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Antibiotic resistance poses a significant threat to human health. While most studies focus on bacteria, interactions between antibiotics and other crucial microbial groups like protists remain uncertain. This study investigates how protists interact with antibiotics and examines how these interactions impact the fate of resistance genes. It reveals that amoebae exhibit high resistance to eight high-risk environmental antibiotics, accumulating significant quantities within their cells. Wild amoeboid strains from distant locations carry substantial antibiotic resistance genes (ARGs) and metal resistance genes (MRGs), with significant heterogeneity within a single species. Amoeboid symbionts and pathogens predominantly carry these genes. Paraburkholderia symbionts have reduced genomes and fewer resistance genes compared to free-living strains, while amoeba-endogenous Stenotrophomonas maltophilia does not exhibit a significantly reduced genome size. This suggests that the amoeboid hosts serve as a temporary medium facilitating its transmission. In summary, the study unveils that soil amoebae represent unexpected hotspots for antibiotics and resistance genes. Future research should assess the effects of antibiotics on often-overlooked protists and explore their role in spreading ARGs and MRGs in ecosystems. Incorporating protists into broader antibiotic resistance research is recommended, highlighting their significance within a One Health perspective.

Acrasids are amoebae with the capacity to form multicellular fruiting bodies in a process known as aggregative multicellularity (AGM). This makes acrasids the only known example of multicellularity among the earliest branches of eukaryotes (the former Excavata). Here, we report the Acrasis kona genome sequence plus transcriptomes from pre-, mid- and post-developmental stages. The genome is rich in novelty and genes with strong signatures of horizontal transfer, and multigene families encode nearly half of the amoeba's predicted proteome. Development in A. kona appears molecularly simple relative to the AGM model, Dictyostelium discoideum. However, the acrasid also differs from the dictyostelid in that it does not appear to be starving during development. Instead, developing A. kona appears to be very metabolically active, does not induce autophagy and does not up-regulate its proteasomal genes. Together, these observations strongly suggest that starvation is not essential for AGM development. Nonetheless, development in the two amoebae appears to employ remarkably similar pathways for signaling, motility and, potentially, construction of an extracellular matrix surrounding the developing cell mass. Much of this similarity is also shared with animal development, suggesting that much of the basic tool kit for multicellular development arose early in eukaryote evolution., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Bioindication, evaluating biological responses to environmental disturbances, is crucial for assessing the ecological status of an ecosystem. While historical bioindication relied on macroscopic organisms, the introduction of environmental DNA (eDNA) techniques allows the application of protists without the necessity of morphological identification. In this study, we propose a novel bioindication methodology utilizing Arcellinida, a group of top predators among protists, as bioindicators of freshwater ecosystems. For that purpose, we first characterized the Arcellinida diversity over 1 year at three different points of Lake Sanabria, an ancient glacier lake known to be subjected to anthropogenic disturbances. We compared this diversity with an undisturbed control site. Second, we characterized the Arcellinida diversity in other ecosystems to generate the ecological background to test the connectivity between them. Results indicate limited connectivity between the different ecosystems and an edge effect between terrestrial and aquatic ecosystems. Disturbed freshwater ecosystems exhibited reduced Arcellinida diversity at both specific and infraspecific levels, providing valuable insight into recent disturbances. Arcellinida-based bioindication provides a sensitive, accurate and easy-to-interpret protocol for monitoring disturbances in freshwater ecosystems. It represents a valuable tool for environmental assessments and conservation strategies., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

