Ultrastructural Study of Gametocytes and Gametogenesis of Haemoproteus metchnikovi

SYNOPSIS. The ultrastructure of gametocytes and microgametogenesis of Haemoproteus metchnikovi , an intracellular malaria parasite of chelonians, was compared with other malarial parasites. Gametocytes resemble those described for other avian, reptilian and mammalian malarial parasites. The process of microgametogenesis is similar in many respects to that reported for Leucocytozoon simondi (Aikawa et al., 1970) and Haemoproteus columbae (Bradbury and Trager, 1968). Atypical centrioles are described from both microgametocytes and macrogametocytes. Axoneme development begins before the microgametocyte leaves the host red blood cell. Fully developed microgametes which bud off from the parent gametocyte contain a single axoneme with the typical 9 + 2 pattern of microtubules.     

Leucocytozoonosis in Canada geese in upper Michigan,. 1. Strain differences among geese from different localities.

Geographic variation in pathogenicity of Leucocytozoon simondi in Canada geese (Branta canadensis maxima) was investigated by exposing goslings to natural infection at three locations in the upper peninsula of Michigan. Examination of blood smears and tissue sections revealed two patterns of development. Hepatic schizogony and secondary megaloschizogony occurred in cells of the reticuloendothelial system, with round and elongate gametocytes, or only hepatic schizonts and round gametocytes. The evidence for strain differences in L. simondi and its implications in wildfowl management practices are discussed.     

Electron Microscope Studies on Eimeria perforans (Sporozoa)

SYNOPSIS. By means of electron microscopy, a study has been made of the fine structure of the macrogametocytes, microgametocytes and oocysts of Eimeria perforans from the intestine of the wild rabbit (Oryctolagus cuniculus). The parasites lie in a vacuole within the host cell. The surface of the gametocytes is not plain, but displays irregular protrusions. A large intranuclear body can be detected within the macrogametocytes. Similar structures are also found within the cytoplasm. Within the latter there exists a large spread out reticulum, the channels and vesicles of which concentrate especially close to the nuclear membrane. Tubuli are seen in the numerous mitochondria, which often have a dumb-bell shape.
In most of the gametocytes irregular, strongly osmiophilic lipid inclusions are observed, which always are surrounded by the endoplasmic reticulum. Strange folded ovoid bodies are found within the cytoplasm of the oocysts. Nothing can be told with certainty of their nature and function. Probably they represent specific storage bodies.     

Freeze-Preservation of Leucocytozoon simondi Sporozoites in Liquid Nitrogen

SYNOPSIS. Sporozoites of L. simondi were maintained in a viable state for 7 months in liquid nitrogen. Comparison of parasite development initiated with fresh and frozen sporozoites showed a delay in development of each stage studied. Comparisons of prepatency, first elongate gametocytes, peak density of round and elongate forms, anemia and disappearance of megaloschizonts were made. In each phase there was a delay of 2–3 days in ducks infected with frozen sporozoites.     

A Comparative Study of Gametocyte Ultrastructure in Avian Haemosporidia*

The fine structure of gametocytes of 3 avian haemosporidian parasites Plasmodium gallinaceum, Haemoproteus columbae, and Leucocytozoon simondi has been studied and compared by electron microscopy. The gametocytes of all 3 species are bounded by a 3-layered limiting membrane system, possess a cytostome during some portion of their residence within host cells, and their sex can be distinguished by both nuclear and cytoplasmic characteristics. L. simondi differs most significantly from P. gallinaceum and H. columbae in possessing large intranuclear granules, mitochondria associated with pocket infoldings of the nuclear envelope near the atypical centriole complex and compartmentalization of the cytoplasm by segments of closely aligned unit membranes. Further, the cytostome of L. simondi does not appear to be a persistent structure as in the other 2 species and pigment is not present within food vacuoles. L. simondi also is capable of infecting a wider variety of host cells and within leukocytes produces striations of the host nucleus and an apparent spiral banding of the host cell surface. The comparison of P. gallinaceum, H. columbae, and L. simondi gametocytes by electron microscopy leads to the conclusion that Plasmodium and Haemoproteus are more closely related to each other than either of them is on Leucocytozoon. The terminology used to describe certain organelles within the gametocyte's cytoplasm has been reexamined and the relationship of the nucleolus to parasite maturation also is described.     

