BIOLOGY

OF THE MALARIA PARASITE

Bulletin of the World Health Organization, 55 (2-3): 139-156 (1977)

Variations in stucture and function during the lifecycle of malarial parasites *M. AIKAWA1

The fine structure of malarial parasites is reviewed and the function of the intracellularorganelles is discussed. When the erythrocytic, exoerythrocytic, and mosquito stages ofplasmodia are compared, substantial differences are seen. The major differences involvethe amount ofsurface coat of the motileforms, the structure andfunction ofthe mitochondria,and the ingestion and digestion of nutrients. Significant structural differences are alsoobserved between comparable stages of mammalian and avian parasites. These differencesindicate that malarial parasites adapt themselves to the different environments in whichthe parasite resides.

When host cell changes induced by malarial parasite infection are reviewed, alterationscharacteristic of the infecting plasmodia are observed in erythrocytes. Erythrocyte changesinclude caveola-vesicle complexes, excrescences, and clefts. The caveola-vesicle complexespossess malarial antigens and exhibit pinocytotic activities. The excrescences form focaljunctions with adjacent cells and may be responsible for infected erythrocyte sequestrationin organs. The significance of these host cell changes specific to certain species of malarialparasite is still unknown.

Light microscopy of malarial parasites has enabledinvestigators to identify different species of malarialparasite and to identify some intracellular organelles.However, the restricted resolving power of the lightmicroscope limits the elucidation of detailed struc-ture and has failed to reveal many organelles thathave now been demonstrated by electron micro-scopy. This review focuses on those aspects of thefine 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 readilyapparent. In this review, the malarial parasites arediscussed in three categories: the motile, extra-cellular forms; the nonmotile, intracellular forms;and the sexual forms. Structural differences amongthe erythrocytic, exoerythrocytic, and mosquitostages are emphasized and structural variations

* This work was partially supported by US Public HealthService research grants AI-10645 and AI-13366 and by USArmy research and development contract DADA-17-70-C-0006. This article is Contribution No. 1444 from the USArmy Malaria Research Program.

1 Professor of Pathology, Institute of Pathology, CaseWestern Reserve University, Cleveland, OH 44106, USA.

among different parasite species are reviewed. Anattempt is made to correlate the functions of theintracellular organelles with their cytochemistry andbiochemistry. Finally, changes in host cells inducedby malarial parasite infection, as revealed by electronmicroscopy, are reviewed.

FINE STRUCTURE

Asexual motile formsThe motile, extracellular forms include sporo-

zoites formed in the oocyst of the mosquito andmerozoites 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 diameterfor the erythrocytic stages and about 2.5 ,um inlength and about 1.5 ,um in diameter for the exo-erythrocytic stage. The sporozoite is more elongatedthan either the erythrocytic or the exoerythrocyticmerozoites. Sporozoites measure about 11 .m inlength and 1.0 ,m in diameter. In general, thesemotile forms have common morphological featuresand possess similar organelles (Fig. 1). They aresurrounded by a pellicle of two membranes and arow of subpellicular microtubules. The lateral side

3590 139

M. AIKAWA

of the pellicle shows a circular structure called thecytostome. The anterior end is a truncated cone-shaped projection demarcated by three polar rings.Electron-dense rhoptries and micronemes are presentin the anterior portion of the parasite. The nucleusis situated in the mid-portion. The posterior portionof the parasite is occupied by a mitochondrion anda 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 nmthick and is the plasmalemma of the motile forms.The inner membrane complex is about 15 nm thickand the erythrocytic and exoerythrocytic merozoiteshave 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 theappearance of the sporozoite's inner membrane hasbeen made. A row of subpellicular microtubulesoriginating from the most distal polar ring is locatedbeneath the inner membrane. In the exoerythrocyticmerozoite of Plasmodium fallax, 24-26 microtubulesradiate in an even pattern from the circumferenceof the polar rings (2). On the other hand, sporo-zoites show a row of several microtubules aroundtwo thirds of the periphery of the sporozoites, plusan additional tubule in the remaining third (23, 60).

