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In this study, we presented the results of our molecular phylogenetic analysis of Archamoebae using a newly obtained sequence of the 18S rRNA gene of Mastigamoeba aspera, the type species of the genus. In addition, we characterized the general organization of the tubulin cytoskeleton of M. aspera by immunofluorescent microscopy and TEM. Our findings allowed us to revise the family Mastigamoebidae and establish within it two subfamilies: Mastigamoebinae for the genus Mastigamoeba and Seraviniinae subfam. nov. for the genera Seravinia gen. nov., Paramastigamoeba gen. nov., Iodamoeba and Endolimax. The type genera Mastigamoeba and Seravinia are clearly distinguished by the structure of the basal apparatus of flagella. In addition, the tubulin cytoskeleton of several Mastigamoeba spp. contains a rim of microtubules around the nuclei.

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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.

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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.

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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.

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The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

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BACKGROUND: Application of next-generation sequencing (NGS) to genomic DNA extracted from sewage offers a unique and cost-effective opportunity to study the genetic diversity of intestinal parasites. In this study, we used amplicon-based NGS to reveal and differentiate several common luminal intestinal parasitic protists, specifically Entamoeba, Endolimax, Iodamoeba, and Blastocystis, in sewage samples from Swedish treatment plants. MATERIALS AND METHODS: Influent sewage samples were subject to gradient centrifugation, DNA extraction and PCR-based amplification using three primer pairs designed for amplification of eukaryotic nuclear 18S ribosomal DNA. PCR products were sequenced using ILLUMINA® technology, and resulting sequences were annotated to species and subtype level using the in-house BION software, sequence clustering, and phylogenetic analysis. RESULTS: A total of 26 samples from eight treatment plants in central/southern Sweden were analysed. Blastocystis sp. and Entamoeba moshkovskii were detected in all samples, and most samples (n = 20) were positive for Entamoeba coli. Moreover, we detected Entamoeba histolytica, Entamoeba dispar, Entamoeba hartmanni, Endolimax nana, and Iodamoeba bütschlii in 1, 11, 4, 10, and 7 samples, respectively. The level of genetic divergence observed within E. nana and E. moshkovskii was 20.2% and 7.7%, respectively, across the ~400-bp region studied, and two clades of E. moshkovskii were found. As expected, Blastocystis sp. subtypes 1-4 were present in almost all samples; however, ST8 was present in 10 samples and was the only subtype not commonly found in humans that was present in multiple samples. CONCLUSIONS: Entamoeba and Blastocystis were identified as universal members of the "sewage microbiome". Blastocystis sp. ST8, which has been rarely reported in humans, was a very common finding, indicating that a hitherto unidentified but common host of ST8 contributed to the sewage influent. The study also provided substantial new insight into the intra-generic diversity of Entamoeba and Endolimax.

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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.

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The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 µm in length and 6.8-15.9 µm in width, whereas the cysts were 8.9-15.8 µm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

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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.

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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.

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