Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

A Small Genome amidst the Giants: Evidence of Genome Reduction in a Small Tubulinid Free-Living Amoeba.

This study investigates the genomic characteristics of Echinamoeba silvestris, a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce. E. silvestris presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in E. silvestris shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. Additionally, our investigation into noncoding and repetitive elements sheds light on the evolution of genome size in amoebae, with E. silvestris distinguished by low percentage of repetitive elements. Furthermore, the analysis reveals that E. silvestris has the lowest mean number of introns per gene among the species studied, providing further support for its observed compact genome. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions E. silvestris as a valuable addition to genomic data sets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.

Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020-2022.

Free-living amoebae (FLA) are capable of inhabiting diverse reservoirs independently, without relying on a host organism, hence their designation as "free-living". The majority of amoebae that infect freshwater or marine fish are amphizoic, or free-living forms that may colonize fish under particular circumstances. Symphysodon aequifasciatus, commonly referred to as the discus, is widely recognized as a popular ornamental fish species. The primary objective of the present study was to determine the presence of pathogenic free-living amoebae (FLA) in samples of discus fish. Fish exhibiting clinical signs, sourced from various fish farms, were transferred to the ornamental fish clinic. The skin, gills, and intestinal mucosa of the fish were collected and subjected to culturing on plates containing a 1% non-nutrient agar medium. The detection of FLA was conducted through morphological, histopathological and molecular methods. The construction of the phylogenetic tree for Acanthamoeba genotypes was achieved using the maximum likelihood approach. The molecular sequence analysis revealed that all cultures that tested positive for FLA were T4 genotype of Acanthamoeba and Acanthamoeba sp. The examination of gill samples using histopathological methods demonstrated the presence of lamellar epithelial hyperplasia, significant fusion of secondary lamellae, and infiltration of inflammatory cells. A multitude of cysts, varying in shape from circular to elliptical, were observed within the gills. The occurrence of interlamellar vesicles and amoeboid organisms could be observed within the epithelial tissue of the gills. In the current study, presence of the Acanthamoeba T4 genotype on the skin and gills of discus fish exhibiting signs of illness in freshwater ornamental fish farms was identified. This observation suggests the potential of a transmission of amoebic infection from ornamental fish to humans, thereby highlighting the need for further investigation into this infection among ornamental fish maintained as pets, as well as individuals who interact with them and their environment.

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.

Anaeramoeba pumila sp. nov. and Anaeramoeba sp. OCE22C represent two novel types of symbiosis of Anaeramoebae and prokaryotes.

Anaeramoebae is a recently described phylum of anaerobic, marine amoebae, and amoeboflagellates belonging to the Metamonada supergroup. So far, six species have been described based on light microscopic morphology and sequences of the SSU rRNA gene. Here we present three new strains of Anaeramoeba with a description of their morphology, ultrastructure, and phylogenetic position based on the analysis of SSU rRNA gene sequences. Two of the strains represent a new species, Anaeramoeba pumila sp. nov., that has the smallest cells of all known Anaeramoeba species, and one that represents a species from the newly recognized Anaeramoeba flamelloides complex. Anaeramoebae are known to have a syntrophic relationship with prokaryotes. Our strains display two novel, remarkable types of symbioses, previously unknown from Anaeramoebae.

The first arctic strain of Rhizochromulina: Morphology, ultrastructure, and position in the evolutionary tree of the order Rhizochromulinales (Heterokontophyta, Dictyochophyceae).

Rhizochromulina marina is a unicellular amoeboid alga capable of forming flagellate cells; it is a single validly named species in the genus. Besides, there are numerous environmental sequences and undescribed strains designated as Rhizochromulina sp. or R. marina. The biogeography of the genus is understudied: rhizochromulines from the Indian, Southern, and Arctic Oceans are unknown. Here, we present the description of Rhizochromulina sp. B44, which was for the first time isolated from an arctic habitat. Biofilms of this microalga grow at the bottom of a culture vessel, where neighbouring amoeboid cells form associations through a common network of pseudopodia, i.e. meroplasmodia. Pseudopodia branch, anastomose mainly during meroplasmodia formation, and are supported by microtubules that arise from the perinuclear zone. Actin filaments are localized in the cytoplasm and can be revealed only near the bases of pseudopodia. We succeeded in inducing the transformation of amoeboid cells into flagellates using a prolonged agitation of cultures. Morphological and molecular analyses revealed that the studied strain is most closely related to the type strain of R. marina. At the same time, 18S rDNA sequences of early branching-off rhizochromulinids differ significantly from Rhizochromulina sp. B44, suggesting a high divergence at the genus level.

