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It is almost 50 years since amoeba-associated granulomatosis was first described in goldfish (Carassius auratus), but the aetiologic agent has never been identified. In this study, a new archamoeba species, Endolimax carassius, is characterised as the causative agent of systemic granulomatous disease in goldfish. Phylogenetic analyses determined the new goldfish parasite species as a genotype sister of Endolimax piscium, the causative agent of systemic granulomatous disease in Senegalese sole. The fish genotypes clustered with other available genotypes from mammals in a well-supported Endolimax clade within the Mastigamoebidae. Endolimax is a genus of intestinal amoebae which stands among the least known human protists. Previous studies on amoebic systemic granulomatosis of a marine fish (Solea senegalensis) resulted in the unexpected characterization of a new organism which was related to Endolimax and named E. piscium. The existence of multiple reports of systemic granulomatosis caused presumptively by unidentified amoebae in goldfish lead us to investigate the organism involved in goldfish disease. Analysed goldfish presented small whitish nodules in the kidney, which correspond to chronic granulomatous inflammatory reactions with a ring-layer of amoebae in the periphery. Amoebae were amitochondriate and were located in a parasitophorous vacuole within macrophages, as previous studies on this condition in goldfish and other freshwater fish pointed out. SSU rDNA characterization confirmed a new Endolimax lineage which appears closely related to E. piscium, but the molecular evidence, distinct pathological features and lack of ecological overlapping between the hosts support their assignment to a new species, E. carassius. The results support the existence of a considerable unexplored diversity of Endolimax spp. among fish, and their proper characterization can contribute to an understanding of Archamoebae evolution and pathogenic potential.

Endolimax nana is a common endobiont of the human intestine, but members of the genus have also been reported in non-human hosts and in non-intestinal organs. Limited information is available regarding the genetic diversity of Endolimax, which is necessary to delineate species, host specificity and potential differences in clinical impact on the host. Here, we used cloning of PCR products followed by Sanger sequencing and next-generation PacBio Sequencing to obtain Endolimax-related nuclear ribosomal gene sequences and undertook a phylogenetic analysis to gain additional insight into the taxonomy of Endolimax and related organisms. The new sequences confirmed that E. nana forms a discrete clade within the Archamoebae and is related to Endolimax piscium and Iodamoeba. However, we identified substantial sequence divergence within E. nana and evidence for two distinct clades, which we propose to name E. nana ribosomal lineage 1 and E. nana ribosomal lineage 2. Both of the sequencing approaches applied in the study helped us to improve our understanding of genetic diversity across Endolimax, and it is likely that wider application of next-generation sequencing technologies will facilitate the generation of Endolimax-related DNA sequence data and help complete our understanding of its phylogenetic position and intrageneric diversity.

The nature and features of organization of reserve polysaccharides in three species of the genus Pelomyxa-P. palustris, P. belevskii, and P. stagnalis-were studied using light and transmission electron microscopy. We applied the periodic acid-Schiff reaction that is a highly selective method for detecting glycogen. The fluorescent dye auramine-SO2 (Au-SO2) was used as a Schiff-type reagent. The densely packed aggregates of glycogen that form the morphologically differentiated organelle-like bodies are revealed in the cytoplasm in all studied species. The organization of these bodies is characterized by the species-specific features, while in most cases, their size and number in the cells vary depending on the season of the year. Although in all the cases we studied, these bodies do not have their own boundary membrane, in fact, they are surrounded by membranous structures. These structures differ in a variety of Pelomyxa species. We concluded that there are two groups of species in the genus Pelomyxa. The first one includes organisms containing glycogen structures in the cytoplasm (P. palustris, P. belevskii, P. stagnalis, P. binucleata, P. corona, P. secunda). No inclusions resembling glycogen bodies were found in P. flava, P. paradoxa, P. gruberi, and P. prima that form the second group.

