Morphology, ultrastructure, and molecular phylogeny of the ciliate Sonderia vorax with insights into the systematics of order Plagiopylida.

BACKGROUND: Ciliates of the family Sonderiidae are common members of the eukaryotic communities in various anoxic environments. They host both ecto- and endosymbiotic prokaryotes (the latter associated with hydrogenosomes) and possess peculiar morpho-ultrastructural features, whose functions and homologies are not known. Their phylogenetic relationships with other ciliates are not completely resolved and the available literature, especially concerning electron microscopy and molecular studies, is quite scarce. RESULTS: Sonderia vorax Kahl, 1928 is redescribed from an oxygen-deficient, brackish-water pond along the Ligurian Sea coastlines of Italy. Data on morphology, morphometry, and ultrastructure are reported. S. vorax is ovoid-ellipsoid in shape, dorsoventrally flattened, 130 x 69 µm (mean in vivo); it shows an almost spherical macronucleus, and one relatively large micronucleus. The ventral kinetom has a "secant system" including fronto-ventral and fronto-lateral kineties. A distinctive layer of bacteria laying between kineties covers the ciliate surface. Two types of extrusomes and hydrogenosomes-endosymbiotic bacteria assemblages are present in the cytoplasm. The phylogeny based on 18S rRNA gene sequences places S. vorax among Plagiopylida; Sonderiidae clusters with Plagiopylidae, although lower-level relationships remain uncertain. The studied population is fixed as neotype and the ciliate is established as type species of the genus, currently lacking. CONCLUSIONS: This is the first description of a representative of Sonderiidae performed with both morphological and molecular data. To sum up, many previous hypotheses on this interesting, poorly known taxon are confirmed but confusion and contradictory data are as well highlighted.

Coexistence of tubulins and ftsZ in different Prosthecobacter species.

Prosthecobacter, one of the few cultivable representatives of the bacterial phylum Verrucomicrobia, is of increasing interest to the scientific community due to the presence of tubulin genes in its genome and the apparent absence of the bacterial homologue FtsZ that is normally involved in prokaryotic cell division. These findings suggested the possibility of a vicarious takeover of the FtsZ function through these novel tubulins and opened new scenarios on the possible evolution of bacterial cytoskeleton and cell division. In the present manuscript, we report the characterization of ftsZ and ftsA homologues in different Prosthecobacter species that also possess tubulin genes. Based on these findings, we propose an FtsZ-based cell division mechanism in Verrucomicrobia. The analysis of available genome data of Verrucomicrobia suggests that tubulins are not a feature common to all members of this phylum. Therefore, it can be assumed that Prosthecobacter acquired tubulins through horizontal gene transfer. The functional role of tubulins in Prosthecobacter remains enigmatic.

Molecular systematics and ultrastructural characterization of a forgotten species: Chattonidium setense (Ciliophora, Heterotrichea).

In the present paper we redescribe the ciliate Chattonidium setense Villeneuve 1937 combining morphological observations (live, stained, scanning, and transmission electron microscope) with behavioral notes and molecular data. Ultrastructural analysis revealed remarkable similarities between Chattonidium and representative members of the class Heterotrichea in cortical structure and cytoplasmic organization. The most similar genus for these aspects appears to be Condylostoma. To verify this relatedness, 18S rRNA genes from Chattonidium and from one Condylostoma species were sequenced. Phylogenetic analysis indicates Chattonidium belongs to the class Heterotrichea defined according to the modern taxonomy, and confirms its relatedness with Condylostoma already hypothesized by Villeneuve-Brachon (1940). The presence of the aboral cavity complex, a unique feature never described in other ciliates, and its peculiar organization revealed by ultrastructural analysis fully justify, in our opinion, the maintenance of Chattonidium in the separate family Chattonidiidae, established by Villeneuve-Brachon in 1940.

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia).

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.

Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of 'Candidatus Devosia euplotis'.

This paper reports the identification of bacterial endosymbionts that inhabit the cytoplasm of the marine ciliated protozoon Euplotes magnicirratus. Ultrastructural and full-cycle rRNA approaches were used to reveal the identity of these bacteria. Based on analysis of 16S rRNA gene sequences, evolutionary trees were constructed; these placed the endosymbiont in the genus Devosia in the alpha-Proteobacteria. The validity of this finding was also shown by fluorescence in situ hybridization with a Devosia-specific oligonucleotide probe. Differences at the 16S rRNA gene level (which allowed the construction of a species-specific oligonucleotide probe) and the peculiar habitat indicate that the endosymbiont represents a novel species. As its cultivation has not been successful to date, the provisional name 'Candidatus Devosia euplotis' is proposed. The species- and group-specific probes designed in this study could represent convenient tools for the detection of 'Candidatus Devosia euplotis' and Devosia-like bacteria in the environment.

A multidisciplinary approach to describe protists: a morphological, ultrastructural, and molecular study on Peritromus kahli Villeneuve-Brachon, 1940 (Ciliophora, Heterotrichea).