Re-isolation and revaluation of boundaries of Thecamoeba terricola Greeff, 1886 (Amoebozoa, Discosea, Thecamoebida), with description of Thecamoeba vicaria n. sp.

We studied a large species of Thecamoeba found in a glass dish with soaked moss and obtained 18s rRNA gene sequence of this organism. Morphologically, the strain was most similar to T. terricola sensu Page, but had significant differences in cell size and nuclear morphology. A more complete similarity was found with the original description of "Amoeba terricola" by Greeff, as well as with Penard's descriptions and slides. The analysis of literature data shows that the strain described by Page in 1977 as a re-isolated T. terricola differs from the original description of this species provided by Greeff in 1866 and data by Penard published in 1902 and 1913. Based on our observations as well as on Greeff's and Penard's data, we reassessed the species boundaries of T. terricola and established T. vicaria n. sp. for the organism described by Page in 1977. The species T. terricola was defined according to its original description. The observations of amoebae on agar have shown that T. terricola cells can form the "standing amoeba" stage, previously described only for Sappinia pedata. This and some other "behaviour" features of T. terricola may be associated with living conditions in terrestrial habitats.

Single-cell transcriptomics supports presence of cryptic species and reveals low levels of population genetic diversity in two testate amoebae morphospecies with large population sizes.

The enormous population sizes and wide biogeographical distribution of many microbial eukaryotes set the expectation of high levels of intraspecific genetic variation. However, studies investigating protist populations remain scarce, mostly due to limited 'omics data. Instead, most genetics studies of microeukaryotes have thus far relied on single loci, which can be misleading and do not easily allow for detection of recombination, a hallmark of sexual reproduction. Here, we analyze >40 genes from 72 single-cell transcriptomes from two morphospecies-Hyalosphenia papilio and Hyalosphenia elegans-of testate amoebae (Arcellinida, Amoebozoa) to assess genetic diversity in samples collected over four years from New England bogs. We confirm the existence of cryptic species based on our multilocus dataset, which provides evidence of recombination within and high levels of divergence between the cryptic species. At the same time, total levels of genetic diversity within cryptic species are low, suggesting that these abundant organisms have small effective population sizes, perhaps due to extinction and repopulation events coupled with efficient modes of dispersal. This study is one of the first to investigate population genetics in uncultivable heterotrophic protists using transcriptomics data and contributes towards understanding cryptic species of nonmodel microeukaryotes.

First report of free-living amoebae in watercourses in southern Brazil: molecular diagnosis and phylogenetic analysis of Vermamoeba vermiformis, Naegleria gruberi, and Acanthamoeba spp.

Free-living amoebae (FLA) are protozoa dispersed in different environments and are responsible for different infections caused to humans and other animals. Microorganisms such as Acanthamoeba spp., Vermamoeba sp., and Naegleria sp. are associated with diseases that affect the central nervous system, in addition to skin infections and keratitis, as occurs in the genus Acanthamoeba and with Vermamoeba vermiformis. Due to the concerns of these FLA in anthropogenic aquatic environments, this work aimed to identify these microorganisms present in waters of Porto Alegre, Brazil. One litre sample was collected in two watercourses during the summer of 2022 and inoculated onto non-nutrient agar plates containing heat-inactivated Escherichia coli. Polymerase chain reaction results indicated the presence of FLA of the genera Acanthamoeba, Vermamoeba, and Naegleria in the study areas. Genetic sequencing indicated the presence of V. vermiformis and Naegleria gruberi. These aquatic and anthropogenic environments can serve as a means of spread and contamination by FLA, which gives valuable information on public health in the city.

Molecular evidence for a new lineage within the Acanthamoeba T4 genotype.

Acanthamoeba is a widespread free-living amoeba capable of causing serious infections in humans and other animals, such as amoebic keratitis, disseminated infections, and fatal encephalitis. Strain identification is usually based on 18S rDNA sequencing, which allows the distinction of over twenty genotypes. Most sequences from environmental and clinical samples belong to the T4 genotype, which can be divided into seven subtypes, T4A to T4G, and by a nearly similar grouping of mitochondrial sequences into T4a to T4j subtypes. The co-clustering of nuclear and mitochondrial groups can be very useful for a better identification of lineages within the very rich T4 genotype. In this study, we provided molecular phylogenetic evidence for the delineation of a new nuclear subtype, hereafter labelled T4H, and its co-clustering with the mitochondrial T4j subtype. At least three cases of amoebic keratitis are due to strains belonging to this new group, present mainly in fresh water and detected in various countries (France, Iran, India and China).

