Chronology of the ultrastructural modifications during the growth of Gregarina blaberae]

In an ultrastructural study the development of the sporozoite as well as the growth and development of the trophozoite of Gregarina blaberae were followed in the course of experimental infections of larvae of the cokroach Blaberus craniifer. The spectacular growth involved the transformation within 18 days of the sporozoite, measuring 15 X 1 micronm, to a cephaline--trophozoite affixed to the intestinal epithelium--of 250 micronm length and 65 micronm diameter. The sporozoite's ultrastructure is not different from that of sporozoites of other Sporozoa studies to date--the conoid and dense bodies are present. The pellicle consists of 3 membranes, but there are some interruptions in the internal membrane complex. The first dictyosomes are formed from the nuclear envelope. The migration of the nucleus and of the dense bodies, followed by the regression of all the structures characteristic of the sporozoites, and the establishment of a cortical zone that comes to cover the epimerite, take place within 48 h after infection and mark the transformation of the sporozoite into the trophozoite. Development of the cephaline involves the formation of the epicytic folds, which occurs at the base of the deutomerite, starting on the 3rd day of development. A regular system of longitudinal or epicytic folds is formed over the entire surface of the gregarine. On the 4th and 5th days of development, a vacuolar system and a chondriome become differentiated in the epimerite, while a fibrillar septum separates the protomerite from the deutomerite. The next stage, starting on the 6th day, is characterized by distribution of polysaccharide reserves between these 2 segments. The model studied allows us to determine the role of the epimerite in the parasite's nutrition, as well as the development of the chondriome and of the cortical membranes in the course of the vegetative growth phase of the cephaline gregarine.     

L'Organisation Ultrastructurale Corticale de la Gregarine Lecudina pellucida; ses Rapports avec l'Alimentation et la Locomotion

SYNOPSIS. The structure of the cortical region (epicyte and ectoplasm) of the gregarine Lecudina pellucida , an intestinal parasite of the polychete worm Perinereis cultrifera was studied by electron microscopy.
The epicitary folds have 3 unit type membranes. Between the 1st and 2nd is a layer probably composed of fine longitudinal fibrils which has an arch-like or gutter-like structure at the crest of the folds. Inside these folds is cytoplasm without any noticeable differentiation or inclusion except for a granular (or finely fibrillar) layer under the limiting inner membrane and close to it.
The ectoplasmic zone of the entocyte is separated from the epicitary region by a lengthwise discontinuous cylindrical opaque layer, inwardly tangential to the folds. The ectoplasm lacks paraglycogen granules but has various organelles: apparently pinocytic vesicles against the wall between the folds, vesicles with myelinic membranes, opaque granules, a few mitochondria with blistered internal vesicles, and a few circular tubular fibers.
The superficial zone of the gregarine is supposed to contribute to nutrition, thru the extensive surface furnished by its folds and thru the pinocytic vesicles; but this alimentary intake is incomplete compared with that of the previously studied anterior region.
Insufficient mucus is discharged to account for locomotion. There are some circular ectoplasmic fibers, but locomotory myonemes are completely absent. However, there are deformations of the folds and corresponding waves that could account for locomotion by creeping or swimming. These movements of the folds might be due to the action of the contractile proteins and correspond with some of the layers seen in the wall.     

Scanning Electron Microscopy of Gregarines (Protozoa, Sporozoa) and Its Contribution to the Theory of Gregarine Movement

SYNOPSIS. Scanning electron microscopy was used to reveal detailed surface structure of 4 septate ( Gregarina cuneata, G. steini, G. rhyparobiae, Pileocephalus blaberae ) and one aseptate species ( Nematocystis elmassiani ) of eugregarines. The epicyte of all these gregarines is differentiated into a system of regular longitudinal folds. In the septate species these folds undulate so that these organisms glide along. The undulatory pattern is absent from Nematocystis , which does not glide. The theories and the mechanism of gregarine gliding are discussed.     

