Relationships of the Insect-Pathogenic Order Entomophthorales (Zygomycota, Fungi) Based on Phylogenetic Analyses of Nuclear Small Subunit Ribosomal DNA Sequences (SSU rDNA)

We sequenced the nuclear small subunit of ribosomal DNA (SSU rDNA) from seven species within the insect-pathogenic order Entomophthorales. These sequences were aligned with other published SSU rDNA sequences and phylogenies were inferred using phenetic and cladistic methods. Based on three different phylogenetic methods the Entomophthorales (excludingBasidiobolus ranarum) is monophyletic;B. ranarumwas more closely related to chytrids from Chytridiales and Neocallimasticales than to Entomophthorales, as was proposed by Nagahamaet al.(Mycologia87:203–209, 1995). Nuclear characters (large nuclei containing conspicuous condensed chromatin and lack of a prominent nucleolus) were of predictive value for the monophyly of the family Entomophthoraceae. Conidial characters separate the Entomophthoraceae, which only includes obligate pathogens, into at least two lineages: one lineage with uninucleate conidia and another with multinucleate conidia. The two species ofConidiobolusstudied were paraphyletic in our analyses and only distantly related to each other. This information may prove to be important in the use of these fungi as biocontrol agents.     

Immunodiffusion test for diagnosing basidiobolomycosis

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ° C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.     

SPHEROSOMES AND MITOCHONDRIA IN THE LIVING FUNGAL CELL

Study of living hyphae of Fusarium oxysporum Schlect., Fomes annosus (Fries) Cooke, Ceratocystis fagacearum (Bretz) Hunt, Basidiobolus ranarum Eidam, and Mycotypha microspora Fenner with phase contrast revealed that these fungi have spherosomes similar to those in vascular plants. The spherosomes are conspicuous in the hyphal tip, suggesting some function other than fat synthesis. It may be that the Woronin bodies reported by other workers are spherosomes. Mitochondria in these fungi are highly pleomorphic and exhibit saltatory movement. They often interact with nuclei in a manner suggesting close membrane contact.     

First culture-proven gastrointestinal entermophthoromycosis in the United States: A case report and review of the literature

The zygomycoses are fungal infections often occurring in compromised hosts. We report the first culture-proven case of a gastrointestinal infection in the United States by Basidiobolus haptosporus (ranarum). The clinical and histological features are noted in order to distinguish this infection from the more widely reported mucormycosis.     

Gastrointestinal zygomycosis: a report of three cases

Three cases of gastrointestinal zygomycosis, probably caused by Basidiobolus ranarum, are described. The diagnosis was based on morphology of the fungal elements in infected tissues and histopathologic findings. All the three patients responded favorably to management strategy that included surgical resection of the infected portion of the bowel and institution of specific antifungal therapy.     

Histological and ultrastructural features of gastrointestinal basidiobolomycosis

Basidiobolus ranarum is a fungus found in the dung of amphibians, reptiles, and insectivorous bats. Its structural elements include both hyphae and zygospores. Patients with B. ranarum infection may present with subcutaneous, gastrointestinal, or systemic lesions. Here we report a case of gastrointesinal badidiomycosis in a 13-year-old male child who presented with acute abdomen. Exploration revealed a mass in the ascending colon. On histology, transmural granulomatous inflammation composed of abundant eosinophils, lymphocytes, histiocytes and giant cells was seen. Histochemical stains revealed broad, non-septate, hyphae-like structures surrounded by an eosinophilic sheath. On an ultrastructural level, fungal hyphae, spores, and macrophage-laden crystalloids were observed. The diagnosis of gastrointestinal basidiobolomycosis was established and the patient received antifungal treatment. This paper reviews the relevant literature regarding basidiomycosis, and discusses its diverse clinicopathological features, as well as distinguishing it from other diseases.     

Basidiobolomycosis of the Nose and Face: A Case Report and a Mini-Review of Unusual Cases of Basidiobolomycosis

Background  

Subcutaneous zygomycosis is a chronic infection caused by fungus of the order Entomophthorales. It can have varying presentations and presents in the nose and face area with gradually progressing subcutaneous swelling that may be difficult to diagnose unless a strong suspicion of fungal involvement is maintained. We present a case of subcutaneous zygomycosis in a 35-year-old male patient, resident of a North Indian state. The patient was diagnosed to be suffering from subcutaneous zygomycosis, the causative agent being Basidiobolus ranarum identified on culture and lactophenol cotton blue mount preparation. He responded well to treatment with Itraconazole and Terbinafine and is asymptomatic on follow-up.     

Taxonomy,morphology, and surface structure of Basidiobolus sp. isolate

The isolate under investigation was determined as Basidiobolus ranarum Eidam, 1886. Scanning electron micrographs elucidated the origin of a cap-like part of the conidiophore adhering to the conidium and showed new details of the surface of the fragment of conidiophore and proper papilla during their gradual separation. The attaching sac of nonattached anadhesive conidia was smooth and very turgid, but after attaching, there appeared depressions due to the loss of turgor.     