The nucleus of some representatives of the genus Pelomyxa (Amoebozoa, Archamoebae, Pelobiontida) contains specific bodies (membrane-less organelles). They may be either embedded in the nucleolar mass or detached from the nucleolus. We termed these nuclear bodies the glomerulosomes for their characteristic ultrastructural appearance. The glomerulosomes are distinct nuclear bodies, about 1 μm in diameter. The morphological and diagnostic unit of a glomerulosome is an electron-dense thread/string, about 30-40 nm in thickness. These threads are not direct continuation of the nucleolar material. The threads create the unique geometric appearance of the glomerulosome by being organized into precisely parallel rows/cords. Each cord of the threads can curve at different angles within the glomerulosome body, but the threads themselves are not coiled. Nowadays, the glomerulosomes have been discovered in P. palustris, P. stagnalis, P. paradoxa, and Pelomyxa sp. Despite the unique appearance of glomerulosomes, their existence may be a more common phenomenon in eukaryotic cells than just a specific feature of the nucleus of elected pelomyxes.

We described Pelomyxa doughnuta sp. nov. and examined it with the use of light, electron, and immunofluorescence microscopy as well as cytochemical methods. The cells of P. doughnuta sp. nov. are usually binuclear, although cells with one, three, or four nuclei are sometimes found in the population. A unique feature of the new species is a dense capsule around the nucleus. It consists of a continuous layer of glycogen 5-20 µm thick. The tubulin cytoskeleton is mainly represented by perinuclear microtubules. P. doughnuta sp. nov. has a filamentous glycocalyx and strongly reduced components of flagellar apparatus. Obligate prokaryotic endocytobionts of two morphotypes are present in the cytoplasm.

Background Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. Results We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5′-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and d-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. Conclusions This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

Pelomyxa schiedti is a free-living amoeba belonging to the group Archamoebae, which encompasses anaerobes bearing mitochondrion-related organelles (MROs) – hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica. Anaerobic peroxisomes, another adaptation to anaerobic lifestyle, were identified only recently in M. balamuthi. We found evidence for both these organelles in the single-cell-derived genome and transcriptome of P. schiedti, and corresponding vesicles were tentatively revealed in electron micrographs. In silico reconstructed MRO metabolism seems similar to that of M. balamuthi harboring respiratory complex II, electron-transferring flavoprotein, partial TCA cycle running presumably in reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, glycine cleavage system, and sulfate activation pathway. The cell disposes with an expanded set of NIF enzymes for iron sulfur cluster assembly, but their localization remains unclear. Quite contrary, out of 67 predicted peroxisomal enzymes, only four were reported also in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Other putative functions of peroxisomes could be pyridoxal 5I⍰-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Future experimental evidence is necessary to define functions of this surprisingly enzyme-rich anaerobic peroxisome.Author summaryA major part of the microbial diversity cannot be cultured in isolation, and so it escapes from traditional ways of investigation. In this paper, we demonstrate the successful approach for generating good-quality genome and transcriptome drafts from a peculiar amoeba Pelomyxa schiedti using single-cell methods. P. schiedti is a member of Archamoebae clade harboring microaerobic protists including a free-living Mastigamoeba balamuthi and a human parasite Entamoeba histolytica. Mitochondria and peroxisomes represent two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform to anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, all of which encompass different enzymatic capacity. Anaerobic peroxisomes have been first noticed in M. balamuthi, but their function remained unclear for now. Data obtained in this study were used for revealing the presence and for the detailed functional annotations of anaerobic derivates of mitochondria and peroxisomes in P. schiedti, which were corroborated by transmission electron microscopy.

Systemic amoebiasis of Senegalese sole Solea senegalensis is caused by Endolimax piscium Constenla, Padrós & Palenzuela, 2014 a cryptic parasitic member of the Archamoebae whose epidemiology is yet unknown. To test whether the parasite can be transmitted horizontally, an experimental trial by cohabitation between non-infected and infected fish was designed. Transmission of the parasite from naturally infected to healthy fish was confirmed in the experiment, with the water as the most likely route of infection. Under the conditions of the study, the infection process was remarkably slow, as parasites could be detected by in situ hybridization within the intestinal mucosa of recipient fish only after 17 wk of cohabitation, and none of the new hosts displayed clinical signs of disease. Long prepatent period and the need for additional triggering factors for the development of the clinical condition are suggested. The intestinal mucosa is proposed as the tissue where the amoeba can survive as endocommensal, but also as an invasion route from which the parasite would disperse to other organs, This work was partially supported by the research grants PROMETEO 2010/006 and ISIC 2012/003 (Generalitat Valenciana). Experiments in ECIMAT were supporting by the Programme for Consolidation of Competitive Research Units (Xunta de Galicia), cofounded by the European Regional Development Fund. M.C. was supported by a PhD student grant from the Universitat Autònoma de Barcelona (‘P.I.F. Programme’).

Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi. We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named “anaerobic” peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.

The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30–50 µm in length and 9–13 µm in width. Observed cysts were uninucleate, ranging in size from 10 to 17.5 µm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis.

Archamoebae is an understudied group of anaerobic free-living or endobiotic protists that constitutes the major anaerobic lineage of the supergroup Amoebozoa. Hitherto, the phylogeny of Archamoebae was based solely on SSU rRNA and actin genes, which did not resolve relationships among the main lineages of the group. Because of this uncertainty, several different scenarios had been proposed for the phylogeny of the Archamoebae. In this study, we present the first multigene phylogenetic analysis that includes members of Pelomyxidae, and Rhizomastixidae. The analysis clearly shows that Mastigamoebidae, Pelomyxidae and Rhizomastixidae form a clade of mostly free-living, amoeboid flagellates, here called Pelobiontida. The predominantly endobiotic and aflagellated Entamoebidae represents a separate, deep-branching lineage, Entamoebida. Therefore, two unique evolutionary events, horizontal transfer of the nitrogen fixation system from bacteria and transfer of the sulfate activation pathway to mitochondrial derivatives, predate the radiation of recent lineages of Archamoebae. The endobiotic lifestyle has arisen at least three times independently during the evolution of the group. We also present new ultrastructural data that clarifies the primary divergence among the family Mastigamoebidae which had previously been inferred from phylogenetic analyses based on SSU rDNA.

Members of the archamoebae comprise free-living and endobiotic amoeboid flagellates and amoebae that live in anoxic/microoxic habitats. Recently, the group has been divided into four separate families, Mastigamoebidae, Entamoebidae, Pelomyxidae, and Rhizomastixidae, whose interrelationships have not been completely resolved. There still are several key members of the archamoebae, notably the genus Mastigella, from which sequence data are missing. We established 12 strains of 5 species of Mastigella and Pelomyxa in culture, examined their morphology and determined their actin gene sequences. In addition, we examined the ultrastructure of three strains and determined and analyzed SSU rDNA sequences of two strains. Our data strongly suggest that Mastigella is specifically related to Pelomyxa, and it is transferred into the family Pelomyxidae. Surprisingly, Mastigella is likely paraphyletic with Pelomyxa forming its internal branch. The two genera share several morphological features that point to their common evolutionary history. Three new species of Mastigella are described: M. erinacea sp. nov., M. rubiformis sp. nov. and M. ineffigiata sp. nov.

Amoebozoa is a key phylum for eukaryote phylogeny and evolutionary history, but its phylogenetic validity has been questioned since included species are very diverse: amoebo-flagellate slime-moulds, naked and testate amoebae, and some flagellates. 18S rRNA gene trees have not firmly established its internal topology. To rectify this we sequenced cDNA libraries for seven diverse Amoebozoa and conducted phylogenetic analyses for 109 eukaryotes (17-18 Amoebozoa) using 60-188 genes. We conducted Bayesian inferences with the evolutionarily most realistic site-heterogeneous CAT-GTR-Γ model and maximum likelihood analyses. These unequivocally establish the monophyly of Amoebozoa, showing a primary dichotomy between the previously contested subphyla Lobosa and Conosa. Lobosa, the entirely non-flagellate lobose amoebae, are robustly partitioned into the monophyletic classes Tubulinea, with predominantly tube-shaped pseudopodia, and Discosea with flattened cells and different locomotion. Within Conosa 60/70-gene trees with very little missing data show a primary dichotomy between the aerobic infraphylum Semiconosia (Mycetozoa and Variosea) and secondarily anaerobic Archamoebae. These phylogenetic features are entirely congruent with the most recent major amoebozoan classification emphasising locomotion modes, pseudopodial morphology, and ultrastructure. However, 188-gene trees where proportionally more taxa have sparser gene-representation weakly place Archamoebae as sister to Macromycetozoa instead, possibly a tree reconstruction artefact of differentially missing data.

