Ultrastructural Studies of Microgametogenesis and Macrogametogenesis of Eimeria truncata of the Lesser Snow Goose1

ABSTRACT. Microgamonts and macrogamonts of Eimeria truncata were observed in renal epithelial cells of collecting tubules and ducts and occasionally in macrophages of experimentally infected lesser snow geese (Anser c. caerulescens) beginning 8.5 days post inoculation. Intraparasitophorous vesicles in parasitophorous vacuoles of both types of gamonts appeared to originate in host cell cytoplasm and enter gamonts through micropores by budding of plasmalemma or by pinocytosis. Within the parasite's cytoplasm, the vesicles were broken down in Golgi-associated vacuoles. The surfaces of microgamonts were highly invaginated to facilitate extrusion of numerous microgametes. Formation and maturation of microgametes were similar to those of other eimerian species. Each microgamete had two flagella, a mitochondrion, and a peculiarly shaped electron-dense nucleus that was oval anteriorly in cross section and somewhat dumbbell-shaped posteriorly. A longitudinally arranged inner membrane complex lay between a portion of the mitochondrion and the plasmalemma. About five subpellicular microtubules extended the length of the microgamete body. Macrogametogony differed little from that described in other eimerian species. Type 1 wall-forming bodies (WFB) formed in Golgi complexes early in macrogametogony, and type 2 WFB formed in cisternae of endoplasmic reticulum in intermediate stages of macrogamont development.     

A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti‐PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission‐blocking vaccine target candidate.     

An electron microscopic study of Cryptosporidium. II. The stages of gametogenesis and sporogony in Cryptosporidium parvum

The ultrastructure of stages of gametogony and sporogony of C. parvum from the intestine of experimentally infected suckling rats was studied by transmission electron microscope. Unlike merogony, in which the whole cytoplasm of the mother meront is used up for the merozoite formation, during microgametogony the large residual mass of gamonts remains in contact with the feeder organelle even after microgamete outbudding. Unlike other coccidia, during the microgametogenesis in C. parvum, the nuclear substance of the daughter nuclei is not separated into osmiophilic (containing the condensed chromatin) and achromatinic parts. The gamete outbudding in C. parvum is accompanied by evagination of the pellicle of the mother gamont whose cytoplasm displays some slit-like canals that seem to sequester the daughter nuclei with some portion of the surrounding cytoplasm. The flagella-free microgametes of C. parvum resemble somatic cells, rather than male sexual cells of other coccidia. The study of thick-walled oocysts of C. parvum made it possible to suggest that the fragile wall of the oocyst proper may be easily destroyed in the course of processing of the material to look eventually as a ghost of electron lucent substance in the parasitophorous vacuole, whereas the structures revealed on the electronograms may presumably represent the outer and inner layers of the sporocyst. If so, the suture described elsewhere in the cryptosporidial oocysts, is to be considered as belonging to the sporocyst wall rather than to the oocyst wall, i.e. likely as in other investigated coccidia. However, the question on the mode of sporozoite excystment in the thin-walled oocysts of C. parvum still remains obscure.     

Further studies on the development of gamonts and oocysts of Eimeria magna in cultured cells

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.     

Ultrastructural observations on fertilization and sporulation in Goussia iroquoina (Molnar and Fernando, 1974) in experimentally infected fathead minnows (Pimephales promelas, Cyprinidae)

The ultrastructural features of fertilization and sporogony of Eimeria iroquoina are described from the intestinal epithelium of experimentally infected fathead minnows (Pimephales promelas). Intact microgametes were observed in the cytoplasm of macrogametes. Within immature macrogametes the microgamete was segregated from the cytoplasm of the former by the plasma membrane of each cell plus additional membranes. Within mature macrogametes, only the plasma membranes separated the gametes. Fertilization by fusion of the limiting membrane of both gametes occurred after the entire microgamete lay within the cytoplasm of the macrogamete. The cytoplasm of the zygote cleaved into sporoblasts within cisternae of endoplasmic reticulum. The sporocystic wall was composed of an outer electron-lucent layer and an inner, thicker layer with periodic striations at right angles to the surface of the sporocyst. The sporocysts were bivalved and joined by a continuous suture. The sporozoites were morphologically similar to sporozoites and merozoites of other Coccidia. Due to the structure of the sporocyst, Eimeria iroquoina Molnar and Fernando, 1974 is amended to Goussia iroquoina (Molnar and Fernando, 1974).     

