An international group of 49 scientists from 12 countries assembled at Brock University, St. Catharines, Ontario, Canada, from June 22–27, 2025, for the 11th International Symposium on Testate Amoebae (ISTA-11 Niagara). The meeting, jointly organized by the International Society of Testate Amoebae Research (ISTAR) and the Canadian Association of Palynologists (CAP), marked a significant milestone as the first ISTA meeting held in North America. Participation included 19 students, underscoring strong engagement from early-career researchers. The largest national delegation was from Canada (17 participants), followed by China, Switzerland, and the United States (six participants each). The scientific program comprised 41 oral and 12 poster presentations, reflecting the continued growth and diversification of research on testate amoebae.
Trachelopyllidae Kent, 1881 is a family of predatory litostomatean ciliates distinguished by a three-rowed dorsal brush composed of two dikinetidal rows and one monokinetid row, and one or more types of organic epicortical scales called lepidosomes. To date, the taxonomy of the nine genera comprising the family has been based, almost exclusively, on the types of scales and their combinations together with cell morphometry. Six of the nine genera are monotypic. To date, molecular data has been available from only four species representing two genera. With the aim of broadening the molecular characterization of the Trachelophyllidae, we present new rRNA cistron sequences of three trachelophyllid species from Idaho, USA, identified by scanning electron microscopy and representing three genera of the family not yet sequenced, and we describe a novel species representing a new genus from the Czech Republic. The results confirm the extraordinarily conserved nature of the 18S rRNA gene and ITS2 regions in the Trachelophyllidae and demonstrate the discordance between lepidosome-based taxonomy and the rRNA cistron-based molecular phylogeny in this group. Additionally, we consider ciliary ultrastructural features unique to members of the Trachelophyllidae and Lingulotrichidae (suborder Trachelophyllina Grain, 1994).
Centrohelid heliozoans are non-motile, single-celled, heterotrophic protists with radiating filose pseudopodia used to capture food prey, that are common components of soil, marine, and freshwater environments. Centrohelids are characterized by a cell covering consisting of overlapping siliceous components referred to as scales that serve as the primary means to distinguish between genera and species. Given the widespread occurrence of heliozoans in aquatic habitats, and the fact that the siliceous scales can fossilize, it is surprising that these organisms have not been previously reported from the fossil record. A rich assemblage of exquisitely preserved fossil centrohelids, including 15 species from six genera, is reported from an early Eocene freshwater locality situated near the Arctic Circle in Canada. Of these, six are described as new species, Pterocystis giraffensis sp. nov., Quendis spatha sp. nov., Choanocystis bulla sp. nov., Raphidocystis reticula sp. nov., Raphidocystis eocenensis sp. nov., and Raphidocystis kanowa sp. nov. The morphologies of the fossil heliozoan scales were remarkably similar to modern congeners, highlighting evolutionary stasis with respect to scale structure over 48 Ma. This is the first account of heliozoans from the fossil record, and supports the idea that the origin of this clade is older than the Eocene.
We present an updated checklist of testate amoebae (TA) in China covering TA records in the literature of the past ∼100 years. In total 363 TA species/subspecies in 53 genera have been included in the current Chinese checklist. The updated Chinese checklist includes five recently discovered TA species (Netzelia mulanensis, Pentagonia zhangduensis, Pontigulasia pentangulostoma, Cornutheca jiuhuensis, and Planhoogenraadia wuchanica) and 24 TA species/subspecies that have been added as new records for China. TA taxonomy and classification in the updated Chinese checklist are based on current knowledge derived from morphological, physiological, and molecular (ribosomal and mitochondrial DNA sequence data, genomics) studies. Corresponding changes in the updated Chinese checklist are outlined and the taxonomy and diversity of (Chinese) TA as well as their biogeography, ecology, and conservation are discussed. As all kinds of TA studies are eventually based on a reliable taxonomy and nomenclature, we are confident that our updated Chinese checklist will be of great value for TA researchers not only in China, but also worldwide.
