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A new species of the "proteus-type" naked amoebae (large cells with discrete tubular pseudopodia) was isolated from tree bark sample of a birch tree in the surrounding of Kislovodsk town, Russia and named Polychaos centronucleolus n. sp. (Amoebozoa, Tubulinea). Amoebae of this species have a filamentous cell coat and a nucleus with a central compact nucleolus. This type of nucleolar organization has not been previously known for the genus Polychaos. A sequence of the 18S rRNA gene of this strain was obtained using whole genome amplification of DNA from the single amoeba cell, followed by NGS sequencing. The analysis of molecular data robustly groups this species with Polychaos annulatum within the family Hartmannellidae. Our results, together with the results of our previous studies, show that the taxonomic assignment of "proteus-type" amoebae species is becoming increasingly complex, and the taxonomic characters that can be used to classify these organisms are becoming more shadowed., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

We report the discovery of a new genus of heterolobosean flagellates, Dactylomonas gen. nov., with two species, D. venusta sp. nov. and D. crassa sp. nov. Phylogenetic analysis of the SSU rRNA gene showed that Dactylomonas is closely related to the amoeba Selenaion, the deepest-branching lineage of Tetramitia. Dactylomonads possess two flagella, and ultrastructural studies revealed an unexpected organization of the flagellar apparatus, which resembled Pharyngomonada (the second lineage of Heterolobosea) instead of Tetramitia: basal bodies were orthogonal to each other and a putative root R1 was present in the mastigont. On the other hand, Dactylomonas displayed several features uncommon in Heterolobosea: a microtubular corset, a distinctive rostrum supported by the main part of the right microtubular root, a finger-like projection on the proximal part of the recurrent flagellum, and absence of a ventral groove. In addition, Dactylomonas is anaerobic and seems to have lost mitochondrial cristae. Dactylomonas and Selenaion are accommodated in the family Selenaionidae fam. nov. and order Selenionida ord. nov. The taxonomy of Tetramitia is partially revised, and the family Neovahlkampfiidae fam. nov. is established., (© 2018 International Society of Protistologists.)

The species Leptomyxa flabellata was described by Goodey in 1915 and re-isolated by Pussard and Pons in 1976. It seems that it was never seen (or never recognized) again since that time. The strain designated as "Leptomyxa flabellata CCAP 1546/2" was studied by Cann in 1984, however the quality of the electron microscopic images of that time was poor. Based on the cyst structure and size characters, Page in 1988 suggested that this strain is not co-specific with Goodey's Leptomyxa flabellata, but represents a species 'Ripidomyxa' australiensis Chakraborty and Pussard, 1985, nowadays known as Rhizamoeba australiensis. In the present paper light- and electron-microscopic images of CCAP 1546/2 strain, which is now lost, are provided. Based on the morphological evidences it is suggested to establish it in a rank of a new species, Leptomyxa ambigua n. sp. Neither "true" L. flabellata Goodey, 1915 nor original R. australiensis Chakraborty et Pussard, 1985 are nowadays represented in the culture collections, and no original type material is available on both these species., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Leptomyxa valladaresi was isolated from soil in a pine forest on the southern flank of Mt Teide in Tenerife, Spain. It feeds on bacteria and on a range of other amoebae, and it was possible to establish bi-axenic cultures with L. valladaresi and Acanthamoeba. It is easily propagated on a E. coli also. 18S rDNA gene sequence analysis suggests that it is most closely related to Leptomyxa variabilis, however this amoeba differs in important detail. L. valladaresi is primarily mononucleate whereas L. variabilis is multinucleate. L. valladaresi is a larger amoeba and although the cysts are similar in size, there is no sign of the pore-like structures described in L. variabilis cysts. L. valladaresi can adopt a rapid monopodal and tubular morphology similar to that described for L. neglecta and Rhizamoeba matisi, and is never reticulated as larger L. variabilis individuals tend to be. The mean generation time was found to be 18 h, in line with amoebae of this size. Like other members of the genus, L. valladaresi is reported to harbour intracellular, presumably endosymbiotic bacteria, and a Delftia sp has been identified by 16S PCR a bacterium which is also known to grow within Acanthamoeba. The availability of this easily cultured species will help to characterize of this little studied genus and family and their relationship with bacteria, both prey and symbionts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Testate amoebae are free-living shelled protists that build a wide range of shells with various sizes, shapes, and compositions. Recent studies showed that xenosomic testate amoebae shells could be indicators of atmospheric particulate matter (PM) deposition. However, no study has yet been conducted to assess the intra-specific mineral, organic, and biologic grain diversity of a single xenosomic species in a natural undisturbed environment. This study aims at providing new information about grain selection to develop the potential use of xenosomic testate amoebae shells as bioindicators of the multiple-origin mineral/organic diversity of their proximal environment. To fulfil these objectives, we analysed the shell content of 38 Bullinularia indica individuals, a single xenosomic testate amoeba species living in Sphagnum capillifolium, by scanning electron microscope (SEM) coupled with X-ray spectroscopy. The shells exhibited high diversities of mineral, organic, and biomineral grains, which confirms their capability to recycle xenosomes. Mineral grain diversity and size of B. indica matched those of the atmospheric natural mineral PM deposited in the peatbog. Calculation of grain size sorting revealed a discrete selection of grains agglutinated by B. indica. These results are a first step towards understanding the mechanisms of particle selection by xenosomic testate amoebae in natural conditions., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Although the presence of test is the most fundamental synapomorphy of arcellinid lobose amoebae, the significance of tests in maintenance of the biological functions of these organisms is still largely unclear. This paper demonstrates the effect of the artificial test removal on the behaviour, ultrastructure and reproduction of the testate lobose amoebae belonging to the two species of the genus Arcella. The studied cells can survive after artificial removal of their test, and many specimens are capable of building a test de novo. We investigated this process and found that test-free cells of Arcella in culture remained alive for weeks; they are able to move and feed, but never undergo normal cell division accompanied by mitosis. Test-free cells can restore the test in three different ways: (1) building almost normal test; (2) building a very small single-chambered test; (3) building a test which consisted of several small chambers attached to each other. Although newly constructed tests are abnormal, cells restore shape, size and other characteristic features of the test in the next one or two generations of descendants. The results obtained suggest that the test may be critically important for completing a life cycle of an amoeba., (Copyright © 2016. Published by Elsevier GmbH.)