The structure of archamoeba Pelomyxa paradoxa Penard, 1902—an organism that has been reisolated for the first time since its redescription—has been studied at the light and electron-microscopy levels. During movement, pelomyxa have a cigarlike body shape, at the sides of which conical and fingerlike hyaline pseudopodia are formed. At the posterior end of the cell, a massive bulb-shaped uroid is exposed. The plasmalemma of cells of P. paradoxa carries om its surface a thin layer of amorphous glycocalyx. The cytoplasm of pelomyxa is filled by numerous so-called “structural” and digestive vacuoles of different sizes. In P. paradoxa, two kinds of obligate prokaryotic endocytobionts lying in individual symbiontophore vacuoles are present. In the life cycle of P. paradoxa, the mononuclear stage is predominant. In multinuclear forms, the number of nuclei usually does not exceeds 12. A 250- to 300-nm-thick multilamellar layer is present outside the nuclear membrane; a layer of numerous small vesicles filled with electron-dense content is located “over” it. The nucleolar material is represented as several large structures of irregular shape, which are located closer to the periphery and occupy a significant part of the inner content of the nucleus. Inside the nucleolus and sometimes directly in the nucleoplasm, there are revealed small rounded bodies formed by densely packed and uniformly interlaced electron-dense fibrils. The relatively numerous nonfunctioning flagella are located predominantly in the posterior part of the protozoan body. The flagellum base contains a single short kinetosome surrounded by a muff of electron-dense material. From 50 to 60 radial microtubules and a lateral root depart from it. Comparative-morphological analysis shows that P. paradoxa occupies an intermediate position between two groups of species of the order Pelomyxa (P. gruberi + P. prima and P. palustris + P. stagnalis + P. belewski) that differ significantly in the character of organization of the basal flagellar apparatus.

We report the light-microscopic morphology and ultrastructure of a novel free-living, heterotrophic protist, Creneis carolina gen. et sp. nov. isolated from marine anoxic sediments. C. carolina is a heterotrophic, obligately anaerobic amoeboid flagellate, and superficially resembles Mastigamoeba (Amoebozoa: Archamoebae) or Breviata (Breviatea) by possessing a single anterior flagellum closely associated with the nucleus, and because it appears to be an anaerobe. However, its life cycle contains multiflagellate cells with an unusual morphology. The structure of the mastigont of C. carolina is unique and not readily comparable with any eukaryotic group. Unexpectedly, phylogenetic analyses of SSU rDNA and of a concatenate of α- and β-tubulin genes with SSU rDNA convincingly showed that C. carolina is a member of Heterolobosea and belongs to the taxon Tetramitia.

The archamoebae form a small clade of anaerobic/microaerophilic flagellates or amoebae, comprising the pelobionts (mastigamoebids and pelomyxids) and the entamoebae. It is a member of the eukaryotic supergroup Amoebozoa. We examined 22 strains of 13 species of Mastigamoeba, Pelomyxa and Rhizomastix by light-microscopy and determined their SSU rRNA gene sequences. The SSU rRNA gene sequences of Pelomyxa palustris and Mastigella commutans in GenBank are shown to belong to P. stagnalis and Mastigamoeba punctachora, respectively. Five new species of free-living archamoebae are described: Mastigamoeba abducta, M. errans, M. guttula, M. lenta, and Rhizomastix libera spp. nov. A species of Mastigamoeba possibly living endosymbiotically in Pelomyxa was identified. Rhizomastix libera, the first known free-living member of that genus, is shown to be an archamoeba. R. libera possesses an ultrastructure unique within archamoebae: a rhizostyle formed from a modified microtubular cone and a flagellum with vanes. While many nominal species of pelobionts are extremely hard to distinguish by light microscopy, transient pseudopodial characters are worthy of further investigation as taxonomic markers.

