Ascus function: From squirt guns to ooze tubes.

Unlike the mechanism of ballistospore discharge, which was not solved until the 1980s, the operation of asci as pressurized squirt guns is relatively straightforward and was understood in the nineteenth century. Since then, mycologists have sought to understand how structural adaptations to asci have allowed the ascomycetes to expel spores of different shapes and sizes over distances ranging from a few millimeters to tens of centimeters. These modifications include the use of valves at the tips of asci that maintain ascus pressure and expel spores at the highest speeds, and gelatinous appendages that connect spores after release and create larger projectiles with greater momentum than single spores. Clever experiments in the twentieth century coupled with meticulous microscopic studies led investigators to understand how asci with complicated apical structures worked and mathematical models produced estimates of launch speeds. With the recent application of high-speed video microscopy, these inferences about ascus function have been tested by imaging the motion of spores on a microsecond timescale. These experiments have established that ascospore discharge is the fastest fungal movement and is among the fastest movements in biology. Beginning with the history of the study of asci, this review article explains how asci are pressurized, how spores are released, and how far spores travel after their release. We also consider the efficiency of ascospore discharge relative to the mechanism of ballistospore discharge and examine the way that the squirt gun mechanism has limited the morphological diversity of ascomycete fruit bodies.

Axial resolution enhancement for planar Airy beam light-sheet microscopy via the complementary beam subtraction method.

Airy beam light-sheet illumination can extend the field of view (FOV) of light-sheet fluorescence microscopy due to the unique propagation properties of non-diffraction and self-acceleration. However, the side lobes create undesirable out-of-focus background, leading to poor axial resolution and low image contrast. Here, we propose an Airy complementary beam subtraction (ACBS) method to improve the axial resolution while keeping the extended FOV. By scanning the optimized designed complementary beam that has two main lobes (TML), the generated complementary light-sheet has almost identical intensity distribution to that of the planar Airy light-sheet except for the central lobe. Subtraction of the two images acquired by double exposure respectively using the planar Airy light-sheet and the planar TML light-sheet can effectively suppress the influence of the out-of-focus background. The axial resolution improves from ∼4µm to 1.2 µm. The imaging performance was demonstrated by imaging specimens of aspergillus conidiophores and GFP labeled mouse brain section. The results show that the ACBS method enables the Airy beam light-sheet fluorescence microscopy to obtain better imaging quality.

Genotypic variation in an ecologically important parasite is associated with host species, lake and spore size.

Genetic variation in parasites has important consequences for host­parasite interactions. Prior studies of the ecologically important parasite Metschnikowia bicuspidata have suggested low genetic variation in the species. Here, we collected M. bicuspidata from two host species (Daphnia dentifera and Ceriodaphnia dubia) and two regions (Michigan and Indiana, USA). Within a lake, outbreaks tended to occur in one host species but not the other. Using microsatellite markers, we identified six parasite genotypes grouped within three distinct clades, one of which was rare. Of the two main clades, one was generally associated with D. dentifera, with lakes in both regions containing a single genotype. The other M. bicuspidata clade was mainly associated with C. dubia, with a different genotype dominating in each region. Despite these associations, both D. dentifera- and C. dubia-associated genotypes were found infecting both hosts in lakes. However, in lab experiments, the D. dentifera-associated genotype infected both D. dentifera and C. dubia, but the C. dubia-associated genotype, which had spores that were approximately 30% smaller, did not infect D. dentifera. We hypothesize that variation in spore size might help explain patterns of cross-species transmission. Future studies exploring the causes and consequences of variation in spore size may help explain patterns of infection and the maintenance of genotypic diversity in this ecologically important system.

Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: Their phylogeny and mycoparasitic activity, including timing and quantifying mycoparasitism of Pseudoidium neolycopersici on tomato.

