Electron Microscopic and Histochemical Studies of Sporozoite Formation in Plasmodium berghei*

SYNOPSIS. Observations were made on the differentiation of fine structure during sporogonic development of Plasmodium berghei. The oocyst in the process of sporozoite formation is an encapsulated structure 30-40 μ in diameter. It typically develops while in an extracellular position, attached to the basement membrane of the mosquito midgut and projecting into the mosquito hemocoel. Occasionally, however, ookinetes passing thru the midgut epithelial cells may become impacted within a cell so that the resulting oocyst develops intracellularly. Each oocyst has a large differentiating region, the sporoblastoid body. This body contains large dividing nuclei which are Feulgenpositive, and a cytoplasm which includes mitochondria, dense rodlike structures, cytoplasmic membranes, cisternae and vacuolar structures, Golgi material, and ribosomes which are both free and membrane-associated. Sporozoite budding takes place along the surface of the sporoblastoid body. Bits of a new membrane condense under the plasma membrane which bounds the sporoblastoid body. These 2-membraned sites then bulge out, continue to elongate, and eventually become sporozoites. The various nuclear and cytoplasmic components of the sporoblastoid body are passed into the sporozoites during their elongation. In addition, the sporozoite develops a system of elogate, subpellicular microtubules, possibly contractile in function. The pellicle of the sporozoite is broken by an opening, the cytostome (micropyle). The anterior end is truncate.     

Fine Structure of Oocyst Transformation and the Sporozoites of Leucocytozoon dubreuili*

SYNOPSIS. The sporogonic stages of Leucocytozoon dubreuili in the midgut and salivary glands of the simuliid vectors was studied by electron microscopy. Young uninucleate oocysts have a pellicle that initially resembles that of the ookinetc. Numerous electron-dense bodies and microtubules in the peripheral cytoplasm may be involved in the formation of the cyst wall. The dense bodies appear to give rise to the amorphous material of the wall. The tubules which run circumferentially beneath the oocyst's boundary probably serve as a skeletal support for the cell surface during deposition of the wall material. A subcapsular “space” which provides area for expansion of the developing sporozoites is formed in early multinucleate oocysts. The subcapsular “space” appears to be formed through a condensation of the peripheral cytoplasm, resulting in an osmotic gradient across the oocyst's limiting membrane. Consequently water diffuses out, creating a fluid-filled space. Sporozoite formation begins with localized thickenings on the oocyst's limiting membrane. Subsequent extension of the thickened regions into the subcapsular “space” marks the onset of sporozoite budding. The process is highly synchronized, and culminates with the production of up to 150 sporozoites about the sporoblastoid body. The structure of sporozoites from mature oocysts and of the salivary glands of the vector is basically similar, although salivary gland sporozoites are more elongate and have numerous electron-dense micronemes. The paired rhoptries in the latter sporozoites are more elongate and uniformly electron-dense than in oocyst sporozoites.     

Identification of Plasmodium berghei Oocyst Rupture Protein 2 (ORP2) domains involved in sporozoite egress from the oocyst

Sporozoites are the infective form of malaria parasites which are transmitted from the mosquito salivary glands to a new host in a mosquito blood meal. The sporozoites develop inside the sporogonic oocyst and it is crucial for the continuation of the life cycle that the oocyst ruptures to release sporozoites. We recently described two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2), localized at the oocyst capsule, that are each essential for rupture of the oocysts. Both ORPs contain a histone fold domain implicated in the mechanism of oocyst rupture, possibly through the formation of a heterodimer between the two histone fold domains. To gain an understanding of the function of the different regions of the ORP2 protein, we generated deletion mutants. We monitored oocyst formation and rupture as well as sporozoites in the salivary gland. Our results show that different regions of ORP2 play independent roles in sporozoite egress. Deleting the N-terminal histone fold domain of ORP2 blocked sporozoite egress from the oocyst. Progressive deletions from the C-terminal resulted in no or significantly impaired sporozoite egress.     

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.     