Planktonic arcellinid testate amoebae exhibit a broad-range of morphological variability but it is currently unclear to what extent this variability represents phenotypic plasticity or if it is genetically determined. We investigated the morphology and phylogenetic relationships of three endemic east-asian Difflugia taxa 1) the vase-shaped D. mulanensis, 2) and a spinose and a spineless morphotypes of D. tuberspinifera using scanning electron microscopy and two ribosomal genetic markers (SSU rDNA and ITS sequences). Our phylogenetic analyses shows that all three taxa are genetically distinct and closely related to D. achlora and Netzelia oviformis. The genetic variations between the spineless and spinose morphotypes of D. tuberspinifera were low at the SSU rRNA level (0.4%), but ten times higher at the ITS level (4.5-6%). Our data suggest that the two forms of D. tuberspinifera are sufficiently differentiated in terms of morphology and genetic characteristics to constitute two separate entities and that the presence of spines does not result from phenotypic plasticity due to environmental selective pressure. However further observational and experimental data are needed to determine if these two forms constitute different biological species., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

We describe here a new species of sphagnicolous testate amoeba found abundantly in the forested part of the Le Cachot peatland (Jura Mountains, Neuchâtel, Switzerland) based on microscopical observations (LM, SEM). The new species, called Nebela gimlii was placed in a phylogenetic tree based on mitochondrial cytochrome oxidase sequences (COI), and branched robustly within the N. collaris complex next to the morphologically similar N. guttata and N. tincta. It is however genetically clearly distinct from these two species, and differs morphologically from them by its smaller size and stouter shape of the shell. This new species completes the phylogeny of the Nebela collaris species complex, with now eight species described, mostly from peatlands and acidic forest litter, and further demonstrates the existence of an unknown diversity within testate amoebae. Improving the taxonomy of testate amoebae in peatlands and clarifying the ecology of newly discovered species should make these organisms even more valuable as bioindicator and for palaeoecological reconstruction., (Copyright © 2014. Published by Elsevier GmbH.)

