Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

CRISPR/Cas9-induced disruption of Bodo saltans paraflagellar rod-2 gene reveals its importance for cell survival.

Developing transfection protocols for marine protists is an emerging field that will allow the functional characterization of protist genes and their roles in organism responses to the environment. We developed a CRISPR/Cas9 editing protocol for Bodo saltans, a free-living kinetoplastid with tolerance to both marine and freshwater conditions and a close non-parasitic relative of trypanosomatids. Our results show that SaCas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex-mediated disruption of the paraflagellar rod 2 gene (BsPFR2) was achieved using electroporation-mediated transfection. The use of CRISPR/Cas9 genome editing can increase the efficiency of targeted homologous recombination when a repair DNA template is provided. Our sequence analysis suggests two mechanisms for repairing double-strand breaks in B. saltans are active; homologous-directed repair (HDR) utilizing an exogenous DNA template that carries an antibiotic resistance gene and likley non-homologous end joining (NHEJ). However, HDR was only achieved when a single (vs. multiple) SaCas9 RNP complex was provided. Furthermore, the biallelic knockout of BsPFR2 was detrimental for the cell, highlighting its essential role for cell survival because it facilitates the movement of food particles into the cytostome. Our Cas9/sgRNA RNP complex protocol provides a new tool for assessing gene functions in B. saltans and perhaps similar protists with polycistronic transcription.

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

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

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing.

Wound Healing Efficacy of Cucurbitaceae Seed Oils in Rats: Comprehensive Phytochemical, Pharmacological, and Histological Studies Tackling AGE/RAGE and Nrf2/Ho-1 Cue.

The Cucurbitaceae family includes several edible species that are consumed globally as fruits and vegetables. These species produce high volumes of seeds that are often discarded as waste. In this study, we investigate the chemical composition and biological activity of three seed oils from Cucurbitaceae plants, namely, cantaloupe, honeydew, and zucchini, in comparison to the widely used pumpkin seed oil for their ability to enhance and accelerate wound healing in rats. Our results showed that honeydew seed oil (HSO) was effective in accelerating wound closure and enhancing tissue repair, as indicated by macroscopic, histological, and biochemical analyses, as compared with pumpkin seed oil (PSO). This effect was mediated by down-regulation of the advanced glycation end products (AGE) and its receptor (RAGE) cue, activating the cytoprotective enzymes nuclear factor erythroid 2 (Nrf2) and heme oxygenase-1 (HO-1), suppressing the inflammatory mediators tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-κB), and nod-like receptor protein 3 (NLRP3), and reducing the levels of the skin integral signaling protein connexin (CX)-43. Furthermore, immunohistochemical staining for epidermal growth factor (EGF) showed the lowest expression in the skin after treatment with HSO, indicating a well-organized and complete healing process. Other seed oils from cantaloupe and zucchini exhibited favorable activity when compared with untreated rats; however, their efficacy was comparatively lower than that of PSO and HSO. Gas chromatographic analysis of the derivatized oils warranted the superior activity of HSO to its high nutraceutical content of linoleic acid, which represented 65.9% of the fatty acid content. This study's findings validate the use of honeydew seeds as a wound-healing fixed oil and encourage further investigation into the potential of Cucurbitaceae seeds as sources of medicinally valuable plant oils.

Ultrasound-assisted synthesis of MSNs/PS nanocomposite membranes for effective removal of Cd2+ and Pb2+ ions from aqueous solutions.