Ultrastructural studies of the zygote and oocyst wall formation of Eimeria truncata of the lesser snow goose

Zygote development and oocyst wall formation of Eimeria truncata occurred in epithelial cells in renal tubules and ducts of experimentally infected lesser snow geese (Anser c. caerulescens). Post-fertilization stages were present throughout the kidneys beginning nine days post-inoculation. Initially, a single plasmalemma enclosed the zygote, and type 1 wall-forming bodies (WF1) became labyrinthine and moved toward the surface. There, WF1 degranulated and formed the outer layer of the oocyst wall between the plasmalemma and a newly formed second subpellicular membrane. Several WF2 fused and formed the inner layer of the oocyst wall between the third and fourth subpellicular membranes. Six subpellicular membranes were observed during wall formation. Other features of oocyst development were similar to those of other eimerian species.     

Gametocyte Maturation,Exflagellation and Fertilization in Parahaemoproteus (=Haemoproteus) velans (Coatney & Roudabush) (Haemosporidia: Haemoproteidae): an Ultrastructural Study*

SYNOPSIS. The structural changes in macro and microgametocytes of Parahaemoproteus velans following removal of infected blood from the avian host were studied in the light and electron microscope. Gametocytes of both sexes round up and soon escape from their host cells. Shortly thereafter they assume a dumbbell shape. The microgametocyte undergoes exflagellation forming 8 slender microgametes. During fertilization the entire microgamete appears to enter the female. The most striking ultrastructural change in the formation of the macrogamete is the condensation and enclosure by a membrane of abundant amophorus dense material seen in the cytoplasm of the immature gametocyte. Maturation of the microgametocyte begins prior to its escape from the host cell. Axonemes are present in the cytoplasm and nuclear reorganization occurs while the parasite is intracellular. Bundles of microtubules associated with condensed chromatin are found in the peripheral cytoplasm of maturing forms and apparently participate in the formation of small compact microgamete nuclei. Each of these filiform structures consists of a dense, centrally located nucleus and a single axoneme lying in flocculent cytoplasm. The nucleus and axoneme of the microgamete are seen free in the cytoplasm of a fertilized macrogamete.     

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.     

Ultrastructural Studies of the Zygote and Oocyst Wall Formation of Eimeria truncata of the Lesser Snow Goose1

ABSTRACT. Zygote development and oocyst wall formation of Eimeria truncata occurred in epithelial cells in renal tubules and ducts of experimentally infected lesser snow geese (Anser c. caerulescens). Post-fertilization stages were present throughout the kidneys beginning nine days post-inoculation. Initially, a single plasmalemma enclosed the zygote, and type 1 wall-forming bodies (WF1) became labyrinthine and moved toward the surface. There, WF1 degranulated and formed the outer layer of the oocyst wall between the plasmalemma and a newly formed second subpellicular membrane. Several WF2 fused and formed the inner layer, of the oocyst wall between the third and fourth subpellicular membranes. Six subpellicular membranes were observed during wall formation. Other features of oocyst development were similar to those of other eimerian species.     

Ultrastructure of gamonts and gametes and fertilization of Sarcocystis sp. from the roe deer (capreolus capreolus) in dogs

Gamonts of Sarcocystis sp. from the roe deer were examined in the intestine of dogs 10 h after inoculation. Early macrogamonts were limited by a three-membranous pellicle, and situated in a parasitophorous vacuole. Female sexual stages during fertilization, the macrogametes, were limited by five membranes, and microgametes were observed in the parasitophorous vacuole. The outer membranes of the microgamete and macrogamete fuse, and the nucleoplasm of the microgamte enters the cytoplasm of the macrogamete. No wall-forming bodies were observed in macrogamonts and macrogametes.     

The Fine Structure of Microgametogenesis in Haemoproteus columbae Kruse*

SYNOPSIS. The structural changes leading to the formation of motile microgametes from a single immobile intracellular gametocyte have been examiued in the electron microscope. After pigeon blood infected with Haemoproteus columbae was exposed to the air at room temperature for a few minutes axonemes appeared in the parasite's cytoplasm and the cytoplasm itself appeared less dense. The axonemes were connected with bundles of intranuclear microtubules that were perhaps spindle fibers. No conventional kinetosomes or centrioles have been observed. After the microgametocyte left the erythrocyte, it assumed the shape of a polarized slug or a dumb-bell. Half of the organism was surrounded by a single membrane and filled by part of the nucleus. The other half was surrounded by the remains of the multiple membranes of the gametocyte and contained pigment granules, mitochondria, axonemes and nuclear extensions. The axonemes and nuclear extensions were segregated at the periphery of the cell, exterior to the gametocyte's inner membrane, and were assembled in situ into microgametes. The mature microgamete appeared to peel off from the gametocyte, leaving a residual body.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