THE TRANSFORMATION OF THE PLASMODIUM GALLINACEUM OOCYST IN AEDES AEGYPTI MOSQUITOES

Sporoblast and sporozoite formation from oocysts of the avian malarial parasite, Plasmodium gallinaceum, after the seventh day of infection in Aedes aegypti mosquitoes offers an interesting example of differentiation involving the appearance and modification of several cellular components. Sporoblast formation is preceded by (a) invaginations of the oocyst capsule into the oocyst cytoplasm, (b) subcapsular vacuolization and cleft formation, (c) the appearance of small tufts of capsule material on the previously noted invaginations, and (d) linear dense areas located just below the oocyst plasma membrane which predetermine the site of emerging sporozoites from the sporoblast. The subcapsular clefts subdivide the once-solid oocyst into sporoblast peninsulae. Within the sporoblast, nuclei migrate from the random distribution seen in the solid oocyst and come to lie at the periphery of the sporoblast just below the linear dense areas noted in the earlier stage. A typical nuclear fiber apparatus occurs in most of the nuclei seen in random sections at this stage although such a fiber apparatus may occasionally be seen in the solid oocyst stage. The nucleus, its associated fiber apparatus, and the overlying dense area appear to induce the onset of sporozoite budding from the sporoblast as well as the formation of the sporozoite pellicular complex and the paired organelle precursor. Several mitochondria are present in each sporozoite, in contrast to the single mitochondrion seen in the merozoites of the erythrocytic and exoerythrocytic stages of avian malaria infection. The paired organelles and associated dense inclusion bodies are formed by condensation of an irregular meshwork of membrane-bound, coarse, dense material. The nature of small, particulate cytoplasmic inclusions is described.     

Sporogonic Development of Leucocytozoon smithi

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30–50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

Sporogonic development of Leucocytozoon smithi.

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30-50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

The Fine Structure of Haemoproteus columbae Sporozoites*

SYNOPSIS. The fine structure of Haemoproteus columbae sporozoites has been studied and compared to sporozoite structure as revealed by the light microscope. The sporozoites are ultrastructurally similar to those of other Haemosporidia in that they possess a 3-layered pellicle, subpellicular microtubules, polar ring, micropore, free ribosome-like particles, micronemes, a structure resembling a Golgi complex, an irregular mitochondrion, and a large nucleus. In the anterior region of the sporozoite there are 21–22 regularly arranged longitudinal subpellicular microtubules located peripherally around the cell. In the apical region the microtubules appear thickened on 1 side. The sporozoite of H. columbae has a microneme system in which 1–3 micronemes are associated with the outer pellicular membrane at the anterior end. Micronemes are found throughout the cytoplasm, but occur in greater concentration in the anterior region of the sporozoite. A clear pellicular cavity, located between the polar ring and the termination of the inner pellicular layer, is present at the anterior end of the sporozoite. Vesicular invaginations of the inner pellicular layer have been observed in the anterior region; their function is unknown. Spherical osmophilic bodies are found throughout the cytoplasm.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Scanning electron microscopic observations of the oocyst, sporoblast and sporozoite of Plasmodium yoelii yoelii

Scanning electron microscopy was used to study the surface characteristics of the oocyst, sporoblast and sporozoite of Plasmodium yoelii yoelii. Observations were made of the sporogonic stages of 6-12 day infections of the malaria parasite in Anopheles stephensi. Oocyst and sporoblast development were not synchronous. The surface of the undifferentiated (early stage) oocyst appeared smooth, whereas that of differentiated (late stage) oocysts were rough or wrinkled. The wall of the differentiated oocysts showed numerous micropores at higher magnification (x15,000-20,000) the biological significance of which is not known. Small, bud-like satellite bodies were seen attached to some oocysts. Various forms of different stages of the sporoblast were described. Sporozoite budding took place on the surface of the sporoblast body. The sporozoite was elongate, curved and with a blunt anterior end.     

