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BACKGROUND: Protists are diverse single-celled eukaryotes found in various habitats. They exhibit a wide range of forms and functions, representing a significant portion of the eukaryotic tree of life, which also includes animals, plants, and fungi. Due to their high sensitivity to environmental changes, these organisms are widely used as biological indicators of organic pollution. METHODS AND RESULTS: We investigated the molecular diversity of ciliate protists at seven strategic points along the Sapucaí River (Itajubá, Minas Gerais State, Brazil), to assess the impact of urban pollution on the richness, abundance, and diversity indexes of these communities. For each sampling point, values of physicochemical parameters were also recorded. DNA sequences were obtained by high-throughput sequencing (HTS) and analyzed using the V4 18S-rRNA molecular marker, employing the DNA metabarcoding method. We recorded 125 ciliate taxonomic units (OTUs), with nearly 80% corresponding to the classes Spirotrichea, Oligohymenophorea, and Litostomatea. At the genus level, 54 OTUs (43.2%) were identified, spanning 28 genera. CONCLUSIONS: The composition of ciliates varied significantly along the river's course, from upstream to downstream of Itajubá city. Samples collected from the urban area displayed the lowest richness and diversity, corroborating the influence of the pollution gradient on these communities. The physicochemical parameters showed little variation among the samples and were not linked to the observed changes in ciliate communities, revealing that these organisms are strongly affected by environmental changes and respond more sensitively to these disturbances than physicochemical parameters, emphasizing their potential as bioindicators.

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In this study, we compared the efficacy of geometric morphometric techniques, including outlines and landmark-based approaches, to support the differentiation of Trichodina bellottii from three co-occurring killifish species. Both methods were able to differentiate trichodinids from different host species. However, discriminat analyses and MANOVA results based on landmarks had greater accuracy possibly because these analyses only provide information on certain points defined by the researcher, while the analyses based on outlines take into account points with less taxonomic information.

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Several hundred ciliate species live in animals' guts as a part of their microbiome. Among them, Muniziella cunhai (Trichostomatia, Pycnotrichidae), the largest described ciliate, is found exclusively associated with Hydrochoerus hydrochaeris (capybara), the largest known rodent reaching up to 90 kg. Here, we present the sequence, structural and functional annotation of this giant microeukaryote macronuclear genome and discuss its phylogenetic placement. The 85 Mb genome is highly AT rich (GC content 25.71 %) and encodes a total of 11 397 protein-coding genes, of which 2793 could have their functions predicted with automated functional assignments. Functional annotation showed that M. cunhai can digest recalcitrant structural carbohydrates, non-structural carbohydrates, and microbial cell walls, suggesting a role in diet metabolization and in microbial population control in the capybara's intestine. Moreover, the phylogenetic placement of M. cunhai provides insights on the origins of gigantism in the subclass Trichostomatia.

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Since their discovery, rumen ciliates of domestic cattle have been reported from various geographic locations. However, until now there is only one taxonomic inventory of ciliates associated with Brazilian cattle. The present study aimed to assess the community composition, relative abundance, richness, and density of rumen ciliates in Brazilian cattle, whose feeding diets were supplemented with crescent urea levels. Across all treatments analyzed, one subclass, two orders, three families, 11 genera, and 31 species of ciliates were identified. The ciliate community composition and species richness varied among the four treatments used. However, the total ciliate density was not affected by the experimental diets. We described a new entodiniid morphotype, Entodinium caudatum m. orbonuclearis, and recorded Oligoisotricha bubali for the second time in Brazil.

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Changes in the biodiversity of aquatic environments over time and space due to human activities are a topic of theoretical and conservational interest in ecology. Thus, variation in taxonomic beta diversity of the planktonic ciliates community was investigated along a temporal and spatial gradient in two subsystems of a Neotropical floodplain, one impacted by dams (Paraná) and the other free of them along its course (Ivinhema). For the spatial analysis, the Paraná subsystem did not show a significant decrease in beta diversity, presenting a pattern like that observed for the Ivinhema subsystem. Therefore, biotic homogenization was not observed for the ciliate's community downstream of the dams. It was noted that there was a fluctuation in the relevance of the components of beta diversity, regardless of the subsystem analyzed. For the temporal analysis there was a significant change in species composition from the first to the last year investigated, essentially for the subsystem impacted by dams, and that this was determined mainly by species loss. Although spatial beta diversity remained high without a clear process of biotic homogenization, dams promoted remarkable changes in ciliate species composition over the years mainly by continuous loss of species.