Phylogenetic analysis of the genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina)

A phylogenetic analysis of species of Hepatozoon Miller, 1908 was performed using 16 morphological, morphometric and developmental characters. An adeleorin parasite of gastropod molluscs, Klossia helicina Schneider, 1875 and four haemogregarines of other genera, Karyolysus lacertae (Danilewsky, 1886), Cyrilia lignieresi (Laveran, 1906), Desseria myoxocephali (Fantham, Porter & Richardson, 1942) and Haemogregarina balli Paterson & Desser, 1976 were used as the outgroup to 12 species of Hepatozoon. A single most parsimonious interpretation of the data was found, with the resulting cladogram having 28 transformations and a consistency index of 0.75. The proposed phylogeny revealed Hepatozoon as a paraphyletic group. One monophyletic lineage contained 10 of the 12 species of Hepatozoon, including all of those species that undergo gametogenesis and sporogony in the haemocoel of arthropods. Within this lineage, gamonts of those species found in vertebrate leucocytes instead of erythrocytes formed a clade basal to the remainder of the species. Species of Hepatozoon which undergo gametogenesis and sporogonic development in the gut epithelium of acarines, and which produce aflagellate microgametes, as well as the four outgroup taxa of haemogregarines, formed a monophyletic group and were the sister group to the remainder of the species of Hepatozoon. The biological and morphological diversity of these parasites suggests that species of the genus Hepatozoon, which are members of the paraphyletic haemogregarine complex, could be partitioned into at least two genera of adeleorin parasites.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

Ultrastructural features of the apical complex, pellicle, and membranes investing the gamonts of Haemogregarina magna (Apicomplexa: Adeleina)

Mature gamonts of Haemogregarina magna lie within a type of parasitophorous vacuole (Pv) apparently unique to the haemogregarines. The cytoplasm of infected erythrocytes was separated from the parasite by two Pv membranes. An additional membrane, coated on both sides with electron-dense material, closely invested the gamonts. The apical complex of the gamonts includes a conoid, two preconoidal rings, and an elaborate polar ring complex. The latter consisted of the polar ring and approximately 78 posteriorly directed, radially arranged, "tine-like" structures which fuse as they merge anteriorly into the polar ring. Freeze fracture replicas demonstrated that the pellicle of gamonts of H. magna was structurally similar to that of other apicomplexans. The closely apposed inner membranes of the pellicle formed plates which were arranged into strips along the long axis of the gamont. Calculations indicated that 13 such strips are found around the circumference of the gamonts with about six subpellicular microtubules associated with the inner surface of each strip. Gamonts of H. magna share many structural similarities with the kinetes, ookinetes, and sporokinetes of other apicomplexans. We propose that the conoid and polar ring complex are fundamental features of all apicomplexan "kinetes."     

Plasmalemma Invaginations in Cultured Callus of Stevia rebaudiana: Ultrastructure and Ultracytochemical Localization of Acid Phosphatase

The plasmalemma of cells within meristematic regions was observed to possess invaginations in cultured callus of Stevia rebaudiana under differentiation. The ultrastructure and acid phosphatase (AcPase) ultracytochemistry Of these invaginations were studied. The plasmalemma invaginations occurred in the cells at various stages of vacuolation. In cells with dense protoplasm, plasmalemma appeared undulated but occasionally spherical and variable in size with conspicuous invaginations that projected into the peripheral cytoplasm. In the partially vacuolated cells, plasmalemma invagination became voluminously enlarged with increased contents and structurally complexed. In vacuolated cells, the enlarged invaginations protruded into the central vacuole but were delimitted from the tonoplast by an intermembrane zone continuous with the peripheral cytoplasm. Complex accumulations of membranes consisting of vesicular and coiled membranous Structures might develop within the plasmalemma invaginations. AcPase localization demonstrated high enzymic activity in the plasmalemma and its associated invagination. It seemed likely that these invaginations were functionally analogous to the vacuoles and therefore constituted part of the lytic compartment in these cells.     

