Variations in structure and function during the life cycle of malarial parasites*

BIOLOGY OF THE MALARIA PARASITE Bulletin of the World Health Organization, 55 (2-3): 139-156 (1977) Variations in stucture and function during the life cycle of malarial parasites * M. AIKAWA1 The fine structure of malarial parasites is reviewed and the function of the intracellular organelles is discussed. When the erythrocytic, exoerythrocytic, and mosquito stages of plasmodia are compared, substantial differences are seen. The major differences involve the amount ofsurface coat of the motileforms, the structure andfunction ofthe mitochondria, and the ingestion and digestion of nutrients. Significant structural differences are also observed between comparable stages of mammalian and avian parasites. These differences indicate that malarial parasites adapt themselves to the different environments in which the parasite resides. When host cell changes induced by malarial parasite infection are reviewed, alterations characteristic of the infecting plasmodia are observed in erythrocytes. Erythrocyte changes include caveola-vesicle complexes, excrescences, and clefts. The caveola-vesicle complexes possess malarial antigens and exhibit pinocytotic activities. The excrescences form focal junctions with adjacent cells and may be responsible for infected erythrocyte sequestration in organs. The significance of these host cell changes specific to certain species of malarial parasite is still unknown. Light microscopy of malarial parasites has enabled investigators to identify different species of malarial parasite and to identify some intracellular organelles. However, the restricted resolving power of the light microscope limits the elucidation of detailed struc- ture and has failed to reveal many organelles that have now been demonstrated by electron micro- scopy. This review focuses on those aspects of the fine structure of malarial parasites and their organ- elles that have been revealed by electron microscopy. There are some structural differences in the intra- cellular organelles among the erythrocytic, exoery- throcytic, and mosquito stages of malarial parasites, although major structural similarities are readily apparent. In this review, the malarial parasites are discussed in three categories: the motile, extra- cellular forms; the nonmotile, intracellular forms; and the sexual forms. Structural differences among the erythrocytic, exoerythrocytic, and mosquito stages are emphasized and structural variations * This work was partially supported by US Public Health Service research grants AI-10645 and AI-13366 and by US Army research and development contract DADA-17-70- C-0006. This article is Contribution No. 1444 from the US Army Malaria Research Program. 1 Professor of Pathology, Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA. among different parasite species are reviewed. An attempt is made to correlate the functions of the intracellular organelles with their cytochemistry and biochemistry. Finally, changes in host cells induced by malarial parasite infection, as revealed by electron microscopy, are reviewed. FINE STRUCTURE Asexual motile forms The motile, extracellular forms include sporo- zoites formed in the oocyst of the mosquito and merozoites formed by the erythrocytic and exo- erythrocytic stages in the vertebrate host. The merozoite is oval or elongated and measures about 1.5 ,um in length and about 1 ,um in diameter for the erythrocytic stages and about 2.5 ,um in length and about 1.5 ,um in diameter for the exo- erythrocytic stage. The sporozoite is more elongated than either the erythrocytic or the exoerythrocytic merozoites. Sporozoites measure about 11 .m in length and 1.0 ,m in diameter. In general, these motile forms have common morphological features and possess similar organelles (Fig. 1). They are surrounded by a pellicle of two membranes and a row of subpellicular microtubules. The lateral side 3590 139 M. AIKAWA of the pellicle shows a circular structure called the cytostome. The anterior end is a truncated cone- shaped projection demarcated by three polar rings. Electron-dense rhoptries and micronemes are present in the anterior portion of the parasite. The nucleus is situated in the mid-portion. The posterior portion of the parasite is occupied by a mitochondrion and a spherical body. The pellicle of these motile forms is composed of an outer and two closely associated inner membranes (2, 27, 35). The outer membrane is about 7.5 nm thick and is the plasmalemma of the motile forms. The inner membrane complex is about 15 nm thick and the erythrocytic and exoerythrocytic merozoites have interrupted foci along their length. With nega- tive staining, the inner membrane of the exoerythro- cytic and erythrocytic merozoites appears labyrin- thine (2). Freeze cleaving has also revealed