间日疟原虫卵囊内成孢子细胞与子孢子形成的超微结构观察

应用透射电镜观察间日疟原虫在大劣按蚊体内发育的卵囊内成孢子细胞及子孢子形成过程形态变化。疟原虫采自带有配子体的间日疟自愿者。蚊虫在感染后8天作解剖。本研究观察结果与前人所描述的柏氏疟原虫和鸣疟原虫的成孢子细胞与子孢子形成过程相似,即成孢子细胞形成开始于卵囊被膜下的周围出现液泡,而随着膜下液泡增大,逐渐向胞质延伸并联接成裂缝,使胞质再分裂而形成。子孢子周围的膜下微管分布不对称,其数目和排列型式,多数为:7+4、7+5、8+4和8+5,少数为10+1,与前人报告不同(10+1)。     

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.     

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.     

The fine structure of the developing oocyst of Pterospora floridiensis (Apicomplexa,Urosporidae)

Developing oocysts of the gregarine Pterospora floridiensis Landers 2001 were examined by transmission electron microscopy. Each oocyst had an outer capsule and an inner capsule that contained 8 sporozoites. In early stages of development the inner capsular wall was separated from the developing sporozoites and residual mass, and was not appressed to the sporozoites. Early stage sporozoites were connected to a residual mass and were filled with endoplasmic reticulum, golgi and numerous developing secretory vesicles. In late stages of oocyst and sporozoite development, the inner capsular wall was closely appressed to the sporozoite surface. The inner capsular wall was ～60-100 nm thick and the outer capsular wall was ～160-320 nm thick. There were no extensions on the outer wall for which the genus was named. Late stage sporozoites had no residual mass connection, were more electron dense, and contained three distinct types of dense secretory structures: 1) small oval/spherical dense vesicles, 2) large (350-400 nm) vesicles near the anterior end, and 3) elongated dense tubular bodies that converged at the apex. Few ultrastructural reports exist of developing gregarine oocysts and sporozoites, and as more studies are completed these morphological characteristics may be important in interpreting molecular phylogenetic analyses.     

Fine Structure of Differentiating Oocysts and Mature Sporozoites of Haemoproteus metchnikovi in its Intermediate Host Chrysops callidus*

The fine structure of the sporogonic stages of Haemoproteus metchnikovi has been investigated by electron microscopy. Young oocysts are found beneath the basement membrane of midgut epithelial cells. These eventually protrude outward into the haemocoel space and are surrounded by a distinct oocyst capsule. Sporozoite formation begins with a subcapsular vacuolation. Evagination of the oocyst cytoplasm occurs in regions of membrane thickenings and 100–200 sporozoites are formed about a single sporoblastoid body. Remnants of the ookinete pellicle can be observed in maturing oocysts and always are found in the residual body. The fine structure of the mature sporozoite is essentially similar to that which has been described for other haemosporidia and a spherical body is described in association with the mitochondrion of the sporozoite. The sporogonic stages of H. metchnikovi have features common to the sporogonic stages of Plasmodium and Leucocytozoon that are not held in common by the latter 2 genera, including pattern of sporozoite formation and number of sporozoites formed, the presence of a cytostome and of “crystalloid” in the sporozoite.     

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.     

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.     

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.     

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.     

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.     

Light and electron microscopy on the sporulation of the oocysts of Eimeria brunetti. II. Development into the sporocyst and formation of the sporozoite

The later stages of sporulation in oocysts of Eimeria brunetti were examined in samples which had been allowed to sporulate at 27 degrees C for 24, 36 and 48 hours. It was observed that the sporoblasts became ellipsoidal and the nucleus underwent the final division. A nucleus with associated Golgi bodies was not observed at either end of the organism. The cytoplasm was limited by two unit membranes and contained rough endoplasmic reticulum, dense bodies, electron translucent vacuoles and mitochondria. The first evidence of sporozoite formation was the appearance of a dense plaque at either end of the organism. This appeared in the vicinity of the nuclei, and adjacent to the limiting membrane of the soroblast. At this stage the sporocyst wall was still unformed. Then the two sporozoites were formed from opposite ends of the organism by growth of the dense plaques and invaginations of the plasmalemma which thus formed the pellicles of the developing sporozoites. A conoid and subpellicular microtubules were observed at this stage as development continued, a number of vacuoles were found between the nucleus and the conoid. These vacuoles constituted the precursors of the rhoptries and micronemes. At the same stage a large dense body had appeared within the forming sporozoite. As the sporozoite developed, this body, anterior refractile body, is followed by the nucleus and another dense body which formed the posterior refractile body. During this period, the thin sporocyst wall was formed and Stieda and sub-Stieda bodies were now present at one end of the sporocyst. Each mature sporocyst contained two sporozoites.     

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.     

The Repeat Region of the Circumsporozoite Protein is Critical for Sporozoite Formation and Maturation in Plasmodium

The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH₂-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH₂-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective.     