Possible functions for the complex pellicle of themotile 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 motileform has to be outside the host cell during host cellinvasion, albeit for only a very short time. Themicrotubules may also give the parasite motility.Although the significance of the peculiar arrange-ment of subpellicular microtubules in the sporozoiteis not known, their arrangement may be associatedwith 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 merozoiteswas first described in P. berghei and P. gallinaceum(32, 33). A similar coat was also described on thesurface of free erythrocytic merozoites of P. knowlesi(36, 40). The coat is electron-dense, compact, andfibrillar and is approximately 20 nm thick (Fig. 2).It appears to consist of protein or glycoproteinsince it is removed by trypsin. The surface coat

appears not to be present on merozoites withinintact erythrocytes (36), but merozoites that aresituated within haemolyzed or ghost erythrocytesshow a small amount of surface coat. It seems,therefore, that the coat is formed when merozoitesbecome extracellular, indicating that an interactionbetween the merozoite and substances in the outerenvironment contributes to the formation of thesurface coat (36). Sporozoites are also reported topossess a very thin (15 nm) surface coat of fibrillarmaterial loosely surrounding the outer sporozoitemembrane (18) (Fig. 2). No description of thesurface coat of the exoerythrocytic merozoites hasyet been made.Although the significance of the surface coat of

the motile forms must be explored further, it appearsto play a role in the parasites' response to theimmune reactions of the host (18, 40). When freemerozoites are exposed to immune serum, a looselypacked coat 60-100 nm thick appears on the surfaceand agglutination of merozoites is caused by adher-ence between the thick surface coats of adjacentparasites (Fig. 3). Similarly, a prominent surfacecoat (250 nm) can be seen in sporozoites incubatedin immune serum (Fig. 3). The circum-sporozoiteprecipitation (CSP) reaction seen in sporozoites in-cubated with immune serum is a result of the for-mation of an electron-dense coat along the surfaceof the sporozoite (Fig. 4). Pretreatment of sporo-zoites with mouse anti-sporozoite serum followedby incubation with rabbit anti-mouse IgG con-jugated to haemocyanin has revealed haemocyaninmolecules on the parasite's surface (Fig. 3). Thisindicates that immunoglobulins participate in theformation of the thick surface coat of sporozoitesand 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 ofavian and reptilian malarial parasites measures170-200 nm in its inner diameter (Fig. 6), whereasthat of mammalian malarial parasites measures50-80 nm (7). The cytostome of the exoerythrocyticmerozoites 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 theerythrocytic stages of avian and reptilian malarialparasites (4, 8, 44), while cytostomes measuring60-100 nm in diameter are found in the erythrocyticstages of mammalian malarial parasites (7) and the

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(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 (fromAikawa, 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 offibrillar surface coat (arrow).

I-,r'* .'. .... ..

'. -.

Fig. 3. Electron micrographs of sporozoites and merozoites incubated in immune sera. (a) A sporozoite of P. cynomolgsurrounded 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 areseen on the surface of the coat (arrow). (c) Merozoites of P. knowlesi surrounded by a prominent coat. Also noteadherence 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 immuneserum, covered with a surface coat.

Fig. 5. Mitochondria of P. berghei (from Aikawa, M. & Sterling, C., ref. 6). (a) Mitochondrion (M) of the mosquitostage with typical protozoan-type cristae (arrow). (b) Mitochondrion (M) of the exoerythrocytic stage withoutprominent cristae.

Fig. 6. Electron micrographs of ingestion and digestion of host cell cytoplasm. (a) Erythrocytic trophozoite ofP. gaflinaceum ingesting host cell cytoplasm by means of a cytostome (Ct). Also present is a food vacuole containingelectron-dense host cell cytoplasm. (b) A food vacuole with malarial pigment particles. The matrix is less dense thanthat of (a). (c) Oocyst of P. berghei. The capsular material is pinched off (arrow) from the internal surface, thussupplying nutrients to the parasite.

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Fig. 7. Electron micrographs of nuclear changes. (a) The metaphase nucleus of P. gallinaceum showing spindlefibres (Sf), kinetochores (Kc), and centriolar plaques (Cp) (from Aikawa, M. & Sterling, C., ref. 6). (b) The nucleusof P. berghei showing a centriolar plaque (Cp) located at the nuclear envelope. Spindle fibres (Sf) extend from theenvelope 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. Theareas covered by segments of thick membrane protrude outwards forming new merozoites. (c) Advanced schizogonyof 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-layeredmembrane (arrow). Notice abundant ribosomes, well developed endoplasmic reticulum, and osmiophilic bodies(from Aikawa, M. & Sterling, C., ref. 6). (b) Extracellular microgametocyte showing axonemes. (c) Microgametocyteforming 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 infectedby P. inui shows cavaolae (arrow). (c) Erythrocytes infected with P. brasilianum. Note numerous excrescences onthe surface (arrow).