Fine Structure of Haemoproteus columbae Kruse During Macrogametogenesis and Fertilization*

When blood is withdrawn from a pigeon (Columba livia) infected with gametocytes of Haemoproteus columbae, differentiation of the gametes begins immediately. This study examines the formation of the macrogamete and its fertilization. The first visible signs of differentiation are the elongation of the nucleus along with the appearance of an intranuclear spindle and atypical centrioles. Then maturation bodies, the products of nuclear reduction, form in both erythrocytic macrogametocytes and macrogametocytes free of their host cells. Penetration of the macrogamete by the microgamete occurs rapidly. Their plasma membranes fuse, and the microgamete's nucleus, axonemes and cytoplasm enter the macrogamete. The nucleus of the microgamete expands and migrates to lie at an angle to the macrogamete nucleus. The 2 fuse across a small area. The nuclear envelope and the plasma membrane of the zygote are a mosaic of the membranes of the 2 gametes.     

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.     

Plasmodium simium: Ultrastructure of erythrocytic phase

An electron microscopic study of Plasmodium simium infections in the squirrel monkey has supplied information on the ultrastructure of erythrocytic trophozoites, schizonts, merozoites, and gametocytes, in addition to an unusual form of host cell pathology. In general, the structural features, as well as certain specialized functions, e.g., hemoglobin ingestion and utilization, nuclear and cytoplasmic division, were found to be similar to those described for other malarial parasites. Some striking features were noted, however. A highly asynchronous mode of merozoite production was observed within single segmenting parasites in spite of the overall developmental synchrony displayed by the population as a whole. Secondly, during parasite segmentation, newly formed merozoites are connected to one another, as well as to the parasitophorous membrane, by periodic surface strands. It is speculated that these interparasite bridges serve as structural support to the segmenting parasite. When merozoites are matured fully, these interconnections break, leaving a uniform array of short surface bristles. In addition, a number of different pathological changes in host cell structure have been noted. Localized surface discontinuities appear in region of infected cells where apical regions of developing or fully mature merozoites are abutted against the plasma membrane. These profiles suggest that these specialized apical regions of the merozoite function in release as well as in host cell penetration. More generalized surface pathology occurs within parasitized erythrocytes in the form of surface blebs, surface clefts, and associated cytoplasmic microvesicles. The severity of this pathology increases as the intraerythrocytic parasite matures. Topographically these altered cells have a “berry-like” surface texture which makes them quite distinctive when viewed by scanning electron microscopy.     

Observations on the ultrastructure of Haemogregarina simondi in the marine flatfish Solea solea (L.).

Developing stages of Haemogregarina simondi from the marine fish Solea solea (L.) were examined by electron microscopy. Merozoites lay in a parasitophorous vacuole and were bound by a pellicle of three unit membranes beneath which lay a ring of 45--61 microtubules. The cytoplasm contained 4--6 rhoptries, more than 169 micronemes, several mitochondria, and amylopectin granules. A conoid and one polar ring were observed at the anterior end. Intraleucocytic and intraerythrocytic schizonts with up to eight merozoites were described also. Intraerythrocytic and free gametocytes were characterized by distinct refractile bodies and a pellicle consisting of only two unit membranes. The number of micronemes was in excess of 194. The results were discussed in comparison with other members of the Haemogregarinidae.     

Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.     

Early Stages in the Differentiation of Gametocytes of Haemoproteus columbae Kruse*

SYNOPSIS The sexes of mature gametocytes of Haemoproteus columbae Kruse circulating in the blood of the domestic pigeon can be identified in the electron microscope by the same criteria that distinguish them in the light microscope. The microgametocyte has a large nucleus and pigment granules restricted to the 2 extremities of its halter-shaped cells. The macrogametocyte has dense granular cytoplasm with scattered pigment granules and a small central nucleus. The sex of young gametocytes cannot yet be recognized. When blood containing mature gametocytes is cooled outside the body of the host visible signs of gametogenesis appear within 30 seconds. The earliest signs are increasing electron lucidity of the cytoplasm and separation of the outer membrane from the body of the parasite. The membrane may form vesicles or whorls or lie free in the erythrocyte's cytoplasm. The middle membrane of the parasite becomes the plasma membrane. Axonemes and microtubules appear in the cytoplasm and nucleoplasm of the microgametocyte. The macrogametocyte lags slightly behind the microgametocyte in development. With the first signs of differentiation, the host cell cytoplasm begins to disappear. The fate of the outer membrane and the erythrocyte's cytoplasm suggests the release of a lytic substance by the parasite.     

