Graziano Di Giuseppe

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a Parasite Infecting the Hypotrichous Ciliate Euplotes woodruffi, with Observations on Microsporidian Infections in Ciliophora

ABSTRACT. 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 × 1.65±0.22 μm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5–5.5 μm 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.

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.

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 Ca2+ 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 (Δψm). 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, Δψm and intracellular free Ca2+, 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.

A Structurally Deviant Member of the Euplotes raikovi Pheromone Family: Er-23

Abstract Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx2–4-Cys-Xxx5–7 -Cys.

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.

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.

Seasonal variations of glycogen synthase and phosphorylase activities in the liver of the frog Rana esculenta

Abstract Glycogen metabolism has been studied in the liver of the frog Rana esculenta throughout the annual cycle. Hepatic glycogen reserves are high during winter and decrease during spring and summer, the changes being reflected by variations in the activities of the rate-limiting enzymes glycogen synthase and phosphorylase. Glycogen deposition is associated with increased levels of synthase D activity and no changes in the relative proportions of the active-inactive form of the enzyme, whereas during glycogen mobilization significant changes in both total phosphorylase and in the percent phosphorylase a are observed. Regression analysis indicates a positive correlation between glycogen content and synthase activity, and an inverse relationship between glycogen content and either total phosphorylase or phosphorylase a . The results suggest that glycogen deposition and/or mobilization could be mainly controlled by mechanisms related to either changes in the amount of enzyme protein or to allosteric effects of the metabolite glucose-6-phosphate on glycogen synthase and phorphorylase.

New records of the pygmy mussel Xenostrobus securis (Bivalvia: Mytilidae) in brackish-water biotopes of the western Mediterranean provide evidence of its invasive potential

The present study reports new records of the invasive pygmy mussel Xenostrobus securis in six brackish-water localities in the western Mediterranean. Until now X. securis , whose native range includes southern Australia and New Zealand, was also known from the northern Adriatic Sea and southern France. Along the coast of northern Tuscany (Italy) X. securis invaded brackish-water canals in the area between the port of Leghorn and the mouth of the Arno River. Moreover, this mussel has been found in one inner site of the Gulf of Olbia (north-eastern Sardinia, Italy) and at the mouth of the Fluvia River (northern Catalonia, Spain). As an ecosystem engineer, X. securis is an ecologically important species, heavily altering pre-existing benthic communities. Given that its presence in other western Mediterranean sites is highly possible, we recommend a closer monitoring of brackish-water biotopes.

Isolation and Structural Characterization of Two Water‐Borne Pheromones from Euplotes crassus, a Ciliate Commonly Known to Carry Membrane‐Bound Pheromones

ABSTRACT. Ciliates comprise species synthesizing water‐diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane‐bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone‐secreting ciliate, like other Euplotes species. From cell‐free filtrate preparations of the E. crassus strain L‐2D, we isolated two distinct pheromones, designated as Ec‐α and Ec‐1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec‐α pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec‐1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full‐length Ec‐α and Ec‐1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec‐α and Ec‐1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome.

Identification of the Tubulin Gene Family and Sequence Determination of One β-Tubulin Gene in a Cold-Poikilotherm Protozoan, the Antarctic Ciliate Euplotes focardii

ABSTRACT. Four different tubulin genes were identified in the somatic nucleus (macronucleus) of Euplotes focardii, a strictly coldadapted, Antarctic ciliate: one of 1,800 bp for α‐tubulin and three of 2,150, 1,900, and 1,600 bp, respectively, for β‐tubulin. Preliminarily analysed for restriction fragment length polymorphisms, these genes showed remarkable differences in organisation from tubulin genes of other ciliates which live in temperate areas and were analysed in parallel with E. focardii. The complete coding sequence of the 1,600 bp β‐tubulin gene was then determined and shown to contain unique structural features of potential importance for E. focardii microtubule organization and activity. Of eight unique substitutions detected, seven were concentrated in the large amino terminal domain of the molecule that directly interacts with the carboxy terminal region of α‐tubulin for heterodimer formation. Sequence analysis of the cloned gene revealed, in addition, a potential new exception in the use of the genetic code by ciliates. A TAG codon was aligned in correspondence with Trp‐21 which is strictly conserved in every tubulin sequence so far determined.