh et al. 1994). The authors suggested that this could be explained by the inability of asymptomatic gametocyte carriers, unlikely to harbour high asexual parasitaemias, to promote the responses. The concept that anti-sexual stage immunity may regulate infection of the moquito vector by gametocyte-infected malarial blood has gained considerable support and must be considered for the development of malaria trasmission blocking vaccines (Kaslow et al. 1992).
c) Antibodies against sporozoites - There is evidence that naturally acquired or experimentally elicited anti-sporozoite antibodies ingested by mosquitoes may affect the dynamics of the sporogonic development in the vector. Several studies with P. falciparum- infected Anopheles species (Vaughan et al. 1988, Beier et al. 1989, Hollingdale & Rosario 1989) showed that (1) ingested human CSP antibodies were detected in the blood meal of field collected mosquitoes up to 36 hr after feeding, (2) antibodies crossing the midgut into hemocoel persist from 4 to 36 hr post-infection in hemolymph, (3) ingested CSP antibodies on day 5 after infection bound to developing oocysts, (4) enhancement of the sporozoite production, (5) ingestion of CSP antibodies on day 10 after feeding had no effect on oocyst maturation or sporozoite production, (6) contact between CSP antibodies and sporozoites in the hemocoel did not block sporozoite invasion of salivary glands, (7) exposure to CSP antibody increased sporozoite infectivity and (8) human IgG antibodies were present on salivary gland sporozoites from field-collected mosquitoes. It has been recently demonstrated that antibodies to P. gallinaceum CSP prevent sporozoites from invading salivary glands of Ae. aegypti (Warburg et al. 1992). Ponnudurai et al. (1989) did not find any influence of anti-sporozoite antibodies on the number of salivary gland sporozoites but concluded that a second blood meal, with or without antibody, simply functions as a nutritional stimulus for faster oocyst maturation. However, when transmission blocking antibodies anti-Pbs21 (a surface antigen present on the surface of P. berghei zygote/ookinete) were added to second bloodfeeds at different stages of parasite development in the mosquito, a significant reduction in oocyst intensity but no detectable change in prevalence occurred. Furthermore, at all times anti-Pbs21 reduced sporozoite number in the thorax but highest gland intensities were obtained when the second bloodfeed was given on day 4 (Ranawaka et al. 1993). These results were interpreted as two opposing roles of second bloodfeeds containing trasmission blocking antibody: (1) inhibition of parasite development and (2) the supply of nutrients which permit more sporozoites to be produced by each oocyst. Despite some controversy these results potentially have significant implications for natural malaria transmission and for a possible vaccine development.
d) Anti-mosquito antibodies - In addition to anti-parasite antibodies, it has been tested experimentally the effect on malaria trasmission of antibodies raised against parts of the mosquito which could be included in a malaria vaccine. Ramasamy and Ramasamy (1990), studying the P. berghei/An. farauti model, found that mosquitoes feeding on mice immunised with midgut antigens exhibited a reduction in mosquito infection rates. Similar results were reported by Billingsley et al. (1990) using monoclonal antibodies produced against mosquito midgut tissue in P. berghei/An. stephensi system.
e) Genetic manipulation of the vector - Experiments with refractory lines of An. gambiae (Collins et al. 1986) and studies on the effects of broad antimicrobial and antiparasitic components, e.g. magainins and cecropins (Gwadz et al. 1989), showed that it may be feasible to induce effective disruption in the normal development of Plasmodium species in the vector by the introduction and expression of appropriate genes into the mosquito genome. Two types of useful target genes can be used in transgenic mosquitoes. First, those that render populations vulnerable to subsequent control measures, such as insecticide susceptibility or temperature sensitivity, and second, those that interrupt disease transmission by replacing vector with non-vector forms (Crampton et al. 1990, Kidwell & Ribeiro 1992, Crampton 1994). Although many technical, and perhaps ethical, problems associated with the wild-release of transgenic insects have yet to be overcome, the potential of this technology has received greater attention recently (Brey 1991, Coluzzi 1992, Collins 1994, Curtis 1994). Introduction and expression of genes coding for antibodies against target antigens present on the ookinete surface into the mosquito embryos is one of the possibilities to examine the potential of this technology (Crampton et al. 1993).
f) Anti-malarial drugs - Sub-therapeutic doses of antimalarial drugs have been reported to enhance infectivity of Plasmodium species to their vectors (Shute & Maryon 1954). Additionally, numerous compounds including chloroquine (Wilkinson et al. 1976), sulphamethoxazole-trimethroprim (Wilkinson et al. 1973), pyrimethamine (Shute & Maryon 1951), Fansidar (Carter & Graves 1988) and Berenil (Ono et al. 1993) have been suggested to induce gametocyte formation but no influence of chloroquine (Jeffery et al. 1956, Smalley 1977, Chutmongkonkul et al. 1992, Hogh et al. 1994) and Fansidar (Hogh et al. 1994) on gametocyte infectivity was observed by some investigators. It was demonstrated that pyrimethamine- and halofantrine-treated gametocytes of P. falciparum are more infective to An. stephensi mosquitoes than untreated controls (Chutmongkonkul et al. 1992). Other studies examined the effects of some schizontocidal agents on the sporogonic cycle of P. falciparum and P. berghei in anopheline mosquitoes (Coleman et al. 1988, do Rosario et al. 1988). It was found that chloroquine, when fed during late sporogony (10-12 days post-infection), may increase the vectorial capacity of some mosquito species. The effects of chloroquine on the infectivity of chloroquine-sensitive and -resistant populations of P. yoelii nigeriensis to An. stephensi mosquitoes were studied by Ichimori et al. (1990). The results showed an enhancement of infectivity in sensitive strains but no effect was detected in resistant clones and sublines. Chloroquine use and the subsequent development of resistance over the past years is associated with an increasing human malaria infectiousness (Lines et al. 1991) which may be indirect effects of parasitaemia on the host. The sporontocidal activity of chloroquine, halofantrine and pyrimethamine was evaluated by administration to An. stephensi mosquitoes, either in the first bloodmeal containing P. falciparum gametocytes from in vitro cultures, or in the second, parasite-free bloodmeal, given four days after infection. A sporontocidal effect was observed only when pyrimethamine was administred with the infective bloodmeal (Chutmongkonkul et al. 1992). It has been demonstrated recently an inhibitory action of the anti- malarial Atovaquone (566C80) against ookinete, oocysts and sporozoites of Plasmodium berghei in An. stephensi (Fowler et al.1994, 1995). Acknowledgments Amauri Braga Simonetti was supported by a scholarship from CNPq (Brazil) during his PhD in the Infection and Immunity Section, Department of Biology at Imperial College of Science, Technology and Medicine (London, U.K.). I would like to acknowlegde Professor Robert E Sinden for his guidance and helpfull advices which made possible this review. References Aikawa M 1971. Plasmodium: The fine structure of malarial parasites. Exper Parasitol 30: 284- 320. Aikawa M Atkinson CT, Beaudoin LM, Sedegah M, Charoenvit Y, Beaudoin R 1990. Localization of CS and Non-CS Antigens in the Sporogonic Stages of Plasmodium yoelii. Bull WHO 68: 165-171. Alano P 1991. Plasmodium sexual stage antigens. Parasitol Today 7: 199-203. Alano P, Carter R 1990. Sexual differentiation in malaria parasites. Ann Rev Microbiol 44: 429-449. Bafort JM 1971. The biology of rodent malaria with particular reference to Plasmodium vinkei vinkei Rhodain 1952. Ann Soc belge de Med Trop 51: 1-204. Ball G H, Chao J 1957. Development in vitro of isolated oocysts of Plasmodium relictum. J Parasitol 43: 409-412.
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192.
The Biology of Malarial Parasite in the Mosquito - A
Review
Amauri Braga Simonetti
Departamento de Microbiologia, Instituto de Biociencias,
Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite
500, 90050-170 Porto Alegre, RS, Brasil
Received 20 December 1995
[TABLES AND FIGURES AT END OF TEXT]
The purpose of this review is to summarize the biology of
Plasmodium in the mosquito including recent data to
contribute to better understanding of the developmental
interaction between mosquito and malarial parasite. The entire
sporogonic cycle is discussed taking into consideration
different parasite/vector interactions and factors affecting
parasite development to the mosquito.
