Ultrastructural details of the xenoma of Loma myrophis (phylum Microsporidia) and extrusion of the polar tube during autoinfection

Xenomas of the recently described new microsporidian species Loma myrophis parasitizing the gut tissue of the Amazonian fish Myrophis platyrhynchus (family Ophichthidae) were described by light- and transmission-electron microscopy. The xenoma consisted of a thin fibrillar wall that surrounded a hypertrophic host cell cytoplasm containing numerous microsporidian developmental stages and spores. Several spores showed different stages of natural extrusion of the polar tube. Numerous longitudinal and transverse sections of the extruded polar tubes were observed in developing life-cycle stages (spores excepted), the nucleus of hypertrophic host cell, the xenoma wall and surrounding fibroblasts. The extruded polar tubes were projected in all directions with no preferential orientation. These aspects suggested that autoinfection occurred within this xenoma.     

Ultrastructural study of the late stages of Loma salmonae development in the gills of experimentally infected rainbow trout

The main objective of this investigation was to examine the ultrastructural features of gills from rainbow trout experimentally infected with Loma salmonae to determine the morphological events that occur during the late stages of development of this parasite. Peripheral distribution of the mature parasites inside round xenomas was observed at weeks 5 and 6 postexposure (PE), but eventually the parasite occupied the entire xenoma. Degenerative changes were observed only in immature parasites at week 7 PE, and eventually an inflammatory reaction with a cellular infiltration was directed against mature spores. Round, flattened, and irregular shaped xenomas were observed at week 8 PE. The round xenomas showed a severe inflammatory response with disintegration of the xenoma membrane. This event was accompanied by eversion of polar tubes within the attacked xenoma and by the simultaneous presence of 2 tubular appendages, the type I and II tubules. Flattened xenomas were observed below the endothelium of gill lamella arteries. The irregular xenomas were located in the connective tissue of the gill filament and showed multiple projections occupied by spores. Both flattened and irregular xenomas showed no evidence of inflammatory reaction. An earlier proposed hypothesis is expanded to explain how L. salmonae is implanted beneath lamellar endothelium and within filament connective tissue.     

Amazonspora hassar n. gen. and n. sp. (phylum Microsporidia,fam. Glugeidae), a parasite of the Amazonian teleost Hassar orestis (fam. Doradidae)

We describe the microsporidian Amazonspora hassar n. gen., n. sp. from the gill xenomas of the teleost Hassar orestis (Doradidae) collected in the estuarine region of the Amazon River. The parasite appeared as a small whitish xenoma located in the gill filaments near the blood vessels. Each xenoma consisted of a single hypertrophic host cell (HHC) in the cytoplasm of which the microsporidian developed and proliferated. The xenoma wall was composed of up to approximately 22 juxtaposed crossed layers of collagen fibers. The plasmalemma of the HHC presented numerous anastomosed, microvilli-like structures projecting outward through the 1-3 first internal layers of the collagen fibrils. The parasite was in direct contact with host cell cytoplasm in all stages of the cycle (merogony and sporogony). Sporogony appears to divide by plasmotomy, giving rise to 4 uninucleate sporoblasts, which develop into uninucleate spores. The ellipsoidal spores measured 2.69 +/- 0.45 x 1.78 +/- 0.18 microm, and the wall measured approximately 75 nm. The anchoring disk of the polar filament was subterminal, being shifted laterally from the anterior pole. The polar filament was arranged into 7-8 coils in a single layer in the posterior half of the spore, surrounding the posterior vacuole. The polaroplast surrounded the uncoiled portion of the polar filament, and it was exclusively lamellar. The spores and different life-cycle stages were intermingled within the cytoplasm of the HHC, surrounding the central hypertrophic deeply branched nucleus. The ultrastructural morphology of this microsporidian parasite suggests the erection of a new genus and species.     