Sporogenesis of a myxosporidan with motile spores

Summary Three types of cells comprise each Fabespora vermicola sporoblast: valvogenic (VAV), capsulogenic (CAP), and germinative (GEM). Walls, polar caps, and sutures are the main assemblages produced by the VAV cells. The unique polar cap organelle extends over the aperture region of the polar capsule component of the CAP cell. The VAV cell also assembles a wall located on the cytoplasmic side of the plasma membrane facing the sporoblast exterior. Bundles of 7 nm microfilaments develop within the extracellular space between the VAV and interior cells of the sporoblast. These microfilaments assemble late in sporogenesis when the spore acquires the capacity for locomotion. Polar filament construction takes place exclusively within the polar capsule primordium (PCP) by apparent self-assembly prior to the PCP being enveloped by membranes. The CAP and GEM cells accumulate considerable glycogen during sporogenesis. The first identifiable GEM cell is single, but has two unpaired nuclei. These GEM cell nuclei later form a paired structure which is sustained into the spore stage.We acknowledge Ann Scarborough and Roswitha Buxton for their expert technical assistance. The study was conducted in cooperation with the U.S. Department of Commerce, NOAA, National Marine Fisheries Service, under PL 88-309, Project No. 2-325-R     

THE GOLGI APPARATUS IN NEURONS AND EPITHELIAL CELLS OF THE COMMON LIMPET PATELLA VULGATA

1. In view of widely diverse views held about the identity and structure of the Golgi apparatus in neurons of Mollusca, particularly gastropods, a study has been made on neurons of the common limpet, Patella vulgata, both by light and electron microscopy. A report is given also of observations made on epithelial cells of Patella by electron microscopy. 2. As revealed by Kolatchev's method, the Golgi apparatus in neurons consists basically of black filaments lying to one side of the nucleus. The filaments generally anastomose to form networks of various complexity. Rarely some cells contain only discrete filaments. Associated with some of the filaments is a weakly osmiophilic substance identified as archoplasm. Kolatchev's method also revealed spheroidal bodies (neutral red bodies, "lipochondria," etc.). 3. It has not been possible to demonstrate the Golgi apparatus using either iron-haematoxylin or Sudan black. 4. Examination of Kolatchev's preparations by electron microscopy has revealed that some of the Golgi filaments consist of chromophilic and chromophobic components. The chromophilic component consists of dense lamellae. 5. After fixation in buffered osmium tetroxide solution and examination by electron microscopy, it has been concluded that (a) the chromophilic component of the Golgi apparatus corresponds to a system of paired membranes (which usually enclose an inner dense substance), (b) the chromophobic component corresponds to a substance lying within small dilations of the paired membrane, and (c) the archoplasm corresponds to numerous small vesicles. 6. The paired membranes branch, anastomose, and can often be traced back to a common source. They are interpreted as lamelliform folds, and occasionally tubular processes, of essentially a single Golgi membrane. In cells containing a Golgi network it is suggested that the membrane extends through the whole of the apparatus in such a way that the substance it encloses may be regarded as being in a continuous phase. 7. Epithelial cells of Patella contain a juxtanuclear Golgi apparatus with an ultrastructure similar to that described for neurons.     

Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ·cm⁻² doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ·cm⁻². With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ·cm⁻² of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ·cm⁻² of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.     

Sporogonic Development of the Gregarine Ascogregarina taiwanensis (Lien and Levine) (Apicomplexa: Lecudinidae) in Its Natural Host Aedes albopictus (Skuse) (Diptera: Culicidae)

ABSTRACT. Sexual reproduction of Ascogregarina taiwanensis occurred in pupal Malpighian tubules of its natural host Aedes albopictus , resulting in the formation of gametocysts within which oocysts developed. Sporogony proceeded in each newly formed unsporulated oocyst; eight sporozoites were formed after completion of nuclear divisions followed by the cytokinesis. Developing oocysts were separated by gradient centrifugation on percoll based on different buoyant densities. The slender sporozoite had a typical apical complex composed of a coiled conoid, polar rings, rhoptries with ductules, subpellicular microtubules and micronemes. An apical cavity was seen in the gland-like rhoptries. Mitochondria of gregarines were not seen in any stage during the sporogony. Howeever, amylopectin granules were frequently seen in the cytoplasm. These starch-related granules became scant when the sporozoite was formed. We assumed they were associated with the energy source. Since the apical complex was only present in the sporozoite stage, it was most likely related to the invasion of host epithelial cells of the midgut during the early phase of infection.     

Crystalloid Inclusions in Species of Leucocytozoon,Parahaemoproteus, and Plasmodium*†

Cytoplasmic vacuoles seen in methanol-fixed, Giemsa's-stained ookinetes of Leucocytozoon simondi, Parahaemoproteus fringillae and Plasmodium gallinaceum, when studied with the electron microscope, were found to correspond with crystalloid inclusions of similar structure, particle size, and arrangement. Cytochemical examination of these “crystalloids” revealed their lipid-protein nature. Morphologically similar inclusions were found also in ookinetes of Leucocytozoon ziemanni and Parahaemoproteus velans. In L. simondi, crystalloid is formed rapidly after fertilization, from amorphous electron dense material seen in mature macrogametocytes. The arrangement and distribution of crystalloids in the zygote, ookinete, oocyst, and sporozoite are described. On the basis of differences in structure and particle size, it is proposed that the crystalloid inclusions in Haemosporina be divided into 2 types. Type I—lipid-protein in nature, characterized by electron dense irregularly spherical particles, 25–40 nm in diameter, with individual particles not invested by membrane. Type II—probably virus, characterized by electron dense, irregularly spherical, membrane-bounded particles, with a diameter usually greater than 40 nm.