p-Cresol and indole are volatile biologically active products of the bacterial degradation of tyrosine and tryptophan respectively. They are typically produced by bacteria in animal intestines, soil and various sediments. Here, we demonstrate that the free-living eukaryote Mastigamoeba balamuthi and its pathogenic relative Entamoeba histolytica produce significant amounts of indole via tryptophanase activity. Unexpectedly, M. balamuthi also produces p-cresol in concentrations that are bacteriostatic to non-p-cresol-producing bacteria. The ability of M. balamuthi to produce p-cresol, which has not previously been observed in any eukaryotic microbe, was gained due to the lateral acquisition of a bacterial gene for 4-hydroxyphenylacetate decarboxylase (HPAD). In bacteria, the genes for HPAD and the S-adenosylmethionine-dependent activating enzyme (AE) are present in a common operon. In M. balamuthi, HPAD displays a unique fusion with the AE that suggests the operon-mediated transfer of genes from a bacterial donor. We also clarified that the tyrosine-to-4-hydroxyphenylacetate conversion proceeds via the Ehrlich pathway. The acquisition of the bacterial HPAD gene may provide M. balamuthi a competitive advantage over other microflora in its native habitat.

Background Intestinal parasites are endemic in many parts of the world where HIV infection is also widespread. Previous studies had shown that the spectrum of opportunistic and common endemic parasitic infections with HIV vary in different regions and usually reflect the infections prevalent in these regions. This present study was aimed at comparing the prevalence and types of intestinal parasitic infections in HIV sero-positive and sero-negative patients in Lagos. Materials and methods Venous blood and stool samples of 1080 patients, recruited from three health care institutions were screened for HIV infection and intestinal parasites using HIV-1, HIV-2 rapid tests, direct wet mount with saline/iodine and formol-ether technique, respectively. Results Results showed that 6% (65/1080) of patients were sero-positive for HIV infection. In addition, 23.3% (252/1080) patients were infected with intestinal parasites and 33.8% (22/65) of patients with HIV had intestinal parasites co-infections. The prevalence of Entamoeba histolytica / Entamoeba dispar , Entamoeba coli , Iodamoeba butschilii , Giardia intestinalis , and Hookworm were statistically significantly higher among HIV sero-positive patients as compared to the HIV sero-negative patients. In addition, HIV sero-positive patients had higher odds of mixed intestinal parasites than the HIV sero-negative patients (9.1% versus 3.9%; adjusted OR 2.05, 95% CI, 1.14–3.72, P = 0.021). Conclusion In this study population, HIV sero-positive patients were more likely to have intestinal parasitic infections. The study underscores the public health significance of intestinal parasitic infections in HIV infected individuals.

Most of the major groups of eukaryotes have microbial representatives that thrive in low oxygen conditions. Those that have been studied in detail generate ATP via pathways involving anaerobically functioning enzymes of pyruvate catabolism that are typically absent in aerobic eukaryotes and whose origins remain controversial. These enzymes include pyruvate:ferredoxin oxidoreductase, pyruvate:NADP(+) oxidoreductase, and pyruvate formate lyase (Pfl). Pfl catalyzes the nonoxidative generation of formate and acetyl-Coenzyme A (CoA) from pyruvate and CoA and is activated by Pfl activating enzyme (Pfla). Within eukaryotes, this extremely oxygen-sensitive pathway was first described in the hydrogenosomes of anaerobic chytrid fungi and has more recently been characterized in the mitochondria and chloroplasts of the chlorophyte alga Chlamydomonas reinhardtii. To clarify the origins of this pathway, we have comprehensively searched for homologs of Pfl and Pfla in publicly available large-scale eukaryotic genomic and cDNA sequencing data, including our own from the anaerobic amoebozoan Mastigamoeba balamuthi. Surprisingly, we find that these enzymes are widely distributed and are present in diverse facultative or obligate anaerobic eukaryotic representatives of the archaeplastidan, metazoan, amoebozoan, and haptophyte lineages. Using maximum likelihood and Bayesian phylogenetic methods, we show that the eukaryotic Pfl and Pfla sequences each form monophyletic groups that are most closely related to homologs in firmicute gram-positive bacteria. Topology tests exclude both α-proteobacterial and cyanobacterial affinities for these genes suggesting that neither originated from the endosymbiotic ancestors of mitochondria or chloroplasts. Furthermore, the topologies of the eukaryote portion of the Pfl and Pfla trees significantly differ from well-accepted eukaryote relationships. Collectively, these results indicate that the Pfl pathway was first acquired by lateral gene transfer into a eukaryotic lineage most probably from a firmicute bacterial lineage and that it has since been spread across diverse eukaryotic groups by more recent eukaryote-to-eukaryote transfer events.

