Spore Wall Research Articles

Ultrastructural characterization of Acarispora falculifera n.gen., n.sp., a new microsporidium (Opisthokonta: Chytridiopsida) from the feather mite Falculifer rostratus (Astigmata: Pterolichoidea).

Only about 20 species of microsporidia have been described from mites. All except one species produce typical spores with a long polar filament and a polaroplast. This paper is the first study of an atypical microsporidium infection in a feather mite (Falculifer rostratus). The infection of the pigeon feather mite is restricted to the colon epithelium where it leads to hypertrophy of the concerned cells. During sporogony, a multinucleate plasmodial aggregate is formed within a sporont (endogenous sporogony resulting in a polysporophorous vesicle). The cisterns delimiting the single sporoblasts later form the spore walls. Sporogonial stages are in direct contact to the host cell cytoplasm. Merogonial stages were not present. Spores are tiny (3.6 μm × 2.6 μm), broad oval in form and monokaryotic. The spore wall of mature spores consists of a three-layered endospore and a thin, electron-dense, wavy exospore. The polar filament is anisofilar and completely coiled in 3-4 turns. In cross-sections, it has a star-like appearance because the electron-dense core forms rounded compartments of lucent material at its surface. In superficial sections, this results in a honeycomb-like pattern. A polaroplast is missing. The polar filament arises subapically at a polar sac that lacks an internal anchoring disk. These atypical spore structures clearly classify the species from the feather mite as a member of the order Chytridiopsida. It could not be clearly affiliated to one of the known genera, so we created a new genus, Acarispora, with the species A. falculifera.

Ultrastructure and phylogeny of Glugea nagelia sp. n. (Microsporidia: Glugeidae), infecting the intestinal wall of the yellowfin hind, Cephalopholis hemistiktos (Actinopterygii: Serranidae), from the Red Sea.

A new microsporidian species of the genus Glugea Thélohan, 1891 parasitising the marine teleost fish Cephalopholis hemistiktos Rüppell, collected from the Red Sea in Saudi Arabia, is described on the basis of microscopic and molecular procedures. Spherical and whitish xenoma were observed adhering to the intestinal wall. The numerous spores contained within these xenoma, were ovoid to pyriform and measured 4.3-6.0 µm (5.1 µm) in length and 1.8-2.9 µm (2.2 µm) in width. The spore's wall was composed of two thick layers, which were thinner in the area contacting the anchoring disk. The latter appeared at the spore's anterior pole, in an eccentric position to the longitudinal axis. A lamellar polaroplast surrounded the uncoiled portion of the polar filament projected to the basal region of the spore, giving rise to 26-29 turns with winding from the base to the anterior zone of the spore. The posterior vacuole, located at the spore's posterior pole, and surrounded by the polar filament coils, was irregular and composed of light material. Molecular analysis of the rRNA genes, including the ITS region, was performed using maximum parsimony, neighbour-joining and maximum likelihood methods. The ultrastructural features observed, combined with the phylogenetic data analysed, suggest this parasite to be a new species of the genus Glugea. This is the first species of this genus to be reported from Saudi Arabia and is herein named Glugea nagelia sp. n.

L-cysteine induced hemin/G-quadruplex concatemers electrocatalytic amplification with Pt-Pd supported on fullerene as a nanocarrier for sensing the spore wall protein of Nosema bombycis.

This work described a pseudobienzyme electrocatalytically amplified immunosensor for sensing the spore wall protein of Nosema bombycis by adopting L-cysteine as the electrocatalytic substance to generate H2O2in situ with the aid of hemin/G-quadruplex concatemers loaded onto C60@Pt-Pd. The hemin/G-quadruplex here is also acting as the probe for electrochemical signal output.

Synthesis of the spore envelope in the developmental life cycle of Streptomyces coelicolor

Members of the family of Streptomycetaceae, the main producer of antibiotics and other secondary metabolites, are Gram-positive multi-cellular soil bacteria with a complex life cycle. By apical tip extension Streptomyces coelicolor forms a multiply branching vegetative mycelium penetrating the substrate. Upon nutrient limitation, a hydrophobic aerial mycelium is erected, which eventually develops into a regular chain of spores that are able to survive detrimental environmental conditions.Morphological differentiation involves a switch in the peptidoglycan synthesizing machinery. Whereas apical tip extension is directed by the so-called polarisome, sporulation septation and synthesis of the thickened spore wall involves a multi-protein complex, which resembles the elongasome of rod-shaped bacteria. The Streptomyces spore wall synthesizing complex (SSSC) does not only direct synthesis of the peptidoglycan layer but is also involved in the incorporation of anionic spore wall glycopolymers, which contribute to the resistance of spores. The SSSC also contains eukaryotic type serine/threonine kinases which might control its activity by protein-phosphorylation.