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An integral analysis of the acute and chronic toxicity, bioaccumulation, sites of entry, and distribution of four trace metals: copper, iron, lead, and nickel, and the non-trace metal mercury were performed in the ciliate Paramecium caudatum. Mercury was the fastest metal accumulated, and the most toxic. The sensitivity of Paramecium caudatum to the five metals tested (Cu, Fe, Hg, Ni, and Zn) falls in the range of other ciliate species. We observed similarities between the toxicity of the five metals to the ciliate P. caudatum with the rotifer Euchlanis dilatata: (a) Mercury was the most toxic metal in terms of acute and body burdens. (b) Acute values were very similar in both species, Hg as the most toxic and Fe as the less toxic, (c) the vacuole/ingestion chronic tests were more sensitive than growth inhibition chronic tests. These analyses would ideally help generate safer guidelines for protecting aquatic biota.

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The presence of colonial and solitary ciliated peritrichous protozoa was determined in a Sequencing Batch Reactor system filled with tezontle, a volcanic rock, economic, and abundant material that can be found in some parts of the world, like Mexico. The presence of these protozoa was related to the removal efficiencies of organic matter. Also, two novel staining techniques are proposed for staining both colonial and solitary peritrichous protozoa. The results show that tezontle promotes the growth of solitary and colonial ciliated peritrichous protozoa, which, once identified, could be used as indicators of the efficiency of the wastewater treatment process. Additionally, the staining techniques established in the current study allowed the precise observation of protozoan nuclei. They can represent a useful complementary methodology for identifying protozoan species present in water treatment processes, along with the already existing identification techniques. The number and variety of protozoa found in the system may be considered potential bioindicators of water quality during biological treatments.

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Trichodina centrostrigeata Basson, Van As et Paperna, 1983 from Oreochromis mossambicus (Peters) and O. niloticus (Linnaeus) from different host populations from Argentina, Mexico and South Africa was reviewed. Although T. centrostrigeata has a distinct denticle structure that makes morphological taxonomic inferences uncomplicated, variation of the denticles within and among individuals and populations were still observed. While traditional taxonomy of mobilines is heavily reliant on morphometrics, and recently even more so on molecular analysis, this paper proposes the use of geometric morphometry, specifically elliptical Fourier analysis, to address morphological conflicts that arise when comparing different populations. By applying this technique, combined with traditional taxonomy, it was found that T. centrostrigeata in this study can be grouped into two separate morphotypes, the first (type a) from aquaculture farms in Argentina and Mexico and the second (type b) from a natural habitat in Glen Alpine Dam, South Africa. This study supports the validity of geometric morphometry as an additional technique to distinguish not only between species but also evolutionary plasticity of the same species from different localities and habitats.

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Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.

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The family Ophryoscolecidae currently comprises 225 species of trichostomatid ciliates, subdivided into three subfamilies (Entodiniinae, Diplodiniinae, and Ophryoscolecinae). The last taxonomic review of the family was performed 55 years ago, but recent morphological and molecular studies indicate the need for a profound review of the systematics of this taxon, since its current taxonomy is insufficient to organize the diversity of the group. Here, we briefly review the systematics of the family Ophryoscolecidae based on information recovered from the literature and new morphological and molecular data. We add four new 18S rDNA sequences of ophryoscolecids to molecular databases, which contributed to improving the comprehension of intrafamily relationships within this group. Finally, we discuss some systematic problems and suggest approaches to resolve such inconsistencies in the future.