Further Observations on the Fine Structure of the Spores of Glugea weissenbergi (Microsporida,Nosematidae)*

SYNOPSIS. A Glugea xenoma sectioned and viewed with the electron microscope contained many spores with everting polar filaments. Several details not seen in previous studies of this species were observed. A specialized area with the appareance of a lattice was commonly present near the anterior end of the polaroplast. The external portion of a partially everted polar filament appeared to have about twice the diameter of the part remaining within the spore. No membrane was seen limiting the external surface of the everted portion. The everting filament had pushed thru the polar cap and the adjacent thin area of the spore wall, making the polar cap into a ring. The ring connected the proximal end of the everting filament to the inner spore membrane, thereby anchoring the filament to the spore. The electron density of some of the membranous organelles of the spore was enhanced by the use of ruthenium red.     

Immunohistochemistry, histopathology and ultrastructure of Gasterosteus aculeatus tissues infected with Glugea anomala

Immunohistochemical and histopathological studies were conducted on a population of 3-spined sticklebacks Gasterosteus aculeatus (L.) from Loch Airthrey (Stirling, Scotland) naturally infected with the microsporean Glugea anomala (Moniez 1887). Of the 55 host specimens that were examined, 16 (29.09%) were infected, the intensity of infection ranging from 1 to 4 xenomas per fish, which were principally located within the central portion of the body lateral flank musculature. All 32 G. anomala xenomas examined were mature, their diameter ranging from 936 to 2232 Pum, and their walls of presented a laminar structure. Subcutaneously situated xenomas protruded from the fish body surface, whilst xenomas encountered within the intestine were seen to cause distortion. Light and electron microscopical observations confirmed a host cellular reaction around the xenoma, seen by the presence of eosinophile granule cells (EGCs), and some neutrophils. The occurrences of rodlet cells among the intestinal epithelial cells, and in close proximity to the xenoma wall, were observed in certain specimens. Outside the xenoma wall, macrophage aggregates (MAs) were commonly encountered. Within the xenoma wall, the presence of eosinophile granular cells immunoreactive to the anti-serotonin serum was also recorded. Further immunohistochemical tests revealed that a high number of nerve fibres running along the white lateral muscle fibres were immunoreactive to bombesin-, galanin-, and leu-enkephalin-antisera. Nerve fibres containing bombesin- and leu-enkephalin-like substances were also observed in the connective inflammatory tissue around the protozoan cyst, while neurons in the spinal ganglia were immunoreactive to met-enkephalin, and serotonin antisera. The control for the specificity of immunohistochemical reactions was performed using preabsorption tests of each antiserum with the corresponding antigen, and no immunoreactivity was noticed. The data presented are discussed in relation to the occurrence of G. anomala, which alters the pattern of nerve fibres present in the host. Specifically, the protozoan induces a response in the stickleback nervous system, the reaction of which is revealed through the application of immunohistochemical techniques.     

Ultrastructure of the Peripheral Zone of a Glugea-Induced Xenoma*

SYNOPSIS. The Glugea stephani-induced xenoma in the winter flounder, Pseudopleuronectes americanus, is a large spherical host-parasite complex, up to 4.0 mm in diameter, with the host and parasite components of the xenoma being most active in the peripheral zone. The xenoma has an extensive periodic acid-silver methenamine-positive surface coat covering the plasma membrane. The surface of this membrane is amplified by the presence of numerous folds and fine tubular extensions. The peripheral zone of the xenoma contains many host-cell mitochondria in addition to numerous microsporidan parasites. At the ultrastructural level, the peripheral zone of the host-cell cytoplasm appears normal. Inside the peripheral region of the 0.4–1.0 mm xenoma, the host-cell component largely disintegrates in the presence of microsporidan parasites undergoing sporogenesis.     