Contamination of heavy metal (Cd2+ & Pb2+) ions in drinking water is producing major impacts on the environment and public health and is considered one of the greatest dangers to humanity. Membrane technology has been chosen over other processing methods due to its simplicity and high capacity for more effective removal of hazardous heavy metals. In the current study, amine, thiol, and bi-thiol functional groups were used to functionalize mesoporous silica nanoparticles (MSNs) to improve the efficiency of the silica nanoparticle. The morphology of the MSNs as well as the existence of amine and thiol on the surface of MSNs was demonstrated by a variety of characterization techniques, including FTIR, TEM, and SEM examination. The impact of surface-modified MSNs on the morphology, properties, and performance of polysulfone (PS) nanofiltration (NF) membranes was also evaluated. The membrane that incorporated amine with thiol-based MSNs (DiMP-MSNs/PS-NF membrane) had the highest pure water permeability (6.7 LMH bar-1). As a result of the functional groups, the surface-modified MSNs/PS nanofiltration are extremely effective at removing heavy metal ions from aqueous solutions. The surface-modified MSNs/PS nano-filtration membranes exhibit unprecedented Cd2+ and Pb2+ removal rates of approximately 82% and 99%, respectively. This research indicates the possible application of the surface-modified MSNs/PS nanofiltration membrane as a promising platform to remove heavy metal ions from polluted water.

How the COVID-19 Pandemic Contributed to Diagnostic Bias.

Diagnosis bias in the medical field is a recognized entity that can contribute to misdiagnoses and incorrect management. It remains a constant challenge that must be recognized and addressed. Several factors play a role in the formation of preconceptions which influence the physicians' decision-making process. The aim of this paper is to present a case that was misdiagnosed and mistakenly managed due to diagnosis bias during the coronavirus disease 2019 (COVID-19) pandemic. We also suggest two ways to reduce the risk of diagnosis bias. Multi-inflammatory syndrome of children (MIS-C) was described during the COVID-19 pandemic. The rise in the incidence of MIS-C masked the diagnosis of other diseases that present in a similar fashion. In this paper, we describe the case of a seven-year-old girl, who presented in 2020, with acute onset respiratory distress. Her chest images were suggestive of COVID-19 pneumonitis which prompted the physicians to complete the MIS-C workup by performing an echocardiogram. A large aneurysm of the left main artery was seen which led to a preliminary diagnosis of MIS-C. A repeat echocardiography, 48 hours after the initiation of MIS-C treatment, was suggestive of a large coronary fistula complicated by infective endocarditis and multiple septic pulmonary emboli. It can be inferred that the misdiagnosis occurred as a result of availability and premature-closure biases. Efforts to decrease such biases include group decision-making and using checklists during the assessment of a patient.

Transition from stromatolite to thrombolite fabric: potential role for reticulopodial protists in lake microbialites of a Proterozoic ecosystem analog.

Prior observations suggest that foraminiferan protists use their reticulopodia (anastomosing pseudopodia) to alter sediment fabric by disrupting laminations of subtidal marine stromatolites, erasing the layered structures in an experimental setting. Because microbialites and foraminifera are found in non-marine settings, we hypothesized that foraminifera living in lakes could also disrupt layered microbialite fabric. With this aim and using a variety of multidisciplinary approaches, we conducted field surveys and an experiment on microbialites from Green Lake (GL; Fayetteville, New York State, United States), which has been studied as a Proterozoic ecosystem analog. The lake is meromictic and alkaline, receiving calcium sulfate-rich water in the monimolimnion; it supports a well-developed carbonate platform that provides access to living and relict microbialites. The living microbialites grow from early spring to autumn, forming a laminated mat at their surface (top ~5 mm), but a clotted or massive structure exists at depth (> ~ 1 cm). We observed a morphotype of "naked" foraminiferan-like protist in samples from GL microbialites and sediments; thus, considered the possibility of freshwater foraminiferan impact on microbialite fabric. Results of an experiment that seeded the cultured freshwater foraminifer Haplomyxa saranae onto the GL microbialite surface indicates via micro-CT scanning and anisotropy analysis that the introduced foraminifer impacted uppermost microbialite layering (n = 3 cores); those cores with an added inhibitor lacked changes in anisotropy for two of those three cores. Thus, it remains plausible that the much smaller, relatively common, native free-form reticulate protist, which we identified as Chlamydomyxa labyrinthuloides, can disrupt microbialite fabrics on sub-millimeter scales. Our observations do not exclude contributions of other possible causal factors.

