Protozoan parasites of birds from the Tremembé formation (Oligocene of the Taubaté Basin), São Paulo, Brazil.

OBJECTIVE: To analyze the presence of protozoan parasites in bird coprolites from the Tremembé Formation (Oligocene of the Taubaté Basin). MATERIALS: Twenty avian coprolites embedded in pyrobituminous shale matrices. METHODS: Samples were rehydrated and subjected to spontaneous sedimentation. RESULTS: Paleoparasitological analyses revealed oocysts compatible with the Eimeriidae family (Apicomplexa) and one single Archamoebae (Amoebozoa) cyst. CONCLUSIONS: The present work increases the amount of information about the spread of infections throughout the Cenozoic Era and reveals that the Brazilian paleoavifauna played an important role in the Apicomplexa and Amoebozoa life cycles. SIGNIFICANCE: This is the first record of protozoans in avian coprolites from the Oligocene of Brazil. These findings can help in the interpretation of phylogenies of coccidian parasites of modern birds, as certain taxonomic characters observed in the Oligocene Protozoa characterize monophyletic groups in current molecular phylogenetic analyses. LIMITATIONS: None of the oocysts were sporulated; therefore, it is not possible to identify the morphotypes to genus or species. SUGGESTIONS FOR FURTHER RESEARCH: Our results create new perspectives related to biogeographic studies of the parasitic groups described and may improve the understanding of the temporal amplitude of parasitic evolutionary relationships between Protozoans and birds.

Living in the cracks: Two novel genera of Variosea (Amoebozoa) discovered on an urban sidewalk.

Biological soil crusts represent a rich habitat for diverse and complex eukaryotic microbial communities. A unique but extremely common habitat is the urban sidewalk and its cracks that collect detritus. While these habitats are ubiquitous across the globe, little to no work has been conducted to characterize protists found there. Amoeboid protists are major predators of bacteria and other microbial eukaryotes in these microhabitats and therefore play a substantial ecological role. From sidewalk crack soil crusts, we have isolated three naked amoebae with finely tapered subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a holistic approach including light, electron, and fluorescence microscopy as well as phylogenetics using the ribosomal small subunit rRNA gene and phylogenomics using 230 nuclear genes, we find that these amoeboid organisms fail to match any previously described eukaryote genus. However, we determined the amoebae belong to the amoebozoan lineage Variosea based on phylogenetics. The molecular analyses place our isolates in two novel genera forming a grade at the base of the variosean group Protosteliida. These three novel varioseans among two novel genera and species are herein named "Kanabo kenzan" and "Parakanabo toge."

Deconstructing Difflugia: The tangled evolution of lobose testate amoebae shells (Amoebozoa: Arcellinida) illustrates the importance of convergent evolution in protist phylogeny.

Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba.

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 µm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

MyTH4-FERM myosins have an ancient and conserved role in filopod formation.

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.

Viable Species of Flamella (Amoebozoa: Variosea) Isolated from Ancient Arctic Permafrost Sediments.

Six viable strains of amoebae belonging to the genus Flamella (Amoebozoa, Variosea) were isolated from permafrost sediments sampled in the Russian Arctic region. Two of them are from late Pleistocene permafrost in North-East Siberia, and four--from Holocene and late Pleistocene in North-West Siberia. Light- and electron-microscopic study and molecular phylogeny show that these isolates represent two new species belonging to the genus Flamella. Both species are cyst-forming. This is a remarkable case of high resistance of protozoan cysts, allowing them to survive and recover an amoebae population after a very long, geologically significant period of rest; a "snapshot" of evolution in time. This study directly shows for the first time that amoeba cysts can be conserved not only for years and decades but for many thousand years and then recover, contributing to the formation of an active microbial community. We propose to name the new species as Flamella pleistocenica n.sp. and Flamella beringiania n.sp. Phylogenetic analysis shows that the genus Flamella is a robust and potentially species-rich group of Variosea.

Phylogeny of the Highly Divergent Echinosteliales (Amoebozoa).