A total of 26 Ampelomyces strains were isolated from mycelia of six different powdery mildew species that naturally infected their host plants in Japan. These were characterized based on morphological characteristics and sequences of ribosomal DNA internal transcribed spacer (rDNA-ITS) regions and actin gene (ACT) fragments. Collected strains represented six different genotypes and were accommodated in three different clades of the genus Ampelomyces. Morphology of the strains agreed with that of other Ampelomyces strains, but none of the examined characters were associated with any groups identified in the genetic analysis. Five powdery mildew species were inoculated with eight selected Ampelomyces strains to study their mycoparasitic activity. In the inoculation experiments, all Ampelomyces strains successfully infected all tested powdery mildew species, and showed no significant differences in their mycoparasitic activity as determined by the number of Ampelomyces pycnidia developed in powdery mildew colonies. The mycoparasitic interaction between the eight selected Ampelomyces strains and the tomato powdery mildew fungus (Pseudoidium neolycopersici strain KTP-03) was studied experimentally in the laboratory using digital microscopic technologies. It was documented that the spores of the mycoparasites germinated on tomato leaves and their hyphae penetrated the hyphae of Ps. neolycopersici. Ampelomyces hyphae continued their growth internally, which initiated the atrophy of the powdery mildew conidiophores 5 days post inoculation (dpi); caused atrophy 6 dpi; and complete collapse of the parasitized conidiphores 7 dpi. Ampelomyces strains produced new intracellular pycnidia in Ps. neolycopersici conidiophores ca. 8-10 dpi, when Ps. neolycopersici hyphae were successfully destroyed by the mycoparasitic strain. Mature pycnidia released spores ca. 10-14 dpi, which became the sources of subsequent infections of the intact powdery mildew hyphae. Mature pycnidia contained each ca. 200 to 1,500 spores depending on the mycohost species and Ampelomyces strain. This is the first detailed analysis of Ampelomyces strains isolated in Japan, and the first timing and quantification of mycoparasitism of Ps. neolycopersici on tomato by phylogenetically diverse Ampelomyces strains using digital microscopic technologies. The developed model system is useful for future biocontrol and ecological studies on Ampelomyces mycoparasites.

Wake-up alarm: virtual time-lapse gene expression landscape illuminates mechanisms underlying dormancy breaking of germinating spores.

Dormancy breaking is a common physiological phenomenon that is shared by eukaryotes. Germination of spores in fungi is one of the most representative cases of dormancy breaking. Understanding the mechanisms of spore germination is therefore fundamental to basic studies on the control of cell proliferation and differentiation, as well as agricultural applications and medical investigation of fungal pathogenesis. In fission yeast, spores are generated as a consequence of sexual differentiation under nutrient starvation, remaining dormant until further nourishment, but little is known about how dormant spores germinate in response to environmental change. In a breakthrough, methods for single-cell-based gene expression profiling have recently been introduced. Several mRNA expression profiles were assembled from single spore cells during dormancy or germination. Single-cell RNA-seq profiles were aligned sequentially according to their similarities. The alignment of transcriptomes visualised how gene expression varies over time upon dormancy breaking. In this review, we revisit knowledge from previous studies on germination, select candidate genes that may be involved in germination, and query their expression from the temporal transcriptomic dataset so that studies on S. pombe germination can be extended further.

Morphology and surface characteristics of the anamorphic stage of powdery mildew Erysiphe australiana on crape myrtle leaves.

The morphology and surface characteristics of the powdery mildew Erysiphe australiana growing on crape myrtle leaves were observed with field emission scanning electron microscopy. The powdery mildew infection caused distortion and withering of the leaves, and nearly all external parts such as flowers, petioles, and branches were covered by the whitish colonies. Hyphal proliferation was prevalent on the adaxial surface of the powdery mildew-infected leaves. Globose ascocarp initials with hyphal aggregations were frequently seen on the leaf surface. Collapsed conidia showed longitudinal striations or ridges on the surface and deep linear wrinkling. Foot-cells were straight and grew at right angles from the vegetative hyphae. The conidiophores had fragmented, cylindrical, non-chained conidia which were produced singly at the apex of the conidiophores. The germ tubes formed intercalary multi-lobed appressoria and the conidia produced filiform protrusions emerging from subterminal positions. This study visualized previously unknown structures of E. australiana such as the ascocarp initials, filiform protrusions on conidia, and multi-lobed appressoria on germ tubes. These observations will facilitate the identification and taxonomy of this fungus and its allied species.