甜菊愈伤组织中的质膜内陷:超微结构和酸性磷酸酶细胞化学定位

对在分化条件下的甜菊 (Stevia rebaudiana)愈伤组织分生区域细胞的质膜内陷进行了超微结构和酸性磷酸酶细胞化学研究。结果表明 ,在不同液泡化状态的细胞中均有质膜内陷存在。在原生质浓密的细胞中 ,质膜呈起伏的波纹状 ,某些部位发生明显内陷 ,大小不等 ,多呈圆球状。在部分液泡化细胞中 ,质膜内陷体积增大 ,内含物增多且结构复杂。在液泡化细胞中 ,质膜内陷嵌入中央液泡 ,但彼此间以一膜间隙隔开。质膜内陷中的内含物以小泡和卷绕的膜结构形式存在。酸性磷酸酶活性定位结果显示 ,质膜及其内陷含高的酶活性。推测质膜内陷在功能上与液泡相似 ,构成了这些细胞水解空间的一部分。     

Gametogenesis and Sporogonic Development of Haemogregarina balli (Apicomplexa: Adeleina: Haemogregarinidae) in the Leech Placobdella ornata

ABSTRACT. The sexual stages and sporogonic development of Haemogregarina balli an apicomplexan blood parasite of snapping turtles, Chelydra serpentina were studied by electron microscopy for 30 days post feeding (PF). Gamonts were invested by an extracellular sheath which fused with intestinal microvilli. All stages of development were observed epicellularly within intestinal epithelial cells of the leech Placobdella ornata. Nuclear division in microgametogenesis was characterized by a trans-nuclear cytoplasmic channel containing the spindle fibers. Basal bodies associated with nuclear division were unpaired with an atypical (8 + 0) microtubular conformation. Four aflagellate microgametes were formed. During fertilization, a single microgamete was enclosed in a pocket of a microgamont. The pocket was lined by a dense layer and underlying ER. In sporogony, nuclei were invested by a trilaminar pellicle as they divided, forming four anlagen. Each anlage divided by longitudinal binary fission forming eight sporozoites in mature oocysts. Sporozoites penetrated the intestinal epithelium by 27 days PF.     

Developmental stages of Hepatozoon hemprichii sp. nov. infecting the skink Scincus hemprichii and the tick Hyalomma impeltatum from Saudi Arabia

The life cycle of Hepatozoon hemprichi n. sp. is described; the vertebrate host is Scincus hemprichii and it is vectored by Hyalomma impeltatum. Erythrocytic stages of 18 ± 1.8 × 4 ± 0.8 μm developed in the hemocoel of ticks to sporozoites within 16-18 days. Schizogony occurred in the liver parenchyma and the endothelial cells of blood capillaries in lung and spleen. Mature schizonts measuring 27 ± 3.11 × 20.13 ± 3.0 μm produced 28 merozoites (on average). The merozoites were 13 ± 1.21 × 1.21 ± 0.72 μm with nuclei 5 ± 0.65 × 2.1 ± 0.51 μm. Syzygy and differentiation of gamonts took place in tick's hemocoel up to the third day post-infection (PI). The microgamont (16 ± 0.31 × 18 ± 0.42 μm) produced 4, uniflagellated microgametes at 4-5 days PI. The microgamete measured 15.2 ± 0.31 μm while the flagellum was always at least 26 μm. The macrogamete was very large in size (31 ± 3.11 μm) with a central nucleus. After fertilization, (5-6 days PI) zygotes developed into oocysts (55 ± 3.41 × 52 ± 4.11 μm) in which repeated mitotic divisions with centripetal invaginations occurred; each contained 18 banana-shaped sporozoites, 13.61 ± 0.8 × 1.2 ± 0.31 μm in size. Experimental transmission was successfully carried out by oral administration or by intra-peritoneal inoculation of the infective stages (sporozoites) to uninfected skinks and led to the appearance of blood stages after 5 wk and 4 wk, respectively.     

Ultrastructural Features of the Apical Complex,Pellicle, and Membranes Investing the Gamonts of Haemogregarina magna (Apicomplexa: Adeleina)1

ABSTRACT. Mature gamonts of Haemogregarina magna lie within a type of parasitophorous vacuole (Pv) apparently unique to the haemogregarines. The cytoplasm of infected erythrocytes was separated from the parasite by two Pv membranes. An additional membrane, coated on both sides with electron-dense material, closely invested the gamonts. The apical complex of the gamonts includes a conoid, two preconoidal rings, and an elaborate polar ring complex. The latter consisted of the polar ring and approximately 78 posteriorly directed, radially arranged, “tine-like” structures which fuse as they merge anteriorly into the polar ring. Freeze fracture replicas demonstrated that the pellicle of gamonts of H. magna was structurally similar to that of other apicomplexans. The closely apposed inner membranes of the pellicle formed plates which were arranged into strips along the long axis of the gamont. Calculations indicated that 13 such strips are found around the circumference of the gamonts with about six subpellicular microtubules associated with the inner surface of each strip. Gamonts of H. magna share many structural similarities with the kinetes, ookinetes, and sporokinetes of other apicomplexans. We propose that the conoid and polar ring complex are fundamental features of all apicomplexan “kinetes.”