a poss- ibly labyrinthine structure (45). No report on the appearance of the sporozoite's inner membrane has been made. A row of subpellicular microtubules originating from the most distal polar ring is located beneath the inner membrane. In the exoerythrocytic merozoite of Plasmodium fallax, 24-26 microtubules radiate in an even pattern from the circumference of the polar rings (2). On the other hand, sporo- zoites show a row of several microtubules around two thirds of the periphery of the sporozoites, plus an additional tubule in the remaining third (23, 60). Possible functions for the complex pellicle of the motile forms have been suggested by many investi- gators. The inner membrane system and micro- tubules have been suggested to be that of a cytoskele- ton imparting rigidity to the parasite (2); this sup- position was supported by the fact that the motile form has to be outside the host cell during host cell invasion, albeit for only a very short time. The microtubules may also give the parasite motility. Although the significance of the peculiar arrange- ment of subpellicular microtubules in the sporozoite is not known, their arrangement may be associated with the crescent shape of the sporozoite. A great deal of attention has recently been paid to the surface structure of merozoites and sporo- zoites. A surface coat on free erythrocytic merozoites was first described in P. berghei and P. gallinaceum (32, 33). A similar coat was also described on the surface of free erythrocytic merozoites of P. knowlesi (36, 40). The coat is electron-dense, compact, and fibrillar and is approximately 20 nm thick (Fig. 2). It appears to consist of protein or glycoprotein since it is removed by trypsin. The surface coat appears not to be present on merozoites within intact erythrocytes (36), but merozoites that are situated within haemolyzed or ghost erythrocytes show a small amount of surface coat. It seems, therefore, that the coat is formed when merozoites become extracellular, indicating that an interaction between the merozoite and substances in the outer environment contributes to the formation of the surface coat (36). Sporozoites are also reported to possess a very thin (15 nm) surface coat of fibrillar material loosely surrounding the outer sporozoite membrane (18) (Fig. 2). No description of the surface coat of the exoerythrocytic merozoites has yet been made. Although the significance of the surface coat of the motile forms must be explored further, it appears to play a role in the parasites' response to the immune reactions of the host (18, 40). When free merozoites are exposed to immune serum, a loosely packed coat 60-100 nm thick appears on the surface and agglutination of merozoites is caused by adher- ence between the thick surface coats of adjacent parasites (Fig. 3). Similarly, a prominent surface coat (250 nm) can be seen in sporozoites incubated in immune serum (Fig. 3). The circum-sporozoite precipitation (CSP) reaction seen in sporozoites in- cubated with immune serum is a result of the for- mation of an electron-dense coat along the surface of the sporozoite (Fig. 4). Pretreatment of sporo- zoites with mouse anti-sporozoite serum followed by incubation with rabbit anti-mouse IgG con- jugated to haemocyanin has revealed haemocyanin molecules on the parasite's surface (Fig. 3). This indicates that immunoglobulins participate in the formation of the thick surface coat of sporozoites and merozoites incubated in immune serum. A cytostome is present in the pellicle of sporo- zoites and of erythrocytic and exoerythrocytic mero- zoites. The cytostome of erythrocytic merozoites of avian and reptilian malarial parasites measures 170-200 nm in its inner diameter (Fig. 6), whereas that of mammalian malarial parasites measures 50-80 nm (7). The cytostome of the exoerythrocytic merozoites measures 80-100 nm and that of sporo- zoites measures about 100 nm (10, 17, 27). The parasites can be classified roughly into two groups based on the size of the cytostome. Cyto- stomes measuring 170-200 nm are found in the erythrocytic stages of avian and reptilian malarial parasites (4, 8, 44), while cytostomes measuring 60-100 nm in diameter are found in the erythrocytic stages of mammalian malarial parasites (7) and the 140 (E .. Fig. 1. Electron micrographs of motile forms. (a) Erythrocytic merozoites of P. cathemerium showing polar rings (Pr), micronemes (Mi), a nucleus (N), a mitochondrion (M), a spherical body (Sb), and pellicular complexes (from Aikawa, M., ref. 3). (b) Exoerythrocytic merozoites of P. fallax. (c) Sporozoites of P. cynomolgi. Fig. 2. Electron micrographs of a merozoite and sporozoite. (a) A merozoite of P. knowlesi showing a uniform' compact surface coat (from Mason, S. et al., ref. 36). (b) A sporozoite of P. cynomolgi covered by a small amount of fibrillar surface coat (arrow). I-,r'* .'