Ultrastructural study of microsporidian development

Summary Vegetative growth of Nosema sp. occurs within the gut submucosal cells of Callinectes sapidus. Vegetative cell morphology is dominated by profiles of endoplasmic reticulum, numerous free ribosomes and aggregates of vesicles enclosed by a membranous sac. The dikaryotic vegetative cell is the earliest stage found in the target area for sporogenesis, the sarcoplasm of the striated muscle cell. The next obvious stage is the sporoblast mother cell; it undergoes karyokinesis without breakdown of the nuclear envelope. Intranuclear mitotic microtubules extend from the chromosomes to the intact nuclear envelope. After repeated nuclear divisions, the sporoblast mother cell undergoes delayed cytokinesis and a series of sporoblast progeny develops.The polar filament is the first visually apparent system to develop during sporogenesis. It appears to be of dual origin: (1) the central core component is condensed in Golgi-like saccules, and (2) the envelopes around the core originate from the endoplasmic reticulum.The polaroplast, which forms after early polar filament development, appears to originate as an elaboration of the endoplasmic reticulum.Supported in part by a training grant from the National Institutes of Health (GM-669-05) and research grants from the National Science Foundation (GB-3036, GB-5235, and GB-7938) to Prof. F. Sogandares-Bernal. The skillful guidance of Prof. F. Sogandares-Bernal is acknowledged. Special thanks are extended to Prof. D. E. Copeland for the use of a Siemens Elmiskop IA electron microscope. I also wish to thank Mr. Julian King, professional fisherman of Irish Bayou, Louisiana, for providing hundreds of blue crabs used in the course of this study.     

Fine structure of the developing spore of Nosema apis zander

Summary The mature spore possesses a thick spore coat and a particle-bearing spore membrane. The highly laminated polaroplast membranes are located at the anterior pole of the spore. Close to its base, the polar filament is surrounded by the polaroplast membrane. The polar filament runs spirally towards the posterior pole of the spore. A large portion of the polar filament is arranged in two layers. A similar arrangement was also observed in immature spores and in the sporoblast stage, although it was not so orderly arranged in the latter. The developing polaroplast membrane was observed in the immature spore, but not in the sporoblast. The sporoblast wall is much thinner than the spore coat, but has the same texture. Endoplasmic reticulum is the most prominent cytoplasmic organelle in the developing stages of Nosema apis. Porous nuclear envelopes are also observed in developing stages. The role of the endoplasmic reticulum in the formation of the polar filament, polaroplast and spore coat, and the function of the spore membrane, are discussed.     

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.     

Light and electron microscopic observations of Leptotheca koreana n. sp. (Myxosporea) in the kidney of cultured rockfish Sebastes schlegeli.

The structure and sporogenesis of Leptotheca koreana n. sp. from cultured rockfish Sebastes schlegeli from South Korea were studied by light and transmission electron microscopy. Broadly oval spores and disporous pseudoplasmodia were observed in the lumen of renal tubules. Spores were 8.59 +/- 1.25 microm in length, 13.42 +/- 1.0 microm in width in sutural view and 8.13 +/- 0.52 pm in thickness in the plane perpendicular to the suture. The width of each valve was always smaller than spore length. Two spherical polar capsules were equal in size (3.86 +/- 0.45 microm in diameter) containing a polar filament with 6 to 7 turns, opening at the anterior end of the spore. Two uninucleate sporoplasms filled the spore cavity. The asynchronous division of secondary and tertiary cells and asynchronous development in spore formation of the present Leptotheca koreana resembled the disporous sphaerosporids. Cytoplasmic projections of pseudoplamodia were considered to be rhizoids, as they seem to strengthen the attachment to the epithelial cells of the renal tubules. The capsulogenic cells in early sporoblast had large amounts of rough endoplasmic reticulum but had a few Golgi apparatus.     

