Spore Structure Research Articles

Bacterial endospores the ultimate survivors

Bacterial endospores are the most resistant living structures, known. Their high degree of resistance to many treatments (including heat and UV) is due to many factors and is governed by the unique spore structure. Spore core dehydration is a primary determinant of heat resistance. A specialised cell wall peptidoglycan layer, termed the cortex maintains dehydration. During germination the spore cortex is hydrolysed to allow outgrowth of the new vegetative cell. The germination-specific lytic enzymes (GSLEs) responsible for cortex hydrolysis have recently begun to be identified. Their position, in the dormant spore, outside the protection of the dehydrated core requires a special mechanism of heat resistance, which is so far unknown. Recent evidence has begun to elucidate the molecular basis not only for resistance but also how it is acquired, maintained and lost during germination. Understanding of resistance mechanisms may allow the design of novel sporicidal treatments.

SpoVID guides SafA to the spore coat in Bacillus subtilis.

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

Antagonism of the Two-Needle Pine Stem Rust FungiCronartium flaccidumandPeridermium pinibyCladosporium tenuissimumIn Vitro and In Planta

ABSTRACT Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degrees C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Palynology of South American Argyrochosma and Notholaena (Pteridaceae) species

The palynology of the South American taxa Argyrochosma chilensis (J. Rémy) Windham, A. nivea (Poir.) Windham, var. nivea , var. flava (Hook.) Ponce, var. tenera (Hook. ex Gillies) Ponce and A. stuebeliana (Hieron.) Windham; N. sulphurea (Cav.) J. Sm. and N. galapagensis Weath. & Svenson has been investigated. These species show differences in spore size, shape, sporoderm thickness and structure. In both genera, the exospore is psilate and the perispore has either cristate-reticulated or reticulated ornamentation, with a complex structure and stratification. The perispore presents several characteristics useful in establishing differences valuable for taxonomic purposes. The investigated species of Argyrochosma have a perispore with a middle stratum composed of radial rodlets, and an outer continuous stratum, which bears the elements of the sculpture. These are independent of the inner structure. The spores of the investigated Notholaena species have a thick, complex, three-stratified perispore, with an outer discontinuous stratum. The middle stratum is composed of threads ramified at several levels and laterally fused. The elements of the sculpture show continuity with those of the structure. Granular material of sporopolleninous composition fills the lumina at the surface. Abnormal spores and 32 spores per sporangium were frequent in specimens of A. nivea var. nivea and N. sulphurea .

Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy

High resolution solid-state nuclear magnetic resonance spectroscopy (NMR) in combination with transmission electron microscopy (TEM) of spores of Bacillus cereus, an outer coatless mutant B. subtilis 322, an inner coatless mutant B. subtilis 325 and of germinated spores of B. subtilis CMCC 604 were carried out. Structural differences in the coats, mainly protein of spores were reflected by NMR spectra which indicated also differences in molecular mobility of carbohydrates which was partially attributed to the cortex. Dipicolinic acid (DPA) of spores of B. cereus displayed a high degree of solid state order and may be crystalline. Heat activation was studied on spores of B. subtilis 357 lux+ and revealed a structural change when analysed by TEM but this was not associated with increases in molecular mobility since no effects were measured by NMR.

A two-dimensional protein gel electrophoresis study of the heat stress response of Bacillus subtilis cells during sporulation.

The heat resistance of spores of Bacillus subtilis formed at 30 degrees C was enhanced by pretreatment at 48 degrees C for 30 min, 60 min into sporulation, for all four strains examined. High-resolution two-dimensional gel electrophoresis showed the generation and/or overexpression of 60 proteins, 11 of which were specific to heat shock, concurrent to this acquired thermotolerance. The greatest number of new proteins was observed between 30 and 60 min after heat shock, and the longer the time between exponential growth and heat treatment, the fewer differences were observed on corresponding protein profiles. The time at which heating produced the maximum increase in spore resistance and the most new proteins on two-dimensional gels occurred before alkaline phosphatase and dipicolinic acid production and corresponded to stage I or II of sporulation. The stress proteins formed disappeared later in sporulation, suggesting that heat shock proteins increase spore heat resistance by altering spore structure rather than by repairing heat damage during germination and outgrowth.