STRUCTURE AND FUNCTION OF MALARIAL PARASITES

exoerythrocytic (27) and mosquito (23) stages of allplasmodia. The difference in the size of the cyto-stomes may be related to the function of thecytostomes (7). It is apparent that a dramatic changeoccurs in the size of the cytostomes of avian andreptilian malarial parasites when exoerythrocyticand mosquito stages of these parasites differentiateinto blood stages.At the periphery of the nucleus of the erythrocytic

and exoerythrocytic merozoites, there is heavilyclumped chromatin material while small dense par-ticles and fine fibrils are observed loosely scatteredin the nucleoplasm (3). On the other hand, thenucleus of the sporozoites does not exhibit promin-ent chromatin clumping (3). The significance of theprominent 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 presenceof classically distinct nucleoli in any of these motileforms.

Organelles of the anterior end are similar amongsporozoites and erythrocytic and exoerythrocyticmerozoites, although the shape and number maybe different (1, 4, 20, 21, 32, 46, 60). Two electron-dense rhoptries and micronemes are found in theseforms. The rhoptries of sporozoites are much largerthan 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 rolein the entry of malarial parasites into the host cells.They may contain surface-active substances thatcause membrane expansion, since the motile formsenter the host cell by invaginating the host cellmembrane (6). Recently histidine-rich protein wasisolated from malarial parasites and it was suggestedthat it is localized in the rhoptries of micronemesand that interaction of this protein with the erythro-cyte membrane causes invagination (31). These ob-servations further support a role of the rhoptriesand micronemes in host cell invasion by the parasite.The typical protozoan type mitochondria are pre-

sent in the merozoites and sporozoites of avian andreptilian malarial parasites. They are composed ofan outer and an inner membrane and microtubularcristae formed by invaginations of the inner mem-brane (Fig. 5). Cristate mitochondria have alsobeen 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 ofmammalian parasites than in those of avian malarial

parasites. Some of the mammalian malarial para-sites lack a structure that can be identified as amitochondrion; instead, they have double-mem-brane-bounded structures that are considered as theequivalent of mitochondria. The evidence suggestingthat these structures represent the mitochondria ofmalarial parasites comes from cytochemical studiesthat have demonstrated the presence of cytochromeoxidase (28, 29, 57). Cytochemical studies havedemonstrated the presence of succinic dehydrogenaseactivity in cristate mitochondria, while no activityhas been found in acristate mitochondria. Thisobservation suggests that malarial parasites withcristate mitochondria utilize a Krebs cycle, while itis not certain whether the Krebs cycle is used byparasites with acristate mitochondria (51). It is inter-esting to note that the mosquito stages of somemammalian malarial parasites possess cristate mito-chondria while the exoerythrocytic and erythrocyticstages of the same species do not (Fig. 5). As sug-gested, the change from acristate mitochondria inthe mosquito stage may be related to differences inthe parasite metabolism in the mammalian andinsect hosts.

Asexual nonmotile formsThe nonmotile or intracellular forms include uni-

nucleate trophozoites and schizonts of the erythro-cytic and exoerythrocytic stages and oocysts andsporoblasts of the mosquito stages.When the erythrocytic merozoite enters a new

host cell, the membrane of the host cell invaginatesand engulfs the merozoite, forming a parasitophor-ous vacuole (16, 33). The penetration of host cellsby the exoerythrocytic merozoite and ookinetes (22)may occur in a manner similar to that of the erythro-cytic merozoites, although a clear demonstration ofthis process is lacking for these forms. The processof entry into host cells by the motile forms ofmalarial parasites is reviewed on pages 157-162 ofthis issue.

