Prostomateans, as common inhabitants in diverse aquatic environments, are among the simplest ciliate lineages, and serve as trophic links in food webs. However, only a few members are well-known and thoroughly studied, and the diversity of this group remains elusive. The unique genus Plagiocampa has a long history of research, but few studies have been performed using up-to-date methods. In the present work, Plagiocampa longis Kahl, 1927 and Plagiocampa minima Kahl, 1927, collected from Chinese coastal habitats, were investigated based on microscopical observation, protargol staining, and SSU rRNA gene sequencing. Their ciliature and morphometric data as well as gene sequences are documented. Phylogenetic analyses revealed that the family Plagiocampidae is likely monophyletic and has a closer relationship with parasitic Cryptocaryon.

Haeckel's 'art book', Kunstformen der Natur, is likely familiar to most protistologists as it is probably Haeckel's best known work, and it prominently featured protists. No doubt many of us have used some of the images from it in our lectures. Most familiar are perhaps the often-reproduced images of nassularian radiolaria, but plates were also devoted to phaeodarians, acantharia, foraminifera, ciliates, diatoms, dinoflagellates and desmids. Despite the fame of Kunstformen der Natur, there are aspects of the work that have received little attention, and are of potential interest to protistologists. These include the overall importance of protists in the work, compared to other taxa, the surprisingly under appreciated role of Haeckel's collaborator, Adolph Giltsch in creating the figures, the disputed fidelity of the illustrations of protists, and Haeckel's discrete inclusion of many descriptions of new species of protists, forcing us to consider Kunstformen a scientific publication. Here these aspects, and the 20 plates featuring protists will be reviewed. The goal of this review is to familiarize protistologists with the most famous work featuring protists, and point out the many particular figures of protists we may wish to avoid showcasing, as the illustrations are questionable. A supplemental file is provided listing the currently accepted names of the protist taxa (for names found in a database), and the reasons for considering some illustrations as questionable.

Iodamoeba is a single-celled intestinal parasite, which is common in humans in certain parts of the world, and also in pigs. For the first time, we provide DNA-based evidence of goat, dromedary, fallow deer, and donkey as hosts of Iodamoeba and show that Iodamoeba-specific nucleotide sequences from these four hosts do not appear to overlap with those of humans, unlike those from pigs. We moreover show that similar strains of Iodamoeba can be found in Madagascar, Western Sahara, and Ecuador and that intra-sample diversity is typically extensive across even small fragments of DNA in both human and non-human hosts.

Mutimo cylindricus gametes have two flagella with different structures : an anterior and a posterior flagellum. Their flagellar waveforms are regulated by calcium ions through various mechanisms, however the factors involved in this regulation remain largely unknown To elucidate the molecular basis underlying the difference between the two flagella, we performed a flagellar proteomic analysis of male M. cylindricus gametes. We identified 848 proteins shared with Ectocarpus siliculosus, including 28 calcium-binding proteins. Among the EF-hand proteins, a 111 kDa protein showed predominant localization along the anterior flagellum. Immunogold localization suggested that this protein is associated with outer doublet microtubules. This is the first report to show heterogeneous localization of a calcium-binding protein between two flagellar axonemes and suggests that calcium-binding proteins are involved in the specific regulation of the anterior flagellum.

Autophagy is an intracellular degradation mechanism by which cytoplasmic materials are delivered to and degraded in the lysosome-fused autophagosome (autolysosome) and proposed to have been established at an early stage of eukaryotic evolution. Dinoflagellates harboring endosymbiotic diatoms (so-called "dinotoms"), which retain their own nuclei and mitochondria in addition to plastids, have been investigated as an intermediate toward the full integration of a eukaryotic phototroph into the host-controlled organelle (i.e., plastid) through endosymbiosis. Pioneering studies systematically evaluated the degree of host governance on several metabolic pathways in the endosymbiotic diatoms (ESDs). However, little attention has been paid to the impact of the endosymbiotic lifestyle on the autophagy operated in the ESDs. In this study, we searched for ATG3, ATG4, ATG5, ATG7, ATG8, ATG10, and ATG12, which are required for autophagosome formation, in the RNA-seq data from dinotoms Durinskia baltica and Kryptoperidinium foliaceum. We detected two evolutionally distinct sets of the ATG proteins in the dinotom species, one affiliated with the dinoflagellate homologs and the other with the diatom homologs in phylogenetic analyses. The results suggest that the ATG proteins descended from the diatom taken up by the dinoflagellate host persist for autophagosome formation and, most likely, autophagy.