Myxomycetes or plasmodial slime molds are widespread and very common soil amoebae with the ability to form macroscopic fruiting bodies. Even if their phylogenetic position as a monophyletic group in Amoebozoa is well established, their internal relationships are still not entirely resolved. At the base of the most intensively studied dark-spored clade lies the order Echinosteliales, whose highly divergent small subunit ribosomal (18S) RNA genes represent a challenge for phylogenetic reconstructions. This is because they are characterized by unusually long variable helices of unknown secondary structure and a high inter- and infraspecific divergence. Current classification recognizes two families: the monogeneric Echinosteliaceae and the Clastodermataceae with the genera Barbeyella and Clastoderma. To better resolve the phylogeny of the Echinosteliales, we obtained three new small subunit ribosomal (18S) RNA gene sequences of Clastoderma and Echinostelium corynophorum. Our phylogenetic analyses suggested the polyphyly of the family Clastodermataceae, as Barbeyella was more closely related to Echinostelium arboreum than to Clastoderma, while Clastoderma debaryanum was the earliest branching clade in Echinosteliales. We also found that E. corynophorum was the closest relative of the enigmatic Semimorula liquescens, a stalkless-modified Echinosteliales. We discuss possible evolutionary pathways in dark-spored Myxomycetes and propose a taxonomic update.

Occurrence and molecular characterization of free-living amoeba species (Acanthamoeba, Hartmannella, and Saccamoeba limax) in various surface water resources of Iran.

This study was conducted to determine the presence and molecular identity of Acanthamoeba species in the surface water resources of four provinces in Iran, namely Guilan, Mazandaran (North of Iran), Alborz, and Tehran (capital city), using culture- and molecular-based methods. During March to November 2014, 49 surface water samples were collected from environmental water sources-the distinct surface waters of Guilan, Mazandaran, Alborz, and Tehran provinces, in Iran. For the isolation of Acanthamoeba species, approximately 500 ml of the water samples were filtered through a cellulose nitrate membrane with a pore size of 0.45 µ. The filter was transferred onto non-nutrient agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. The presence of Acanthamoeba was confirmed by the genus-specific primer pair JDP1 and 2, and/or NA primers were used to identify Acanthamoeba and certain other free-living amoebae. In total, 38 out of 49 samples were positive by culture and/or PCR for Acanthamoeba and other free-living amoebae from all three provinces. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba (16 isolates belonged to T4 and 2 isolates belonged to T5), Hartmannella vermiformis (3/24), and Saccamoeba limax (2/24). The T4 and T5 genotypes were detected in Guilan and Mazandaran provinces. Two isolates from Guilan and Tehran provinces belonged to S. limax, and H. vermiformis was detected in Guilan province. The results of this study highlight the need to pay more attention to free-living amoebae, as human activity was observed in all of the localities from which these samples were taken. These surface waters can be potential sources for the distribution and transmission of pathogenic Acanthamoeba in the study areas, and free-living amoebas (FLA) (particularly the Acanthamoeba species) can serve as hosts for and vehicles of various microorganisms.

Evidence of parasexual activity in "asexual amoebae" Cochliopodium spp. (Amoebozoa): extensive cellular and nuclear fusion.

The majority of microbial eukaryotes have long been considered asexual, though new evidence indicates sex, or sexual-like (parasexual) behaviors that deviate from the usual union of two gametes, among other variant aspects. Over a dozen amoebozoans are implicated to have sexual stages. However, the exact mechanism by which sex occurs in these lineages remains elusive. This is mainly due to the diverse quality and cryptic nature of their life cycle. In this study we present evidence of some previously unreported aspects of the life cycle of an amoeba, Cochliopodium, that undergoes unusual intraspecific interactions using light microscopy and immunocytochemistry. Similar to other amoebozoans, Cochliopodium, is considered asexual with no published reports of sex or parasexuality. We also investigated environmental conditions that govern the observed intraspecific interactions. Both light microscopic and immunocytochemistry evidence demonstrates Cochliopodium undergoes cellular fusion (plasmogamy) and nuclear fusion (karyogamy). Large plasmodia eventually undergo karyogamy and contain large fused, polyploid, nuclei. These are observed to fragment, subsequently, by karyotomy (nuclear fission) and cytoplasmic fission to yield uninucleated amoebae. This process could lead to a non-meiotic, parasexual exchange of chromosomes in Cochliopodium. These findings strongly suggest that Cochliopodium is involved in parasexual activity and should no longer be considered strictly asexual.

Development of an in vitro model system for studying bacterially expressed dsRNA-mediated knockdown in Neoparamoeba genus.