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There is a significant gap in research and knowledge on the diversity and distribution of Chilean ciliates. To tackle these issues, we used cultures and protargol preparations to describe the ciliates present in poorly explored areas. At these sites, we identified 45 ciliate morphospecies, 35 of which represent unprecedent records to Chile. Then, we brought together our records with literature data to construct a species checklist. This checklist summarises 132 years of data and describes the identity, habitat and distribution of 207 species, including 15 species potentially endemic to Chile. This checklist is far from complete: a diversity estimate suggests that at least two-thirds of the ciliate species occurring in Chile have yet to be described. The checklist is dominated by freshwater taxa because ciliates from marine, brackish and terrestrial environments have rarely been investigated in Chile. Finally, after controlling for sampling artefacts, we found that ciliates exhibit a bell-shaped latitudinal diversity gradient in Chile. This peculiar biogeographical pattern is common in Chile. Plants, animals and testate amoebae also exhibit a bell-shaped latitudinal diversity gradient in Chile. This finding suggests that the historical contingencies that drove the biogeography of the Chilean biota also shaped ciliate biogeography.

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Copemetopus Villeneuve-Brachon, 1940 is a rare, poorly known sapropelic ciliate genus composed of only two valid nominal species. Over time, Copemetopus was taxonomically assigned to Heterotrichea and Armophorea classes, but its phylogenetic affinities remained unknown. Until the present study, there were no molecular data available for Copemetopus representatives. Here, we present the 18S and 28S-rDNA sequences and the phylogenetic position of Copemetopus verae sp. nov., as well as its detailed morphological description based on live observations, protargol impregnation, and scanning electron microscopy. Transmission electron micrographs of the type species C. subsalsus Villeneuve-Brachon, 1940 reveal new morphological traits and a unique somatic ciliature pattern of Copemetopus, composed by short segments of dikinetids with one or two supplementary kinetosomes. The phylogenetic trees recovered Copemetopus as the sister group of the genus Protocruzia, both constituting early-divergent lineages that split first from a common ancestor of Intramacronucleta. Morphological and molecular evidence suggest that Copemetopus is neither a heterotrichean nor an armophorean ciliate, but a distinct clade related to Protocruzia.

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Ancestral adaptations to tropical-like climates drive most multicellular biogeography and macroecology. Observational studies suggest that this niche conservatism could also be shaping unicellular biogeography and macroecology, although evidence is limited to Acidobacteria and testate amoebae. We tracked the phylogenetic signal of this niche conservatism in far related and functionally contrasted groups of common soil protists (Bacillariophyta, Cercomonadida, Ciliophora, Euglyphida and Kinetoplastida) along a humid but increasingly cold elevational gradient in Switzerland. Protist diversity decreased, and the size of the geographic ranges of taxa increased with elevation and associated decreasing temperature (climate), which is consistent with a macroecological pattern known as the Rapoport effect. Bacillariophyta exhibited phylogenetically overdispersed communities assembled by competitive exclusion of closely related taxa with shared (conserved) niches. By contrast, Cercomonadida, Ciliophora, Euglyphida and Kinetoplastida exhibited phylogenetically clustered communities assembled by habitat filtering, revealing the coexistence of closely related taxa with shared (conserved) adaptations to cope with the humid but temperate to cold climate of the study site. Phylobetadiversity revealed that soil protists exhibit a strong phylogenetic turnover among elevational sites, suggesting that most taxa have evolutionary constraints that prevent them from colonizing the colder and higher sites of the elevation gradient. Our results suggest that evolutionary constraints determine how soil protists colonize climates departing from warm and humid conditions. We posit that these evolutionary constraints are linked to an ancestral adaptation to tropical-like climates, which limits their survival in exceedingly cold sites. This niche conservatism possibly drives their biogeography and macroecology along latitudinal and altitudinal climatic gradients.