. .... .. '. -. Fig. 3. Electron micrographs of sporozoites and merozoites incubated in immune sera. (a) A sporozoite of P. cynomolg surrounded by a fibrillar coat. (b) A sporozoite of P. berghei incubated first with immune mouse serum and sub- sequently with rabbit anti-mouse immunoglobulin conjugated with haemocyanin. The haemocyanin molecules are seen on the surface of the coat (arrow). (c) Merozoites of P. knowlesi surrounded by a prominent coat. Also note adherence between the surface coat of adjacent parasites (arrow). Fig. 4. Scanning electron micrographs of sporozoites (from Cochrane, A. et al., ref. 18). (a) Sporozoite of P. cyno- molgi incubated in normal serum; note the smooth surface. (b) Sporozoite of P. cynomolgi, incubated in immune serum, covered with a surface coat. Fig. 5. Mitochondria of P. berghei (from Aikawa, M. & Sterling, C., ref. 6). (a) Mitochondrion (M) of the mosquito stage with typical protozoan-type cristae (arrow). (b) Mitochondrion (M) of the exoerythrocytic stage without prominent cristae. Fig. 6. Electron micrographs of ingestion and digestion of host cell cytoplasm. (a) Erythrocytic trophozoite of P. gaflinaceum ingesting host cell cytoplasm by means of a cytostome (Ct). Also present is a food vacuole containing electron-dense host cell cytoplasm. (b) A food vacuole with malarial pigment particles. The matrix is less dense than that of (a). (c) Oocyst of P. berghei. The capsular material is pinched off (arrow) from the internal surface, thus supplying nutrients to the parasite. v - *M..'IO .... Fig. 7. Electron micrographs of nuclear changes. (a) The metaphase nucleus of P. gallinaceum showing spindle fibres (Sf), kinetochores (Kc), and centriolar plaques (Cp) (from Aikawa, M. & Sterling, C., ref. 6). (b) The nucleus of P. berghei showing a centriolar plaque (Cp) located at the nuclear envelope. Spindle fibres (Sf) extend from the envelope into the nucleus. Pellicular inner membranes (arrow) are seen along the plasmalemma. Fig. 8. Electron micrographs showing schizogony in the erythrocytic, exoerythrocytic, and mosquito stages. (a) Beginning of schizogony in the erythrocytic stage of P. cathemerium. (b) Advanced stage of schizogony. The areas covered by segments of thick membrane protrude outwards forming new merozoites. (c) Advanced schizogony of the exoerythrocytic stages of P. fallax. (d) Advanced sporogony of the mosquito stage of P. berghei. Fig. 9. Electron micrograph of sexual forms. (a) Macrogametocyte of P. gallinaceum surrounded by a three-layered membrane (arrow). Notice abundant ribosomes, well developed endoplasmic reticulum, and osmiophilic bodies (from Aikawa, M. & Sterling, C., ref. 6). (b) Extracellular microgametocyte showing axonemes. (c) Microgametocyte forming microgametes (arrow). A cross section of the microgamete shows a nucleus (N) and an axoneme (A). :I Fig. 10. Electron micrograph of host cell changes infected with malarial parasites. (a) A caveola-vesicle complex (arrow) is seen in an erythrocyte infected by P. vivax. (b) A scanning electron micrograph of an erythrocyte infected by P. inui shows cavaolae (arrow). (c) Erythrocytes infected with P. brasilianum. Note numerous excrescences on the surface (arrow). STRUCTURE AND FUNCTION OF MALARIAL PARASITES exoerythrocytic (27) and mosquito (23) stages of all plasmodia. The difference in the size of the cyto- stomes may be related to the function of the cytostomes (7). It is apparent that a dramatic change occurs in the size of the cytostomes of avian and reptilian malarial parasites when exoerythrocytic and mosquito stages of these parasites differentiate into blood stages. At the periphery of the nucleus of the erythrocytic and exoerythrocytic merozoites, there is heavily clumped chromatin material while small dense par- ticles and fine fibrils are observed loosely scattered in the nucleoplasm (3). On the other hand, the nucleus of the sporozoites does not exhibit promin- ent chromatin clumping (3). The significance of the prominent chromatin clumping is not known, al- though it may indicate that the nucleus of the mero- zoite is inactive. There is no report on the presence of classically distinct nucleoli in any of these motile forms. Organelles of the anterior end are similar among sporozoites and erythrocytic and exoerythrocytic merozoites, although the shape and number may be different (1, 4, 20, 21, 32, 46, 60). Two electron- dense rhoptries and micronemes are found in these forms. The rhoptries of sporozoites are much larger than those of other forms and extend to the mid- portion. There are more micronemes in the sporo- zoites than in the other forms. Rhoptries and micronemes appear to play a role in the entry of malarial parasites into the host cells. They may contain surface-active substances that cause membrane expansion, since the motile forms