Anthropogenic nutrient enrichment and plant diversity loss reshape soil biodiversity, yet disentangling their individual and combined effects on key groups such as fungi and protists remains a major challenge. Here, we investigated soil microeukaryote communities using long-read amplicon rRNA gene sequencing in a temperate grassland experiment with 11 years of moderate NPK fertilization and manipulated plant diversity (1, 2, or 4 plant species). Our results indicate that fertilization generally had a stronger influence on microeukaryote communities than plant species richness. Fertilization altered the community composition of fungi and protists, with an increase in OTU richness by 20.8% and 52.7%, respectively, and shifted community dominance from fungi to protists. Plant diversity exclusively affected protists with a shift in community composition. Community changes were largely driven by increases in plant biomass (resulting from both fertilization and plant diversity), alongside higher soil phosphorus and lower soil pH, which were exclusively influenced by fertilization. Moreover, the experimental treatments exerted distinct effects on the different life strategies of fungi and protists. Fertilization enhanced fungal saprophytes (only richness), fungal animal pathogens, and protist consumers, whereas a decline in plant diversity increased phototrophic protists and decreased protist animal pathogens. Notably, fertilization and the decline in plant diversity together led to a cumulative increase in fungal plant pathogens. In conclusion, our results show that fertilization and reduced plant species richness exert distinct yet interacting effects on soil microeukaryotic communities. This highlights the need for integrated assessments of these two factors, rather than studying them in isolation, when evaluating global change effects.
Protists are an underexplored but functionally important component of aerobic-activated granular sludge under pollution stress. Using metagenomics, we profiled protistan responses to ciprofloxacin, triclosan, and Cu2+ (alone or in combination). Protists remained a stable 6.35%-7.88% of the bacterial community, and the consumers were the most abundant groups. Ciprofloxacin showed little effect on protist abundance, while Cu2+ increased protist abundance, especially consumers. Stress conditions also strengthened predominantly positive protist-bacteria associations, suggesting cross-domain interactions that may enhance community resilience. These results demonstrate that protists are key determinants in stabilizing microbial communities under multiple stressors.
Grazing disturbance strongly affects grassland ecosystem structure and function, but its impacts on soil protist communities across vertical soil profiles remain unclear in alpine grasslands. We investigated soil protist community composition, alpha diversity, and their relationships with environmental factors under four grazing intensities (no grazing, light, moderate, heavy grazing) at three depths (0-10, 10-20, 20-30 cm) on the eastern Qinghai-Tibet Plateau. Our results showed that grazing significantly altered the relative abundance of dominant protist orders, including Pyrenomonadales, Kinetoplastida, and Amoebida. The Shannon-Wiener and Pielou indices of protists decreased significantly with increasing soil depth, while grazing mainly reduced the Margalef index. Soil total nitrogen, bulk density, and pH were the dominant factors shaping soil protist community structure. Overall, soil protists were more sensitive to soil depth than grazing intensity. These findings improve our understanding of vertical protist distribution under grazing disturbance and provide a scientific basis for sustainable grassland management.
John James Wild, born Jean-Jacques Wild, was an artist and linguist. He was a member of the scientific crew of the historic Challenger Expedition (1863-1876), employed as the artist of the expedition, and secretary to the Expedition Director, Charles Wyville Thomson. During the expedition, two preliminary reports of progress and findings, containing illustrations of protists by Wild, were sent to the Royal Society in London and published in the Proceedings of the Royal Society in 1874 and 1876. Wild's figures of protists in the preliminary report of 1874 were the first illustrations of new organisms published in the scientific press. The figures of protists, and those of the second preliminary report, and an article in Nature, both in 1876, were very widely reproduced at the time, and over the years until present times, in both the scientific and popular press. Some of the figures of protists are now iconic. However, the artist who created them remains obscure as his name appeared rarely in the figure legends or plate inscriptions. Here the story of Wild is told, and his remarkable figures of protists, drawn while on board the Challenger, are shown. Some attention is given to revealing the use of his artwork by a variety of writers, including notable protistologists.
