Scanning Electron Microscopy of Spore Germination in Dictyostelium discoideum

The ellipsoidal dormant spores of Dictyostelium dicoideum prepared by freeze-drying had a uniform, compact appearance with fine wrinkles or ridges on the surface. Swollen spores were uneven in appearance, without fine wrinkles but with a seemingly expanded surface covering. The surfaces of the postgermination spore husks appeared unaltered except for a single straight exit slit along the longitudinal plane.     

Species of the genus Chloromyxum Mingazzini (Myxozoa: Myxosporea) infecting burbot (Lota lota L.)

Three different species of the genus Chloromyxum Mingazzini were found in burbot (Lota lota L.) collected in south-west Bohemia (Czechoslovakia). Comparison with existing records revealed that one species could be identified as C. pseudomucronatum Kashkovskiy, 1966. Found in the urinary bladder, it had subspherical spores with fine surface ridges and polysporic plasmodia. The two other myxosporea were established as new species: C. lenorae n. sp. was found in the kidney, renal corpuscles, renal tubules and interstitium, and had ellipsoid spores with surface ridges barely perceptible in the light microscope but clearly revealed by transmission electron microscopy. In the polysporic plasmodia, spores developed in pansporoblasts. C. reticulatum n. sp. was found in the gall bladder. It had polysporic plasmodia and spherical spores (average size 8.1 m in diameter) with a unique surface structures: elevated crests marking off irregular fields which appeared as a reticulum. In five of the fish infected with C. lenorae, bloodstream myxosporean stages of an extrasporogonic cycle were found. Further research is needed to determine whether they are stages of Sphaerospora cristata Shulman, 1962, a species also found in two of the burbot examined, or stages belonging to the Chloromyxum life cycle.     

A MUTANT OF DICTYOSTELIUM DISCOIDEUM CAPABLE OF DIFFERENTIATING WITHOUT MORPHOGENESIS

A mutant which is capable of differentiating into spores and stalk cells without forming a cell aggregate was isolated from the cellular slime mould, Dictyostelium discoideum. The mutant stopped developing at various stages, before formation of mature fruits, and the cells differentiated into spores and stalk cells at whichever stage the development stopped. Unaggregated cells also differentiated into spores or stalk cells, depending on the culture conditions; differentiation into spores predominated in nutrient rich medium, while differentiation into stalk cells predominated in nutrient poor medium. The ratio of spores to stalk cells or of prespores to total cells in cell masses depended on the terminal structures formed; the ratio was unusually high or unusually low in a structure which stopped developing before papilla formation, while the ratio was normal in a structure formed after that stage. When isolated from a cell mass, prespore cells of the mutant did not dedifferentiate or resumed vegetative growth, indicating that they had lost plasticity of differentiation. The conditioned medium in which the mutant cells had grown was effective in inducing differentiation of wild type slug cells into spore-like or stalk-like cells.     

THE EFFECTS OF CYCLIC AMP ON DIFFERENTIATION OF A MUTANT DICTYOSTELIUM DISCOIDEUM CAPABLE OF DEVELOPING WITHOUT MORPHOGENESIS

The dev 1510 mutant of Dictyostelium discoideum differs from the wild type in that unaggregated cells are capable of differentiating into either spores or stalk cells depending on the culture conditions (12). Taking advantage of this fact, the effects of cyclic AMP (cAMP) on differentiation of the mutant cells were examined under conditions that prevent normal morphogenesis. In the presence of low concentrations of exogenous cAMP, the cells differentiated into only stalk cells, whereas in the presence of high concentrations they differentiated into only spores. Untreated cells formed stalk cells, but this was inhibited by addition of phosphodiesterase, indicating that it was induced by a low concentration of cAMP which they produced themselves. Cyclic GMP and dibutyryl cAMP also induced spore formation though less effectively, while 5'AMP, ADP and ATP had no effect. During development, the cells increased in sensitivity to cAMP in that spore formation was induced at lower concentration of cAMP after 4 hr of starvation. Treatment of cells that had been starved for 6hr with 10⁻⁴M cAMP for as short a time as 30 min was enough to induce 8% of the cells to form spores.
The effects on cAMP-induced differentiation of chemicals that are known to influence development of the wild type were also examined. Both NH₄Cl and KCl inhibited cAMP-induced stalk formation, but had no effect on spore formation. In the presence of arginine, spore formation was induced at a lower concentration of cAMP with higher efficiency. CaCl₂, LiCl and KF had no effect on cAMP-induced differentiation.     