Key words: malaria - mosquito - sporogonic cycle - cell
biology
The control and prevention of malaria has been pursued for a
long time. Although campaigns against malaria were initially
successful in some areas, the emergence of resistance of the
parasite to drugs and of the mosquito vector to insecticides,
combined with the difficulties in implementing and maintaining
effective control schemes have led to a resurgence of the
disease in many parts of the world (Wernsdorfer 1991, Schapira
et al. 1993, Roush 1993). Much of the current work on malaria
focuses on immunological aspects of the disease and there has
been remarkable progress in the identification of a variety of
parasite antigens in different stages of the parasite
development, some of which may be included in a future vaccine
(Nussenzweig & Nussenzweig 1989, Mitchel 1989, Targett 1989,
Nussenzweig 1990, Hommel 1991, Good 1991a). However,
experimental and field studies have shown the complexity of
the immune response to parasites, indicating that an efficient
malaria vaccine is difficult to achieve (Cattani 1989, Good
1991b, 1992, Philips 1992). Therefore, for control of the
disease greater understanding of the biological mechanisms
involved in host/parasite/vector interactions is essential.
In recent years an increasing number of studies have
concentrated on the mosquito stages of the parasite
development. Comprehensive reviews on biological,
ultrastructural, biochemical, molecular and immunological
aspects of the parasite and the vector can be obtained in the
literature (Sinden 1978, 1983, 1984, Carter & Graves 1988,
Carter et al. 1988, Alano & Carter 1990, Billingsley 1990,
Crampton et al. 1990, 1994, Alano 1991, Brey 1991, Coluzzi
1992, Kaslow et al. 1994a, Sinden et al. 1996). This review
foccuses on the cell biology of malarial parasites at
different stages of its development in the mosquito and also
includes factors that may affect the parasite infectivity.
The sporogonic cycle
Some erythrocytic parasites differentiate into sexual forms
called gametocytes. Mature and functional gametocytes ingested
by an appropriate species of mosquito in a bloodmeal are
stimulated to transform into the stages which establish the
parasites in their vector (Garnham 1966). Under the influence
of changes in the mosquito midgut environment the gametocytes
become extracellular within 8-15 min of ingestion. After
emergence from the red blood cell (exflagellation) the male
gametes fertilize the female gametes within 60 min of
ingestion of blood. The fertilized macrogamete (zygote)
differentiates into a single motile ookinete over the next 10-
25 hr, which migrates from the bloodmeal through the midgut
wall to form an oocyst underneath the basal lamina of the
midgut. Each oocyst produces many thousands of invasive
sporozoites over a period of 7-12 days. The sporozoites escape
from the oocyst and then invade the salivary glands, here they
stay for possibly very long periods until injected into
another vertebrate host when the next bloodmeal is taken
(Sinden 1984, Carter & Graves 1988). A diagram of the
sporogonic cycle in the mosquito is shown in Figure.
Development of gametocytes (gametocytogenesis)
As with other members of the order Haemosporina, gametocytes
of Plasmodium can arise from merozoites from pre-
erythrocytic parasites. Nevertheless, during natural
infections, most gametocytes arise from merozoites of blood-
stage origin (Alano & Carter 1990). Two alternative
possibilities by which malaria parasites could become
committed to either sexual or asexual development have been
proposed (Inselburg 1983, Mons 1985, 1986, Bruce et al. 1990):
first, the merozoite is not committed at the time of invasion
of a red blood cell. During early growth (as a ring form), the
parasite is suceptible to factors that will commit it to
either sexual or asexual development. Second, during growth of
an asexual parasite, environmental factors influence it so
that at maturity the schizont produces merozoites that are
precommitted to form asexual parasites or committed to form
gametocytes upon subsequent invasion of a red blood cell.
A number of studies have attempted to identify factors that
may influence the differentiation and production of sexual
stage parasites including cAMP, presence of antimalarial drugs
and other environmental factors associated to the host such as
the presence of serum undetermined components (reviewed by
Sinden 1983, Mons 1986, Carter & Graves 1988, Dearsly et al.
1990). In a recent report (Schneweis et al. 1991) it was
claimed that the production of infective gametocytes in
vitro can be enhanced by products of haemolysis but the
nature of the erythrocytic factor was not identified.
Gametocyte development takes longer than asexual schizogony,
e.g. 26 hr as opposed to 22.5 hr in P. berghei (Mons et
al. 1985) or in the more extreme case of P. falciparum
8-12 days as opposed to 48 hr (Sinden 1983). The proportion
of parasites that develop into gametocytes varies greatly
during the course of natural infections even at its peak but
is very low in relation to the total parasitaemia (Smalley et
al. 1981). The growth and differentiation of gametocytes of
P. falciparum has been divided into five stages (I to
V) covering about eight days from merozoite invasion to mature
gametocyte, each stage being distinguished by successive
changes in the organization of the cell (Hawking et al. 1971).
Little is known about commitment of gametocytes to be male or
female. The fact that both female and male can be produced by
haploid blood-stage parasite, the sex of a gametocyte does not
result from chromosomal differences between both types of cell
but their development must be due to selective gene
expression. In general, the number of female gametocytes
predominates over the number of male, but this predominance
may vary at different times between cloned infections
(Cornelissen 1988, Schall 1989).
Gametogenesis
Mature and functional gametocytes ingested by the mosquito in
a bloodmeal are stimulated by the midgut environment to
transform into gametes. Studies indicate that various triggers
induce gametocytes to undergo differentiation. Microgameto-
genesis in vitro is, optimally, dependent upon a rise
in pH (Nijhout & Carter 1978), a rise in pCO2 and bicarbonate
levels (Carter & Nijhout 1977, Nijhout & Carter 1978), a fall
in temperature of a few degrees below that of the vertebrate
host (Sinden & Croll, 1975) or a very potent factor termed
mosquito exflagellation factor (MEF). The latter is a small
heat stable molecule from the mosquito head and gut which
stimulates gametogenesis via a bicarbonate- and pH-independent
mechanism (Nijhout 1979). Recently, Kawamoto et al. (1991)
showed in vitro that induction of exflagellation of
P. berghei is triggered by a rise in the intracellular
pH (pHi) which is mediated by Ca^++ and cGMP regulation. pHi
can be modulated by alkaline media and is controlled by a
complex series of interdependent ion pumps and channels
controlling Na^+, K^+, Cl^- and HCO3^- transport between the
parasite and the environment. Other influential factors
described include cAMP analogues and inhibitors of
phosphodiesterase (Martin et al. 1978). The duration of
microgametogenesis is both temperature and species dependent,
e.g. at 20-22 C it may take 7-15 min for P. falciparum
in vitro, although exflagellation may be detected after
shorter periods in the fluid excreted by feeding
Anopheles (Sinden 1983). There is no evidence that
exflagellation is influenced by factors released by digestion
of the blood meal since digestion normally begins several
hours later (Graf et al. 1986). Microgamete formation involves
three mitotic divisions with a rapid assembly of eight
axonemes on the single microtubule organizing centre that
divides and passes to the spindle poles. This division
simultaneously segregates the genome and the axoneme so that
each of the eight emergent gametes receives a single axoneme
and haploid genome, both being connected to a common
microtubule organizing centre. After exflagellation the
microgametes, normally bearing a single axoneme, a single
condensed nucleus and a single kinetosome with its sphere and
granule at the distal end, are torn from the microgametocyte
surface and rapidly move away into the blood meal (Sinden &
Croll 1975).
Macrogametogenesis at the morphological level involves little
more than escape from the host cell (Sinden 1984). At the
cellular level there is de novo synthesis of the
proteins which are expressed on the surface of macrogamete
(Kumar & Carter 1984). It was recently identified a gametocyte
specific protein of P. falciparum called Pf11-1 and
there is some evidence that this protein might be involved in
the emergence of gametes from the infected erythrocyte (Scherf
et al. 1992, 1993).
Interactions between the vertebrate host and the parasite do
not cease when the blood meal is taken. Following induction of
gametogenesis the parasite is liberated into the blood meal.
It has been shown that the gametes are susceptible to the
phagocytes in the host's blood (Rutledge et al. 1969, Sinden &
Smaley 1976) and that they are susceptible to activation of
the components of the complement pathway (Grotendorst et al.
1986, 1987).
Zygote formation (fertilization) and ookinete
development
Fertilization is rapid usually occurring within 1 hr of gamete
formation, the plasmalemma of the two gametes fuse and the
microgamete axoneme and nucleus enter the macrogamete
cytoplasm (Sinden 1983). During zygote development, structural
changes occur and its transformation to ookinete is in part
determined by the gradual assembly of the apical complex
including the collar and pollar rings, rhoptries and
micronemes (Canning & Sinden 1973, Davies 1974, Sinden 1978).
