Fine Structure of the Endogenous Stages of Eimeria labbeana. 5. Schizonts and Merogony,with Special Reference to the Rhoptry-Microneme System*

SYNOPSIS The fine structure of the 3 generations of meronts, merogony, and merozoites of Eimeria labbeana Pinto from the ileal mucosa of experimentally infected pigeons, Columba livia Linnaeus, was described and compared to that of similar stages in other species of Eimeria. Sporozoite-trophozoite transition stages, trophozoites (5.8 × 4.2 μm), young meronts (10.1 × 8.4 μm), and mature meronts with free merozoites of the first generation, were observed at 20, 28, 36, and 48 hr post-infection, respectively. The 2nd and 3rd generation merogony were completed at 96 and 144 hr. Merogony was essentially of the ectomerogonous type without cytomere formation, as in most species. The average number of merozoites per meront in the 3 generations was 10 (5–15), 14 (8–19), and 7.5 (6–16); and the average size was 4.4 × 2.1 (4.1–5.9 × 1.8–2.2) μm, 4.2 × 1.8 (4.0–4.8 × 1.5–2.0) μm, and 5.4 × 1.8 (5.2–7.8 × 1.6–2.0) μm, respectively. Aggregation and subsequent degeneration of micronemes within membrane-bounded vesicles in the sporozoite-trophozoite stage, was observed as a possible mode of eliminating certain organelles present in the motile stages. Centrioles with (9 + 1) microtubular composition, and centrocones, were frequently seen in early meronts. Anlagen of micronemes, without any apparent association with the Golgi complex and the merozoite bud, were seen to develop in the cytoplasm of the meront. A single, median structure, probably representing the anlage of the rhoptry-microneme system was observed within the conoid of an early merozoite bud. Connections between the micronemes and the bulbous portion of the rhoptries, and a branched (interconnected ?) structure of the rhoptries observed in the present study, substantiate the present contention that the micronemes and rhoptries are functional forms of the same complex of organelles, the rhoptry-microneme system.     

Stages of Merogony in Multinucleate Merozoites of Eimeria magna Pérard, 1925*

SYNOPSIS. Stages of merogony of Eimeria magna were observed with the electron microscope in schizonts in ultrathin intestinal sections from white rabbits killed 4 days after inoculation. Some of the mature schizonts observed had uninucleate merozoites, whereas others had multinucleate ones. In each of the latter were rough endoplasmic reticulum, mitochondria, micronemes, refractile bodies, and occasionally, lipid droplets; some nuclei had a nucleolus. In the interior of some multinucleate merozoites, anlagen of daughter merozoites were observed. Each anlage was associated closely with a nucleus. In some of these nuclei, a cone-shaped pole of a spindle was directed toward the anlage. Each early anlage consisted of an inner membrane complex with a rhoptry anlage. A Golgi complex frequently was seen at the base of the anlage. One multinucleate merozoite, still attached to the residual body, had a merozoite anlage. In later stages of merogony, the anlagen were longer and each had a conoid. In one such merozoite, 2 merozoite anlagen were observed in close association with an eccentric intranuclear spindle, and 1 anlage had a Golgi adjunct. Another merozoite had an eccentric spindle and associated centrioles, but no visible anlagen. The finding of stages of merogony in multinucleate merozoites of E. magna indicates that these might represent a further schizogonic generation occurring in the original parasitophorous vacuole.     

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.     

Fine Structure of the Oocyst Wall of Eimeria nieschulzi

SYNOPSIS. Oocysts of Eimeria nieschulzi from the laboratory rat, Rattus, norvegicus , were studied by scanning and transmission electron microscopy. Oocysts had a rough outer wall with apparent random depressions. The oocyst wall is composed of 2 layers: an osmiophilic outer layer consisting of a rough external and smooth internal surface, and a relatively thick, electron-lucent inner layer. The outer layer is composed of a dense, coarsely granular matrix. The inner layer consists of homogeneous fine granular material interspersed with coarse osmiophilic granules and contains one closely applied membrane on the outermost surface. Several raised lenticular areas are seen on the coarse outer surface of the inner layer. These layers are 102 (75–128) and 176 (135–204) nm thick, respectively.
The sporocyst wall is thin, consisting of 3 to 4 unit membranes, and measures 27 (18–34) nm thick.     