Using light and electron microscopy, the morphology of a new species of pelobionts Pelomyxa flava was studied. The coverings of P. flava are represented by plasma membrane bearing the thick layer of weakly structured glycocalyx on its outer surface. Numerous flagella are often located on the tops of short conical pseudopodia. Kinetosomes of flagella reach a length of 0.9 microm and are hollow with a pronounced central filament. Rootlet system is represented by three groups of microtubules: the radial, basal and microtubules of lateral root. The transition zone is short and does not exceed the level of cell surface; the axoneme is characterized by an unstable set of microtubules. Trophic stages of P. flava life cycle are presented by binuclear cells; plasmotomy is performed at the tetranuclear stage. Nuclei have a granular structure. Fibrillar nuclear bodies were revealed in karyoplasm. The nuclei shell has a complex organization. On its surface, the outer membrane has a layer of electron-dense material which contacts with short microtubules, located in a row at the surface of the nuclear envelope. The bubbles and cisterns of endoplasmic reticulum, which are the derivatives of the nuclear envelope, are located outward from the microtubules. The presence of structural and digestive vacuoles and grains of glycogen was noticed in P. flava endoplasm. Three types of prokaryotic cytobionts were revealed. Large multi-membranous organelles reaching 5 pm in diameter were described for the first time. We discuss morphology and biology features of P. flava in comparison with the previously studied Pelomyxa species.

The structure of a new pelomyxa species was investigated on the level fo light and electron microscopy. The length of locomotive forms of Pelomyxa stagnalis reaches 800 μm. The thin layer of amorphous glycocalyx is located on the cell surface. Numerous nonfunctioning flagellae are revealed predominantly in the uroidal zone. The axoneme has a nonstable set of microtubules. No additional structures are present in the transition zone. The length of P. stagnalis flagella kinetosomes does not exceed 150 nm. Fifteen to twenty microtubules extend from the side surface of each kinetosome at a small angle to the cell surface. One of main components of the P. stagnalis cytoplasm are structural vacuoles. Glycogen bodies in cells are surrounded by flattened ER cisterns, which are often filled with electron-dense material. Cells of P. stagnalis were found to contain two species of prokaryote endobionts that differ in the peculiarities of their fine structure. The number of nuclei in cells of the P. stagnalis adult individuals can reach 50 or more. The nuclei are surrounded by a bilayer envelope formed by the multilaminar layer and by the outer layer composed of vesicles often filled with an electron-dense material. The nucleolus is usually single and is located in the center of the nucleus. In nuclei, predominantly in connection with nucleoli, bodies are formed that are formed by interlacing electron-dense strands.

The genus Rhizomastix contains five species of amoeboflagellates with a single anterior flagellum, which live as intestinal symbionts of insects and amphibians. Though established in 1911, Rhizomastix has been neglected for decades and its phylogenetic position is uncertain. This paper describes the morphology of the first cultivated strain of Rhizomastix. The organism was isolated from an argentine horned frog and differs from the known Rhizomastix species by the presence of biflagellate cells. The isolate is described as Rhizomastix biflagellata sp. nov. A possible relationship of Rhizomastix to Archamoebae is discussed.

Members of the group Archamoebae are free-living or endobiotic amoeboid flagellates and amoebae. They live in anoxic or microoxic habitats, and their mitochondria have been reduced. They were originally thought to lack mitochondria and represent one of the earliest eukaryotes. However, this hypothesis has been refuted, and now it is evident that the Archamoebae belongs to the lineage Conosa within the supergroup Amoebozoa, together with aerobic slime molds (Macromycetozoa) and variosean amoebae and flagellates. Relatively simple microtubular cytoskeleton is a characteristic feature of Archamoebae. It consists of a single basal body from which a flagellum arises, lateral root, and microtubular cone. Cytoskeleton of aflagellated genera has been completely reduced. About 350 species names of Archamoebae have been created so far. However, most descriptions were based on inadequate morphological features. The identity of numerous species is uncertain, and many of them are likely synonymous. Another problem is a small amount of available molecular data. During our project, we have substantially improved the dataset of DNA sequences of archamoebae. On the basis of molecular and morphological data, we described 13 new species. We showed that genus Rhizomastix belongs to Archamoebae and displays a new type...