A new species of Sphaeromyxa (Myxosporea: Sphaeromyxina: Sphaeromyxidae) in devil firefish, Pterois miles (Scorpaenidae), from the northern Red Sea: morphology, ultrastructure, and phylogeny

Sphaeromyxa zaharoni n. sp. (Myxosporea) is described from the gallbladder of devil firefish, Pterois miles (Scorpaenidae), from coral reefs of the Gulf of Eilat, Israel, northern Red Sea. The parasite was found also in bearded scorpionfish, Scorpaenopsis barbata, from the same area. This is the first report on Sphaeromyxa sp. from this zoogeographical region. The plasmodia are amoeboid when young, becoming disc-shaped and elongated when mature. In paraffin sections, the plasmodium periphery appears as a finely granulated, strongly eosinophilic layer with an intricate surface membrane network. Sphaeromyxa zaharoni n. sp. is polysporous, disporoblastic, and has asynchronous sporogenesis. The mature spore is elongated and fusiform, has delicately ridged valves, and contains a single, binucleated sporoplasm. In valvular view, the tips are truncated. The mean spore size is length 14.5 microm, width 4.8 microm, and polar capsule 4.8 x 3.4 microm. The 2 equally sized ovoid polar capsules are positioned at opposite ends of the spore, each containing a filament loosely folded in 2 loops. The fine structure of the sporoblast and spore corresponded with previously studied Sphaeromyxa species. According to small-subunit ribosomal DNA gene sequence analysis, S. zaharoni n. sp. is most closely related to 2 Myxidium spp. The close phylogenetic relatedness of Sphaeromyxa and Myxidium and similar spore morphology raises the question whether these 2 genera should be maintained in separate families and suborders.     

Light and electron microscope study of a new species of Urosporidium (Haplosporida), hyperparasite of trematode sporocysts in the clam Abra ovata

Abra ovata, collected at Bcauduc, France, contained sporocysts of Gymnophallus nereicola and another trematode of the family Monorchiidae. Frequently the former trematode and occasionally the latter was infected with a species of Urosporidium. Stages observed were mostly in the sporogenesis sequence. The sporoblast, an elongated body of uncertain origin, differentiates into two parts delimited by a girdle-shaped constriction between them. These are an anterior part, or sporoplasm primordium, containing a vesicular nucleus and a posterior part, or envelope primordium, containing a “parietal apparatus” (possibly a transformed nucleus). Cytoplasm of the envelope primordium (just behind the constriction) advances to enclose the sporoplasm primordium while it differentiates into endospore, exospore and an internally situated cover over the orifice. These two primordia separate late in the sporogenesis sequence. Thus, the typical haplosporidan spore may, as Cépède reported in 1911, consist of 2 cells, a generative cell enveloped by a somatic cell. Evidence that the Haplosporida have bicellular spores raises fundamental questions regarding the taxonomy of this group.     

Notes on the ultrastructure and sporoblast development in fish parasitizing myxosporidian of the genusSphaeromyxa

Summary The ultrastructure of trophic stage (plasmodium), spore and changes in fine structure during morphogenesis of the spore were studied by electron microscopy in two representatives of the genusSphaeromyxa. Plasmodium has a highly differentiated structure; there is an outer layer of homogeneous plasm, the endoplasm consisting of a vacuolated mass in which float generative cells and sporoblasts in different degree of development. Generative cells have well developed pseudopodia. Sporoblasts arise from the union of two cells, out of which the inner one forms all cells of the spore, the outer one has only an enveloping function. Polar capsule develops in a way identical with other myxosporidia; the wide filament, however, has a longitudinally folded structure and is located within the capsule in two loose loops. The mitochondria of an early sporoblast are characterized by a high content of DNA. The identity of polar capsule development with the nematocyst morphogenesis is too conspicuous to be taken for a mere convergence. Studies on ultrastructure give no evidence for a protozoan character of myxosporidia; together with other findings, they are in favor of the nonprotozoan nature of this group of organisms.The essential part of this work was carried out in the Department of Biology, University of Illinois at Chicago Circle, where this study was aided by a grant from the National Science Foundation (NSF GB-2800) to Dr.J. Corliss. The author is also indebted to Dr.R. Fernald, University of Washington, for the use of all facilities at the Friday Harbour Laboratories of the University of Washington.     