Amoebae are environmental predators feeding on bacteria, fungi, and other eukaryotic microbes. Predatory interactions alter microbial communities and impose selective pressure toward phagocytic resistance or escape which may, in turn, foster virulence attributes. The ubiquitous fungivorous amoeba Protostelium aurantium has a wide prey spectrum in the fungal kingdom but discriminates against members of the Saccharomyces clade, such as Saccharomyces cerevisiae and Candida glabrata . Here, we show that this prey discrimination among fungi is solely based on the presence of ubiquinone as an essential cofactor for the predator. While the amoeba readily fed on fungi with CoQ presenting longer isoprenyl side chain variants CoQ8-10, such as those from the Candida clade, it failed to proliferate on those with shorter CoQ variants, specifically from the Saccharomyces clade (CoQ6). Supplementing non-edible yeast with CoQ9 or CoQ10 rescued the growth of P. aurantium , highlighting the importance of a long isoprenyl side chain. Heterologous biosynthesis of CoQ9 in S. cerevisiae by introducing genes responsible for CoQ9 production from the evolutionary more basic Yarrowia lipolytica complemented the function of the native CoQ6. The results suggest that the use of CoQ6 among members of the Saccharomyces clade might have originated as a predatory escape strategy in fungal lineages and could be retained in organisms that were able to thrive by fermentation., Importance: Ubiquinones (CoQ) are universal electron carriers in the respiratory chain of all aerobic bacteria and eukaryotes. Usually 8-10 isoprenyl units ensure their localization within the lipid bilayer. Members of the Saccharomyces clade among fungi are unique in using only 6. The reason for this is unclear. Here we provide evidence that the use of CoQ6 efficiently protects these fungi from predation by the ubiquitous fungivorous amoeba Protostelium aurantium which lacks its own biosynthetic pathway for this vitamin. The amoebae were starving on a diet of CoQ6 yeasts which could be complemented by either the addition of longer CoQs or the genetic engineering of a CoQ9 biosynthetic pathway., Competing Interests: The authors declare no conflict of interest.

The photosynthetic amoeba, Paulinella provides a recent (ca. 120 Mya) example of primary plastid endosymbiosis. Given the extensive data demonstrating host lineage-driven endosymbiont integration, we analysed nuclear genome and transcriptome data to investigate mechanisms that may have evolved in Paulinella micropora KR01 (hereinafter, KR01) to maintain photosynthetic function in the novel organelle, the chromatophore. The chromatophore is of α-cyanobacterial provenance and has undergone massive gene loss due to Muller's ratchet, but still retains genes that encode the ancestral α-carboxysome and the shell carbonic anhydrase, two critical components of the biophysical CO 2 concentrating mechanism (CCM) in cyanobacteria. We identified KR01 nuclear genes potentially involved in the CCM that arose via duplication and divergence and are upregulated in response to high light and downregulated under elevated CO 2 . We speculate that these genes may comprise a novel CO 2 delivery system (i.e., a biochemical CCM) to promote the turnover of the RuBisCO carboxylation reaction and counteract photorespiration. We posit that KR01 has an inefficient photorespiratory system that cannot fully recycle the C 2 product of RuBisCO oxygenation back to the Calvin-Benson cycle. Nonetheless, both these systems appear to be sufficient to allow Paulinella to persist in environments dominated by faster-growing phototrophs., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin., (© 2024 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

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., (© 2023 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n = 13) and Allovahlkampfia spp. (n = 3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species., (© M. Yu et al., published by EDP Sciences, 2024.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

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., (© 2023 International Society of Protistologists.)

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.

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)., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

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 O 2 -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 O 2 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 O 2 . JcdI, a nonheme dioxygenase shown to have physiological O 2 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 O 2 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 O 2 is tempered by homeostatic downregulation via PhyA and association with Skp1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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. 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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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.

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.

To date, genomic analyses in amoebozoans have been mostly limited to model organisms or medically important lineages. Consequently, the vast diversity of Amoebozoa genomes remain unexplored. A draft genome of Cochliopodium minus, an amoeba characterized by extensive cellular and nuclear fusions, is presented. C. minus has been a subject of recent investigation for its unusual sexual behavior. Cochliopodium's sexual activity occurs during vegetative stage making it an ideal model for studying sexual development, which is sorely lacking in the group. Here we generate a C. minus draft genome assembly. From this genome, we detect a substantial number of lateral gene transfer (LGT) instances from bacteria (15%), archaea (0.9%) and viruses (0.7%) the majority of which are detected in our transcriptome data. We identify the complete meiosis toolkit genes in the C. minus genome, as well as the absence of several key genes involved in plasmogamy and karyogamy. Comparative genomics of amoebozoans reveals variation in sexual mechanism exist in the group. Similar to complex eukaryotes, C. minus (some amoebae) possesses Tyrosine kinases and duplicate copies of SPO11. We report a first example of alternative splicing in a key meiosis gene and draw important insights on molecular mechanism of sex in C. minus using genomic and transcriptomic data., (© 2022. The Author(s).)