Quantifying abundances of unicellular eukaryotes (protists) remains a central challenge in microbial ecology, as methodological differences can strongly influence abundance estimates and ecological interpretation. Although molecular tools have thus far greatly improved our understanding of protists, high rRNA gene copy numbers limit quantitative inferences. Digital PCR (dPCR) has emerged as a promising tool for absolute quantification, yet its application for unicellular eukaryotes and its comparability to established cell-based methods remain insufficiently explored. Here, we develop species-specific dPCR assays for two important freshwater ciliates (Urotricha castalia and Urotricha pseudofurcata) and establish gene copy number correction factors to enable highly accurate quantitative abundance estimates. We assess assay performance using controlled laboratory experiments and apply the approach to environmental samples, directly benchmarking dPCR against catalyzed reporter deposition-FISH (CARD-FISH). Under controlled conditions, dPCR and CARD-FISH yielded comparable accuracy, with dPCR showing superior precision. In field applications, method-dependent differences emerged, reflecting both methodological constraints and biological variability. Notably, dPCR provided an overall higher sensitivity, enabling robust detection of low-abundance taxa. Our results highlight dPCR as a scalable and sensitive approach that, when combined with appropriate correction strategies, represents a significant step towards more reliable molecular quantification of protists. At the same time, differences between methods underscore the value of integrating molecular and microscopy-based approaches. We propose that combining dPCR with tools such as CARD-FISH can offer complementary insights into protist population dynamics. Such integrative frameworks provide a powerful path forward for improving abundance estimates and advancing quantitative microbial ecology.
Protists form the foundation of aquatic food webs and drive global nutrient cycles, yet distinguishing which species photosynthesize, graze or do both remains a major challenge because most are uncultivable and community surveys seldom resolve species-level traits. We developed a field grazing-scPCR framework that integrates short-term grazing assays, single-cell microscopy and 18S rRNA sequencing to link morphology, fluorescence-based trophic indicators, ingestion evidence and phylogenetic identity in 21 individually isolated protistan cells spanning freshwater to oceanic ecosystems. Using a conservative, phylogeny-informed classification, this approach confirmed constitutive mixotrophs (Cryptomonas curvata, Poterioochromonas malhamensis), identified a candidate non-constitutive mixotroph within Katablepharidaceae, and showed that prey-derived fluorescence can overestimate mixotrophy in natural assemblages. Two C. curvata isolates exhibited contrasting states, an active grazer with plastid autofluorescence and a non-grazing, aflagellate cyst retaining plastid fluorescence, highlighting the limits of single-time-point assays. Linking single-cell observations to MetaPR2 and Tara Oceans exact-match records further placed trophically characterized taxa in a broader biogeographic context. This framework advances species-level resolution of protistan trophic diversity in nature while underscoring the need to interpret fluorescence and ingestion signals in phylogenetic and ecological context.
In containing the first six-to-seven glycolytic enzymes within the organelle matrix, the glycosomes of trypanosomatid protists and their nearest relatives represent extreme forms of peroxisome specialisation. How or why such extreme peroxisomes evolved is not known. Many proteins are peroxisome targeted following recognition of a C-terminal type-1 peroxisome targeting signal (PTS1). Here, we identified in Naegleria gruberi, an amoeboflagellate distantly related to trypanosomatids, cryptic PTS1 motifs in a variety of metabolic enzymes, including in some glycolytic enzymes. These signals arise from stop-codon read-through or alternative splicing and are conserved in opportunistic pathogen N. fowleri. We show selected cryptic N. gruberi PTS1 motifs function in protein import into glycosomes of trypanosomatid Crithidia fasciculata. Further analysis revealed similar cryptic PTS1 motifs in evolutionarily diverse protists, including some from Discoba - the broad eukaryotic group to which Naegleria and trypanosomatids belong. Fragmentary data allowed only a cursory, equivocal glimpse of peroxisome biochemistry within Euglenida, the protists most closely related to trypanosomatids. Collectively, however, our data indicate Naegleria displays more versatility within its unusual metabolism than previously appreciated but moreover suggest dual protein localisation may have been used to diversify peroxisome function early in eukaryotic evolution and point towards at least two stages in glycosome evolution.
The Quaternary glaciations profoundly shaped the biogeography of plants and animals, yet their impact on microbial eukaryotes remains largely unexplored. We tested the "genetic legacy of the Quaternary" (GLQ) paradigm in terrestrial protists using Arcellinida testate amoebae diversity distribution across the Iberian Peninsula, a well-established glacial refugium. To do so, we compiled the most extensive Arcellinida metabarcoding dataset to date (ArKOI), including 615 samples from multiple continents and ecosystems. Our results showed that hotspots of intra-OTU genetic diversity align with known Iberian refugia, supporting the concept of "refugia within refugia," and displaying clear ecoregion-specific climatic niches. Patterns of spatial clustering, niche breadth, and historical demographic reconstruction revealed repeated range contractions and expansions during the Pleistocene, mirroring those observed in macro-organisms. These findings extend the GLQ paradigm to protists, highlighting shared historical and ecological processes across the eukaryotic tree of life and contributing to a unified theory of biogeographic responses to climatic change.