enter the host cell by invaginating the host cell membrane (6). Recently histidine-rich protein was isolated from malarial parasites and it was suggested that it is localized in the rhoptries of micronemes and that interaction of this protein with the erythro- cyte membrane causes invagination (31). These ob- servations further support a role of the rhoptries and micronemes in host cell invasion by the parasite. The typical protozoan type mitochondria are pre- sent in the merozoites and sporozoites of avian and reptilian malarial parasites. They are composed of an outer and an inner membrane and microtubular cristae formed by invaginations of the inner mem- brane (Fig. 5). Cristate mitochondria have also been observed in certain primate plasmodia, includ- ing P. falciparum, P. malariae, P. berghei, P. brasi- lianum, P. inui, P. vivax, and P. cynomolgi (51). How- ever, fewer cristae are seen in the mitochondria of mammalian parasites than in those of avian malarial parasites. Some of the mammalian malarial para- sites lack a structure that can be identified as a mitochondrion; instead, they have double-mem- brane-bounded structures that are considered as the equivalent of mitochondria. The evidence suggesting that these structures represent the mitochondria of malarial parasites comes from cytochemical studies that have demonstrated the presence of cytochrome oxidase (28, 29, 57). Cytochemical studies have demonstrated the presence of succinic dehydrogenase activity in cristate mitochondria, while no activity has been found in acristate mitochondria. This observation suggests that malarial parasites with cristate mitochondria utilize a Krebs cycle, while it is not certain whether the Krebs cycle is used by parasites with acristate mitochondria (51). It is inter- esting to note that the mosquito stages of some mammalian malarial parasites possess cristate mito- chondria while the exoerythrocytic and erythrocytic stages of the same species do not (Fig. 5). As sug- gested, the change from acristate mitochondria in the mosquito stage may be related to differences in the parasite metabolism in the mammalian and insect hosts. Asexual nonmotile forms The nonmotile or intracellular forms include uni- nucleate trophozoites and schizonts of the erythro- cytic and exoerythrocytic stages and oocysts and sporoblasts of the mosquito stages. When the erythrocytic merozoite enters a new host cell, the membrane of the host cell invaginates and engulfs the merozoite, forming a parasitophor- ous vacuole (16, 33). The penetration of host cells by the exoerythrocytic merozoite and ookinetes (22) may occur in a manner similar to that of the erythro- cytic merozoites, although a clear demonstration of this process is lacking for these forms. The process of entry into host cells by the motile forms of malarial parasites is reviewed on pages 157-162 of this issue. After penetration of the host cell, the intracellular merozoite loses its polar rings, rhoptries, micro- nemes, inner pellicular membrane, subpellicular microtubules, and spherical body and transforms into a uninucleate trophozoite (1, 9, 32). Although there is no report on the process of transformation of an ookinete to an oocyst, it is reasonable to assume that a similar process occurs since intra- cellular organelles specific to the ookinete cannot be seen in the oocyst. The disappearance after pene- tration of the organelles, which are specific to the 151 M. AIKAWA motile forms, suggests that these organelles have specific functions that are not required for the stages concerned with growth and multiplication. The two major activities of the nonmotile, intra- cellular forms are feeding and multiplication. It is well established that the intraerythrocytic stages ingest host cell cytoplasm through the cytostome (1, 7, 8, 34, 51). The host cell cytoplasm enters a cyto- stomal cavity (Fig. 6), is ingested in vacuoles, and is pinched off from the cytostomal cavity. Food ingestion processes similar to those occurring in the erythrocytic stages have been reported to occur in exoerythrocytic stages of P. elongatum (9), P. gal- linaceum (10), and P. lophurae (17). It is also poss- ible that simple diffusion of nutrients through the parasite membrane supplies the exoerythrocytic stages with nutrients (7). A cytostome has not been observed in oocysts of malarial parasites. Instead, the oocysts are surrounded by a thick, electron-dense capsule (6, 56). On its internal surface, masses of capsular material appear to be pinched off, suggest- ing that the capsular material may supply nutrients to the oocyst (Fig. 6). Digestion of erythrocyte cytoplasm occur within the food vacuoles (1, 5, 41, 42). The process of digestion in the erythrocytic stages is indicated by the formation of malarial pigment particles and the concomitant decreased density of the host cell