After penetration of the host cell, the intracellularmerozoite loses its polar rings, rhoptries, micro-nemes, inner pellicular membrane, subpellicularmicrotubules, and spherical body and transformsinto a uninucleate trophozoite (1, 9, 32). Althoughthere is no report on the process of transformationof an ookinete to an oocyst, it is reasonable toassume that a similar process occurs since intra-cellular organelles specific to the ookinete cannot beseen in the oocyst. The disappearance after pene-tration of the organelles, which are specific to the

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M. AIKAWA

motile forms, suggests that these organelles havespecific functions that are not required for thestages concerned with growth and multiplication.The two major activities of the nonmotile, intra-

cellular forms are feeding and multiplication. It iswell established that the intraerythrocytic stagesingest 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, andis pinched off from the cytostomal cavity. Foodingestion processes similar to those occurring in theerythrocytic stages have been reported to occur inexoerythrocytic stages of P. elongatum (9), P. gal-linaceum (10), and P. lophurae (17). It is also poss-ible that simple diffusion of nutrients through theparasite membrane supplies the exoerythrocyticstages with nutrients (7). A cytostome has not beenobserved in oocysts of malarial parasites. Instead,the oocysts are surrounded by a thick, electron-densecapsule (6, 56). On its internal surface, masses ofcapsular material appear to be pinched off, suggest-ing that the capsular material may supply nutrientsto the oocyst (Fig. 6).

Digestion of erythrocyte cytoplasm occur withinthe food vacuoles (1, 5, 41, 42). The process ofdigestion in the erythrocytic stages is indicated bythe formation of malarial pigment particles and theconcomitant decreased density of the host cell cyto-plasm within the food vacuoles (Fig. 6). There is aninverse relationship between the amount of malarialpigment and the density of the host cell cytoplasmin the food vacuoles. The malarial pigment particlesin avian and reptilian malarial parasites are uni-formly electron-dense and do not have a clearcrystalloid appearance (7), whereas malarial pigmentparticles seen in mammalian malarial parasites arecrystalloid and rectangular in shape. The differencemay be due to variations in the haemoglobin com-position of the avian, reptilian, and mammalianhost cells that is ingested by the parasites. Foodvacuoles seen in the exoerythrocytic parasites aresmall and are electron transparent, although somecontain granular material (17).

Mitochondria with microtubular cristae are foundin the oocysts of those mammalian malarial parasitesthat do not possess such mitochondria in the erythro-cytic stage in the vertebrate host (3). The trans-formation of mitochondria without cristae to thosewith cristae apparently occurs with the change ofhost (6).During multiplication, nuclear division and dif-

ferentiation of the cytoplasmic organelles are the

two major events. Nuclear division is accompaniedby considerable morphological change (Fig. 7). Oneof the most noticeable changes is the appearanceof spindle fibres or intranuclear microtubules (12).Bundles of the microtubules radiate in a fan-shapedfashion from poorly delineated electron-opaque re-gions located on opposite sides of the nuclearmembrane. They radiate towards each other andmeet midway; at this point paired, electron-densestructures occur. They appear to be kinetochores,since they cannot be extracted by DNase and sinceduring nuclear division they are located at irregularintervals along the spindle fibres (12). Betweenkinetochores is an ill-defined electron-dense zonethat can be extracted with DNase and thereforeprobably comprises the chromosomes. As nucleardivision progresses, the nucleus becomes dumbbellshaped and finally two daughter nuclei are formed.During nuclear division, the nuclear membrane doesnot disappear but interruption occurs where theintranuclear 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 alongthe plasmalemma of the parasite). The process ofdaughter cell formation is similar among the erythro-cytic (1), exoerythrocytic (27), and mosquito stages(53, 54, 55, 59). One significant difference foundamong these stages is the number of daughter cellsthat 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 beginningof intracellular development reappear. The organellesthat were first noted in the parasites are randomlydistributed segments of the inner membrane of thepellicle of motile forms. These segments are usuallyseen opposite the centriolar plaques. The subpelli-cular microtubules, rhoptries, micronemes, and polarrings subsequently appear beneath the segments ofthe inner membrane. Areas covered by the segmentsof the thick inner membrane, together with the sub-pellicular microtubules, begin to protrude into thewidened parasitophorous vacuole (Fig. 8). The mito-chondrion increases in size with changes of the cyto-plasm and it becomes irregular, forming severalbuds (1). Finally, it undergoes fission to yield manymitochondria. With the progression of daughtercell budding, a nucleus and other organelles migrateinto the developing daughter cells. As developmentof the daughter cells advances, the size of the

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STRUCTURE AND FUNCTION OF MALARIAL PARASITES

mother cell decreases and finally only a residualbody remains.