Hartmannellid amoebae are an unnatural assemblage of amoeboid organisms that are morphologically difficult to discern from one another. In molecular phylogenetic trees of the nuclear-encoded small subunit rDNA, they occupy at least five lineages within Tubulinea, a well-supported clade in Amoebozoa. The polyphyletic nature of the hartmannellids has led to many taxonomic problems, in particular paraphyletic genera. Recent taxonomic revisions have alleviated some of the problems. However, the genus Saccamoeba is paraphyletic and is still in need of revision as it currently occupies two distinct lineages. Here, we report a new clade on the tree of Tubulinea, which we infer represents a novel genus that we name Ptolemeba n. gen. This genus subsumes a clade of hartmannellid amoebae that were previously considered in the genus Saccamoeba, but whose mitochondrial morphology is distinct from Saccamoeba. In accordance with previous research, we formalize the clade as distinct from Saccamoeba. Transmission electron microscopy of our isolates illustrate that both molecularly discrete species can be further differentiated by their unique mitochondrial cristal morphology., (© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.)

Difflugia is a morphologically diverse genus of testate amoebae, which are common components of freshwater ecosystems. We observed a new morphotype similar to Difflugia tuberspinifera but without spine in four Xiamen reservoirs, Fujian Province, southeast China. We investigated its morphology and biometry using light and scanning electron microscopy. The linear discriminant analysis and principal component analysis of biometric characters revealed that the spiny and spineless forms of D. tuberspinifera differed only in the presence or absence of spine. Shell height, shell diameter, aperture diameter and collar height did not differ significantly between the two morphotypes. The number of conical spines varies from 0 to 8. However, the distribution of spine numbers showed two main modes at 0 (spineless form 45.1% of individuals) and 4-6 (38.9%), suggesting the possible existence of two genetically distinct lineages. Spines may have ecological and evolutionary significance. Our results suggested that the spiny and spineless morphotypes of D. tuberspinifera represent either a single variable taxon with different ecotypes or sibling species. Further morphological studies on clonal variations and molecular approaches are needed to clarify if the spineless morph represents an independent species or not., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Testate amebae are unicellular shelled protozoa commonly used as indicators in ecological and paleoecological studies. We explored the potential application of three-dimensional (3D) X-ray micro-tomography used in addition to 2D techniques (environmental scanning electron microscopy, electron probe micro-analysis, and cathodoluminescence) for detailed characterization of agglutinated shells of protozoa. We analyzed four specimens of the aquatic testate ameba Difflugia oblonga (Arcellinida), to test whether size distribution and mineral composition of shell grains diverged from sediment size distribution and mineralogical composition. From the 3D images, the geometry of the specimens (size and mass) and of the individual grains forming the specimen (grain size distribution and volume) were calculated. Based on combined chemical, mineralogical, and morphological analyses we show that D. oblonga is able to selectively pick up the small size fraction of the sediment with a preference for low-density silicates close to quartz density (~2.65). The maximum size of the grains matches the size of the pseudostome (shell aperture), suggesting the existence of a physical limit to grain size used for building the shell. This study illustrates the potential of this combined approach to characterize agglutinated shells of protozoa. This data can be useful for detailed morphological studies with applications in taxonomy and ecology.

Flagship species are defined as microbial eukaryote species with characteristic morphologies and restricted geographic distributions. These are proposed as ideal systems to elucidate patterns of geographical distribution in microbial eukaryotes. Here we present new records of the putative flagship species Pseudonebela africana, a lobose testate amoeba (Arcellinida) characterized by a cross or clover-shaped aperture and geographic distribution previously believed to be restricted to Africa. We have sampled P. africana from 5 separate ponds in the Central and Southwest Brazillian "cerrado", and characterized individuals both by light and electron microscopy. We provide a brief description to facilitate further studies on this poorly understood taxon, and show that light microscopy is sufficient for identification, an important feature for ecological and biogeographical studies., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

An amoeba isolated from an aquatic biotope, identified morphologically as Saccamoeba limax, was found harbouring mutualistic rod-shaped gram-negative bacteria. During their cultivation on agar plates, a coinfection also by lysis-inducing chlamydia-like organisms was found in some subpopulations of that amoeba. .Here we provide a molecular-based identification of both the amoeba host and the two bacterial endosymbionts. Analysis of the 18S rRNA gene revealed that this strain is the sister-group to Glaeseria, for which we proposed the name Saccamoeba lacustris. The rod-shaped endosymbiont was identified as a member of Variovorax paradoxus group (Comamonadaceae, Beta-Proteobacteria). No growth on bacteriological agars was recorded, hence this symbiont might be strictly intracellular. The chlamydia-like parasite was unable to infect Acanthamoeba and other amoebae in coculture, showing high host specificity. Phylogenetic analysis based on the 16S rDNA indicated that it is a new member of the family Parachlamydiaceae (order Chlamydiales), for which we proposed the name 'Candidatus Metachlamydia lacustris'., (Copyright (c) 2009 Elsevier GmbH. All rights reserved.)

Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans. Furthermore, cysts, but not trophozoites, fluoresced when stained with calcofluor white. Calcofluor white and DCB, a cellulose synthesis inhibitor, inhibited B. mandrillaris encystment. This is the first report suggesting possible glycan biosynthesis in B. mandrillaris encystment, and this pathwaymay provide a potentially useful drug target and help improve treatment.

The morphology of a new testate amoeba Pentagonia zhangduensis nov. spec. was investigated using light and scanning electron microscopy. The new species was discovered in the sediments of Lake Zhangdu, Hubei Province, China. The low coefficients of variation and normal size frequency distribution suggest that P. zhangduensis is a size-monomorphic species. P. zhangduensis differs from the one other species in this genus (P. maroccana), by its larger size and its quadrangular cross section with two parallel longitudinal ridges on each of the two flattened sides of the test.

Morphological identification of protists remains an expert task, especially for little known and poorly described species. Culture collections normally accept organisms under the name provided by depositors and are not responsible for identification. Uncritical acceptance of these names by molecular phylogeneticists may result in serious errors of interpretation of phylogenetic trees based on DNA sequences, making them appear more incongruent with morphology than they really are. Several cases of misidentification in a major culture collection have recently been reported. Here we provide evidence for misidentifications of two more gymnamoebae. The first concerns "Gephyramoeba sp." ATCC 50654; it is not Gephyramoeba, a leptomyxid with lobose pseudopods, but a hitherto undescribed branching amoeba with fine, filamentous subpseudopods named here Acramoeba dendroida gen. et sp. nov. We also sequenced 18S rRNA of Page's strain of Rhizamoeba saxonica (CCAP 1570/2) and show that it is the most deeply branching leptomyxid and is not phylogenetically close to 'Rhizamoeba saxonica' ATCC 50742, which was misidentified. Correcting these misidentifications improves the congruence between morphological diversity of Amoebozoa and their rRNA-based phylogenies, both for Leptomyxida and for the Acramoeba part of the tree. On morphological grounds we transfer Gephyramoebidae from Varipodida back to Leptomyxida and remove Flamella from Leptomyxida; sequences are needed to confirm these two revisions.

The genus Sappinia, a taxon of free-living amoebae with trophozoites that typically have two closely appressed nuclei, contains two named species, Sappinia pedata, the type species, and S. diploidea. The amoebae of both species are essentially identical according to the literature. The two species are distinguished by S. pedata having a standing amoeba stage, incorrectly interpreted as a cyst, and S. diploidea having sessile, bicellular cysts. Using four isolates of S. pedata collected from around the world, we present detailed light micrographic illustrations of all stages of its life cycle. We confirm that the standing amoeba lacks a cell wall. In two isolates of S. pedata, there are bicellular cysts indistinguishable from those of S. diploidea. Using sequence data from the nuclear small subunit ribosomal RNA gene, we conclude that S. pedata and the published neotype of S. diploidea are congeneric but not conspecific. The genus branches within Thecamoebidae. Sequencing of the actin gene confirms the inclusion of Sappinia in Thecamoebidae. Resolving the taxonomy of Sappinia is gaining importance because it has recently been attributed as an opportunistic human pathogen.