Fine Structure of Zygotes and Oocysts of Eimeria nieschulzi*

SYNOPSIS. Mature macrogamonts were present in the small intestine of rats 5.5 to 7.5 days postinoculation with Eimeria nieschulzi oocysts; oocysts were present at 6 to 7.5 days. Types I and II wall-forming bodies in macrogamonts began to undergo ultrastructural changes within zygotes to form the outer and inner layers of the oocyst wall. Before and during oocyst wall formation a total of 5 membranes (M1–5) were formed at or near the surface of the zygote. The outer and inner oocyst wall layers formed between M2 and M3, and M4 and M5, respectively. The mature oocyst was loosely surrounded by M1 and M2, had an electron-dense outer layer, 100–275 nm thick, and an electron-lucent inner layer, 160–180 nm thick. It also contained an electron-lucent line consisting of M3 and M4 interposed between the outer and inner layers of the oocyst wall. The micropyle, measuring 935 × 47 nm, was located in the outer layer of the oocyst wall and consisted of 10–14 alternating layers of electron-dense and lucent material. The sporont of mature oocysts was covered by M5, immediately beneath which were M6 and M7. The sporont contained a nucleus and nucleolus, lipid and amylopectin bodies, mitochondria, ribosomes, as well as smooth and rough endoplasmic reticulum. Canaliculi, Golgi complexes, and types I and II wall-forming bodies were absent.     

The Fine Structure of the Centriolar Apparatus and Associated Structures in the Flagellates Deltotrichonympha and Koruga. I. Interphase

SYNOPSIS. An electronmicroscopic study was made of the centriolar apparatus in the rostrum of Deltotrichonympha operculata and Koruga bonita , 2 closely related hypermastigote flagellates from the Australian termite, Mastotermes darwiniensis. In interphase flagellates, the centriolar apparatus consists of 2 similar parts with a mutually perpendicular orientation. Each part contains a large, club-shaped centriolar body consisting of fibrillar and granular material, without recognizable internal symmetry or microtubules. The anterior centriolar body extends from the inner rostral wall, which is structurally related to the fibrous wall surrounding the posterior centriolar body. The 2 centriolar bodies are joined by connecting branches, which meet at 3 barren kinetosome-like structures located inside the rostrum. Thus, an interphase flagellate has 2 centriolar bodies oriented at a 90° angle to each other, like a pair of typical centrioles in an interphase metazoan cell.     

Fine Structure of the Cyst and Some Sporulation Stages of Ichthyosporidium (Microsporida)*

SYNOPSIS. Ichthyosporidium sp. Schwartz, 1963, apparently identical with the type species, I. giganteum (Thélohan, 1895) Swarczewsky, 1914, was studied with the electron microscope. Only late stages, a mature cyst containing sporulation stages and a cyst in the terminal (necrotic) stage were observed. The cyst, originating from host tissue, is a highly organized structure that is integrated with the surrounding connective tissue by means of numerous conspicuous processes. It is interpreted as essentially a manifestation of a defensive reaction of the host that is elicited by the parasite and then used to its advantage. Eventually the cyst dies and disintegrates. This type of cyst, peculiar among those associated with microsporidia, may be regarded as a distinctive character of the poorly defined genus Ichthyosporidium. Other observations let to an hypothesis which reconciles several different views regarding the identity of the Golgi complex. According to this new interpretation, these different views concern different aspects af the total complex. When all such views are integrated, a “classical Golgi” can be recognized in the presporoblastic stages and the “primitive Golgi” concept disappears. This “classical Golgi” then becomes highly modified during spore morphogenesis, giving rise to many of the internal organelles that are peculiar to the spore.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. I. Fine Structure and Cytochemistry*†

