Pathogenic free-living amoebae from water sources in Cape Verde.

Free-living amoebae (FLA) are protozoa which have been reported in different countries worldwide from diverse sources (water, soil, dust, air), contributing to the environmental microbiological contamination. Most of the FLA species present a life cycle with two different phases: an active vegetative and physiologically form named trophozoite, and an extremely resistant phase called cyst. Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Sapinia pedata, Vahlkampfia spp., Paravahlkampfia spp. and Vermamoeba vermiformis have been reported not only as causal agents of several opportunistic diseases including fatal encephalitis or epithelial disorders, but also as capable to favour the intracellular survival of common pathogenic bacteria, which could avoid the typical water disinfection systems, non-effective against FLAs cysts. Even though Santiago Island possesses high levels of humidity compared to the rest of the archipelago of Cape Verde, the water resources are scarce. Therefore, it is important to carry out proper microbiological quality controls, which currently do not contemplate the FLA presence in most of the countries. In the present work, we have reported the presence of Acanthamoeba spp. (69.2%); Vannella spp. (15.4%); Vermamoeba vermiformis (7.7%) and the recently discovered Stenamoeba dejonckheerei (7.7%) in different water sources of Santiago Island.

Superficially described and ignored for 92 years, rediscovered and emended: Apodera angatakere (Amoebozoa: Arcellinida: Hyalospheniformes) is a new flagship testate amoeba taxon from Aotearoa (New Zealand).

Eukaryotic microbial diversity is known to be extensive but remains largely undescribed and uncharted. While much of this unknown diversity is composed of inconspicuous flagellates and parasites, larger and morphologically distinct protists are regularly discovered, most notably from poorly studied regions. Here we report a new flagship species of hyalospheniid (Amoebozoa; Arcellinida; Hyalospheniformes) testate amoeba from New Zealand and an unusual story of overlooked description under a preoccupied name and subsequent oversight for nearly one century. Through a process involving The Maori Language Commission, we named the species Apodera angatakere, meaning "a shell with a keel." This species resembles Apodera vas but differs by the presence of a distinctive hollow keel. Cytochrome Oxidase Subunit 1 (COI) sequence data show that this species forms a distinct clade nested within genus Apodera. This conspicuous species is so far known only from New Zealand and is restricted to peatlands. It is one of the few examples of endemic microorganisms from this biodiversity hotspot and biogeographer's paradise. As over 90% of New Zealand's peatlands have been lost since European colonization and much of the remaining surfaces are threatened, Apodera angatakere could be a flagship species not only for microbial biogeography but also for island biodiversity conservation.

The increasing importance of Vermamoeba vermiformis.

Vermamoeba vermiformis are one of the most prevalent free-living amoebae. These amoebae are ubiquitous and also thermotolerant. Of concern, V. vermiformis have been found in hospital water networks. Furthermore, associations between V. vermiformis and pathogenic bacteria have been reported, such as Legionella pneumophila. Moreover, V. vermiformis are well known to host viruses, bacteria, and other microorganisms and cases of keratitis due to V. vermiformis in conjunction with other amoebae have been reported. Despite the preceding, the medical importance of V. vermiformis is still an ongoing discussion and its genome has been only recently sequenced. Herein, we present a review of the current understanding of the biology and pathogenesis pertaining to V. vermiformis, as well as its' role as an etiological agent and trojan horse. An approach known as theranostics which combines both diagnosis and therapy could be utilized to eradicate and diagnose keratitis cases caused by such amoebae. Given the rise in global warming, it is imperative to investigate these rarely studied amoebae and to understand their importance in human health.

Three New Freshwater Cochliopodium Species (Himatismenida, Amoebozoa) from the Southeastern United States.

Cochliopodium is a lens-shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often-used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum-likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, "crystal UK-YT2" (Cochliopodium crystalli n. sp.) and "crystal-like UK-YT3" (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical-shaped crystals. The "Marrs Spring UK-YT4" isolate, C. marrii n. sp., was 100% identical to "Cochliopodium sp. SG-2014 KJ569724." These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.

A Comparative Characterization of the Mitochondrial Genomes of Paramoeba aparasomata and Neoparamoeba pemaquidensis (Amoebozoa, Paramoebidae).

