Platyamoeba pseudovannellida n. sp., a naked amoeba with wide salt tolerance isolated from the Salton Sea, California

A new species of naked amoeba, Platyamoeba pseudovannellida n.sp., is described on the basis of light microscopic and fine structural features. The amoeba was isolated from the Salton Sea, California, from water at a salinity of ca. 44%. Locomotive amoebae occasionally had a spatulate outline and floating cells had radiating pseudopodia, sometimes with pointed tips. Both these features are reminiscent of the genus Vannella. However, the surface coat (glycocalyx) as revealed by TEM indicates that this is a species of Platyamoeba. Although salinity was not used as a diagnostic feature, this species was found to have remarkable tolerance to fluctuating salinity levels, even when changes were rapid. Amoebae survived over the range 0 per thousand to 150 per thousand salt and grew within the range 0 per thousand to 138 per thousand salt. The generation time of cells averaged 29 h and was not markedly affected by salt concentration. This is longer than expected for an amoeba of this size and suggests a high energetic cost of coping with salinity changes. The morphology of cells changed with increasing salinity: at 0 per thousand cells were flattened and active and at the other extreme (138 per thousand) amoebae were wrinkled and domed and cell movement was very slow. At the ultrastructural level, the cytoplasm of cells grown at high salinity (98 per thousand was considerably denser than that of cells reared at 0 per thousand.     

Pseudoloma neurophilia n. g., n. sp., a new microsporidium from the central nervous system of the zebrafish (Danio rerio)

An unusual xenoma-forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 x 2.7 microm with 13-16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the "Ichthyosporidium group" along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.     

Morphological,biochemical, and molecular characterization of Oscillatoria kawamurae (Oscillatoriales,Cyanobacteria) isolated from different geographical regions

Oscillatoria kawamurae is an unusual freshwater cyanobacterium because of its large trichome and ambiguous gas vacuole. Because little is known about its phenotypic or genotypic characteristics, this study conducted morphological, biochemical, and genetic characterization of O. kawamurae strains isolated from Japan, Laos, and Myanmar. All strains displayed similar morphological characteristics; however some differences were observed in vegetative cell widths, trichome colors, and the distribution patterns of their gas vacuole‐like structures. The in vivo and phycobiliprotein absorption spectra revealed the two different trichome colors found in the four representative strains of O. kawamurae (Inle1, Lao7, Biwa6, and Inba3). These different trichome colors corresponded to the different ratios of phycoerythrin and phycocyanin, the two types of phycobilin pigments: 0.25 for olive‐green strain (Inle1) and 0.65–0.73 for brown‐green strains (Biwa6, Inba3, and Lao7). Cellular fatty acid compositions of the four strains were C14:0, C15:0, C16:0, C16:1c, C17:0, C18:0, C18:1c, C18:3α and C18:4, whereas two strains (Biwa6 and Inba3) lacked C17:0. Of the fatty acids, palmitic acid (C16:0) was predominant. PCR experiments using primers targeting a gas vesicle gene (gvpA) recovered gvpA fragments from all O. kawamurae strains, suggesting that this species has true gas vacuoles. The 16S rDNA sequences of all of the strains were identical regardless of their different trichome colors and/or geographic origins. Phylogenetic analyses based on the 16S rDNA sequences indicated that O. kawamurae forms a monophyletic clade with O. princeps CCALA 1115 clB1 and O. duplisecta ETS‐06. We discuss the taxonomy of O. kawamurae based on the data obtained in this study.     

Morphologic, genetic, and biochemical characterization of Helicobacter magdeburgensis, a novel species isolated from the intestine of laboratory mice

Background: The presence of enterohepatic Helicobacter species (EHS) is commonly noted in mouse colonies. These infections often remain unrecognized but can cause severe health complications or more subtle host immune perturbations and therefore can confound the results of animal experiments. The aim of this study was to isolate and characterize a putative novel EHS that has previously been detected by PCR screening of specific‐pathogen‐free mice. Materials and Methods: Biochemical analysis of enzyme activities (API campy), morphologic investigation (Gram‐staining and electron microscopy) and genetic analyses (16SrRNA and 23SrRNA analyses, DNA fingerprinting, restriction fragment polymorphisms, and pulsed‐field gel electrophoresis) were used to characterize isolated EHS. Genomic DNA fragments were sequenced to develop a species‐specific PCR detection assay. Results: Scanning electron microscopy revealed the presence of spiral‐shaped EHS, which varied in length (2.5–6 μm) and contained single monopolar or single bipolar sheathed flagella. The bacteria were grown under anaerobic conditions, preferably on agar plates containing serum or blood. The 16SrRNA, genetic, and biochemical analyses indicated the identification of a novel EHS species, named Helicobacter magdeburgensis. We also examined the genome content using pulsed‐field gel electrophoresis. Based on the pattern produced by two restriction enzymes, BamIII and KspI, the genome size was determined to be about 1.7–1.8 Mbp. Conclusion: We isolated and characterized a novel EHS species, H. magdeburgensis, morphologically, biochemically, and genetically. These results are important for future studies on the prevalence and pathophysiologic relevance of such infections. Our PCR assay can be used to detect and discriminate H. magdeburgensis from other Helicobacter species.     

