Sergei I. Fokin

Phylogenetic Relationships of the Subclass Peniculia (Oligohymenophorea, Ciliophora) Inferred from Small Subunit rRNA Gene Sequences

Abstract Peniculine ciliates have been recognized as a distinct higher taxon of ciliates for almost 50 years. However, phylogenetic relationships within the Subclass Peniculia are still unsettled. To contribute to our understanding of their phylogeny and provide evidence for the position of Urocentrum turbo, we sequenced its small subunit (SS) rRNA gene and the SSrRNA genes from Lembadion bullinum, Frontonia sp., Paramecium caudatum, Paramecium multimicronucleatum, Paramecium putrinum, and Paramecium woodruffi. Urocentrum turbo was the only one of these species not to exhibit a shortened Helix E10_1, which we conclude characterizes the “higher” peniculines. Except for U. turbo, the peniculines are strongly supported as a monophyletic clade with Lembadion, Frontonia, and Paramecium species forming separate and strongly supported clades by bootstrap analysis using distance matrix, maximum parsimony, and maximum likelihood methods. Urocentrum turbo is associated with different lineages, depending upon the analysis used. The Paramecium species form at least four clades with the Paramecium aurelia subgroup being the most derived. We conclude that the Subclass Peniculia should be divided into two orders, the Order Urocentrida and Order Peniculida, with the latter order having two suborders, the Suborder Frontoniina and Peniculina. We place U. turbo with the peniculines because of shared morphological and stomatogenetic features.

Holospora species infecting the nuclei of Paramecium appear to belong into two groups of bacteria

Summary Ten Holospora bacteria in various Paramecium species, namely P. novaurelia, P. caudatum, P. bursaria, P. woodruffi , and P. putrinum were found to be similar concerning their morphology and life cycle. However, four species (group 1): Holospora caryophila, H. bacillata, H. curvata , and a species newly found in P. putrinum differed from the other bacteria (group 2), among them H. obtusa, H. undulata, H. elegans, H. recta , as they had a different behavior during the division of the host nucleus; in group 2 the infectious forms were collected in the connecting piece of the dividing host nucleus and were then released into the medium, whereas in group 1 the infectious forms remained distributed over the whole nucleus during its division. The bacteria of the group 1 also were not labelled by in situ hybridization using an oligonucleotide probe hybridizing with the rRNA of bacteria of group 2. The data indicate that Holospora bacteria may at least belong into two groups.

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

ABSTRACT. 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.

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.

Morphological Diversity of the Micronuclei in Paramecium

Summary Morphological analysis of the micronuclei in 51 stocks of 10 Paramecium species was carried out to examine the diversity of the nuclear morphology in these organisms. Instead of the two general structural types of micronuclei which are well-known in the literature (“caudatum” and “aurelia”), 4 types of nuclei are proposed. They are the “vesicular” type ( P. polycaryum, P. arcticum, P. aurelia, P. multimicronucleatum ), the “endosomal” type ( P. calkinsi, P. woodruffi, P. duboscqui ), the “chromosomal” type ( P. jenningsi, P. wichtermani ) and the “compact” type (3 varieties) — P. caudatum (a), P. bursaria (b) and P. putrinum (c).

Flagellar Movement in Two Bacteria of the Family Rickettsiaceae : A Re-Evaluation of Motility in an Evolutionary Perspective

Bacteria of the family Rickettsiaceae have always been largely studied not only for their importance in the medical field, but also as model systems in evolutionary biology. In fact, they share a recent common ancestor with mitochondria. The most studied species, belonging to genera Rickettsia and Orientia, are hosted by terrestrial arthropods and include many human pathogens. Nevertheless, recent findings show that a large part of Rickettsiaceae biodiversity actually resides outside the group of well-known pathogenic bacteria. Collecting data on these recently described non-conventional members of the family is crucial in order to gain information on ancestral features of the whole group. Although bacteria of the family Rickettsiaceae, and of the whole order Rickettsiales, are formally described as non-flagellated prokaryotes, some recent findings renewed the debate about this feature. In this paper we report the first finding of members of the family displaying numerous flagella and active movement inside their host cells. These two new taxa are hosted in aquatic environments by protist ciliates and are described here by means of ultrastructural and molecular characterization. Data here reported suggest that the ancestor of Rickettsiales displayed flagellar movement and re-evaluate the hypothesis that motility played a key-role in the origin of mitochondria. Moreover, our study highlights that the aquatic environment represents a well exploited habitat for bacteria of the family Rickettsiaceae. Our results encourage a deep re-consideration of ecological and morphological traits of the family and of the whole order.

Frequency and biodiversity of symbionts in representatives of the main classes of Ciliophora.

Representatives of all classes of Ciliophora have been studied for the detection and investigation of both prokaryotic and eukaryotic (not algal) endo- (EnS) and ectosymbionts (EcS). Different methods including transmission electron microscopy (TEM) and fluorescence in situ hybridisation (FISH) have been used. Apparently, the capability of keeping symbionts varies among the different ciliate groups as it generally is the case in different protist taxa. Most of the prokaryotic EnSs detected belong to Alphaproteobacteria. Holospora or Holospora-like infectious bacteria of this group were found in representatives of Heterotrichea, Armophorea, Phyllopharyngea, Prostomatea and mainly of Oligohymenophorea. Bacteria associated with bacteriophages were found in species of Heterotrichea and Oligohymenophorea. This holds true also for bacteria with R-bodies. A quite rare type of EnS - motile bacteria - was found in ciliates of the same two classes as well, either in the cytoplasm (Heterotrichea) or in the macronucleus and its perinuclear space (Oligohymenophorea). EcSs are more common in Heterotrichea, Armophorea and Plagiopylea, but were never found in other groups. Among the eukaryotic EnSs of ciliates, very few representatives of Microsporidia and Trypanosomatidae were recorded. In conclusion, heterotrichs and oligohymenophoreans are the most promising groups of Ciliophora for the investigation of symbiosis.

Rediscovery of Paramecium nephridiatum Gelei, 1925 and its Characteristics

Paramecium nephridiatum Gelei. 1925, was rediscovered. It is a euryhaline brackish‐water species that morphologically resembles Paramecium woodruffi. but with multiple contractile vacuole pores. The general morphology, morphometry. and random amplified polymorphic DNA (RAPD) fingerprint patterns are presented for a number of the stocks collected around the world.

Caedibacter macronucleorum sp. nov., a Bacterium Inhabiting the Macronucleus of Paramecium duboscqui

Summary The macronucleus of Paramecium duboscqui was observed to contain bacteria. Two morphologically different types of bacteria were found. Type 1 was 1.5-3.0 µm long and 0.5-0.8 µm wide. This type sometimes contained R bodies, which were associated with capsids of bacteriophages. The capability to producing R bodies was, however, lost in some host cell lines in laboratory culture. Type 2 had a length of 0.8 to 2.0 µm and a width of 0.4 µm, but sometimes occurred as much longer rods, which were straight, curved or crescent-shaped. Because of the morphological features of type 1 and of its ability to produce R bodies, the bacterium was named Caedibacter macronucleorum sp.n. Both types of bacteria were maintained by their host cells under all conditions tested. It cannot be decided, whether type 2 of the bacteria, which did not produce R bodies, represents a different stage or morph of the same species or belongs to a different species of bacteria.

Candidatus Paraholospora nucleivisitans, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host.

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological-botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.