Systemic amoebiasis of sole is caused by Endolimax piscium, a cryptic parasitic archamoeba whose epidemiology and pathogeny are yet unknown. To establish reliable detection methods for this parasite, a battery of molecular diagnostic tools (ISH, PCR and qPCR) were developed and evaluated with a panel of clinical samples from symptomatic diseased fish and from apparently normal animals of different stocks. As there is neither enough background information on the epidemiology of the disease nor a validated reference method, comparison of tests used a composite reference method approach. The ISH technique was the most specific and sensitive in intestine samples and particularly useful as a reference confirmatory method, while the best method in muscle samples was qPCR. Application of the tests to asymptomatic fish demonstrated presence of parasites in a large proportion (>25%) of their intestines, suggesting that this is the point of entry of the amoebae and the initial stage in the development of the disease. The triggering factors that facilitate the breaching of the intestinal barrier by E. piscium, causing granulomatous lesions in other organs and systemic spreading, are not completely understood but our results point to the connective tissue as a preferential target for parasite development and migration., This work was funded through CSIC Internal Research Programmes (Intramural project 201330E025). Additional funding was granted by the regional Government (Generalitat Valenciana PROMETEO 2010/006 and ISIC 2012/003). M. Constenla was supported by a Ph.D. student grant of the Universitat Autònoma de Barcelona (‘P.I.F. Programme’).

Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle.

Morphology of a pelobiont Pelomyxa secunda (Gruber, 1884) comb. nov. was investigated at light- and electron-microscopical levels. Locomotive forms are elongated or cigar-shaped. The size of active forms varies from 200 to 300 μm. Larger individuals (up to 400 μm) are not able to directed movement. Organism can produce short, usually finger-shaped hyaline pseudopodia at the frontal side or laterally. The cell coat is represented by amorphous glycocalix, up to 300 nm in thickness. A thin periphery cytoplasmic zone is deprived of any organelles, vacuoles, endocytobionts and other inclusions and separated from main cytoplasm by a layer of arranged microfilaments. P. secunda is multinucleate organism; nuclei are of granular type. The nucleolar material is represented by two forms of discrete structures differing in size and electron density. Two or three layers of short microtubules organized in the parallel arrangement are associated with outer side of the nuclear envelop. P. secunda possess two types of obligate prokaryotic endocytobionts lying in individual symbiontophoric vacuoles. Undulipodia, kinetosomes and root microtubular derivatives are not observed in P. secunda cells as well as any developed cytoplasmic microtubular cytoskeleton.

The genus Rhizomastix is a poorly known group of amoeboid heterotrophic flagellates living as intestinal commensals of insects, amphibians or reptiles, and as inhabitants of organic freshwater sediments. Eleven Rhizomastix species have been described so far, but DNA sequences from only a single species have been published. Recently, phylogenetic analyses confirmed a previous hypothesis that the genus belongs to the Archamoebae; however, its exact position therein remains unclear. In this study we cultured nine strains of Rhizomastix, both endobiotic and free-living. According to their light-microscopic morphology and SSU rRNA and actin gene analyses, the strains represent five species, of which four are newly described here: R. bicoronata sp. nov., R. elongata sp. nov., R. vacuolata sp. nov. and R. varia sp. nov. In addition, R. tipulae sp. nov., living in the intestine of crane flies, is separated from the type species, R. gracilis. We also examined the ultrastructure of R. elongata sp. nov., which revealed that it is more complicated than the previously described R. libera. Our data show that either the endobiotic lifestyle of some Rhizomastix species has arisen independently from other endobiotic archamoebae, or the free-living members of this genus represent a secondary switch from the endobiotic lifestyle.

Recent molecular phylogenetic studies have led to the erection of the phylum Amoebozoa, uniting naked and testate lobose amoebae, the mycetozoan slime moulds and amitochondriate amoeboid protists (Archamoebae). Molecular data together with ultrastructural evidence have suggested a close relationship between Mycetozoa and Archamoebae, classified together in the Conosea, which was named after the cone of microtubules that, when present, is characteristic of their kinetids. However, the relationships of conoseans to other amoebozoans remain unclear. Here, we obtained the complete small-subunit (SSU) rRNA gene sequence (2746 bp) of the enigmatic, multiflagellated protist Multicilia marina, which has formerly been classified either in a distinct phylum, Multiflagellata, or among lobose amoebae. Our study clearly shows that Multicilia marina belongs to the Amoebozoa. Phylogenetic analyses including 60 amoebozoan SSU rRNA gene sequences revealed that Multicilia marina branches at the base of the Conosea, together with another flagellated amoebozoan, Phalansterium solitarium, as well as with Gephyramoeba sp., Filamoeba nolandi and two unidentified amoebae. This is the first report showing strong support for a clade containing all flagellated amoebozoans and we discuss the position of the root of the phylum Amoebozoa in the light of this result.