Phylogenetic placement of the protosteloid amoeba Microglomus paxillus identifies another case of sporocarpic fruiting in Discosea (Amoebozoa).

Protosteloid amoebae are a paraphyletic assemblage of amoeboid protists found exclusively in the eukaryotic assemblage Amoebozoa. These amoebae can facultatively form a dispersal structure known as a fruiting body, or more specifically, a sporocarp, from a single amoeboid cell. Sporocarps consist of one to a few spores atop a noncellular stalk. Protosteloid amoebae are known in two out of three well-established major assemblages of Amoebozoa. Amoebae with a protosteloid life cycle are known in the major Amoebozoa lineages Discosea and Evosea but not in Tubulinea. To date, only one genus, which is monotypic, lacks sequence data and, therefore, remains phylogenetically homeless. To further clarify the evolutionary milieu of sporocarpic fruiting we used single-cell transcriptomics to obtain data from individual sporocarps of isolates of the protosteloid amoeba Microglomus paxillus. Our phylogenomic analyses using 229 protein coding markers suggest that M. paxillus is a member of the Discosea lineage of Amoebozoa most closely related to Mycamoeba gemmipara. Due to the hypervariable nature of the SSU rRNA sequence we were unable to further resolve the phylogenetic position of M. paxillus in taxon rich datasets using only this marker. Regardless, our results widen the known distribution of sporocarpy in Discosea and stimulate the debate between a single or multiple origins of sporocarpic fruiting in Amoebozoa.

Comparative Genomics for Evolutionary Cell Biology Using AMOEBAE: Understanding the Golgi and Beyond.

Taking an evolutionary approach to cell biology can yield important new information about how the cell works and how it evolved to do so. This is true of the Golgi apparatus, as it is of all systems within the cell. Comparative genomics is one of the crucial first steps to this line of research, but comes with technical challenges that must be overcome for rigor and robustness. We here introduce AMOEBAE, a workflow for mid-range scale comparative genomic analyses. It allows for customization of parameters, queries, and taxonomic sampling of genomic and transcriptomics data. This protocol article covers the rationale for an evolutionary approach to cell biological study (i.e., when would AMOEBAE be useful), how to use AMOEBAE, and discussion of limitations. It also provides an example dataset, which demonstrates that the Golgi protein AP4 Epsilon is present as the sole retained subunit of the AP4 complex in basidiomycete fungi. AMOEBAE can facilitate comparative genomic studies by balancing reproducibility and speed with user-input and interpretation. It is hoped that AMOEBAE or similar tools will encourage cell biologists to incorporate an evolutionary context into their research.

Stenamoeba aeronauta n. sp., a new case of sibling species in the order Thecamoebida (Amoebozoa, Discosea).

A new speciesStenamoeba aeronautan. sp.was isolated from a culture of large thecamoebids during laboratory studies. Our study of this species showed almost complete morphological identity with the well-known speciesStenamoeba stenopodia. Despite the morphological similarity and proximity in the phylogenetic tree, significant differences in the sequence of the 18S rRNA gene forced us to recognize it as a new species. Known species ofStenamoebahave noticeable morphological differences, but the discovery of the new speciesshows that cryptic speciation appears in this amoeba genus as well as in many others, likeThecamoebaorVannella. In contrast with many other amoebae genera, the number of available 18S rRNA gene sequences exceeds that of morphologically described isolates. So, it is not yet possible to suggest the application of the names of monophyletic species groups, as it was recently proposed forThecamoebaspecies, since every clade ofStenamoebacontains both sequences of species with known morphology and with unknown ones.Overall, the present study further confirms that probably almost all "classical" morphospecies of amoebae may represent a cluster of a sibling species, showing remarkable differences at the molecular level.

Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

Oxygen-dependent regulation of E3(SCF)ubiquitin ligases and a Skp1-associated JmjD6 homolog in development of the social amoeba Dictyostelium.