Electron microscope investigation of the evolutionary stages of the trophozoite of Didymophyes gigantea (Sporozoa, Gregarinida). III. The fine structure of the epicyte with emphasis on the contractile elements (author's transl)

The fine structure of the epicyte of D. gigantea was investigated. The motility of the gregarine and the contractile elements are described. Four essential types of movements can be observed in this gregarine: (1) rolling up and pendular movements, (2) locomotion by gliding forward, (3) cytoplasmic streaming (Fig. 1), (4) peristaltic contractions (Fig. 2) which seem to be accompanied by the contraction of annular myonemes (Fig. 2). The epicyte is formed by the folding of the parasitic cell wall which is made from three membranes (Figs. 3 and 4). At the top of each fold one can see apical struts between the outer and middle membrane and apical filaments under the inner membrane (Fig. 3). In addition, the epicytic folds are covered by a cell coat which is made from tubular structures (Fig. 5). At the base of the epicytic folds can be observed the basal lamina (Fig. 3) composed of very fine fibrillar material with an average thickness of 2.5 nm (Fig. 6). These fibrils are oriented in the longitudinal axis of the gregarine. Beneath the epicytic fold in the ectoplasm are found the annular myonemes with a width of up to 0.5 micrometers (Fig. 7). They are composed of many fine fibrils with an average thickness of 5 nm. In young trophozoites, the myonemes also contain microtubuli (Fig. 8). Between the epicytic folds, the cell wall is interrupted by three different types of vesicles: the vesicles with an electrondense content (Fig. 9), the three-membranous vesicles (Fig. 10), and the hose-shaped vesicles (Fig. 11). Glycerol-extraction of the parasites was performed in order to define the contractile structures. After extraction the annular myonemes are difficult to recognize (Fig. 13). When ATP is added, the gregarine does not contract but the myonemes reappear after 3 to 4 min (Fig. 14). Differences can also be observed in the myoneme structure using electron microscopy: After extraction, the myonemes are composed of a very limp fibrillar network (Fig. 15) which becomes very dense after the action of ATP (Fig. 16). Glycerol extraction does not disturb either the apical struts and apical filaments or the fibrils of the basal lamina (Figs. 15--17). In addition, cytoplasmic fibrillar structures appear after glycerol extraction (Figs. 15 and 16). The experimental and electron microscope results indicate that the motility of the gregarine depends upon four different systems: (1) the ectoplasmic annular myonemes, (2) the apical structures in the undulating epicytic folds, (3) the cytoplasmic fibrils, and (4) the basal lamina.     

Chronologie des Modifications Ultrastructurales au Cours de la Croissance de Gregarina blaberae*

SYNOPSIS. In an ultrastructural study the development of the sporozoite as well as the growth and development of the trophozoite of Gregarina blaberae were followed in the course of experimental infections of larvae of the cockroach Blaberus craniifer. The spectacular growth involved the transformation within 18 days of the sporozoite, measuring 15 × 1 μm, to a cephaline—trophozoite affixed to the intestinal epithelium—of 250 μm length and 65 μm diameter. The sporozoite's ultrastructure is not different from that of sporozoites of other Sporozoa studied to date—the conoid and dense bodies are present. The pellicle consists of 3 membranes, but there are some interruptions in the internal membrane complex. The first dictyosomes are formed from the nuclear envelope. The migration of the nucleus and of the dense bodies, followed by the regression of all the structures characteristic of the sporozoites, and the establishment of a cortical zone that comes to cover the epimerite, take place within 48 hr after infection and mark the transformation of the sporozoite into the trophozoite. Development of the cephaline involves the formation of the epicytic folds, which occurs at the base of the deutomerite, starting on the 3rd day of development. A regular system of longitudinal or epicytic folds is formed over the entire surface of the gregarine. On the 4th and 5th days of development, a vacuolar system and a chondriome become differentiated in the epimerite, while a fibrillar septum separates the protomerite from the deutomerite. The next stage, starting on the 6th day, is characterized by distribution of polysaccharide reserves between these 2 segments. The model studied allows us to determine the role of the epimerite in the parasite's nutrition, as well as the development of the chondriome and of the cortical membranes in the course of the vegetative growth phase of the cephaline gregarine.     

Morphology and phylogenetic position of two novel marine gregarines (Apicomplexa,Eugregarinorida) from the intestines of North‐eastern Pacific ascidians

Eugregarine apicomplexans parasitize marine, freshwater and terrestrial invertebrates, and have lifecycles involving trophozoites (feeding stages) with complex morphologies and behaviour. The genus Lankesteria refers to marine aseptate eugregarines that parasitize ascidians. We described the surface ultrastructure of two new gregarine species, L. chelyosomae sp. n. and L. cystodytae sp. n. that inhabit the intestines of Chelyosoma columbianum and Cystodytes lobatus, respectively, collected from the North‐eastern Pacific Ocean. Apart from inhabiting different hosts and major differences in the cell size of L. chelyosomae sp. n. (mean length 182 µm) and L. cystodytae sp. n. (mean length 70 µm), the morphology of both gregarine species was quite similar. The trophozoites ranged from elliptoid to obdeltoid in shape and were brownish in colour. The nucleus was situated at the anterior end of the cell just behind a pointed mucron. A dense array of epicytic knobs was present over the entire surface of trophozoites in both species, and longitudinal epicytical folds were only weakly developed. We also sequenced the small subunit rDNA from the gregarines collected from both hosts, which supported the establishment of two new Lankesteria species. Phylogenetic analyses of the new DNA sequences and those derived from other alveolates, demonstrated that both new species clustered in a strongly supported clade consisting of other Lankesteria species, Lecudina species, and some environmental sequences. These morphological and molecular phylogenetic data suggested that improved knowledge of gregarine diversity could lead to the recognition of more than one distinct clade (genus) of gregarines within ascidian hosts.     

