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This paper highlights and honors the connectivity among protistan researchers, using my own research journey as a backdrop, with attention to the supply chain of ideas, supporters, and other influencers who helped to shape and guide my career by sharing their ideas, protocols, skills, and enthusiasm. In looking back at the journey, the supply chain in my career has also included changes in the conceptual framework for my research studies, converging with a continuous flow of ideas and support from colleagues and mentors. To illustrate the complex map of ideas and supporters, this paper will examine technological advances, paradigm shifts in ecological constructs, geographical considerations, breakthroughs in peritrich biology, and the importance of an integrated perspective as we navigate the changing realities of today's scientific challenges., (© 2024 International Society of Protistologists.)

Challengerids, phaeogromids rhizarian protists, are emblematic protists of the deep sea but are also enigmatic as they occur in very low concentrations. In previous studies, we reported on temporal changes in abundance at a near-shore mesopelagic site, but only as part of sampling of the entire microplankton assemblage, not well-suited for examining phaeogromids. Consequently, we turned to using a closing plankton net to provide material from large volumes of seawater, thus allowing for more robust estimates of concentrations and material for observations of living cells, to our knowledge the first made. Here, we report our results on the four most commonly occurring species: Challengeranium diadon, Challengereron willemoesii, Challengeria xiphodon, and Euphysetta lucani. In contrast to our previous report, we found that changes in concentrations were not related to water column stratification, and the four species roughly co-varied with time. Observations of live cells revealed that all four species deploy tentacle-like pseudopods and also very large unstructured webs of fine pseudopods. The similarities in feeding webs suggest similar prey are exploited, and the similar temporal changes in abundances suggest a common factor or factors (unknown at this time) govern their concentrations. Films of live cells are provided in Supplementary Files., (© 2023 International Society of Protistologists.)

The bulk of knowledge on marine ciliates is from shallow and/or sunlit waters. We studied ciliate diversity and distribution across epi- and mesopelagic oceanic waters, using DNA metabarcoding and phylogeny-based metrics. We analyzed sequences of the 18S rRNA gene (V4 region) from 369 samples collected at 12 depths (0-1000 m) at the Bermuda Atlantic Time-series Study site of the Sargasso Sea (North Atlantic) monthly for 3 years. The comprehensive depth and temporal resolutions analyzed led to three main findings. First, there was a gradual but significant decrease in alpha-diversity (based on Faith's phylogenetic diversity index) from surface to 1000-m waters. Second, multivariate analyses of beta-diversity (based on UniFrac distances) indicate that ciliate assemblages change significantly from photic to aphotic waters, with a switch from Oligotrichea to Oligohymenophorea prevalence. Third, phylogenetic placement of sequence variants and clade-level correlations (EPA-ng and GAPPA algorithms) show Oligotrichea, Litostomatea, Prostomatea, and Phyllopharyngea as anti-correlated with depth, while Oligohymenophorea (especially Apostomatia) have a direct relationship with depth. Two enigmatic environmental clades include either prevalent variants widely distributed in aphotic layers (the Oligohymenophorea OLIGO5) or subclades differentially distributed in photic versus aphotic waters (the Discotrichidae NASSO1). These results settle contradictory relationships between ciliate alpha-diversity and depth reported before, suggest functional changes in ciliate assemblages from photic to aphotic waters (with the prevalence of algivory and mixotrophy vs. omnivory and parasitism, respectively), and indicate that contemporary taxon distributions in the vertical profile have been strongly influenced by evolutionary processes. Integration of DNA sequences with organismal data (microscopy, functional experiments) and development of databases that link these sources of information remain as major tasks to better understand ciliate diversity, ecological roles, and evolution in the ocean., (© 2023 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Foraminifera include diverse shell-building lineages found in a wide array of aquatic habitats from the deep-sea to intertidal zones to brackish and freshwater ecosystems. Recent estimates of morphological and molecular foraminifera diversity have increased the knowledge of foraminiferal diversity, which is critical as these lineages are used as bioindicators of past and present environmental perturbation. However, a comparative analysis of foraminiferal biodiversity between their major habitats (freshwater, brackish, intertidal, and marine) is underexplored, particularly using molecular tools. Here, we present a metabarcoding survey of foraminiferal diversity across different ecosystems using newly designed foraminifera-specific primers that target the hypervariable regions of the foraminifera SSU-rRNA gene (~250-300 bp long). We tested these primer sets on four foraminifera species and then across several environments: the intertidal zone, coastal ecosystems, and freshwater vernal pools. We retrieved 655 operational taxonomic units (OTUs); the majority of which are undetermined taxa that have no closely matching sequences in the reference database. Furthermore, we identified 163 OTUs with distinct habitat preferences. Most of the observed OTUs belonged to lineages of single-chambered foraminifera, including poorly explored freshwater foraminifera which encompass a clade of Reticulomyxa-like forms. Our pilot study provides the community with an additional set of newly designed and taxon-specific primers to elucidate foraminiferal diversity across different habitats., (© 2022 International Society of Protistologists.)

