Co-Isolation and Characterization of Two Pandoraviruses and a Mimivirus from a Riverbank in Japan.

Giant viruses, like pandoraviruses and mimiviruses, have been discovered from diverse environments, and their broad global distribution has been established. Here, we report two new isolates of Pandoravirus spp. and one Mimivirus sp., named Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. isolate styx, co-isolated from riverbank soil in Japan. We obtained nearly complete sequences of the family B DNA polymerase gene (polB) of P. hades and P. persephone; the former carried two known intein regions, while the latter had only one. Phylogenetic analysis revealed that the two new pandoravirus isolates are closely related to Pandoravirus dulcis. Furthermore, random amplified polymorphic DNA analysis revealed that P. hades and P. persephone might harbor different genome structures. Based on phylogenetic analysis of the partial polB sequence, Mimivirus sp. isolate styx belongs to mimivirus lineage A. DNA staining suggested that the Pandoravirus spp. asynchronously replicates in amoeba cells while Mimivirus sp. replicates synchronously. We also observed that P. persephone- or Mimivirus sp. isolate styx-infected amoeba cytoplasm is extruded by the cells. To the best of our knowledge, we are the first to report the isolation of pandoraviruses in Asia. In addition, our results emphasize the importance of virus isolation from soil to reveal the ecology of giant viruses.

Selection of Legionella Virulence-Related Traits by Environmental Protozoa.

Predation by protozoa provides a strong selective pressure for Legionella to develop and maintain mechanisms conferring resistance to digestion and ability to replicate within both amoebae and mammalian macrophages. Here we describe how to isolate environmental protozoa that prey on virulent Legionella. These protists are extremely useful models to study the cellular mechanisms employed by Legionellae to survive and grow in its natural environment. We present here procedures that are available to study the interactions between environmental protozoa and Legionella and thus increase our current understanding of Legionella virulence and the infection process.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Parvularia atlantis gen. et sp. nov., a Nucleariid Filose Amoeba (Holomycota, Opisthokonta).

The opisthokonts constitute a eukaryotic supergroup divided into two main clades: the holozoans, which include animals and their unicellular relatives, and the holomycotans, which include fungi, opisthosporidians, and nucleariids. Nucleariids are phagotrophic filose amoebae that phenotypically resemble more their distant holozoan cousins than their holomycotan phylogenetic relatives. Despite their evolutionary interest, the diversity and internal phylogenetic relationships within the nucleariids remain poorly studied. Here, we formally describe and characterize by molecular phylogeny and microscopy observations Parvularia atlantis gen. et sp. nov. (formerly Nuclearia sp. ATCC 50694), and compare its features with those of other nucleariid genera. Parvularia is an amoebal genus characterized by radiating knobbed and branching filopodia. It exhibits prominent vacuoles observable under light microscopy, a cyst-like stage, and completely lacks cilia. P. atlantis possesses one or two nuclei with a central nucleolus, and mitochondria with flat or discoid cristae. These morphological features, although typical of nucleariids, represent a combination of characters different to those of any other described Nuclearia species. Likewise, 18S rRNA-based phylogenetic analyses show that P. atlantis represents a distinct lineage within the nucleariids.

Cubic membrane formation supports cell survival of amoeba Chaos under starvation-induced stress.

Cubic membranes (CM) are highly organized membrane structures found in biological systems. They are mathematically well defined and reveal a three-dimensional nano-periodic structure with cubic symmetry. These membrane arrangements are frequently induced in cells under stress, disease conditions, or upon viral infection. In this study, we investigated CM formation in the mitochondria of amoeba Chaos carolinense and observed a striking correlation between the organism's ability to generate CM and the cell survival under starvation. Since starvation also induces autophagy, rapamycin was used to pharmacologically induce autophagy, and interestingly, CM formation was observed in parallel. Conversely, inhibition of autophagy reverted the cubic mitochondrial inner membrane morphology to tubular structure. In starved Chaos cells, mitochondria and autophagosomes did not co-localize and ATP production was sustained. CM transition in the mitochondria during starvation or upon induction of autophagy might prevent their sequestration by autophagosomes, thus slowing their rate of degradation. Such sustained mitochondrial activity may allow amoeba Chaos cells to survive for a longer period upon starvation.

Neovahlkampfia nana n. sp. Reinforcing an Underrepresented Subclade of Tetramitia, Heterolobosea.

The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.

Heterolobosean amoebae from Arctic and Antarctic extremes: 18 novel strains of Allovahlkampfia, Vahlkampfia and Naegleria.

