Analysis of autapomorphic point mutations provides a key for the tangled taxonomic distinction of the closely related species, Euplotes crassus, E. minuta and E. vannus (Ciliophora, Euplotida).

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.

Evidence for gene duplication and allelic codominance (not hierarchical dominance) at the mating-type locus of the ciliate, Euplotes crassus.

The high-multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating-type substance (pheromone). In strain L-2D, now known to be homozygous at the mating-type locus, we previously identified two pheromones (Ec-α and Ec-1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR-73 characterized by a heterozygous genotype and strong mating compatibility with L-2D strain. It was found that POR-73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec-α pheromone of L-2D cells. The other two pheromones were shown to be new and were designated Ec-2 and Ec-3 to denote their structural homology with the Ec-1 pheromone of L-2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating-type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.

Improved description of the bipolar ciliate, Euplotes petzi, and definition of its basal position in the Euplotes phylogenetic tree.

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.

Phylogeographical pattern of Euplotes nobilii, a protist ciliate with a bipolar biogeographical distribution.

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.

Ciliate-adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment.

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.

Coding genes and molecular structures of the diffusible signalling proteins (pheromones) of the polar ciliate, Euplotes nobilii.

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.

Sequenced RAPD markers to detect hybridization in the barbary partridge (Alectoris barbara, Phasianidae).

In the Alectoris partridges (Phasianidae), hybridization occurs occasionally as a result of the natural breakdown of isolating mechanisms but more frequently as a result of human activity. No genetic record of hybridization is known for the barbary partridge (A. barbara). This species is distributed mostly in North Africa and, in Europe, on the island of Sardinia (Italy) and on Gibraltar. The risk of hybridization between barbary and red-legged partridge (A. rufa: Iberian Peninsula, France, Italy) is high in Sardinia and in Spain. We developed two random amplified polymorphic DNA (RAPD) markers to detect A. barbara × A. rufa hybrid partridges. We tested them on 125 experimental hybrids, sequenced the relative species-specific bands and found that the bands and their corresponding sequences were reliably transmitted through a number of generations (F1, F2, F3, BC1, BC2). Our markers represent a highly valuable tool for the preservation of the A. barbara genome from the pressing threat of A. rufa pollution.

Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating.

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.

Structures, biological activities and phylogenetic relationships of terpenoids from marine ciliates of the genus Euplotes.

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.

Changes in the non-protein thiol pool and production of dissolved gaseous mercury in the marine diatom Thalassiosira weissflogii under mercury exposure.

Two detoxification mechanisms working in the marine diatom Thalassiosira weissflogii to cope with mercury toxicity were investigated. Initially, the effect of mercury on the intracellular pool of non-protein thiols was studied in exponentially growing cultures exposed to sub-toxic HgCl(2) concentrations. T. weissflogii cells responded by synthesizing metal-binding peptides, named phytochelatins (PCs), besides increasing the intracellular pool of glutathione and gamma-glutamylcysteine (gamma-EC). Intracellular Hg and PC concentrations increased with the Hg concentration in the culture medium, exhibiting a distinct dose-response relationship. However, considerations of the PCs-SH:Hg molar ratio suggest that glutathione could also be involved in the intracellular mercury sequestration. The time course of the non-protein thiol pool and Hg intracellular concentration shows that PCs, glutathione and gamma-EC represent a rapid cellular response to mercury, although their role in Hg detoxification seems to lose importance at longer incubation times. The occurrence of a process of reduction of Hg(II) to Hg degrees and subsequent production of dissolved gaseous mercury (DGM) was also investigated at lower Hg concentrations, at which the PC synthesis doesn't seem to be involved. The significant (P<0.01) correlation between the cellular density in solution and the production of DGM suggests that this diatom is capable of directly producing DGM, both in light and dark conditions. This finding has been confirmed by the absence of DGM production in the culture media containing formaldehyde-killed cells. Finally, the relationship between these two different pathways of Hg detoxification is discussed.

Biophysical effects of the natural product euplotin C on the Paramecium membrane.

The effect of euplotin C--a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus--on the voltage-dependent Ca(2+) channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca(2+) channel blockers, we demonstrate that euplotin C modulates Ca(2+) channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca(2+) channel functions through the reduction of cell membrane fluidity.

The secondary metabolite euplotin C induces apoptosis-like death in the marine ciliated protist Euplotes vannus.

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.

"Candidatus Cryptoprodotis polytropus," a novel Rickettsia-like organism in the ciliated protist Pseudomicrothorax dubius (Ciliophora, Nassophorea).