Developea is a poorly studied group of flagellated protists, with only seven species known to date. It is closely related to parasitic oomycetes, hyphochytriomycetes, Pirsoniales, and photosynthetic ochrophytes (e.g., diatoms and brown algae), altogether forming the large clade Gyrista within supergroup Stramenopiles. Due to their deep phylogenetic position and phagotrophic feeding mode, developeans might have preserved ancestral characteristics shared with related large and important sister groups. Despite their cosmopolitan distribution, only few environmental 18S rRNA gene sequences related to Developea are known. Here we describe 12 new strains which represent eight new species and two new genera, as well as the previously described species Developayella elegans. We provide feeding experiments on diverse eukaryotic prey, including red algae, diatoms, and heterotrophic flagellates. The ability of the new developean species to successfully consume red algae represents missing piece of the previously postulated developean-like phagoheterotrophic model for the symbiotic ancestor of photosynthetic stramenopiles. Three species, including D. elegans, are omnivorous, i.e. able to survive on either eukaryotic or prokaryotic prey. Finally, we observe new and rare morphological features for Developea, such as facultative multiflagellated life stages, cysts and self-aggregation. These features might have been present in the ancestor of Stramenopiles.
The morphology, reproduction, molecular phylogeny and chemical physiology of a new genus and species of thraustochytrid, Caledochytrium aldermanii gen. et sp. nov is described here. Caledochytrium aldermanii has, amongst its means of reproduction, a novel method not previously reported, in which a mature cell vacuolates and produces secondary cell(s) within the vacuole. Daughter cells are released by rupture of the mother cell. The daughter cell may already contain a tertiary cell within it. Caledochytrium aldermanii displays a range of dispersal mechanisms from sporangia, including unflagellated aplanospores moving on ectoplasmic nets, and ovoid spores with paired flagellae typical of thraustochytrids. In media cultures, pyriform spores with flagellae are also produced, as well as round, flagellated cells resembling gametes. In pollen cultures, some sporangia release an amoeba, which produces two aplanospores, which then divide further. The development of residual elements as thraustochytrid cells die is also described. The plasticity of growth and reproductive strategies in these thraustochytrids is discussed, together with their pathogenicity, which suggests the organism is an environmental saprotroph and an opportunist pathogen in the presence of stressed animal hosts. Initial investigations of compounds of commercial importance produced by C. aldermanii are also described.
The genus Ceratium comprises freshwater dinoflagellates with worldwide distribution, some of which are rapidly expanding their non-native range and altering community dynamics. Understanding their genetic diversity and population structure is essential for assessing dispersal mechanisms and geographic distribution patterns. Using complete and partial sequences of the 18S rRNA gene, we reconstructed a phylogenetic tree that revealed high genetic variability within the genus Ceratium and identified nine well-differentiated clades with distinct geographic signals. Haplotype network analysis further resolved two main clusters: one comprising sequences of C. furcoides and another formed almost exclusively by C. hirundinella. While C. hirundinella showed greater sequence dispersion consistent with higher diversity and population structuring, C. furcoides sequences clustered tightly around a central node with few mutational steps, a pattern indicative of recent expansion from ancestral lineages distributed across multiple geographic regions. Together, these results provide novel insights into the evolutionary relationships and global spread of Ceratium, supporting a scenario of recent range expansion in C. furcoides contrasting with the stronger historical structuring observed in C. hirundinella.
Free-living amoebae (FLA) are key components of aquatic and terrestrial ecosystems, but their diversity and distribution in central-western Brazil remain poorly characterized. Some FLA are pathogenic and may harbor bacteria of public health relevance. We aimed to identify and to characterize the FLA biodiversity in the Cuiabá River basin and to infer their pathogenic potential. Water samples were collected at eight sites across two hydrological periods, and physicochemical and microbiological parameters were quantified. FLA were isolated on non-nutrient agar seeded with heat-inactivated Escherichia coli and identified by using morphology and molecular markers. We obtained 39 isolates (six genera and 14 species); Acanthamoeba (51.3%), Vannella (23.1%), and Vermamoeba (12.8%) were predominated. Only V. vermiformis was detected during both the high-water (when four exclusive species were detected) and low-water (when nine exclusive species were detected) periods. Seventeen FLA strains (n = 35) killed zebrafish larvae (Danio rerio); 11 of these strains (64.7%) were Acanthamoeba species, and the remaining strains belonged to the genera Flamella, Naegleria, Ptolemeba, Vannella, and Vermamoeba. Thermo- and osmotolerance did not correlate with zebrafish larval mortality. These findings expand current knowledge of FLA biodiversity in Brazil and reinforce that integrated FLA monitoring in the Cuiabá River basin is needed.