Structural analysis of spores of Bacillus subtilis during germination and outgrowth

Morphological changes of spores of Bacillus subtilis 604 during germination were investigated by phase contrast, interference contrast, environmental electron microscopy and light scattering. Dormant, phase bright spores displayed no significant size difference to germinated, phase-dark spores. After completion of an optical phase change, collapse of the whole spore structure was observed by interference contrast. Similar morphological changes were seen by environmental electron scanning microscopy where phase bright spores appeared as rods and phase dark spores displayed a dark, collapsed halo in the centre. Microscopical techniques and light scattering indicated further no significant swelling of spores during germination. Outgrowth of vegetative cells was monitored with individual spores by light microscopy. Interference contrast was more suitable than phase contrast to study outgrowth stages because of better differentiation between germinated spores, outgrowing cells and residual spore coats.

Structural changes of spores of tree fungal pathogens after treatment with the designed antimicrobial peptide D2A21

In vitro effects of the antimicrobial synthetic D2A21 peptide on the structure of spores of four fungal pathogens causing important tree diseases were examined by microscopy in parallel with tests to measure inhibition of spore germination. With light microscopy, the use of SYTOX®green stain indicated that the peptide rapidly altered the plasma membrane of conidia of three Ascomycetes: Gremmeniella abietina (Lagerberg) Morelet var. abietina Petrini et al., Ophiostoma ulmi (Buism.) Nannf., and Nectria galligena Bres. With basidiospores of Cronartium ribicola J.C. Fisch., a difference between control and treated spores was also found, but it was less pronounced than with conidia of the Ascomycetes. In transmission electron microscopy, untreated conidia showed typical cytoplasmic contents with the regular presence of mitochondria, ribosomes, and nuclei, at times accompanied by vacuoles of various sizes. At concentrations of the peptide inhibitory to spore germination, plasma membranes, as well as nuclear and mitochondrial envelopes, were either generally difficult to discern or were distorted and swollen. At more advanced stages, the cytoplasm of treated spores contained numerous vesicles and was in places more electron-dense than in controls. Cytoplasm leakage was also regularly observed. Present observations strongly suggest that the primary site of action of this peptide is located at the plasma membrane level.Key words: Cronartium ribicola, Gremmeniella abietina, Nectria galligena, Ophiostoma ulmi, D2A21, peptide.

Structural changes of spores of tree fungal pathogens after treatment with the designed antimicrobial peptide D2A21

In vitro effects of the antimicrobial synthetic D2A21 peptide on the structure of spores of four fungal pathogens causing important tree diseases were examined by microscopy in parallel with tests to measure inhibition of spore germination. With light microscopy, the use of SYTOX® green stain indicated that the peptide rapidly altered the plasma membrane of conidia of three Ascomycetes: Gremmeniella abietina (Lagerberg) Morelet var. abietina Petrini et al., Ophiostoma ulmi (Buism.) Nannf., and Nectria galligena Bres. With basidiospores of Cronartium ribicola J.C. Fisch., a difference between control and treated spores was also found, but it was less pronounced than with conidia of the Ascomycetes. In transmission electron microscopy, untreated conidia showed typical cytoplasmic contents with the regular presence of mitochondria, ribosomes, and nuclei, at times accompanied by vacuoles of various sizes. At concentrations of the peptide inhibitory to spore germination, plasma membranes, as well as nuclear an...