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Spirostomum is a widely distributed heterotrichean genus composed of well-known species with described ecology and phylogenetic affinities. The morphological classification of Spirostomum species is mostly based on the body size/shape, number of cortical granule rows and macronuclear characteristics. These features along with molecular phylogenies based on ribosomal genes divide the genus into two phylogroups, one including species with a compact macronucleus, and another including species with a moniliform macronucleus. Here, we present our observations on atypical Spirostomum specimens with unusually two distinct macronuclei and shortened adoral zone of membranelles. These atypical forms appeared in the cultures of S. minus and S. yagiui, sampled at different sites in South America (Chile and Brazil) and associated with unrelated substrate types. Morphological observations of living and stained cells, 18S rRNA gene analyses, and a thorough investigation of the literature suggest that the atypical phenotype may be a result of uncommon pathways during the conjugative process. Thus, we demonstrate that studies of ciliate natural populations and their morphological variations, especially from undersampled biogeographical regions, can reveal the boundaries of widely used morphological characters for Spirostomum taxonomy and species identification.

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Eight genera are included in the family Zoothamniidae, and at the present the data of the epibiotic species on crustaceans is not updated. Thus, the main goal of the present work is to provide the checklist of zoothamniids worldwide reported as associated to several groups of crustaceans, including some data of their geographic distribution, and also to provide the complete list of species of this peritrich group. We obtained all available data of family Zoothamniidae and checked their taxonomic status. Then we provided the complete list of species including those reported as epibionts on crustacean hosts. We obtained 156 species included in eight genera of Zoothamniidae, being 85 species recorded as symbionts on 86 species of hosts. Genus Zoothamnium contained the higher number (56) of epibiotic species on crustaceans, and genus Pseudohaplocaulus has not been reported for any crustacean. The reviewed data highlights the concentration of records in Europe, and stands out the need to amplify sampling and studies in the Southern Hemisphere. The main groups of crustaceans infested by zoothamniids are the amphipods, isopods and decapods.

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Tintinnids are loricated ciliates found in coastal and oceanic waters that play a key role in the transference of energy to highest trophic levels. The purpose of this article is intended to integrate the available information about the taxonomy and distribution of Colombian tintinnids. An annotated distributional checklist based on published data is presented. One hundred and seven valid species of tintinnids, belonging to 12 families and 33 genera were listed. The genera Tintinnopsis and Eutinntinnus presented the highest number of species. Eighty-three species were recorded in the Caribbean basin and 51 in the Pacific, with a higher number in coastal waters.

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We aimed to investigate spatial and temporal scales, abundance, and factors that structure the communities of protozoans in a tropical urban stream. Methods: Samples of water for analysis of biological communities (testate amoebae, ciliates and bacteria) and limnological variables were taken in the Mandacaru stream located in the Conservation Unit of Parque do Cinquentenário, in the city of Maringá, Paraná State, Brazil, in two hydrological periods (dry and rainy). We calculated the Water Quality Index (WQI) composed by nine parameters of water quality (dissolved oxygen, thermotolerant coliforms, pH, BOD, water temperature, total nitrogen, total phosphorous, turbidity, and total solids), Trophic State Index (TSI) for phosphorous and we used a Redundancy Analysis (RDA) to verify the influence of environmental variables in the protozoan community. Results: The WQI showed that water quality was considered good in some points and considered bad in other points. The TSI for phosphorus classified the stream as mesotrophic in the majority of sampled sites points (mean between 53.09 and 58.35). We identified 19 taxa of testate amoebae, belonging to six families, being Difflugiidae, Centropyxidae, and Arcellidae those with more species and 71 infrageneric taxa of ciliates, distributed in 12 orders being Peniculida the most representative order, followed by Euplotida. According to RDA analysis, samples of the dry period were characterized by higher values of bacteria density and concentrations of chlorophyll-a, total phosphorous, and total nitrogen. Ciliates and testate amoebae presented higher abundance values in some of the months characterized by higher precipitation and in conditions of higher system productivity. Conclusion: we may conclude that the Mandacaru stream, although strongly influenced by anthropic action, still presents an acceptable water quality. Lastly, we emphasize that protists abundance was strongly influenced by system productivity. This was evidenced by elevated protozoan densities where there was higher primary and bacterial productivity. Thus, these organisms must be considered in studies that aim at the identification of organisms that may indicate anthropic impacts and environmental quality.(AU)