Rapid urbanization has significantly influenced urban aquatic ecosystems, with impacts manifested primarily in phytoplankton communities. However, the mechanisms driving the spatiotemporal dynamics of phytoplankton functional group (FGs) dynamics within megacities remain limited. In this study, Chaoyang District in Beijing was selected as the research site, the spatial-temporal patterns of physicochemical parameters, phytoplankton density, and FGs across 17 sites in summer and autumn were investigated. 116 species spanning 7 phyla and 57 genera were identified. Phytoplankton density peaked in autumn (4.7 × 107 cells L-1) versus summer (3.5 × 107 cells L-1), driven by Bacillariophyta dominance (58.5-61.9%). Synedra sp. and Microcystis sp. were perennial dominants (dominance: 0.4-0.9). Nutrient gradients governed FG succession: COD and TP peaked in summer (34.7 and 0.5 mg L-1), while inorganic nitrogen surged in autumn (TN: 3.4 mg L-1, NH4+-N: 1.4 mg L-1) due to temperature-mediated shifts in nitrogen cycling. The Redundancy analysis and partial least squares path modeling suggested that NH4+-N was the primary driver of FG dynamics (β = 0.6, p < 0.01), with water temperature indirectly modulating communities via nutrient cycling. This study presents an FGs-based framework to guide water use management and ecological resilience in rapidly developing cities.
Dinoflagellates are unicellular and generally marine protists and have been used as a model for the study of the biochemical bases of daily rhythms. Rhythms can result from changes in gene expression, and in the dinoflagellate Lingulodinium polyedra this occurs at a translational rather than a transcriptional level. However, the implication of other post-transcriptional mechanisms has not been previously examined. We report here that the abundance of phosphorylated proteins can also be rhythmic over the course of a diurnal cycle in L. polyedra. LC-MS/MS analyses of enriched phosphopeptide preparations identified 2485 unique peptides of which 348 showed significant rhythms in abundance over a 24-h light-dark cycle as judged by JTK_Cycle. The abundance of these rhythmic peptides typically showed peaks at either midday and midnight, and intriguingly, some peptides showed two peaks of abundance. Many of the phosphopeptides were found to be derived from proteins with functions related to RNA/DNA binding, metabolism, and phosphatase/kinase activities. The sequence motif surrounding the phosphosite was used to predict the kinase involved, and the kinases CK2, CDK9, and CDK4 appeared responsible for phosphorylation of most of the rhythmic phosphorylated peptides.
Immunophilins are a conserved family of proteins present in all organisms, including bacteria, fungi, plants, and animals. Immunophilins are sub-classified into three subfamilies: cyclophilins, FK-506 binding proteins, and parvulins, and are involved in essential cellular functions, especially protein folding. Despite their broad biological relevance, little is known about immunophilins in Phytomyxea, a group of obligate biotrophic pathogens of oomycetes, diatoms, brown algae, and plants. In this study, we analysed the immunophilins in Maullinia ectocarpii that infects brown algae, and compared them to the immunophilins of the plant pathogens Plasmodiophora brassicae and Spongospora subterranea. Through protein domain, structural, and phylogenetic analyses, we identified and characterized immunophilins. Also, the expression of selected immunophilins in M. ectocarpii was validated throughout the full life cycle. Our results reveal a conserved nature of immunophilins across phytomyxid lineages infecting evolutionarily distinct hosts. We found a strong conservation of functional domains and protein structure in selected immunophilins in both plant- and algae-associated pathogens. This conservation suggests that immunophilins play fundamental roles in the biology of phytomyxids, but also a potentially conserved role of these proteins in host adaptation nd infection strategies, offering insights into pathogen-host interactions.