The role of NbTMP1, a surface protein of sporoplasm, in Nosema bombycis infection.

BACKGROUND: Nosema bombycis is a unicellular eukaryotic pathogen of the silkworm, Bombyx mori, and is an economic and occupational hazard in the silkworm industry. Because of its long incubation period and horizontal and vertical transmission, it is subject to quarantine measures in sericulture production. The microsporidian life-cycle includes a dormant extracellular phase and intracellular proliferation phase, with the proliferation period being the most active period. This latter period lacks spore wall protection and may be the most susceptible stage for control. METHODS: In order to find suitable target for the selective breeding of N. bombycis-resistant silkworm strains, we screen highly expressed membrane proteins from the transcriptome data of N. bombycis. The subcellular localization of the candidate protein was verified by Indirect immunofluorescence analysis (IFA) and immunoelectron microscopy (IEM), and its role in N. bombycis proliferation was verified by RNAi. RESULTS: The N. bombycis protein (NBO_76g0014) was identified as a transmembrane protein and named NbTMP1. It is homologous with hypothetical proteins NGRA_1734 from Nosema granulosis. NbTMP1 has a transmembrane region of 23 amino acids at the N-terminus. Indirect immunofluorescence analysis (IFA) results suggest that NbTMP1 is secreted on the plasma membrane as the spores develop. Western blot and qRT-PCR analysis showed that NbTMP1 was expressed in all developmental stages of N. bombycis in infected cells and in the silkworm midgut. Downregulation of NbTMP1 expression resulted in significant inhibition of N. bombycis proliferation. CONCLUSIONS: We confirmed that NbTMP1 is a membrane protein of N. bombycis. Reduction of the transcription level of NbTMP1 significantly inhibited N. bombycis proliferation, and this protein may be a target for the selective breeding of N. bombycis-resistant silkworm strains.

Evolutionary relationships of Metchnikovella dogieli Paskerova et al., 2016 (Microsporidia: Metchnikovellidae) revealed by multigene phylogenetic analysis.

The species Metchnikovella dogieli (Paskerova et al. Protistology 10:148-157, 2016) belongs to one of the early diverging microsporidian groups, the metchnikovellids (Microsporidia: Metchnikovellidae). In relation to typical ('core') microsporidia, this group is considered primitive. The spores of metchnikovellids have no classical polar sac-anchoring disk complex, no coiled polar tube, no posterior vacuole, and no polaroplast. Instead, they possess a short thick manubrium that expands into a manubrial cistern. These organisms are hyperparasites; they infect gregarines that parasitise marine invertebrates. M. dogieli is a parasite of the archigregarine Selenidium pygospionis (Paskerova et al. Protist 169:826-852, 2018), which parasitises the polychaete Pygospio elegans. This species was discovered in samples collected in the silt littoral zone at the coast of the White Sea, North-West Russia, and was described based on light microscopy. No molecular data are available for this species, and the publicly accessible genomic data for metchnikovellids are limited to two species: M. incurvata Caullery & Mesnil, 1914 and Amphiamblys sp. WSBS2006. In the present study, we applied single-cell genomics methods with whole-genome amplification to perform next-generation sequencing of M. dogieli genomic DNA. We performed a phylogenetic analysis based on the SSU rRNA gene and reconstructed a multigene phylogeny using a concatenated alignment that included 46 conserved single-copy protein domains. The analyses recovered a fully supported clade of metchnikovellids as a basal group to the core microsporidia. Two members of the genus Metchnikovella did not form a clade in our tree. This may indicate that this genus is paraphyletic and requires revision.