The surface of the malaria parasite: I. Scanning electron microscopy of the oocyst

Scanning electron microscopy has been used to study surface characteristics of the early sporogonic stages of Plasmodium gallinaceum and Plasmodium berghei. Observations upon oocysts from 9-day old infections of P. gallinaceum in Aedes aegypti and 14-day old infections of P. berghei in Anopheles stephensi indicate the oocyst surface is relatively smooth, although an outline of the underlying sporozoites can easily be recognized in the mature oocyst. Although all infections with each species were the same age, the stage of development of each oocyst appeared highly variable even within an individual mosquito. Oocysts appear to be covered by the overlying basement membrane which separates them from direct contact with the hemolymph as well as hemocytes. Small buds designated as satellite bodies were often seen attached to large oocysts of P. gallinaceum. Neither their origin nor their significance is yet known. During the study, numerous observations were made of the liberated sporozoites of both species. In each species the sporozoites are comma-shaped; however, those of P. gallinaceum are shorter, more strongly curved and stouter than those of P. berghei.     

Development of Babesiosoma stableri (Dactylosomatidae; Adeleida; Apicomplexa) in its Leech Vector (Batracobdella picta) and the Relationship of the Dactylosomatids to the Piroplasms of Higher Vertebrates

The sporogonic and merogonic development of Babesiosoma stableri Schmittner & McGhee, 1961 within its definitive host and vector, a leech Batracobdella picta (Verrill, 1872), was studied by light and electron microscopy. Gamonts released from frog erythrocytes in the blood meal of the leech associated in syzygy and fused; the gamonts were isogamous and only 1 microgamete was formed. The ultrastructural appearance of the resulting zygote was similar to that of the gamonts, but it was larger. The zygote had an apical complex (including a polar ring, conoid and 2 pre-conoidal rings and micronemes, but no recognizable rhoptries), triple-membraned pellicle, about 40 subpellicular microtubules and prominent stores of amylopectin. Zygotes penetrated the cells of the intestine and underwent sporogony directly within the cytosplasm of the ieech epithelial cell without the formation of a parasitophorous vacuole. Eight sporozoites budded simultaneously around the periphery of an irregularly shaped oocyst. No oocyst wall was formed. Each sporozoite had a complete apical complex (including rhoptries), abundant amylopectin inclusions and a triple-membraned pellicle with about 32 subpellicular microtubules. The sporozoites initiated merogonic replication primarily within the salivary cells of the leech although other tissues, such as muscle, were infected. Each meront produced 4 merozoites by simultaneous budding, forming a cruciform meront typical of the intraerythrocytic development of this parasite. The meront was located directly within the cytoplasm of the host cell. Merozoites, with abundant amylopectin, had a complete apical complex and triple-membraned pellicle with about 40 subpellicular microtubules. The merozoites either initiated a further cycle of replication, or they moved into the ductules of the leech salivary cells which extend to the tip of the proboscis. Observations on gametogenesis. syngamy and sporogony of B. stableri in its leech host indicate that the family Dactylosomatidae should be placed in the suborder Adeleina (Eucoccidiida: Apicomplexa). Babesiosoma stableri was transmitted to uninfected frogs (Rana spp.) by the bite of infected leeches. Prepatent periods ranged from 26 to 38 days at 25° C. Despite a directed search in laboratory reared tadpoles which had each been injected intraperitoneally with 150,000 merozoites, no pre-erythrocytic developmental stages were observed. Similarities in their biology suggest close phylogenetic affinities of the dactylosomatids, and other adeleid blood parasites, with the piroplasms of higher vertebrates.     

Eimeria tenella (Eucoccidiorida): A Quantitative Assay for Sporozoite Infectivity In Vivo1

ABSTRACT. A quantitative technique for the assessment of sporozoite infectivity in vivo, using intra-cecal inoculation of Eimeria tenella sporozoites, has been developed. Evaluation of the infection using cecal lesion scores and oocyst counts showed that this technique should be useful for the quantitation of sporozoite viability and thus for the anti-sporozoite activity of different treatments prior to inoculation. Pre-treatment of sporozoites with heat-inactivated hyperimmune antisera neutralized sporozoite infectivity in vivo and indicated that antibodies in the absence of complement inhibited sporozoite infectivity in vivo.     