Pioneering phylogenetic studies recovered the sister relationship between two distinct types of DNA polymerase (DNAP)—one is nucleus-localized Polθ with a pan-eukaryotic distribution, and the other is mitochondrion-localized PolIA found exclusively in Euglenozoa. Nevertheless, previous phylogenetic analyses considered Polθ sequences from a highly restricted set of eukaryotes, leaving the precise evolutionary trajectory of PolIA unsettled. In this study, we conducted phylogenetic analyses using a large collection of Polθ sequences from diverse eukaryotes to examine the evolutionary relationship between Polθ and PolIA more rigorously than previously reported. We recovered the maximum likelihood tree in which the clade of PolIA was nested within the clade of Polθ. In the radiation of Polθ sequences, the PolIA clade branched specifically with one of the two types of Polθ identified in euglenozoans. These results suggest that a gene duplication of one of the two Polθ types in Euglenozoa produced PolIA specific to the members of this phylum. We also propose, based on the putative distributions of the two Polθ types and PolIA, the vertical inheritance of the three DNAPs from the ancestral euglenozoan to its descendants with multiple losses of one of the two Polθ types.
Dinoflagellate algae are a diverse group of single-celled eukaryotes, often living in marine environments. The majority of species are entirely free-living, but many can become symbionts with corals, jellyfish and other marine organisms. With rising sea temperatures, the function of the dinoflagellate photosynthetic machinery, and the redox state of the photosynthetic electron transport chain are impaired. This photosynthetic impairment is likely to be an important cause of coral bleaching. In the chloroplasts of plants and many algae, disturbance of the chloroplast redox state can be in part alleviated by the Plastid Terminal Oxidase protein (PTOX). Here, we made use of our newly developed genetic modification tools in the free-living dinoflagellate species Amphidinium carterae, which is found in both in temperate and tropical waters. We test if the introduction of PTOX to the chloroplast would allow A. carterae to withstand temperature stress. We find that the expression of the PTOX gene caused a lethal phenotype. Genetic engineering of dinoflagellate algae has long been problematic, and the ability to express heterologous proteins represents a significant advance in the long-term quest to engineer a heat-tolerant dinoflagellate.
The genus Stylocephalus Ellis, 1912 (Apicomplexa: Eugregarinorida) exhibits wide distribution and morphological diversity; however, species delimitation has traditionally relied on light microscopy. In the present study, a septate gregarine parasitising Gonocephalum depressum from West Bengal, India, is investigated using DIC microscopy, SEM, and SSU rDNA sequencing. Trophozoites (446.98-596.15 μm) are elongated, bearing a distinctive stupa-shaped epimerite proper with a cylindrical diamerite and basal tumidus; epicytic folds (3-4 folds/μm) extend along the trophozoite except the epimerite proper. Gamonts (562.50-856.21 μm) are elongated with fold densities ranging from 3-4/μm to 4-4.5/μm. Associations are frontal. Gametocysts are roughly orbicular (178.51-219.73 μm), papillated, and dehisce by simple rupture. Oocysts (9.78-12.61 × 8.10-10.40 μm) are obovoid and smooth. Phylogenetic analysis of SSU rDNA places the species within Stylocephaloidea, where it forms a strongly supported clade (PP = 1; BP = 100) with congeners, confirming Stylocephalus epistupaformis n. sp. as a distinct species within Stylocephalus. The present study bridges a critical gap in septate gregarine research by providing the first integrative account combining light microscopy, SEM, and molecular phylogeny for a Stylocephalus species.
Blastocystis is a highly prevalent protist infecting both humans and a wide range of animal hosts. Despite its global distribution and frequent association with gastrointestinal symptoms infections, its pathogenicity remains controversial. In this study, we report the de novo genome assembly of Blastocystis subtype 1 strain AMC (ST1 AMC) and subtype 8 strain AMC (ST8 AMC) generated from Illumina paired-end reads. The haploid genome sizes were approximately 15 Mbp and 13.9 Mbp, with N50 values of 11.2 Kbp and 19.8 Kbp, and encoded 6603 and 5579 predicted genes, respectively. Comparative genomic analyses with previously annotated genome from ST1 strain NandII, ST4 strain WR1 and ST7 strain B revealed conserved gene synteny among subtypes, supported the putative pathogenic potential of Blastocystis, and highlighted the expansion of specific gene families previously described. In addition, we reported a high and variable abundance of transposable elements across Blastocystis genomes, suggesting a dynamic and plastic genome architecture that may contribute to genomic diversification and adaptation.