RNA interference (RNAi) has been extensively used to study gene function in non-model organisms and has the potential to identify parasite target molecules in order to develop alternative treatment strategies. This technology could assist in further development of preventive methods against amoebic gill disease (AGD), the main health problem affecting the Atlantic salmon aquaculture industry in Tasmania (Australia) and now a significant emerging issue in Europe. Using ß-actin and EF1-α as candidate genes, we investigated the feasibility of gene knockdown by double-stranded RNA (dsRNA) in Neoparamoeba pemaquidensis, the non-infective strain closely related to the causative agent of AGD, Neoparamoeba perurans. Bacterially expressed dsRNA targeting the selected target genes was administered by soaking (2, 20 and 50 µg/mL) and a time course sampling regime performed. Quantitative real-time PCR analysis showed that candidate genes were successfully downregulated with silencing efficiency and duration both target and dose-dependent. Additionally, ß-actin deficient trophozoites unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. An effective RNAi model system for N. pemaquidensis was validated in the current study. Such findings will greatly facilitate further application of RNAi in the aetiological agent of AGD. To our knowledge, this is the first time that RNAi-mediated technology has been successfully employed in a member of the Neoparamoeba genus.

Relationships between free-living protozoa, cultivable Legionella spp., and water quality characteristics in three drinking water supplies in the Caribbean.

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).

Balamuthia mandrillaris transmitted through organ transplantation --- Mississippi, 2009.

On December 14, 2009, a physician in Mississippi contacted CDC to report possible transplant-transmitted encephalitis in two kidney transplant recipients who shared the same organ donor. Histopathologic testing of donor autopsy brain tissue at CDC showed amebae, and subsequent testing of specimens from the donor and the two kidney recipients confirmed transmission by transplantation of Balamuthia granulomatous amebic encephalitis (GAE), a rare disease caused by Balamuthia mandrillaris, a free-living ameba found in soil. One kidney recipient, a woman aged 31 years, died; the other recipient, a man aged 27 years, survived with neurologic sequelae. Recipients of the heart and liver from the same donor received preemptive therapy and have shown no signs of infection. The donor, a previously healthy boy aged 4 years, was presumed to have died from acute disseminated encephalomyelitis (ADEM), an autoimmune neurologic disease, after infection with influenza A. An investigation was conducted by the state health departments in Mississippi, Kentucky, Florida, and Alabama and CDC to characterize the cases, elucidate possible exposures in the donor, and develop recommendations for early detection and prevention. This is the first reported transmission of Balamuthia by organ transplantation. Clinicians should be aware of Balamuthia infection as a potentially fatal cause of encephalitis. Organ procurement organizations (OPOs) and transplant centers should be aware of the potential for Balamuthia infection in donors with encephalitis of uncertain etiology, and OPOs should communicate this elevated risk for infection to transplant centers so they can make an informed risk assessment in the decision to accept an organ.

A phylogenetic survey of myotubularin genes of eukaryotes: distribution, protein structure, evolution, and gene expression.

BACKGROUND: Phosphorylated phosphatidylinositol (PtdIns) lipids, produced and modified by PtdIns kinases and phosphatases, are critical to the regulation of diverse cellular functions. The myotubularin PtdIns-phosphate phosphatases have been well characterized in yeast and especially animals, where multiple isoforms, both catalytically active and inactive, occur. Myotubularin mutations bring about disruption of cellular membrane trafficking, and in humans, disease. Previous studies have suggested that myotubularins are widely distributed amongst eukaryotes, but key evolutionary questions concerning the origin of different myotubularin isoforms remain unanswered, and little is known about the function of these proteins in most organisms. RESULTS: We have identified 80 myotubularin homologues amidst the completely sequenced genomes of 30 organisms spanning four eukaryotic supergroups. We have mapped domain architecture, and inferred evolutionary histories. We have documented an expansion in the Amoebozoa of a family of inactive myotubularins with a novel domain architecture, which we dub "IMLRK" (inactive myotubularin/LRR/ROCO/kinase). There is an especially large myotubularin gene family in the pathogen Entamoeba histolytica, the majority of them IMLRK proteins. We have analyzed published patterns of gene expression in this organism which indicate that myotubularins may be important to critical life cycle stage transitions and host infection. CONCLUSIONS: This study presents an overall framework of eukaryotic myotubularin gene evolution. Inactive myotubularin homologues with distinct domain architectures appear to have arisen on three separate occasions in different eukaryotic lineages. The large and distinctive set of myotubularin genes found in an important pathogen species suggest that in this organism myotubularins might present important new targets for basic research and perhaps novel therapeutic strategies.