Detection of Tilletia controversa using immunofluorescent monoclonal antibodies.

Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries. The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 2.0 μg ml(-1) of the D-1 monoclonal antibody. This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores. This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved.

Trichioides iridescens, a new genus and new species (incertae sedis in Myxomycetes)

A new monotypic myxomycete genus Trichioides, with type species T. iridescens, originating from the Northwestern Caucasus and Sakhalin Island is described, discussed and illustrated. In the absence of molecular sequence data, the genus Trichioides is temporarily placed in an incertae sedis position among Myxomycetes. This proposed enigmatic genus has morphological affinities with some genera of Trichiales and Liceales from the phylogenetic clade Lucisporidia, and shows the following unique combination of characters: a thick transparent gelatinous layer covering the whole sporocarp up to the final stage of sporogenesis; tubular, slightly curved, simple or forked bristle-like capillitium threads attached to the base of the sporotheca, sometimes to the inner surface of the peridium, ornamented by poorly or well-defined spiral bands; light greyish yellowish brown by transmitted light and smooth spores with spore wall of uniform thickness. Light and scanning electron micrographs of relevant morphological details are included.

Spore wall traits of ectomycorrhizal and saprotrophic agarics may mirror their distinct lifestyles

Although fungal spores are tiny compared to plant seeds, their morphological variability is enormous, which points toward selective forces. We investigated the frequency of ornamentation, thick walls, pigmentation and germ pores of spores of ectomycorrhizal and saprotrophic agarics. We hypothesised that these traits are shaped by the needs of these distinct lifestyles. All traits showed a strong phylogenetic signal; we therefore applied a phylogenetically informed statistical analysis. There was a significantly higher occurrence of spore ornamentation in ectomycorrhizal agarics and a higher occurrence of thick-walled spores in saprotrophic agarics. The interplay between thick-walled and pigmented spores and the occurrence of germ pores was only significant for saprotrophs. We argue that ornamentation is probably important to ectomycorrhizal fungi for dispersal by soil invertebrates, whereas pigmented thick walls and germ pores would be more advantageous for predominantly r-selected saprotrophic agarics exposed to hazardous environments and in need of quick germination success.

Does spore ultrastructure mirror different dispersal strategies in mosses? A study of seven iberian orthotrichum species.

Most mosses have xerochastic dispersal (i.e., they open their capsules when conditions are dry), which is thought to favor long-distance dispersal. However, there are several species that use a hygrochastic strategy: spores are dispersed when conditions are wet. The significance of this strategy in the Mediterranean region is unknown. In this study, we explored whether ultrastructural features related to differences in spore resistance may explain these different strategies of spore dispersal. To this end, we examined the ultrastructural features of the spores of seven closely related species in the moss genus Orthotrichum. These species all grow as epiphytes in sub-Mediterranean forests, and the group includes both xerochastic and hygrochastic members. First, we found that the spore wall layers exhibit several features previously undescribed in mosses. Second, we discovered that there are only subtle differences in spore ultrastructure with regards to spore wall thickness, the degree of plastid development, or the storage substances used. We suggest that the hygrochastic dispersal in mosses from Mediterranean environments might be related to a safe-site strategy, rather than to drought avoidance, and we underscore the necessity of conducting spore ultrastructural studies on a greater number of bryophyte species.

Callose is integral to the development of permanent tetrads in the liverwort Sphaerocarpos.