The taxonomic position of the uniciliate, unicentriolar zooflagellate Phalansterium is problematic; its distinctive ultrastructure with a pericentriolar microtubular cone placed it in its own order and suggested phenotypic closeness to the eukaryote cenancestor. We sequenced the 18S rRNA of a unicellular Phalansterium. Phylogenetic analysis shows that it belongs to Amoebozoa, decisively rejecting a postulated relationship with the cercozoan Spongomonas; Phalansterium groups with Varipodida ord. nov. (Gephyramoeba/Filamoeba) or occasionally Centramoebida emend. (Acanthamoebidae/Balamuthiidae fam. nov.), centrosomes of the latter suggesting flagellate ancestors. We also studied Phalansterium solitarium cyst ultrastructure; unlike previously studied P. solitarium, this strain has pentagonally symmetric walls like P. consociatum. We also sequenced 18S rRNA genes of further isolates of Hyperamoeba, an aerobic unicentriolar amoeboflagellate with conical microtubular skeleton; both group strongly with myxogastrid Mycetozoa. However, the four Hyperamoeba strains do not group together, suggesting that Hyperamoeba are polyphyletic derivatives of myxogastrids that lost fruiting bodies independently. We revise amoebozoan higher-level classification into seven classes, establishing Stelamoebea cl. nov. for Protosteliida emend. plus Dictyosteliida (biciliate former ‘protostelids’ comprise Parastelida ord. nov. within Myxogastrea), and new subphylum Protamoebae to embrace Variosea cl. nov. (Centramoebida, Phalansteriida, Varipodida), Lobosea emend., Breviatea cl. nov. for ‘Mastigamoeba invertens’ and relatives, and Discosea cl. nov. comprising Glycostylida ord. nov. (vannellids, vexilliferids, paramoebids, Multicilia), Dermamoebida ord. nov. (Thecamoebidae) and Himatismenida. We argue that the ancestral amoebozoan was probably unikont and that the cenancestral eukaryote may have been also.

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyse actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, as well as the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the “Gymnamoebia sensu stricto” and the Archamoebae (pelobionts+entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

Morphology of a pelobiont Pelomyxa paradoxa Penard, 1902 was investigated at light- and electron-microscopical levels. Locomoting cells are cigar-shaped. The cells produce many hyaline pseudopodia of digital and conical form at lateral sides of the body. The organism has a pronounced hyaline bulbous uroid with broad peripheral zone of hyaloplasm and many conical hyaline villi. There is a thin layer of amorphous glycocalix at the cell surface. "Structure" and food vacuoles of different size are very abundant in the endoplasm. Two different species of prokaryote endocytobionts are peculiar for P. paradoxa. Uninucleate stage dominates in the life cycle of P. paradoxa. Usually there are no more than 10-12 nuclei in multinucleate forms of P. paradoxa. Pelomyxae nuclei are closely surrounded by thick multilaminar layer and additionally by one more layer, which is formed by small vesicles with electron-dense content. Several irregular-shaped nucleoli are situated at the nucleus periphery. Inside the nucleoli, and sometimes directly in nucleoplasm the small round bodies are revealed, these bodies being formed by tightly packed electron-dense fibrils. Many non-motile flagellae are located mainly in the uroidal zone of the cell. Pronounced lateral root and 50-60 radial microtubules originate from the electrone-dense muft around the kinetosome. All elements of the rootlet system of flagella are limited by peripheral layers of cytoplasm. P. paradoxa occupy an intermediate position between two groups of species of Pelomyxa genus--P. gruberi + P. prima and P. palustris + P. stagnalis + P. belewski, which differ greatly by the organization of their flagella basal apparatus.