Microbial communities entering wastewater treatment plants (WWTPs) through untreated sewage represent an important interface between human, environmental, and treatment-associated microbiomes, yet our understanding of their biogeography remains poorly resolved, particularly for microbial eukaryotes. Using shotgun metagenomic time-series data from influent samples of seven WWTPs across a European latitudinal gradient, we analyzed the taxonomic composition and dynamics of bacteria, protists, fungi, and microscopic metazoa. Influent community composition varied with geographic location and season, with a pronounced north-south divergence driven by dominant taxa and stronger seasonal shifts observed at higher latitudes. Cross-domain associations were pervasive, suggesting that co-varying bacterial and eukaryotic components structure the incoming microbial pool. Our findings provide a pan-European baseline for whole-microbiome wastewater surveillance and highlight that influent communities differ regionally and seasonally. These patterns may be relevant for downstream treatment-stage microbiomes, but direct effects on reactor community assembly and treatment performance require targeted sampling across treatment stages.
Haplosporidians are protistan parasites of aquatic invertebrates. Described here is the first report of the zebra mussel (Dreissena polymorpha) parasite Haplosporidium raabei in the River Thames, London, UK. The prevalence of this protist in the upper tidal Thames, Richmond, was higher than previously reported in other studies from the UK and mainland Europe of infected D. polymorpha populations, with molecular detection of H. raabei DNA in all D. polymorpha specimens collected. Also reported is the molecular detection (DNA and RNA) of H. raabei in other invasive Thames bivalves: the Asian clam (Corbicula fluminea) and quagga mussel (Dreissena bugensis). The molecular sequence of a closely related, but genetically distinct H. raabei-like sequence associated with D. polymorpha is also reported. Two new PCR assays are presented for the specific and sensitive detection of H. raabei and H. raabei-like sequences, enabling independent screening of these two lineages and also confirming the molecular presence of H. raabei in native unionid mussel species. A diversity of novel haplosporidian lineages associated with other invasive bivalves C. fluminea and D. bugensis and associated eDNA from the River Thames is also reported, demonstrating haplosporidian diversity associated with freshwater bivalves that is yet to be explored.
The genus Goussia Labbé 1896 represents a polyphyletic assemblage of piscine coccidia whose systematics remain poorly resolved due to morphological convergence and limited genomic data. Here we describe Goussia sairae n. sp. from the liver of Pacific saury (Cololabis saira) in the Northwestern Pacific, characterized through integrated morphological, histopathological, and molecular analyses. Long-read amplicon sequencing of single-oocyst isolates enabled the first complete mitochondrial genome assembly for a marine Goussia species, revealing two co-existing mitogenome genotypes (99.3% identity) within individual oocysts-the first robust evidence consistent with intra-oocyst mitochondrial heterogeneity in the Eimeriidae. Phylogenetic reconstructions based on nuclear 18S ribosomal RNA and concatenated mitochondrial protein-coding genes robustly supported the polyphyly of Goussia sensu lato, recovering five major lineages. A time-calibrated Bayesian phylogeny dated the marine-freshwater divergence to the Early Jurassic (median 197 million years ago; 95% HPD: 124-275 Mya), congruent with the ancient divergence of their respective teleost host groups. These findings support re-evaluation of the current generic boundaries of Goussia and suggest that the hyper-divergent Epicellular lineage may warrant recognition as a distinct family basal to the Eimeriorina.
Sandy beaches are dynamic ecosystems where terrestrial and marine organisms coexist, resulting in diverse communities where microeukaryotes (protists, microalgae, and meiofauna) are key drivers. Understanding their diversity patterns and responses to stressors is essential for elucidating ecological processes, yet information on this subject remains limited. Herein, microeukaryotic diversity in a sandy beach in Acapulco, Guerrero, was analyzed via ITS1 metabarcoding assessing community structure changes following hurricane Otis. Overall, the number of ASVs decreased substantially from 260 before the hurricane to 93 after. Groups like Porifera, Platyhelminthes, Rotifera, Amoeboza, and Choanoflagellata disappeared after the hurricane. Ochrophyta, Cnidaria, Ctenophora, and metazoans in general decreased in richness and abundance, whereas Ciliophora proliferated, particularly toward the supralittoral zone. ASVs turnover varied among zones; middle and lower coastal zones lost over 50% ASVs, and the supralittoral zone almost doubled its richness. These changes in community structure were related to total phosphorus and total nitrogen concentrations, suggesting possible bottom-up effects where nutrients available influence microbial resources for heterotrophic microeukaryotes. The observed patterns reflect high sensitivity of microeukaryotes to high-impact natural disturbances and suggest low recovery potential within the first 6 months following the event. Whether recovery occurs over longer interannual scales remains to be evaluated.