A striking feature of the liverwort Sphaerocarpos is that pairs of male and female spores remain united in permanent tetrads. To identify the nature of this phenomenon and to test the hypothesis that callose is involved, we examined spore wall development in Sphaerocarpos miche lii, with emphasis on the appearance, location and fate of callose vis-à-vis construction of the sculptoderm. All stages of sporogenesis were examined using differential interference contrast optics, and aniline blue fluorescence to locate callose. For precise localization, specimens were immunogold labeled with anti-callose antibody and observed in the transmission electron microscope. Callose plays a role in Sphaerocarpos spore wall development not described in any other plant, including other liverworts. A massive callose matrix forms outside of the sculptured sporocyte plasmalemma that predicts spore wall ornamentation. Consequently, layers of exine form across adjacent spores uniting them. Spore wall development occurs entirely within the callose and involves the production of six layers of prolamellae that give rise to single or stacked tripartite lamellae (TPL). Between spores, an anastomosing network of exine layers forms in lieu of intersporal septum development. As sporopollenin assembles on TPL, callose progressively disappears from the inside outward leaving layers of sporopollenin impregnated exine, the sculptoderm, overlying a thick fibrillar intine. This developmental mechanism provides a direct pathway from callose deposition to sculptured exine that does not involve the intermediary primexine found in pollen wall development. The resulting tetrad, encased in a single wall, provides a simple model for development of permanent dyads and tetrads in the earliest fossil plants.

Characterization of a novel spore wall protein NbSWP16 with proline-rich tandem repeats from Nosema bombycis (microsporidia).

Nosema bombycis, a pathogen of silkworm pebrine, is an obligate unicellular eukaryotic parasite. It is reported that the spore wall proteins have essential functions in the adherence and infection process of microsporidia. To date, the information related to spore wall proteins from microsporidia is still limited. Here, a 44 kDa spore wall protein NbSWP16 was characterized in N. bombycis. In NbSWP16, a 25 amino acids signal peptide and 3 heparin binding motifs were predicted. Interestingly, a region that contains 3 proline-rich tandem repeats lacking homology to any known protein was also present in this protein. The immunofluorescence analysis (IFA) demonstrated that distinct fluorescent signals were detected both on the surface of mature spores and the germinated spore coats. Immunolocation by electron microscopy revealed that NbSWP16 localized on the exospore regions. Finally, spore adherence analysis indicated that spore adherence to host cell was decreased more than 20% by anti-NbSWP16 blocking compared with the negative control in vitro. In contrast with anti-NbSWP16, no remarkable decrement inhibition was detected when antibodies of NbSWP16 and NbSWP5 were used simultaneously. Collectively, these results suggest that NbSWP16 is a new exospore protein and probably be involved in spore adherence of N. bombycis.

Glomus tetrastratosum, a new species of arbuscular mycorrhizal fungi (Glomeromycota)

Morphological studies of spores and mycorrhizal structures and phylogenetic analyses of sequences of the partial small subunit (SSU) rRNA gene, the whole internal transcribed spacer (ITS) rDNA region, including the 5.8S rRNA gene, and the partial large subunit (LSU) rRNA gene of an arbuscular mycorrhizal fungus (Glomeromycota) proved that it is an undescribed Glomus sp. of the family Glomeraceae. The species, named here G. tetrastratosum, forms spores in loose clusters and singly in soil. The spores are pastel yellow to brownish yellow, globose to subglobose, 55–240 μm diam (mean 136 μm), rarely ovoid, 90-135 × 110-150 μm, and have a subtending hypha with a pore that is open or occluded by a septum. The spore wall of G. tetrastratosum consists of four layers: a mucilaginous, hyaline layer 1 which stains in Melzer's reagent, a unit, hyaline, permanent layer 2, a laminate, colored layer 3 and a flexible to semi-flexible, colored layer 4. In the field G. tetrastratosum was frequently associated with roots of four plant species colonizing maritime sand dunes of the Słowiński National Park located in northern Poland. In single-species cultures with Plantago lanceolata as host plant G. tetrastratosum formed mycorrhiza with arbuscules and vesicles.

A new species of Glugea Thélohan, 1891 in the red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae) from China.