Sterilization by Low-Pressure Plasma: The Role of Vacuum-Ultraviolet Radiation

Low-pressure plasma is a promising method for destroying microorganisms, an alternative to “conventional” methods, which have numerous drawbacks. Several plasma-based sterilization technologies are presently under development, even though the exact role of the various plasma constituents, for example ultraviolet radiation, on the sterilization mechanism is still unknown and subject to controversy. In this study, we first report high sporicidal activity of a microwave (MW) plasma compared to its radio-frequency (RF) counterpart, which we believe to be due to the higher concentration of reactive particles in the former plasma. We then report a relatively low sporicidal efficacy of vacuum ultraviolet (VUV) radiation (between 115 and 170 nm) emitted by an hydrogen MW plasma, in spite of the high effectiveness of these photons to break chemical bonds. We discuss these results in terms of etching (ablation), which we have observed for both synthetic polymers and spores, and in terms of other possible mechanisms proposed in the literature. The sporicidal effectiveness of VUV/UV radiation appears to vary markedly with wavelength and intensity, on account of spore structure and molecular absorption.

Ontogeny of Glomus clarum, in Ri T-DNA transformed roots

The spore ontogeny of Glomus clarum was monitored during the nonsymbiotic growth phase and after the colonization in transformed roots of clover (Trifolium repens) and carrot (Daucus carota). The germinated spore in water agar produced sporelike vesicles during the nonsymbiotic phase. It has been shown that these structures were in fact juvenile spores, that after root colonization were able to form mature spores. G. clarum began the spore formation in its nonsymbiotic phase. However, the juvenile spore structures senesced without establishment of an effective symbiosis. Spores of the genus Glomus are assumed to form terminally from sporogenic hyphae, but the ontogeny of G. clarum in transformed roots demonstrates that sporogenesis can also involve intercalary sporelike vesicles formed along the sporogenic hyphae. Using root organ culture system, all steps of spore formation can be followed in vivo without disturbing the system. This technique provides a better way to visualise events that occur during the pre-and postcolonization phase of arbuscular mycorrhiza, allowing the comprehension of full life cycle of this fungi. There are morphological variations in the ontogeny of G. clarum spores which differentiate its pattern from other Glomus species.

Ultrastructure of laevigate hilate spores in sporangia and spore masses from the Upper Silurian and Lower Devonian of the Welsh Borderland

Spore masses and isolated sporangia, containing laevigate hilate cryptospores attributable to the dispersed taxonLaevolancis divellomedia sensu lato, have been recovered on bulk maceration of Upper Silurian (Pridoli) and Lower Devonian (Lochkovian) deposits from the Welsh Borderland. Detailed morphological, anatomical and ultrastructural analysis, using light microscope, scanning electron microscope and transmission electron microscope techniques, reveals subtle differences between the specimens and they can be grouped into five distinct types. The different groups are distinguished principally by using sporangia–spore mass characteristics, presence or absence of extra–exosporal material and nature of spore–wall ultrastructure. Of the groups, one has a uniformly homogeneous exospore and the other four groups have a bilayered exospore. In the former the spores lack extra–exosporal material and occur in a discoidal sporangium. Of the bilayered groups, two have exospores of homogeneous composition but with the two layers differing in electron density. They occur in discoidal sporangia and spore masses and are distinguished on the presence or absence of extra–exosporal material and differences in the widths of the two layers. Finally, two bilayered groups possess a lamellate inner layer, but vary in presumed sporangial shape. Elongate sporangia have spores with concentric continuous lamellae, lacking further ultrastructure. In contrast, spores from a discoidal spore mass have white–line–centred, presumably tripartite, lamellae which are laterally discontinuous, overlapping and irregularly spaced. These findings, which suggest that morphologically similar spores were produced by a number of plant taxa, have important implications regarding the assessment of early land–plant diversity. The affinities of hilate cryptospore–producing plants are unknown and problematic, particularly as no extant non–angiosperm plants produce dyads, other than through meiotic irregularity, and spore–sporangial characters have no exact counterpart in coeval plants. Studies of specimens within situhilate cryptospores suggest that they derive from rhyniophytoids, i.e. plants that resemble the simplest of vascular plants but lack evidence of vascular tissue, although hilate cryptospore–containing examples show no axial branching. It might be argued, based on evidence from spore wall ultrastructure, that some of the plants have more in common with lycopsids and filicopsids than bryophytes, a surprising finding bearing in mind the stratigraphic distribution of hilate cryptospores–dyads and inferences that the producers were bryophyte–like. Detailed studies of wall structure in the hilate cryptospores permit consideration of spore wall development. It is suggested that extra–exosporal material derives from a tapetum and is thus produced by the diploid sporophyte. The white–line–centred lamellae in a single specimen provide the earliest evidence for the presence of such structures in early land plant spores and provide further evidence that sporopollenin deposition on such structures is the most primitive mode of sporopollenin deposition among land plants.