Morphological,cytological and molecular characterization of a novel symmetric somatic hybrid between N. tabacum and N. glauca

Abstract

By using the protoplast fusion technique, we have obtained 44 regenerated plants, phenotypically different and distinct from their parents, among which we have identified a fertile symmetric somatic hybrid, designated as TG-32, between N. tabacum var. Gexin No.1 and N. glauca. The morphology, fertility, chromosome number and nuclear constitution of the somatic hybrid have been studied in detail. Unlike other asymmetric interspecific somatic hybrids, the chromosome number of the symmetric somatic hybrid is 72, equal to the sum of chromosomes of both parents. The TG-32 plant has flowers similar to those of N. tabacum, but with petals similar to those of N. glauca. Interestingly the offspring of TG-32 vary in seed production ability with temperature, and produce more seeds under a relatively low temperature. Two SCAR markers were used to evaluate genetic variability and structure. The hybrid amplified the expected fragment, but the parents showed only one of two markers. This experimental result supports the hypothesis of the co-existence of two parental genomes in the somatic hybrid.     

Bonamia perspora n. sp. (Haplosporidia), a parasite of the oyster Ostreola equestris, is the first Bonamia species known to produce spores

Examination of the oyster Ostreola equestris as a potential reservoir host for a species of Bonamia discovered in Crassostrea ariakensis in North Carolina (NC), USA, revealed a second novel Bonamia sp. Histopathology, electron microscopy, and molecular phylogenetic analysis support the designation of a new parasite species, Bonamia perspora n. sp., which is the first Bonamia species shown to produce a typical haplosporidian spore with an orifice and hinged operculum. Spores were confirmed to be from B. perspora by fluorescent in situ hybridization. Bonamia perspora was found at Morehead City and Wilmington, NC, with an overall prevalence of 1.4% (31/2,144). Uninucleate, plasmodial, and sporogonic stages occurred almost exclusively in connective tissues; uninucleate stages (2-6 microm) were rarely observed in hemocytes. Spores were 4.3-6.4 microm in length. Ultrastructurally, uninucleate, diplokaryotic, and plasmodial stages resembled those of other spore-forming haplosporidians, but few haplosporosomes were present, and plasmodia were small. Spore ornamentation consisted of spore wall-derived, thin, flat ribbons that emerged haphazardly around the spore, and which terminated in what appeared to be four-pronged caps. Number of ribbons per spore ranged from 15 to 30, and their length ranged from 1.0 to 3.4 microm. Parsimony analysis identified B. perspora as a sister species to Bonamia ostreae.     

Phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy

AIMS: Identification and biotyping of lactic acid bacteria (LAB) isolated from raw-milk Pecorino cheese manufactured in the Marche region (central Italy) for selection of suitable starter cultures or adjuncts. METHODS AND RESULTS: Preliminary characterization with morphological and biochemical assays were undertaken for 112 Gram-positive and catalase-negative isolates. Unequivocal identification of the isolates was obtained through restriction analysis of the amplified 16S rRNA gene and sequencing of 360-380 bp amplicons. Fifty-nine isolates belonging to LAB species generally recognized as safe and potentially utilized as starters or flavour-producing adjuncts were preselected and tested for their acidifying, proteolitic and autolytic activities. Fifty-five of these isolates were also subject to RAPD (randomly amplified polymorphic DNA) fingerprinting and unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis for the estimation of genotypic intra-species variation. As a result, in Pecorino cheese, a heterogeneous lactic acid bacteria population, which includes strains with metabolic characteristics of technological interest, was characterized. CONCLUSIONS: The polyphasic approach proposed allows the bacterial ecology of Pecorino cheese to be investigated and allows to assess the potential role of autochthonous LAB strains for the dairy industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The great economic importance of Pecorino cheese encouraged a deeper knowledge of its microbiota, which is known to influence the peculiar sensory properties of this cheese, also in view of its exploitation.     