Effects of high-pressure processing on fungi spores: Factors affecting spore germination and inactivation and impact on ultrastructure.

Food contamination with heat-resistant fungi (HRF), and their spores, is a major issue among fruit processors, being frequently found in fruit juices and concentrates, among other products, leading to considerable economic losses and food safety issues. Several strategies were developed to minimize the contamination with HRF, with improvements from harvesting to the final product, including sanitizers and new processing techniques. Considering consumers' demands for minimally processed, fresh-like food products, nonthermal food-processing technologies, such as high-pressure processing (HPP), among others, are emerging as alternatives to the conventional thermal processing techniques. As no heat is applied to foods, vitamins, proteins, aromas, and taste are better kept when compared to thermal processes. Nevertheless, HPP is only able to destroy pathogenic and spoilage vegetative microorganisms to levels of pertinence for food safety, while bacterial spores remain. Regarding HRF spores (both ascospores and conidiospores), these seem to be more pressure-sensible than bacterial spores, despite a few cases, such as the ascospores of Byssochlamys spp., Neosartorya spp., and Talaromyces spp. that are resistant to high pressures and high temperatures, requiring the combination of both variables to be inactivated. This review aims to cover the literature available concerning the effects of HPP at room-like temperatures, and its combination with high temperatures, and high-pressure cycling, to inactivate fungi spores, including the main factors affecting spores' resistance to high-pressure, such as pH, water activity, nutritional composition of the food matrix and ascospore age, as well as the changes in the spore ultrastructure, and the parameters to consider regarding their inactivation by HPP.

Extracts from Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. Roots: A New Hope Against Honeybee Death Caused by Nosemosis.

Pollinators, the cornerstones of our terrestrial ecosystem, have been at the very core of our anxiety. This is because we can nowadays observe a dangerous decline in the number of insects. With the numbers of pollinators dramatically declining worldwide, the scientific community has been growing more and more concerned about the future of insects as fundamental elements of most terrestrial ecosystems. Trying to address this issue, we looked for substances that might increase bee resistance. To this end, we checked the effects of plant-based adaptogens on honeybees in laboratory tests and during field studies on 30 honeybee colonies during two seasons. In this study, we have tested extracts obtained from: Eleutherococcus senticosus, Garcinia cambogia, Panax ginseng, Ginkgo biloba, Schisandra chinensis, and Camellia sinensis. The 75% ethanol E. senticosus root extract proved to be the most effective, both as a cure and in the prophylaxis of nosemosis. Therefore, Eleutherococcus senticosus, and its active compounds, eleutherosides, are considered the most powerful adaptogens, in the pool of all extracts that were selected for screening, for supporting immunity and improving resistance of honeybees. The optimum effective concentration of 0.4 mg/mL E. senticosus extract responded to c.a. 5.76, 2.56 and 0.07 µg/mL of eleutheroside B, eleutheroside E and naringenin, respectively. The effect of E. senticosus extracts on honeybees involved a similar adaptogenic response as on other animals, including humans. In this research, we show for the first time such an adaptogenic impact on invertebrates, i.e., the effect on honeybees stressed by nosemosis. We additionally hypothesised that these adaptogenic properties were connected with eleutherosides-secondary metabolites found exclusively in the Eleutherococcus genus and undetected in other studied extracts. As was indicated in this study, eleutherosides are very stable chemically and can be found in extracts in similar amounts even after two years from extraction. Considering the role bees play in nature, we may conclude that demonstrating the adaptogenic properties which plant extracts have in insects is the most significant finding resulting from this research. This knowledge might bring to fruition numerous economic and ecological benefits.

Adiaspore development and morphological characteristics in a mouse adiaspiromycosis model.