Scanning Electron Microscope Observations on Some Life History Stages of Carchesium polypinum (Ciliata,Peritrichia)1

SYNOPSIS. Sessile zooids, and mobile telotrochs and microgamonts of Carchesium polypinum (Protozoa, Ciliata, Peritrichia), were examined by scanning electron microscopy. The results were compared to earlier light and electron microscope studies in order to investigate structural changes concerned with adaptation and differentiation. Telotrochs and microgamonts always had a contracted peristome and usually had a long phalange of cilia. Striae around the contracted buccal apparatus in all 3 stages were convoluted and often had thickened margins; those in telotrochs and microgamonts had oral-aboral ectoplasmic cross-connections. Nonbuccal striae of telotrochs and microgamonts varied in structure and height differences between epiplasmic peaks and alveoli surface membranes. The number of striae were constant in all 3 stages. Pellicular pore structure did not vary in any of the stages examined and resembled parasomal sacs located near buccal structures. Fully relaxed sessile zooids had ectoplasmic ridges coursing from polykinety kinetosomes and cilia to an area in front of the ciliated portion of the haplokinety; these ridges were interpreted to be the interkinetal fibers. Telotroch bands of sessile zooids consisted of 2 or 3 parallel ectoplasmic ridges which circled the aboral region and contained structures resembling pores. Telotroch bands in telotrochs and microgamonts had 2 enlarged, parallel ectoplasmic ridges circling the aboral region; telotroch band cilia were found between these ridges. In addition, a fold-like, ectoplasmic structure extended beyond the 2 ridges and was located between the telotroch band cilia and the aboral ridge. The epiplasmic shelf surrounding the stalk in sessile zooids was enlarged in telotrochs, and cilia were seen in the scopula depression. No scopula organelle was seen in any microgamont.     

Disruption of multicellular organization in the cellular slime molds by cyclic AMP.

Addition of cyclic AMP causes disorder in the multicellular stage of a number of species of cellular slime molds. In those which produce fruits with cellular stalks, the addition of cyclic AMP stimulates prestalk cells to differentiate into mature stalk cells. Prespore cells do not differentiate into spores under the influence of cyclic AMP, most degenerate and seem to die. I hypothesize that the normal course of differentiation from vegetative cells is one leading to spores, but that cyclic AMP can divert this course to one leading to the stalk cell. Dibutyryl cyclic AMP, cyclic GMP and cyclic AMP disrupt slugs of Polysphondylium pallidum, while species of Dictyostelium are disrupted by only cyclic AMP. The multicellular stage of P. violaceum is unaffected by high concentrations of exogenous cyclic nucleotides. Cell organization of Acytostelium ellipticum, a species with an acellular stalk, was disrupted by cyclic AMP, but no stalk cells were formed; only spores.     

Scanning electron microscopy of spores of the myxosporidans Chloromyxum trijugum Kudo and Chloromyxum catostomi Kudo

Spores of Chloromyxum trijugum Kudo from Lepomis macrochirus Rafinesque and Chloromyxum catostomi Kudo from Notropis dorsalis (Agassiz) were obtained from infected gall bladders, glass-bead sonicated, and examined by scanning electron microscopy. Valves of C trijugum spores each have a thick ridge running parallel to the sutural ridge. Uncapped cnidocyst pores open into the extrasutural ridges. A pyriform structure of unknown function was observed at the posterior surface in some spores. Spore valves of C catostomi are sculptured with ridged striations running in various parallel and converging patterns over the entire surface. Cnidocyst pores open into ridges adjacent to the sutural plane. Glass bead sonication was found effective in polar filament extrusion. Discharged filaments were twisted along the long axis and partially coated with mucoid globules.     