O objetivo foi investigar, em escalas espacial e temporal, a abundância das comunidades de protozoários bem como os fatores intervenientes na estruturação das mesmas em um córrego urbano. Métodos: Amostras de água para análise de comunidades biológicas (amebas testáceas, ciliados e bactérias) e para análise de variáveis limnológicas foram tomadas no córrego Mandacaru localizado na Unidade de Conservação do Parque do Cinquentenário, na cidade de Maringá, Paraná State, Brasil, em dois períodos hidrológicos (seco e chuvoso). Calculamos o Índice de Qualidade de Água (IQA), o Índice de Estado Trófico (ETI) para fósforo e utilizamos uma Análise de Redundância (RDA) para verificar a influência de variáveis ambientais na comunidade de protozoários. Resultados: O IQA mostrou que a qualidade da água foi considerada boa em alguns pontos e considerada ruim em outros pontos, e o ETI para o fósforo classificou o córrego como mesotrófico na maioria dos locais amostrados (média entre 53,09 e 58,35). Foram identificados 19 táxons de amebas testáceas, pertencentes a seis famílias, sendo Difflugiidae, Centropyxidae e Arcellidae as mais especiosas, e 71 táxons de ciliados, distribuídos em 12 ordens, sendo Peniculida a ordem mais representativa, seguida por Euplotida. De acordo com a análise RDA, amostras do período seco foram caracterizadas por maiores valores de densidade bacteriana e concentrações de clorofila-a, fósforo total e nitrogênio total. As amebas testáceas e os ciliados apresentaram valores de abundância maiores em alguns dos meses caracterizados por maior precipitação e em condições de maior produtividade do sistema. Podemos concluir que o córrego Mandacaru, embora fortemente influenciado pela ação antrópica, ainda apresenta uma qualidade de água aceitável. Por fim, enfatizamos que a abundância de protistas foi fortemente influenciada pela produtividade do sistema. Isto foi evidenciado pelas elevadas densidades de protozoários onde houve maior produtividade primária e bacteriana. Assim, esses protistas devem ser considerados em estudos que visem à identificação de organismos que possam indicar impactos antrópicos e qualidade ambiental.(AU)

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Abstract We aimed to investigate spatial and temporal scales, abundance, and factors that structure the communities of protozoans in a tropical urban stream. Methods: Samples of water for analysis of biological communities (testate amoebae, ciliates and bacteria) and limnological variables were taken in the Mandacaru stream located in the Conservation Unit of Parque do Cinquentenário, in the city of Maringá, Paraná State, Brazil, in two hydrological periods (dry and rainy). We calculated the Water Quality Index (WQI) composed by nine parameters of water quality (dissolved oxygen, thermotolerant coliforms, pH, BOD, water temperature, total nitrogen, total phosphorous, turbidity, and total solids), Trophic State Index (TSI) for phosphorous and we used a Redundancy Analysis (RDA) to verify the influence of environmental variables in the protozoan community. Results: The WQI showed that water quality was considered good in some points and considered bad in other points. The TSI for phosphorus classified the stream as mesotrophic in the majority of sampled sites points (mean between 53.09 and 58.35). We identified 19 taxa of testate amoebae, belonging to six families, being Difflugiidae, Centropyxidae, and Arcellidae those with more species and 71 infrageneric taxa of ciliates, distributed in 12 orders being Peniculida the most representative order, followed by Euplotida. According to RDA analysis, samples of the dry period were characterized by higher values of bacteria density and concentrations of chlorophyll-a, total phosphorous, and total nitrogen. Ciliates and testate amoebae presented higher abundance values in some of the months characterized by higher precipitation and in conditions of higher system productivity. Conclusion: we may conclude that the Mandacaru stream, although strongly influenced by anthropic action, still presents an acceptable water quality. Lastly, we emphasize that protists abundance was strongly influenced by system productivity. This was evidenced by elevated protozoan densities where there was higher primary and bacterial productivity. Thus, these organisms must be considered in studies that aim at the identification of organisms that may indicate anthropic impacts and environmental quality.