Lesions of adiaspiromycosis, a respiratory disease affecting wild animals, have been found mainly in dead mammals and free-living mammals captured for surveillance. No report has described an investigation of adiaspore formation progress in the lung. After establishing an experimental mouse model of intratracheal adiaspiromycosis infection with the causative agent Emmonsia crescens, we observed adiaspore development. The spores grew and reached a plateau of growth at 70 days post-infection. The median adiaspore diameter showed a plateau of around 40 µm. The characteristic three-layer cell-wall structure of adiaspores was observed in the lung at 70 days post-infection. We examined infection with a few spores, which revealed that adiaspores in the mouse lung progressed from intratracheal infection of at least 400 spores. Moreover, we developed adiaspores in vitro by culture in fetal bovine serum. Although most spores broke, some large spores were intact. They reached about 50 µm diameter. Thick cell walls and dense granules were found as common points between in vitro adiaspores and in vivo adiaspores. These models are expected to be useful for additional investigations of E. crescens adiaspores and adiaspiromycosis.

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana.

Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.

Chitosan-citric acid edible coating to control Colletotrichum gloeosporioides and maintain quality parameters of fresh-cut guava.

This study aimed to verify the action of edible chitosan-citric acid (CHI-CA) coating to control Colletotrichum gloeosporioides and maintain quality parameters of fresh-cut guava. Chitosan was obtained from Litopenaeus vannamei shells using high temperature and short exposure times. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of CHI-CA against C. gloeosporioides were determined by macrodilutions at 28 °C/120 h in the absence/presence of CHI-CA (0-10 mg/mL). Scanning electron microscopy was used to evaluate morphological changes in the fungus. Guava slices were coated with CHI-CA (MIC) or 5 mg/mL glycerol (control). Rot incidence and physicochemical, physical, and microbiological factors were determined at 0, 3, 7, and 14 days at 24 °C and 4 °C. Chitosan presented typical structural characterization, 64% deacetylation, and a molecular weight of 1.6 × 104 g/mol. CHI-CA exhibited MIC and MFC values of 5 mg/mL and 10 mg/mL, respectively, and promoted changes in the morphology and cell surface of fungal spores. The fresh-cut guava coated with CHI-CA maintained quality parameters during storage and preserved their sensorial characteristics. Therefore, the use of CHI-CA as a coating is a promising strategy for improving postharvest quality of fresh-cut fruits.

Fungal spore adhesion on glycidoxypropyltrimethoxy silane modified silica nanoparticle surfaces as revealed by single cell force spectroscopy.

Thin film coatings prepared from commercially available glycidoxypropyltrimethoxysilane (GPS) modified silica nanoparticles (SiNPs) (Bindzil® CC301 and Bindzil® CC302) have previously shown excellent antifouling performance against a broad range of microbes [Molino et al., "Hydration layer structure of biofouling-resistant nanoparticles," ACS Nano 12, 11610 (2018)]. In this work, single cell force spectroscopy (SCFS) was used to measure the biological interactions between Epicoccum nigrum fungal spores and the same silica nanoparticle-based surfaces used in the aforementioned study, including a: glass coverslip, unmodified SiNP coatings, and both low (Bindzil® CC301) and high density (CC302) GPS functionalized SiNP coatings as a function of NaCl concentration. From the SCFS curves, the spore adhesion to the surface was greatest on the glass coverslip (20-80 nN) followed by the unmodified SiNP (3-5 nN) across all salt concentrations. Upon approach to both surfaces, the spores showed a long-range attraction generally with a profile characteristic of biointeractions and likely those of the outer cell wall structures or biological constituents. The attractive force allowed the spores to initially adhere to the surface and was found to be linearly proportional to the spore adhesion. In comparison, both high and low density GPS-SINP significantly reduced the spore adhesion (0.5-0.9 nN). In addition, the spore adhesion on high density GPS-SiNP occurred in only 14%-27% of SCFS curves (40%-48% for low density GPS-SiNP) compared to 83%-97% for the unmodified SiNP, indicating that in most cases the GPS functionalization completely prevented spore adhesion. The GPS-SiNP surfaces conversely showed a long-range electrostatic repulsion at low 1mM NaCl that was replaced by short-range repulsion at the higher salt concentrations. From the findings, it is proposed that the attractive force is a critical step in initial adhesion processes of the spore. The effective antifouling properties of the GPS are attributed to the ability to negate the attractive forces, either through electrostatic repulsion in low salt conditions and primarily from short-range repulsion correlating to the previously reported combined steric-hydration effect of the GPS functionalization on SiNP coatings.