SYNOPSIS. An extracellular surface coat was observed at the fine-structural level on the outer lamina of the pellicular and flagellar membranes of intact Trypanosoma musculi bloodstream forms. The surface coat had a mean width of 9.2 nm, and was composed of a somewhat electron dense, uneven, fibrillar-like matrix. Brief trypsin treatment of living blood forms completely removed the cell surface coat. Several cytochemical methods applicable to electron microscopy were used to detect the presence and distribution of carbohydrates in the trypanosome's surface coat and pellicular membrane. The polycationic dye compounds employed were: ruthenium red, ruthenium violet, Alcian blue chloride, and lanthanum nitrate. Electron-dense stain reaction products, indicative of polysaccharides, were evident in the surface coat of cells treated with these dyes, which also agglutinated both living and glutaraldehyde fixed cells. Like the surface coat, the pellicular membrane of trypsinized cells gave strong positive staining reactions with the several dyes, indicating the presence of membrane bounded carbohydrates, and living and glutaraldehyde-fixed trypsinized cells were agglutinated with the polycationic stains. Bloodstream forms were treated with the enzymes, α-amylase, dextranase, and neuraminidase. No obvious morphologic difference, however, was apparent between the surface coat of untreated cells and those subjected to treatment with any of the various glycoside hydrolase enzymes. Further, these enzymes had no apparent gross effect on the staining affinity of the surface coat for the several polycationic dyes. Cationized ferritin was used to visualize the negative cell surface charge of T. musculi bloodstream forms. Large quantities of cationized ferritin were bound in the surface coat matrix. Glycoside hydrolase enzyme treatments had no apparent effect on the amount of ferritin bound in the surface coat. Cationized ferritin was bound also to the outer lamina of the pellicular membrane in trypsinized cells, which had quantitatively less ferritin bound per surface unit area than bloodstream forms untreated by the enzyme. Living and glutaraldehyde-fixed cells were agglutinated with cationized ferritin. The results obtained in the various experiments indicated that polyanionic polysaccharides were constituent terminal ligands of the surface coat matrix and pellicular membrane in T. musculi bloodstream forms.     

Eimeria crotalviridis sp. n. from Prairie Rattlesnakes, Crotalus viridis viridis, in New Mexico with Data on Excystation of Sporozoites and Ultrastructure of the Oocyst Wall

SYNOPSIS Oocysts of Eimeria crotalviridis sp. n. are described from prairie rattlesnakes, Crotalus viridis viridis in New Mexico on the basis of light and electron microscopy and in vitro excystation of sporozoites. Sporulated oocysts of E. crotalviridis are elliptical, 26.4 × 22.3 (23–29 × 20–24) μm with ovoid sporocysts 11.7 × 8.1 (11–13 × 7–9) μm. A micropyle, micropyle cap and polar bodies are absent, but oocyst and sporocyst residua and Stieda and substieda bodies are present. Excysted sporozoites are 12.4 × 2.8 (11–13 × 2–3) μm and have 1 large posterior refractile body and a nucleus with a prominent nucleolus. Ultrastructurally, the oocyst wall has 2 layers, a thick, electron-dense, highly sculptured outer layer composed of a fine granular matrix and a thin, granular, osmiophilic inner layer, separated from the outer layer by at least one unit membrane. These layers are 441 (353–510) and 21.6 (19–29) nm thick, respectively. Within 15 min after exposure to a trypsin-sodium taurocholate fluid, sporozoites of E. crotalviridis excysted from 5-month-old sporocysts.     

The Fine Structure of the Centriolar Apparatus and Associated Structures in the Flagellates Deltotrichonympha and Koruga. II. Division

SYNOPSIS. At division of Deltotrichonympha operculata and Koruga bonita from the Australian termite, Mastotermes darwiniensis , the 2 centriolar bodies separate, each becoming a mitotic center. Spindle microtubules develop from the lower end of each centriolar body and radiate towards the elongating nucleus. A new rostrum is formed in association with each centriolar body. Thus, centriolar bodies which lack the structure of typical centrioles can nevertheless function as division centers during mitosis.     

The Fine Structure of the Phoront of the Apostomatous Ciliate,Hyalophysa chattoni*

The phoront of the apostomatous ciliate, Hyalophysa chattoni, is an encysted stage that is carried on the exoskeleton of its crustacean host until the ecdysis of the host. At molting the phoront rapidly metamorphoses to the feeding stage, excysts, and immediately begins to feed on exuvial fluid trapped in the cast-off exoskeleton. The fine structure of the resting phoront resembles that of the preceding migratory stage, the tomite. A prominent ventral tuft of cilia, the ogival field, has vanished, and the trichocysts that paralleled the kinetics have disappeared. The dense inclusion bodies that were concentrated around the mouth and falciform fields have dispersed and greatly decreased in number. The cytoplasm and its membranous organelles do not appear visibly condensed or altered from the preceding stage in the life cycle. The phoront is merely quiescent instead of dormant. Unlike the few ciliate cysts previously examined by electron microscopy, the phoront's cyst is not divisible into separable layers. It resembles the loricae of certain suctoria in being formed principally of a fibrous substance, the outer surface of which has a paracrystalline pattern. The peduncle attaching the cyst to the crab's gill is a continuation of the cyst wall although its structure is somewhat modified. The most conspicuous innovation in the phoront's fine structure is the massive tracts of microtubules that run longitudinally through the macronucleus. The microtubules are in intimate contact with Feulgen-positive chromatin masses which are crowded toward the periphery of the macronucleus.     

