Testate amoebae (Arcellinida, Amoebozoa) community diversity in New England bogs and fens assessed through lineage-specific amplicon sequencing.

Testate amoebae (order Arcellinida) are abundant in freshwater ecosystems, including low pH bogs and fens. Within these environments, Arcellinida are considered top predators in microbial food webs and their tests are useful bioindicators of paleoclimatic changes and anthropogenic pollutants. Accurate species identifications and characterizations of diversity are important for studies of paleoclimate, microbial ecology, and environmental change; however, morphological species definitions mask cryptic diversity, which is a common phenomenon among microbial eukaryotes. Lineage-specific primers recently designed to target Arcellinida for amplicon sequencing successfully captured a poorly-described yet diverse fraction of the microbial eukaryotic community. Here, we leveraged the application of these newly-designed primers to survey the diversity of Arcellinida in four low-pH New England bogs and fens, investigating variation among bogs (2018) and then across seasons and habitats within two bogs (2019). Three OTUs represented 66% of Arcellinida reads obtained across all habitats surveyed. 103 additional OTUs were present in lower abundance with some OTUs detected in only one sampling location, suggesting habitat specificity. By establishing a baseline for Arcellinida diversity, we provide a foundation to monitor key taxa in habitats that are predicted to change with increasing anthropogenic pressure and rapid climate change.

It's time to consider the Arcellinida shell as a weapon.

The shells of testate amoebae are morphologically diverse and persistent in the environment. Accordingly, the examination of the morphology and composition of shells became a standard tool in ecological, palaeoecological, and evolutionary studies. However, so far the function of the shell remains poorly understood and, although based on limited evidence, the shell was considered as a defense mechanism. Based on recent evidence, we propose that the shell of arcellinid testate amoebae is a crucial component facilitating the amoebae's attack of large prey. Accordingly, the shell is not purely protective, but must be considered also as a weapon. This change in perspective opens up numerous new avenues in protistology and will lead to a substantial change in ecological, palaeoecological, and evolutionary research.

Two new species of the genus Leptomyxa (Goodey, 1915) - Leptomyxa botanica n. sp. and Leptomyxa monrepos n. sp. from the soil habitats of Northwestern Russia.

Amoebae of the genus Leptomyxa have variable morphologies and can only be reliably identified using molecular data. However, species distinction based on the 18S rRNA gene sequence is difficult due to the very low level of sequence divergence among morphologically different species. The database for other genes is much smaller, and genomic data on Leptomyxa is almost absent. In this study, we describe two new terrestrial species of the genus Leptomyxa isolated from Northwestern Russia, Leptomyxa botanica n. sp. and Leptomyxa monrepos n. sp. Both species easily adopt an expanded fan-shaped form and have a complex structure of the nucleolar material. Phylogenetic analyses show a derived status of these two species. They form a clade with Leptomyxa valladaresi. Our tree confirms that the 18S rRNA gene sequences of Leptomyxa species are split into two large clades. The morphological synapomorphies of these clades are not obvious. This analysis is complicated by the lack of reliable morphological data on many sequenced strains and probable misidentification of some isolates.

Expansion of the cytochrome C oxidase subunit I database and description of four new lobose testate amoebae species (Amoebozoa; Arcellinida).

Arcellinida is ascending in importance in protistology, but description of their diversity still presents multiple challenges. Furthermore, applicable tools for surveillance of these organisms are still in developing stages. Importantly, a good database that sets a correspondence between molecular barcodes and species morphology is lacking. Cytochrome oxidase (COI) has been suggested as the most relevant marker for species discrimination in Arcellinida. However, some major groups of Arcellinida are still lacking a COI sequence. Here we expand the database of COI marker sequences for Arcellinids, using single-cell PCR, transcriptomics, and database scavenging. In the present work, we added 24 new Arcellinida COI sequences to the database, covering all unsampled infra- and suborders. Additionally, we added six new SSUrRNA sequences and described four new species using morphological, morphometrical, and molecular evidence: Heleopera steppica, Centropyxis blatta, Arcella uspiensis, and Cylindrifflugia periurbana. This new database will provide a new starting point to address new research questions from shell evolution, biogeography, and systematics of arcellinids.

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea).

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.

Coronamoeba villafranca gen. nov. sp. nov. (Amoebozoa, Dermamoebida) challenges the correlation of morphology and phylogeny in Amoebozoa.