Exploring the protist microbiome: The diversity of bacterial communities associated with Arcella spp. (Tubulina: Amoebozoa).

Research on protist-bacteria interactions is increasingly relevant as these associations are now known to play important roles in ecosystem and human health. Free-living amoebae are abundant in all environments and are frequent hosts for bacterial endosymbionts including pathogenic bacteria. However, to date, only a small fraction of these symbionts have been identified, while the structure and composition of the total symbiotic bacterial communities still remains largely unknown. Here, we use the testate amoeba Arcella spp. as model organisms to investigate the specificity and diversity of Arcella-associated microbial communities. High-throughputamplicon sequencing from the V4 region of the 16S rRNA gene revealed high diversity in the bacterial communities associated with the wild Arcella spp. To investigate the specificity of the associated bacterial community with greater precision, we investigated the bacterial communities of two lab-cultured Arcella species, A. hemispherica and A. intermedia, grown in two different media types. Our results suggest that Arcella-bacteria associations are species-specific, and that the associated bacterial community of lab-cultured Arcella spp. remains distinct from that of the surrounding media. Further, each host Arcella species could be distinguished based on its bacterial composition. Our findings provide insight into the understanding of eukaryotic-bacterial symbiosis.

Willingness and Attitude of the Arab World Population Toward Solid-Organ Donation.

OBJECTIVES: Awareness regarding organ donation has been steadily growing in the Arab world yet is still far from the current demand. A thorough analysis of population behavior toward organ donation can improve organ transplant education. Therefore, we designed this study to assess the knowledge, attitude, donation desires, and views on organ donation among adults in Arab countries. MATERIALS AND METHODS: An observational cross- sectional study approach was used by assessing 1004 adult survey respondents from 22 Arab countries through the snowball sampling technique via social media platforms and emails. A fact sheet was used to collect demographic information, which was followed by a predesigned questionnaire to assess the attitude and willingness of participants toward solid-organ donation. RESULTS: Results showed that only 17.0% of respondents had willingness to donate in the future, and only 2.0% respondents were already organ donors or registered as organ donors. Respondents indicated that the most acceptable organs to be donated after death were kidneys (57.8%), followed by liver (45.1%) and heart (42.3%). Regarding the type of surgery for living donation, 48.1% of the respondents had no surgery type preference, whereas 12.9% would only agree to laparoscopic intervention. A significant difference (P < .001) was noted among respondents with transplant experience and without experience regarding organ donation willingness. In terms of paired exchange and list exchange donation, 18.0% indicated that they would refuse to donate, 23.0% would accept, and 19.0% would accept if no alternative was available. CONCLUSIONS: This study highlighted the psychology of the Arab world and factors influencing decisions toward solid-organ donation and transplant. The biggest factor for unwillingness to donate organs was posttransplant health-related risks; almost 50% of respondents were afraid of health complications. A need for awareness and education regarding the importance of organ donation and transplant emerged as common themes in this study.

Multiple integrated metabolic strategies allow foraminiferan protists to thrive in anoxic marine sediments.

Oceanic deoxygenation is increasingly affecting marine ecosystems; many taxa will be severely challenged, yet certain nominally aerobic foraminifera (rhizarian protists) thrive in oxygen-depleted to anoxic, sometimes sulfidic, sediments uninhabitable to most eukaryotes. Gene expression analyses of foraminifera common to severely hypoxic or anoxic sediments identified metabolic strategies used by this abundant taxon. In field-collected and laboratory-incubated samples, foraminifera expressed denitrification genes regardless of oxygen regime with a putative nitric oxide dismutase, a characteristic enzyme of oxygenic denitrification. A pyruvate:ferredoxin oxidoreductase was highly expressed, indicating the capability for anaerobic energy generation during exposure to hypoxia and anoxia. Near-complete expression of a diatom's plastid genome in one foraminiferal species suggests kleptoplasty or sequestration of functional plastids, conferring a metabolic advantage despite the host living far below the euphotic zone. Through a unique integration of functions largely unrecognized among "typical" eukaryotes, benthic foraminifera represent winning microeukaryotes in the face of ongoing oceanic deoxygenation.

