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A well-defined clade of the Euplotes phylogenetic tree is represented by marine species characterized by a single-type dargyrome and ten fronto-ventral cirri. Three of them, namely Euplotes crassus, E. minuta and E. vannus, form a complex of closely related species of large use in experimental ciliatology. Despite morphometric and genetic analyses having substantiated their taxonomic separation, ambiguities still persist in strain assignments to one or another species. In addition to objective reasons intrinsic to significant overlapping of most morphological parameters, ambiguities also result from divergences (inherited from past literature) in deciding which of the two morphotypes, E. crassus or E. vannus, is characterized by a larger or a medium cell body size (E. minuta being clearly distinct by a smaller morphotype). By analysing nuclear SSU-rRNA gene and ITS region sequences from 37 strains, previously assigned to E. crassus, E. minuta and E. vannus based on conventional taxonomic parameters, we identified and used ITS autapomorphic point mutations to design three species-specific primers. In combination with an Euplotes-generic primer, they proved to be very effective in running polymerase chain reactions that produce amplicons of species-specific size that reliably resolve ambiguities in assigning strains to E. crassus, E. minuta or E. vannus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Data improving the characterization of the marine Euplotes species, E. petzi Wilbert and Song, 2008, were obtained from morphological, ecological and genetic analyses of Antarctic and Arctic wild-type strains. This species is identified by a minute (mean size, 46 μm × 32 μm) and ellipsoidal cell body which is dorsally decorated with an argyrome of the double-patella type, five dorsal kineties (of which the median one contains 8-10 dikinetids), five sharp-edged longitudinal ridges, and a right anterior spur. Ventrally, it bears 10 fronto-ventral, five transverse, two caudal and two marginal cirri, 30-35 adoral membranelles, and three inconspicuous ridges. Euplotes petzi grows well at 4 °C on green algae, does not produce cysts, undergoes mating under the genetic control of a multiple mating-type system, constitutively secretes water-borne pheromones, and behaves as a psychrophilic microorganism unable to survive at >15 °C. While the α-tubulin gene sequence determination did not provide useful information on the E. petzi molecular phylogeny, the small subunit rRNA (SSU rRNA) gene sequence determination provided solid evidence that E. petzi clusters with E. sinicus Jiang et al., 2010a, into a clade which represents the deepest branch at the base of the Euplotes phylogentic tree., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild-type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool., (© 2013 John Wiley & Sons Ltd.)

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

In protozoan ciliates, diffusible signalling proteins (pheromones) regulate the vegetative growth and mating interactions. Here, the coding genes and the structures of the encoded pheromones were studied in genetically distinct wild-type strains representing interbreeding Antarctic and Arctic populations of the marine ciliate Euplotes nobilii. Determination of seven allelic pheromone-coding DNA sequences revealed that an unusual extension and high structural conservation of the 5' non-coding region are peculiar traits of this gene family, implying that this region is directly involved in the mechanism of pheromone gene expression, possibly through phenomena of intron splicing and/or frame-shifting. For four pheromones, the three-dimensional structures were determined by nuclear magnetic resonance spectroscopy in solution. These structures show that the pheromones represent a protein family which adapts to its polar environment by combining a structurally stable core of a three-helix bundle with extended polypeptide segments that are devoid of regular secondary structures and concomitantly show enhanced structural flexibility., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Wild-type strains of the protozoan ciliate Euplotes collected from different locations on the coasts of Antarctica, Tierra del Fuego and the Arctic were taxonomically identified as the morpho-species Euplotes nobilii, based on morphometric and phylogenetic analyses. Subsequent studies of their sexual interactions revealed that mating combinations of Antarctic and Arctic strains form stable pairs of conjugant cells. These conjugant pairs were isolated and shown to complete mutual gene exchange and cross-fertilization. The biological significance of this finding was further substantiated by demonstrating that close homology exists among the three-dimensional structures determined by NMR of the water-borne signaling pheromones that are constitutively secreted into the extracellular space by these interbreeding strains, in which these molecules trigger the switch between the growth stage and the sexual stage of the life cycle. The fact that Antarctic and Arctic E. nobilii populations share the same gene pool and belong to the same biological species provides new support to the biogeographic model of global distribution of eukaryotic microorganisms, which had so far been based exclusively on studies of morphological and phylogenetic taxonomy.