The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the host cell and the engulfed prokaryote became genetically codependent, with the integration of genes from the endosymbiont into the host nuclear genome and subsequent gene loss from the endosymbiont. This process required that horizontally transferred genes become active and properly regulated despite inherent differences in genetic features between donor (endosymbiont) and recipient (host). Although this genetic reorganization is considered critical for early stages of organellogenesis, we have little knowledge about the mechanisms governing this process. The photosynthetic amoeba Paulinella micropora offers a unique opportunity to study early evolutionary events associated with organellogenesis and primary endosymbiosis. This amoeba harbors a “chromatophore,” a nascent photosynthetic organelle derived from a relatively recent cyanobacterial association (∼120 million years ago) that is independent of the evolution of primary plastids in plants (initiated ∼1.5 billion years ago). Analysis of the genome and transcriptome of Paulinella revealed that retrotransposition of endosymbiont-derived nuclear genes was critical for their domestication in the host. These retrocopied genes involved in photoprotection in cyanobacteria became expanded gene families and were “rewired,” acquiring light-responsive regulatory elements that function in the host. The establishment of host control of endosymbiont-derived genes likely enabled the cell to withstand photo-oxidative stress generated by oxygenic photosynthesis in the nascent organelle. These results provide insights into the genetic mechanisms and evolutionary pressures that facilitated the metabolic integration of the host–endosymbiont association and sustained the evolution of a photosynthetic organelle.

Plastid primary endosymbiosis has occurred twice, once in the Archaeplastida ancestor and once in the Paulinella (Rhizaria) lineage. Both events precipitated massive evolutionary changes, including the recruitment and activation of genes that are horizontally acquired (HGT) and the redeployment of existing genes and pathways in novel contexts. Here we address the latter aspect in Paulinella micropora KR01 (hereafter, KR01) that has independently evolved spliced leader (SL) trans-splicing (SLTS) of nuclear-derived transcripts. We investigated the role of this process in gene regulation, novel gene origination, and endosymbiont integration. Our analysis shows that 20% of KR01 genes give rise to transcripts with at least one (but in some cases, multiple) sites of SL addition. This process, which often occurs at canonical cis-splicing acceptor sites (internal introns), results in shorter transcripts that may produce 5'-truncated proteins with novel functions. SL-truncated transcripts fall into four categories that may show: (i) altered protein localization, (ii) altered protein function, structure, or regulation, (iii) loss of valid alternative start codons, preventing translation, or (iv) multiple SL addition sites at the 5'-terminus. The SL RNA genes required for SLTS are putatively absent in the heterotrophic sister lineage of photosynthetic Paulinella species. Moreover, a high proportion of transcripts derived from genes of endosymbiotic gene transfer (EGT) and HGT origin contain SL sequences. We hypothesize that truncation of transcripts by SL addition may facilitate the generation and expression of novel gene variants and that SLTS may have enhanced the activation and fixation of foreign genes in the host genome of the photosynthetic lineages, playing a key role in primary endosymbiont integration., (© 2022 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Entamoeba moshkovskii Tshalaia, 1941 is prevalent in developing countries and it is considered to be primarily a free-living amoeba, which is morphologically indistinguishable, but biochemically and genetically different from the human infecting, pathogenic Entamoeba histolytica Schaudinn, 1903. The pathogenic potential of this organism is still under discussion. Entamoeba moshkovskii in human stool samples has been reported in different countries such as the United States, Italy, Australia, Iran, Turkey, Bangladesh, India (Pondicherry), Indonesia, Colombia, Malaysia, Tunisia, Tanzania and Brazil, but no data are available about the occurrence of E. moshkovskii in farm animals. This study provides data on the occurrence of E. moshkovskii in pigs in a total of 294 fresh faecal samples collected from five different regions in Kolkata, West Bengal, India. Stool samples were tested by nested PCR using primers targeting SSU rDNA of E. moshkovskii. The amplified PCR products were further confirmed by RFLP technique. Purified nested PCR products were also sequenced and identified via BLAST program run on the NCBI website to confirm species along with their genetic characteristics of the E. moshkovskii isolates. Overall 5.4 % samples were identified as E. moshkovskii positive. Results of this study demonstrate that swine can host E. moshkovskii and should be considered as a potential natural reservoir for E. moshkovskii. However, the occurrence of E. moshkovskii infection in pigs was not statistically associated with their faecal consistency, sex and developmental stage.