Meringosphaera is an enigmatic marine protist without clear phylogenetic affiliation, but it has long been suggested to be a chrysophyte-related autotroph. Microscopy-based reports indicate that it has a worldwide distribution, but no sequence data exist so far. We obtained the first 18S rDNA sequence for M. mediterranea (identified using light and electron microscopy) from the west coast of Sweden. Observations of living cells revealed granulated axopodia and up to 6 globular photosynthesizing bodies about 2 μm in diameter, the nature of which requires further investigation. The ultrastructure of barbed undulating spine scales and patternless plate scales with a central thickening is in agreement with previous reports. Molecular phylogenetic analysis placed M. mediterranea inside the NC5 environmental clade of Centroplasthelida (Haptista) along with additional environmental sequences, together closely related to Choanocystidae. This placement is supported by similar scales in Meringosphaera and Choanocystidae. We searched the Tara Oceans 18S V9 metabarcoding dataset, which revealed four OTUs with 94.8%-98.2% similarity, with oceanic distribution similar to that based on morphological observations. The current taxonomic position and species composition of the genus are discussed. The planktonic lifestyle of M. mediterranea contradicts the view of some authors that centrohelids enter the plankton only temporarily., (© 2021 International Society of Protistologists.)

We have obtained a sequence of the 18S rRNA gene of the species Polychaos annulatum (Penard 1902) Smirnov et Goodkov 1998 using the isolation of a single nucleus from an amoeba cell. Attempts to amplify the 18S rRNA gene from the DNA of this species by conventional PCR were not successful, so we applied the whole genome amplification of the nuclear DNA followed by NGS sequencing. The 18S rRNA gene was found among the resulting contigs. The analysis unexpectedly shows that P. annulatum robustly groups within the family Hartmannellidae, but not Amoebidae. This finding warrants revision of the basic morphological criteria used to classify Euamoebida families and show that "proteus-type" amoebae may belong to other families rather than Amoebidae. This makes taxonomic assignments of such species more complex and the borders between Euamoebida families more nuanced. It is getting evident that molecular data are necessary to clarify the position of species even in this most "classical" order of naked lobose amoebae., (© 2020 International Society of Protistologists.)

Heterotrophic protists Rotosphaerida, Thaumatomonadida, and centrohelid heliozoans are among the less studied silicified protists in terms of their biogeography and ecology. These organisms inhabit fresh and brackish water, and leave behind siliceous structural elements after death that are species-specific and amenable to electron microscopic analysis. This paper is the first to present data on species richness and taxonomic structure of silica-scaled heterotrophic protists-rotosphaerids, colorless free-living thaumatomonad flagellates and centrohelid heliozoans-in the large continuous water system of Siberia connecting Lake Baikal to the Kara Sea. In the study area, electron microscopy revealed 21 centrohelid heliozoan species from the genera Raphidiophrys (1), Acanthocystis (7), Choanocystis (3), Raineriophrys (2), Raphidocystis (6), and Pterocystis (2), seven rotosphaerid species from the genera Pinaciophora (3), Turriplaca (2), Rabdiophrys (1), and Pompholyxophrys (1), and one thaumatomonad flagellate species Thaumatomastix. Two species of rotosphaerids, Rabdiophrys cf. anulifera and Pinaciophora tridentata, and two species of centrohelid heliozoans, Acanthocystis cf. tubata, and A. cf. cornuta, were found in the waters of Russia for the first time. The most widespread species in fresh water from Lake Baikal to the Lower Yenissei River were Pinaciophora fluviatilis and Raineriophrys cf. fortesca. These species disappeared from the protist assemblages of the Yenissei gulf of the Kara Sea due to higher salinity, with only three species, Acanthocystis cf. mylnikovi (at 3.12‰ salinity) and A. pectinata and Raphidocystis sp. 2 (at 8‰ salinity), found in the area. The sensitivity of silica-scaled heterotrophic protists to changes in habitat parameters suggests that these microeukaryotes could be new indicator organisms., (© 2021 International Society of Protistologists.)