The Fine Structure of the Colonial Kinetoplastid Flagellate Cephalothamnium cyclopum Stein

Cell structure, cell adhesion, and stalk formation have been examined by electron microscopy in the colonial flagellate, Cephalothamnium cyclopum. Each cell is obconical or spindle-shaped, pointed posteriorly and truncated anteriorly. The cell membrane is underlain by epiplasm 0.1 μm thick in the posterior region, but bands of microtubules support the anterior region which is differentiated into a flagellar pocket, oral apparatus and contractile vacuole. Each of 2 flagella, joined a short way above their bases by an interflagellar connective, has a paraxial rod and mastigonemes. One flagellum is free and is important in food gathering while the other is recurrent and lies in a shallow groove on the ventral cell surface but projects posteriorly into the stalk. The basal bodies of these flagella are bipartite structures connected by a pair of striated rootlets with accessory microtubular fibers. The oral apparatus consists of a funnel-shaped buccal cavity and cytostome. It is supported by helical and longitudinal microtubules and also has nearby striated and microtubular fibers. Possible roles of associated oral vesicles in relation to ingestion are discussed. A reticulate mitochondrion houses a massive kinetoplast which has a fibrillar substructure resembling that of dinoflagellate chromosomes. Adjacent flagellates adhere by laminate extensions of their posterior regions and attach by their recurrent flagella to a communally secreted stalk composed of finely fibrillar material. This study indicates that Cephalothamnium belongs in the order Kinetoplastida, and has many features in common with members of the family Bodonidae.     

The Fine Structure of the Cytostome of the Apostomatous Ciliate Hyalophysa chattoni*

SYNOPSIS. The fine structure of the organelles concerned with the ingestion of exuvial fluid by the trophont of the apostome ciliate, Hyalophysa chattoni, has been examined. One of the taxonomic characteristics of the order Apostomatida is that cytostomes of ciliates within the taxon are reduced and evolving toward astomy. When examined by electron microscopy the cytostome of H. chattoni appears as a small region of active pinocytosis which is continuous with a very large cortical area, the extended cytostome. The fine structure of the extended cytostome resembles that of the cytostomes of ciliates from other orders in that it is covered by a single membrane underlain with microtubular ribs. Beneath the extended cytostome are accumulations of peculiar organelles that may represent stored membrane for recycling during food vacuole formation. Associated with the site of pinocytosis is a complex fiber that may be contractile.     

On the Fine Structure of the Nucleus in Trypanosoma cruzi in Tissue Culture Forms. Spindle Fibers in the Dividing Nucleus*

The fine structure of the dividing nucleus in the intracellular amastigote forms of Trypanosoma cruzi from tissue cultures has been described. In the first phase of division the nucleus shows a homogenous structure owing to the dispersion of its chromatin and nucleolar material. Microtubules similar to those of a mitotic spindle in metozoan cells then appear, running from one pole to the other. They disappear when the division of the nucleus is complete and the chromatin and the nucleolar material reorganize into their former positions.     

Fine Structure of an Unidentified Protozoon in the Epithelium of Rainbow Trout Exposed to Water with Myxosoma cerebralis*

SYNOPSIS. An intracellular protozoon was discovered in the epithelium of young rainbow trout (Salmo gairdneri) exposed for as short a time as 1 hr to water known to contain infective stages of Myxosoma cerebralis. Light- and electron-microscopic examination of this tissue revealed what appeared to be a proliferative stage (presumptive schizont) of a sporozoon; other possible stages in the life cycle were also observed. The relationship of this unidentified protozoon to M. cerebralis remains unresolved.     

On the Fine Structure of Trypanosoma cruzi in Tissue Cultures of Pigment Epithelium from the Chick Embryo. Uptake of Melanin Granules by the Parasite*

Trypanosoma cruzi has been cultured in pigment epithelial cells of the iris from the chick embryo. Melanin granules, identical with those of the host cells were found in the intracellular, amastigote (leishmania) forms. In many of the intracellular forms cytostome-like structures were seen, often in intimate contact with the pigment granules of the host cells, which suggests the uptake of melanin granules through the cytostomes by the process of intracellular phagotrophy.     

Fine Structure of the Oocyst Walls of Isospora serini and Isospora canaria and Excystation of Isospora serini From the Canary, Serinus canarius L.