Multiple isolations of a culturable, motile Ichthyosporean (Mesomycetozoa, Opisthokonta), Creolimax fragrantissima n. gen., n. sp., from marine invertebrate digestive tracts

A fragrant, spherical, osmotrophic eukaryote was isolated 27 times from the digestive tracts of marine invertebrates collected from the Northeast Pacific. The isolates were cultured from 7 animal collections over a 2-year period, most from the peanut worm, Phascolosoma agassizii. A small subunit ribosomal DNA phylogeny placed the spherical organism within the ichthyosporea, closest to Sphaeroforma arctica and Pseudoperkinsus tapetis. Supporting the close relationship of isolates, the sequences of ribosomal gene internal transcribed spacers determined for 26 isolates were identical, as were the elongation factor 1-alpha-like gene fragments from 7 isolates. Dispersal via amoeboid cells distinguished this species from its closest relatives and led to the erection of a new genus and species, "Creolimax fragrantissima." Vegetative cells reproduced asexually in vitro after they reached 30-60 microm in diameter by producing amoebae or endospores, which escaped through openings in the parent cell wall. Ultrathin sections of vegetative cells prepared by high-pressure-freeze substitution provided some of the first images of ichthyosporean spindle pole bodies and document, for the first time, tubular extensions of the plasma membrane into an electron-translucent inner layer of the cell wall. Ichthyosporeans are parasites and commensals of animals and culturable species are few. Because "C. fragrantissima" can be isolated regularly and repeatedly from nature and then grown easily through cycles of asexual reproduction, it has the potential to serve as a model organism for further research into marine ichthyosporeans.     

Morphological and molecular characterization of Tulasnella spp. fungi isolated from the roots of Epidendrum secundum,a widespread Brazilian orchid

Tulasnella spp. are the main fungal symbionts of Brazilian Epidendrum orchids. The taxonomy of these fungi is largely based on ITS rDNA similarity, but culture dependent techniques are still essential to establish the true biological entity of the mycobiont. The aim of this study was to characterize morphologically and molecularly 16 Tulasnella spp. fungi isolated from three different populations of E. secundum and to test the coincidences between morphological and molecular characterization. Two uninucleate rhizoctonia fungi, obtained from Oncidium barbaceniae, and two phytopathogenic isolates were included as outgroups. Qualitative and quantitative morphological characteristics were analyzed using multivariate statistics and were able to distinguish Ceratobasidium, Tulasnella and Thanatephorus genera and separate the isolates of Tulasnella spp. into two groups. Analysis of RAPD (Random Amplified Polymorphic DNA) and ITS rDNA sequences validated the morphological data. Symbionts of O. barbaceniae presented identity to ITS sequences of Ceratobasidium genus, while E. secundum isolates presented identity to two species of Tulasnella. We observed homogeneity among Tulasnella spp. obtained from a single population and from neighboring populations, but there was higher variability among isolates obtained from populations of regions that were farther apart. Morphological data associated with multivariate statistics proved to be a useful tool in the multi-level taxonomy of these orchid-associated fungi and in estimating the diversity of orchid mycorrhizal fungi.     

A new microsporidian parasite, Potaspora morhaphis n. gen., n. sp. (Microsporidia) infecting the Teleostean fish, Potamorhaphis guianensis from the River Amazon. Morphological, ultrastructural and molecular characterization

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2.8+/-0.3 x 1.5+/-0.2 microm. In mature spores the polar filament was arranged into 9-10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.     

Morphological,immunological and biochemical characterization of purified transverse tubule membranes isolated from rabbit skeletal muscle

Summary A microsomal fraction consisting of membranes of transverse tubule origin has been purified by a modification of the calcium-loading procedure initially described by Rosemblatt et al. (J Biol Chem 256:8140–8, 1981). Enzymatic analysis of this fraction shows an enrichment of the vesicles in the Mg⁺⁺ATPase (basal) activity characteristic of the T-tubules and an absent or very low Ca⁺⁺-dependant ATPase activity. Stereological analysis of freeze fracture replica of the membranes in the purified fraction indicates that they have a very low density of particles in their P faces and lack the structural manifestation of the caveolae typical of the sarcolemma. Immunological analysis performed with monoclonal antibodies prepared against purified T-tubule and sarcoplasmic reticulum membranes define some T-tubule specific antigens and confirm the morphological and biochemical data regarding the origin and purity of the Ttubule preparation.     