Outside-in signaling of cellulose synthesis by a spore coat protein in Dictyostelium

The spore coat of Dictyostelium is formed de novo from proteins secreted from vesicles and cellulose synthesized across the plasma membrane as differentiating spores rise up the stalk. The mechanism by which these events are coordinated is not understood. In the course of experiments designed to test the function of the inner layer coat protein SP85 (PsB), expression of a specific partial length fragment was found to interrupt coat assembly after protein secretion and prior to cellulose synthesis in 85% of the cells. This fragment consisted of SP85's N-terminal domain, containing prespore vesicle targeting information, and its Cys-rich C1 domain. The effect of the NC1 fusion was not cell autonomous in interstrain chimeras, suggesting that it acted at the cell surface. SP85-null spores presented an opposite phenotype in which spores differentiated prematurely before reaching the top of the stalk, and cellulose was slightly overproduced in a disorganized fashion. A similar though less severe phenotype occurred when a fusion of the N and C2 domains was expressed. In a double mutant, absence of SP85 was epistatic to NC1 expression, suggesting that NC1 inhibited SP85 function. Together, these results suggest the existence of an outside-in signaling pathway that constitutes a checkpoint to ensure that cellulose synthesis does not occur until coat proteins are properly organized at the cell surface and stalk formation is complete. Checkpoint execution is proposed to be regulated by SP85, which is in turn under the influence of other coat proteins that interact with SP85 via its C1 and C2 domains.     

A new myxozoan from feral goldfish (Carassius auratus)

In February 2004, a mass die-off of common goldfish Carassius auratus L., presumptively caused by bacterial coldwater disease (Flavobacterium psychrophilum), occurred at Fern Ridge Reservoir, Oregon. A range of size classes was affected, but all mature fish were female and all fish were infected with a single myxozoan, Chloromyxum auratum n. sp. No histological changes were observed associated with the parasite. Infection was represented by mictosporic plasmodia and free-floating spores in the gall bladder. Parasite spores were nearly spherical, 13.6 microm long x 12.6 microm wide x 13.1 microm thick, and possessed 4 equal-sized polar capsules. Spores had a coglike appearance in apical view because of distinct ridges 2.1 microm high protruding from the valve cells. There were 6-9 extrasutural ridges per valve (15-20 ridges per spore), aligned along the longitudinal axis, with some branching, and convergence at both poles. Morphologically, spores identified most closely with Chloromyxum cristatum Léger, 1906; however, 18S rDNA sequence data indicated only 97.5% similarity over 2,076 bp with Chloromyxum cyprini, the only synonym of C. cristatum for which DNA data are available; additional sequence data may reveal the other synonyms to be distinct species. This is the first record of a species of Chloromyxum from goldfish.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Studies on the bacterial spore coat 6 effects of alkali extraction on the spore of Bacillus thiaminolyticus.

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three district layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and than the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.     

Studies on the bacterial spore coat. 6. Effects of alkali extraction on the spore of Bacillus thiaminolyticus

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three distinct layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and then the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.     

Conditions that elevate intracellular cyclic AMP levels promote spore formation in Dictyostelium