CHLOROPLAST DEVELOPMENT DURING SPOROGENESIS IN SIX SPECIES OF MOSSES

Chloroplast development during sporogenesis in Mnium cuspidatum, M. medium, M. rostratum, Aulacomnium heterostichum, Bartramia pomiformis, and Timmia megapolitana is as follows: During the early mitotic divisions in the sporogenous area of the capsule the number of plastids is reduced from many to one cup-shaped plastid per sporogenous cell. This single plastid divides during the early spore-mother-cell stage. A second division of plastids produces four plastids within each spore-mother-cell. A massive accumulation of starch occurs within each of the four plastids. Following meiosis, the single plastid allocated to each spore produces distinct lobes that are “blebbed” off as proplastids. A photosynthetic membrane system is established within the many proplastids as each spore matures.     

吸鱼粘体虫在异育银鲫心脏中的孢子发生

吸鱼粘体虫主要寄生在异育银鲫的心肌纤维间。随着营养体的长大,营养体内的两型生殖细胞相聚,小生殖细胞包围大生殖细胞,形成泛孢子母细胞。大生殖细胞进行连续的核分裂,成为产孢体。核分裂达12核时,产孢体内分化为10个细胞:4个成极囊细胞,4个成壳片细胞和2个双核的孢子质细胞。这些细胞均分为两组,从而形成双生孢子型的泛孢子母细胞。     

Fine Structure of Developing Spores of Thelohania bracteata (Strickland, 1913) (Microsporida,Nosematidae) Emphasizing Polar-Filament Formation*

SYNOPSIS. In the microsporidian, Thelohania bracteata, the polar filament, as it starts to develop in the sporoblast, apparently receives material synthesized by the granular endoplasmic reticulum and Golgi vesicles. In immature spores many dilated sacs are observed in areas where there is less endoplasmic reticulum. These sacs, that persist into the almost mature spore, are probably Golgi-type vesicles and may be related to the formation of the spore coat. The polar filament of the mature spore possesses 8 coils and in cross section or cross-fractured face the electron-dense central portion of the polar filament contains a tubular structure, ringed by 12–14 cylindrical structures. In thin sections, an electron-lucid zone is observed between the core and membrane of the polar filament. The polar filament runs through the highly laminated polaroplast which occupies the anterior portion of the spore. In cross-fractured face the lamellae of the polaroplast are arranged like the petals of a flower. The basal portion of the polar filament is enlarged, appearing arrow-shaped in thin sections and pear-shaped in frozen-etched preparations. Frozen-etched membranes differ in the size and distribution of the surface particles.     

Fine Structure of the Sporogonic Stages of Nosema parkeri

SYNOPSIS The fine structure of sporogonic stages of Nosema parkeri Krinsky, a microsporidan from the argasid tick, Ornithodoros parkeri Cooley, is described. Developmental changes in the spore coat and cytoplasmic organelles are discussed. As a sporoblast transforms into a spore, the organelles become more compact and the membranes surrounding them appear to become more taut. It is suggested that the polaroplast complex is involved in fluid transport during development of the spore. Organelles in the mature spore include 2 contiguous nuclei enveloped in a lattice containing ribosome-like particles, a polaroplast complex composed of laminar and saccular regions, and a coiled tubular polar filament attached to a polar sac. Sporogonic stages do not appear to have mitochondria, Golgi apparatus, or a posterior vacuole. The fine structure of the spore of N. parkeri is very similar to that of species of Nosema found in insects, crustaceans, and trematodes.     

Organization of statocysts in the Otoplanidae (Plathelminthes): an ultrastructural analysis with implications for the phylogeny of the Proseriata