Spontaneous alternation behavior (SAB) is a robust paradigm to investigate short-term spatial memory across diverse taxa. While extensively studied in animals, its presence in unicellular aneural organisms remains poorly understood. Here, we tested SAB in the slime mold Physarum polycephalum using 3D-printed T-mazes with forced turns at varying distances (3 mm, 7 mm, and 14 mm), as well as in a double-turn design. A total of 1274 plasmodia from a clonal line were examined under controlled laboratory conditions. Our results reveal significant alternation behavior only in the short-distance maze (3 mm), independent of turn direction. Neither medium nor long distances, nor the double-turn de-sign, yielded significant effects after correction for multiple testing. These findings suggest that SAB in Physarum depends on spatial scale, with decision-making localized to the active moving front of the plasmodium. It is yet unclear if the observed behavior is induced by the topography of the used mazes or, as in other organisms, a result of memory. Further study on possible mechanisms guiding this behavior are required.
Dinoflagellate algae are an extremely important group of eukaryotic algae, found primarily in marine environments. They are responsible for a significant proportion of global primary productivity. Whilst some species are essential symbionts in coral reefs, others form toxic blooms and ‘red tides’, causing mass fish mortality. Understanding the basic biology of these organisms is therefore important not only for studying ecology and the environment, but also for food safety. Despite the fact that genetic engineering tools are at the cornerstone of modern biological research, the ability of researchers to carry out genetic modification in dinoflagellate algae is limited. Here, we examine the challenges facing molecular research in dinoflagellate algae, showing which genetic transformation techniques have worked and which have been less successful. We suggest that further research into nuclear gene expression in these enigmatic algae would greatly aid genetic engineering endeavours.
Bioluminescence is a striking feature of many dinoflagellates, yet the origin of the luciferin substrate that underlies light emission remains unresolved. Previous studies have noted that luciferin is structurally similar to chlorophyll and related catabolites, but its presence in heterotrophs is puzzling, as such organisms have no need to produce chlorophyll. Hypothetical and sometimes conflicting views on several luciferin biosynthesis reactions have been proposed, but a conclusive template for the pathway is missing. Here, we integrate existing evidence into a model for luciferin biosynthesis based on three testable hypotheses. First, we posit that phototrophic and heterotrophic dinoflagellates are capable of de novo luciferin synthesis through a plastid-derived pathway. Secondly, we surmise that luciferin is derived from a specific pathway for chlorophyll degradation that includes the structurally similar pyropheophorbide a. Finally, we revisit the role of P630 as a likely biosynthetic precursor rather than a mere oxidation artefact. We then outline experimental strategies to test these hypotheses within the broader framework of tetrapyrrole metabolism. Resolving luciferin biosynthesis will elucidate not only the biochemical, spatial, and regulatory underpinning of bioluminescence but also illuminate its evolutionary origins and how ancestral metabolic pathways can be retooled for novel cellular functions.
Protein transport between the cytoplasm and the nucleus is a fundamental process for the survival and proper functioning of all eukaryotic cells. This transport is regulated by adaptor proteins that recognize nuclear localization signals (NLSs) present in essential nuclear proteins. The data compiled in this review provide an overview of the classification and diversity of NLSs across various organisms. As in all eukaryotes, NLS-mediated transport is essential for Leishmania spp. For this parasite, there is evidence that NLS-mediated transport is essential for its physiology, allowing key proteins to perform crucial functions in maintaining intrinsic nuclear processes. Beyond classical nuclear proteins, this review indicates that some Leishmania proteases also contain NLSs, suggesting that these enzymes may have multifaceted roles: acting within the parasite itself and functioning as a strategy by the parasite to modulate the nuclear dynamics of the host cell. NLS-tagged metalloproteases of the GP63 family are known to interact with host nuclear components and inactivate critical transcription factors, thereby modulating cellular responses and promoting parasite survival. This function persists during the parasite's differentiation from the promastigote to the amastigote form, highlighting a continuous and sophisticated survival strategy. However, this mechanism still requires experimental confirmation. A deeper understanding of these processes offers promising perspectives for the development of novel therapeutic approaches against leishmaniasis.