Fine structure of the microsporidan Abelspora portucalensis gen.n., sp.n. (Microsporida) parasite of the hepatopancreas of Carcinus maenas (Crustacea,Decapoda)

A new species of a microsporidan, Abelspora portucalensis, was found in the hepatopancreas of Carcinus maenas, forming white xenomas. Each xenoma seems to consist of an aggregate of hypertrophic host cells in which the parasite develops and proliferates. This cytozoic microsporidan being characterized by one uninucleate schizont giving rise to two sporonts, each originating two sporoblasts, resulting in two spores within a persistent sporophorous vacuole (pansporoblast) should be included in a new family Abelsporidae. In fresh smears most spores were 3.1–3.2 μm long and 1.2–1.4 μm wide. Fixed, stained, and observed in SUS mature spores measured 3.1 ± 0.08 × 1.3 ± 0.06 μm (n = 25 measurements). Spore cytoplasm was dense and granular, polyribosomes were arranged in helicoidal tape form. The polar filament was anisofilar and consisted of a single coil with 5–6 turns. The anchoring disc and and the anterior zone of the filament are surrounded by the polaroplast composed of two usual zones. In the anterior zone, the membrane of the polar filament is in continuity with the membranes of the polaroplast. The appearance of a microsporidan with described nuclear divisions in life cycle, spores shape and size, polaroplast and polar filament morphology and identity of the host suggests that we may erect a new genus Abelspora and a new species A. portucalensis (Portugal = Portucalem).     

The microsporidian spore invasion tube. III. Tube extrusion and assembly

The polar filaments within microsporidian spores discharges as tubes with subsecond velocity. Populations of discharging tubes of Glugea hertwigi spores pulse-labeled with latex particles for 1-3 s were consistently devoid of label at the distal ends; discharging tubes were completely labeled after 30- to 60-s exposure to latex. This experiment indicates that discharge tubes grow at the tip. Completely assembled discharge tubes consisted of single, empty cylinders; however, incompletely discharged tubes had a cylinder-within-a-cylinder profile at the distal ends. This observation indicates that the discharge tube material emerges at the distal end by an eversion process. Finally, studies with cinematic Nomarski interference optics of spore tubes extruding across a water-air interphase indicate that all the material emerging from the growing tip of the tube is incorporated into the wall of the discharge tube. Evidence indicates that the polar filament of undischarged spores is a homogeneous coil of polar tube protein equivalent to the polar tube protein in discharged tubes.     

Observations on Ichthyosporidium giganteum (Microsporida) with particular reference to the host-parasite relations during merogony

Connective tissue cells, particularly fibroblasts, of the fish Leiostomus xanthurus Lacépède respond to the invading microsporidian parasite Ichthyosporidium sp. [assumed to be identical with Ichthyosporidium giganteum (Thélohan)] by proliferating themselves, coalescing into a syncytium, synthesizing copious amounts of cytoplasm around the parasites, and walling off the parasitized islands of cytoplasm with fibrous capsules. The resulting cysts are xenoparasitic complexes of the syncytial xenoma type, clearly different from the cell hypertrophy tumor (xenoma sensu Weissenberg) exemplified by the Glugea cyst. These findings involve a new concept of the structure and host-parasite relations of Ichthyosporidium. Formerly, the parasitized masses of cytoplasm were interpreted as extracellular plasmodial stages of the parasite (stages uncharacteristic of the microsporidia), while the parasites themselves were interpreted as nuclei of the "plasmodia." Actually, the parasite undergoes merogony in parasitophorous vacuoles which coalesce before sporogony begins. The nuclei of the mermonts are very small chromatin granules, becoming transformed into large basophilic diplokarya of the sporonts. Sporulation is diplokaryotic throughout, the diplokarya becoming reduced in size through 2 steps during sporogony.     

A redescription of Nosema herpobdellae (Microspora: Nosematidae), a parasite of the leech Erpobdella octoculata (Hirudinea: Erpobdellidae)

Nosema herpobdellae was recorded in populations of the leech Erpobdella octoculata from lakes in northwest England and North Wales and is redescribed using light and transmission electron microscopy. It differes from N. glossiphoniae in the nature of the infection and tissues parasitized and from N. tractabile in its larger spore size, longer polar filament, in the angle of the anterior coils of the polar filament to the spore long axis, and apparently in its developmental cycle. The infection was found in a massive xenoma, in the connective tissue surrounding the gut, which was presumed to be formed from a single hypertrophied cell. Its developmental cycle included merogony and sporogony.     