Effects of griseofulvin on the mitotic cycle of the fungusBasidiobolus ranarum

Summary Griseofulvin has been shown to block mitosis in the fungusBasidiobolus ranarum. A wide range of metabolic inhibitors were used to investigate the relationship between growth rate and mitotic index. On the basis of such experiments inhibitors could be divided into two groups; the first affect the cell during the interphase period, the second affect the cell during mitosis. Griseofulvin is comparable with other known antimitotic agents in that it increases the mitotic index when the organisms growth rate is decreased.     

Croissance et sporulation de 6 espèces d'Entomophthorales II. Influence de diverses sources d'azote

Résumé La croissance et la sporulation d'Entomophthora sp. aff. obscura, E. destruens, E. sp. aff. thaxteriana, E. virulenta, Basidiobolus ranarum et Conidiobolus osmodes sont étudiées sur des milieux gélosés contenant différentes sources azotées: 3 nitrates, 6 sels d'ammonium, 16 acides aminés et 6 hydrolysats de protéine sont testés chacun à la concentration de 0,33 g d'azote par litre dans un milieu à base de glucose et de sels minéraux. E. sp. aff. obscura ne se développe pas sur ces milieux. Les 5 autres espèces métabolisent bien les sels d'ammonium, les hydrolysats de protéine et 10 des 16 acides aminés testés. Les nitrates ne sont pas utilisés. Les sources azotées qui assurent une bonne croissance sont également celles qui entraînent une formation importante de spores durables.

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Growth and sporulation of Entomophthora near obscura, E. destmens, E. near thaxteriana, E. virulenta, Basidiobolus ranarum and Conidiobolus osmodes were studied in solid synthetic media containing various nitrogen sources at 0,33 g N/liter. Thirty two nitrogen sources representing nitrates, ammonium salts, amino acids and protein hydrolysates were tested. E. near obscura did not grow on any medium. Protein hydrolysates, ammonium salts and ten of the sixteen amino acids tested were the best nitrogen sources for growth and resting spores production of the five other species.

     

Ultrastructure of Mitochondria and Crystal-containing Bodies in Mature Ballistospores of the Fungus Basidiobolus ranarum as Revealed by Freeze-etching

By using the freeze-etch technique, a regular pattern on both sides of the outer mitochondrial membrane can be demonstrated in mature spores of Basidiobolus ranarum. Bands consisting of 5 to 10 parallel ridges, each of which is 20 nm wide, envelop the outer as well as the inner side of this membrane. Hexagonal crystals, probably representing stored protein, are enclosed in special bodies with a very smooth surrounding unit membrane. The crystals are formed by parallel rods which consist of globular subunits, 6.5 to 7.0 nm wide.     

OBSERVATIONS ON CELL GROWTH, MITOSIS, AND DIVISION IN THE FUNGUS BASIDIOBOLUS RANARUM

The paper describes the forward streaming, growth, and division of the vegetative cell of Basidiobolus ranarum. The cell is several hundred microns long and has a single large nucleus. Mitosis is invariably followed by cell division. Both processes have been studied in the living cell by ordinary and phase contrast microscopy. Mitosis is accompanied by a temporary coarsening of the organisation of the cytoplasm and a considerable slowing down of the rate of growth of the cell wall tube. Fixed and stained preparations have shown that there is a large number of small chromosomes and that the mitotic spindle is formed from the nucleolus. Basidiobolus appears suitable for observations on the cell duplication cycle and the physiology of mitosis.     

Action de quelques fongicides sur la croissance mycélienne de trois espèces d'Entomophthorales

Résumé L'action,in vitro, de 34 fongicides sur la croissance mycélienne de 3 espèces d'Entomophthorales:Basidiobolus ranarum (Eidam),Conidiobolus osmodes (Drechsler) etEntomophthora virulenta (Hall & Dunn) [désignéEntomophthora nr.thaxteriana (Petch) parSoper & Bryan, 1974] a été examinée. Dans l'ordre croissant de leur sensibilité aux fongicides, les souches étudiées se classent comme suit:B. ranarum, E. virulenta etC. osmodes. Il existe une grande disparité dans l'activité des différents fongicides sur la croissance mycélienne des 3 espèces d'Entomophthorales. Certains fongicides sont peu ou pas actifs (chlorothalonil, éthirimol, oxychlorure de cuivre, soufre, etc.), d'autres agissent moyennement (captafol, dichlofluanide, mancozèbe, thirame, etc.); d'autres encore se révèlent très toxiques (bénodanil, chinométhionate, chloronèbe, triarimol). Mais il n'est pas simple de classer les fongicides en fonction de leur activité; un bon nombre d'entre eux possède un comportement hétérogène devant les 3 souches (carbendazime, oxycarboxine, 26019 RP, etc.). Dans l'ensemble, les fongicides systémiques se montrent plus efficaces sur la croissance mycélienne que les non systémiques.