Mixotrophic protists capable of both photosynthesis and phagotrophy are key members of marine plankton communities. Yet, little is known about their responses to the combination of ocean acidification and warming. A marine mixotrophic chrysophyte, Ochromonas CCMP2951, was subjected to two levels of pCO2 (300 and 800 ppm, resulting in pH of 8.2 and 7.8) and temperature (21°C and 26°C) in a factorial design. Enhanced growth rates were observed in both the high CO2 and high temperature treatments, while cell size significantly decreased with temperature. Strongly decreased cellular phosphorus content led to increased N : P and C : P ratios of Ochromonas with temperature. Furthermore, warming increased grazing rates, while elevated CO2 reduced the Chl content but increased photosynthetic carbon acquisition, albeit only at low temperature. The combination of warming and elevated CO2 had antagonistic effects on the balance between autotrophic and heterotrophic carbon acquisition, keeping the net role of this mixotroph in the marine carbon cycle stable. Altogether, both the direct stimulation of growth and the indirect effects of altered stoichiometry may favor mixotrophs under future ocean conditions. However, their contribution to future carbon cycling in complex natural communities will need further study.
Microbial eukaryotes are prevalent members of host-associated and free-living microbial communities, but are routinely excluded from studies of these communities. Existing methods for eukaryote detection from whole metagenome sequencing are limited by contamination of eukaryotic reference genomes and incomplete taxonomic coverage. Our previously published tool EukDetect addressed these challenges using a curated database of universal BUSCO marker genes, but lacked validated quantitative abundance metrics and was built from a limited number of genomes. Here we present EukDetect2, incorporating a database containing 6,948 microbial eukaryotic genomes representing 6,594 unique species, 2,339 of which are newly added since EukDetect version 1, alongside quantitative metrics for estimating absolute and relative abundance of microbial eukaryotes. Using simulated data, we demonstrate accurate abundance estimation, no false positives from bacterial or host-derived reads, and equivalent or greater sensitivity and specificity than alternative taxonomic profiling tools across a range of microbial abundances and community compositions. Applying EukDetect2 across globally distributed human gut microbiome cohorts, we find that Blastocystis spp. and Dientamoeba fragilis are the most prevalent gut eukaryotes across cohorts, while host-associated fungi are consistently less prevalent than commensal protists. Blastocystis abundance is positively associated with a gut microbial community enriched for fiber-fermenting microbes and depleted for pro-inflammatory and industrialization-associated taxa. EukDetect2 provides sensitive, accurate, and quantitative metrics for investigating microbial eukaryotes from metagenomic samples.
Cellular form and function are inextricably linked. Close integration of experiment and theory in the intensive study of a suctorian ciliate reveals generalizable regulatory principles for precisely yet adaptively controlling the size and number of subcellular structures.
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As humans, we bring biases based on our own vertebrate, mammalian biology into the way we conceptualize the world. This includes how we discuss the range of concepts we reference with the term sex and the implicit assumptions that are often embedded in how we discuss life cycles and reproduction. These assumptions situate diplontic life cycles, unitary body plans, and amphigonic reproduction as normative and frame sex (meiosis and syngamy) and reproduction (the creation of new individuals) as inherently intertwined. We draw from the social science theoretical framework of master narrative theory to discuss these implicit assumptions. Master narrative theory typically is used to articulate normative expectations for humans in specific socio-cultural contexts. Here we use it to reveal normative expectations about organisms that are common in western modern science. Following this explication, we discuss frameworks and language that biologists working with modular metazoans and plants have developed to discuss complex sexual variation, addressing multi-level modularity and continuous, quantitative variation in investment into or reproductive success via male and female gametes. We consider ways these "specialized" approaches can be useful for broadly comparative work, across organisms with a range of sexual simplicity and complexity, and identify challenges to greater conceptual integration. As a step toward tools for improving conceptual integration across sexual systems and body plans, we introduce a graphical system for mapping anisogamete-production strategy across levels of modularity. We call for further conversations that build on the "Sex Across Origins" symposium at the 2026 Society for Integrative Biology conference, considering key concepts in the biology of sex and reproduction through the prism of different kinds of organisms. This practice can help break apart common oversimplifications and overgeneralizations, reveal the breadth or limits of concepts, and generate insights from the ways that concepts fail for subsets of organisms, suggesting paths forward to better understand and articulate the complexity and variation of sex and reproduction.