Marine amebae of the genus Paramoeba (Amoebozoa, Dactylopodida) normally contain a eukaryotic endosymbiont known as Perkinsela-like organism (PLO). This is one of the characters to distinguish the genera Neoparamoeba and Paramoeba from other Dactylopodida. It is known that the PLO may be lost, but PLO-free strains of paramoebians were never available for molecular studies. Recently, we have described the first species of the genus Paramoeba which has no parasome-Paramoeba aparasomata. In this study, we present a mitochondrial genome of this species, compare it with that of Neoparamoeba pemaquidensis, and analyze the evolutionary dynamics of gene sequences and gene order rearrangements between these species. The mitochondrial genome of P. aparasomata is 46,254 bp long and contains a set of 31 protein-coding genes, 19 tRNAs, two rRNA genes, and 7 open reading frames. Our results suggest that these two mitochondrial genomes within the genus Paramoeba have rather similar organization and gene order, base composition, codon usage, the composition and structure of noncoding, and overlapping regions.

Intra-Lake Arcellinida (Testate Lobose Amoebae) Response to Winter De-icing Contamination in an Eastern Canada Road-Side "Salt Belt" Lake.

Salt contamination of lakes, due to the application of winter de-icing salts on roads, presents a significant environmental challenge in the "salt belt" region of eastern North America. The research reported here presents the first deployment of a previously published proxy tool based on Arcellinida (testate lobose amoebae) for monitoring road salt contamination. The research was conducted at Silver Lake in Eastern Ontario, a 4-km-long lake with the heavily traveled Trans-Canada Highway (HWY 7) transiting the entire southern shore. The lake showed elevated conductivity (297-310 µS/cm) and sub-brackish conditions (0.14-0.15 ppt). Sodium levels were also elevated near the roadside (median Na = 1020 ppm). Cluster analysis and nonmetric multidimensional scaling results revealed four distinct Arcellinida assemblages: "Stressed Cool Water Assemblage (SCWA)," "Deep Cold Water Assemblage (DCWA)," both from below the 8-m thermocline, and the shallower water "Shallow Water Assemblage 1 (SWA-1)" and "Shallow Water Assemblage 2 (SWA-2)". Redundancy analysis showed a minor response of Arcellinida to road salt contamination in shallower areas of the lake, with confounding variables significantly impacting assemblage distribution, particularly beneath the thermocline (e.g., water temperature, water depth, sediment runoff from catchment [Ti], sediment geochemistry [Ca, S]). The results of this study indicate that the trophic structure of the lake has to date only been modestly impacted by the cumulative nature of road salt contamination. Nonetheless, the Silver Lake results should be considered of concern and warrant continued arcellinidan biomonitoring to gauge the ongoing and long-term effects of road salt on its ecosystem.

Behind the Organic Veil: Assessing the Impact of Chemical Deflocculation on Organic Content Reduction and Lacustrine Arcellinida (Testate Amoebae) Analysis.

Arcellinida (testate lobose amoebae) are widely used as bio-indicators of lacustrine environmental change. Too much obscuring organic material in a gridded wet Petri dish preparation makes it difficult to observe all specimens present and slows quantification as the organic material has to be carefully worked through with a dissection probe. Chemical deflocculation using soda ash (Na2CO3·H2O), potassium hydroxide (KOH), or sodium hexametaphosphate ((NaPO3)6) has previously been shown to disaggregate and reduce organic content in lake sediments, but to date, no attempt has been made to comparatively evaluate the efficiency of these deflocculants in disaggregating organic content and their impact on Arcellinida analysis in lacustrine sediments. Here, we assess the effectiveness of soda ash, potassium hydroxide, and sodium hexametaphosphate treatments on removing organic content and the impact of those digestions on Arcellinida preservation in 126 sample aliquots subdivided from three sediment samples (YK-20, YK-25, and YK-57) collected from three lakes near Yellowknife, Northwest Territories, Canada. Following treatment, cluster analysis and Bray-Curtis dissimilarity matrix (BCDM) were utilized to determine whether treatments resulted in dissolution-driven changes in Arcellinida assemblage composition. Observed Arcellinida tests in aliquots increased drastically after treatment of organic-rich samples (47.5-452.7% in organic-rich aliquots and by 14.8% in aliquots with less organic matter). The BCDM results revealed that treatment with 5% KOH resulted in the highest reduction in observed organic content without significantly affecting Arcellinida assemblage structure, while soda ash and sodium hexametaphosphate treatments resulted in marginal organic matter reduction and caused severe damage to the arcellinidan tests.