Summary The statocyst of otoplanids is enveloped by a bipartite capsule which consists of two different extracellular matrices. This capsule encircles three different types of aciliary cells: several peripherally located flattened parietal cells, one central statolith forming cell (lithocyte) and two clusters of accessory cells. Intracapsular lumina exist which are different from extracapsular intercellular spaces. The accessory cells most probably represent those structures that are mainly involved in nervous conduction. These cells extend cytoplasmatic processes towards different peripheral regions of the statocyst where processes of outer nerve cells penetrate the capsule. The statocyst does not seem to represent a more evolved equilibrium receptor system but may function as a relatively simple aciliary sense organ suitable for positive geotactic behaviour. The otoplanid statocyst corresponds to statocysts in other lithophorous proseriates but not to statocysts in other taxa of the free-living Plathelminthes. The monophyly of a taxon Lithophora within the Proseriata is corroborated by this autapomorphic characteristic.Abbreviations ac accessory cell(s) - c capsule of the statocyst - ce cerebrum - ci cephalic intestine - co capsule opening - cp cell process(es) of accessory cell(s) and cell(s) containing filaments - ecm extracellular matrix - fc cell(s) containing filaments - ic intercellular spaces within the capsule - mc muscle cell(s) - n lobed nucleus of the lithocyte - nac nucleus (nuclei) of accessory cell(s) - nc nerve cell(s) - npc nucleus (nuclei) of parietal cell(s) - pc parietal cell(s) - s statolith - sc statolith cell (lithocyte)     

Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium Wall and of Sporogenesis*

SYNOPSIS Ultrastructure of the plasmodium wall and of sporogenesis were studied in Myxosoma funduli Kudo infecting the gills of Fundulus kansae (Garman). Plasmodia were located within the lamellar tissues adjacent to sinuses and capillaries. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite ectoplasm. The plasmodium membrane was covered by a surface coat of almost uniform thickness which prevented direct parasite-host cell contact. Numerous generative cells and cell aggregates, representing early stages of spore development, were seen in immature plasmodia. Later stages of spore development, including mature spores, were observed in older plasmodia. Sporogenesis was initiated by envelopment of one generative cell, the sporont, by a 2nd, nondividing cell, the envelope cell. The sporont and its progeny proceeded through a series of divisions until there were 10 cells, all compartmentalized within the envelope cell. Subsequently, the 10 cells became structurally differentiated and arranged into two 5-celled spore-producing units, each consisting of 1 binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Observations of later developmental stages revealed the major events of capsulogenesis, valvogenesis, and sporoplasm maturation, which occurred concomitantly during spore construction.     

SPORE ULTRASTRUCTURE OF THE MYXOMYCETE PHYSARUM POLYCEPHALUM

Spores of the true slime mold Physarum polycephalum were examined at several stages of their development by means of scanning and transmission electron microscopy. The spores were globose, spine-covered structures produced within a sporangium enclosed in a tough, noncellular peridium. Cytologically, the spore represented a typical eukaryotic cell, having discrete organelles similar to spores of other myxomycetes. The presence of dictyosomes, helical filaments, and microbodies in these cells, as well as the further elucidation of the cell wall and the “polysaccharide-containing” areas, represent new contributions to the ultrastructure of the myxomycete spore. Of special interest were observations of metaphase nuclei just prior to spore cleavage, interphase nuclei in young spores, and nuclei in mature spores containing synaptonemal complexes. These observations indicate that in Physarum polycephalum mitosis occurs just prior to spore cleavage, and meiosis takes place after spore cleavage.     

The Presence of Keratin-like Substance in Spore Coat of Bacillus subtilis

Unique crystalline structures were found by X-ray diffractometry to be present in spore coats of Bacillus subtilis. By crystallographical and chemical studies of the purified spore coats it was found that these crystalline structures of the spore coats were essentially similar to those of α- and β-keratin, and that the spore coats were composed of keratin-like substance (or keratin). This keratin-like substance was found to be synthesized during sporogenesis from sulfur-containing water-soluble substances in the cells.     

Light and Electron Microscope Observations on Nosema nelsoni Sprague, 1950 (Microsporida,Nosematidae) with Particular Reference to its Golgi Complex*

SYNOPSIS. A species of Nosema in the muscles of the North American white shrimp, generally known as Penaeus setiferus but also known as P. fluviatilis, appears identical with type specimens of N. nelsoni Sprague, 1950, in P. aztecus. Its Golgi apparatus, as seen in the sporoblast, is a complex system of cisternae, small vesicles and expanded sacs which plays a major role in spore morphogenesis. It transforms directly into the polaroplast complex, certain membranous investments of the polar filament, the polar sac and perhaps part of the posterior vacuolar system. Probably the polar sac contains the polar cap. The PAS-positive material in both the cap and the filament may be a component of the Golgi complex. This new concept of the Golgi complex supplements our earlier view of spore morphogenesis according to which the polar filament is of nuclear origin. It also reconciles the idea with Vávra's identification of Golgi vesicles associated with the developing polar filament.     