We have been using sporogenous mutants of Dictyostelium discoideum strain V12M2 to study regulation of cell fate during terminal differentiation of spores and stalk cells. Analyses of intracellular cAMP accumulation, cAMP secretion, cAMP binding to cell surface receptors, and chemotactic sensitivity to exogenous cAMP during aggregation showed that all of these functions were identical in V12M2 and HB200, a sporogenous mutant. We used several methods of altering intracellular cAMP levels in HB200 cells to test the hypothesis that intracellular cAMP levels affect cell fate. First, HB200 amoebae were treated with 5 mM caffeine for 4 h during growth, washed, and allowed to develop in the absence of caffeine. Treated cells had normal levels of intracellular cAMP and adenylate cyclase activities at the beginning of differentiation; by 6 h development, they contained two to three times more intracellular cAMP and two times more GTP-dependent adenylate cyclase activity than untreated cells. However, their level of basal Mn++-dependent adenylate cyclase activity was the same as untreated controls. Thus, treatment of growing HB200 amoebae with caffeine for only 4 h leads to hyperinduction of a GTP-dependent regulator (or inhibition of a negative regulator) of adenylate cyclase during subsequent differentiation, without induction of basal activity. The fraction of amoebae forming spores increased twofold when HB200 amoebae were treated with caffeine during growth. Spore (but not stalk cell) differentiation by such treated cells was blocked by inhibitors of cAMP accumulation. Second, cells grown on nutrient agar accumulated higher levels of intracellular cAMP and formed more spores in vitro than cells grown in shaken suspension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structure of Chloromyxum menticirrhi n. sp. (Myxozoa) infecting the urinary bladder of the marine teleost Menticirrhus americanus (Sciaenidae) in Southern Brazil

A myxosporidian was found in the urinary bladder of the teleost Menticirrhus americanus Linnaeus, 1758 (Sciaenidae) collected from the South Atlantic coast of Brazil. Polysporic amoeboid plasmodia containing sporoblasts, developing pansporoblasts and spores were free in the bladder lumen. The prevalence of infection was 17.64% (15/85). Unfixed spores were spherical to subspherical, on average 10.5 μm long, 9.8 μm wide and 10.1 μm thick (n=25), and fixed spores measured 10.1×9.5×9.7 μm. The two spore valves were of equal size and each possessed prominent sutural lines and about 41 (37–45) surface ridges aligned parallel with the suture line. These ridges gave transverse sections a cog-wheel-like outline. The spores contained four pyriform polar capsules of equal size (3.20×2.0 μm) (n=25) (fixed), each with a polar filament having 3–4 (rarely 5) coils. The binucleate sporoplasm was irregular in shape, with granular matrix and randomly distributed dense bodies. The shape and dimensions of the spore, as well as the number, position and arrangement of the surface ridges, polar capsules and polar filament indicate that this is a new species, herein designated Chloromyxum menticirrhi. The gill, liver, gall bladder and intestine of the host showed no abnormalities.     

Development of the Mycetozoan Echinosteliopsis oligospora*

SYNOPSIS. The mycetozoan genus Echinosteliopsis, resembling the myxomycete Echinostelium in some of its features, is described. The single species, E. oligospora Reinhardt & Olive, forms small sporocarps which consist of a basal disk, stalk and a sporangium with only 1–8 spores. Spores form progressively, not simultaneously, by segmentation. The spores germinate to release non-flagellate amebae which, in liquid, assume a characteristic broad, fan shape. Each ameba has one or more nuclei. The nucleus is distinctive because of refractile, globular to elongate peripheral bodies which cytochemical tests indicate to be primarily RNA. At the time of nuclear division the characteristic RNA bodies disappear and, as observed with the phase microscope and in stained preparations, optically dense material accumulates in the middle area of the nucleus. Threads, either a spindle or actual chromatin, can be seen attached to the nuclear membrane. The threads separate to opposite poles as the nucleus elongates. During this division process the nuclear membrane apparently remains intact. Synchronous binucleate divisions, as well as a tripolar nuclear division, have been observed. Uninucleate and synchronous binucleate divisions may or may not be followed by cytokinesis. The absence of cell division after nuclear division leads to the production of cells with varying numbers of nuclei. Nuclear divisions in early sporangial stages and in spores have not been observed. The spores are uni- to multinucleate. In 8-spored sporangia and in most 4-spored sporangia there is a characteristic small “stalk spore” at the apex of the stalk. The stalk spore germinates slowly, if at all, but the larger spores germinate readily. No evidence of a sexual process has been found.     