Rickettsia-like organisms (RLO) are obligate, often highly fastidious, intracellular bacterial parasites associated with a variety of vertebrate and invertebrate hosts. Despite their importance as causative agents of severe mortality outbreaks in farmed aquatic species, little is known about their life cycle and their host range. The present work reports the characterization of "Candidatus Cryptoprodotis polytropus," a novel Rickettsia-like bacterium associated with the common ciliate species Pseudomicrothorax dubius by means of the "Full-Cycle rRNA Approach" and ultrastructural observations. The morphological description by in vivo and scanning electron microscopy and the 18S rRNA gene sequence of the host species is provided as well. Phylogenetic analysis based on the 16S rRNA gene supports the inclusion of "Candidatus Cryptoprodotis polytropus" within the family Rickettsiaceae (cl. Alphaproteobacteria) together with the genera Rickettsia and Orientia. Observations on natural ciliate populations account for the occasional nature of this likely parasitic association. The presence of a previously unknown RLO in ciliates sheds a new light on the possible role of protists as transient hosts, vectors or natural reservoir for some economically important pathogens.

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii.

Allelic genes encoding water-borne signal proteins (pheromones) were amplified and sequenced from the somatic (macronuclear) sub-chromosomic genome of Antarctic and Arctic strains of the marine ciliate, Euplotes nobilii. Their open reading frames appeared to be specific for polypeptide sequences of 83 to 94 amino acids identifiable with cytoplasmic pheromone precursors (pre-pro-pheromones), requiring two proteolytic steps to remove the pre- and pro-segments and secrete the mature pheromones. Differently from most of the macronuclear genes that have so far been characterized from Euplotes and other hypotrich ciliates, the 5' and 3' non-coding regions of all the seven E. nobilii pheromone genes are much longer than the coding regions (621 to 700 versus 214 to 285 nucleotides), and the 5' regions in particular show nearly identical sequences across the whole set of pheromone genes. These structural peculiarities of the non-coding regions are likely due to the presence of intron sequences and provide presumptive evidence that they are site of basic, conserved activities in the mechanism that regulates the expression of the E. nobilii pheromone genes.

Action mechanisms of the secondary metabolite euplotin C: signaling and functional role in Euplotes.

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.

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in ciliophora.

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.

Effect of the bioactive metabolite euplotin C on phagocytosis and fluid-phase endocytosis in the single-celled eukaryote Paramecium.

The effect of euplotin C -- a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus -- on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent (p<0.001), even if euplotin C did not completely block the phagocytosis. Following a 15 min treatment with a euplotin C (0.5 microg/ml), the latex particle uptake was inhibited up to 25%. Furthermore, the pretreatment of cells with taxol strongly counteracted euplotin C effect. The amount of extracellularly provided dextran, which is internalized exclusively by fluid-phase uptake, was quantified in cells whose phagocytic activity was blocked by trifluoperazine. The amount of the internalized dextran was about 50% of that in controls after 15 min incubation in the presence of euplotin C. Fluorescence confocal images showed that no endosomes were formed on the surface of these cells. The effect of euplotin C on the food vacuole formation and fluid-phase endocytosis is apparently mediated by a modification of microtubule network.

Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases.

The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca(2+) stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Deltapsi(m)). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Deltapsi(m) and intracellular free Ca(2+), indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects.

Genetic insight into Mediterranean chukar (Alectoris chukar, Galliformes) populations inferred from mitochondrial DNA and RAPD markers.

The chukar (Alectoris chukar, Galliformes) is one of the most important game birds as it is widely distributed and hunted over the whole of its range. The aim of this work was to assess the genetic differentiation as well as the possible presence of hybrid specimens in A. chukar populations from Italy, Greece and Cyprus. To provide phylogenetic context, conspecific, allopatric specimens from Israel, Georgia, Armenia, Kazakhstan, Afghanistan, Pakistan, Mongolia, China and USA were compared. Sequencing of the mitochondrial DNA (mtDNA) Control Region supplied information on the ancestry of A. chukar populations, whereas Random Amplified Polymorphic DNA (RAPD) fingerprinting was used to assess whether hybridization had occurred. The Italian population was found to be an inter-specific mixture of A. chukar and A. rufa (i.e., the red-legged partridge) mtDNA lineages, whereas the representatives from Greece and Cyprus showed only the A. chukar maternal line. RAPD markers revealed introgression with A. rufa genes in the Italian population, whereas no A. chukar x A. rufa hybrid specimens were detected in the eastern Mediterranean populations. The genetic data obtained from the Italian A. chukar population as well as from a few Greek specimens pointed against their Mediterranean kinship, suggesting relationships with A. chukar subspecies from the easternmost part of the Asian continent.