Vancomycin MIC Distribution among Methicillin-Resistant Staphylococcus Aureus. Is Reduced Vancomycin Susceptibility Related To MIC Creep?

AIM: To determine the distribution of vancomycin MIC and the frequency of S. aureus strains with reduced vancomycin susceptibility among Methicillin-Resistant Staphylococcus aureus (MRSA) isolates. METHODS: MRSA isolates (n = 100) were tested for reduced susceptibility to vancomycin using MIC broth microdilution method (BMD), vancomycin screening agar with different vancomycin concentrations with and without casein, and Vitek 2 system. RESULTS: BMD detected (22%) vancomycin-intermediate S. aureus (VISA) and (78%) vancomycin-susceptible S. aureus (VSSA) but couldn't detect nine (Heterogeneous VISA) (hVISA) isolates (9%) with MIC ≤ 2 µg/ml that grew on screening agar 4 µg/ml or 6 µg/ml. Adding casein to vancomycin screening agar increased detection rate of VISA by 4.5%. Screening agar with 6 µg/ml vancomycin overall detection rate for VISA was 95.45%. Probable 'pre-hVISA'isolates (17%) showed growth on vancomycin screening agar 2 µg/ml with casein. Vitek 2 system failed to detect any VISA isolates. CONCLUSION: Vancomycin screening agar; 2 µg/ml and (4 and 6 µg/ml) were able to detect; probable "pre hVISA and (hVISA and VISA) isolates respectively based on their BMD MIC values. Decreased vancomycin susceptibility in MRSA isolates might be related to MIC creep. Analysis of vancomycin MIC values over longer periods is recommended to further study this phenomenon and its impact on vancomycin treatment failure.

Experimental colitis reduces microglial cell activation in the mouse brain without affecting microglial cell numbers.

Inflammatory bowel disease (IBD) patients frequently suffer from anxiety disorders and depression, indicating that altered gut-brain axis signalling during gastrointestinal inflammation is a risk factor for psychiatric disease. Microglia, immune cells of the brain, is thought to be involved in a number of mental disorders, but their role in IBD is largely unknown. In the current work, we investigated whether colitis induced by dextran sulphate sodium (DSS), a murine model of IBD, alters microglial phenotypes in the brain. We found that colitis caused a reduction of Iba-1 and CD68 immunoreactivity, microglial activation markers, in specific brain regions of the limbic system such as the medial prefrontal cortex (mPFC), while other areas remained unaffected. Flow cytometry showed an increase of monocyte-derived macrophages during colitis and gene expression analysis in the mPFC showed pronounced changes of microglial markers including cluster of differentiation 86 (CD86), tumour necrosis factor-α, nitric oxide synthase 2, CD206 and chitinase-like protein 3 consistent with both M1 and M2 activation. Taken together, these findings suggest that experimental colitis-induced inflammation is propagated to the brain altering microglial function.

A contribution to the phylogeny of agglutinating Arcellinida (Amoebozoa) based on SSU rRNA gene sequences.

Arcellinid testate amoebae include a wide variety of amoeboid organisms whose test (shell) varies in shape, composition and size. A decade ago, we initiated molecular phylogenetic analyses based on SSU rRNA gene sequences and a taxonomic revision of Arcellinida. However, many lineages within Arcellinida still lack molecular data, and the phylogeny of this group is largely incomplete. In this study, we obtained SSU rRNA gene sequences from seven taxa, of which six have agglutinated shell (Difflugia oblonga, D. labiosa, D. gramen, Mediolus corona, Netzelia wailesi, and N. tuberculata), and one has an entirely proteinaceous shell (Arcella intermedia). All species but Difflugia oblonga branched within the recently erected suborder Sphaerothecina, confirming the synapomorphic value of an oviform or discoid shell. Thus, we propose that species with an oviform or discoid shell currently classified within genus Difflugia must be transferred to other genera, thus continuing the process of taxonomic revision of genus Difflugia, the largest Arcellinida genus. We therefore transferred the current and the previously sequenced oviform Difflugia spp. to Netzelia spp., based on the shared globular/oviform shell shape and their monophyly. Another species, D. labiosa, formed an independent lineage that branched as a sister clade to Arcella spp.; based on the shell morphology and their phylogenetic position, we considered D. labiosa as incertae sedis.