The ability to distinguish between viable and non-viable protozoan parasites is central to improved human and animal health management. While conceptually simple, methods to differentiate cell viability in situ remain challenging. Amoebic gill disease, caused by Neoparamoeba perurans is a parasitic disease impacting Atlantic salmon aquaculture globally. Although commercial freshwater treatments alleviate AGD, viable amoebae remain on gills or in used treatment water. Existing PCR-based assays are able to quantify N. perurans abundance but cannot discriminate amoeba viability. We investigated the use of propidium monoazide (PMA) application, prior to real-time PCR, to distinguish between alive and dead cells. We demonstrate that 200 μM PMA can significantly reduce amplification from non-viable (isopropanol treated) cultured amoebae across at least three logs of cell concentrations. Using a serial dilution of viable and non-viable cells, we show that non-PMA PCR amplifies both viable and non-viable amoebae, while PMA exposure suppresses (but does not completely inhibit) amplification from non-viable amoebae. The effect of freshwater treatment on N. perurans viability was assessed using the PMA-PCR. Following PMA exposure, amplification from freshwater treated amoebae was reduced by approximately 94-97 %. Taken together this study demonstrates that PMA combined with traditional real-time PCR can estimate amoeba viability.

Protosteloid amoebae are slime molds characterized by simple sporocarp comprised only of a stalk and typically single spore. Recent molecular phylogenetic studies showed that protosteloid amoebae are scattered among the Amoebozoa, but most of them belongs to the Variosea. The Cavosteliida is one of the largest protosteloid group of the Variosea. We have established novel protosteloid amoeba strain YIP-63 from the fruiting body of jelly fugus Auricularia sp. The strain YIP-63 is unique to have a tiny sporocarp and amoeba comparing to the other protosteloid species, and trophic amoebae forming both round and branched shape. The molecular phylogenetic analyses based on 18S rRNA gene suggest that YIP-63 represents a novel lineage in the Cavosteliida. Therefore, we propose the new genus and new species for the strain YIP-63 as Nannostelium ampullaceum gen. et sp. nov. We provide morphological and molecular data on this novel protosteloid amoeba.

This study presents the results of a complex survey of freshwater heterotrophic euglenoids in the Czech Republic, including both literature data and own field surveys of 469 sites visited in the course of three years. The checklist includes 189 taxa in 28 genera: Anisonema (10), Astasia (26), Atraktomonas (1), Calycimonas (2), Chasmostoma (1), Dinematomonas (3), Distigma (8), Dylakosoma (1), Entosiphon (4), Euglena (1), Gyropaigne (1), Heteronema (19), Jenningsia (11), Khawkinea (1), Lepocinclis (1), Menoidium (7), Neometanema (3), Notosolenus (18), Petalomonas (40), Phacus (1), Ploeotia (2), Pseudoperanema (7), Rhabdomonas (5), Scytomonas (1), Sphenomonas (5), Teloprocta (1) Tropidocyphus (1), Urceolus (4), and 4 species of uncertain identity. In addition, a general description of habitat types in which the taxa were found and a review of the current taxonomy and nomenclature of included taxa are provided. Several taxonomic and nomenclatural novelties are proposed, based on the review of morphological features, mostly applying to the genera Notosolenus and Jenningsia.