Visualization of Arbuscular Mycorrhizal Fungal Extraradical Hyphae and Spores Vitality and Activity.

The hyphae and spores of arbuscular mycorrhizal (AM) fungi represent an essential component in the extraradical zone due to their role in nutrients and water uptake and as propagules that allow the perpetuation of the AM symbiosis over time, respectively. However, the attention of scientific literature is usually more focused on root colonization than on the study of the extraradical components of AM fungi, especially their vital, active, or functional fractions. This chapter presents some easy-to-use alternatives for staining vital, active, or functional structures of AM fungi for their subsequent microscopic visualization, such as the application of enzyme-based stains, NADPH formation, and also nucleus staining. Some modified methods for the extraction of mycelium from the soil are also presented.

Morphology and Transcriptome Analysis of Nosema bombycis Sporoplasm and Insights into the Initial Infection of Microsporidia.

Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro The sporoplasm was spherical, 3.64 ± 0.41 µm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [Padj] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism.IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm.

Surface, adhesiveness and virulence aspects of Candida haemulonii species complex.

The emerging opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii [Ch], C. duobushaemulonii [Cd] and C. haemulonii var. vulnera[Chv]) are notable for their intrinsic antifungal resistance. Different clinical manifestations are associated with these fungal infections; however, little is known about their biology and potential virulence attributes. Herein, we evaluated some surface properties of 12 clinical isolates of Ch (n = 5), Cd (n = 4) and Chv (n = 3) as well as their virulence on murine macrophages and Galleria mellonella larvae. Scanning electron microscopy demonstrated the presence of homogeneous populations among the species of the C. haemulonii complex, represented by oval yeasts with surface irregularities able to form aggregates. Cell surface hydrophobicity was isolate-specific, exhibiting high (16.7%), moderate (25.0%) and low (58.3%) hydrophobicity. The isolates had negative surface charge, except for one. Mannose/glucose- and N-acetylglucosamine-containing glycoconjugates were evidenced in considerable amounts in all isolates; however, the surface expression of sialic acid was poorly detected. Cd isolates presented significantly higher amounts of chitin than Ch and Chv. Membrane sterol and lipid bodies, containing neutral lipids, were quite similar among all fungi studied. All isolates adhered to inert surfaces in the order: polystyrene > poly-L-lysine-coated glass > glass. Likewise, they interacted with murine macrophages in a quite similar way. Regarding in vivo virulence, the C. haemulonii species complex were able to kill at least 80% of the larvae after 120 hours. Our results evidenced the ability of C. haemulonii complex to produce potential surface-related virulence attributes, key components that actively participate in the infection process described in Candida spp.

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia).

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed-sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5-6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

Conidiation in Neurospora crassa: vegetative reproduction by a model fungus.

Asexual development, conidiation, in the filamentous fungus Neurospora crassa is a simple developmental process that starts with the growth of aerial hyphae. Then, the formation of constrictions and subsequent maturation gives rise to the mature conidia that are easily dispersed by air currents. Conidiation is regulated by environmental factors such as light, aeration and nutrient limitation, and by the circadian clock. Different regulatory proteins acting at different stages of conidiation have been described. The role of transcription factors such as FL, and components of signal transduction pathways such as the cAMP phosphodiesterase ACON-2 suggest a complex interplay between differential transcription and signal transduction pathways. Comparisons between the molecular basis of conidiation in N. crassa and other filamentous fungi will help to identify common regulatory elements.