Scanning electron microscopic study of the gut mucosa of the rainbow trout Salmo gairdneri Richardson.

The topological characteristics of the entire gut surface of the rainbow trout were investigated utilizing the scanning electron microscope. The mucosa exhibited longitudinal ridges in the oesophagus and stomach, villi in the intestine, fine longitudinal ridges in the caeca and annular folds in the rectum, arranged as a stack of caudally-directed funnels starting from the intestino-rectal valve to the vent. Detailed study revealed taste pores in the anterior oesophagus and the sculpting of the luminal plasmalemma of the surface cells into micro-ridges with complicated patterns. The surfaces of the posterior oesophagus and stomach were demarcated into polygons by rows of stubby microvilli—each polygon representing the luminal surface of an epithelial cell. Each rectal fold consisted of a smooth, caudally-directed apex and a base which was supported by perpendicular buttress-like secondary folds. The functional significance of these features which emphasize the vast difference in the physical length of the gut and the effective surface area is discussed.     

Organization of the musculature of schistosome cercariae

Phalloidin-fluorescein isothiocyanate staining of filamentous actin was used to identify muscle systems within the cercariae of Schistosoma mansoni. Examination of labeled cercariae by confocal scanning laser microscopy revealed distinct organizational levels of myofiber arrangements within the body wall, anterior cone, acetabulum, and esophagus. The body wall throughout showed a typical latticelike arrangement of outer circular and inner longitudinal myofibers, with an additional innermost layer of diagonal fibers in the anterior portion of the body. Circular and longitudinal fibers were also evident in the anterior organ and esophagus and, to some extent, the ventral acetabulum. Most striking was the striation of the cercarial tail musculature.     

Ultrastructure de la Grégarine Callynthrochlamys phronimae Frenzel; Étude Comparée de son Noyau avec Celui de Thalicola salpae (Frenzel) (Eugregarina)

SYNOPSIS. The structure of the gregarine Callynthrochlamys phronimae has been studied with the electron microscope. The cuticular complex is not different from those previously described in other species of gregarines. It is composed of 2 layers of different thickness delimited by 3 unit membranes and constitutes series of oblique folds at the surface of the deutomerite. Longitudinal rods of dense material surrounded by a slight pellicle are seen under the cuticle. Pinocytic vacuoles are present under the surface of the gregarine. Cytoplasmic organelles include mitochondria, Golgi complexes, endoplasmic reticulum, vacuoles and dense bodies from different sizes. There is a connection between the different features of the cytoplasm in the protomerite and deutomerite and the corresponding cuticular organization.
A characteristic feature of the species is the peculiar differentiation of the nuclear membrane. The nucleus is surrounded by a typical double membrane of which the inner one has a dense fibrillar layer apposed to it. In mature trophozoites, tubular expansions without inner layer arise from the double membrane; the same type of nuclear membrane occurs in another species, Thalicola salpae.     

The Ultra‐Structural Similarities between Cryptosporidium parvum and the Gregarines

Using a transmission electron microscopy‐based approach, this study details the striking similarities between Cryptosporidium parvum and the gregarines during in vitro axenic development at high ultra‐structural resolution. C. parvum zoites displayed three unusual regions within uninucleated parasites: epimerite‐like, protomerite‐like, and the cell body; these regions exhibited a high degree of morphological similarity to gregarine‐like trophozoites. The presence of a mucron‐like bulging structure at the side of the free ovoid gregarine‐like zoites was observed after 2 h of cultivation. An irregular pattern of epicytic‐like folds were found to cover the surface of the parasites 24 h postcultivation. Some extracellular stages were paired in laterocaudal or side‐side syzygy, with the presence of a fusion zone between some of these zoites. The present findings are in agreement with phylogenetic studies that have proposed a sister relationship with gregarines. Cryptosporidium appears to exhibit tremendous variety in cell structure depending on the surrounding environment, thereby mimicking the “primitive” gregarines in terms of the co‐evolution strategy between the parasites and their environments. Given this degree of similarity, different aspects of the evolutionary biology of Cryptosporidium need to be examined, considering the knowledge gained from the study of gregarines.     