A new microsporidian species is described from farmed red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae). Large numbers of spherical whitish xenomas were observed throughout the visceral organs of the host. Histological examination showed that the microsporidia caused several xenomas that were embedded in the intestinal muscularis externa or submucosa. Light and transmission electron microscopy examination of the spores also revealed morphological features typical of species of Glugea Thélohan, 1891. This microsporidian parasite has two different types of mature spores: microspores and macrospores. The spores are elongate-ovoid, with a large posterior vacuole. The polaroplast is bi-partite, with anterior and posterior parts comprising densely packed lamellae and loose membranes, respectively, and occupies approximately the anterior half of the spore. The polar filament is anisofilar, with 12-13 coils in a single layer almost touching the posterior spore wall. Comparison of the small subunit rDNA sequences revealed 92.7-98.1% identity with the sequences available from other Glugea spp. from piscine hosts. Phylogenetic analysis demonstrated that the microsporidian species studied clustered within the Glugea clade with strong support. Based on the differences in the morphological characteristics and molecular data, the microsporidian infecting P. major is considered to represent a species new to science, Glugea pagri n. sp.

Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway

The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

Developmental stage dependent metabolic regulation during meiotic differentiation in budding yeast.

BackgroundThe meiotic developmental pathway in yeast enables both differentiation of vegetative cells into haploid spores that ensure long-term survival, and recombination of the parental DNA to create genetic diversity. Despite the importance of proper metabolic regulation for the supply of building blocks and energy, little is known about the reprogramming of central metabolic pathways in meiotically differentiating cells during passage through successive developmental stages.ResultsMetabolic regulation during meiotic differentiation in budding yeast was analyzed by integrating information on genome-wide transcriptional activity, 26 enzymatic activities in the central metabolism, the dynamics of 67 metabolites, and a metabolic flux analysis at mid-stage meiosis. Analyses of mutants arresting sporulation at defined stages demonstrated that metabolic reprogramming is tightly controlled by the progression through the developmental pathway. The correlation between transcript levels and enzymatic activities in the central metabolism varies significantly in a developmental stage-dependent manner. The complete loss of phosphofructokinase activity at mid-stage meiosis enables a unique setup of the glycolytic pathway which facilitates carbon flux repartitioning into synthesis of spore wall precursors during the co-assimilation of glycogen and acetate. The need for correct homeostasis of purine nucleotides during the meiotic differentiation was demonstrated by the sporulation defect of the AMP deaminase mutant amd1, which exhibited hyper-accumulation of ATP accompanied by depletion of guanosine nucleotides.ConclusionsOur systems-level analysis shows that reprogramming of the central metabolism during the meiotic differentiation is controlled at different hierarchical levels to meet the metabolic and energetic needs at successive developmental stages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0060-x) contains supplementary material, which is available to authorized users.

Cross-species functional complementation of cellulose synthase during the development of cellular slime molds.

Cellulose is a major and important component of the extracellular matrix during the development of Dictyostelium discoideum. Upon starvation, solitary amoebae of D.discoideum gather and form fruiting bodies in which cells differentiate into stalk cells and spores. The stalk tubes and walls of spores and stalk cells are made of cellulose. In the genus Acytostelium, however, all cells are destined to become spores and the stalks comprise only a cellulose tube, suggesting species-specific regulation of cellulose synthesis. In this study, we cloned a putative cellulose synthase gene (cesA) of Acytostelium subglobosum and performed comparative analyses with the D.discoideum cellulose synthase gene (dcsA). Although the deduced amino acid sequences were highly conserved between cesA and dcsA, the numbers of transmembrane spans preceding the catalytic domain were dissimilar; 2 and 3, respectively. Since ectopic expression of cesA in dcsA(-) null cells failed to restore the developmental defects of the mutant, we constructed a series of chimerical genes for complementation analyses and found that the catalytic domain of cesA was functional in D.discoideum cells if the preceding transmembrane region was swapped with dcsA. The non-functional products that contained the cesA-derived transmembrane region were localized to lysosomes. These results indicate that the transmembrane region of cellulose synthase is essential for proper accumulation of cellulose during the development of D.discoideum and that its differential localization in A.subglobosum may be related to the characteristic morphogenesis in this species.

Glomus rubiforme, an arbuscular mycorrhizal fungus new to the mycota of Poland

<i>Glomus rubiforme</i> is described and illustrated. as well as its occurrence in Poland and in the world is presented. <i>Glomus rubiforme</i> forms pale yellow to light brown spores arranged in blackberry-like sporocarps. The spores develop from a centrally positioned, inflated, thick-walled cell. The spore wall consists of two layers: a sloughing, hyaline outer layer adherent to a coloured, laminated layer. <i>Glomus rubiforme</i> is a new arbuscular mycorrhizal fungus to the mycota of Poland.