cyto- plasm within the food vacuoles (Fig. 6). There is an inverse relationship between the amount of malarial pigment and the density of the host cell cytoplasm in the food vacuoles. The malarial pigment particles in avian and reptilian malarial parasites are uni- formly electron-dense and do not have a clear crystalloid appearance (7), whereas malarial pigment particles seen in mammalian malarial parasites are crystalloid and rectangular in shape. The difference may be due to variations in the haemoglobin com- position of the avian, reptilian, and mammalian host cells that is ingested by the parasites. Food vacuoles seen in the exoerythrocytic parasites are small and are electron transparent, although some contain granular material (17). Mitochondria with microtubular cristae are found in the oocysts of those mammalian malarial parasites that do not possess such mitochondria in the erythro- cytic stage in the vertebrate host (3). The trans- formation of mitochondria without cristae to those with cristae apparently occurs with the change of host (6). During multiplication, nuclear division and dif- ferentiation of the cytoplasmic organelles are the two major events. Nuclear division is accompanied by considerable morphological change (Fig. 7). One of the most noticeable changes is the appearance of spindle fibres or intranuclear microtubules (12). Bundles of the microtubules radiate in a fan-shaped fashion from poorly delineated electron-opaque re- gions located on opposite sides of the nuclear membrane. They radiate towards each other and meet midway; at this point paired, electron-dense structures occur. They appear to be kinetochores, since they cannot be extracted by DNase and since during nuclear division they are located at irregular intervals along the spindle fibres (12). Between kinetochores is an ill-defined electron-dense zone that can be extracted with DNase and therefore probably comprises the chromosomes. As nuclear division progresses, the nucleus becomes dumbbell shaped and finally two daughter nuclei are formed. During nuclear division, the nuclear membrane does not disappear but interruption occurs where the intranuclear microtubules are attached. The merozoites and sporozoites are formed from respective mother cells by schizogony (Fig. 8) (schizogony is defined as an asexual form of multi- plication whereby new progeny are formed along the plasmalemma of the parasite). The process of daughter cell formation is similar among the erythro- cytic (1), exoerythrocytic (27), and mosquito stages (53, 54, 55, 59). One significant difference found among these stages is the number of daughter cells that develop from a single mother cell. While nuclear division is taking place, the cyto- plasm shows dramatic changes (Fig. 8). The cyto- plasmic organelles that disappeared at the beginning of intracellular development reappear. The organelles that were first noted in the parasites are randomly distributed segments of the inner membrane of the pellicle of motile forms. These segments are usually seen opposite the centriolar plaques. The subpelli- cular microtubules, rhoptries, micronemes, and polar rings subsequently appear beneath the segments of the inner membrane. Areas covered by the segments of the thick inner membrane, together with the sub- pellicular microtubules, begin to protrude into the widened parasitophorous vacuole (Fig. 8). The mito- chondrion increases in size with changes of the cyto- plasm and it becomes irregular, forming several buds (1). Finally, it undergoes fission to yield many mitochondria. With the progression of daughter cell budding, a nucleus and other organelles migrate into the developing daughter cells. As development of the daughter cells advances, the size of the 152 STRUCTURE AND FUNCTION OF MALARIAL PARASITES mother cell decreases and finally only a residual body remains. Sexual forms Studies by electron microscopy of the gametocytes of various malarial parasites have revealed that not only can they be differentiated from the parasites of the asexual stages but also that the microgameto- cytes can be distinguished from the macrogameto- cytes on the basis of their fine structure (11, 30, 43). The parasite that is identifiable as a gametocyte by means of the electron microscope is a uninucleate parasite surrounded by three membranes (Fig. 9). These membranes are particularly pronounced among the gametocytes of avian and reptilian malarial para- sites, but are not quite so apparent in mammalian parasites. The outer of the three membranes appears to be the host cell membrane forming a parasit- ophorous vacuole. Therefore, the gametocyte's pel- licle is actually composed of two membranes. This observation led to a hypothesis that the gametocyte originates from a merozoite that failed to break down its inner membrane after infecting a new host cell (11). The cytoplasm of the mature macrogametocytes is filled with ribosomes, while the microgametocyte contains fewer ribosomes (Fig. 9). This difference in number of ribosomes may indicate a difference in the amount of protein synthesis between the two forms (11). Another feature that distinguishes the macrogametocyte from the microgametocyte is the number of osmiophilic bodies. These are more fre- quent in the macrogametocyte than in the micro- gametocyte. A narrow ductule extends from the osmiophilic body to the inner membrane of the pellicle. The osmiophilic bodies resemble rhoptries and may contain material similar to that in the rhoptries (11). They may assist in the escape of the gametocyte from the host cell by attaching to the plasmalemma of the parasite and then causing dis- solution of the host cell. Gametocytes of avian and reptilian parasites possess several mitochondria but those of mammalian parasites do not possess typical mitochondria. Cytostomes are present in gameto- cytes (8). Because the gametocyte does not show any invaginations or protrusions along the pellicular complex, there seems to be no doubt that ingestion of host cell cytoplasm takes place at the cytostome. The food vacuoles are scattered in the cytoplasm and malarial pigment particles are found within these vacuoles. A single large nucleus is present in both macro- and microgametocytes. When micro- gametogenesis commences, the host cell ruptures and the intracellular microgametocyte becomes extra- cellular. In microgametogenesis, nuclear division occurs by a process similar to that in the asexual stages (48). Kinetosomes are seen near centriolar plaques located at the nuclear membrane and axonemes assemble from the kinetosome. Condensation of chromatin becomes evident at the periphery of the nucleus and the condensed masses of nuclear chromatin even- tually envelop axonemes. The axonemes and at- tached nuclear chromatin migrate to the surface of the microgametocyte and participate in the for- mation of flagellar buds (Fig. 9). The buds pass through interruptions of the inner membrane and are surrounded by the outer membrane of the micro- gametocyte, thus forming the microgametes. A free microgamete contains a single axoneme, a kineto- some, and a nucleus intertwined with the axoneme (24, 47, 48). Also present are perikinetosomal and juxtakinetosomal spheres and granules (48). At fertilization of a macrogamete with a micro- gamete, the microgamete approaches the surface of the macrogamete and fusion occurs (48). In these macrogametes, an axoneme and a nucleus of the microgamete can be seen. The ookinete developed from a zygote has a structure similar to that of the merozoite and sporozoite (22, 25, 54). In addition, an aggregate of virus-like particles has been reported in the cytoplasm of the ookinete (54). Host cell changes Dramatic changes are seen in erythrocytes infected by some species of malarial parasites (13, 14, 15, 19, 26, 37, 38, 39, 49, 52, 58). The structures that occur in infected erythrocytes and that may be seen by light microscopy have been given various names such as Schuiffner's dots, Maurer's clefts, Zieman's stippling, or Sinton & Mulligan's stippling. Electron microscopic examination of erythrocytes infected with various species of malarial parasites has clarified our knowledge of these structures. Schiiffner's dots seen by light microscopy in the erythrocytes infected by vivax-type and ovale-type malarial parasites are demonstrated by electron microscopy to be caveola- vesicle complexes along the erythrocyte plasma mem- brane (14, 15, 52) (Fig. 10). They consist of caveolae surrounded by vesicles in an alveolar fashion. Horse- radish-peroxidase-labelled immunoglobulin from monkeys infected with P. vivax has been shown to bind to the vesicle membrane (16). Cationized fer- ritin has appeared within the vesicles after incu- 153 M. AIKAWA bation with viable parasitized erythrocytes, suggest- ing that these vesicles are pinocytotic in origin (16). The presence of horseradish peroxidase within the vesicles indicates the presence of malarial antigens within them. Maurer's clefts seen by light micro- scopy appear to correspond to narrow slit-like struc- tures in the cytoplasm. Another prominent change seen in erythrocytes infected with malarial parasites is excrescences on the erythrocyte plasmalemma (13, 14, 37, 43, 51, 58) (Fig. 10). These excrescences form focal junctions with the membrane of endo- thelial cells or with excrescences on other erythro- cytes, suggesting that they are responsible for the sequestration of infected erythrocytes in the organs (13, 35, 38). Alterations characteristic of the infect- ing plasmodia may be observed by electron micro- scopy in erythrocytes (Table 1). Erythrocytes infected by vivax- and ovale-type parasites show caveola- Table 1. Changes in erythrocytes