Ultrastructure of asci,ascospores, and spore release inLophodermella sulcigena (Rostr.) v. Hohn.

Summary The croziers were formed from large multinucleate cells at the base of the hysterothecium. The diploid ascus had basal and apical vacuoles and there was prominant endoplasmic reticulum near the extending tip of the ascus. The spore delimiting membranes were continuous with the plasmalemma and possibly arose from it. The spore walls were formed between the two membranes. The ascus had a simple apical ring around a thinner region of the wall which became the pore through which the spores were released. Just before spore release the outer layer of the ascospore wall became vesiculated and eventually mucilagenous. The long clavate ascospores were released one at a time, stretching the neck of the ascus as they emerged.     

The fine structure of ascospore wall formation in Sordaria fimicola

Summary The fine structure of ascopore wall development in the pyrenomycete Sordaria fimicola has been studied in asci fixed in either glutaraldehyde-OsO₄ or KMnO₄. Three distinct wall layers are deposited between the outer spore investing membrane and the inner spore plasma membrane which initially delimit each of the eight ascopores. Primary wall deposition occurs initially, followed by the deposition of secondary wall material to the outside of the primary wall. The tertiary wall is initiated as an electron dense layer along the interface between the primary and secondary walls. Pigment accumulates in this region. Increase in overall thickness of the ascospore wall occurs mainly in the secondary and tertiary layers, the former becoming fibrous and gelationous, and the latter multilayered, at maturity.     

Electron microscopy of sporulation inSchwanniomyces alluvius

Sporulation inSchwanniomyces alluvius appeared to be preceded by fusion of a mother and a daughter cell. Meiosis probably occurred in the mother cell and one or two spores were formed in the latter. A study of thin sections showed that the spore wall developed from a prospore wall. The mature spore wall consisted of a broad light inner layer and a thinner dark outer layer including warts. An equatorial ledge was present. During germination in the ascus, a new light inner layer was formed and the old layers of the spore wall partly broke up. Ascospores in a strain ofS. persoonii had a different wall structure in that the dark layer had changed into light areas separated by dark material which formed bulges at the surface.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

Ultrastructure of ascosporogenesis in Nannizzia gypsea.

Ascosporogenesis in Nannizzia gypsea was studied by electron microscopy. Development of ascospores began with the formation of an ascus vesicle composed of two paired unit membranes. Myelin figures consisting of coiled or concentric membranes were regularly connected with the growing ascus vesicle. Both the ascus vesicle and the myelin figures possessed an electron-dense line between paired membranes, and both were stained by the periodic acid-silver methenamine technique. Invagination of the ascus vesicle about the haploid nuclei resulted in eight uninucleate prospores bounded by two concentric membranes. Spore wall material was deposited between the two membranes of the prospores, and deposition was greatest in areas of the wall overlying stacked elements of endoplasmic reticulum. A single myelin figure surrounded by a polysaccharide halo was observed in the spore.     

Electron microscopy of ascus formation in the yeast debaryomyces hansenii.

Ascus formation in Debaryomyces hansenii includes fusion of two cells, usually mother and daughter while still attached to each other, through short protuberances developed from the cross wall between them. Nuclear fusion takes place in the channel connecting the two cells; meiosis apparently occurs in the mother cell. Generally, only one lobe of the meiotic nucleus is surrounded by a prospore wall and it becomes the nucleus of a spore with a warty wall. The rest of the nucleus disappears. The spores germinate by swelling in the ascus and forming one or more buds.     

Formation of spore ornamentation in two Sphaerophorus species

Two different ways of achieving a spore ornamentation have been demonstrated in Sphaereophorus , belonging to the Caliciales. In S. globosus the ornamentation is formed within the ascus by an external secondary spore wall in an ontogenetic process with several unique features. In S. murrayi the ornamentation is formed at a late stage, when the spores have been released from the asci. Carbonaceous material formed among the asci and paraphyses is added to the surface of the primary wall, and a very irregular ornamentation is formed. The name Sphaerophorus murrayi Ohlsson is validated.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Microsporidian Spore Discharge and the Transfer of Polaroplast Organelle Membrane into Plasma Membrane