Complete mitochondrial genomes were sequenced and annotated from three Klossia species (Apicomplexa; Adeleorina) infecting snails in North America and Europe. Mitochondrial genomes of K. helicina (type species-NC_058857.1; 6728 bp), K. razorbacki (NC_058856.1; 6775 bp), and K. vaderbriani (OM891106.1; 6757 bp) were circular-mapping with the same gene contents and organization with 3 CDS regions (cytochrome c subunits I [COI] and III [COIII], and cytochrome b [CytB]) interspersed with 37 large and small ribosomal DNA fragments. Content and organization of mitochondrial genomes of Klossia species were shared with Legerella nova (monoxenous adeleorinid protist infecting pill millipedes) except for the reversed coding direction of one rDNA fragment; Haemogregarina species (heteroxenous adeleorinid protists with leech definitive hosts) mitochondrial genomes shared similar genome organization but lacked 2 rDNA fragments found in those monoxenous genera. In contrast, mitochondrial genomes of the Klossia species differed markedly in organization from Klossiella equi, a monoxenous adeleorinid protist parasite of vertebrates. Pairwise comparisons of the CDS regions of the Klossia species to other adeleorinid protists confirmed the close relatedness of monoxenous L. nova (Legerellidae) to monoxenous Klossia species (Adeleidae). The diversity of mitochondrial genomes of monoxenous adeleorinid protists infecting invertebrates has only begun to be explored.
Soil acidification is a widespread consequence of intensive agriculture and represents a major abiotic stress affecting plant performance, nutrient availability, and ecosystem functioning. Long-term tea (Camellia sinensis) plantations provide model systems of chronic acidification, where sustained low pH imposes strong environmental filtering on soil microbial communities. Although microbial responses to acidification have been extensively studied, research has focused predominantly on bacteria and fungi, leaving other key functional groups, particularly protists, largely overlooked. Here, we synthesize current knowledge on microbial communities in acidified soils and highlight trophic interactions, especially protist-mediated regulation, as a potentially critical but underexplored dimension linking abiotic stress to plant-soil processes. We propose that soil acidification may not only filter microbial community composition but also reshape trophic interactions. Based on evidence from other soil systems, protist-mediated trophic interactions could influence nutrient cycling, pathogen suppression, and ultimately plant responses under stress conditions. Integrating environmental filtering with trophic perspectives provides a conceptual framework for understanding microbiome dynamics in acidified soils. However, direct evidence linking protist-mediated trophic regulation to ecosystem functioning and plant performance in tea plantation soils remains limited and requires experimental validation. We further suggest that these systems provide unique opportunities to investigate how abiotic constraints and biotic interactions jointly shape plant performance. Addressing this gap is essential for advancing predictive understanding of plant-microbiome interactions under ongoing environmental change.
The gut microbiome of termites plays a crucial role in lignocellulose degradation and nutrient recycling. This study presents the first metagenomic characterization of the gut microbiota in two lower termite species, Anacanthotermes ahngerianus and Anacanthotermes turkestanicus, collected from distinct ecological habitats. In Uzbekistan, the first lives in building a mound in nature in the West part while the second mainly lives in contact with human constructions in the East part without building a proper mound. Both species showed similar bacterial dominance (~53%) in their guts but A. ahngerianus exhibited higher overall microbial diversity (Shannon index: 4.046 vs. 3.363; Simpson's index: 0.927 vs. 0.776). Moreover, both termite species showed differences in microbial profiles, including bacterial taxa and eukaryotic groups relevant to lower-termite gut symbiosis. Protist-associated eukaryotic reads were retained because flagellated protists are essential symbionts of lower termites, whereas unexpected non-protist eukaryotic assignments were interpreted cautiously and were not used as evidence of functional gut symbionts or host adaptation. Functional profiling revealed enrichment of pathways related to carbohydrate metabolism, amino acid transport, and energy production in both species. However, A. turkestanicus exhibited stronger bacterial dominance associated with lignocellulose degradation and nitrogen cycling, while A. ahngerianus maintained a more balanced representation of bacteria, fungi, and viruses. These findings suggest that species identity and ecological habits may be associated with differences in gut microbiome structure and predicted functional potential.