Spirostomum is a genus of large ciliates, and its species are distributed worldwide. However, there has been limited research conducted on their geographical distribution and genomics. We obtained nine samples of ciliates from eight regions in Liaoning Province, China, and conducted a study on their geographical distribution and characteristics. Morphological and second-generation high-throughput sequencing methods were applied to identify the species, and a phylogenetic tree was established to gain a deeper understanding of the geographical distribution and evolutionary relationships of Spirostomum in Northeast China. The results identified Spirostomum yagiui and Spirostomum subtilis as a newly recorded species in Northeast China region. There are now five species of Spirostomum that have been recorded in China, and new details on the genomic characteristics of Spirostomum yagiui were provided. In addition, this study also identified the main branches of Spirostomum teres and Spirostomum minus in northern China, and provided a theoretical basis for the existence of hidden species. Spirostomum yagiui is the first species in the family Spirostomidae to have undergone mitochondrial genome sequencing.

The genomes of peridinin-containing dinoflagellate chloroplasts have a very unusual organisation. These genomes are highly fragmented and greatly reduced, with most of the usual complement of chloroplast genes relocated to the nucleus. Dinoflagellate chloroplasts highlight evolutionary changes that are found to varying extents in a number of other organelle genomes. These include the chloroplast genome of the green alga Boodlea and other Cladophorales, and the mitochondrial genomes of blood-sucking and chewing lice, the parasitic plant Rhopalocnemis phalloides, the red alga Rhodosorus marinus and other members of the Stylonematophyceae, diplonemid flagellates, and some Cnidaria. Consideration of the coding content of the remnant chloroplast genomes indicates that organelles may preferentially retain genes for proteins important in initiating assembly of complexes, and the same is largely true for mitochondria. We propose a new principle, of CO-location for COntrol of Assembly (COCOA), indicating the importance of retaining these genes in the organelle. This adds to, but does not invalidate, the existing hypotheses of the multisubunit completion principle, CO-location for Redox Regulation (CORR) and Control by Epistasy of Synthesis (CES).

Ciliates of the family Nyctotheridae (Armophorea: Clevelandellida) are frequent intestinal symbionts of various invertebrates and some poikilotherm vertebrates. Depending on the classification scheme, there are between 15 and 18 recognized genera of Nyctotheridae, the majority of which exhibit a rather uniform morphology. They have round to ellipsoidal cells with an adoral zone of membranelles that begins anteriorly in an adoral groove and continues posteriorly into the buccal cavity where it extends deep into the cell in the peristomial funnel. The taxonomy of the Nyctotheridae is primarily based on the number and location of kinetal sutures. The only known divergence from the relatively conservative nyctortherid body plan are the bizarre symbionts of Panesthiinae cockroaches, ciliates of the family Clevelandellidae, which forms a clade nested within the Nyctotheridae genus Nyctotherus. In this study we report another ciliate that diverges morphologically from the canonical Nyctotheridae body plan, and which is also found in Panesthiinae hosts. The novel ciliate Reductitherus cryptostomus n. gen., n. sp. differs from the rest of Nyctotheridae by absence of the anterior adoral groove, a shortened adoral zone completely enclosed in a notably small buccal cavity, and two strongly reduced kinetal sutures, one left anterodorsal and the other right posterodorsal.

Over the past decade, the autotrophic and heterotrophic protist Euglena gracilis (E. gracilis) has gained popularity across the studies of environmental science, biosynthesis experiments, and nutritional substitutes. The unique physiology and versatile metabolism of E. gracilis have been a recent topic of interest to many researchers who continue to understand the complexity and possibilities of using E. gracilis biomolecule production. In this review, we present a comprehensive representation of recent literature outlining the various uses of biomolecules derived from E. gracilis across the fields of natural product biosynthesis, as a nutritional substitute, and as bioremediation tools. In addition, we highlight effective strategies for altering metabolite production using abiotic stressors and growth conditions. To better understand metabolite biosynthesis and its role in E. gracilis, integrated studies involving genomics, metabolomics, and proteomics should be considered. Together, we show how the ongoing advancements in E. gracilis related research continue to broaden applications in the biosynthetic sector and highlight future works that would strengthen our understanding of overall Euglena metabolism.