Piriformospora indica, gen. et sp. nov., a new root-colonizing fungus

A new fungus isolate was discovered in an arbuscular mycorrhizal fungal spore from a desert soil in India. It could easily be cultivated on various synthetic media, and formed pear-shaped chlamydospores. Inoculation of maize showed that the fungus colonized the root cortex. Since it did not resemble any known fungus based on morphology and ultrastructure, a new genus was described. For its characteristic spore structure the isolate was named Piriformospora indica. Electron microscopy revealed the presence of typical dolipores with continuous parenthesomes, which indicated that P. indica belongs to the Hymenomycetes (Basidiomycota). DNA was extracted and the 5′ end of the 18S rRNA was amplified and sequenced. Comparison with sequences from the Genbank data base indicated that P. indica is related to the Rhizoctonia group.

Piriformospora indica, gen. et sp. nov., a New Root-Colonizing Fungus

A new fungus isolate was discovered in an arbuscular mycorrhizal fungal spore from a desert soil in India. It could easily be cultivated on various synthetic media, and formed pear-shaped chlamydospores. Inoculation of maize showed that the fungus colonized the root cortex. Since it did not resemble any known fungus based on morphology and ultrastructure, a new genus was described. For its characteristic spore structure the isolate was named Piriformospora indica. Electron microscopy revealed the presence of typical dolipores with continuous parenthesomes, which indicated that P. indica belongs to the Hymenomycetes (Basidiomycota). DNA was extracted and the 5′ end of the 18S rRNA was amplified and sequenced. Comparison with sequences from the Genbank data base indicated that P. indica is related to the Rhizoctonia group.

CotM of Bacillus subtilis, a member of the alpha-crystallin family of stress proteins, is induced during development and participates in spore outer coat formation.

We cloned and characterized a gene, cotM, that resides in the 173 degrees region of the Bacillus subtilis chromosome and is involved in spore outer coat assembly. We found that expression of the cotM gene is induced during development under sigma K control and is negatively regulated by the GerE transcription factor. Disruption of the cotM gene resulted in spores with an abnormal pattern of coat proteins. Electron microscopy revealed that the outer coat in cotM mutant spores had lost its multilayered type of organization, presenting a diffuse appearance. In particular, significant amounts of material were absent from the outer coat layers, which in some areas had a lamellar structure more typical of the inner coat. Occasionally, a pattern of closely spaced ridges protruding from its surface was observed. No deficiency associated with the inner coat or any other spore structure was found. CotM is related to the alpha-crystallin family of low-molecular-weight heat shock proteins, members of which can be substrates for transglutaminase-mediated protein cross-linking. CotM was not detected among the extractable spore coat proteins. These observations are consistent with a model according to which CotM is part of a cross-linked insoluble skeleton that surrounds the spore, serves as a matrix for the assembly of additional outer coat material, and confers structural stability to the final structure.

