No 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.

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.

Apofrontonia dohrni, a new peniculine ciliate, was discovered in a slightly brackish water sample from Mediterranean coastline puddle in Naples, Italy. Its morphology was studied in vivo, in silver- and Feulgen-stained preparations, as well as at the scanning electron microscope; 18S rRNA gene sequence was also determined. The species is characterized by a medium cell size (118 x 61 mu m - fixed cell) and an oval-extended body, flattened dorsoventrally; a very long sausage-like macronucleus rolled up into almost a ring; a comparatively large compact micronucleus; relatively small number of vestibular kineties (4) and a peculiar structure of 3 peniculi, consisting of 5-6, 4-5 and 3 ciliary rows, respectively. The ciliate always has about 20 contractile vacuoles distributed beneath its entire body surface. Using these features, A. dohrni, definitely, can be easily discriminated from the two other species of the genus: Apofrontonia lametschwandtneri and Apofrontonia obtusa. T..

Karyorelictea is a class of traditionally unculturable ciliates characterized by a non-dividing macronucleus. Their classification has been recently revised and morphological redescription of many species has been performed as well. On the contrary, molecular data of karyorelictean ciliates are largely underrepresented in public databases. In the present article we resumed and improved a method to characterize 18S rRNA gene sequences through direct amplification and sequencing of single cells. Using this approach, we characterized 12 different karyorelictean molecular operational taxonomic units (MOTUs: nine trachelocercids, one Geleia, one Remanella and one Loxodes), most of which were also photo-recorded. These molecular data were used to reconstruct phylogenetic relationships among the three orders (Protostomatida, Loxodida and Protoheterotrichida) in which the class is traditionally subdivided. The most supported tree topology shows an association between orders Loxodida (Loxodes, Remanella) and Protoheterotrichida (Geleia), in contrast with previous works associating orders Loxodida and Protostomatida (trachelocercids) on a morphological basis.

BackgroundCiliates 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.ResultsSonderia 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.ConclusionsThis 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.

Recent culture‐independent studies based on small‐subunit ribosomal RNA (SSU rRNA) gene analysis revealed the existence of completely new clades of protists. The main problems with this approach are to correlate sequences from environmental rRNA genes with the organisms they belong to and then to detect the ecological role of these organisms in the environment. In order to overcome such problems we chose an alternative approach allowing us both a molecular characterization of uncultivable organisms with a low relative abundance in environmental samples, and a morphological analysis, even if restricted. The experimental protocol consists of two steps: an initial observation and photo‐taking of the single cell under the DIC (Differential Interferential Contrast) microscope and then PCR amplification and direct sequencing of the 18S rRNA gene of the same cell. The advantages of this method are the possibility to: (1) establish a precise link between morphology and gene sequence; (2) detect the possible occurrence of highly similar species within the studied population; (3) avoid the insertion of Taq‐polymerase errors in the gene sequence; and (4) detect possible polymorphisms in the gene under examination. Such an approach was used to sequence the 18S rRNA gene of organisms belonging to the class Karyorelictea that comprises several uncultivable ciliates with limited distinctive features. Gene sequences analysis revealed an unexpected genetic variability in trachelocercids and, in particular, the existence of polymorphisms in the SSU rRNA gene of a group of them. Such specimens show a similar morphology and, as a result of phylogenetic analysis, they form a constant clade.