This study reports a comprehensive analysis of photoautotrophic euglenids' distribution and biodiversity in 16 small water bodies of various types (including fish ponds, field ponds, rural ponds and park ponds) located in three regions of Poland: Masovia, Masuria and Pomerania during a period of three years. By employing a euglenid specific barcode marker and a curated database of V2 18S rDNA sequences it was possible to identify 97.7 % of euglenid reads at species level. A total of 152 species classified in 13 genera were identified. The number of euglenid species found in one pond varied from 40 to 102. The most common species were Euglena agilis and Euglenaria caudata, found in every analysed waterbody. The highest number of observed species belonged to Trachelomonas and Phacus. Certain species exhibited a tendency to coexist, suggesting the presence of distinct species assemblages. Among them, the most distinctive cluster was associated with water bodies located in the Masuria region, characterized also by the greatest species richness, including many very rare species: Euglenaformis chlorophoenicea, Lepocinclis autumnalis, L. marssonii, Trachelomonas eurystoma, T. manschurica, T. mucosa, T. zuberi, T. zuberi var. nepos.

The relationships of the mainly free living, obligately anaerobic ciliated protists belonging to order Metopida continue to be clarified and now comprise three families: Metopidae, Tropidoatractidae, and Apometopidae. The most species-rich genus of the Metopidae, Metopus has undergone considerable subdivision into new genera in recent years as more taxa are characterized by modern morphologic and molecular methods. The genus, Castula, was established to accommodate setae-bearing species previously assigned to Metopus: C. setosa and C. fusca, and one new species, C. flexibilis. Another new species, C. specialis, has been added since. Here we redescribe another species previously included in Metopus, using morphologic and molecular methods, and transfer it to Castula as C. strelkowi n. comb. (original combination Metopus strelkowi). We also reassess the monotypic genus, Pileometopus, which nests within the strongly supported Castula clade in 18S rRNA gene trees and conclude that it represents a morphologically divergent species of Castula.

Extrachromosomal circular DNA (eccDNA) enhances genomic plasticity, augmenting its coding and regulatory potential. Advances in high-throughput sequencing have enabled the investigation of these structural variants. Although eccDNAs have been investigated in numerous taxa, they remained understudied in euglenids. Therefore, we examined eccDNAs predicted from Illumina sequencing data of Euglena gracilis Z SAG 1224-5/25, grown under optimal photoperiod and exposed to UV irradiation. We identified approximately 1000 unique eccDNA candidates, about 20% of which were shared across conditions. We also observed a significant enrichment of mitochondrially encoded eccDNA in the UV-irradiated sample. Furthermore, we found that the heterogeneity of eccDNA was reduced in UV-exposed samples compared to cells that were grown in optimal conditions. Hence, eccDNA appears to play a role in the response to oxidative stress in Euglena, as it does in other studied organisms. In addition to contributing to the understanding of Euglena genomes, our results contribute to the validation of bioinformatics pipelines on a large, non-model genome.

The protist Euglena gracilis has various trophic modes including heterotrophy and photoheterotrophy. To investigate how cultivation mode influences metabolic regulation, the chemical composition of cellular metabolites of Euglena gracilis grown under heterotrophic and photoheterotrophic conditions was monitored from the early exponential phase to the mid-stationary phase using two different techniques, i.e, nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). The combined metabolomics approach allowed an in-depth understanding of the mechanism of photoheterotrophic and heterotrophic growth for biomolecule production. Heterotrophic conditions promoted the production of polar amino and oxygenated compounds such as proteins and polyphenol compounds, especially at the end of the exponential phase while photoheterotrophic cells enhanced the production of organoheterocyclic compounds, carbohydrates, and alkaloids.