A new microsporidian parasite, Potaspora morhaphis n. gen., n. sp. (Microsporidia) infecting the Teleostean fish, Potamorhaphis guianensis from the River Amazon. Morphological, ultrastructural and molecular characterization

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2.8+/-0.3 x 1.5+/-0.2 microm. In mature spores the polar filament was arranged into 9-10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.     

Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes

Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.     

Impact of a water temperature shift on xenoma clearance and recovery time during a Loma salmonae (Microsporidia) infection in rainbow trout Oncorhynchus mykiss

Previous studies have modelled the relationship between water temperature and the rate of sporulation as defined by xenoma formation during microsporidial gill disease (MGD) in salmon caused by Loma salmonae. Although offering insight into the epidemiology of MGD, a key unexplored area is the role of temperature in the rate of xenoma dissolution including spore release into the environment, and this is crucial to our ability to model horizontal transmission of MGD within confined net-pen populations of farmed salmon. Results from a previous trial suggested that xenoma dissolution may be dramatically hastened as water temperature declines, thus introducing a critical anomaly into any predictive exercise. The data generated herein was evaluated using the statistics of survival analysis to re-establish the baseline relationship of xenoma formation and dissolution relative to water temperature and to compare these results with those of previous studies. We infected 30 individuals of Oncorhynchus mykiss (Walbaum) with macerated xenoma-laden gill material, and afterwards allocated them to tanks with water temperatures of 11, 15, or 19 degrees C and monitored them through a disease cycle. Xenoma onset and clearance times were similar to previous findings with both events being accelerated at higher water temperatures, thereby suggesting a similar temperature response in the current strain to those used in previous studies. Another group of 45 fish was infected with L. salmonae and held at 15 degrees C until xenomas formed, and were subsequently shifted to 11, 15, or 19 degrees C. The median xenoma dissolution time in these tanks was 49, 35 and 28 d, respectively, similar to rates observed when water temperature remained constant. Thus we rejected the hypothesis that a sudden change in water temperature triggers rapid or anomalous xenoma dissolution.     

Microgemma vivaresi n. sp. (Microsporidia, Tetramicridae), infecting liver and skeletal muscle of sea scorpions, Taurulus bubalis (Euphrasen 1786) (Osteichthyes, Cottidae), an inshore, littoral fish

The ultrastructure of a new microsporidian species Microgemma vivaresi n. sp. causing liver cell xenoma formation in sea scorpions, Taurulus bubalis, is described. Stages of merogony, sporogony, and sporogenesis are mixed in the central cytoplasm of developing xenomas. All stages have unpaired nuclei. Uninucleate and multinucleate meronts lie within vacuoles formed from host endoplasmic reticulum and divide by binary or multiple fission. Sporonts, no longer in vacuoles, deposit plaques of surface coat on the plasma membrane that cause the surface to pucker. Division occurs at the puckered stage into sporoblast mother cells, on which plaques join up to complete the surface coat. A final binary fission gives rise to sporoblasts. A dense globule, thought to be involved in polar tube synthesis, is gradually dispersed during spore maturation. Spores are broadly ovoid, have a large posterior vacuole, and measure 3.6 microm x 2.1 microm (fresh). The polar tube has a short wide anterior section that constricts abruptly, then runs posteriad to coil about eight times around the posterior vacuole with granular contents. The polaroplast has up to 40 membranes arranged in pairs mostly attached to the wide region of the polar tube and directed posteriorly around a cytoplasm of a coarsely granular appearance. The species is placed alongside the type species Microgemma hepaticusRalphs and Matthews 1986 within the family Tetramicridae, which is transferred from the class Dihaplophasea to the class Haplophasea, as there is no evidence for the occurrence of a diplokaryotic phase.     

