Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum

We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.     

Onychomycosis in Tehran,Iran: Prevailing fungi and treatment with itraconazole

A total of 187 Patients with suspected onychomycosis were examined for causative fungal agents between 1996 and 1997. Laboratory examination confirmed onychomycosis in 115 patients, of which 97 cases were presented with positive microscopic and cultural examinations, and they were selected for itraconazole pulse therapy. From an etiological point of view, 48.4% of the nail infections, mainly toenail infections, were caused by dermatophytes, 43.3% were infected with Candida spp, specially infected fingernails, and 8.2% by non-dermatophytic molds. Trichophyton mentagrophytes var. interdigital and T. violaceum were the most prevalent species. Candida albicans and C. parapsilosis were the predominant species of the Genus Candida. Scopolariopsis brevicaulis was the most common non-dermatophyte molds observed. Female affected more frequently than male and in both sexes, those who were 30–49 years old, more infected. Toenails were affected more frequently than fingernails. In this study, itraconazole pulse therapy (400 mg daily) gave during the first week of per month for 3 months. The study included 51 patients with toenail onychomychosis (group 1) and 46 patients with fingernail infections (group 2). Patients were followed up for 9 months after the last treatment. Clinical response rates were 83% in the group 1, 95% in the group 2 at month 12; the corresponding mycological cure rates were 71 and 87%, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Successful Demonstration of an Elusive Cell Coat in Amebae*

SYNOPSIS. Cell coats were cytochemically demonstrated for the first time in myxamebae of Fuligo septica, Didymium iridis, Dictyostelium discoideum, Cavostelium apophysatum, and amebae of Naegleria gruberi. The stain enhances the cell coats of Physarum polycephalum plasmodia, Ceratiomyxa fruticulosa myxamebae, and Acanthamoeba sp. Cell coats usually unstained by cationic dyes stain intensely with the aid of the new cytochemical protocol utilizing 0.5% Alcian blue in the primary fixative and 0.05% ruthenium red in the secondary fixative.     

The Breeding System of Polysphondylium pallidum,a Cellular Slime Mold*

SYNOPSIS. A mating type analysis was performed on 231 isolates of the cellular slime mold, Polysphondylium pallidum found in 61 samples collected in eastern North America between northern Florida and southern Canada. Seventy-eight percent of the isolates belonged to one of 2 mating types; 18% were incapable of mating with any partner; 3% were homothallic; and 1%, consisting of 2 isolates from a Florida sample, belonged to a separate breeding group. It is suggested that the majority of isolates represent a species capable of local genetic adaptation to a niche, the parameters of which undergo considerable variation over space and time.     

Species-specific cell mobility of bacteria-feeding myxamoebae in plasmodial slime molds

On decaying wood or litter in forests, plasmodial slime molds (myxomycetes) represent a large fraction of eukaryotic protists that feed on bacteria. In his seminal book Experimental Physiology of Plants (1865), Julius Sachs referred to the multinucleate plasmodium of myxomycetes, which were considered at that time as primitive plants (or fungi). Today it is well established that myxomycetes are members of the Amoebozoa (Protista). In this study we compare the mobility of myxamoebae of 3 European species, Lycogala epidendrum (order Liceales), Tubulifera arachnoidea, and Trichia decipiens (order Trichiales). Using agar plates, on which 3 separate bacterial species were cultivated as prey organisms (Methylobacterium mesophilicum, Escherichia coli, Agrobacterium tumefaciens), we document large differences in cell motility between the myxomycetes investigated. In addition, we show that the 3 species of myxamoebae can be distinguished based on their average cell size. These data shed light on the mode of co-occurrence via differential substrate utilization in these members of the Amoebozoa.     

Novel chlorinated dibenzofurans isolated from the cellular slime mold,Polysphondylium filamentosum,and their biological activities

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster.     

Changes in fungi and mycotoxins in pearl millet under controlled storage conditions

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25°C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25°C in either air (aerobic) or N₂ (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25°C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), “Helminthosporium”-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25°C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20–22%. Aflatoxin contamination averaged 174 ng g⁻¹ over all treatments, and increased up to 798 ng g⁻¹ in high-moisture grain at stored at 25°C.     

Genetic structure of the protist Physarum albescens (Amoebozoa) revealed by multiple markers and genotyping by sequencing

Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1–3 genetic markers (SSU, EF1A, COI) and analysed 98 specimens with genotyping by sequencing. The structure of the three-gene phylogeny, SNP-based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well-studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes.     

Slimes in the city: The diversity of myxomycetes from inner-city and semi-urban parks in Sydney,Australia

Natural and agricultural areas are rapidly becoming urbanised, causing changes in habitat structure and diversity. Although the effect of urbanisation on the diversity of terrestrial plants and animals has been well studied, there is a significant gap in our understanding of how urbanisation impacts diversity in protists. Here, we measure the diversity of plasmodial slime moulds (a group of large, macroscopic protists also known as myxomycetes) in inner-city and semi-urban parks. We studied the impact of a range of environmental characteristics (pH, temperature, canopy cover, area of green space and substrate type) on species richness and composition of myxomycetes. We also examined the influence of different degrees of urban development surrounding these parks. Species composition was significantly different between substrate types but not between inner-city and semi-urban parks. Temperature was the only environmental characteristic that affected diversity, having a negative effect on myxomycete presence. Our findings suggest that myxomycete diversity in urban parks is driven by factors at the substrate level, and not by the park's location within the city (inner city or semi-urban).     

Community of dark-spored myxomycetes in ground litter and soil of taiga forest (Nizhne-Svirskiy Reserve,Russia) revealed by DNA metabarcoding

The diversity of dark-spored myxomycetes in forest ground litter and soil from four study plots across a fire chronosequence in Nizhne-Svirskiy Nature Reserve (northwest of Russia) was assessed by a DNA metabarcoding approach. The history of fire disturbances and the season of sampling influenced the composition of myxomycete assemblages. Several species showed substrate preferences different from what is known from fruiting body records. A high abundance of nivicolous species in the majority of the samples suggests that this ecological group constitutes a common element of soil microbial communities in lowland taiga forests. Four species are reported for the Leningrad region for the first time. Our results support the hypothesis that amoebae populations of myxomycetes may have much wider ecological niches than indicated by records of fructifications, and further environmental studies may provide valuable new insights on myxomycete ecology and distribution.