Labyrinthulomycetes are a class of fungus-like heterotrophic protists from the Stramenopiles lineage, recognized for their ecological role as decomposers and contributors to nutrient cycling. They colonize various substrates, from seaweed to terrestrial environments, utilizing ectoplasmic networks for nutrient absorption. This study characterized a novel Labyrinthula strain associated with the marine diatom Biddulphia. Phylogenetic analysis of the full-length 18S rRNA gene positioned this strain as a new species, Labyrinthula merlionensis sp. nov. Scanning electron and light microscopy observations revealed bi-flagellated zoospores and spindle-shaped vegetative cells with ectoplasmic networks. Time-series observations of the interactions between L. merlionensis and Biddulphia were categorized into different phases: establishment, infection, and aggregation. Scanning electron and confocal microscopy observations during the infection phase established the use of ectoplasmic nets to target the marginal ridge regions between diatoms, and the detection of labyrinthulid cells within diatom frustules. These findings enhance the understanding of the diversity, morphology, and ecological roles of Labyrinthulomycetes, particularly their intra- and extra-cellular interactions with diatom hosts.
Although it had been reported that the translation product of the functional aquaporin gene, aqp1, is localized to the contractile vacuole complex of Paramecium multimicronucleatum, the molecules that pass through this channel protein had not been identified. In the present study, we introduced and expressed this protein in Xenopus oocytes and identified the molecules that pass through this channel protein using a swelling assay. The aqp1 mRNA injected into Xenopus oocytes was found to be sufficiently expressed and functional within the oocytes. As a result, P. multimicronucleatum AQP1 (PmAQP1) allowed water to pass through at a relatively slow rate of approximately 50 × 10-4cm/s, a rate comparable to that of aquaporins present in the contractile vacuoles of other protists. This water permeability was almost completely inhibited by tetraethylammonium, an orthodox aquaporin inhibitor, and phloretin, an aquaglyceroporin inhibitor. PmAQP1 also had the ability to pass glycerol or urea through, but the rates of these processes could not be accurately measured in this study. These findings demonstrate that PmAQP1 is an atypical multifunctional channel, suggesting that the contractile vacuole of free-living protists plays a crucial role not only in osmoregulation but also in the clearance of metabolic wastes.
The dinoflagellate order Prorocentrales comprises two genera: Prorocentrum, which has been extensively studied, and the little-known Mesoporos. We established a monoclonal culture of Mesoporos perforatus from Long Island Sound, USA. The high variability in cell size (12-27 μm) and outline, together with irregular chloroplast morphology, suggests that previously described congeneric species may represent synonyms. The principal generic diagnostic character, commonly referred to as a pore, is not a true perforation but a conical depression located at the center of the theca; its function remains unknown. Cell morphology (cell size and shape, thecal ornamentation, and short apical spine) suggests affinities with P. cordatum and related species. However, a first detailed study of the periflagellar structure reveals differences such as platelet 8 separating platelets 2 and 3, a feature unreported in Prorocentrum species. Our first report of molecular data (SSU, LSU rDNA, and ITS regions) places M. perforatus as a species nested within Prorocentrum sensu stricto, with P. nux as the closest relative. Accordingly, we consider Mesoporos a synonym of Prorocentrum, and this species should be referred to as Prorocentrum perforatum (Gran) Krachmalny 1993, despite the absence of a true thecal perforation.