Ribosomal RNA (rRNA) genes serve as foundational markers for microbial eukaryotic diversity assessment, yet their responses to environmental stressors remain underexplored. This study examined the effects of sublethal oxytetracycline and CuCl2 on phenotypic and ribotypic traits in the ciliated protists Paramecium bursaria and Euplotes vannus using single-cell quantitative PCR and high-throughput sequencing of 18S rDNA/rRNA. We found that both pollutants inhibited growth, enlarged cell volume, and elevated per-cell rDNA and rRNA copy numbers, but oxytetracycline selectively elevated rRNA: rDNA ratios. These effects increased the variance around established allometric scaling relationships between rDNA copy number and cell volume, without significantly altering the scaling slope. Intragenomic polymorphisms also increased, with the amplicon sequence variant (ASV) number of rRNA transcripts exceeding that of rDNA, and these elevated polymorphisms persisted post-stress relief. Re-analysis of field 18S metabarcoding data from CuCl2-polluted marine biofilms revealed dose-dependent increases in the number of ASVs per operational taxonomic unit (OTU, defined at a cutoff of 97% sequence identity) in many protistan groups, suggesting that copper-induced mutagenic pressure on ribosomal RNA genes may prevail across diverse protistan taxa. Our findings imply that: (1) when interpreting ASV-based microbial diversity patterns, the potential for ASV richness inflation and elevated per-cell rDNA and rRNA abundances should be recognized as a likely consequence of exposure to environmental pollutants; (2) there is a potential for using the ASV-to-OTU number ratio in assessing environmental stress; and (3) metabarcoding of rRNA likely introduces more artificial ASVs than targeting rDNA. A combination of double metabarcoding of both rDNA and rRNA to identify active microbial members is recommended.
Afforestation is vital for combating land degradation and restoring ecosystem multifunctions in karst desertification regions. However, the connections between multiple characteristics of microbial communities and soil multifunctionality (SMF) remain poorly understood in the different plantations within subtropical karst regions. Here, using the single function, and averaging method, we calculated the SMF of the two typical types of plantation (i.e., Pinus armandii Franch. PAF, and Alnus nepalensis, ANP) based on 20 variables associated with soil nutrient provisioning and cycling, soil water retention, microbial growth efficiency, and soil organic matter decomposition. The results showed that the carbon and nitrogen cycling indexes, soil water regulation capacity, and SMF were all significantly greater in the PAF compared to the ANP. The fungal and protistan communities, along with multitrophic microbial β-diversity, positively influenced SMF, while the complexity and stability of bacterial-fungal-protistan networks have a negative impact. Remarkably, changes in the abundance of sensitive taxa within Module 3 adversely impacted the SMF, with sensitive taxa of fungi (e.g., Trichocladium) and protists (e.g., Spongospora, Hartmannellidae_X and Trinematidae_X), as opposed to bacteria, playing a crucial role in driving the SMF. Furthermore, carbon and nitrogen sources, exchangeable Mg, and soil water content were determined to be pivotal forcing factors of SMF. Overall, these findings offer a framework for predicting ecosystem functions in subtropical karst forests and valuable insights for managing and conserving fragile ecosystems globally.
Hydrological regimes of river ecosystems are increasingly altered by climate change and other human-induced stressors, yet how these alterations affect aquatic metacommunity assembly remains unclear. Here, we investigated seasonal zooplankton dynamics in the subtropical Chishui River in southern China by sampling 80 sites across the dry and wet seasons. Using the morphology-based approach, we identified 146 zooplankton taxa, including protists, rotifers, cladocerans, and copepods, with protists accounting for nearly half of the total taxa and being dominated by ciliates. By integrating physicochemical, climatic, and land-use data with neutral community models and co-occurrence network analysis, we found a clear seasonal shift in assembly mechanisms: stochastic dispersal dominated during the hydrologically connected wet season, whereas deterministic environmental filtering prevailed during the dry season. Unexpectedly, higher α-diversity was associated with lower cohesion-based network stability, suggesting that seasonal community assembly processes may influence the diversity-cohesion relationship. During the dry season, deterministic assembly processes were more prominent, and community composition was associated with nutrient-related variables. These findings suggest that maintaining natural flow variability and reducing nutrient inputs may help sustain habitat heterogeneity and seasonal metacommunity dynamics in river ecosystems.