The diversity of heterolobosean amoebae, important members of soil, marine and freshwater microeukaryote communities in the temperate zones, is greatly under-explored in high latitudes. To address this imbalance, we studied the diversity of this group of free-living amoebae in the Arctic and the Antarctic using culture dependent methods. Eighteen strain representatives of three heterolobosean genera, Allovahlkampfia Walochnik et Mulec, 2009 (1 strain), Vahlkampfia Chatton et Lalung-Bonnaier, 1912 (2) and Naegleria Alexeieff, 1912 (15) were isolated from 179 samples of wet soil and fresh water with sediments collected in 6 localities. The Allovahkampfia strain is the first representative of the genus from the Antarctic; 14 strains (7 from the Arctic, 7 from the Antarctic) of the highly represented genus Naegleria complete the 'polar' cluster of five Naegleria species previously known from the Arctic and Sub-Antarctic regions, whereas one strain enriches the 'dobsoni' cluster of Naegleria strains of diverse origin. Present isolations of Naegleria polarisDe Jonckheere, 2006 from Svalbard, in the Arctic and Vega Island, in the Antarctic and N. neopolarisDe Jonckheere, 2006 from Svalbard and Greenland in the Arctic, and James Ross Island, the Antarctic demonstrate their bipolar distribution, which in free-living amoebae has so far only been known for Vermistella Morand et Anderson, 2007.

A New Freshwater Naked Lobose Amoeba Korotnevella venosa n. sp. (Amoebozoa, Discosea).

A new freshwater species of naked lobose amoebae Korotnevella venosa n. sp. isolated from freshwater pond in St. Petersburg, Russia was studied with light and transmission electron microscopy. Basket scales of this species have six vertical columns supporting perforated rim. The latter has tongue-like broadening with membranous region. Vertical columns bifurcate at both ends so that neighboring columns are connected by their bifurcations forming combined structure. Basket scales of K. venosa are similar to those of Korotnevella hemistylolepis in having six full-length vertical columns and perforated rim. At the same time, they are different in having tongue-like broadening of perforated rim with membranous region and absence of six half-length columns and an intermediate crosspiece. Phylogenetic trees based on 18S rDNA gene placed K. venosa either at the base of the whole Korotnevella clade, next to K. hemistylolepis, or as a sister to the clade comprising Korotnevella species with latticework basket in large scales.

Vermamoeba vermiformis-Aspergillus fumigatus relationships and comparison with other phagocytic cells.

Free living amoebae (FLA) are protists ubiquitously present in the environment. Aspergillus fumigatus is a mould responsible for severe deep-seated infections, and that can be recovered in the same habitats as the FLA. By conducting coculture experiments and fungal incubation with amoebal supernatants, we report herein that Vermamoeba vermiformis, a FLA present in hospital water systems, promotes filamentation and growth of A. fumigatus. This finding is of particular importance to institutions whose water systems might harbor FLA and could potentially be used by immunocompromised patients. Also, the relationships between V. vermiformis and A. fumigatus were compared to those between this fungus and two other phagocytic cells: Acanthamoeba castellanii, another FLA, and macrophage-like THP-1 cells. After 4 h of coincubation, the percentages of the three phagocytic cell types with adhered conidia were similar, even though the types of receptors between FLA and macrophagic cell seemed different. However, the percentage of THP-1 with internalized conidia was considerably lower (40 %) in comparison with the two other cell types (100 %). Thus, this study revealed that interactions between A. fumigatus and these three phagocytic cell types show similarities, even though it is premature to extrapolate these results to interpret relationships between A. fumigatus and macrophages.

Trachyrhizium urniformis n. g., n. sp., a Novel Marine Filose Thecate Amoeba Related to a Cercozoan Environmental Clade (Novel Clade 4).

A novel cercozoan filose thecate amoeba, Trachyrhizium urniformis n. g., n. sp., was isolated from a marine sediment sample collected at Agenashiku Island, Okinawa, Japan. We performed light and electron microscopic observations, and a molecular phylogenetic analysis using the small subunit ribosomal RNA gene of the isolate. Cells of T. urniformis are spherical in shape and are covered by a thin theca possessing a wide rounded aperture. Branching and occasionally anastomosing filopodia with small granules emerge from the aperture. The granules are transported in the filopodia bidirectionally. Transmission electron microscopy showed that cells of T. urniformis possess nucleus with permanently condensed chromatin, Golgi apparatuses, microbodies, mitochondria with tubular cristae, and extrusomes. Several morphological and ultrastructural features of T. urniformis (the presence of thecae and nucleus with permanently condensed chromatin) show similarities with those of Thecofilosea. In a phylogenetic analysis, T. urniformis included in Thecofilosea with weak statistical supports and formed a clade with two sequences that constitutes a cercozoan environmental clade, novel clade 4. On the basis of morphological and ultrastructural information and the results of the phylogenetic analysis, we propose T. urniformis as a new member of class Thecofilosea.