E3-SCF (Skp1/cullin-1/F-box protein) polyubiquitin ligases activate the proteasomal degradation of over a thousand proteins, but the evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged their elucidation in protists. Here, we expand the FBP candidate list in the social amoeba Dictyostelium and show that the Skp1 interactome is highly remodeled as cells transition from growth to multicellular development. Importantly, a subset of candidate FBPs was less represented when the posttranslational hydroxylation and glycosylation of Skp1 was abrogated by deletion of the O2-sensing Skp1 prolyl hydroxylase PhyA. A role for this Skp1 modification for SCF activity was indicated by partial rescue of development, which normally depends on high O2 and PhyA, of phyA-KO cells by proteasomal inhibitors. Further examination of two FBPs, FbxwD and the Jumonji C protein JcdI, suggested that Skp1 was substituted by other factors in phyA-KO cells. Although a double-KO of jcdI and its paralog jcdH did not affect development, overexpression of JcdI increased its sensitivity to O2. JcdI, a nonheme dioxygenase shown to have physiological O2 dependence, is conserved across protists with its F-box and other domains, and is related to the human oncogene JmjD6. Sensitization of JcdI-overexpression cells to O2 depended on its dioxygenase activity and other domains, but not its F-box, which may however be the mediator of its reduced levels in WT relative to Skp1 modification mutant cells. The findings suggest that activation of JcdI by O2 is tempered by homeostatic downregulation via PhyA and association with Skp1.

Free-Living Amoeba Vermamoeba vermiformis Induces Allergic Airway Inflammation.

The high percentage of Vermamoeba was found in tap water in Korea. This study investigated whether Vermamoeba induced allergic airway inflammation in mice. We selected 2 free-living amoebas (FLAs) isolated from tap water, which included Korean FLA 5 (KFA5; Vermamoeba vermiformis) and 21 (an homolog of Acanthamoeba lugdunensis KA/ E2). We axenically cultured KFA5 and KFA21. We applied approximately 1 × 106 to mice's nasal passages 6 times and investigated their pathogenicity. The airway resistance value was significantly increased after KFA5 and KFA21 treatments. The eosinophil recruitment and goblet cell hyperplasia were concomitantly observed in bronchial alveolar lavage (BAL) fluid and lung tissue in mice infected with KFA5 and KFA21. These infections also activated the Th2-related interleukin 25, thymic stromal lymphopoietin, and thymus and activation-regulated chemokines gene expression in mouse lung epithelial cells. The CD4+ interleukin 4+ cell population was increased in the lung, and the secretion of Th2-, Th17-, and Th1-associated cytokines were upregulated during KFA5 and KFA21 infection in the spleen, lung-draining lymph nodes, and BAL fluid. The pathogenicity (allergenicity) of KFA5 and KFA21 might not have drastically changed during the long-term in vitro culture. Our results suggested that Vermamoeba could elicit allergic airway inflammation and may be an airway allergen.

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.

Molecular identification and phylogenetic analysis of free-living amoeba in the water resources of Arak, Iran.

The aim of the present study was to detect free-living amoeba (FLA) in the water resources of Arak, Iran using molecular tools. A total of 154 samples were collected from different water supplies. Molecular analyses, sequencing, and phylogenetic study were conducted to confirm the species and genotypes of FLA. Fisher exact test was used to determine the significance. Of 154 water samples, 19 (12.3%) samples were tested positive for FLA. Three genotypes of Acanthamoeba including T4, subtype D, and T5 were identified among the isolates. The pathogenicity assay showed that the isolate of Acanthamoeba in drinking water was highly pathogenic. Three species of Naegleria, including N. australiensis, N. pagei, and N. gruberi were found among the samples. Six isolates of Vermamoeba were identified as V. vermiformis. Meanwhile, three other species including Vannella sp., Vahlkampfia avara, and Stenamoeba polymorpha were also recovered from the water samples. Statistical analysis showed a significant difference between the various water resources contaminated with FLA. This is the first study to reveal the presence of S. polymorpha in water sources in Iran. According to the findings of the present study, health officials should be beware of potential public health impacts of FLA in water resources.

Isolation and identification of potentially pathogenic free-living amoeba in dental-unit water samples.

Although the presence of free-living amoebae (FLAs) in various water sources has been reported, few studies have been surveyed on their abundance in medical-unit and dental-unit water samples. The current study aimed to identify morphological and molecular characteristics of FLA isolates in the water samples of the dental unit in Iran. A total of 232 water samples were collected from 17 dental units. Then, filtration and cultivation were conducted on a non-nutrient agar (NNA) medium. Also, polymerase chain reaction (PCR) assay and sequencing were performed by using the genus/species-specific primers plus a common primer set on positive samples. One hundred and sixty-six samples were positive for FLA by the microscopic method, whereas 114 samples were positive by the molecular method with a common primer set. Considering the PCR assay with genus/species-specific primers, 23.27% (54/232) samples were identified as Acanthamoeba spp. (belonging to T4 genotype), 36.63% (85/232) as Vermamoeba vermiformis, and 1.72% (4/232) as Vahlkampfiidae family (Naegleria lovaniensis). These results highlight a need to improve filtration systems in dental units and periodic screenings for FLA in dental-unit water.

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.