infected Aikawa, M. et al., ref. 14) vesicle complexes and cytoplasmic clefts, while those infected by malariae-type parasites exhibit excres- cences and cytoplasmic clefts (14). Erythrocytes infected by falciparum-type parasites show excres- cences and cytoplasmic clefts. In addition, the caveolae are seen in the erythrocytes infected with P. fragile and P. coatneyi (falciparum-type parasites), although they are lacking in erythrocytes infected with P. falciparum (16). It is still not clear why a given group of malarial parasites produce changes in the erythrocytes specific to that given group. Exoerythrocytic malarial parasites also produce changes in the host cell. When a parasitophorous vacuole enlarges, the parasite occupies a major part of the host cell cytoplasm (10, 50), the cytoplasmic organelles and the nucleus of the host being pushed to one side of the host cell. However, the organelles of the host cell do not show any significant changes. by some primate malarial parasites (from Electron microscopy a Parasites Light Plasma membrane microscopy Cytoplasmic Excrescences Caveolae Caveola- clefts vesicle complexes vivax type P. vivax Schuffner's dots + - + + P. simium Schuffner's dots + - + + P. cynomolgi Schuffner's dots + - + + ovale type P. simiovale Schuffner's dots + + + P. fieldi Schuffner's dots + + + falciparum type P. falciparum Maurer's clefts + +b P. fragile faint stippling + + + P. coatneyi Maurer's clefts + +b + malariae type P. malariae Ziemann's stippling + +b P. brasilianum Ziemann's stippling + +b P. inu Ziemann's stippling + - + + others P. knowlesi Sinton & Mulligan's + - + stippling a + = structure present; ± = structure occasionally present; - = structure absent.b Excrescences are found on erythrocytes infected by asexual forms and on those infected by gametocytes of P. malariae and P. brasilianum but are only found on erythrocytes infected with asexual forms of P. falci- parum and P. coatneyi. 154 STRUCTURE AND FUNCTION OF MALARIAL PARASITES 155 These observations may indicate that the host cell changes are due to the presence of malarial parasites as foreign bodies and not to " toxin " produced by the parasite. There is no report on the fine structural changes of host cells in the mosquito stage of malarial parasites. CONCLUSIONS Since the first report on the electron microscopy of malarial parasites was made in 1942, much data on morphology has accumulated and many intra- cellular organelles have been discovered. When the fine structures of the erythrocytic, exoerythrocytic, and mosquito stages of malarial parasites are compared, substantial structural dif- ferences are seen. These differences indicate that the parasite must adapt itself to the different environ- ments in which the parasite resides. In addition, sig- nificant structural differences are found between comparables stages of mammalian and avian para- sites. This suggests that experimental data on avian malarial parasites may not necessarily apply to mammalian malarial parasites. This review on the fine structure of malarial para- sites has demonstrated that electron microscopy has contributed greatly to the understanding of the biology of malarial parasites. With current progress in cytochemical and biochemical research, we have begun to understand the function of the intracellular organelles of malarial parasites; this will make it possible to take further steps forward in the analysis of malarial parasites. ACKNOWLEDGEMENTS The author wishes to thank Dr J. Kreier of Ohio State University for his criticism of the manuscript, Mr C. L. Hsieh for technical assistance, and Mrs J. Pirina for typing the manuscript. RtSUMIE MODIFICATIONS STRUCTURALES ET FONCTIONNELLES PENDANT LE CYCLE BIOLOGIQUE DES PARASITES DU PALUDISME Le present travail traite de la structure fine des para- sites du paludisme et de la fonction des organites intra- cellulaires. Lorsque l'on compare les stades erythrocy- taire, exoerythrocytaire et sporogonique des plasmo- diums, on observe des differences appreciables. Les prin- cipales de ces differences interessent l'epaisseur de l'enveloppe de surface des formes mobiles, la structure et la fonction des mitochondries et l'ingestion et la digestion des nutriments. On observe egalement des differences de structure importantes lorsque l'on com- pare les parasites mammaliens et aviaires a des stades correspondants. Ces differences indiquent que les para- sites du paludisme s'adaptent aux differents environne- ments dans lesquels ils sejournent. Lorsque l'on etudie les modifications de la cellule h6te provoquees par l'infection parasitaire, on observe dans les erythrocytes des modifications, caracteristiques du plasmodium infectant: notamment de l'apparition de complexes caveoles-vesicules d'excroissances et de fissures. 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