The Journey of Malaria Sporozoites in the Mosquito Salivary Gland

The life cycle of malaria parasites in the mosquito vector is completed when the sporozoites infect the salivary gland and are ready to be injected into the vertebrate host. This paper describes the fine structure of the invasive process of mosquito salivary glands by malaria parasites. Plasmodium gallinaceum sporozoites start the invasion process by attaching to and crossing the basal lamina and then penetrating the host plasma membrane of the salivary cells. The penetration process appears to involve the formation of membrane junctions. Once inside the host cells, the sporozoites are seen within vacuoles attached by their anterior end to the vacuolar membrane. Mitochondria surround, and are closely associated with, the invading sporozoites. After the disruption of the membrane vacuole, the parasites traverse the cytoplasm, attach to, and invade the secretory cavity through the apical plasma membrane of the cells. Inside the secretory cavity, sporozoites are seen again inside vacuoles. Upon escaping from these vacuoles, sporozoites are positioned in parallel arrays forming large bundles attached by multilammelar membrane junctions. Several sporozoites are seen around and inside the secretory duct. Except for the penetration of the chitinous salivary duct, our observations have morphologically characterized the entire process of sporozoite passage through the salivary gland.     

La Sporogenèse chez la Coccidie Aggregata eberthi. Etude en Microscopic Electronique

SYNOPSIS. The structure of the oocyst and formation of sporozoites of Aggregata eberthi were studied with the electron microscope. After penetration of the microgamete, a cyst wall containing fine projections is formed beneath the "anhist" layer which is pushed away. The cytoplasm is retracted beneath the cyst wall and is irregular in outline. Lipid inclusions are abundant, while paraglycogen is less so. Vacuoles present in the early stages of development may be instrumental in elaboration of the cyst wall. Granulations appear in the early oocyst cytoplasm and form large compact masses in the sporoblasts, assuming a crystalline appearance (crystalloid) in the sporozoites. The sporoblasts are separated by the coalescence of vesicles. Each sporoblast is surrounded by an epispore and a striated endospore which is perforated by the "dehiscence device." Three sporozoites of classical structure are formed in each sporoblast.     

Transformation of the Plasmodium cynomolgi Oocyst1

SYNOPSIS. The transformation of the P. cynomolgi oocyst into definitive sporozoite forms occurs 8–10 days after an infective blood meal by Anopheles, stephensi mosquitoes. Vacuolization divides the oocyst cytoplasm into sporoblast sub-units from which sporozoites bud. The role of sporoblast nuclear and cytoplasmic components in the complex differentiative process is discussed.     

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.     

Ultrastructural and Cytologic Studies of the Sporozoites of Four Eimeria Species*

SYNOPSIS. Studies were made with the light microscope of live sporozoites of E. ninakohlyakimovae and E. ellipsoidalis as well as sporozoites fixed with Schaudinn's, Stieve's and Zenker's fluids, methanol and ethanol saturated with picric acid. Sporozoites were stained with Giemsa, bromphenol blue, modified PAS-AO, Feulgen, Harris’hematoxylin and eosin Y, and iron hematoxylin. Sporozoites of the above species as well as those of E. auburnensis and E. bovis were also fixed with glutaraldehyde and osmium tetroxide or negatively stained for study with the electron microscope. Living sporozoites had gliding, pivoting, flexing, and probing movements. Each sporozoite of each species was covered by a pellicle consisting of an outer limiting unit membrane that was continuous around the sporozoite and an inner membrane that terminated at the polar ring. Twenty-four subpellicular microtubules were longitudinally arranged just beneath the inner membrane. At the anterior end of the sporozoites was a protruded or retracted conoid composed of spirally-arranged fibrillar structures, 2 rings anterior to the conoid, and the polar ring, a thickening at the anterior termination of the microtubules and inner membrane. Other organelles observed with the electron microscope were a nucleus with or without a net-like nucleolus, club-shaped organelles, refractile bodies, micronemes, endoplasmic reticulum, Golgi apparatus, mitochondria with tubular cristae, micropores, lipoid-like bodies, oval polysaccharide bodies and ribosomes. The fine structure of these sporozoites is compared to that of related Sporozoa.