The Social Amoeba Polysphondylium pallidum Loses Encystation and Sporulation, but Can Still Erect Fruiting Bodies in the Absence of Cellulose

Amoebas and other freely moving protists differentiate into walled cysts when exposed to stress. As cysts, amoeba pathogens are resistant to biocides, preventing treatment and eradication. Lack of gene modification procedures has left the mechanisms of encystation largely unexplored. Genetically tractable Dictyostelium discoideum amoebas require cellulose synthase for formation of multicellular fructifications with cellulose-rich stalk and spore cells. Amoebas of its distant relative Polysphondylium pallidum (Ppal), can additionally encyst individually in response to stress. Ppal has two cellulose synthase genes, DcsA and DcsB, which we deleted individually and in combination. Dcsa- mutants formed fruiting bodies with normal stalks, but their spore and cyst walls lacked cellulose, which obliterated stress-resistance of spores and rendered cysts entirely non-viable. A dcsa-/dcsb- mutant made no walled spores, stalk cells or cysts, although simple fruiting structures were formed with a droplet of amoeboid cells resting on an sheathed column of decaying cells. DcsB is expressed in prestalk and stalk cells, while DcsA is additionally expressed in spores and cysts. We conclude that cellulose is essential for encystation and that cellulose synthase may be a suitable target for drugs to prevent encystation and render amoeba pathogens susceptible to conventional antibiotics.

Anthracoidea transberingiana, a new smut species on Carex pauciflora from Beringia

The new smut fungus, Anthracoidea transberingiana attacking ovaries of Carex pauciflora, is described and illustrated from Beringia (Alaska and Kamchatka). The new species is compared with Anthracoidea caricis-pauciflorae infecting the same host plant species predominantly in northern Europe, from which it differs in having smaller spores, thinner spore walls, 2–3 weakly visible internal swellings and somewhat less prominent spore ornamentation.

English

Microsporidia are obligate intracellular, eukaryotic, spore-forming parasites. The environmentally resistant spores, which harbor a rigid cell wall, are critical for their survival outside their host cells and host-to-host transmission. The spore wall comprises two major layers: the exospore and the endospore. In Nosema bombycis, five spore wall proteins have been identified which contain two endosporal proteins (SWP25 and SWP30) and three exosporal proteins (NbSWP5, SWP26 and SWP32). In the current study, we identified a novel endosporal protein NbSWP12 with calculated molecular mass of 25.56 kDa and pI of 6.69 using SDS-PAGE and MALDI-TOF MS technique. Followed by gene cloning and protein expression, polyclonal antibody production, indirect immunofluorescence antibody test, and immunoelectron microscopy analysis, the results indicate that this protein is localized to the endospore and has no obvious enhancement on adherence to host cells. The characterization of this novel spore wall protein from N. bombycis may facilitate our further investigation of the relationship between N. bombycis and its host, Bombyx mori.   Key words: Microsporidia, Nosema bombycis, Spore wall protein, NbSWP12.

Applied usage of yeast spores as chitosan beads.

In this study, we present a nonhazardous biological method of producing chitosan beads using the budding yeast Saccharomyces cerevisiae. Yeast cells cultured under conditions of nutritional starvation cease vegetative growth and instead form spores. The spore wall has a multilaminar structure with the chitosan layer as the second outermost layer. Thus, removal of the outermost dityrosine layer by disruption of the DIT1 gene, which is required for dityrosine synthesis, leads to exposure of the chitosan layer at the spore surface. In this way, spores can be made to resemble chitosan beads. Chitosan has adsorptive features and can be used to remove heavy metals and negatively charged molecules from solution. Consistent with this practical application, we find that spores are capable of adsorbing heavy metals such as Cu(2+), Cr(3+), and Cd(2+), and removal of the dityrosine layer further improves the adsorption. Removal of the chitosan layer decreases the adsorption, indicating that chitosan works as an adsorbent in the spores. Besides heavy metals, spores can also adsorb a negatively charged cholesterol derivative, taurocholic acid. Furthermore, chitosan is amenable to chemical modifications, and, consistent with this property, dit1Δ spores can serve as a carrier for immobilization of enzymes. Given that yeast spores are a natural product, our results demonstrate that they, and especially dit1Δ mutants, can be used as chitosan beads and used for multiple purposes.