The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis

Polar cell expansion in differentiating tissues is critical for the development and morphogenesis of plant organs and is modulated by hormonal and developmental signals, yet little is known about signaling in this fundamental process in plants. In contrast to tip-growing cells, such as pollen tubes and root hairs, cells in developing tissues are thought to expand by diffuse growth. In this study, we provide evidence that these cells expand in two phases with distinct mechanisms. In the early phase, cell expansion can occur in both longitudinal and radial or lateral directions and is mediated by Rop GTPase signaling, a mechanism known to control tip growth. The expression of a dominant-negative mutant for ROP2 (DN-rop2) inhibited polar cell expansion, whereas the expression of a constitutively active mutant (CA-rop2) caused isotropic expansion in the early phase. In the late phase, expansion occurs only in the longitudinal direction and is not affected by DN-rop2 or CA-rop2 expression. The transition from the early to the late phase coincides with the reorientation of cortical microtubules from random to transverse arrangements. Thus, cell expansion in the late phase is consistent with polar diffuse growth, in which polarity probably is defined by transverse cortical microtubules. We show that the direction of cell expansion in the early phase is associated with the localization of diffuse fine cortical F-actin in leaf epidermal cells. DN-rop2 expression specifically inhibited the formation of this F-actin, but not actin cables, whereas CA-rop2 expression caused delocalized distribution of this fine F-actin throughout the cell cortex. Furthermore, green fluorescent protein-ROP2 was localized preferentially to the cortical region of the cell, where expansion apparently occurs. These observations suggest that ROP2 control of the polar expansion of cells within tissues is analogous to the Rop control of tip growth and of tip-localized F-actin in pollen tubes and root hairs and that the tip growth mechanism also may modulate polar cell expansion in differentiating tissues.     

Influence of applied electrical fields on yeast and hyphal growth of Candida albicans

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.     

微丝骨架的构成及其对花粉管极性生长的调控作用

微丝骨架是细胞骨架的重要组成部分,它由肌动蛋白和肌动蛋白结合蛋白组成,广泛存在于真核细胞中。近年来,大量研究表明植物花粉及花粉管中存在丰富的微丝骨架。目前,在微丝骨架作为信号转导途径的靶标参与对花粉管极性生长的调控、微丝骨架在花粉和花粉管中的分布及其在花粉管生长过程中与其他信号分子之间的相互作用等方面取得了一系列突破性进展。     

Overexpression of an Arabidopsis formin stimulates supernumerary actin cable formation from pollen tube cell membrane

Formins, actin-nucleating proteins that stimulate the de novo polymerization of actin filaments, are important for diverse cellular and developmental processes, especially those dependent on polarity establishment. A subset of plant formins, referred to as group I, is distinct from formins from other species in having evolved a unique N-terminal structure with a signal peptide, a Pro-rich, potentially glycosylated extracellular domain, and a transmembrane domain. We show here that overexpression of the Arabidopsis formin AFH1 in pollen tubes induces the formation of arrays of actin cables that project into the cytoplasm from the cell membrane and that its N-terminal structure targets AFH1 to the cell membrane. Pollen tube elongation is a polar cell growth process dependent on an active and tightly regulated actin cytoskeleton. Slight increases in AFH1 stimulate growth, but its overexpression induces tube broadening, growth depolarization, and growth arrest in transformed pollen tubes. These results suggest that AFH1-regulated actin polymerization is important for the polar pollen cell growth process. Moreover, severe membrane deformation was observed in the apical region of tip-expanded, AFH1-overexpressing pollen tubes in which an abundance of AFH1-induced membrane-associated actin cables was evident. These observations suggest that regulated AFH1 activity at the cell surface is important for maintaining tip-focused cell membrane expansion for the polar extension of pollen tubes. The cell surface-located group-I formins may play the integrin-analogous role as mediators of external stimuli to the actin cytoskeleton, and AFH1 could be important for mediating extracellular signals from female tissues to elicit the proper pollen tube growth response during pollination.     

Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha,O. kisutch,O. mykiss) but not by salinity

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.