Background: Free-living amoeba (FLA), including Acanthamoeba, Naegleria, Balamuthia and Vermamoeba, have been isolated from water, sand, soil, dust and air. Numerous studies considered that FLA are a significant cause of neurological and ocular complications in high-risk groups, including immunocompromised individuals. The present study aimed to identify morphological and molecular characteristics of FLA isolates in dialysis fluid samples of hydraulic systems in hemodialysis units in Iran., Methods: A total of 328 dialysis fluid samples were collected from 16 dialysis machines, including 164 samples before hemodialysis sessions (after cleaning) and 164 samples after hemodialysis sessions (before cleaning). Filtration and cultivation were performed on non-nutrient agar medium. Also, PCR and sequencing were applied by using the genus-specific primers along with a common primer set on positive samples., Results: Both morphology and molecular investigations showed that 22.5% (74/328) of dialysis fluid samples were positive for FLA. There was a positive relationship between the high frequency of FLA after hemodialysis sessions (before cleaning) compared with before hemodialysis sessions (after cleaning) (OR=2.86; 95% CI 1.5 to 5.45). Considering the PCR assay, 16.46% (54/328) samples were identified as Acanthamoeba spp. (belonging to T3 and T4 genotypes), 5.18% (17/328) as Vermamoeba vermiformis and 0.91% (3/328) as Vahlkampfiidae family (Naegleria australiensis, Naegleria pagei and Allovahlkampfia)., Conclusion: The present results support a need to improve filtration and purification methods for dialysis fluid of hydraulic systems in hemodialysis units. They also highlight the relevance of periodic screenings for FLA-related diseases in hemodialysis patients., (© The Author(s) 2021. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Paulinella represents the only known case of an independent primary plastid endosymbiosis, outside Archaeplastida, that occurred c. 120 (million years ago) Ma. These photoautotrophs grow very slowly in replete culture medium with a doubling time of 6-7 d at optimal low light, and are highly sensitive to photodamage under moderate light levels. We used genomic and biophysical methods to investigate the extreme slow growth rate and light sensitivity of Paulinella, which are key to photosymbiont integration. All photosystem II (PSII) genes except psb28-2 and all cytochrome b 6 f complex genes except petM and petL are present in Paulinella micropora KR01 (hereafter, KR01). Biophysical measurements of the water oxidation complex, variable chlorophyll fluorescence, and photosynthesis-irradiance curves show no obvious evidence of PSII impairment. Analysis of photoacclimation under high-light suggests that although KR01 can perform charge separation, it lacks photoprotection mechanisms present in cyanobacteria. We hypothesize that Paulinella species are restricted to low light environments because they are deficient in mitigating the formation of reactive oxygen species formed within the photosystems under peak solar intensities. The finding that many photoprotection genes have been lost or transferred to the host-genome during endosymbiont genome reduction, and may lack light-regulation, is consistent with this hypothesis., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. The captivating spine-bearing taxa in Thecofilosea were not yet molecularly characterized due to being very rare. There are only two known spine-bearing species, Pamphagus armatus and Lecythium spinosum, which were synonymized by Penard in 1902. Due to a morphological difference of those taxa, we discuss here that we disagree with this taxonomical act. We further isolated single cells of Pamphagus armatus directly from their habitat and successfully sequenced their SSU rDNA, which we subjected to phylogenetic analyses. We show that Pamphagus armatus branches within the Rhizaspididae (Tectofilosida, Thecofilosea). Accordingly, we transfer Pamphagus armatus and the assumingly closely related species Lecythium spinosum to Rhizaspis., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized., Results: We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5'-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two., Conclusions: This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle., (© 2022. The Author(s).)