The Cutosea represent a deep-branching lineage within the phylum Amoebozoa that is still relatively poorly explored. Currently, there are four cutosean representatives known - the monotypic genera Armaparvus, Idionectes, Sapocribrum, and Squamamoeba - with marked genetic distances. Idionectes vortex is the deepest-branching species and differs markedly from the other Cutosea in ecology, life history, and most importantly, in its ability to form a flagellated swarmer with an exceptional swimming mechanism. As far as we know, the other Cutosea lack flagella and rather represent small, marine amoebae with a characteristic cell coat. The present paper focuses on the amoeboid life history stage of the algivorous amoeboflagellate Idionectes vortex to provide data for a first in-depth comparison with other Cutosea and to document structural specialties. The amoeboid stage of Idionectes is mainly associated with the specific feeding process, that is, the interaction with algal prey cells and phagocytosis of protoplast material. Yet, the present data from time-lapse microscopy, cytochemical stainings, and electron microscopy demonstrate clear similarities with the other cutosean species concerning amoeboid locomotion and cell coat ultrastructure. Furthermore, Idionectes amoebae exhibit a well-developed microtubular cytoskeleton, and an unusual basal apparatus that seems to undergo marked changes during the life history of this exceptional amoebozoan., (© 2021 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Most discussions of human microbiome research have focused on bacterial investigations and findings. Our target is to understand how human eukaryotic microbiome research is developing, its potential distinctiveness, and how problems can be addressed. We start with an overview of the entire eukaryotic microbiome literature (578 papers), show tendencies in the human-based microbiome literature, and then compare the eukaryotic field to more developed human bacterial microbiome research. We are particularly concerned with problems of interpretation that are already apparent in human bacterial microbiome research (e.g. disease causality, probiotic interventions, evolutionary claims). We show where each field converges and diverges, and what this might mean for progress in human eukaryotic microbiome research. Our analysis then makes constructive suggestions for the future of the field., (© 2019 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

It has been long thought that RNA Polymerase (Pol) II transcriptional regulation does not operate in trypanosomes. However, recent reports have suggested that these organisms could regulate RNA Pol II transcription by epigenetic mechanisms. In this paper, we investigated the role of TbRRM1 in transcriptional regulation of RNA Pol II-dependent genes by focusing both in genes located in a particular polycistronic transcription unit (PTU) and in the monocistronic units of the SL-RNA genes. We showed that TbRRM1 is recruited throughout the PTU, with a higher presence on genes than intergenic regions. However, its depletion leads both to the decrease of nascent RNA and to chromatin compaction only of regions located distal to the main transcription start site. These findings suggest that TbRRM1 facilitates the RNA Pol II transcriptional elongation step by collaborating to maintain an open chromatin state in particular regions of the genome. Interestingly, the SL-RNA genes do not recruit TbRRM1 and, after TbRRM1 knockdown, nascent SL-RNAs accumulate while the chromatin state of these regions remains unchanged. Although it was previously suggested that TbRRM1 could regulate RNA Pol II-driven genes, we provide here the first experimental evidence which involves TbRRM1 to transcriptional regulation., (© 2019 International Society of Protistologists.)

In recent years, Tetrahymena species have been used as model organisms for research in a wide range of fields, highlighting the need for a fuller understanding of the taxonomy of this group. It is in this context that this paper uses living observation and silver staining methods to investigate the morphology and infraciliature of one Tetrahymena species, T. setosa (Schewiakoff 1892 Verh. Naturh. Med. Ver. Heidelb., 4:544) McCoy (1975) Acta Protozool., 14:253; the senior subjective synonym of T. setifera Holz and Corliss (1956) J. Protozool., 3:112; isolated from a freshwater pond in Harbin, north-eastern China. This organism can be distinguished from other described Tetrahymena species mainly by its single caudal cilium, which is about twice the length of the somatic ciliature. While the Harbin isolate appears similar to the population described by Holz and Corliss (1956) J. Protozool., 3:112, an improved diagnosis for T. setosa is given based on the previous descriptions and the Harbin population. In summary, this species can be recognized mainly by the combination of the following characters: body in vivo approximately 40 μm × 25 μm, 21-26 somatic kineties, one to four contractile vacuole pores associated with meridians 6-11 and a single caudal cilium. The small subunit ribosomal (SSU) rRNA gene and the cox1 gene sequences of Harbin population are also characterized in order to corroborate that the isolated species branches in phylogenetic trees as a T. setosa species. The phylogenetic analysis also indicated that sequences of populations of Tetrahymena species should be published with detailed morphological identifications., (© 2018 International Society of Protistologists.)