New Taxa and a New Rank of Selaginella (Selaginellaceae) from Madagascar and the Comoros

Two new species of Selaginella from Madagascar, S. bemarahensis and S. marinii, are described and illustrated, as are a new subspecies of S. nivea Alston and a new form of S. fissidentoides (Hooker & Greville) Spring var. fissidentoides. The Malagasy species S. fissidentoides is segregated into two varieties, one of which has been previously known as S. amphirrhizos A. Braun ex Hieronymus. The taxonomic position and closest relationships of each taxon are briefly discussed. The monogeneric family Selaginellaceae comprises about 700 species distributed throughout the world, but for the most part concentrated in tropical and subtropical areas. The most recent infrageneric classification of the genus Selaginella (Jermy, 1986) recognized five subgenera. The last complete account of the family in Madagascar and the Comoros was made by A. H. G. Alston (1932), who listed eleven species of Selaginella as occurring in that area. Two additional Malagasy species were established, one by R. M. Tryon (1955) and one by W. Rauh and W. Hagemann (1991). During revision of this family for the Flore de Madagascar et des Comores, made possible by increased collecting in the last 40 years (specimens have been studied from B, BM, K, P), one existing taxon has been divided and four distinct taxa have been noted. They are described in this paper, so that the names can be used in the Flora treatment. Two species, S. bemarahensis and S. marinii, belong to the subgenus Heterostachys Baker, and one subspecies, S. nivea subsp. humbertii, to the subgenus Tetragonostachys Jermy. Selaginella fissidentoides var. amphirrhizos and S. Jissidentoides fo. ovata (of var. fissidentoides) belong to the subgenus Stachygynandrum (P. Beauvois) Baker. At present, fifteen species of Selaginella are recognized in Madagascar and the Comoros, one of them represented by two subspecies and one by two varieties and one form. The descriptive terms referring to shape, surface, and structure of spores follow terminology used by A. F. Tryon and B. Lugardon (1991). 1. Selaginella bemarahensis S. Stefanovid & Rakotondrainibe, sp. nov. TYPE: Madagascar. Mahajanga: Reserve Naturelle de Bemaraha, au sud-est d'Antsalova, 18?45'S, 44?48'E, 400 m, 23 Aug. 1987, Phillipson 2257 (holotype, P; isotype, MO not seen). Figure 1A, B. Species haec, inter species subgeneris Heterostachys Baker, habitu parvulo et muscoso ad S. perpusillam Baker accedens, sed foliis lateralibus et medianis similaribus (praeter staturam), foliorum mediorum apice acuto et foliorum lateralium marginibus et sporophyllorum ventralium denticulatis (non ciliatis) ab ea differt. Plants tiny, moss-like, slender, without rhizome, forming mats. Main stem suberect to erect, 2-4.5 cm long, outline lanceolate, rarely or not branched, provided with rhizophores. Branches, when present, short and unbranched. Rhizophores dorsal, situated on the lower half of the stem, 2-8 mm long and very fine, straw-yellow, dichotomizing 2-3 times. Leaves dimorphic, pale green, herbaceous, widely spaced on the main stem, contiguous toward the tips of the main stem and branches. Lateral leaves subequal, lanceolate, 1.8-3 mm long, 0.6-1 mm wide, midrib apparent, base symmetrical and blunt, apex acute, margin subentire to denticulate all around the leaf. Axillary leaves similar to lateral but equal. Median leaves also similar to lateral leaves but smaller, 1.2-1.8 mm long, 0.5-0.8 mm wide. Strobili solitary, inserted terminally on main stem and branch tips, bilateral, resupinate, 5-8 mm long and 1.5-2 mm wide. Sporophylls dimorphic, spreading, margins denticulate. Dorsal sporophylls ovate-lanceolate, 1-1.5 mm long and 0.4-0.6 mm wide, keeled, keel well developed and denticulate, base blunt, apex acute. Ventral sporophylls ovate, 0.9-1.1 mm long and 0.4-0.6 mm wide, slightly or not keeled, base blunt, apex acute-acuminate. Megaspores light lemon yellow, 278-287 C.m diam., spherical, trilete, with laesura nearly equaling the radius, surface finely tuberculate on both proximal and distal face (Fig. 2A). Microspores bright orange, 35-38 /m diam., tetrahedral-globose, trilete, with laesura nearly equaling the radius, surface laevigate to finely granulate on both proximal and NovoN 6: 203-209. 1996. This content downloaded from 207.46.13.72 on Sun, 07 Aug 2016 07:12:16 UTC All use subject to http://about.jstor.org/terms

Alternation of actinosporean and myxosporean phases in the life cycle of Zschokkella nova (Myxozoa).