Free-living amoebae (FLA) occur ubiquitously in many aquatic habitats and humid soils as well as in "artificial" water samples. In addition to their role as pathogens, FLA are known to serve as natural hosts and vehicles of transmission for various intracellular organisms. An otherwise healthy 24-year-old female patient presented with keratitis in her inflamed left eye. She was a contact lens wearer and had no history of corneal trauma. No acanthamoebae could be determined by culture methods. A Vannella strain (called VanAun0) isolated from corneal scrapings showed intracellular aggregating organisms. Within 1-2 days, the host amoebae ruptured, and numerous coccoid organisms (called Kaun1) were released. We succeeded in detecting the mechanisms of infection and intrusion of this eukaryotic organism, growing within the nucleus of the FLA, by light and electron microscopy. It could be shown that the spores at the cell membrane of strain KAun1 resemble Microsporidia and were taken up into the Amoeba by phagocytosis after adhesion of the spores and food cup formation (infective phase). The spores were transported into the cytoplasm of the vannellae in food vacuoles. Phase contrast microscopy revealed early stages of the parasites moving through the cytoplasm into the nucleus of the host amoeba. Electron microscopy showed the proliferation of polymorphic stages within the karyoplasm. The life cycle of these microsporidian-like organisms ended up with a sporogenic phase in which a terminal differentiation took place and numerous spores were released by rupture of the host cell wall. With the rupture of the host amoeba's cell membrane, the cycle started again from the beginning, the released infectious spores being ingested by other host amoebae. In particular, the morphology of the organelles made visible by electron microscopy finally allowed us to classify the endocytobionts as a microsporidan-like organism. Infection of Vannella sp. with the microsporidia-like organism strain KAun1 is a suitable model for studying the host-parasite relations of organisms using their hosts as so-called Trojan horses.

Seven marine gymnamoebae were isolated from different environments of seawater, slush (pack ice meltwater), and sediment in the Ross Sea area of Antarctica. All amoebae were isolated and maintained at temperatures below 4 degrees C. Growth, rate of locomotion, and general morphology were observed at an environmentally appropriate temperature (1 degrees C) and at room temperature (approximately 25 degrees C). Molecular (srDNA sequences) and microscopical techniques were used to identify the gymnamoebae and establish their phylogenetic affinities. Three isolates (S-131-2, SL-200, and W4-3) were assigned to a psychrophilic subspecies of Neoparamoeba aestuarina, N. aestuarina antarctica n. subsp., one isolate (S-205) was assigned to a new species of Platyamoeba, P. oblongata n. sp., two isolates (W51C#4 & W51C#5) were also assigned to a new species of Platyamoeba, P. contorta n. sp., and one isolate (S-241) was a novel psychrophilic gymnamoeba Vermistella antarctica n. gen. n. sp. Molecular and morphological results revealed that V. antarctica was not related to any described family of gymnamoebae. Strains S-205, W51C#4, and W51C#5 were capable of locomotion at room temperature, while strains SL-200, S-131-2, W4-3, and S-241 exhibited locomotion only below approximately 10 degrees C. Our results imply that the Antarctic environment is host both to cosmopolitan gymnamoebae that have acquired adaptations for existence at low environmental temperature and to apparently novel psychrophilic amoebae described here for the first time.

We characterised 9 strains selected from primary isolates referable to Paramoeba/Neoparamoeba spp. Based on ultrastructural study, 5 strains isolated from fish (amoebic gill disease [AGD]-affected Atlantic salmon and dead southern bluefin tuna), 1 strain from netting of a floating sea cage and 3 strains isolated from invertebrates (sea urchins and crab) were assigned to the genus Neoparamoeba Page, 1987. Phylogenetic analyses based on SSU rDNA sequences revealed affiliations of newly introduced and previously analysed Neoparamoeba strains. Three strains from the invertebrates and 2 out of 3 strains from gills of southern bluefin tunas were members of the N. branchiphila clade, while the remaining, fish-isolated strains, as well as the fish cage strain, clustered within the clade of N. pemaquidensis. These findings and previous reports point to the possibility that N. pemaquidensis and N. branchiphila can affect both fish and invertebrates. A new potential fish host, southern bluefin tuna, was included in the list of farmed fish endangered by N. branchiphila. The sequence of P. eilhardi (Culture Collection of Algae and Protozoa [CCAP] strain 1560/2) appeared in all analyses among sequences of strain representatives of Neoparamoeba species, in a position well supported by bootstrap value, Bremer index and Bayesian posterior probability. Our research shows that isolation of additional strains from invertebrates and further analyses of relations between molecular data and morphological characters of the genera Paramoeba and Neoparamoeba are required. This complexity needs to be considered when attempting to define molecular markers for identification of Paramoeba/Neoparamoeba species in tissues of fish and invertebrates.