Stenamoeba polymorpha, a New Species Isolated from Domesticated Horse Equus ferus caballus.

A new species of lobosean amoeba, Stenamoeba polymorpha n. sp., was isolated from the diarrheic stool of a domesticated horse in Great Falls Virginia, U.S. It shares characteristics with the five other described Stenamoeba species. However, electron microscopy revealed S. polymorpha has a substantially thickened cell surface lamina. Under light microscopy, the amoebae had a dynamic polymorphic appearance because hyaloplasm readily formed and resorbed subpseudopodia from any peripheral region of the cell. While in locomotion, the amoebae produced subpseudopodia that led and alternated the direction of movement with an apparent zigzag path. Sometimes, stationary amoebae had a vertical cell posture that was substantially taller than wide or long. The new species description is also supported by small subunit ribosomal ribonucleic acid gene analyses using phylogenies with both broad and narrow taxon sampling, pairwise nucleotide comparisons, and in-silico secondary structure predictions. The latter suggested Stenamoeba spp. discriminatory motifs in variable region eight. Stenamoeba polymorpha n. sp. is the first of its genus to be recovered from mammal feces, the first to yield a potential group one intron and the second described from North America. Here, the phylogenies suggest Stenamoeba limacina is this new species' nearest known relative.

Testate Amoebae (Amebozoa: Arcellinida) in Tropical Lakes of Central Mexico / Amebas testadas (Amebozoa, Arcellinida) en lagos tropicales del centro de México

AbstractTestate amoebae are common single-celled eukaryotic organisms in aquatic ecosystems. Despite their important role in these ecosystems, and their potential as bioindicators and paleoindicators, they remain poorly studied in Mexico. The major objectives of this study were to: 1) increase knowledge of testate amoebae in Mexico's tropical lakes, and 2) create a catalog of high-quality scanning electron micrographs that can be used for future ecological and paleoenvironmental studies. We collected surface-sediment samples from 29 lakes, located in the Transmexican Volcanic Belt, one at each lake during June and October 2011, and March 2013. Sediments were collected with an Ekman grab and preserved in anhydrous ethanol. Sub-samples were observed under a stereomicroscope and morphometric data for each species were recorded. Total diameter and aperture diameter were measured on circular tests. Irregularly shaped tests were measured for length and width of the aperture and for the size of the whole test. If a specimen possessed spines, the length of one randomly selected spine was measured. The best-preserved specimen of each taxon was photographed with an optical microscope and a scanning electron microscope (SEM). We found 41 taxa of testate amoebae belonging to the genera: Arcella, Argynnia, Centropyxis, Cucurbitella, Cyclopyxis, Cyphoderia, Difflugia, Euglypha, Lesquereusia, Pentagonia, Pseudodifflugia and Scutiglypha. Twelve species not previously reported for Mexico were recorded, along with 13 varieties. The average number of taxa recorded in each lake was eight, and the highest taxonomic richness was 18. The taxon found in the greatest number of lakes was Centropyxis aculeata var. aculeata. Taxonomic richness varied among lakes in the same region. This could reflect lake-specific differences in environmental conditions, underscoring the need for more detailed studies that include collection of data on physical and chemical variables in the lakes. Our results highlighted the need of further studies for the distribution patterns and ecology of lacustrine testate amoebae. Rev. Biol. Trop. 64 (1): 377-397. Epub 2016 March 01.