In-depth analysis of the replication cycle of Orpheovirus.

BACKGROUND: After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. METHODS: In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. RESULTS: We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. CONCLUSIONS: The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Dactylomonas gen. nov., a Novel Lineage of Heterolobosean Flagellates with Unique Ultrastructure, Closely Related to the Amoeba Selenaion koniopes Park, De Jonckheere & Simpson, 2012.

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.

Small-scale Variation of Testate Amoeba Assemblages: the Effect of Site Heterogeneity and Empty Shell Inclusion.

Studies on testate amoeba species distribution at small scales (i.e., single peatland sites) are rare and mostly focus on bogs or mineral-poor Sphagnum fens, leaving spatial patterns within mineral-rich fens completely unexplored. In this study, two mineral-rich fen sites of contrasting groundwater chemistry and moss layer composition were selected for the analysis of testate amoeba compositional variance within a single site. At each study site, samples from 20 randomly chosen moss-dominated plots were collected with several environmental variables being measured at each sampling spot. We also distinguished between empty shells and living individuals to evaluate the effect of empty shell inclusion on recorded species distribution. At the heterogeneous-rich Sphagnum-fen, a clear composition turnover in testate amoebae between Sphagnum-dominated and brown moss-dominated samples was closely related to water pH, temperature and redox potential. We also found notable species composition variance within the homogeneous calcareous fen, yet it was not as high as for the former site and the likely drivers of community assembly remained unidentified. The exclusion of empty shells provided more accurate data on species distribution as well as their relationship with some environmental variables, particularly moisture. Small-scale variability in species composition of communities seems to be a worthwhile aspect in testate amoeba research and should be considered in future sampling strategies along with a possible empty shell bias for more precise understanding of testate amoeba ecology and paleoecology.

Salt-Enrichment Impact on Biomass Production in a Natural Population of Peatland Dwelling Arcellinida and Euglyphida (Testate Amoebae).

Unicellular free-living microbial eukaryotes of the order Arcellinida (Tubulinea; Amoebozoa) and Euglyphida (Cercozoa; SAR), commonly termed testate amoebae, colonise almost every freshwater ecosystem on Earth. Patterns in the distribution and productivity of these organisms are strongly linked to abiotic conditions-particularly moisture availability and temperature-however, the ecological impacts of changes in salinity remain poorly documented. Here, we examine how variable salt concentrations affect a natural community of Arcellinida and Euglyphida on a freshwater sub-Antarctic peatland. We principally report that deposition of wind-blown oceanic salt-spray aerosols onto the peatland surface corresponds to a strong reduction in biomass and to an alteration in the taxonomic composition of communities in favour of generalist taxa. Our results suggest novel applications of this response as a sensitive tool to monitor salinisation of coastal soils and to detect salinity changes within peatland palaeoclimate archives. Specifically, we suggest that these relationships could be used to reconstruct millennial scale variability in salt-spray deposition-a proxy for changes in wind-conditions-from sub-fossil communities of Arcellinida and Euglyphida preserved in exposed coastal peatlands.

Vermamoeba vermiformis in hospital network: a benefit for Aeromonas hydrophila.

Aeromonas hydrophila, considered as an emerging pathogen, is increasingly involved in opportunistic human infections. This bacterium, mainly present in aquatic environments, can therefore develop relationships with the free-living amoeba Vermamoeba vermiformis in hospital water networks. We showed in this study that the joint presence of V. vermiformis and A. hydrophila led to an increased bacterial growth in the first 48 h of contact and moreover to the protection of the bacteria in adverse conditions even after 28 days. These results highlight the fact that strategies should be implemented to control the development of FLA in hospital water systems.

Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream.