Only two species of Cochliopodium below 20mum in size are currently known. These are the recently described marine Cochliopodium gallicum Kudryavtsev and Smirnov, 2006 and a freshwater C. minutum West, 1901, an incompletely described species that has never been re-isolated since its initial description. In this paper, the descriptions of three independent species of similar size below 20microm, C. minutoidum, C. kieliense, and C. maeoticum are presented. In the light microscope they all resemble the description of C. minutum, C. minutoidum being most similar to this species. However, the available description of C. minutum is inadequate to permit the assignment of any strain to this species with confidence. Therefore, a status of nomen dubium is proposed for C. minutum. Furthermore, C. minutoidum appears to be very similar to a fish endobiont identified by Dyková et al. (1998. Cochliopodium minus, a scale-bearing amoeba isolated from organs of perch Perca fluviatilis. Dis. Aquat. Org. 34, 205-210) as C. minus. Direct comparison of my strain of C. minutoidum with the type strain of C. minus suggests that it is not identical to C. minus. The obtained data enabled me to add the ultrastructural features to the diagnosis of C. minus for more precise identification of this species.

Cochliopodium gallicum n. sp., isolated from cyanobacterial mats in the Camargue (France) is the smallest marine species of Cochliopodium to date. Its unusual tectum consists of flat plate-shaped scales with honeycomb-like centres, underlain by a layer of filamentous structures connected to each other in the basal and apical parts. The tectum is very fine and can be easily lost under inappropriate EM fixation. In its light-microscopical features, this species resembles Ovalopodium carrikeri Sawyer, 1980, a himatismenid that is believed to possess a scaleless, fuzzy or hairy "glycocalyx". We suggest that O. carrikeri might have been a similar species that lost scales under fixation. Our finding makes desirable a re-investigation of the genus Ovalopodium.

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.

A total of 18 Neoparamoeba strains were characterized both morphologically and using the SSU rRNA gene sequences as molecular markers. Nine were isolated from gills of farmed Atlantic salmon, Salmo salar L., six from sediments sampled in areas of sea-cage farms and three from net material of sea-cages. The newly obtained sequences extended substantially the dataset of Neoparamoeba strains available for phylogenetic analyses, which were used to infer taxonomic relatedness among 32 strains morphologically assigned to this genus. In addition to the N. pemaquidensis and N. aestuarina clades, phylogenetic analyses clearly distinguished a third clade with sequences from six strains. Members of this clade are characterized as representatives of a new species, N. branchiphila n. sp. The diagnostic primers for the identification of this species are introduced.

Free-living amoebae infecting freshwater and marine fish include those described thus far as agents of fish diseases, associated with other disease conditions and isolated from organs of asymptomatic fish. This survey is based on information from the literature as well as on our own data on strains isolated from freshwater and marine fish. Evidence is provided for diverse fish-infecting amphizoic amoebae. Recent progress in the understanding of the biology of Neoparamoeba spp., agents responsible for significant direct losses in Atlantic salmon and turbot industry, is presented. Specific requirements of diagnostic procedures detecting amoebic infections in fish and taxonomic criteria available for generic and species determination of amphizoic amoebae are analysed. The limits of morphological and non-morphological approaches in species determination are exemplified by Neoparamoeba, Vannella and Platyamoeba spp., which are the most common amoebae isolated from fish gills, Acanthamoeba and Naegleria spp. isolated from various organs of freshwater fish, and by other unique fish isolates of the genera Nuclearia, Thecamoeba and Filamoeba. Advances in molecular characterisation of SSU rRNA genes and phylogenetic analyses based on their sequences are summarised. Attention is particularly given to specific diagnostic tools for fish-infecting amphizoic amoebae and ways for their further development.

Phylogenetic analysis of small-subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair-like filaments without any apparent organization (Neoparamoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguiform while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Clydonella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.

Members of the genus Platyamoeba are among the most common of the free-living brackish and marine amoebae; yet, to date only twelve species have been documented in the literature and only a limited number of habitats have been sampled globally. During the course of a systematic survey of salt-marsh amoebae along the east coast of the United States, a new species of Platyamoeba was discovered in sediment samples obtained from a salt marsh at Assateague Island, VA. The species can be distinguished from all other described species within the genus by the presence of a nucleus with a single parietal nucleolus and a floating form with long tapering pseudopods. Its shape varies from flabellate to spatulate as described for species of Platyamoeba and Vannella. The fine structure of the glycocalyx, however, is characteristic of Platyamoeba.