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Summary The effect of 34 fungicides on the mycelial growth of 3 Entomophthorales species:Basidiobolus ranarum (Eidam),Conidiobolus osmodes (Drechsler) andEntomophthora virulenta (Hall & Dunn) was investigatedin vitro. The examination of growth was conducted in test tubes and five fungicidal concentrations were tested. The amount of growth of the various fungi in all treatments was expressed as a percentage of the growth of the control in order to evaluate the concentrations which reduce the growth speed by half. In the increasing order of their sensitivity to fungicides the strains under study could be classified in the following manner:B. ranarum, E. virulenta andC. osmodes. There is a great disparity between the action of the different fungicides upon the mycelial growth of the 3 Entomophthorales species. Some fungicides have little or no action (chlorothalonil, ethirimol, copper oxychloride, sulphur, etc.); others are more or less active (captafol, dichlofluanid, mancozeb, thiram, etc.); others again showthemselves to be highly toxic (benodanil, quinomethionate, chloroneb, triarimol). However it is not simple to classify the fungicides as a function of their activity; many of them react differently on the 3 species (carbendazim, oxycarboxin, 26019 RP, etc.). Overall, systemic fungicides are more effective on mycelial growth than non-systemic fungicides.

     

Chonopeltis australis (Crustacea) male reproductive system morphology; Sperm transfer and review of reproduction in Branchiura

The morphology of the male reproductive system as well as sperm transfer in Branchiura has been described for Dolops ranarum and Argulus japonicus. In this study, the reproductive system and accessory structures are described for male Chonopeltis australis using histology, light microscopy, and scanning electron microscopy. For the first time, we describe sperm transfer by means of a spermatophore in this genus. The internal and external morphology and mechanism of sperm transfer is compared with other Branchiura, where it has been described. The morphology of the reproductive system of C. australis is similar to that of D. ranarum while the accessory structures and the spermatophore produced are similar to that of A. japonicus. A revision of the definition of Branchiura with respect to reproduction is provided. J. Morphol. 276:209–218, 2015. © 2014 Wiley Periodicals, Inc.     

Mitosis in the fungusBasidiobolus ranarum as revealed by electron microscopy

Summary Mitosis of nuclei in vegetative hyphae of the fungusBasidiobolus ranarum has been studied by electron microscopy. Cells fixed with glutaraldehyde and OsO⁴ were embedded in Vestopal. Sections were obtained of single cells whose mitotic status was known. Attention was paid to the behaviour of the microtubules, the nuclear envelope and the nucleolus. Nuclear division begins with the dilution and rearrangement of nucleolar material and the gradual breakdown of the nuclear envelope. At this stage the nucleus is surrounded by a sheet of closely packed microtubules. Some of these penetrate into the nucleus through gaps in the envelope. Dissolution of the envelope is followed or accompanied by the development of an extensive labyrinth of membranous cisternae which persists at the periphery of the division site through mitosis and probably contributes material to the envelopes of the daughter nuclei. The drum-shaped spindle of metaphase is composed of large numbers of microtubules aligned parallel to each other. Many of them are associated with chromosomes. Metaphase is soon followed by the movement of dense masses of nucleolar material and chromosomes to the poles of the division figure to form the socalled end plates. Microtubules extend into the end plates but not beyond. Neither centrioles nor centriolar plaques have been seen.     

Flavonoids in plant nuclei: detection by laser microdissection and pressure catapulting (LMPC), in vivo staining, and uv–visible spectroscopic titration

Previous studies in our laboratory have indicated that the nuclei of a number of trees are associated with flavonoids, especially flavan‐3‐ols. In the present study, three techniques were applied to verify that flavonoids are naturally incorporated into nuclei. These were histochemistry, UV–visible (UV‐VIS) titration and laser microdissection. Nuclei from intact seed wings of Tsuga canadensis were isolated from their cells using laser microdissection and pressure catapulting (LMPC). Thereafter, the excised nuclei were stained with p‐dimethylamino‐cinnamaldehyde (DMACA), which resulted in a blue coloration due to the presence of flavanols. Thus, there is no doubt that the nuclei were, prior to staining, associated with flavanols. The nuclei of the coniferous species Abies lasiocarpa, Cedrus deodara, Cedrus libani, Juniperus communis, Picea abies, Picea orientalis and Pseudotsuga menziessii(Douglas fir) showed a yellow fluorescence typical for flavonols from the beginning of bud break over the entire growing season. However, after the bud‐breaking period, the nuclei of all species, except for Cedrus deodara, showed additionally a blue reaction for flavanols. Rather late, in midsummer, blue‐stained flavanols in nuclei were found in Picea orientalis. Generally, zeatin intensified the flavanol association with the nuclei. The main components of nucleosomes are DNA and the histone proteins. The nature of their association with the flavonols quercetin and rutin was investigated by UV‐VIS spectroscopic titration. The data were evaluated by means of the Mauser (A and AD) diagrams. The results indicate that DNA shows largely no spectroscopically detectable association equilibria under the experimental conditions chosen. However, association (aggregation) equilibria can be observed with rutin or quercetin and histone sulphate in Tris buffer (pH 8.0, 7.4 and 7.0). In phosphate buffer, rutin shows spectroscopically no or only weak association with histone sulphate, in contrast to its behaviour towards quercetin.     