Description of Trichotokara nothriae n. gen. et sp. (Apicomplexa, Lecudinidae) - An intestinal gregarine of Nothria conchylega (Polychaeta, Onuphidae)

The trophozoites of a novel gregarine apicomplexan, Trichotokara nothriae n. gen. et sp., were isolated and characterized from the intestines of the onuphid tubeworm Nothria conchylega (Polychaeta), collected at 20 m depth from the North-eastern Pacific Coast. The trophozoites were 50-155 μm long with a mid-cell indentation that formed two prominent bulges (anterior bulge, 14-48 μm wide; posterior bulge, 15-55 μm wide). Scanning electron microscopy (SEM) demonstrated that approximately 400 densely packed, longitudinal epicytic folds (5 folds/μm) inscribe the surface of the trophozoites, and a prominently elongated mucron (14-60 μm long and 6-12 μm wide) was covered with hair-like projections (mean length, 1.97 μm; mean width, 0.2 μm at the base). Although a septum occurred at the junction between the cell proper and the mucron in most trophozoites, light microscopy (LM) demonstrated that the cell proper extended into the core of the mucron as a thin prolongation. A spherical nucleus (8-20 μm) was situated in the middle of the trophozoites, and gamonts underwent caudal syzygy. The small subunit (SSU) rDNA sequence and molecular phylogenetic position of T. nothriae was also characterized. The sequence from this species was the most divergent of all SSU rDNA sequences currently known from gregarines and formed a weakly supported clade with Lecudina polymorpha, which also possesses densely packed epicyctic folds (3-5 folds/μm) and a prominently elongated mucron.     

Characterization of a coelomic gregarine parasite from Thitarodes pui (Lepidoptera: Hepialidae) in the Tibetan Plateau

Thitarodes pui, one of the host species of entomopathogenic fungus Ophiocordyceps sinensis, has great economic importance in the Tibetan Plateau. We report here, for the first time, a gregarine parasite found in the coelom of 7th instar and adults of T. pui. Gregarine gamonts (ovoid, ～15×8μm) underwent syzygy to produce reproductive gametocysts in T. pui larval hemolymph. All infected T. pui carried 2-17 mature gametocysts filled with numerous oocysts (lemon-shaped, 17.17±0.73×6.49±0.4μm). Transmission electron microscopy showed that these oocysts contained vacuoles of various sizes and amylopectin granules in the cytoplasm; scanning electron microscopy revealed a number of small bumps all over the surface of these oocysts. Small subunit ribosomal DNA sequence analysis showed a close relationship between the gregarine and the species of Ascogregarina (Eugregarinorida: Lecudinidae). Internal transcribed spacers and 5.8S ribosomal DNA from this gregarine exhibited 76% highest sequence identity with that from Ascogregarina culicis Ross.     

Oviducal anatomy and sperm storage structures in lizards

Gross and histological examination of lizard oviducts was made in 11 species of the family Iguanidae, and in one species of each of the families Gekkonidae and Eublepharidae. Lizard oviducts are bound dorsally by a mesentery which is continuous with the peritoneum, and ventrally by a smooth muscle band which extends from the posterior segment of the vagina to the base of the infundibular ostium. The musculature of the vagina consists of an inner circular smooth muscle layer which is thickened posteriorly, and an outer longitudinal layer which is arranged into longitudinal folds at about the utero-vaginal transition. In iguanid lizards the vaginal mucosa is arranged into longitudinal folds that extend the entire length of the vagina. Posteriorly, the folds are high and reduced in number. Anteriorly, they decrease gradually in height and become more numerous. In Phyllodactylus homolepidurus fold height and number remain essentially constant through the vagina. Seminal receptacles in the iguanids occur principally in the anterior segment of the vagina. Receptacles in P. homolepidurus (Gekkonidae) and Coleonyx variegatus (Eublepharidae) appear to be confined to the tube between the uterus and the infundibulum. Most receptacles are located adjacent to the oviducal mesentery and to the smooth muscle band.     

Immunohistochemical analysis of connexin26 and 43 expression in the mouse alimentary tract

The spatial pattern of connexins26 (Cx26) and 43 (Cx43) expressions were investigated in the mouse digestive tract by immunocytochemistry. High levels of connexin43 in the epithelium of the oesophagus, non-glandular part of the stomach, and the circular layer of duodenal and ilea muscularis externa were detected. Cx26 was expressed in stratum granulosum of oesophagal folds and in the non-glandular part of the stomach. A low level of immunoreactivity of Cx43 was observed in the circular, and very low in the longitudinal layer of the muscularis externa in the stomach and colon. No immunoreactivity for Cx26 and Cx43 was found in the entire muscularis externa of the oesophagus or in the longitudinal muscle layers of the duodenum and ileum.     