Leucocytozoon in the Avian Order Columbiformes, with a Description of L. marchouxi Mathis and Leǵer 1910 from the Mourning Dove

Leucocytozoon marchouxi is described from the mourning dove, Zenaidura macroura carolinensis in Illinois. The macrogametocytes are rounded or elliptical, measuring 7 to 11 by 8 to 15 microns (mean, 9 by 12 microns), and stain dark blue with Giemsa stain. The microgametocytes are often distorted or ruptured by the smearing process, but if not badly injured measure 5 to 11 by 8 to 15 microns, (mean, 8 by 11 microns). They stain pale blue with Giemsa stain. Host cell cytoplasm is rarely seen surrounding the microgametocytes, but was found in 26% of cells parasitized by the macrogametocytes. When present it forms a narrow border around part or all of the parasite's periphery, and is not drawn out to form elongate "horns."
The parasite was found in ten birds, of which five were adults, one was a juvenile, and four were nestlings. The infections were relatively light in the adults. The youngest bird in which the protozoon was found was 14 days old.
The literature on Leucocytozoon in the avian order Columbiformes is reviewed. All forms with rounded gametocytes so far reported are considered to be L. marchouxi Mathis and Leger, 1910, of which L. turtur Covaleda Ortega and Gallego Berenguer, 1946, is considered a synonym.     

DNA synthesis,reduction, and elimination during life cycles of the eimeriine coccidian, Eimeria tenella and the haemogregarine, Hepatozoon domerguei

The occurrence of zygotic nuclear reduction and the haploid state of postzygotic stages were demonstrated in Eimeria tenella (Coccidia, Eimeriina) and Hepatozoon domerguei (Coccidia, Adeleina) by direct measurement of DNA by microdensitometry. Nuclear divisions, which were probably mitotic, were demonstrated in the macrogametocytes of a species of Hepatocystis (Coccidia, Haemosporina). The macrogametocytes of E. tenella were Feulgen negative but gametocytes of both sexes of H. domerguei were Feulgen positive and had DNA values estimated at about twice the haploid amount. This latter finding was interpreted as DNA synthesis preceding a maturation process which involved mitotic divisions and led to micro- and macrogamete formation. In macrogametogenesis this may be an atavistic trait, recapitulating the evolution of sexuality or a method for increased RNA synthesis.     

Elongate and Round Gametocytes of Leucocytozoon simondi (Mathis & Léger) in Ducks Inoculated with Megaloschizonts

SYNOPSIS. Single megaloschizonts give rise to elongate and round gametocytes, the former outnumbering the latter. Male and female elongate gametocytes develop from merozoites of a single megaloschizont. Elongate gametocytes were seen 2–7 days and round gametocytes 6–11 days after megaloschizonts had been inoculated into ducklings. Experimental evidence indicates that merozoites of megaloschizonts invade blood cells and develop into elongate gametocytes. Other merozoites infect tissue cells and develop into secondary exoerythrocytic schizonts which give rise to round gametocytes. Relapse in Leucocytozoon simondi infections is discussed in relation to megaloschizont-induced exoerythrocytic schizogony.     

Gametogenesis, fertilization and ookinete differentiation of Leucocytozoon smithi

Development of Leucocytozoon smithi during gametogenesis, fertilization, and ookinete differentiation was studied by light and electron microscopy. Gametogenesis occurred rapidly, within 1-2 min after gametocytes were ingested by black flies. Usually one axoneme, but not infrequently two, was observed in microgametes. The macrogamete nucleus was characteristically elongated and fragmented, with a convoluted nuclear envelope. Fertilization occurred within five min after ingestion of gametocytes by the vector. The entire axoneme and nucleus of the microgamete entered the cytoplasm of the macrogamete. Zygote differentiation resembled sporozoite formation in that a thickened inner membrane and subpellicular microtubules developed beneath the plasmalemma, followed by cytoplasmic protrusion or evagination to form the anterior end. Extension of the inner thickened membrane continued as the zygote elongated. Development of sausage-shaped ookinetes was completed within 6-8 h after ingestion of a blood meal by a black fly. Mature ookinetes possessed a single nucleus, double-layered pellicle, canopy, apical pore, polar ring complex, subpellicular microtubules, micronemes, crystalloids, abundant mitochondria, endoplasmic reticulum, and ribosomes. Comparison of development of L. smithi with species of Plasmodium and Haemoproteus revealed general similarities in both sexual and asexual development within the insect vector. A diagram summarizing life cycle events for L. smithi is included.     

Resistance of early midgut stages of natural Plasmodium falciparum parasites to high temperatures in experimentally infected Anopheles gambiae (Diptera: Culicidae)

We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparum development in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/microl blood (range: 16-1,536/microl). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 +/- 1.6 at 27 C, 9.85 +/- 2.32 at 30 C, and 3.89 +/- 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons.     

Fallisia parasites (Haemosporidia: Plasmodiidae) from the flying lizard, Draco maculatus (Agamidae) in Thailand

Plasmodium (Fallisia) siamense n. sp. was described from the flying lizard Draco maculatus (Agamidae) collected near Bangkok, Thailand. Parasitic in thrombocytes, it produces 18-64 merozoites in schizonts that are larger than the usually oval gametocytes. Both gametocyte sexes showed a prominent nucleus, elongate in macrogametocytes and triangular in microgametocytes. This is the first Fallisia parasite described from a host outside the Neotropical Region, although presence of the genus in Australasia was reported 40 years ago, without formal taxonomic designation.     