Coronamoeba villafranca gen. nov. sp. nov. is a small amoeba isolated from the surface planktonic biotope in the Bay of Villefranche (Mediterranean Sea). It has a confusing set of morphological and molecular characters. Its locomotive form is subcylindrical and monopodial with monoaxial cytoplasmic flow and occasional hyaline bulging at the anterior edge (a monotactic morphotype). Based on this set of characters, this amoeba is most similar to members of the genus Nolandella (Tubulinea, Euamoebida). However, molecular phylogenetic analysis based on only the small subunit ribosomal RNA (SSU rRNA) gene and on two concatenated markers (SSU rRNA gene and actin) robustly places this species in the Discosea, specifically, in a clade with Dermamoeba and Paradermamoeba (Dermamoebida) as the closest described relatives, and several SSU rRNA clones from environmental DNA. A unique glycocalyx of the studied amoeba consisting of complex separate units with pentameric symmetry may be considered a unifying character of this species with other dermamoebids. The monotactic morphotype demonstrated by these amoebae primarily occurs in Tubulinea but was recently confirmed in other clades of Amoebozoa (e.g. Dactylopodida and Variosea). This morphotype may be the plesiomorphic mode of cell organization in Amoebozoa that might have evolved in the last amoebozoan common ancestor (LACA) and conserved in several lineages of this group. It may reflect basic characteristics of the cytoskeletal structure and functions in Amoebozoa.

New insights on the evolutionary relationships between the major lineages of Amoebozoa.

The supergroup Amoebozoa unites a wide diversity of amoeboid organisms and encompasses enigmatic lineages that have been recalcitrant to modern phylogenetics. Deep divergences, taxonomic placement of some key taxa and character evolution in the group largely remain poorly elucidated or controversial. We surveyed available Amoebozoa genomes and transcriptomes to mine conserved putative single copy genes, which were used to enrich gene sampling and generate the largest supermatrix in the group to date; encompassing 824 genes, including gene sequences not previously analyzed. We recovered a well-resolved and supported tree of Amoebozoa, revealing novel deep level relationships and resolving placement of enigmatic lineages congruent with morphological data. In our analysis the deepest branching group is Tubulinea. A recent proposed major clade Tevosa, uniting Evosea and Tubulinea, is not supported. Based on the new phylogenetic tree, paleoecological and paleontological data as well as data on the biology of presently living amoebozoans, we hypothesize that the evolution of Amoebozoa probably was driven by adaptive responses to a changing environment, where successful survival and predation resulted from a capacity to disrupt and graze on microbial mats-a dominant ecosystem of the mid-Proterozoic period of the Earth history.

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.

Examining the Relationship Between the Testate Amoeba Hyalosphenia papilio (Arcellinida, Amoebozoa) and its Associated Intracellular Microalgae Using Molecular and Microscopic Methods.

Symbiotic relationships between heterotrophic and phototrophic partners are common in microbial eukaryotes. Among Arcellinida (Amoebozoa) several species are associated with microalgae of the genus Chlorella (Archaeplastida). So far, these symbioses were assumed to be stable and mutualistic, yet details of the interactions are limited. Here, we analyzed 22 single-cell transcriptomes and 36 partially-sequenced genomes of the Arcellinida morphospecies Hyalosphenia papilio, which contains Chlorella algae, to shed light on the amoeba-algae association. By characterizing the genetic diversity of associated Chlorella, we detected two distinct clades that can be linked to host genetic diversity, yet at the same time show a biogeographic signal across sampling sites. Fluorescence and transmission electron microscopy showed the presence of intact algae cells within the amoeba cell. Yet analysis of transcriptome data suggested that the algal nuclei are inactive, implying that instead of a stable, mutualistic relationship, the algae may be temporarily exploited for photosynthetic activity before being digested. Differences in gene expression of H. papilio and Hyalosphenia elegans demonstrated increased expression of genes related to oxidative stress. Together, our analyses increase knowledge of this host-symbiont association and reveal 1) higher diversity of associated algae than previously characterized, 2) a transient association between H. papilio and Chlorella with unclear benefits for the algae, 3) algal-induced gene expression changes in the host.

New data on the fine structure of Deuteramoeba mycophaga CCAP 1586/1 (Amoebozoa, Tubulinea).

The genus Deuteramoeba is one of the six amoebae genera belonging to the best-known amoeba family - Amoebidae (Amoebozoa, Tubulinea), containing such a popular species as Amoeba proteus. However, members of other genera of the family Amoebidae are much less known, and most of the studies of their morphology and ultrastructure date back to the 1970s and 1980s. Since these "classical" species are believed to be "well studied", their morphology and fine structure rarely become a subject of re-investigation. The absence of modern morphological data may be critical when molecular data of the type strain are not available, and the only way to identify a species is by morphological comparison. For this paper, we performed an ultrastructural study of the strain CCAP 1586/1 - the type strain of the species Deuteramoeba mycophaga. Our study revealed new details of the nuclear structure, including a peripheral layer of filaments and a heterogeneous nucleolus, and provided new data on the cytoplasmic inclusions of this species. We performed a whole-genome amplification of the DNA from a single amoeba cell followed by NGS sequencing and searched for genetic evidence for the presence of a putative nuclear parasite detected in 2017, but found no evidence for the presence of Opisthosporidia.