Experimental evidence has been gathered to show that the life cycle of the myxozoan gallbladder parasite Zschokkella nova Klokacewa, 1914, which infects the fish Carassius carassius, has a complex life cycle with alternation of two hosts (fish and Oligochaeta) and two developmental phases (myxosporean and actinosporean). The gut epithelium of the oligochaete, Tubifex tubifex, exposed experimentally to Z. nova, obtained from C. carassius, became infected with organisms resembling Actinosporea. The spore structure and cube-like network of the interconnected spores is reminiscent of Siedleckiella silesica Janiszewska, 1952, although the spores are very different in size and number of sporoplasm nuclei. The life cycle of Z. nova resembles that of the whirling disease agent Myxosoma cerebralis described by Wolf and Markiw, which also alternates between fish and oligochaete hosts.

Genetic and immunological characterization of the microsporidianSeptata intestinalisCali, Kotler and Orenstein, 1993: reclassification toEncephalitozoon intestinalis

The relationships between the Encephalitozoon-like Septata intestinalis and other microsporidia that occur in humans; notably Encephalitozoon cuniculi and Encephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects of S. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of the S. intestinalis rrs gene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplified rrs genes and establishment of the degree of sequence identity between rrs genes of various microsporidian species. The unique sequence of rrs of S. intestinalis as well as the distinct RFLP and SDS-PAGE profiles indicate that S. intestinalis is clearly different from other human microsporidian species. However, its rrs gene shared about 90% sequence identity with rrs of both Encephalitozoon spp., E. cuniculi and E. hellem. This is remarkably higher than the about 70% identity observed between rrs of microsporidian species which belong to different genera and thus suggests that S. intestinalis should be regarded as a species of the genus Encephalitozoon.(ABSTRACT TRUNCATED AT 250 WORDS)

Taxonomic evaluation of the myxomycete Calonema luteolum

The new combination Perichaena luteola (Kowalski) Gilert is proposed. An ultrastructural study of Calonema luteolum using SEM and TEM and supplemented with X-ray microanalysis has revealed close similarities with species of Perichaena in structure and chemical composition of peridium, capillitium and spores. The internal septation of capillitial threads characteristic of Perichaena has been demonstrated in C. luteolum as well. The taxonomic value of the ‘double wall’, often emphasized as one of the characteristics of Perichaena , is questioned.

Morphological and Immunological Studies on Microsporidia: Antigene Properties of Nosema mesnili Spores

Immunological investigation of two sequences of the life cycle (with mono- and octospores) of presumably one microsporidian species, Nosema mesnili, included: application of a murine anti-monospores serum in an immunofluorescent assay (IFA), electron microscopy (IEM) analysis and Western blotting of antigenes of both spore types; hybridoma production and testing of hybridoma clones by IFA. The spore surface, polar filaments and sporoplasm gave strong positive immune reaction against the antiserum in IFA test. The antiserum reacted with spore antigens ranging in molecular weight from 20 to 100 kDA. Four positive hybridoma clones reacted only with the surface of monospores. Polyclonal antibodies recognized the inner structures of spores and vegetative stages of both sequences of development as it has been revealed by IEM. Immunization with spores lead to antibody production against the spore wall, polar filament and sporoplasm. Cloning the hybridoma cells promised to deliver useful antibodies for investigations of the spore morphogenesis. The strong crossreaction of the antibodies with two developmental stages of the life cycle provides good evidence in favour of their belonging to one species.