Cryptocaryon irritans is a lethal parasitic ciliate of marine fishes whose tomont stage is marked by repeated nuclear division and the morphogenesis of infective theronts, both dependent on dynamic microtubule reorganization. To investigate cytoskeleton-associated regulation during this proliferative process, we cloned and characterized the γ-tubulin gene (Ci-TUBG) and γ-tubulin complex protein 3 gene (Ci-TUBGCP3) from C. irritans. Sequence analysis showed that Ci-TUBG contains a canonical dual tubulin domain architecture and is highly conserved relative to homologs from Ichthyophthirius multifiliis and Tetrahymena thermophila. Structural modeling and molecular docking suggested a stable putative interaction interface between Ci-TUBG and Ci-TUBGCP3. Spatiotemporal expression analysis and immunofluorescence revealed stage-dependent upregulation and pronounced perinuclear enrichment of Ci-TUBG during tomont division, with persistent perinuclear localization in theronts. RNA interference showed that Ci-TUBG knockdown markedly reduced tomont hatching rate, decreased theront yield, and caused defects in theront morphogenesis accompanied by abnormal nuclear development. These findings indicate that Ci-TUBG is essential for tomont division and theront morphogenesis in C. irritans and provide a basis for understanding cytoskeleton-associated developmental regulation in this parasitic ciliate.
The nassophorean ciliates are a key group for understanding morphological diversification, but their phylogenetic relationships remain elusive. Using morphological characteristics, molecular data, and phylogenetic analyses, we attempt to cast light on species relationships within the order Microthoracida Jankowski, 1967. The present multifaceted approach reveals that (1) Leptopharynx costatus costatus Mermod, 1914 exhibits polymorphism, occurring in a large morph (35-55 × 25-40 μm) with a narrow oral basket and an intermediate mean of 228 basal bodies, supporting basal body count as a key diagnostic character; (2) comprehensive morphological and corresponding molecular information for Pseudomicrothorax dubius Maupas, 1883 was provided for the first time; (3) comparisons among different populations and morphs of L. costatus costatus and P. dubius show that the former is more morphologically variable, while the latter exhibits higher molecular variability; (4) SSU rDNA sequences provided in this work cluster with the sequences of conspecific populations, supporting the conclusion that both the families Microthoracidae Wrześniowski, 1870 (containing Leptopharynx Mermod, 1914) and the family Pseudomicrothoracidae Jankowski, 1967 (containing Pseudomicrothorax Mermod, 1914) are monophyletic.
Although species diversity has been extensively studied in anaerobic ciliates, their full diversity is incompletely understood. More research is required to gain further insights into their diversity. In this study, two new metopid ciliates, namely, Bothrostoma oryzaforme nov. sp. and Bothrostoma turgidoryzaforme nov. sp., isolated from the sediments of freshwater ponds in South Korea, were investigated using live observations and protargol impregnation. Bothrostoma oryzaforme nov. sp. is characterized by an elongated ellipsoidal shape, an anterior end with a rounded snout, and a posterior end that is either rounded or truncated. Cortical granules are arranged in 4-5 short, oblique rows; perizonal stripe even-type. Bothrostoma turgidoryzaforme nov. sp. is distinguished from its congeners by its small body size, body shape broadly ellipsoidal to oval with a rounded snout, and posterior end rounded. Cortical granules are arranged in 5-7 short, oblique rows; perizonal stripe 4 + 1 type. SSU rDNA sequence-based phylogeny revealed the evolutionary positions of the two new species, further supporting their novelty.
Giardia lamblia, a protozoan parasite that causes diarrhea in humans, contains two centrins implicated in cellular morphology and cell division. To identify proteins interacting with each G. lamblia centrin (GlCent), we performed a yeast two-hybrid assay. The coding sequences of the two centrins, GlCent1 (GL50803_6744) and GlCent2 (GL50803_104685), were cloned into pGBKT7 as bait constructs. A G. lamblia cDNA library, driven by pGADT7, was transformed into yeast strains expressing each bait protein. Initial screening on selective media yielded 17 and 23 candidate clones for GlCent1 and GlCent2, respectively. After retransformation and interaction strength analysis using growth assays, 10 GlCent1-interacting clones and 5 GlCent2-interacting clones were selected. Their interactions were validated using co-immunoprecipitation and immunofluorescence assays in transgenic Giardia cells co-expressing hemagglutinin-tagged centrins and myc-tagged candidate proteins. The results showed that ubiquitin protein ligase 1 (GL50803_9779) and preimplantation protein 3 (GL50803_3417) were GlCent1-binding proteins, whereas two hypothetical proteins (GL50803_4239 and _27739) interacted with GlCent2. Proteins interacting with the two centrins transiently co-localized during cell division. These findings suggest that GlCent1 and GlCent2 might participate in cell division by associating with distinct partner proteins in Giardia.