Intensive protein synthesis also begins in the fertilized
macrogamete and continues in the zygote and developing
ookinete as reported for different Plasmodium species
(Kaushal et al. 1983a, Kaushal & Carter 1984, Kumar & Carter
1985, Vermeulen et al. 1985a, 1986, Sinden et al. 1987).
Included in this repertoir of proteins are many of the targets
of proposed transmission blocking immunity. It has been
reported that An. gambiae can concentrate the bloodmeal
by a factor of 1.2 to 1.8 reducing the production of ookinetes
when compared to An. freeborni which can not
concentrate (Sinden et al. 1996). The mature ookinete is a
motile cell that varies from 7 to 18 mm in length and 2 to 4
mm in diameter (Sinden, 1978). Locomotion of the ookinete has
been described in different Plasmodium species as a
linear or snake-like gliding motion (Freyvogel 1966). Studying
P. berghei ookinetes in primary mosquito cell cultures,
Speer et al. (1974) described spiral waves on the surface of
some ookinetes, especially in the anterior half of the body,
which might be involved in ookinete locomotion.
Some factors could influence the development, survival and
infectivity of the parasite during its residence in the midgut
lumen. Eyles (1952) has showed that the parasite development
ceases at the ookinete stage unless a macromolecular (non-
dialyzable) component is present in the blood meal. Studying
the influence of red blood cells on the ability of P.
gallinaceum zygotes fertilized in vitro to infect
Aedes aegypti Rosenberg et al. (1984) found a linear
relationship between erythrocyte density and the number of
oocysts up to a 50% hematocrit. Furthermore, they deduced that
there are one or more nondialyzable substances (erythrocytic
factors) contained in normal erythrocytes, and released by
mosquito digestion, that are essential for ookinete invasion
of the gut epithelium. When they added trypsin inhibitor to
the bloodmeal there was an inhibition of midgut penetration by
ookinetes. Recent studies have shown that when mosquitoes are
fed with cultured P. falciparum (Ponnudurai et al.
1989) and P. berghei (Sinden 1989) gametocytes, upon
dilution with fresh red cells, more oocysts result at initial
(low) dilutions whereas further dilution reduces oocyst
counts. The involvement of blood factors and/or its digestive
products in infectivity has been studied in different
parasite-vector models. Using a selected line of An.
stephensi, Feldmann and Ponnudurai (1989) found mature
P. falciparum ookinetes in the midgut lumen of refractory
mosquitoes but no further penetration of the gut epithelium
was observed. The reasons for this limited development in non-
compatible mosquitoes could be related to digestive function
since early initiation of hemoglobin degradation and higher
aminopeptidase activity have been described in refractory
strains of An. stephensi (Feldmann et al. 1990). It has
also been shown that P. gallinaceum develops up to the
ookinete stage in the non-compatible mosquito An.
stephensi, this development occurring over the same time
period and with the same success as in the compatible vector
Ae. aegypti. However, P. gallinaceum ookinetes
did not escape from the midgut lumen in An. stephensi
mosquitoes (Rudin et al. 1991).
Possible mechanism inhibiting parasite development involves
damage of the parasite by digestive enzymes present in the
vector. Trypsin and aminopeptidases are the major proteolytic
enzymes involved in blood digestion by female mosquitoes, and
are produced by the midgut cells in direct response to blood
feeding (Briegel & Lea 1975, Graf & Briegel 1982, Billingsley
1990, Billingsley & Hecker 1991). P. gallinaceum
ookinetes 0-10 hr old (i.e. zygote to ookinete transition)
were shown to be susceptible to mosquito enzymes in double
feeding experiments (Gass 1977) and in vitro damage was
observed to cultured ookinetes by proteinases from Ae.
aegypti (Gass & Yeates, 1979). However, results found by
Shahabuddin et al. (1993) using the same parasite/vector
system suggest that the parasite secretes an inactive or
partially active chitinase that is activated by a mosquito-
produced serine protease. In a recent study Chege et al.
(1996) examined the effect of digestive enzymes on the
kinetics of P. falciparum ookinete development and
oocyst infection rates in An. albimanus, An.
freeborni and An. gambiae. Their data indicated
that proteolytic enzymes alone do not limit the early stages
of sporogonic development in these vector species of
Anopheles.
Peritrophic layer
The peritrophic layer (PL) of the insect midgut forms a
cylindrical sheet separating the midgut contents from the
single cell-layered midgut endothelium. It is secreted within
hours of the blood meal at different rates depending on the
mosquito species (Billingsley 1990). Apical secretion granules
are present in the midgut cells of Anopheles species,
which are released into the periphery of the posterior midgut
lumen during the feeding process in response to stretching of
the gut wall. The vesicle contents coalesce and then condense
to form a compacted PL between 8 and 24 hr (Hecker 1977,
Berner et al. 1983). In culicine mosquitoes the PL is formed
de novo by the posterior midgut cells in the
proventriculus. In A. aegypti, formation starts
immediately after the blood meal, but about 12 hr later it is
a mature structure (Perrone & Spielman, 1988).
The role of the mosquito PL as a barrier to ookinete invasion
of the gut wall and pathogenic effects of Plasmodium
species upon the vector are controversial. In ultrastructural
studies on the interaction of P. falciparum ookinetes
with the midgut epithelium of An. stephensi Meis &
Ponnudurai (1987) frequentely found parasites trapped in the
membrane. They also observed that if the PL was dissected out
36 hr after the blood meal many ookinetes were attached to its
external surface. In the same study it was mentioned that the
ookinete was capable of penetrating the newly formed, but not
the thickened and hardened PL 36 hr after the feeding. A
failure to cross this barrier or retarded penetration might
increase the length of exposure of ookinetes to mosquito
trypsin to which it is known to be sensitive. In contrast,
using P. berghei-infected An. atroparvus
mosquitoes, Sluiters et al. (1986) concluded that the PL would
not function as physical barrier against migrating ookinetes
which can pass through fenestrations. Billingsley and Rudin
(1992) observed that infectivity of An. stephensi by
P. berghei, measured by oocyst counts, was unnaffected
by the presence or absence of the PL. However, in A.
aegypti infected with P. gallinaceum its presence
serves to reduce rather than prevent infection (Ponnudurai et
al. 1988, Billingsley & Rudin 1992). These observations
suggested to the authors that in compatible vector-parasite
combinations, the PL acts as a limiting, rather than absolute
barrier to the penetrating ookinete while in incompatible
combinations the PL appears to be an absolute barrier to
ookinete penetration.
To reach the midgut epithelium the ookinetes must first cross
the partially or fully formed PL. The PL may act as the
recognition site for the penetrating ookinete via lectin-
mediated mechanisms. The occurrence of sugar residues which
could be involved in vector recognition by the parasite has
been demonstrated in the PL of An. stephensi and Ae.
aegypti (Berner et al. 1983). Rudin and Hecker (1989)
showed the presence of binding sites for different lectins in
midguts of P. berghei-infected An. stephensi,
with high specificity for N-acetyl-D-galactosamine
(GalNAc), and P. gallinaceum-infected Ae. aegypti,
with high specificity for N-acetyl-D-glucosamine (GlcNAc).
The authors concluded that it seems likely that lectin-binding
phenomena play a role in the orientation of the parasites on
their way out of the midgut lumen and that the PM and/or
glycocalyx may be crucial structures for the penetration of
the gut epithelium by the ookinete. This does not exclude the
possibility that ookinetes penetrate the PL by an enzymatic
process (see below). Ultrastructural observations on Ae.
aegypti infected with P. gallinaceum showing an
electron dense amorphous material in front of the parasite
were consistent with a blockade of parasites within the PL
(Sieber et al. 1991). The ookinetes appeared to disrupt the
layers of the PL, suggesting an enzymatic mechanism for
penetration. These observations were further investigated by
Huber et al. (1991) who, using the same vector/parasite
system, also suggested a possible role for GlcNAc in the
binding of the ookinete to the PL, and demonstrated the
presence of chitin in the PL. They observed that mature
ookinetes transiently secreted a soluble chitinase, thought to
be responsible for the digestion of the PL. Recently,
Shahabuddin et al. (1993) reported an inhibition of P.
gallinaceum chitinase and a transmission blocking activity
of a chitinase inhibitor, allosamidin, on the sporogonic
development of P. gallinaceum and P. falciparum
in the respective vectors Ae. aegypti and An.
freeborni. However, enzymatic mechanisms of penetration
may differ in other vector/parasite systems since the PL of
An. stephensi appears not to contain chitin (Berner et
al. 1983).