The traditional green algal genus Chloromonas accommodates mesophilic, cold-tolerant and cold-adapted microorganisms. In this paper, we studied a new strain isolated from a wet hummock meadow in the High Arctic. We used morphological, ultrastructural and molecular data to assess the taxonomic position and phylogenetic relationships of the new isolate. The observed morphological features generally corresponded to the cold-tolerant Chloromonas characteristics. However, ellipsoidal or wide ellipsoidal vegetative cells, a massive parietal cup-shaped chloroplast with a number of continuously connected lobes, a thick cell wall, a prominent hemispherical papilla and the anterior position of an oblong or round eyespot distinguished the alga from all previously described Chloromonas species. Analyses of rbcL and 18S rRNA genes showed that the new strain formed an independent lineage within a clade containing mesophilic and psychrotolerant Chloromonas species. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker supported a separate species identity of the new isolate. Considering the morphological and molecular differences from its relatives, a new psychrotolerant species, Chloromonas svalbardensis, is proposed. Further, our results demonstrated the paraphyletic origin of Chloromonas within Chloromonadinia with genetically, morphologically and ecologically well-defined clades. We discuss a scenario of a possible Chloromonas split and revision., (© 2018 International Society of Protistologists.)

The redescription of Opalina undulata Nie 1932, collected from the rectum of the frog Fejervarya limnocharis, is presented in this paper based on detailed morphological information and molecular data. Our results revealed that specimens collected from Diaocha Lake in late August were larger and had more nuclei than those collected from the same site in early May. We sequenced their SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA (5' end) and found that they were completely identical, which means that the two populations belonged to the same species. These facts gave us a hint that body dimension and number of nuclei are not reliable taxonomic parameters for opalinids during their life cycle. Therefore, we recommended that the specific identification of opalinids based on morphological features should be carried out during seasons except spring. Meanwhile, our molecular phylogenetic analysis confirmed the monophyly of Opalinata. Within Opalinata, Opalinea were monophyletic with all opalinid species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data., (© 2017 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

The morphology of Nyctotheroides hubeiensis (Acta Hydrobiol. Sin. 1998, 22(suppl.):187), collected from the rectum of Phelophylax nigromaculatus, is presented in this paper based on detailed morphological information and molecular data. Our phylogenetic analysis showed that N. hubeiensis fell into the Nyctotheroides clade, which was strongly supported as monophyletic and clustered as basal to the genera Nyctotherus and Clevelandella. Also, the monophyly of the Order Clevelandellida and the affinity of parasitic nyctotherids and free-living metopids were indicated in our work. The origin of clevelandellid ciliates as well as their possible evolutionary history was also discussed here; however, the analysis of more species from other vertebrate hosts (fish, reptiles) should be made before a well-supported conclusion can be drawn., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Acanthamoeba genus is divided into 20 genotypes (T1-T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near-complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

Three peritrichous ciliates, Zoothamnium arcuatum n. sp., Z. grossi n. sp., and Z. parahentscheli Sun et al., 2009, were collected from an estuary of the Taehwagang River, Korea. All these species were investigated based on live observations and silver staining, and their small subunit (SSU) rRNA gene was also sequenced. Zoothamnium arcuatum can be identified by a goblet-shaped colony, double-layered peristomial lip, and abstomally shortened row 3 of infundibular polykinety 3 (P3). Zoothamnium grossi is morphologically characterized by an alternately branched stalk with the lowest secondary stalk diverging from the main part of colony, asymmetrically bell-shaped zooids, and three short, parallel ciliary rows in P3. Phylogenetic analysis revealed that the three Zoothamnium species described in this paper clustered with other members of the family Zoothamniidae, as expected., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)