This study represents the first extended report on a species of the ciliate genus Peritromus, widespread in marine biotopes, characterized by a dorso-ventral differentiation peculiar among Heterotrichea. Morphological observations (live, stained, scanning, and transmission electron microscope) were combined with behavioral and molecular data. On the basis of the whole body of observations, the species was recognized as Peritromus kahli. Scanning and transmission electron microscopy have revealed a number of features such as peculiar chalice-like structures external to the dorsal surface, two types of extrusomes, and differences between dorsal and ventral somatic ciliature. The almost complete SSrDNA gene sequence was also determined. A molecular phylogenetic analysis indicated that Peritromus diverged early from other members of the Class Heterotrichea. The dorso-ventral differentiation that certainly influences the behavior of P. kahli (e.g. preference for crawling and thigmotaxis) may have been selected as an adaptation to the constraints of the interstitial habitat.

Growth medium recycling in Nannochloropsis sp. mass cultivation.

During cell division Nannochloropsis releases the thick and multilayered parent cell wall [Phycologia 35 (1996) 253]. The excretion of autoinhibitory substances in Nannochloropsis cultures has been also reported [J. Appl. Phycol. 11 (1999) 123]. Both wall remains and autoinhibitors may negatively affect culture growth and limit the recycling of the exhaust culture medium, a necessity in commercial microalgae plants to reduce production costs. The effect of medium recycling on growth and productivity of Nannochloropsis sp. cultures grown in 120 l annular reactors was investigated. The use of exhaust medium replenished with nutrients decreased significantly culture productivity. The partial removal of the cell walls alleviated, but did not solve the problem. In addition, medium recycling caused a massive formation of cell aggregates accompanied by a progressive deterioration of the culture. The structure of these aggregates was investigated by transmission electron microscopy. The images showed that the aggregates were held together by cell wall remains, which entrapped cells, bacteria and debris resulting from cell decay. Thus, in high density Nannochloropsis cultures, cell walls might play a key role in reducing productivity, favoring contamination and making the biomass unsuitable as aquaculture feed.

A multidisciplinary approach to describe protists: redescriptions of Novistrombidium testaceum Anigstein 1914 and Strombidium inclinatum Montagnes, Taylor, and Lynn 1990 (Ciliophora, Oligotrichia).

We combined behavioral, morphological (live, stained, scanning and transmission electron-microscope), and molecular data to redescribe two common, intertidal oligotrich ciliates, Novistrombidium testaceum and Strombidium inclinatum. Both species were collected from a rocky shore near Leghorn, Ligurian Sea. A literature review revealed four morphotypes of Novistrombidium testaceum that differ in subtle ways, including oral morphology. These differences may be diagnostic, but we do not consider them sufficient to distinguish different taxa. Although other studies have synonymised Strombidium inclinatum and S. sulcatum, based on oral structures, size, and nuclear structure, there are morphological distinctions between them. In particular, the present study supports a lack of anterior protuberance in both live and preserved S. inclinatum, while S. sulcatum possesses a protuberance. The 18S rDNA molecular data, in accordance with morphological and ultrastructural observations, indicate that the Strombidiida (Oligotrichia) constitute a well-supported clade. The separation of the genera within this clade, even between Novistrombidium and Strombidium, remains unresolved, and the analysis of more species is required. Finally, we recommend that when possible, ecologists, morphological taxonomists, and molecular biologists combine their expertise to provide comprehensive taxonomic descriptions.

Extrusomes in ciliates: diversification, distribution, and phylogenetic implications.

Exocytosis is, in all likelihood, an important communication method among microbes. Ciliates are highly differentiated and specialized micro-organisms for which versatile and/or sophisticated exocytotic organelles may represent important adaptive tools. Thus, in ciliates, we find a broad range of different extrusomes, i.e ejectable membrane-bound organelles. Structurally simple extrusomes, like mucocysts and cortical granules, are widespread in different taxa within the phylum. They play the roles in each case required for the ecological needs of the organisms. Then, we find a number of more elaborate extrusomes, whose distribution within the phylum is more limited, and in some way related to phylogenetic affinities. Herein we provide a survey of literature and our data on selected extrusomes in ciliates. Their morphology, distribution, and possible function are discussed. The possible phylogenetic implications of their diversity are considered.

Genes for the cytoskeletal protein tubulin in the bacterial genus Prosthecobacter.

Tubulins, the protein constituents of the microtubule cytoskeleton, are present in all known eukaryotes but have never been found in the Bacteria or Archaea. Here we report the presence of two tubulin-like genes [bacterial tubulin a (btuba) and bacterial tubulin b (btubb)] in bacteria of the genus Prosthecobacter (Division Verrucomicrobia). In this study, we investigated the organization and expression of these genes and conducted a comparative analysis of the bacterial and eukaryotic protein sequences, focusing on their phylogeny and 3D structures. The btuba and btubb genes are arranged as adjacent loci within the genome along with a kinesin light chain gene homolog. RT-PCR experiments indicate that these three genes are cotranscribed, and a probable promoter was identified upstream of btuba. On the basis of comparative modeling data, we predict that the Prosthecobacter tubulins are monomeric, unlike eukaryotic alpha and beta tubulins, which form dimers and are therefore unlikely to form microtubule-like structures. Phylogenetic analyses indicate that the Prosthecobacter tubulins are quite divergent and do not support recent horizontal transfer of the genes from a eukaryote. The discovery of genes for tubulin in a bacterial genus may offer new insights into the evolution of the cytoskeleton.

A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea).

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.