Relations between phenotypic changes of spores and biofilm production by Bacillus atrophaeus ATCC 9372 growing in solid-state fermentation

Bacillus spp. spores are usually obtained from strains cultivated in artificial media. However, in natural habitats, spores are predominantly formed from bacteria present in highly surface-associated communities of cells. Solid-state fermentation (SSF) is the culture method that best mimetizes the natural environment of many microorganisms that grow attached to the surface of solid particles. This study aims to confirm that sporulation through SSF of Bacillus atrophaeus occurs by biofilm formation and that this model of fermentation promotes important phenotypic changes in the spores. Sporulation on standard agar and by SSF with sand and sugarcane bagasse as support was followed by a comparative study of the formed spores. Growth characteristics, metabolic and enzymatic profiles confirmed that sporulation through SSF occurs by biofilm formation promoting important phenotypic changes. It was possible to demonstrate that spores coat had different structure and the presence of ridges only on SSF spores' surface. The sporulation conditions did not affect the dry-heat spore resistance. The type of support evaluated also influenced in the phenotypic alterations; however, the used substrates did not cause interference. This work provides novel information about B. atrophaeus response when submitted to different sporulation conditions and proposes a new concept about bacterial biofilm formation by SSF.     

SPORE WALL ULTRASTRUCTURE IN FOUR SPECIES OF THE LIVERWORT RICCIA

Mature spores of Riccia californica, R. campbelliana, R. sorocarpa, and R. trichocarpa (Marchantiales, Ricciaceae) were examined with both scanning and transmission electron microscopes. The thick-walled spores have species-specific surface sculpturing and similar internal structure. Major surface features include ridges and depressions in areolate or irregular configurations. Finer surface features such as tubercles or papillae may ornament the ridges or depressions. Internally, the sporoderm consists of a thin intine and a three-layered, lamellate exine. The innermost exine lamellae are thin and closely spaced, giving this layer a fibrillar appearance. Lamellae of the middle region are thick in section and have a relatively thick, electron-dense coating. The outermost lamellae are of intermediate thickness and also have a relatively thick electron-dense coating. Lamellae of all regions frequently split, branch and anastomose. The outermost lamella forms the surface features of the spore. Interior lamellae follow the contour of the outermost lamella. Pores usually occur in these four species at the junctures of the triradiate ridge arms and equatorial rim. Pores do not penetrate the entire exine, and they represent localized areas where the outermost group of lamellae have not been formed.     

水蕨孢子壁的形成和发育

利用光镜、扫描电镜和透射电镜对水蕨科(Parkeriaceae)水蕨(Ceratopteris thalictroides (L.) Brongn.)孢子壁的形成和发育进行了研究。结果表明, 水蕨孢子呈辐射对称, 三裂缝, 表面具肋条状纹饰。孢子壁由内壁、外壁和周壁三部分构成。在四分体阶段外壁已基本形成, 其外壁显著, 表面光滑, 质地均匀, 由孢粉素形成, 外壁厚约3-5 μm, 脊高约5-7 μm。周壁由绒毡层残余物在外壁表面沉积形成, 较薄, 厚度只有0.1 μm, 表面具有杆状突起。研究结果对揭示孢子纹饰和孢子壁各层的形成过程、来源和稳定性有一定的意义, 并为蕨类植物孢粉学和系统学研究提供基础资料。     

水蕨孢子壁的形成和发育

利用光镜、扫描电镜和透射电镜对水蕨科（Parkeriaceae）水蕨（Ceratopteris thalictroides（L．）Brongn．）孢子壁的形成和发育进行了研究。结果表明,水蕨孢子呈辐射对称,三裂缝,表面具肋条状纹饰。孢子壁由内壁、外壁和周壁三部分构成。在四分体阶段外壁已基本形成,其外壁显著,表面光滑,质地均匀,由孢粉素形成,外壁厚约3—5μm,脊高约5—7μm。周壁由绒毡层残余物在外壁表面沉积形成,较薄,厚度只有0．1μm,表面具有杆状突起。研究结果对揭示孢子纹饰和孢子壁各层的形成过程、来源和稳定性有一定的意义,并为蕨类植物孢粉学和系统学研究提供基础资料。