Fecal indicator bacteria (FIB) are the basis for water quality regulations and are considered proxies for waterborne pathogens when conducting human health risk assessments. The direct detection of pathogens in water and simultaneous identification of the source of fecal contamination are possible with microarrays, circumventing the drawbacks to FIB approaches. A multigene target microarray was used to assess the prevalence of waterborne pathogens in a fecally impaired mixed-use watershed. The results indicate that fecal coliforms have improved substantially in the watershed since its listing as a 303(d) impaired stream in 2002 and are now near United States recreational water criterion standards. However, waterborne pathogens are still prevalent in the watershed, as viruses (bocavirus, hepatitis E and A viruses, norovirus, and enterovirus G), bacteria (Campylobacter spp., Clostridium spp., enterohemorrhagic and enterotoxigenic Escherichia coli, uropathogenic E. coli, Enterococcus faecalis, Helicobacter spp., Salmonella spp., and Vibrio spp.), and eukaryotes (Acanthamoeba spp., Entamoeba histolytica, and Naegleria fowleri) were detected. A comparison of the stream microbial ecology with that of sewage, cattle, and swine fecal samples revealed that human sources of fecal contamination dominate in the watershed. The methodology presented is applicable to a wide range of impaired streams for the identification of human health risk due to waterborne pathogens and for the identification of areas for remediation efforts.IMPORTANCE The direct detection of waterborne pathogens in water overcomes many of the limitations of the fecal indicator paradigm. Furthermore, the identification of the source of fecal impairment aids in identifying areas for remediation efforts. Multitarget gene microarrays are shown to simultaneously identify waterborne pathogens and aid in determining the sources of impairment, enabling further focused investigations. This study shows the use of this methodology in a historically impaired watershed in which total maximum daily load reductions have been successfully implemented to reduce risk. The results suggest that while the fecal indicators have been reduced more than 96% and are nearing recreational water criterion levels, pathogens are still detectable in the watershed. Microbial source tracking results show that additional remediation efforts are needed to reduce the impact of human sewage in the watershed.

Diverse Legionella-Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa).

Diverse species of Legionella and Legionella-like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella-like bacteria with 94-98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella-like Arcella-associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella-specific probe. This study demonstrates that the host range of Legionella and Legionella-like bacteria in the Amoebozoa extends beyond members of "naked" amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.

Genetic Determinism vs. Phenotypic Plasticity in Protist Morphology.

Untangling the relationships between morphology and phylogeny is key to building a reliable taxonomy, but is especially challenging for protists, where the existence of cryptic or pseudocryptic species makes finding relevant discriminant traits difficult. Here we use Hyalosphenia papilio (a testate amoeba) as a model species to investigate the contribution of phylogeny and phenotypic plasticity in its morphology. We study the response of H. papilio morphology (shape and pores number) to environmental variables in (i) a manipulative experiment with controlled conditions (water level), (ii) an observational study of a within-site natural ecological gradient (water level), and (iii) an observational study across 37 European peatlands (climate). We showed that H. papilio morphology is correlated to environmental conditions (climate and water depth) as well as geography, while no relationship between morphology and phylogeny was brought to light. The relative contribution of genetic inheritance and phenotypic plasticity in shaping morphology varies depending on the taxonomic group and the trait under consideration. Thus, our data call for a reassessment of taxonomy based on morphology alone. This clearly calls for a substantial increase in taxonomic research on these globally still under-studied organisms leading to a reassessment of estimates of global microbial eukaryotic diversity.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

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.

Leptomyxa valladaresi n. sp. (Amoebozoa, Tubulinea, Leptomyxida), from Mount Teide, Tenerife, Spain.

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.

Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran.