In the last two decades, large scale axenic cell cultures of the marine species comprising the family Euplotidae have resulted in the isolation of several new classes of terpenoids with unprecedented carbon skeletons including the (i) euplotins, highly strained acetylated sesquiterpene hemiacetals; (ii) raikovenals, built on the bicyclo[3.2.0]heptane ring system; (iii) rarisetenolides and focardins containing an octahydroazulene moiety; and (iv) vannusals, with a unique C30 backbone. Their complex structures have been elucidated through a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, molecular mechanics and quantum chemical calculations. Despite the limited number of biosynthetic experiments having been performed, the large diversity of ciliate terpenoids has facilitated the proposal of biosynthetic pathways whereby they are produced from classical linear precursors. Herein, the similarities and differences emerging from the comparison of the classical chemotaxonomy approach based on secondary metabolites, with species phylogenesis based on genetic descriptors (SSU-rDNA), will be discussed. Results on the interesting ecological and biological properties of ciliate terpenoids are also reported.

The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.

Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies that does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca(2+) and Na(+), suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environment.

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.

Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs.

Strains of the marine ciliate protist Euplotes crassus produce exclusive terpenoids called euplotins that play an ecological role. Among these derivatives, euplotin C is the main of four secondary metabolites isolated from cultures of this protozoon and represents the sesquiterpene taxonomic marker from E. crassus. Because different terpenoid metabolites of plant origin showed a certain antimicrobial activity, we assessed the compound euplotin C, purified by high-pressure liquid chromatography and solubilized in two solubility enhancers, against the protozoa Leishmania major and Leishmani infantum, the fungus Candida albicans, and nine strains of gram-positive and gram-negative microorganisms. An activity of euplotin C against Leishmania promastigotes was demonstrated (50% lethal doses were 4.6 or 8.1 microg/ml depending on the agent used to solubilize the compound), while the effect was less evident on Candida and nearly absent on bacteria. A nonsignificant cytotoxicity (50% lethal dose, >200 microg/ml) against the J774 cell line was observed. A leishmanicidal activity was also shown by the living, euplotin-producing cells of E. crassus cultured together with promastigotes; this activity increased with time from 10 min to 6 h of incubation. This study provides an initial rationale for the evaluation of euplotin C and other similar natural products as alternative or possibly synergistic compounds for current antiprotozoon chemotherapeutics.

Ciliate protozoa are important members of microbial communities in which they play specific ecological roles. The determination of single species distribution is fundamental for food web analysis, but species recognition, which is mainly based on morphological characters, is often difficult between closely related species. The use of species-specific, purposely designed, fluorescently labeled probes for in situ hybridization is here presented as an easy and fast identification method for three closely related species belonging to the widespread genus Euplotes, namely E. crassus, E. vannus, and E. minuta, that in spite of their remarkable morphological similarity have significant metabolic and ecological differences. These three species can be detected simultaneously, provided the probes employed are bound to different fluorescent dyes: in this way their relative abundance and their population dynamics in the natural environment can be evaluated. As more ciliate sequences become available in databases, species-specific probes can be designed for other ciliates, thus rendering the application of the method of more general importance. The probes used in this study may also provide a tool to prevent erroneous species identification in future studies.

The cosmopolitan genus Euplotes is remarkable among ciliates for its species richness. To understand the still tangled taxonomy and phylogenetic relationships within the genus, small subunit rRNA sequences of 11 morphologically defined species colonizing different habitats were determined. Euplotes 18S rRNA is unique among ciliates for its anomalous length and high evolutionary rate. Phylogenetic reconstruction pointed to a high divergence between this genus and the other Spirotrichea, together with a high variability within the genus. Some of the relationships within the Euplotes group were also resolved. Data from the literature, based on morphological features, habitat, and symbiotic relationships, have been compared with our results and are critically discussed. In many cases, the molecular phylogenetic analysis disagreed with species relatedness established on morphological and ecological grounds. The occurrence of a radiation phenomenon in the evolution of the genus is postulated.

The life styles of ciliated protists are particularly suitable for experimental analyses of certain aspects of developmental and genetic biology. The progression from sexual immaturity to maturity to senescence represents one of the most intriguing aspects of developmental programs. The extent to which progeny clones, their subclones, and testers used in the assay result in different lengths of immaturity has been investigated in Euplotes crassus. Six subclones from each of 12 progeny clones from a cross between stocks EC1 and EC2 were tested for maturity with stocks EC3, EC4, and EC5 on every transfer. Analysis of variance was used to partition the total variation in fissions to maturity into parts due to clones, subclones, and testers and the interactions between these levels. The error, interaction of subclones and testers, corresponds to a standard deviation of only 4.1 fissions, while the within clone within tester means range from 15.2 to 46.7 fissions; all levels except testers contribute significantly to the total variation. Most of the variability is attributable to clones (66%), the next most to error (16%), the next most to interaction of clones by testers (13%), and the least to subclones (5%). An a posteriori analysis examined whether the differences among clones were due to the cytoplasm of the clone ancestor (exconjugant), its mat (mating-type) locus genotype, or the mated pair it came from. None of these characteristics was able to interpret simply the large variability among clones. These results provide evidence that the transition from immaturity to maturity is quantitative and complex rather than a jump from one well-defined state to another.