High Prevalence of blaNDM-1, blaVIM, qacE, and qacEΔ1 Genes and Their Association with Decreased Susceptibility to Antibiotics and Common Hospital Biocides in Clinical Isolates of Acinetobacter baumannii.

The objective of this study was to evaluate the susceptibility of metallo-ß-lactamase (MBL)-producing Acinetobacter baumannii (A. baumannii) clinical isolates to biocides. We also determined the prevalence and correlation of efflux pump genes, class 1 integron and MBL encoding genes. In addition, blaVIM, blaNDM-1, qacE and qacEΔ1 nucleotide sequence analysis was performed and compared to sequences retrieved from GenBank at the National Center for Biotechnology Information database. A. baumannii had a resistance rate to carbapenem of 71.4% and 39.3% and was found to be a MBL producer. The minimum inhibitory concentrations (MICs) of chlorhexidine and cetrimide were higher than the recommended concentrations for disinfection in 54.5% and 77.3% of MBL-positive isolates respectively and their MICs were significantly higher among qac gene-positive isolates. Coexistence of qac genes was detected in 68.1% and 50% of the isolates with blaVIM and blaNDM-1 respectively. There was a significant correlation between the presence of qac genes and MBL-encoding blaVIM and blaNDM-1 genes. Each of the blaNDM-1, blaVIM, qacE and qacEΔ1 DNA sequences showed homology with each other and with similar sequences reported from other countries. The high incidence of Verona integron-encoded metallo-ß-lactamases (VIM) and New-Delhi-metallo-ß-lactamase (NDM) and qac genes in A.baumannii highlights emerging therapeutic challenges for being readily transferable between clinically relevant bacteria. In addition reduced susceptibility to chlorhexidine and cetrimide and the potential for cross resistance to some antibiotics necessitates the urgent need for healthcare facilities to periodically evaluate biocides efficacy, to address the issue of antiseptic resistance and to initiate a "biocidal stewardship".

Current and future perspectives on the systematics, taxonomy and nomenclature of testate amoebae.

Testate amoebae are a polyphyletic assemblage of at least three major, unrelated taxonomic groups of unicellular amoeboid eukaryotes exhibiting a test. The focus on testate amoebae in scientific research has greatly increased in the past 20 years: from an average of about 5 papers a year in the mid-1990s to the current rate of more than 50 papers published yearly. The application range of these organisms is rapidly expanding as well: from the traditional fields of environmental monitoring and paleoecology, to forensic sciences and ecotoxicology studies. These developments are nevertheless strongly dependent on reliable taxonomy and nomenclature. However, scientometric data reveal that despite an ever-increasing necessity for the use of names (the product of taxonomy), the corresponding effort has not been achieved for improving testate amoebae systematics. As a consequence, inaccurate taxonomy yields to misinterpretations in the diversity of the organisms and to potentially incorrect conclusions. These and related problems are discussed in this study, highlighting the outcome of poor taxonomic expertise in accurate classification and phylogeny of testate amoebae, and the consequences derived from it. Additionally, this study is aimed to discuss the current status of testate amoebae classification, and to present all nomenclature and taxonomic changes in higher and lower taxonomic levels of testate amoebae, as a result of recent molecular reconstructions. Finally, we conclude with a list of the needs and suggestions toward a unified and modernized taxonomy of testate amoebae.

Morphological and molecular diversification of Asian endemic Difflugia tuberspinifera (Amoebozoa, Arcellinida): a case of fast morphological evolution in protists?