Research on anaerobic ciliates has intensified over the past decades, with Armophorea being most studied, but members of Armophorida remaining relatively understudied. Previously, the genus Sulfonecta Jankowski, 1978 was monotypic, with S. uniserialis (Levander, 1894) Jankowski, 1978 as the type species. Our morphological and molecular investigation of two Korean populations of freshwater caenomorphid ciliates identified S. uniserialis from Jeju and a new species, S. multiserialis n. sp., isolated from Ulsan. The new species is characterized by one to three macronuclei and a pronounced expansion of the posterior region, with posterior kineties (PK) comprising 7-11 rows, each row possessing more than 25 dikinetids. We consider populations previously reported by Schmall (1976) and Foissner et al. (1992) as S. uniserialis to be synonymous with the new species. By contrast, S. uniserialis has a more slender posterior region bearing only 3 or 4 PK. The separation of Sulfonecta multiserialis n. sp. and S. uniserialis is also supported by clear phylogenetic divergence. Their SSU rRNA gene sequences differ by 22-34 nucleotides, reinforcing recognition of two distinct species within Sulfonecta.
Goniomonads are representatives of Cryptista. They are morphologically and genetically diverse, comprising nine genera described so far. 18S rDNA data has revealed at least one more monophyletic genus-level lineage, as well as novel species within currently described genera. The molecular diversity of goniomonads is not fully investigated, and addition of novel sequences can be crucial for understanding the evolution and diversity of goniomonads and for providing a basis for taxonomic descriptions. In this study, we establish new cultured strains of goniomonads and describe the new genus and species Ebisugoniomonas hispanica n. g. et n. sp., and two novel species within Poseidogoniomonas: P. posteriocircus n. sp. and P. zopfkaesiformis n. sp. We report a range of observations on the cellular features of goniomonads, most importantly, the periplast plate arrangement patterns. We update the 18S rDNA phylogeny of goniomonads by adding sequences from novel isolates and environmental DNA studies indicating highly divergent lineages.
The class Karyorelictea is characterized by simplified ciliary patterns and the inability of somatic nuclei to divide. Despite its importance for understanding ciliate life history, research in this group remains scarce and focused on species from the Northern Hemisphere. Here, we generated and analyzed sequences of SSU rDNA of six species of Karyorelictea collected in the coastal region of southern Brazil. Four of these species (morphologically assigned to Tracheloraphis) comprised a novel, well-supported clade within the family Trachelocercidae. Moreover, we observed extensive non-monophyly of genera Trachelocerca and Tracheloraphis, highlighting the need for a taxonomic revision of these taxa.
In Tetrahymena, autophagy appears to be involved in the integrity of the mitochondria which supply energy for active ciliary movement on the cell surface, the maintenance of various structures such as cilia, and cell shape changes associated with starvation. In this organism, which is evolutionarily distant from yeast and animal cells, the mechanisms controlling autophagy remain largely unknown. mTOR is a major regulatory factor that suppresses autophagy induction when cells are under nutrient-rich conditions. Previous studies suggest that T. thermophila contains two mTOR orthologs, but lacks the canonical TORC1 subunits, while retaining TORC2-associated components. To determine whether mTOR controls autophagy in T. thermophila, we examined Torin1, an ATP-competitive inhibitor that blocks mTOR kinase activity regardless of complex composition. It markedly triggered ATG8 puncta formation and mitochondrial degradation under nutrient-rich conditions. In contrast, rapamycin, a TORC1-specific inhibitor widely used to induce autophagy, did not affect cell growth or autophagy. These findings demonstrate that mTOR functions as a negative regulator of autophagy in T. thermophila, likely through a rapamycin-insensitive TORC2 or noncanonical complex. Our results highlight lineage-specific divergence in mTOR signaling architecture and expand our understanding of the diversity of autophagy regulation across eukaryotes.