Sexual forms

Studies by electron microscopy of the gametocytesof various malarial parasites have revealed that notonly can they be differentiated from the parasitesof 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 bymeans of the electron microscope is a uninucleateparasite surrounded by three membranes (Fig. 9).These membranes are particularly pronounced amongthe gametocytes of avian and reptilian malarial para-sites, but are not quite so apparent in mammalianparasites. The outer of the three membranes appearsto be the host cell membrane forming a parasit-ophorous vacuole. Therefore, the gametocyte's pel-licle is actually composed of two membranes. Thisobservation led to a hypothesis that the gametocyteoriginates from a merozoite that failed to breakdown its inner membrane after infecting a new hostcell (11).The cytoplasm of the mature macrogametocytes

is filled with ribosomes, while the microgametocytecontains fewer ribosomes (Fig. 9). This differencein number of ribosomes may indicate a difference inthe amount of protein synthesis between the twoforms (11). Another feature that distinguishes themacrogametocyte from the microgametocyte is thenumber of osmiophilic bodies. These are more fre-quent in the macrogametocyte than in the micro-gametocyte. A narrow ductule extends from theosmiophilic body to the inner membrane of thepellicle. The osmiophilic bodies resemble rhoptriesand may contain material similar to that in therhoptries (11). They may assist in the escape of thegametocyte from the host cell by attaching to theplasmalemma of the parasite and then causing dis-solution of the host cell. Gametocytes of avian andreptilian parasites possess several mitochondria butthose of mammalian parasites do not possess typicalmitochondria. Cytostomes are present in gameto-cytes (8). Because the gametocyte does not showany invaginations or protrusions along the pellicularcomplex, there seems to be no doubt that ingestionof host cell cytoplasm takes place at the cytostome.The food vacuoles are scattered in the cytoplasmand malarial pigment particles are found withinthese vacuoles. A single large nucleus is present inboth macro- and microgametocytes. When micro-

gametogenesis commences, the host cell ruptures andthe intracellular microgametocyte becomes extra-cellular.

In microgametogenesis, nuclear division occurs bya process similar to that in the asexual stages (48).Kinetosomes are seen near centriolar plaques locatedat the nuclear membrane and axonemes assemblefrom the kinetosome. Condensation of chromatinbecomes evident at the periphery of the nucleus andthe condensed masses of nuclear chromatin even-tually envelop axonemes. The axonemes and at-tached nuclear chromatin migrate to the surface ofthe microgametocyte and participate in the for-mation of flagellar buds (Fig. 9). The buds passthrough interruptions of the inner membrane andare surrounded by the outer membrane of the micro-gametocyte, thus forming the microgametes. A freemicrogamete contains a single axoneme, a kineto-some, and a nucleus intertwined with the axoneme(24, 47, 48). Also present are perikinetosomal andjuxtakinetosomal spheres and granules (48).At fertilization of a macrogamete with a micro-

gamete, the microgamete approaches the surface ofthe macrogamete and fusion occurs (48). In thesemacrogametes, an axoneme and a nucleus of themicrogamete can be seen. The ookinete developedfrom a zygote has a structure similar to that ofthe merozoite and sporozoite (22, 25, 54). In addition,an aggregate of virus-like particles has been reportedin the cytoplasm of the ookinete (54).

Host cell changes

Dramatic changes are seen in erythrocytes infectedby some species of malarial parasites (13, 14, 15, 19,26, 37, 38, 39, 49, 52, 58). The structures that occurin infected erythrocytes and that may be seen bylight microscopy have been given various namessuch as Schuiffner's dots, Maurer's clefts, Zieman'sstippling, or Sinton & Mulligan's stippling. Electronmicroscopic examination of erythrocytes infectedwith various species of malarial parasites has clarifiedour knowledge of these structures. Schiiffner's dotsseen by light microscopy in the erythrocytes infectedby vivax-type and ovale-type malarial parasites aredemonstrated by electron microscopy to be caveola-vesicle complexes along the erythrocyte plasma mem-brane (14, 15, 52) (Fig. 10). They consist of caveolaesurrounded by vesicles in an alveolar fashion. Horse-radish-peroxidase-labelled immunoglobulin frommonkeys infected with P. vivax has been shown tobind to the vesicle membrane (16). Cationized fer-ritin has appeared within the vesicles after incu-