Penetration of midgut epithelium
Ookinetes found in the epithelial cell layer between 24 and 48
hr post-infection have successfully evaded the obstacles
presented by the midgut lumen; however, they still might fail
to develop at the normal site of development (Sinden 1984,
Ponnudurai et al. 1988). It has been shown that the midgut
wall is negatively charged (Houk et al. 1986), but there is no
evidence of electrical charge interactions between ookinetes
and epithelial cells. It has been a matter for discussion as
to whether the ookinete follows an intra- or intercellular
route to reach the ultimate site of development and encystment
in the outer wall of the midgut epithelium.
Intercellular movement of P. gallinaceum ookinetes was
first described by Stohler (1957), but Mehlhorn et al. (1980)
have found the same parasite in an intracellular position.
Recently, Torii et al. (1992a) observed P. gallinaceum
ookinetes in both intracellular and intercellular
positions in the midgut epithelium of the mosquito Ae.
aegypti, which they interpret as that they first enter
into the epithelium, then exit into the intercellular space
and move to the basal lamina. Garnham et al. (1962) showed
that the ookinete of P. cynomolgi bastianelli enters
the epithelial cell by liquifying the cell membrane. Davies
(1974) again postulated intercellular movement by P.
berghei nigeriensis ookinetes. Although describing the
ookinete of P. berghei in an intracellular position, as
did Garnham et al. (1969), Canning and Sinden (1973) stated
that the parasite might also migrate by an intercellular
route. More recently, P. yoelii nigeriensis ookinetes
have been described to take an intracellular route to the
external wall of the midgut (Maier et al. 1987). However, when
the same parasite was used to infect An. omorii, an
intercellular route was mostly undertaken, although the
intracellular occurrence was also observed (Syaffruddin et al.
1991). Meis and Ponnudurai (1987) presented evidence that
P. falciparum ookinetes migrate between the epithelial
midgut cells. Using a specific monoclonal antibody they also
observed a track in the PL, which is related to the shedding
of a 25 kD surface protein (Pfs25) during movement. The
authors suggested that this protein may bind to receptors on
the epithelial cells prior to an intercellular invasion, since
it is reported to have epidermal growth factor (EGF)-like
domains (Kaslow et al. 1988). It was recently shown that Pfs25
persits in the oocyst wall during parasite development in the
mosquito (Lensen et al. 1993). The same group studied the
migration of P. falciparum and P. berghei
ookinetes through the midgut epithelium in An.
stephensi by using ruthenium red staining (Meis et al.
1989). The results of previous studies were confirmed: P.
falciparum ookinetes penetrated by intercellular route,
but the rodent parasite P. berghei appeared to take an
intracellular position, confirming that both mechanisms occur
and are species-dependent. In the case of P. berghei a
protein of 21 kD (Pbs21) present on the surface of the
ookinete (Sinden et al. 1987) could play a role during the
intracellular invasion of the midgut epithelium of An.
stephensi mosquitoes. It was demonstrated in the midgut of
P. berghei infected mosquitoes that expression of Pbs21
was predominantly localized on the ookinete surface one day
after the infectious blood meal and thereafter expression
declined to a minimum on days 2 and 3, the time of onset of
oocyst development (Simonetti et al. 1993).
The mode of penetration by ookinetes can perhaps be related to
damage of the epithelial lining of the midgut. Intercellular
migration may not damage cell membranes, and increased
mortality does not occur in P. falciparum-infected
An. stephensi and An. gambiae during this
period, even with very heavy parasite loads (Meis &
Ponnudurai, 1987). Similar observations have been reported in
P. gallinaceum-infected Ae. aegypti (Freier &
Friedman, 1987). They observed similar mortality rates in
infected and uninfected batches of mosquitoes. However, when
ookinetes use an intracellular route, as described previously,
increased damage to midgut cells might occur, resulting in
higher mosquito mortality. This is probably mediated by
invasion of the hemolymph by opportunistic gram-negative
bacteria and/or microsporidia (Maier et al. 1987, Seitz et al.
1987).
Ultimately the ookinete penetrates the basement membrane, but
fails to pass through the basal lamina of the midgut adjacent
to hemocoele (Sinden 1984). Whether this is due to the
inability of the ookinete to penetrate the basal lamina, or to
the specific recognition of the lamina and consequent shut-
down of the incisive process is not known. Interactions of
parasites with vector extracellular matrix proteins (ECM)
cannot be discounted (Kaslow et al. 1994b).
Oocyst development
Oocyst development is predominantly extracellular (Duncan et
al. 1960, Garnham et al. 1969, Howells & Davies 1971, Sinden
1975), but occasionally occurs within the midgut epithelial
cell (Vanderberg et al. 1967, Bafort 1971, Beaudoin et al.
1974). The ookinete usually comes to rest beneath the basal
lamina 18-24 hr after the infective blood meal (Sinden 1978).
It rapidly rounds up between 18 and 72 hr after feeding and
the apical complex is resorbed into the oocyst cytoplasm
(Garnham et al. 1969). There is some evidence suggesting a
significant role of the basal lamina in the development of the
ookinete (Kaslow et al. 1994b). It was found that in vitro-
cultured ookinetes injected directly into the hemolymph
form clusters of oocysts adherent to the basal lamina
throughout the hemocoele. Furthermore, binding of ookinetes to
artificial surfaces, such as plastic, is enhanced at least 10-
fold by addition of various components of basal lamina such as
matrigel, collagen IV, and laminin (Warburg & Miller, 1992).
The young oocyst is enveloped by a thick plasmalemma that is
covered on the hemocoelomic surface by a fibrous basal lamina.
Oocysts from the second day onwards are also covered by an
amorphous capsule which becomes reduced in thickness at
maturity (Vanderberg et al. 1967, Aikawa 1971, Strome &
Beaudoin 1974, Sinden 1975).
Despite the usual growth of the oocyst under the basal lamina
of the midgut, oocyst development is not site specific.
Weathersby (1952, 1954, 1960) has shown by injection of
gametocytes directly into the hemocoele of susceptible
mosquitoes that oocysts would develop to maturity if attached
to other parts of the body than the stomach or even if they
were floating freely in the hemocoel fluid. In his experiments
he used different parasite-host combinations and concluded
that the site of oocyst development is probably not a critical
factor in the maturation process. Furthermore, the factors
that are responsible for the death of parasite in refractory
lines are not confined to the stomach wall. These results were
supported by those reported by Ball and Chao (1957, 1960,
1961, 1976) who showed that oocysts of P. relictum may
develop in vitro away from the intact stomach of the
mosquito. The overall results of this series of studies by
Ball and Chao demonstrated in vitro development of all
stages from ookinetes to fully infective sporozoites without
attachment of oocysts to the midgut. However, it was not
possible to obtain complete sporogonic development in a single
preparation. Rosenberg and Koontz (1984) injected cultured
P. gallinaceum zygotes into the hemocoele of Ae.
aegypti mosquitoes and observed development of ectopic
oocysts in approximately 50% of the mosquitoes, with
sporozoites being found in the salivary glands. These
observations suggest that oocyst metabolism is not dependent
upon direct transfer of nutrients from the midgut epithelium.
Occasional intracellular oocyst development has been reported
for P. berghei in An. stephensi and An.
quadrimaculatus (Vanderberg et al. 1967). The same
localization was described in P. vinckei by Bafort
(1971) who concluded that both mechanical pressure and
physiological mechanisms play a role in the movement of
oocysts to the hemocoelomic surface. Studying the sporogonic
development of P. berghei in An. stephensi,
Beaudoin et al. (1974) found oocysts developing ectopically
within the midgut epithelium following normal infection,
eventually emptying their sporozoite content into the tissue
itself or the midgut lumen. In addition, they observed no
morphological and structural abnormalities in the luminal
parasites which displayed good viability. In contrast to P.
berghei, no ectopic development was seen in P.
falciparum-infected An. stephensi mosquitoes (Meis
et al. 1992b), confirming previous results observed with P.
falciparum in naturally infected An. gambiae
(Sinden & Strong 1978). Recent reports described an
enhancement of oocyst development in vitro for P.
berghei (Syaffruddin et al. 1992), P. gallinaceum
(Warburg & Miller, 1992) and P. falciparum (Warburg &
Schneider, 1993), when insect cell lines were added into the
culture medium. From these observations it appears that
nutritional or other regulatory requirements of the developing
parasite can be met without a direct contact with midgut
epithelium or haemolymph. The question of how the oocyst is
supplied with nutritive material is an intriguing one.