ResumenLas amebas testadas son organismos eucariontes unicelulares frecuentes en ecosistemas acuáticos. A pesar de su importante rol en estos ecosistemas, y su potencial como bioindicadores y paleoindicadores, son poco estudiados en México. Los principales objetivos de este estudio fueron: 1) incrementar el conocimiento de las amebas testadas en lagos tropicales de México y 2) crear un catálogo de microfotografías electrónicas de barrido de alta calidad que pueda ser usado en futuros estudios ecológicos y paleoambientales. Recolectamos muestras de sedimentos superficiales de 29 lagos, ubicados en la Faja Volcánica Transmexicana, una vez en cada lago durante junio y octubre 2011 y marzo 2013. Los sedimentos se recolectaron con una draga tipo Ekman y se preservaron en etanol anhidro. Las sub-muestras se observaron al microscopio estereoscópico. Para cada especie fueron registrados sus datos morfométricos. En testas circulares fueron medidos el diámetro y la apertura total. En testas irregulares fueron medidos el largo y ancho de la apertura y el tamaño de toda la testa. Si un ejemplar poseía espinas, se seleccionó una al azar y se midió su longitud. El ejemplar mejor preservado de cada taxón fue fotografiado con un microscopio óptico y en microscopio electrónico de barrido (MEB). Encontramos 41 taxa de amebas testadas pertenecientes a los géneros Arcella, Argynnia, Centropyxis, Cucurbitella, Cyclopyxis, Cyphoderia, Difflugia, Euglypha, Lesquereusia, Pentagonia, Pseudodifflugia y Scutiglypha. Se registraron 12 especies y 13 variedades que no estaban reportadas previamente en México. El número promedio de taxa registrados en cada lago fue de ocho, y la riqueza taxonómica mayor fue de 18. El taxón encontrado en el mayor número de lagos fue Centropyxis aculeata var. aculeata. La riqueza taxonómica compartida por lagos de la misma región no fue muy similar. Esto puede reflejar diferencias en las condiciones ambientales, evidenciando la necesidad de elaborar estudios más detallados que incluyan la recolección de datos de las variables físico-químicas en los lagos. Nuestros resultados resaltan la necesidad de estudios posteriores acerca de los patrones de distribución y la ecología de las amebas testadas lacustres.

Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

Testate Amoebae (Amebozoa: Arcellinida) in Tropical Lakes of Central Mexico.

Testate amoebae are common single-celled eukaryotic organisms in aquatic ecosystems. Despite their important role in these ecosystems, and their potential as bioindicators and paleoindicators, they remain poorly studied in Mexico. The major objectives of this study were to: 1) increase knowledge of testate amoebae in Mexico's tropical lakes, and 2) create a catalog of high-quality scanning electron micrographs that can be used for future ecological and paleoenvironmental studies. We collected surface-sediment samples from 29 lakes, located in the Transmexican Volcanic Belt, one at each lake during June and October 2011, and March 2013. Sediments were collected with an Ekman grab and preserved in anhydrous ethanol. Sub-samples were observed under a stereomicroscope and morphometric data for each species were recorded. Total diameter and aperture diameter were measured on circular tests. Irregularly shaped tests were measured for length and width of the aperture and for the size of the whole test. If a specimen possessed spines, the length of one randomly selected spine was measured. The best-preserved specimen of each taxon was photographed with an optical microscope and a scanning electron microscope (SEM). We found 41 taxa of testate amoebae belonging to the genera: Arcella, Argynnia, Centropyxis, Cucurbitella, Cyclopyxis, Cyphoderia, Difflugia, Euglypha, Lesquereusia, Pentagonia, Pseudodifflugia and Scutiglypha. Twelve species not previously reported for Mexico were recorded, along with 13 varieties. The average number of taxa recorded in each lake was eight, and the highest taxonomic richness was 18. The taxon found in the greatest number of lakes was Centropyxis aculeata var. aculeata. Taxonomic richness varied among lakes in the same region. This could reflect lake-specific differences in environmental conditions, underscoring the need for more detailed studies that include collection of data on physical and chemical variables in the lakes. Our results highlighted the need of further studies for the distribution patterns and ecology of lacustrine testate amoebae.

Viruses in close associations with free-living amoebae.

As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses.

Checklist, diversity and distribution of testate amoebae in Chile.

Bringing together more than 170 years of data, this study represents the first attempt to construct a species checklist and analyze the diversity and distribution of testate amoebae in Chile, a country that encompasses the southwestern region of South America, countless islands and part of the Antarctic. In Chile, known diversity includes 416 testate amoeba taxa (64 genera, 352 infrageneric taxa), 24 of which are here reported for the first time. Species-accumulation plots show that in Chile, the number of testate amoeba species reported has been continually increasing since the mid-19th century without leveling off. Testate amoebae have been recorded in 37 different habitats, though they are more diverse in peatlands and rainforest soils. Only 11% of species are widespread in continental Chile, while the remaining 89% of the species exhibit medium or short latitudinal distribution ranges. Also, species composition of insular Chile and the Chilean Antarctic territory is a depauperated subset of that found in continental Chile. Nearly, the 10% of the species reported here are endemic to Chile and many of them are distributed only within the so-called Chilean biodiversity hotspot (ca. 25° S-47° S). These findings are here thoroughly discussed in a biogeographical and evolutionary context.