The fluorescence brightener Rylux BSU induces dimorphism inBasidiobolus ranarum

The fluorescence brightener Rylux BSU (RBSU) showed an affinity for polysaccharide components of cell walls and accumulated in the extension zones of hyphal apices inBasidiobolus ranarum. It inhibited the polarized growth of mycelial hyphae and induced isotropic growth resulting in spherical thick-walled cells up to 456 μm in diameter. On the inner cell wall surface, massive protuberances were formed. The cell wall and protuberances were positive in PAS and the Grocott method and stained with fluorochromes Blankophor BA, Calcofluor, Uvitex 2B, Rylux BSU and FITC-labeled WGA- and ConA-lectins. The WGA-FITC fluorescence intensity of the wall’s outermost layer, if not connected with neighbouring cells, and the fluorescence intensity of the innermost layer and of some protuberances mainly in their apical parts were on the average twice higher than the fluorescence intensity of the remaining wall material. RBSU binding to the cell wall material was stable. The process of converting from polarized to isotropic growth was reversible, depending upon contact with RBSU-containing medium. Repeated transfers of cells from RBSU-containing medium to an RBSU-free medium resulted in the development of apical swollen dumbbell-shaped cells.     

A simple and rapid nuclear staining method for Rhizoctonia solani Kuhn

Abstract

We developed a modified staining technique using acridine orange to stain the nuclei of Rhizoctonia solani. Acridine orange solution was prepared in acetic acid buffer, pH 7.2. Staining for 15 min was critical for observing the nuclei. All of the isolates were found to be multinucleated. The nuclei appeared bright green with light orange background. This method is simple, rapid and reproducible.     

Nuclei isolation protocols for flow cytometry allowing nuclear DNA content estimation in problematic microalgal groups

Microalgae are fundamentally important organisms for global ecosystem functioning with high potential in biotechnology and its applications. The knowledge of their nuclear DNA content has become a prerequisite for many areas of microalgal research. Due to common presence of various pigments, secondary metabolites and complex cell walls, the nuclear DNA content estimation using flow cytometry (FCM) is, however, often laborious or even impossible with the currently used protocols. In this study the performance of six nuclei isolation protocols was compared on various problematic microalgae using FCM. The nuclei isolation methods involved osmotic bursting of cells, razor blade chopping of fresh biomass and two newly introduced protocols, razor blade chopping of desiccated biomass and bead beating. These techniques also involved the use of two different nuclei isolation solutions, Otto I + II solutions, and LB01 buffer. Performance of the particular protocols differed greatly, depending on the used nuclei isolation solution and microalgal group. The most successful method was a newly adopted chopping of desiccated biomass in LB01 buffer. This method seems more appropriate for nuclei isolation in filamentous microalgae; on the other hand, bead beating appears to be more suitable for nuclei isolation in solitarily living algae. Using the optimal protocol for a given species, their nuclear DNA content was estimated, resulting in first DNA content estimates for four investigated taxa (Chlamydomonas noctigama, Gonyostomum semen, Microglena sp. and Stigeoclonium sp.). The estimated DNA content spanned from 0.15 to 32.52 pg.

     

The effect of starvation on the ultrastructure of the digestive cells of Dolops ranarum (Stuhlmann, 1891) (Crustacea: Branchiura)

Transmission electron microscopy was conducted on the digestive epithelium of the crustacean ectoparasite Dolops ranarum to elucidate its ultrastructure for the first time, both in a nourished and starved condition. Specimens were collected from the Limpopo Drainage System in South Africa, and the specimens were killed and dissected in Todd's fixative. The anterior midgut is composed mostly of absorptive cells or R cells, while the diverticula are composed of R cells and of F cells, which are moderately abundant in rough endoplasmic reticulum. They are probably responsible for producing digestive enzymes. The posterior midgut is composed of papilliform B cells with large apically located vesicles and R′ cells devoid of cell inclusions. Under starvation, specimens survive for a maximum of 12 days; R cells show the most conspicuous changes in ultrastructural characteristics. It is concluded that D. ranarum has adapted to short-term survival only without a host.