Electron microscopic examinations of Glugea hertwigi and Spraguea lophii spores indicated the presence of a single plasma membrane; however, this membrane remained in the spore during the discharge of the sporoplasm from the spore. Although discharged spores retained the old plasma membrane, the extruded sporoplasms acquired a new plasma membrane. In order to determine where the new plasma membrane came from, we used two fluorescent probes with membrane affinities. The markers were tested on unfired and discharged spores. The probe, N-phenyl-1-naphthylamine (NPN), labeled the polaroplast membrane in addition to the apolar groups in the posterior vacuoles of unfired spores. After spore discharge, NPN label disappeared from the spore ghosts except for a slight fluorescence on residual plasma membranes. Much of the NPN-labeled membrane reappeared after spore discharge on the outer envelope of discharged sporoplasms. The probe chlorotetracycline (CTC) labeled calcium-associated membranes of spore polaroplasts. During spore discharge, the CTC fluorescence shifted from the polaroplast organelle of unfired spores to the outer envelope of discharged sporoplasms. These results indicate that the polaroplast organelle may provide the new plasma membrane for discharged microsporidian sporoplasms.     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall     

A light and electron microscopic study of sporulation in the myxomyceteStemonitis virginiensis

Summary The conversion of the plasmodium ofS. virginiensis into sporophores has been examined at both the light and electron microscopic levels. Particular attention has been paid to stalk and columella formation, capillitial formation, nuclear behavior during sporulation and spore formation. Both the stalk and columella are formed within the sporangial initial as intraprotoplasmic secretions. A portion of the capillitium arises directly from the columella while the remainder forms within an anastomosing system of tubular vacuoles. As spore cleavage begins the nuclei within the sporangium begin to divide mitotically. The protoplasmic content of the sporangium is first divided into small protospores which typically contain a single dividing nucleus. Following the completion of mitosis each of these segments cleaves into yet smaller segments which develop into spores. Meiosis occurs in the spores some 12–16 hours after cleavage.     

A new genus: Sporopachydermia

In two species of the yeast genus Cryptococcus ascospores have been found. A genus is described to accommodate the two species, because the spore wall is extraordinarily thick and its ultrastructure differs from that found in all other yeast genera. The spores are easily liberated from the ascus at maturation. The name Sporopachydermia is proposed for the genus and the names S. lactativora and S. cereana are proposed for the species.     

Ultrastructural Changes Associated with Spore Formation in Sporangia and Sporangiola of Thamnidium elegans Link

Fully formed pre-cleavage sporangia and sporangiola of Thamnidiumelegans Link were bounded by a primary wall plus a thick, internalsecondary wall layer. In sporangia in late pre-cleavage, Golgi-likecisternae were associated with groups of cytoplasmic vesiclesof characteristic size and appearance which were not found insporangia containing large cleavage vesicles. In both sporangia and sporangiola, protoplast cleavage was effectedby enlargement of endogenous cleavage vesicles each containinga lining layer of variable appearance, mutual fusion of cleavagevesicle membranes and fusion of cleavage vesicle membranes withthe plasmalemma. Golgi-like cisternae and small vesicular profileswere present in sporangium protoplasts at all stages of cleavagevesicle enlargement. In sporangia, the columella zone was delimitedby cleavage vesicles and separated from the sporogenous zoneby a fibrillar wall. A similar wall, which sometimes protrudedto form a small columella, was formed in sporangiola. Recently delimited spore protoplasts were bounded by plasmalemmamembrane derived from cleavage vesicle bounding membrane andsporangium or sporangiolum plasmalemma and surrounded by aninvesting layer derived from cleavage vesicle lining material.The investing layer at first appeared single, but later twoelectron opaque profiles were discernible. The spore wall wasformed between the investing layer and the plasmalemma. Wallsof sporangia and sporangiola which contained fully formed sporesconsisted of the primary layers only.     

The Teliospores of Puccinia smyrnii (Uredinales)

The morphology of the teliospores of Puccinia smyrnii has been variously described as warted, or reticulate, or a combination of both patterns. Spores were examined by LM and SEM, and shown to be irregularly warted. The sequence of development of the spores was examined by TEM. Four phases of wall differentiation were recognised. The ornamentation results from a differential deposition of secondary wall components, which are concentrated into invaginations of the cytoplasm. The subsequent exsertion of these invaginations, and concomitant disappearance of the primary wall, reveal the irregular warts of the mature spore. This mode of ornament formation is compared with other rust spore forms, and contrasted with that already outlined for Puccinia chaerophylli, a truly reticulatespored Umbelliferous rust. Combined SEM and TEM observations suggest an explanation for the erroneous LM interpretations.     