Little information is available on the uptake and source of
nutrients for oocyst development. It is assumed that in
vivo the source of nutrients is the hemolymph. Mack and
Vanderberg (1978) analyzed hemolymph of An. stephensi
collected from uninfected and P. berghei-infected
mosquitoes at different stages of the parasite development. It
was found that four days after the blood meal the osmotic
pressure and the specific gravity were lower in infected
mosquitoes compared with uninfected ones. The difference,
however, was attributed to indirect effects of the quality of
the ingested blood meal. These studies were complemented by
analysis of the concentration of free amino acids in the
hemolymph collected in similar conditions with results showing
significantly lower concentrations in infected mosquitoes with
decreases in valine and histidine, and a total loss of
detectable methionine suggesting it is incorporated (Mack et
al. 1979). This difference could be due to the utilization of
some of these amino acids by the developing oocyst as
suggested by Ball and Chao (1976) who analyzed the uptake of
amino acids by P. relictum oocysts in vitro,
comparing growth of uninfected and infected guts of Culex
tarsalis in Grace's insect culture medium. They found
significant decreases in the concentration of certain amino
acids including arginine, asparagine, proline and histidine,
and less marked decrease in concentrations of others like
methionine, valine, leucine and isoleucine. From these studies
it appears that the reduced amount of free aminoacids in the
hemolymph is due to oocyst metabolism. Autoradiographic
studies with P. gallinaceum in Ae. aegypti
mosquitoes indicated that ^3H-leucine is uniformly
incorporated throught the oocyst within 15 min of injection
into hemocoele (Vanderberg et al. 1967).
Sporogony
With increasing maturation the oocyst undergoes considerable
cytoplasmic subdivision. Initially the plasmalemma forms
invaginations and clefts that penetrate even deeper into the
cytoplasm, thus subdividing the cell (Vanderberg et al. 1967,
Terzakis 1971, Posthuma et al. 1988). In a transmission
electron microscopy study of P. falciparum oocysts it
was suggested that cleft formation was due to dilation of
endoplasmic reticulum (Sinden & Strong 1978). Using immunogold
labelling technique during sporogonic stage of the same
parasite, Posthuma et al. (1988) considered the latter
explanation unlikely. With increasing activity the cytoplasmic
clefts become extended and the expanding vacuolar space more
pronounced leading to the sporoblast formation. Along the
clefts sporozoites are formed by a budding process at the
surface of the limiting membrane (Vanderberg et al. 1967,
Howells & Davies 1971, Canning & Sinden 1973, Sinden & Strong
1978).
As the sporozoite continues to bud off, a nucleus and various
cytoplasmic components are passed into it from the sporoblast.
The membranes of the developing sporozoite pellicle are formed
and other organelles like microtubules and rhoptries become
discernible (Vanderberg et al. 1967, Sinden & Garnham 1973).
When sporogony is completed (about 10-12 days after the
infective feed), the oocyst is filled with sporozoites and one
or more residual bodies (Sinden 1984). Estimations of number
of sporozoites per oocyst have ranged widely. Garnham (1966)
reported that the number in a single P. vivax oocyst
varies from 1,000 to 10,000. Pringle (1965) estimated that a
single oocyst of P. falciparum contains nearly 10,000
sporozoites. Studies carried out with mosquitoes fed on
infected volunteers from Thailand showed a mean count of
approximately 3,700 sporozoites per oocyst for P.
vivax and 3,400 for P. falciparum, whereas for
P. cynomolgi-infected mosquitoes a single oocyst
contained about 7,500 (Rosenberg & Rungsiwongse 1991).
Dependent upon species, the mature sporozoite varies from 9 to
16.5 mm in length and from 0.4 to 2.7 mm in diameter; aberrant
forms have been described up to 40 mm long (Sinden 1978).
So far, studies on synthesis and expression of proteins during
sporogonic development have foccused on a polypeptide called
the circumsporozoite protein (CSP) found on the mature
sporozoite. Observations on the origin of CSP and its
distribution through the mosquito stage were reported by
several authors. It is now well established that these
proteins are synthesized in maturing oocysts of different
Plasmodium species from 6-7 days after the infective
blood meal, before sporozoites are visible (Nagasawa et al.
1987, 1988, Posthuma et al. 1988, Hamilton et al. 1988,
Boulanger et al. 1988, Torii et al. 1992b, Meis et al. 1992a).
At this stage, CSP is present on the plasmalemma and at
various sites within the cytoplasm and endoplasmic reticulum
of the sporoblast. When the sporozoites bud from the
sporoblast they are already covered with CSP which is also
found in salivary gland sporozoites (Yoshida et al. 1981,
Santoro et al. 1983, Tsuji et al. 1992).
Humoral encapsulation of oocysts, which in malaria infected
mosquitoes is known as Ross' black spores, has been described
in P. berghei nigeriensis and P. vivax and seems
to occur mainly in older oocysts which have begun to produce
sporozoites (Sinden & Garnham, 1973). This phenomenon was
studied in a selected line of An. gambiae that
encapsulates different Plasmodium species (Collins et
al. 1986). The authors demonstrated that refractoriness is
manifested by melanization of the ookinete after its passage
through the midgut epithelium. Paskewitz et al. (1989)
localized phenoloxidase activity in the basal lamina of the
epithelial cells of both encapsulating and susceptible
mosquitoes prior to blood feeding. However, after an infective
blood meal, this activity was still observed close to invading
ookinetes in refractory mosquitos but it was reduced or absent
in susceptible mosquitoes. When the non-compatible vector
An. gambiae was fed with ookinetes of P.
gallinaceum, invasion of the midgut epithelium by the
ookinetes occurred but oocysts were infrequentely formed.
Using the same system Vernick et al. (1989) and Vernick and
Collins (1989) tried to elucidate mechanisms involved in
vector-parasite incompatibility by injecting in vitro-
cultured ookinetes into the hemocoel of mosquitoes and
monitoring parasite development using specific rRNA probes. As
no differences were found between susceptible and refractory
mosquitoes the authors suggested that the specific lytic
factor(s) in the refractory line are intracellular.
The sporozoite and salivary gland invasion
The motile sporozoites emerge into the hemocoele through holes
from an area of weakness in the oocyst wall. Holes are
possibly produced by a combination of the muscular action of
the gut wall and the activity of the sporozoites (Sinden 1978,
Meis et al. 1992b). Within the hemocoel the sporozoites are
distributed throughout the mosquito and can initially be found
in many parts of the insect, even in the maxillary palps;
within a day or two of their release from oocysts the
sporozoites invade the salivary glands where they accumulate
and remain until delivery (Vaughan et al. 1992). Thus,
sporozoites do not adhere to most tissues, except for the
salivary glands and rarely the midgut wall, hemocytes or
thoracic muscles (Vanderberg 1974, Sinden 1975, 1978, Golenda
et al. 1990, Vaughan et al. 1992). The latter observation,
especially in heavily infected mosquitoes, could be associated
with an impairment of flight activity in malaria-infected
An. stephensi vectors demonstrated by some authors
(Rowland & Boersma 1988).
It has been estimated that in mosquitoes fed on individuals
with naturally acquired P. vivax about 850 sporozoites
per oocyst reached the glands (Sattabongkot et al. 1991) which
follows, by calculation, that approximately 23 % of all P.
vivax sporozoites released into the hemocoel subsequentely
reach the salivary glands (Rosenberg & Rungsiwongse 1991).
Vaughan et al. (1992), using regression analysis, calculated
the approximately 650 salivary gland sporozoites were produced
per oocyst and reported that virtually all oocyst infections
produced salivary gland infections in An. gambiae
infected with P. falciparum by membrane feeding. The
same group has found a similar number by studying the
sporogonic development of cultured P. falciparum in six
species of laboratory-reared Anopheles mosquitoes
(Vaughan et al. 1994). This is in contrast to observations on
wild-caught An. gambiae from Burkino-Faso (Lombardi et
al. 1987) and western Kenya (Beier et al. 1990) where
sporozoites failed to enter the salivary glands in 43% and 10%
of infected mosquitoes, respectively. However, when
sporozoites from P. gallinaceum oocysts were injected
into Ae. aegypti female mosquitoes only 6.5 to 10.4% of
inoculated sporozoites invaded the salivary glands.