Planktonic arcellinid testate amoebae exhibit a broad-range of morphological variability but it is currently unclear to what extent this variability represents phenotypic plasticity or if it is genetically determined. We investigated the morphology and phylogenetic relationships of three endemic east-asian Difflugia taxa 1) the vase-shaped D. mulanensis, 2) and a spinose and a spineless morphotypes of D. tuberspinifera using scanning electron microscopy and two ribosomal genetic markers (SSU rDNA and ITS sequences). Our phylogenetic analyses shows that all three taxa are genetically distinct and closely related to D. achlora and Netzelia oviformis. The genetic variations between the spineless and spinose morphotypes of D. tuberspinifera were low at the SSU rRNA level (0.4%), but ten times higher at the ITS level (4.5-6%). Our data suggest that the two forms of D. tuberspinifera are sufficiently differentiated in terms of morphology and genetic characteristics to constitute two separate entities and that the presence of spines does not result from phenotypic plasticity due to environmental selective pressure. However further observational and experimental data are needed to determine if these two forms constitute different biological species.

One alga to rule them all: unrelated mixotrophic testate amoebae (amoebozoa, rhizaria and stramenopiles) share the same symbiont (trebouxiophyceae).

Endosymbiosis is a central and much studied process in the evolution of eukaryotes. While plastid evolution in eukaryotic algae has been extensively studied, much less is known about the evolution of mixotrophy in amoeboid protists, which has been found in three of the five super groups of Eukaryotes. We identified the green endosymbionts in four obligate mixotrophic testate amoeba species belonging to three major eukaryotic clades, Hyalosphenia papilio and Heleopera sphagni (Amoebozoa: Arcellinida), Placocista spinosa (Rhizaria: Euglyphida), and Archerella flavum (Stramenopiles: Labyrinthulomycetes) based on rbcL (ribulose-1,5-diphosphate carboxylase/oxygenase large subunit) gene sequences. We further investigated whether there were different phylotypes of algal endosymbionts within single H. papilio cells and the degree of host-symbiont specificity by amplifying two genes: COI (mitochondrial cytochrome oxydase subunit 1) from the testate amoeba host, and rbcL from the endosymbiont. Results show that all studied endosymbionts belong to genus Chlorella sensu stricto, closely related to Paramecium bursaria Chlorella symbionts, some lichen symbionts and also several free-living algae. Most rbcL gene sequences derived from symbionts from all testate amoeba species were almost identical (at most 3 silent nucleotides difference out of 780 bp) and were assigned to a new Trebouxiophyceae taxon we named TACS (Testate Amoeba Chlorella Symbionts). This "one alga fits all mixotrophic testate amoeba" pattern suggests that photosynthetic symbionts have pre-adaptations to endosymbiosis and colonise diverse hosts from a free-living stage.

Amphitremida (poche, 1913) is a new major, ubiquitous labyrinthulomycete clade.

Micro-eukaryotic diversity is poorly documented at all taxonomic levels and the phylogenetic affiliation of many taxa - including many well-known and common organisms - remains unknown. Among these incertae sedis taxa are Archerella flavum (Loeblich and Tappan, 1961) and Amphitrema wrightianum (Archer, 1869) (Amphitremidae), two filose testate amoebae commonly found in Sphagnum peatlands. To clarify their phylogenetic position, we amplified and sequenced the SSU rRNA gene obtained from four independent DNA extractions of A. flavum and three independent DNA extractions of A. wrightianum. Our molecular data demonstrate that genera Archerella and Amphitrema form a fully supported deep-branching clade within the Labyrinthulomycetes (Stramenopiles), together with Diplophrys sp. (ATCC50360) and several environmental clones obtained from a wide range of environments. This newly described clade we named Amphitremida is diverse genetically, ecologically and physiologically. Our phylogenetic analysis suggests that osmotrophic species evolved most likely from phagotrophic ancestors and that the bothrosome, an organelle that produces cytoplasmic networks used for attachment to the substratum and to absorb nutrients from the environments, appeared lately in labyrithulomycete evolution.