The present study tested 80 samples of municipal, geothermal and recreational water samples for the occurrence of waterborne free living amoebae (FLA) including Acanthamoeba, Balamuthia mandrillaris, Vahlkampfiids and Vermamoeba in Semnan province, North half of Iran. Four sets of primers including JDP1,2 primers, ITS1,2 primers (Vahlkampfiids), 16S rRNABal primers (Balamuthia mandrillaris) and NA1,2 primers (Vermamoeba) were used to confirm the morphological identification. From the 80 water samples tested in the present study, 16 (20%) were positive for the outgrowth of free living amoebae based on the morphological page key. Out of the 34 municipal water samples, 7 (20.6%) were positive for outgrowth of Free living amoeba, belonging to Vermamoeba, Naegleria and Acanthamoeba using molecular tools. Three out of the six investigated hot springs were also contaminated with Naegleria spp. Sequencing of the ITS1,2 region of the Vahlkampfiid isolates revealed the highest homology with N. gruberi (2 isolates), N. australiensis (1 isolate) and N. pagei (3 isolates). This is the first report of N. gruberi in the country. Using morphological and molecular analysis, Balamuthia mandrillaris was undetected in all the water samples. The present study further confirmed the occurrence of potentially pathogenic waterborne free living amoebae in habitats with high human activity. It is of utmost importance that more studies are conducted to evaluate the niches of B. mandrillaris and N. fowleri in Iran and worldwide. Such investigations regarding the relevance of FLA as a hazard to humans, should be brought to the notice of the health authorities.

Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae.

UNLABELLED: Giant viruses are protist-associated viruses belonging to the proposed order Megavirales; almost all have been isolated from Acanthamoeba spp. Their isolation in humans suggests that they are part of the human virome. Using a high-throughput strategy to isolate new giant viruses from their original protozoan hosts, we obtained eight isolates of a new giant viral lineage from Vermamoeba vermiformis, the most common free-living protist found in human environments. This new lineage was proposed to be the faustovirus lineage. The prototype member, faustovirus E12, forms icosahedral virions of ≈ 200 nm that are devoid of fibrils and that encapsidate a 466-kbp genome encoding 451 predicted proteins. Of these, 164 are found in the virion. Phylogenetic analysis of the core viral genes showed that faustovirus is distantly related to the mammalian pathogen African swine fever virus, but it encodes ≈ 3 times more mosaic gene complements. About two-thirds of these genes do not show significant similarity to genes encoding any known proteins. These findings show that expanding the panel of protists to discover new giant viruses is a fruitful strategy. IMPORTANCE: By using Vermamoeba, a protist living in humans and their environment, we isolated eight strains of a new giant virus that we named faustovirus. The genomes of these strains were sequenced, and their sequences showed that faustoviruses are related to but different from the vertebrate pathogen African swine fever virus (ASFV), which belongs to the family Asfarviridae. Moreover, the faustovirus gene repertoire is ≈ 3 times larger than that of ASFV and comprises approximately two-thirds ORFans (open reading frames [ORFs] with no detectable homology to other ORFs in a database).

Nebela jiuhuensis nov. sp. (Amoebozoa; Arcellinida; Hyalospheniidae): A New Member of the Nebela saccifera - equicalceus - ansata Group Described from Sphagnum Peatlands in South-Central China.

Hyalospheniids are among the most common and conspicuous testate amoebae in high-latitude peatlands and forest humus. These testate amoebae were widely studied as bioindicators and are increasingly used as models in microbial biogeography. However, data on their diversity and ecology are still very unevenly distributed geographically: notably, data are lacking for low-latitude peatlands. We describe here a new species, Nebela jiuhuensis, from peatlands near the Middle Yangtze River reach of south-central China with characteristic morphology. The test (shell) has hollow horn-like lateral extensions also found in N. saccifera, N. equicalceus (=N. hippocrepis), and N. ansata, three large species restricted mostly to Sphagnum peatlands of Eastern North America. Mitochondrial cytochrome oxidase (COI) data confirm that N. jiuhuensis is closely related to the morphologically very similar North American species N. saccifera and more distantly to N. ansata within the N. penardiana group. These species are all found in wet mosses growing in poor fens. Earlier reports of morphologically similar specimens found in South Korea peatlands suggest that N. jiuhuensis may be distributed in comparable peatlands in Eastern Asia (China and Korea). The discovery of such a conspicuous new species in Chinese peatlands suggests that many new testate amoebae species are yet to be discovered, including potential regional endemics. Furthermore, human activities (e.g., drainage, agriculture, and pollution) have reduced the known habitat of N. jiuhuensis, which can thus be considered as locally endangered. We, therefore, suggest that this very conspicuous micro-organism with a probably limited geographical distribution and specific habitat requirement should be considered as a flagship species for microbial biogeography as well as local environmental conservation and management.