The dynamics of the actin cytoskeleton during sporogenesis in Psilotum nudum L.

The actin cytoskeleton (microfilaments, MFs) accompanies the tubulin cytoskeleton (microtubules) during the meiotic division of the cell, but knowledge about the scope of their physiological competence and cooperation is insufficient. To cast more light on this issue, we analysed the F-actin distribution during the meiotic division of the Psilotum nudum sporocytes. Unfixed sporangia of P. nudum were stained with rhodamine-phalloidin and 4′,6-diamidino-2-phenylindole dihydrochloride, and we monitored the changes in the actin cytoskeleton and nuclear chromatin throughout sporogenesis. We observed that the actin cytoskeleton in meiotically dividing cells is not only part of the kariokinetic spindle and phragmoplast but it also forms a well-developed network in the cytoplasm present in all phases of meiosis. Moreover, in telophase I F-actin filaments formed short-lived phragmoplast, which was adjacent to the plasma membrane, exactly at the site of future cell wall formation. Additionally, the meiocytes were pre-treated with cytochalasin-B at a concentration that causes damage to the MFs. This facilitated observation of the effect of selective MFs damage on the course of meiosis and sporogenesis of P. nudum. Changes were observed that occurred in the cytochalasin-treated cells: the daughter nuclei were located abnormally close to each other, there was no formation of the equatorial plate of organelles and, consequently, meiosis did not occur normally. It seems possible that, if the actin cytoskeleton only is damaged, regular cytokinesis will not occur and, hence, no viable spores will be produced.     

Anti-Tumour Efficacy of Capecitabine in a Genetically Engineered Mouse Model of Pancreatic Cancer

Capecitabine (CAP) is a 5-FU pro-drug approved for the treatment of several cancers and it is used in combination with gemcitabine (GEM) in the treatment of patients with pancreatic adenocarcinoma (PDAC). However, limited pre-clinical data of the effects of CAP in PDAC are available to support the use of the GEMCAP combination in clinic. Therefore, we investigated the pharmacokinetics and the efficacy of CAP as a single agent first and then in combination with GEM to assess the utility of the GEMCAP therapy in clinic. Using a model of spontaneous PDAC occurring in Kras^G12D; p53^R172H; Pdx1-Cre (KPC) mice and subcutaneous allografts of a KPC PDAC-derived cell line (K8484), we showed that CAP achieved tumour concentrations (∼25 µM) of 5-FU in both models, as a single agent, and induced survival similar to GEM in KPC mice, suggesting similar efficacy. In vitro studies performed in K8484 cells as well as in human pancreatic cell lines showed an additive effect of the GEMCAP combination however, it increased toxicity in vivo and no benefit of a tolerable GEMCAP combination was identified in the allograft model when compared to GEM alone. Our work provides pre-clinical evidence of 5-FU delivery to tumours and anti-tumour efficacy following oral CAP administration that was similar to effects of GEM. Nevertheless, the GEMCAP combination does not improve the therapeutic index compared to GEM alone. These data suggest that CAP could be considered as an alternative to GEM in future, rationally designed, combination treatment strategies for advanced pancreatic cancer.     

Henneguya episclera sp. n. (Protozoa: Myxosporida), a new myxosporidan from the eye of the pumpkinseed sunfish.

Henneguya episclera sp. n. is described from the episclera of the eye of the pumpkinseed sunfish (Lepomis gibbosus). Mean spore dimensions (in micrometers) are: total length 62.6; spore length 21.7; spore thickness 8.0; spore breadth 8.7; tail lengths 37.1 and 40.9; polar capsule lengths 6.0 and 6.5; polar capsule breadths 2.7 and 3.0; polar capsule thickness 3.5; and vacuole 5 by 5.     

Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta)

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of γ-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from γ-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.