SYNOPSIS. Oocysts of Isospora serini and Isospora canaria , from the canary Serinus canarius , were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied in the electron microscope. The oocyst wall of each species had an electronlucent inner layer, a more osmiophilic middle layer and an outer layer of electron-lucent ( I. serini ) or electron-dense material interspersed with some electron-lucent material ( I. canaria ). A few, relatively large lipid-like bodies were present in the outer or middle layer of the oocyst wall of I. canaria. As many as 9 membranes were present in the oocyst wall of I. canaria and 3 in that of I. serini. When exposed to a trypsin-sodium taurocholate fluid, sporozoites of I. serini excysted from 5-month-old sporocysts in vitro , but not from sporocysts stored for more than 6 months. No excystation occurred in 15-month-old I. canaria sporocysts. Similarities and differences in excystation between I. serini and other Isospora, Eimeria , and Sarcocystis species are discussed.     

Study of Dientamoeba fragilis Jepps & Dobell I. Electronmicroscopic Observations of the Binucleate Stages II. Taxonomic Position and Revision of the Genus*

Light- and electronmicroscopic observations on Dientamoeba fragilis strain A (Bi) 1 dealing primarily with the binucleate (arrested telophase) stages, predominant in all populations, revealed the microtubular nature of the extranuclear spindle which extends between the 2 polar complexes each adjacent to one of the nuclei. The spindle microtubules originate in paired, nonperiodic structures apparently homologous to the “atractophores” described from hypermastigotes. To the external surface of the atractophores are applied periodic elements, which extend laterally as the parabasal filaments. Extensive Golgi complexes overlie the filaments, these structures corresponding to the components of the parabasal apparatus known from trichomonads and hypermastigotes. The 2-layered structures, consisting of the atractophores and periodic layers, together with the proximal parts of the Golgi complex and the spindle microtubules constitute the polar complex. No kinetosome- or centriole-like organelles have been found in the polar complexes or elsewhere in the organism. The extranuclear spindle is composed of 2 microtubule bundles, each with ～30-40 microtubules. One of the bundles always appears at some distance from the nucleus; the other is juxtanuclear and is seen often to course within a groove of the nuclear envelope. A 3rd bundle of ～35-45 microtubules is seen on occasion to arise from the atractophores and to pass toward the nucleus at a wide angle to the other parts of the spindle. In some instances these microtubules traverse the nucleus within channels delimited by the nuclear envelope. The double-layered nuclear envelope contains numerous pores. Two morphologic types of rounded inclusions, one microbody-like, and the other with a more electron-translucent matrix, as well as digestive vacuoles containing rice starch, bacteria, and/or myelin configurations are distributed in the cytoplasm, which abounds also in glycogen granules. The fine structure of Dientamoeba is compared with those of trichomonads and of Entamoeba spp. The taxonomic position of Dientamoeba is discussed and emended; in view of its affinities, this genus is placed among trichomonads in the family Dientamoebidae Grassé, emend.     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.     

Fine Structure of the Feeding Stage of A Sorogenic Ciliate, Sorogena Stoianovitchae Gen. N., Sp. N.

SYNOPSIS. A new species of kinetophragminophoran ciliate, collected from dried vegetation and capable of forming an aerial sorocarp, is described and named Sorogena stoianovitchae gen. n., sp. n. This ciliate is a voracious predator that feeds on species of Colpoda , and, when the latter is depleted in numbers, aggregates to forms sorogens. Each sorogen rises into the air from the surface of the water, forming a secreted stalk with a sorus of cysts at its apex. the feeding stage of the ciliate resembles an Enchelys in that it has an apical, slit-like mouth surrounded by a lip, a somewhat dorso-ventrally flattened body, and meridional kineties. Its length ranges from 40–75 μm and width from 23–55 μm. It has a typical rhabdos type of cytopharynx, but no specialized oral ciliature. the somatic kineties are formed of rows of paired kinetosomes with associated microfibrils, the arrangement of which differs a little from that of other ciliates of this subclass. Sorogena has tentatively been placed in the order Haptorida although it lacks toxicysts, recognizable mucocysts, and clavate cilia. Its unique life cycle and some of the details of its fine structure indicate differences between Sorogena and other haptorids so profound that a new family, SOROGENIDAE, is created for it. the type species (PNG76-73) was collected on dry figs at the Wau Ecology Institute, Papua New Guinea.