Free-living amoebae often harbor obligate intracellular bacterial symbionts. Amoebophilus (A.) asiaticus is a representative of a lineage of amoeba symbionts in the phylum Bacteroidota. Here, we analyse the transcriptome of A. asiaticus strain 5a2 at four time points during its infection cycle and replication within the Acanthamoeba host using RNA sequencing. Our results reveal a dynamic transcriptional landscape throughout different A. asiaticus life cycle stages. Many intracellular bacteria and pathogens utilize eukaryotic-like proteins (ELPs) for host cell interaction and the A. asiaticus 5a2 genome shows a particularly high abundance of ELPs. We show the expression of all genes encoding ELPs and found many ELPs to be differentially expressed. At the replicative stage of A. asiaticus, ankyrin repeat proteins and tetratricopeptide/Sel1-like repeat proteins were upregulated. At the later time points, high expression levels of a type 6 secretion system that likely prepares for a new infection cycle after lysing its host, were found. This study reveals comprehensive insights into the intracellular lifestyle of A. asiaticus and highlights candidate genes for host cell interaction. The results from this study have implications for other intracellular bacteria such as other amoeba-associated bacteria and the arthropod symbionts Cardinium forming the sister lineage of A. asiaticus., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a 'brain-eating' disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections., (© 2021. The Author(s).)

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D'Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage ("clade C") of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for "clade C.", (© 2021 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Aims: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria., Methods and Results: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture., Significance and Impact of the Study: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures., (© 2020 The Society for Applied Microbiology.)

Ancient pathways promoting unicellularity and multicellularity are associated with cancer, the former being pro-oncogenic and the latter acting to suppress oncogenesis. However, there are only a limited number of non-vertebrate models for studying these pathways. Here, we review Dictyostelium discoideum and describe how it can be used to understand these gene networks. D. discoideum has a unicellular and multicellular life cycle, making it possible to study orthologs of cancer-associated genes in both phases. During development, differentiated amoebae form a fruiting body composed of a mass of spores that are supported atop a stalk. A portion of the cells sacrifice themselves to become non-reproductive stalk cells. Cheating disrupts the principles of multicellularity, as cheater cells alter their cell fate to preferentially become spores. Importantly, D. discoideum has gene networks and several strategies for maintaining multicellularity. Therefore, D. discoideum can help us better understand how conserved genes and pathways involved in multicellularity also influence cancer development, potentially identifying new therapeutic avenues., (© 2021 Wiley Periodicals LLC.)

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Dziani Dzaha is a hypersaline lake (Mayotte island), whose microbial community is dominated by photosynthetic microorganisms. Here, we describe two new free-living heteroloboseans. One belonging to the Pharyngomonas genus and the other, whose 18S rRNA gene sequence shares only 85% homology to its closest relatives Euplaesiobystra hypersalinica, was proposed as a new species of this genus being called Euplaesiobystra dzianiensis. Both strains were salt tolerant to 75‰ and grew between 25 and 37°C. Their distribution patterns varied seasonally and depended also on depth. Noticeably, both free-living amoebae isolates were able to graze on Arthrospira filaments, which are found within the same water layer. In conclusion, we document for the first time the presence and ecology of free-living amoebae in the thalassohaline lake Dziani Dzaha, and describe a new species of the Euplaesiobystra genus., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Of the single-celled eukaryotic microbes, Naegleria fowleri , Balamuthia mandrillaris , and Acanthamoeba spp. are known to cause fatal encephalitis in humans. Being eukaryotes, these cells have been used as a model for studying and understanding complex cellular processes in humans like cell motility, phagocytosis, and metabolism. The ongoing pandemic caused by SARS-CoV-2 that infects multiple organs has emerged as a challenge to unravel its mode of infection and the pathogenicity resulting in eukaryotic cell death. Working with these single-celled eukaryotic microbes provided us the opportunity to plan bioinformatic approaches to look into the likelihood of studying the known and alternative mode of infection of the SARS-CoV-2 in eukaryotic cells. Genome databases of N. fowleri , B. mandrillaris , and Acanthamoeba spp. were used to explore the expression of angiotensin-converting enzyme 2 (ACE2), androgen-regulated serine protease precursor (TMPRSS2), CD4, CD147, and furin that are known to be cardinal for SARS-CoV-2 in recognition and binding to human cells. It was hypothesized that if a receptor-dependent or phagocytosis-assisted SARS-CoV-2 uptake does occur in free-living amoebae (FLA), this model can provide an alternative to human cells to study cellular recognition and binding of SARS-CoV-2 that can help design drugs and treatment modalities in COVID-19. We show that, of the FLA, ACE2 and TMPRSS2 are not expressed in Acanthamoeba spp. and B. mandrillaris , but primitive forms of these cell recognition proteins were seen to be encoded in N. fowleri. Acanthamoeba spp. and N. fowleri encode for human-like furin which is a known SARS-CoV-2 spike protein involved in host cell recognition and binding.