Studies on Hepatocystis sp. in rhesus monkeys from Yunnan, China

A survey for the prevalence of Hepatocystis sp. was done on 10 rhesus monkeys (M. mulatta) from Yunnan, China. Examinations were performed on blood smears to detect erythrocytic gametocytes of the parasite, and on livers to detect merocysts. In 6 out of 10 monkeys, Hepatocystis sp. infection was confirmed. The gametocytes took about 6 days to reach maturity. The fully grown macrogametocytes were about 8 microns in diameter. The cytoplasm was stained pale lilac and the nucleus consisted of dense chromatin dots lying in an unstained zone. The microgametocytes had cytoplasm dyed a biscuit color and large nuclei dyed pink. In the pathological section of livers, the merocyst measured about 3 mm in diameter and had a smooth outline without any projecting limb. The characteristics of this protozoan species were very similar to those of H. taiwanensis. However, more work should be done to clarify the relationship between this species and H. taiwanensis.     

The Fine Structure of the Macrogametocytes of Adelina tribolii Bhatia, 1937 (Eucoccidia, Telosporea) from the Fat Body of the Beetle Tribolium castaneum Hbst.

SYNOPSIS. Macrogametocytes of the coccidium Adelina tribolii Bhatia, 1937 are described from the time when they settle in the fat body of the host and form periparasitic vacuoles around them to the stage of microgametocyte occurrence and the beginning of syzygy formation.
The macrogametocyte is surrounded by a 2-layered pellicle 50 mμ thick. Its continuity is interrupted by one or several micropores 40 mμ across and 86 mμ deep.
The cytoplasm of the parasite contains numerous vesicles and lamellae of rough and smooth endoplasmic reticulum. Mitochondria of various sizes have short tubules. The macrogametocyte contains a variable number of dark bodies 1.4-2.4 μ in diameter. It also contains several vacuoles up to 1.2 μ which are covered with a 3-layered membrane and enclose a granular material.
In old macrogametocytes in syzygy multivesicular bodies develop which measure up to 2.4 by 1.6 μ. Several smaller vacuoles containing granular material are also a constituent of the electrondense basic substance of these corpuscles.
Paraglycogen granules 1.4 by 0.9 A occur in old macrogametocytes and are situated inside the vacuoles which are not bordered by a membrane. The numbers and size of these granules increase with the age of the parasite. The Golgi complex lies close to the nucleus.
The nucleus, 6-8.5 μ in diameter, is in the center of the macrogametocyte and contains a large eccentric nucleolus. The nuclear membrane is 2-layered and has many pores.     

Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida: Haemoproteidae) of sylviid birds, with a description of Haemoproteus parabelopolskyi sp. nov

Haemoproteus spp., with circumnuclear gametocytes and tentatively belonging to Haemoproteus belopolskyi, are widespread and prevalent in warblers belonging to the Sylviidae, with numerous mitochondrial cytochrome b (cyt b) lineages detected among them.We sampled the hemoproteids from 6 species of warblers adjacent to the Baltic Sea. Parasites were identified to species based on morphology of their gametocytes, and a segment of the parasite's cyt b gene was sequenced. Sixteen mitochondrial cyt b lineages of hemoproteids with circumnuclear gametocytes were recorded. Two clades of lineages (clade A in species of Acrocephalus and Hippolais and clade B in species of Sylvia) with sequence divergence between their lineages >5% are distinguished in the phylogenetic tree. Within the clades A and B, the genetic distance between the lineages is < or = 3.9 and < and = 2.8%, respectively. We compared the morphology of gametocytes of 3 lineages (hHIICT1, hMW1, and hSYAT2) in detail. The lineages hHIICTI and hMW1 (clade A) belong to the morphospecies H. belopolskyi. Parasites of the lineage hSYAT2 (clade B) are described as a new species Haemoproteus parabelopolskyi, which can be readily distinguished from H. belopolskyi by the significantly smaller nuclei of its macrogametocytes. Lineages closely related to H. belopolskyi and H. parabelopolskyi are identified. The sequence divergence between lineages of these 2 morphospecies ranges between 5.3 and 8.1%. It seems probable that avian Haemoproteus spp. with a genetic differentiation of > or =5% in mitochondrial cyt b gene might be morphologically differentiated at the stage of gametocytes. This study establishes the value of both PCR and morphology in identification of avian hemoproteids.