Interestingly, injected salivary gland sporozoites did not
reinvade the glands (Touray et al. 1992). Recently, a
laboratory study on An. tesselatus mosquitoes infected
with different isolates of P. vivax and P.
falciparum from patients living in Sri Lanka showed that
approximately 15% of mosquito batches in which oocysts
developed failed to produce salivary gland sporozoites
(Gamage-Mendis et al. 1993). This discrepancy between
naturally and laboratory-infected mosquitoes could be
attributed to different environmental conditions or mixed
mosquito populations.
It could be expected sporozoites would elicit a humoral
response in the mosquito by activation of the prophenoloxidase
cascade and as a result be killed by melanization. However,
sporozoites in the hemocoele are rarely seen to be melanized
(Brey 1991). Sporozoites might be protected from mosquito
defense reactions against non-self' by antigen sharing. This
has been demonstrated as a potential mechanism for avoidance
of mosquito defence reactions by microfilariae of Brugia
pahangi (Maier et al. 1987). Immunoreactivity to CSP was
observed on the midgut wall of mosquitoes infected with P.
falciparum (Boulanger et al. 1988) and P. yoelii
(Beaudoin et al. 1990). The latter authors also detected
reactivity on uninfected midguts and suggest the presence of a
common determinant between the parasite and the mosquito.
The migration of sporozoites from oocysts to salivary glands
could be active (in contact with basal lamina), passive (in
suspension) or both. If active, the parasite would use a
gliding motility to move across the basal lamina and reach the
gland. Vanderberg (1974) described circular gliding and
attached waving locomotion and movement in sporozoites of
different species when bovine serum albumin was added to
Medium 199. Sporozoites that move over a substratum in
vitro leave behind trails of CS proteins (Stewart &
Vanderberg 1988, 1991, 1992). Soluble CSP was shown to be
present throughout the hemocoel (Robert et al. 1988). Beier et
al. (1992a) concluded that the release of CSP by sporozoites
is a normal but complex mechanism that they interpreted to be
associated with sporozoite survival in the host, is not site-
specific and it would be regulated in response to background
levels of soluble CSP in the environment (negative feedback
mechanism). This idea is supported by previous results showing
that the parasites cease to synthesize CSP during their
journey through the hemolymph but shedding still occurs
(Posthuma et al. 1988, 1989). There is no evidence yet for
chemotaxis but the congregation of sporozoites in the vicinity
of the glands before invasion may support, according to some
workers, a tactile mechanism (Golenda et al. 1990, Meis et al.
1992b). Several polysacharides have been identified that may
orient protozoa towards or away from a stimulus (Van Houten
1988).
The selective invasion of mosquito salivary glands by malarial
parasites is not well understood. Specific recognition of the
glands was shown in P. knowlesi. Oocysts developed
normally on the gut of An. freeborni but sporozoites
were never found in salivary glands. When glands from the
susceptible vector An. dirus were implanted into the
abdomen, they did become infected (Rosenberg 1985). These
experiments demonstrated that the salivary glands themselves
determined specificity. Although invasion of salivary glands
seems to require specific recognition, the mechanism by which
sporozoites recognize, attach to, and penetrate the glands
remains to be determined.
Sporozoites preferentially invade the medial lobe and the
distal portions of the lateral lobes of the salivary glands
where the salivary duct is not chitinous in Anopheles
species (Sterling et al. 1973, James & Rossignol 1991,
Ponnudurai et al. 1991). Hence, the occurrence of a specific
receptor-mediated invasion by sporozoites is plausible.
Perrone et al. (1986) used lectins to characterize
carbohydrate moieties on the basal lamina of Ae.
aegypti salivary glands. As the median and distal lateral
lobes bound a common lectin, RCA 120, whose substrate is b-D-
galactose, the authors suggested that sugars that bind this
lectin serve as candidate residues to which sporozoites may
attach. In contrast, the sporozoite coat binds some lectins
with very low efficiency (Schulman et al. 1980, Turner &
Gregson 1982). However, in a recent scanning electron
microscopic study Meis et al. (1992b) localized P.
falciparum sporozoites in proximal and distal parts but
were unable to identify any specific regions on the glands
where sporozoites penetrate. These authors also showed sites
where the sporozoites have pierced the basal lamina, which
probably explains the presence of CSP on the basal lamina
induced by shedding during penetration (Posthuma et al. 1989)
and the presence of immunoreactive spots of 1-2 mm on the
surface of infected glands (Golenda et al. 1990). Recently,
Touray et al. (1994) developed an in vivo salivary
gland invasion assay and have found that anti-salivary gland
antibodies, sulfated glycosaminoglycans and some lectins,
particularly Suc-WGA, block invasion of sporozoites. Although
the mechanism of blocking is not yet known, those lectins that
blocked invasion bound to salivary glands but did not bind to
sporozoites. Beier et al. (1992a) and Beier (1993) proposed
that as sporozoites invade the salivary glands, the build-up
of CSP is the signal for sporozoites to halt their active
motility, and thus their release of CSP (down-regulation). The
involvement of CSP and other proteins distinct from CSP such
as PySSP2 (Charoenvit et al. 1987) and PfSSP2 (Robson et al.
1988, Rogers et al. 1992a,b) in binding of sporozoites to the
basal lamina of mosquito salivary glands is speculative. As
they share similarities in their structure they might be
related to this process since it has been suggested region II
of CSP is involved in hepatocyte invasion (Cerami et al.
1992a,b, Pancake et al. 1992).
Rosenberg (1985) and King (1988) have suggested that
sporozoites invade the salivary gland cells using a mechanism
similar to invasion of erythrocytes by merozoites although
there is no evidence of parasitophorous vacuole membrane
formation observed in other stages of the parasite cycle
(Sinden & Strong 1978, Meis & Verhave 1988). Membrane-limited
vacuoles beneath the plasma membrane in An. stephensi
distal salivary gland cells invaded by P. berghei have
been described (Sterling et al. 1973). Penetration could also
involve an unusual intercellular route as suggested by Golenda
et al. (1990) who detected CSP in the region between cells of
the median lobe of the gland. Posthuma et al. (1989) observed
many sporozoites on the basal side of the cells, but also
found trail-like CSP immunoreactivity at the lateral space
between the cells.
After penetration, sporozoites are present in bundles in the
accini of gland cells in both proximal and distal areas. Most
probably, the sporozoites which are present in proximal areas
of the glands are unable to reach the draining duct because of
the chitinized layer in that area whereas distally localized
sporozoites reach the draining duct via the large unchitinized
collecting tube (Meis et al. 1992b). This explanation would
not be valid for Aedes species since the salivary ducts
are lined with chitin and extend the full length of the glands
(James & Rossignol 1991). Penetration by sporozoites could
cause pathological vesiculation and cytoskeletal changes in
salivary gland tissues as the infected cells are often
deformed and swollen (Sterling et al. 1973, Maier et al.
1987). The efficiency of salivary gland invasion is poorly
understood. It has been estimated that the median sporozoite
load in the glands is less than 10,000 in colonized or wild
Anopheles species (Shute 1945, Pringle 1966, Wirtz et
al. 1987, Beier et al. 1991b, Sattabongkot et al. 1991) or
slightly higher (Ponnudurai et al. 1991).
The development of infectivity by the sporozoites appears to
be asynchronous, in some cases taking place in the hemocoele,
while in others not occuring until after they have invaded the
salivary glands. Thus, it seems to be time-dependent rather
than site-dependent (Vanderberg 1975, Daher & Krettli 1980).
It was demonstrated in P. berghei-infected mice that
populations of salivary gland sporozoites were more than
10,000 times as infective to the vertebrate host as
populations of oocyst sporozoites from the same mosquitoes
(Vanderberg 1975). Touray et al. (1992) found that as few as
10-50 salivary gland P. gallinaceum sporozoites were
required to induce infection in chickens compared to 5,000
oocyst sporozoites. Sporozoite infectivity increases with time
during the first week after the invasion of the salivary gland
(Vanderberg 1975). Degeneration of sporozoites is not frequent
in nature as observed by Barber (1936) who studied anophelines
collected in Mediterranean areas. When degeneration occurred
in a salivary gland or a lobe of a gland, in another gland or
lobe the sporozoites were normal or in a different stage of
degeneration.