DAPI staining and DNA content estimation of nuclei in uncultivable microbial eukaryotes (Arcellinida and Ciliates).

Though representing a major component of eukaryotic biodiversity, many microbial eukaryotes remain poorly studied, including the focus of the present work, testate amoebae of the order Arcellinida (Amoebozoa) and non-model lineages of ciliates (Alveolata). In particular, knowledge of genome structures and changes in genome content over the often-complex life cycles of these lineages remains enigmatic. However, the limited available knowledge suggests that microbial eukaryotes have the potential to challenge our textbook views on eukaryotic genomes and genome evolution. In this study, we developed protocols for DAPI (4',6-diamidino-2-phenylindole) staining of Arcellinida nuclei and adapted protocols for ciliates. In addition, image analysis software was used to estimate the DNA content in the nuclei of Arcellinida and ciliates, and the measurements of target organisms were compared to those  of well-known model organisms.The results demonstrate that the methods we have developed for nuclear staining in these lineages are effective and can be applied to other microbial eukaryotic groups by adjusting certain stages in the protocols.

Arcellinida testate amoebae as climate miner's canaries in Southern Spain.

Southern Spain is currently under threat of desertification as a consequence of global climate change, which pressures on fragile ecosystems such as caves. The organisms living in these extremely stable environments are particularly sensitive and prone to extinction, therefore they can be used as bioindicators for climate change. Cyanobacterial mats form peculiar and vulnerable micro-ecosystems at the entrance of caves and house a diversity of protists. Amongst them, Arcellinida testate amoebae have been traditionally used as bioindicators for environmental quality, notably because their narrow ecological tolerance and their key ecological position as top predators of the microbial foodwebs. We report here two new species of Arcellinida found in the cyanobacterial mats of cave Hundidero, in Sierra de Grazalema, Malaga province, whose traits suggest a narrow tolerance for changes in humidity. We provide a formal description for Difflugia alhadiqa sp. nov. and Heleopera baetica sp. nov. based on morphometrics and 18S rRNA gene data, and propose using the presence of these species to indicate the good health of the cyanobacterial mats, like miner's canaries for local climate.

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.

New record of Arcella gandalfi (Amoebozoa: Arcellinida) from Colombia.

The reports of testate amoebae species in Colombia are limited to a few studies in lentic and peat bog systems; however, the diversity in lotic systems has been poorly investigated. Zooplankton sampling was performed in lotic environments of the Piedemonte Llanero in Colombia. Ten individuals of the species Arcella gandalfi were identified to document the first record of A. gandalfi from the Piedemonte Llanero in Colombia, expanding its distribution from Brazil to Colombia. This finding corroborates the status of A. gandalfi as a flagship species from the South America continent.

The Kaumoebavirus LCC10 Genome Reveals a Unique Gene Strand Bias among "Extended Asfarviridae".

Kaumoebavirus infects the amoeba Vermamoeba vermiformis and has recently been described as a distant relative of the African swine fever virus. To characterize the diversity and evolution of this novel viral genus, we report here on the isolation and genome sequencing of a second strain of Kaumoebavirus, namely LCC10. Detailed analysis of the sequencing data suggested that its 362-Kb genome is linear with covalently closed hairpin termini, so that DNA forms a single continuous polynucleotide chain. Comparative genomic analysis indicated that although the two sequenced Kaumoebavirus strains share extensive gene collinearity, 180 predicted genes were either gained or lost in only one genome. As already observed in another distant relative, i.e., Faustovirus, which infects the same host, the center and extremities of the Kaumoebavirus genome exhibited a higher rate of sequence divergence and the major capsid protein gene was colonized by type-I introns. A possible role of the Vermamoeba host in the genesis of these evolutionary traits is hypothesized. The Kaumoebavirus genome exhibited a significant gene strand bias over the two-third of genome length, a feature not seen in the other members of the "extended Asfarviridae" clade. We suggest that this gene strand bias was induced by a putative single origin of DNA replication located near the genome extremity that imparted a selective force favoring the genes positioned on the leading strand.

Polychaos centronucleolus n. sp. - a new terrestrial species of the genus Polychaos (Amoebozoa, Tubulinea) with nontypical nuclear structure.

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

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.

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.

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.