Sex is a hallmark of eukaryotes but its evolution in microbial eukaryotes is poorly elucidated. Recent genomic studies revealed microbial eukaryotes possess a genetic toolkit necessary for sexual reproduction. However, the mechanism of sexual development in a majority of microbial eukaryotes including amoebozoans is poorly characterized. The major hurdle in studying sex in microbial eukaryotes is a lack of observational evidence, primarily due to its cryptic nature. In this study, we used a tractable fusing amoeba, Cochliopodium, to investigate sexual development using stage-specific Differential Gene Expression (DGE) and cytological analyses. Both DGE and cytological results showed that most of the meiosis and sex-related genes are upregulated in Cochliopodium undergoing fusion in laboratory culture. Relative gene ontology (GO) category representations in unfused and fused cells revealed a functional skew of the fused transcriptome toward DNA metabolism, nucleus and ligases that are suggestive of a commitment to sexual development. However, the GO categories of unfused cells were dominated by metabolic pathways and other processes indicative of a vegetative phase. Our study provides strong evidence that the fused cells represent a sexual stage in Cochliopodium. Our findings have further implications in understanding the evolution and mechanism of inheritance involving multiparents in other eukaryotes with a similar reproductive strategy., Competing Interests: The authors have declared that no competing interests exist.

Objective: These amoebas can cause dangerous illnesses when they accidentally enter the human body, so it is necessary to determine various forms of organisms in water resources to prevent the danger they can cause and risks to human health. Currently, in Bandar Abbas, there is no sufficient information about the distribution of Acanthamoeba, and we intended to study its frequency and determine the related genotypes., Results: Out of 83 water samples collected from different resources in the city, 31 plates (37.3%) were found to be positive for free-living amoebae. Of these, five were identified as Acanthamoeba (6%) by culture method and 8 (9.6%) by molecular method. Positive sample sequence analysis enabled us to distinguish two genotypes of T4 (7 cases) and T15 (1 case) in this study.

Symbiotic interactions exist within a parasitism to mutualism continuum that is influenced, among others, by genes and context. Dynamics of intracellular invasion, replication, and prevalence may underscore both host survivability and symbiont stability. More infectious symbionts might exert higher corresponding costs to hosts, which could ultimately disadvantage both partners. Here, we quantify infection patterns of diverse Paraburkholderia symbiont genotypes in their amoeba host Dictyostelium discoideum and probe the relationship between these patterns and host outcomes. We exposed D. discoideum to thirteen strains of Paraburkholderia each belonging to one of the three symbiont species found to naturally infect D. discoideum : Paraburkholderia agricolaris, Paraburkholderia hayleyella, and Paraburkholderia bonniea . We quantified the infection prevalence and intracellular density of fluorescently labeled symbionts along with the final host population size using flow cytometry and confocal microscopy. We find that infection phenotypes vary across symbiont strains. Symbionts belonging to the same species generally display similar infection patterns but are interestingly distinct when it comes to host outcomes. This results in final infection loads that do not strongly correlate to final host outcomes, suggesting other genetic factors that are not a direct cause or consequence of symbiont abundance impact host fitness.