The number of sporozoites injected into the tissue or
capillary of the vertebrate is very small compared to that
found in the salivary glands. It has been estimated, by
employing different methods, that each bite delivers fewer
than 50 sporozoites with a tendency for most sporozoites to be
ejected in the first droplets of saliva (Vanderberg 1977,
Rosenberg et al. 1990, Ponnudurai et al. 1991, Beier et al.
1991a,b, Beier et al. 1992b, Li et al. 1992). Although a
correlation between salivary gland sporozoite load and
sporozoite inoculum has been reported (Rosenberg et al. 1990)
ejection of sporozoites is probably a random process, more
related to the architecture of the salivary gland duct system
than to the number of sporozoites in this organ (Ponnudurai et
al. 1991).
Some studies reported that P. falciparum-infected
mosquitoes deliver sporozoites in an unpredictable fashion,
sometimes not at all (Ponnudurai et al. 1991), and others
transmit inconsistently (Rickman et al. 1990). Clumping of
sporozoites has been reported in infected salivary glands
(Sterling et al. 1973) and clusters of sporozoites were
detected after delivery when An. stephensi mosquitoes
infected with P. falciparum were allowed to feed
through fresh mouse skin (Ponnudurai et al. 1991). It has been
observed that salivary glands are not depleted of sporozoites
even in vectors that feed up to 15 times (Shute 1945), which
allows infected mosquitoes to remain potentially infectious
for life.
The low sporozoite inoculum and the low entomological
inoculation rates in natural conditions (Mendis et al. 1990a,
Gordon et al. 1991) demonstrates the high efficiency with
which injected sporozoites will develop malaria.
Factors affecting parasite development to the
mosquito
The process of infection of mosquitoes is exceedingly complex
and regulated by a range of factors originating from the
parasite, the vertebrate host and the mosquito vector, and
from the interactions between all three (Sinden 1991). Many of
these and other factors are known to affect fertilization and
subsequent stages of parasite development, thus having great
influence on transmission of the disease. To have an idea,
when sporogonic development of cultured P. falciparum
was evaluated in six species of Anopheles mosquitoes
there was a total loss of approximately 31,600-fold in the
parasite population from macrogametocyte to oocyst stage
(Vaughan et al. 1994).
a) Host location and feeding behaviour -
Of primary importance in malaria transmission is the
proportion of mosquito feeds taken from humans and the
proportion of these feeds taken from infected individuals. In
nature a vector needs to survive longer than the sporogonic
period after taking an infective blood meal; during this
period the mosquito probably takes blood meals every 2-3 days,
depending on its gonotrophic cycle and the availability of
breeding sites (Ponnudurai et al. 1989). Parasitemic hosts
tend to be sick and often less irritated by mosquito feeding.
Additionally, the thrombocytopenia which is commonly
associated with blood-borne parasitic diseases leads to
facilitation of vessel location, resulting in increased
feeding success by mosquitoes on parasitemic hosts (Rossignol
et al. 1985). Salivary glands and saliva contain a whole range
of components with pharmacological activities important for
blood feeding success and subsequent bloodmeal processing,
including anticoagulants, anti-inflammatory, vasodilatory and
immunosuppressive compounds (Ribeiro et al. 1984, 1989,
Ribeiro 1987, Titus & Ribeiro 1990, James & Rossignol 1991).
Rossignol et al. (1984) concluded that the lesions caused by
P. gallinaceum sporozoites in the salivary glands of
Ae. aegypti result in reduced levels of salivary
apyrase. Mosquitoes deficient in salivary apyrase experience
difficulty in locating host blood and engorging; they
therefore probe more often and may attempt to feed on several
hosts in succession (Rossignol et al. 1984, 1986, Ribeiro et
al. 1985). Li et al. (1992) demonstrated that probing time of
P. berghei-infected An. stephensi is not
affected by sporozoite invasion of salivary glands.
Blood meal size may influence infectivity since it determines
the number of gametocytes ingested and therefore subsequent
infection. It has been shown that larger An. dirus
females took larger bloodmeals by artificial feeding with
cultured P. falciparum and developed significantly more
oocysts (Kitthawee et al. 1990). In a field study, Lyimo and
Koella (1992) reported that the proportion of An.
gambiae mosquitoes infected with P. falciparum
during a blood meal was independent of size but the number of
oocysts harboured by infected mosquitoes increased with size
of the mosquito.
b) Gametocyte carriers/transmission blocking immunity -
Malarial infections induce host responses to both asexual and
sexual stage malaria parasites that may modulate gametocyte
infectivity. Great heterogeneity in infectiousness of
different carriers has been noted, with apparently poor
correlation between infectiousness and gametocyte density
(Graves et al.1988). However, it remains unclear whether
symptomatic and asymptomatic asexual infections differ in
their ability to influence gametocyte infectivity. It has been
reported that asymptomatic P. falciparum patients were
more infectious than symptomatic patients (Boyd & Kitchen
1937, Jeffery & Eyles 1955, Muirhead-Thomson 1957, Carter &
Graves 1988) while, in contrast, another report suggested that
asymptomatic and symptomatic P. malariae patients were
equally infective (Young & Burgess 1961).
Specific and non-specific responses are believed to modulate
parasite transmission. The induction of specific immunological
responses to Plasmodium shows marked heterogeneity
within human populations (Mendis et al. 1987, Graves et al.
1988, Targett 1990, Snow et al. 1993). The sexual stages can
induce trasmission blocking immunity (TBI) and effective
targets include antigens identified on the surface of the
macro- and/or microgemetes ( pre-fertilization'antigens) as
well as antigens present on the surface of the gamete, zygote
and ookinete ( post-fertilization' antigens). Antibodies to
pre-fertilization' antigens of P. falciparum such as
Pfs230, Pfs48/50 and Pfs16 have been detected in humans
(Graves et al. 1988, Premawansa et al. 1994, Hogh et al.
1994). A significant association between lacking of
infectivity of P. falciparum gametocyte carriers and
recognition of epitope IIa on Pfs48/50 by antibodies in their
sera has been observed (Graves et al. 1992). Naturally
acquired TBI to P. vivax sexual stage antigens has also
been demonstrated (Mendis et al. 1987, 1990b, Mendis & Carter
1991, Ranawaka et al. 1988, Goonewardene et al. 1990, Gamage-
Mendis et al. 1992) and appears to play a role on transmission
of the disease (de Zoysa et al. 1988). In P. vivax
malaria antibodies, at low concentrations, can also have a
transmission-enhancing effect on infectivity of malarial
parasite to mosquitoes (Mendis et al. 1987, Peiris et al.
1988, Naotunne et al. 1990, Gamage-Mendis et al. 1992). A
recent report described TBI in P. vivax malaria when
antibodies raised against a peptide blocked parasite
development in the mosquito An. tesselatus (Snewin et
al. 1995). As shown in a variety of Plasmodium species,
within the mosquito vector antibody to the pre-fertilization'
antigens may prevent fertilization by any of four mechanisms:
(1) the agglutination of macro/microgametes limiting their
mobility; (2) antibody coating of macro/microgamentes
inhibiting cell-cell recognition; (3) opsonization in the
bloodmeal or (4) complement dependent/independent lysis (Gwadz
1976, Carter & Chen 1976, Carter et al. 1979, 1985, 1990,
Kaushal et al. 1983a,b, Rener et al. 1983, Harte et al. 1985a,
Vermeulen et al. 1985b, Grotendorst et al. 1986, Grotendorst &
Carter 1987, Quakyi et al. 1987, Peiris et al. 1988,
Premawansa et al. 1990). Cellular responses are also involved
in TBI and may have some influence on parasite infectivity
(Harte et al. 1985b, Mendis et al. 1990b, Riley & Greenwood
1990, Goonewardene et al. 1990).
Immunity to `post-fertilization' antigens such as Pfs25,
Pbs21, Pgs25 and Prs25 also plays an important role on
transmission of the disease by suppressing parasite
infectivity at different stages of its development in the
mosquito as demonstrated by many workers in different
Plasmodium species (Grotendorst et al. 1984, Vermeulen
et al. 1985a, Sinden et al. 1987, Winger et al. 1988, Fries et
al. 1989, Carter & Kaushal 1984, Carter et al. 1989a,b, Kaslow
et al. 1991, 1992, 1994b, Foo et al. 1991, Sieber et al. 1991,
Tirawanchai et al. 1991, Duffy et al. 1993, Paton et al. 1993,
Ranawaka et al. 1993, 1994). The mechanisms of blockade could
be the same four described above and/or the antibody may act
by damaging the parasite surface coat (Ponnudurai et al.