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M. AIKAWA

bation with viable parasitized erythrocytes, suggest-ing that these vesicles are pinocytotic in origin (16).The presence of horseradish peroxidase within thevesicles indicates the presence of malarial antigenswithin them. Maurer's clefts seen by light micro-scopy appear to correspond to narrow slit-like struc-tures in the cytoplasm. Another prominent changeseen in erythrocytes infected with malarial parasitesis 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 thesequestration 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 infectedby vivax- and ovale-type parasites show caveola-

Table 1. Changes in erythrocytes infectedAikawa, M. et al., ref. 14)

vesicle complexes and cytoplasmic clefts, while thoseinfected by malariae-type parasites exhibit excres-

cences and cytoplasmic clefts (14). Erythrocytesinfected by falciparum-type parasites show excres-

cences and cytoplasmic clefts. In addition, thecaveolae are seen in the erythrocytes infected withP. fragile and P. coatneyi (falciparum-type parasites),although they are lacking in erythrocytes infectedwith P. falciparum (16). It is still not clear why a

given group of malarial parasites produce changesin the erythrocytes specific to that given group.

Exoerythrocytic malarial parasites also producechanges in the host cell. When a parasitophorousvacuole enlarges, the parasite occupies a major partof the host cell cytoplasm (10, 50), the cytoplasmicorganelles and the nucleus of the host being pushedto one side of the host cell. However, the organellesof the host cell do not show any significant changes.

by some primate malarial parasites (from

Electron microscopy a

Parasites Light Plasma membranemicroscopy Cytoplasmic Excrescences Caveolae Caveola-

clefts vesiclecomplexes

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.

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STRUCTURE AND FUNCTION OF MALARIAL PARASITES 155

These observations may indicate that the host cellchanges are due to the presence of malarial parasitesas foreign bodies and not to " toxin " produced bythe parasite. There is no report on the fine structuralchanges of host cells in the mosquito stage ofmalarial parasites.

CONCLUSIONS

Since the first report on the electron microscopyof malarial parasites was made in 1942, much dataon morphology has accumulated and many intra-cellular organelles have been discovered.When the fine structures of the erythrocytic,

exoerythrocytic, and mosquito stages of malarialparasites 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 betweencomparables stages of mammalian and avian para-sites. This suggests that experimental data on avianmalarial parasites may not necessarily apply tomammalian malarial parasites.

This review on the fine structure of malarial para-sites has demonstrated that electron microscopy hascontributed greatly to the understanding of thebiology of malarial parasites. With current progressin cytochemical and biochemical research, we havebegun to understand the function of the intracellularorganelles of malarial parasites; this will make itpossible to take further steps forward in the analysisof malarial parasites.

ACKNOWLEDGEMENTS

The author wishes to thank Dr J. Kreier of Ohio State University for his criticism of the manuscript, Mr C. L. Hsiehfor technical assistance, and Mrs J. Pirina for typing the manuscript.

RtSUMIEMODIFICATIONS 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 del'enveloppe de surface des formes mobiles, la structureet la fonction des mitochondries et l'ingestion et ladigestion des nutriments. On observe egalement desdifferences de structure importantes lorsque l'on com-pare les parasites mammaliens et aviaires a des stadescorrespondants. 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 h6teprovoquees par l'infection parasitaire, on observe dansles erythrocytes des modifications, caracteristiques duplasmodium infectant: notamment de l'apparition decomplexes caveoles-vesicules d'excroissances et defissures. Les complexes caveoles-vesicules possedent desantigenes palud&ens et presentent des activites pinocyto-tiques. Les excroissances forment des jonctions focalesavec les cellules adjacentes et peuvent etre responsablesde la sequestration d'erythrocytes infectes dans lesorganes. L'importance de ces modifications des cellulesh6tes, specifiques de certaines especes de parasite dupaludisme, n'est pas encore connue.

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