Are sexes equally parasitized in damselflies and dragonflies?

Parasitism plays an essential part in ecology and evolution of host species and understanding the reasons for differential parasitism within and among hosts species is therefore important. Among the very important factors potentially affecting parasitism is the gender of the host. Here, we studied whether either females or males are more likely to harbour parasites among Odonatan insects, by relying on an extensive literature review and new field data. We collected data on numerous dragonfly and damselfly species and their ectoparasites (water mites) and endoparasites (gregarines) to examine the generality of similarities and differences in prevalence, intensity and maximum number of parasites of male and female hosts. We found three main results. Firstly, most of the odonate host species showed no differences between sexes in either gregarine or water mite prevalence and intensity. The only exception was female damselflies’ higher gregarine prevalence and intensity compared to conspecific males. These inequalities in gregarine parasitism may be due to behavioral and physiological differences between conspecific males and females. In comparison, there were no differences in dragonflies between sexes in water mite or gregarine prevalence and intensity. Secondly, damselflies had higher prevalence and intensity levels of both gregarine and water mite parasites compared to dragonflies. Finally, we found a strong species level pattern between female and male parasitism: a certain level of gregarine or water mite parasitism in one sex was matched with a similar parasitism level for the other. This indicates similar exposure and susceptibility to parasites on both sexes. Even though significant differences of parasite levels between the sexes were observed within certain host species, our results strongly suggest that on a general level a more parasitized sex does not exist in the order, Odonata.     

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

     

Ultrastructural comparison of the tailed and tailless P-protein crystals respectively of runner bean (Phaseolus multiflorus) and garden pea (Pisum sativum) with tilting stage electron microscopy

Summary The periodicity of striation, discovered to measure 12 nm in the tailless P-protein crystal ofPisum sativum, is compared with that known in the central body of the tailed P-protein crystal ofPhaseolus multiflorus. A preliminary model advanced to explain the coarse striation in tails accounts for both the wavy outline in longitudinal section and the nearcircular outline in transverse section.     

The microscopic anatomy of the oesophagus, stomach and intestine of the channel catfish, Ictalurus punctatus

An anatomical study of the digestive tract of the channel catfish revealed that the oesophageal mucosa was longitudinally folded and that secondary folds were occasionally located on the primary longitudinal folds. The infoldings were more numerous near the stomach. The stratified squamous epithelium covering the folds was made up of a basal layer, large mucous cells and simple squamous cells on the surface. The epithelium on the side of the folds consisted primarily of mucous secreting cells. Taste buds were observed between mucous cells on the apical portion of the oesophageal folds and were more prevalent in the cranial part of the oesophagus. The remaining layers of the oesophagus were: a lamina propria-submucosa, tunica muscularis and adventitia or serosa.
The J-shaped stomach had two regions: a large sac-shaped region containing gastric glands and a smaller, nonglandular pyloric region. The large rugae of the stomach became gradually smaller near the pylorus. There was a well developed pyloric sphincter. The mucosa included a simple columnar epithelium, a lamina propria and adventitia or serosa.
The intestine could be differentiated into a thick ascending segment, a descending segment, a thin convoluted segment and a thicker terminal segment, the rectum. Many mucosal folds containing branched villi characterized the ascending segment of the intestine. The descending and convoluted segments contained fewer folds with shorter and less-branching villi and were smaller in diameter and thinner walled. Descending and convoluted segments were also mildly convoluted and accounted for 80% of the total length of the intestine. An intestinal valve with a sphincter marked the beginning of the rectum. There was an approximately four-fold increase in the thickness of the tunica muscularis of the terminal segment of the intestine.     

Cell surface interaction of the protozoan Gregarina with concanavalin A beads - implications for models of gregarine gliding

Con A Sepharose beads can be translocated over the surface of the protozoan Gregarina and in (forward) moving gregarines, the bead may be moved backwards relative to the substrate. The speed of bead movement is not constant over the surface of the cell, but has a maximum value in the central region of the deutomerite. The mass of the individual beads used in this study was about the same order of magnitude as the mass of a gregarine, i.e. considerable force must be generated at the gregarine cell surface. The implications of these experiments to models of gregarine locomotion are considered. The close similarities between this system and flagellar surface motility of Clamydomonas studied by particle movement and gliding motility are discussed.