[CHROMATIN ORGANIZATION IN CELL CYCLE OF AMOEBA PROTEUS ACCORDING TO OPTICAL TOMOGRAPHY DATA].

For the first time the nuclear cycle of large freshwater amoeba Amoeba proteus was studied by the method of optical tomography. The nuclei were fixed in situ in the cells of synchronized culture, stained by DAPI and examined by confocal laser scanning microscope. 3D-images of intranuclear chromatin were studied in details at different stages of nuclear cycle. The obtained data, together with literary ones allow represent the dynamics of structural organization of the nucleus in Amoeba proteus cell cycle in a new fashion. It was concluded that in this species the two-stage interphase takes place, as well as mitosis of peculiar type which does not correspond to any known type of mitosis according to classification existing now. It is presumed that in the course of nuclear cycle the chromosomes and/or their fragments are amplified, this presumption being in a good correspondence with the data about nuclear DNA hyperreplication in the cell cycle of A. proteus. As a result of chromosomes amplification their number may vary at different stages of cell cycle, and it allows to explain the contradictory data concerning the exact number of chromosomes in this species. The elimination of extra-DNA occurs mainly at the stage between prophase and prometaphase. We presume the majority of chromosomes, or may be even all of them to be referred to cholocentric type according to their behaviour during the mitosis.

Isolation of mitochondria with cubic membrane morphology reveals specific ionic requirements for the preservation of membrane structure.

Biological membranes with cubic symmetry are a hallmark of virus-infected or diseased cells. The mechanisms of formation and specific cellular functions of cubic membranes, however, are unclear. The best-documented cubic membrane formation occurs in the free-living giant amoeba Chaos carolinense. In that system, mitochondrial inner membranes undergo a reversible structural change from tubular to cubic membrane organization upon starvation of the organism. As a prerequisite to further analyze the structural and functional features of cubic membranes, we adapted protocols for the isolation of mitochondria from starved amoeba and have identified buffer conditions that preserve cubic membrane morphology in vitro. The requirement for high concentration of ion-chelating agents in the isolation media supports the importance of a balanced ion milieu in establishing and maintaining cubic membranes in vivo.

Article for the "Free-living amoebae special issue": Isolation and characterisation of various amoebophagous fungi and evaluation of their prey spectrum.

This article gives an overview on the isolation and characterisation of endoparasitic fungi invading free-living amoebae (FLA), including the ones forming thalli inside their hosts such as Cochlonema euryblastum and also the predatory fungi which capture amoebae by adhesive hyphae. Acaulopage spp. and Stylopage spp. trap, intrude, and exploit amoebal trophozoites. Previous phylogenetic studies proved Cochlonema to be a member of the Zoopagales. The genetic investigation of Acaulopage tetraceros demonstrated its close relationship to Cochlonema. Co-cultivation of A. tetraceros with a number of FLA revealed a great prey spectrum of this amoebophageous fungus. In addition it was shown that solitary amoebal stages of slime moulds such as Dictyostelium sp. and Physarum sp. are also suited as welcome prey amoebae.

Survey on diversity of marine/saline anaerobic Heterolobosea (Excavata: Discoba) with description of seven new species.

Diversity of the anaerobic Heterolobosea (Excavata: Discoba) is only poorly understood, especially in marine environments. We have isolated and cultured 16 strains of anaerobic heteroloboseid amoebae and flagellates from brackish, marine and saline anoxic habitats worldwide. Phylogenetic analyses of SSU rDNA sequences and light-microscopic observations showed that all the strains belong to the family Psalteriomonadidae, the main anaerobic lineage of Heterolobosea, and that they represent eight species from the genera Monopylocystis, Harpagon and Pseudoharpagon. Seven species are newly isolated and described here as Monopylocystis minor n. sp., Monopylocystis robusta n. sp., Monopylocystis elegans n. sp., Monopylocystis disparata n. sp., Harpagon salinus n. sp., Pseudoharpagon longus n. sp. and Pseudoharpagon tertius n. sp. Amoebae, cysts and the ultrastructure of the genus Pseudoharpagon are presented for the first time.