1987).
Non-specific responses to the asexual stages are believed to
modulate parasite trasmission (Naotunne et al. 1991,
Kwiatkowski 1992). Numerous non-specific factors may correlate
with changes in gametocyte infectivity. Acute phase reactants
like C-reactive protein (CRP), which are non-specific
indicators of inflammatory activity, are elevated in patients
with P. falciparum malaria (Ree 1971, Naik & Voller
1984, Chagnon et al. 1992). Some cytokines such as interferon
(IFN-g), tumor necrosis factor (TNF-a) and interleukin 6 (IL-
6) are elevated in sera fom patients with P. falciparum
and P. vivax malaria (Grau et al. 1989, Kern et al.
1989, Kwiatkowski et al. 1990, Mendis et al. 1990c,
Karunaweera et al. 1992). Both IFN-g and TNF-a appear to cause
a transient but marked drop in the infectivity of gametocytes
to mosquitoes due to the intraerythrocytic killing of
parasites (Naotunne et al. 1991, Karunaweera et al. 1992).
However, a recent study has shown no elevation in blood levels
of cytokines IL-2, IL-6, TNF-a and IFN-g nor reactive nitrogen
intermediates (Hogh et al. 1994). The authors suggested that
this could be explained by the inability of asymptomatic
gametocyte carriers, unlikely to harbour high asexual
parasitaemias, to promote the responses.
The concept that anti-sexual stage immunity may regulate
infection of the moquito vector by gametocyte-infected
malarial blood has gained considerable support and must be
considered for the development of malaria trasmission blocking
vaccines (Kaslow et al. 1992).
c) Antibodies against sporozoites -
There is evidence that naturally acquired or experimentally
elicited anti-sporozoite antibodies ingested by mosquitoes may
affect the dynamics of the sporogonic development in the
vector. Several studies with P. falciparum-
infected Anopheles species (Vaughan et al. 1988, Beier
et al. 1989, Hollingdale & Rosario 1989) showed that (1)
ingested human CSP antibodies were detected in the blood meal
of field collected mosquitoes up to 36 hr after feeding, (2)
antibodies crossing the midgut into hemocoel persist from 4 to
36 hr post-infection in hemolymph, (3) ingested CSP antibodies
on day 5 after infection bound to developing oocysts, (4)
enhancement of the sporozoite production, (5) ingestion of CSP
antibodies on day 10 after feeding had no effect on oocyst
maturation or sporozoite production, (6) contact between CSP
antibodies and sporozoites in the hemocoel did not block
sporozoite invasion of salivary glands, (7) exposure to CSP
antibody increased sporozoite infectivity and (8) human IgG
antibodies were present on salivary gland sporozoites from
field-collected mosquitoes. It has been recently demonstrated
that antibodies to P. gallinaceum CSP prevent
sporozoites from invading salivary glands of Ae. aegypti
(Warburg et al. 1992). Ponnudurai et al. (1989) did not
find any influence of anti-sporozoite antibodies on the number
of salivary gland sporozoites but concluded that a second
blood meal, with or without antibody, simply functions as a
nutritional stimulus for faster oocyst maturation. However,
when transmission blocking antibodies anti-Pbs21 (a surface
antigen present on the surface of P. berghei
zygote/ookinete) were added to second bloodfeeds at different
stages of parasite development in the mosquito, a significant
reduction in oocyst intensity but no detectable change in
prevalence occurred. Furthermore, at all times anti-Pbs21
reduced sporozoite number in the thorax but highest gland
intensities were obtained when the second bloodfeed was given
on day 4 (Ranawaka et al. 1993). These results were
interpreted as two opposing roles of second bloodfeeds
containing trasmission blocking antibody: (1) inhibition of
parasite development and (2) the supply of nutrients which
permit more sporozoites to be produced by each oocyst. Despite
some controversy these results potentially have significant
implications for natural malaria transmission and for a
possible vaccine development.
d) Anti-mosquito antibodies -
In addition to anti-parasite antibodies, it has been tested
experimentally the effect on malaria trasmission of antibodies
raised against parts of the mosquito which could be included
in a malaria vaccine. Ramasamy and Ramasamy (1990), studying
the P. berghei/An. farauti model, found that
mosquitoes feeding on mice immunised with midgut antigens
exhibited a reduction in mosquito infection rates. Similar
results were reported by Billingsley et al. (1990) using
monoclonal antibodies produced against mosquito midgut tissue
in P. berghei/An. stephensi system.
e) Genetic manipulation of the vector -
Experiments with refractory lines of An. gambiae
(Collins et al. 1986) and studies on the effects of broad
antimicrobial and antiparasitic components, e.g. magainins and
cecropins (Gwadz et al. 1989), showed that it may be feasible
to induce effective disruption in the normal development of
Plasmodium species in the vector by the introduction
and expression of appropriate genes into the mosquito genome.
Two types of useful target genes can be used in transgenic
mosquitoes. First, those that render populations vulnerable to
subsequent control measures, such as insecticide
susceptibility or temperature sensitivity, and second, those
that interrupt disease transmission by replacing vector with
non-vector forms (Crampton et al. 1990, Kidwell & Ribeiro
1992, Crampton 1994). Although many technical, and perhaps
ethical, problems associated with the wild-release of
transgenic insects have yet to be overcome, the potential of
this technology has received greater attention recently (Brey
1991, Coluzzi 1992, Collins 1994, Curtis 1994). Introduction
and expression of genes coding for antibodies against target
antigens present on the ookinete surface into the mosquito
embryos is one of the possibilities to examine the potential
of this technology (Crampton et al. 1993).
f) Anti-malarial drugs -
Sub-therapeutic doses of antimalarial drugs have been reported
to enhance infectivity of Plasmodium species to their
vectors (Shute & Maryon 1954). Additionally, numerous
compounds including chloroquine (Wilkinson et al. 1976),
sulphamethoxazole-trimethroprim (Wilkinson et al. 1973),
pyrimethamine (Shute & Maryon 1951), Fansidar (Carter & Graves
1988) and Berenil (Ono et al. 1993) have been suggested to
induce gametocyte formation but no influence of chloroquine
(Jeffery et al. 1956, Smalley 1977, Chutmongkonkul et al.
1992, Hogh et al. 1994) and Fansidar (Hogh et al. 1994) on
gametocyte infectivity was observed by some investigators. It
was demonstrated that pyrimethamine- and halofantrine-treated
gametocytes of P. falciparum are more infective to
An. stephensi mosquitoes than untreated controls
(Chutmongkonkul et al. 1992). Other studies examined the
effects of some schizontocidal agents on the sporogonic cycle
of P. falciparum and P. berghei in anopheline
mosquitoes (Coleman et al. 1988, do Rosario et al. 1988). It
was found that chloroquine, when fed during late sporogony
(10-12 days post-infection), may increase the vectorial
capacity of some mosquito species. The effects of chloroquine
on the infectivity of chloroquine-sensitive and -resistant
populations of P. yoelii nigeriensis to An.
stephensi mosquitoes were studied by Ichimori et al.
(1990). The results showed an enhancement of infectivity in
sensitive strains but no effect was detected in resistant
clones and sublines. Chloroquine use and the subsequent
development of resistance over the past years is associated
with an increasing human malaria infectiousness (Lines et al.
1991) which may be indirect effects of parasitaemia on the
host. The sporontocidal activity of chloroquine, halofantrine
and pyrimethamine was evaluated by administration to An.
stephensi mosquitoes, either in the first bloodmeal
containing P. falciparum gametocytes from in
vitro cultures, or in the second, parasite-free bloodmeal,
given four days after infection. A sporontocidal effect was
observed only when pyrimethamine was administred with the
infective bloodmeal (Chutmongkonkul et al. 1992). It has been
demonstrated recently an inhibitory action of the anti-
malarial Atovaquone (566C80) against ookinete, oocysts and
sporozoites of Plasmodium berghei in An.
stephensi (Fowler et al.1994, 1995).
Acknowledgments
Amauri Braga Simonetti was supported by a scholarship from
CNPq (Brazil) during his PhD in the Infection and Immunity
Section, Department of Biology at Imperial College of Science,
Technology and Medicine (London, U.K.).
I would like to acknowlegde Professor Robert E Sinden for his
guidance and helpfull advices which made possible this
review.
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