Soil recovery after contamination relies on the surviving microbiota to reconstruct microbial food webs. The ciliate Colpoda aspera and Brevundimonas sp., Rhizobium sp1, Rhizobium sp2 (gram-negative), Bacillus sp1, Bacillus sp2, and Microbacterium sp.(gram-positive) remain active after pulses of light petroleum contamination. We used phase contrast microscopy to assess excystation time and cyst production by C. aspera after placing 65 cysts in petri dishes with 0.5% LNA medium and adding 100 μL of a 5.2 × 108 cell bacterial inoculum from contaminated soil. C. aspera excystation was monitored by triplicate every hour for 72 h. Excystation occurred faster and resulted in a higher number of cysts when fed with gram-negative bacteria, particularly Rhizobium sp2 and Brevundimonas sp. In contrast, C. aspera cysts formed only two daughter cells when fed with Microbacterium sp., reducing the quantity and speed of reproduction. This reproductive behavior may allow other predatory ciliates to coexist when Microbacterium sp. becomes prevalent in a soil hotspot. Ciliates' feeding preferences and nutritional value are required to understand resource partitioning and understanding these interactions can inform strategies to promote resilient microbial communities and accelerate recovery after contamination.
Accumulating evidence suggests that the Last Eukaryotic Common Ancestor (LECA) resembled contemporary excavates, a non-monophyletic group of deep branching flagellates. Here we explore the functional ecology of distantly related deep-branching "excavate-like" flagellates (in Alveolata and Provora) that share with many excavates a vane-bearing posterior flagellum beating in association with a ventral groove. This arrangement is key to generating a feeding current in the bacterivorous typical excavates. However, the four species examined here are motile "hunters" that randomly encounter eukaryotic prey. Initial prey attachment may be facilitated by the force generated by the vaned flagellum but eventually by firing extrusomes. Propulsion is driven by the two flagella that are directed backwards. Despite minor differences, the propulsion and hunting strategies are similar between these two deep-branching groups and distinctly different from those found in typical excavates. While the vane-and-groove morphology may help prey attachment, simulations show that it seems energetically disadvantageous to propulsion. Thus, its overall role, origin, and phylogenetic implications remain unclear. This study adds to our knowledge of the functional ecological range of living vane- and groove-bearing flagellates. Given their significance to understanding early eukaryote evolutionary history, this may in turn shed light on the deep history of eukaryote life.
Cockroaches are known reservoirs for diverse bacterial microbiomes. However, comprehensive analyses of the eukaryotic communities within cockroaches remain limited. In this study, we selected three long-term laboratory-reared cockroach species (Blattella germanica, Periplaneta fuliginosa, and Periplaneta japonica) and performed metabarcoding of the 18S rRNA V9 region using the iSeq 100 platform. The nematode Blatticola blattae was identified in B. germanica, and Leidynema appendiculata was found in both P. fuliginosa and P. japonica. The amplicon sequence variant (ASV) of Nyctotherus (Ciliophora) was detected in all three species of cockroach, while Entamoeba sp. was detected in P. fuliginosa and P. japonica. Compared with the other two species, B. germanica exhibited a higher prevalence of the fungus Nephridiophaga. Our findings showed that laboratory-reared cockroaches belonging to different species harbored distinct commensal and parasitic eukaryotic taxa. While most ASVs were of reduced clinical concern for humans as opposed to studies using wild-caught specimens, the detection of commensals, arthropod parasites and potential human pathogens illustrates the complexity of the cockroach-associated eukaryome and the influence of host identity. Our study emphasizes the importance of host-specificity in the eukaryotic community of laboratory-reared cockroach models.
Most known halophilic and halotolerant eukaryotes belong to the taxon Heterolobosea sensu lato. Within this group, the Tulamoebidae-Aurem clade comprises exclusively halophiles, supporting the hypothesis that this clade represents an adaptive radiation of halophilic eukaryotes. In this study, we describe a novel amoeboflagellate, strain TA, isolated from a sample with a salinity level of 200 practical salinity units (PSU). Morphological and molecular analyses indicate that strain TA belongs to the Heterolobosea, forming a sister lineage to Aurem hypersalina and clustering with Tulamoebidae. The amoeboid stage exhibits a monopodial limax form with eruptive movement and numerous inclusions, whereas the flagellate form differs distinctly from that of A. hypersalina, exhibiting homodynamic flagella beating. Strain TA grows optimally at 100-150 PSU. We designate it as Halosymphonia sogumizoara gen. nov., et sp. nov., and expand the Tulamoebidae by including Halosymphonia together with Aurem. Based on our qualitative estimation, H. sogumizoara displayed a steady increase in intracellular Na+ concentration with increasing salinity (100-200 PSU), while K+ levels remained relatively stable, suggesting a primarily salt-out strategy. Therefore, Tulamoebidae represents a core halophilic eukaryotic clade for comparative evolutionary and physiological studies with halophilic prokaryotes.