Though endosymbioses between protists and prokaryotes are widespread, certain host lineages have received disproportionate attention what may indicate either a predisposition to such interactions or limited studies on certain protist groups due to lack of cultures. The euglenids represent one such group in spite of microscopic observations showing intracellular bacteria in some strains. Here, we perform a comprehensive molecular analysis of a previously identified endosymbiont in the Eutreptiella sp. CCMP3347 using a single cell approach and bulk culture sequencing. The genome reconstruction of this endosymbiont allowed the description of a new endosymbiont Candidatus Grellia alia sp. nov. from the family Midichloriaceae. Comparative genomics revealed a remarkably complete conjugative type IV secretion system present in three copies on the plasmid sequences of the studied endosymbiont, a feature missing in the closely related Grellia incantans. This study addresses the challenge of limited host cultures with endosymbionts by showing that the genomes of endosymbionts reconstructed from single host cells have the completeness and contiguity that matches or exceeds those coming from bulk cultures. This paves the way for further studies of endosymbionts in euglenids and other protist groups. The research also provides the opportunity to study the diversity of endosymbionts in natural populations.

Karenia longicanalis, an athecate dinoflagellate, was first described during a bloom in Victoria Harbour (Hong Kong, China). This study confirms the presence of K. longicanalis as a bloom former in the eastern Pacific Ocean. Specimens were collected in March 2019 at three sampling stations in Acapulco Bay, Mexico. Water temperature, salinity, dissolved oxygen, and pH were measured in situ at the time of sample collection. Cell morphology was examined by optical and scanning electron microscopy. A molecular analysis based on the amplification of the large subunit (LSU) rDNA region revealed that the LSU sequences formed a monophyletic group with other GenBank sequences belonging to K. longicanalis. The resulting phylogeny demonstrates that Karenia is closely related to Asterodinium, Gertia, and Shimiella. The morphology of the specimens was consistent with previous descriptions.

Although copper (Cu) is a micronutrient, the metal may be toxic if present in high concentrations in soil ecosystems and subsequently affect various organisms, ranging from microorganisms to earthworms. We performed a microcosm study with an array of Cu concentrations, with a specific focus on Cercozoa, an important protozoan group in most soil food webs. Research on Cercozoa is still scarce in terms of both diversity and ecology; hence, to explore this group in more depth, we used high-throughput sequencing to detect Cu induced community changes. Increased levels of Cu caused a shift in the cercozoan community, and we observed decreased cercozoan relative abundance across the majority of orders, families and genera. Due to their key role in soil food webs, especially as bacterial predators and providers of nutrients to plants, the reduction of cercozoan abundance and diversity may seriously affect soil functionality. Our results indicate that the increase of Cu concentrations in the soil could potentially have this effect and the consequences need exploration.

The nivicolous species of the genus Diderma are challenging to identify, and there are several competing views on their delimitation. We analyzed 102 accessions of nivicolous Diderma spp. that were sequenced for two or three unlinked genes to determine which of the current taxonomic treatments is better supported by molecular species delimitation methods. The results of a haplotype web analysis, Bayesian species delimitation under a multispecies coalescent model, and phylogenetic analyses on concatenated alignments support a splitting approach that distinguishes six taxa: Diderma alpinum, D. europaeum, D. kamchaticum, D. meyerae, D. microcarpum and D. niveum. The first two approaches also support the separation of Diderma alpinum into two species with allopatric distribution. An extended dataset of 800 specimens (mainly from Europe) that were barcoded with 18S rDNA revealed only barcode variants similar to those in the species characterized by the first data set, and showed an uneven distribution of these species in the Northern Hemisphere: Diderma microcarpum and D. alpinum were the only species found in all seven intensively sampled mountain regions. Partial 18S rDNA sequences serving as DNA barcodes provided clear signatures that allowed for unambiguous identification of the nivicolous Diderma spp., including two putative species in D. alpinum.