Resumo O objetivo foi investigar, em escalas espacial e temporal, a abundância das comunidades de protozoários bem como os fatores intervenientes na estruturação das mesmas em um córrego urbano. Métodos: Amostras de água para análise de comunidades biológicas (amebas testáceas, ciliados e bactérias) e para análise de variáveis limnológicas foram tomadas no córrego Mandacaru localizado na Unidade de Conservação do Parque do Cinquentenário, na cidade de Maringá, Paraná State, Brasil, em dois períodos hidrológicos (seco e chuvoso). Calculamos o Índice de Qualidade de Água (IQA), o Índice de Estado Trófico (ETI) para fósforo e utilizamos uma Análise de Redundância (RDA) para verificar a influência de variáveis ambientais na comunidade de protozoários. Resultados: O IQA mostrou que a qualidade da água foi considerada boa em alguns pontos e considerada ruim em outros pontos, e o ETI para o fósforo classificou o córrego como mesotrófico na maioria dos locais amostrados (média entre 53,09 e 58,35). Foram identificados 19 táxons de amebas testáceas, pertencentes a seis famílias, sendo Difflugiidae, Centropyxidae e Arcellidae as mais especiosas, e 71 táxons de ciliados, distribuídos em 12 ordens, sendo Peniculida a ordem mais representativa, seguida por Euplotida. De acordo com a análise RDA, amostras do período seco foram caracterizadas por maiores valores de densidade bacteriana e concentrações de clorofila-a, fósforo total e nitrogênio total. As amebas testáceas e os ciliados apresentaram valores de abundância maiores em alguns dos meses caracterizados por maior precipitação e em condições de maior produtividade do sistema. Podemos concluir que o córrego Mandacaru, embora fortemente influenciado pela ação antrópica, ainda apresenta uma qualidade de água aceitável. Por fim, enfatizamos que a abundância de protistas foi fortemente influenciada pela produtividade do sistema. Isto foi evidenciado pelas elevadas densidades de protozoários onde houve maior produtividade primária e bacteriana. Assim, esses protistas devem ser considerados em estudos que visem à identificação de organismos que possam indicar impactos antrópicos e qualidade ambiental.

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Photosynthetic species of the dinoflagellate genus Dinophysis are known to retain temporary cryptophyte plastids of the Teleaulax/Plagioselmis/Geminigera clade after feeding the ciliate Mesodinium rubrum. In the present study, partial plastid 23S rDNA sequences were retrieved in Southern Chilean waters from oceanic (Los Lagos region), and fjord systems (Aysén region), in single cells of Dinophysis and accompanying organisms (the heliozoan Actinophrys cf. sol and tintinnid ciliates), identified by means of morphological discrimination under the light microscope. All plastid 23S rDNA sequences (n = 23) from Dinophysis spp. (Dinophysis acuta, D. caudata, D. tripos and D. subcircularis) belonged to cryptophytes from clade V (Rhinomonas, Rhodomonas and Storeatula), although they could not be identified at genus level. Moreover, five plastid sequences obtained from heliozoans (Actinophryida, tentatively identified as Actinophrys cf. sol), and tintinnid ciliates, grouped together with those cryptophyte sequences. In contrast, two additional sequences from tintinnids belonged to other taxa (chlorophytes and cyanobacteria). Overall, the present study represents the first time that red cryptophyte plastids outside of the Teleaulax/Plagioselmis/Geminigera clade dominate in wild photosynthetic Dinophysis spp. These findings suggest that either Dinophysis spp. are able to feed on other ciliate prey than Mesodinium and/or that cryptophyte plastids from clade V prevail in members of the M. rubrum species complex in the studied area.

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Information regarding trichodinid ectoparasites on marine fishes of North America is relatively scarce. In this study, 5 ciliate species from the family Trichodinidae were found associated with 8 fish hosts from the rocky intertidal zone of the western coast of the Baja California Peninsula (BCP), Mexico. All of the host-parasite relationships recorded here are new. Furthermore, 3 of the trichodinid species found are recorded for the first time for Mexico. Trichodinids taxa do not show a noticeable distributional gradient along the BCP, which suggests a wide-continuous distribution of the species throughout the study area.

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