SSP2 and OSW1, two sporulation-specific genes involved in spore morphogenesis in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae requires the synthesis of prospore membranes (PSMs) followed by the assembly of spore walls (SWs). We have characterized extensively the phenotypes of mutants in the sporulation-specific genes, SSP2 and OSW1, which are required for spore formation. A striking feature of the osw1 phenotype is asynchrony of spore development, with some spores displaying defects in PSM formation and others spores in the same ascus blocked at various stages in SW development. The Osw1 protein localizes to spindle pole bodies (SPBs) during meiotic nuclear division and subsequently to PSMs/SWs. We propose that Osw1 performs a regulatory function required to coordinate the different stages of spore morphogenesis. In the ssp2 mutant, nuclei are surrounded by PSMs and SWs; however, PSMs and SWs often also encapsulate anucleate bodies both inside and outside of spores. In addition, the SW is not as thick as in wild type. The ssp2 mutant defect is partially suppressed by overproduction of either Spo14 or Sso1, both of which promote the fusion of vesicles at the outer plaque of the SPB early in PSM formation. We propose that Ssp2 plays a role in vesicle fusion during PSM formation.     

A MORPHOMETRIC ANALYSIS OF CYTOLOGICAL CHANGES DURING SPORE MATURATION IN THE MYXOMYCETE DIDYMIUM IRIDIS

Ultrastructure of spore maturation in the myxomycete Didymium iridis was investigated using morphometric analytical techniques. Changes in actual volume (μm³) and relative volume (Vv) of nuclei, autophagic vacuoles, mitochondria, microbodies, lipid droplets, and spore wall were described for spores in three stages of development. Stage I spores were newly formed, surrounded only by the cell membrane. Stage II spores were approximately 1 hr older than Stage I spores and possessed surface spines, but little if any additional wall material. Stage III spores were 24 hr old and possessed a fully formed, multilayered wall. The results of this study indicate that spore maturation in D. iridis is a multistep process involving a decrease in spore volume and coordinated changes in specific organelle compartments. From Stage I to Stage III, mean spore volume decreased by more than 50%. Percent volume data (Vv) showed that Stage I spores allocated volume equally to all measured organelles except microbodies and the spore wall, the latter of which had not yet begun to develop. By Stage II, only the nucleus and spore wall showed significant changes in Vv values, both increasing. In terms of actual volume, the nucleus, autophagic vacuole and spore wall increased by Stage II. Between Stages II and III the cell wall was the only component to increase in volume, all others decreased in volume. Our data indicate a close relationship between a decrease in organelle volume and an increase in cell wall volume in the Stage III spore. The autophagic vacuole and the cell wall dominated the volume of the Stage III spore while the remaining volume was allocated unequally to the other components.     

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.     

Vacuole Partitioning during Meiotic Division in Yeast

We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y, were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.     

Ultrastructure and elemental composition of dormant and germinating Diplodia maydis spores.

The ultrastructure of Diplodia maydis spores was studied in thin sections with a transmission electron microscope. Storage vacuoles were evenly distributed in the two cells. Some of the vacuoles that contained a dense osmiophilic sphere(s) were surrounded by a membrane, and had membranous aggregates around their periphery. The sport wall was composed of an electron-dense layer and an electron-translucent layer. An inner cytoplasmic membrane was present. Dormant and germinating spores were studied with scanning electron microscopy and also with a Si (Li) energy-dispersive X-ray analyzer. The dormant spore was ovate and usually two-celled with a central septum. Germination proceeded via a germ tube from the side of one end of the cell. Of several methods for preparation of specimens for X-ray analysis studied, freeze-dried spores mounted on carbon stubs and then further carbon coated gave the best results. X-ray analyses revealed that spore populations contained large amounts of Si, P, Cl, and K, smaller amounts of S and Ca, and trace amounts of Mg and Al. Analyses of single spores revealed high K and Cl and low P and Mg at one end of the cell with concomitant low K and Cl and high P and Mg in the central portion and other end of the cell. In two-celled germinating spores, high K and Cl occurred in the end of the nongerminating spore cell, whereas the germinating cell contained high P and Mg and low K and Cl. X-ray image maps revealed that K and Cl were located together at one end of the spore.