Cryptic diversity of free-living parabasalids, Pseudotrichomonas keilini and Lacusteria cypriaca n. g., n. sp., as inferred from small subunit rDNA sequences

Ultrastructural and molecular phylogenetic evidence indicate that the Parabasalia consists of seven main subgroups: the Trichomonadida, Honigbergiellida, Hypotrichomonadida, Tritrichomonadida, Cristamonadida, Spirotrichonymphida, and Trichonymphida. Only five species of free-living parabasalids are known: Monotrichomonas carabina, Ditrichomonas honigbergii, Honigbergiella sp., Tetratrichomonas undula, and Pseudotrichomonas keilini. Phylogenetic analyses show that free-living species do not form a clade and instead branch in several different positions within the context of their parasitic relatives. Because the diversity of free-living parabasalids is poorly understood, the systematics of these lineages is in a significant state of disarray. In order to better understand the phylogenetic distribution of free-living parabasalids, we sequenced the small subunit rDNA from three different strains reminiscent of P. keilini; the strains were isolated from different geographical locations: (1) mangrove sediments in Japan and (2) sediments in Cyprus. These data demonstrated that the free-living parabasalids P. keilini and Lacusteria cypriaca n. g., n. sp., form a paraphyletic assemblage near the origin of a clade consisting mostly of parasitic trichomonadids (e.g. Trichomonas vaginalis). This paraphyletic distribution of similar morphotypes indicates that free-living trichomonadids represent a compelling example of morphostasis that provides insight into the suite of features present in the most recent free-living ancestor of their parasitic relatives.     

Morphological and molecular characterization of Pisolithus occurring in Hokkaido Island, Northern Japan

Pisolithus basidiomes were found under different forest trees in Hokkaido Island, Japan. These basidiomes were characterized morphologically and molecularly. Although presenting different basidiome morphology and growing under different hosts, specimens presented similar spores ornamentation, and diameters. These spores had coarse, crowded, and blunted spines with three to eight basidiospores per basidium. Ribosomal DNA-based phylogenetic analysis indicated that variability of Pisolithus in this area is low. Phylogenetic analysis showed that Pisolithus analyzed in this study did not group with Pisolithus specimens from other geographical origins. These results suggest that Pisolithus from this area should be taxonomically distinguished from other Pisolithus.     

Characterization of Kudoa monodactyli n. sp. (Myxosporea: Multivalvulida) from the Muscle of Monodactylus argenteus (Teleostei: Monodactylidae) from Moreton Bay, Queensland, Australia

Kudoa monodactyli n. sp. is described from the somatic musculature of Monodactylus argenteus from several localities in southern Queensland, Australia. This is the first record of a myxozoan parasite from the family Monodactylidae. The spores typically have five polar capsules, making this species similar to the four other five-valved Kudoa species (K. neurophila, K. muscularis, K. shulmani, K. cutanea) that have been described to date. However, morphometric measurements particularly of spore length and width make the species from M. argenteus distinct from the other species. Comparison of the small subunit ribosomal DNA sequence of this species with its congeners for which sequence data are available, provides further evidence of novelty. Kudoa monodactyli n. sp. displays 38 (of 1,554) nucleotide differences compared with rDNA sequence of Kudoa neurophila, which on phylogenetic analysis places these species in clades exclusive of each other. Phylogenetic analyses also provide evidence that the number of valves per spore in this genus is an imperfect indicator of relatedness.     

Distribution and phylogenetic relationships of freshwater Thaumatomonads with a description of the new species Thaumatomonas coloniensis n. sp

The order Thaumatomonadida includes biflagellated heterotrophic flagellates that form filopodia and typically possess siliceous surface scales. We found thaumatomonads to contribute on average about 5%-10% to flagellate abundance in different benthic habitats. A new species of thaumatomonads, Thaumatomonas coloniensis n. sp., is described on the basis of morphological and molecular biological features. This new species was isolated both from groundwater at Appeldorn near Rees (Germany) and from the Rhine River at Cologne (Germany). We have sequenced the small subunit rRNA (ssu rRNA) gene and a fragment of the large subunit rRNA (lsu rRNA) gene (D3-D5 region) from the isolates of the new species, including the first sequence of a representative of the thaumatomonad genus Gyromitus. In agreement with previous studies, the differences in ribosomal genes of different thaumatomonad species are very small. For understanding the phylogenetic relationships of Thaumatomonadida and to explore their sister group relationships, we have created three sequence data sets (ssu rRNA, partial lsu rRNA, concatenated alignment of both) with the same composition of isolates (from Thaumatomonadida, Euglyphida, Cercomonadidae, and Heteromitidae). According to a Kishino-Hasegawa test, Thaumatomonadida evolved within the Cercozoa as a sister taxon to the Heteromitidae. A possibly close relationship to the Euglyphida, recently grouped together with the Thaumatomonadida in the class Imbricatea/Silicofilosea based on the rRNA data sets was not supported by our analyses.