Franco Verni

A Bacterium Belonging to the Rickettsiaceae Family Inhabits the Cytoplasm of the Marine Ciliate Diophrys appendiculata (Ciliophora, Hypotrichia)

AbstractBacteria of the family Rickettsiaceae (order Rickettsiales, α-Proteobacteria) are mainly known to be endosymbionts of arthropods with the capability to infect also vertebrate cells. Recently, they have also been found as leech endocytobionts. In the present paper, we report the first finding of a bacterium belonging to the family Rickettsiaceae in a natural population of a marine ciliate protozoan, namely Diophrys appendiculata, collected in the Baltic Sea. Bacteria were unambiguously identified through morphological characterization and the “full-cycle rRNA approach” (i.e., 16S rRNA gene characterization and use of specifically designed oligonucleotide probes for in situ detection). Symbionts are rod-shaped bacteria that grow freely in the cytoplasm of the host cell. They present two different morphotypes, similar in size, but different in cytoplasmic density. These are typical morphological features of members of the family Rickettsiaceae. 16S rRNA gene sequence showed that Diophrys symbionts share a high similarity value (>92%) with bacteria belonging to the genus Rickettsia. Phylogenetic analysis revealed that these new endosymbionts are clearly included in the clade of the family Rickettsiaceae, but they occupy an independent phylogenetic position with respect to members of the genus Rickettsia. This is the first report of a member of this family from a host protozoan and from a marine habitat. This result shows that this bacterial group is more diversified and widespread than supposed so far, and that its ecological relevance could until now have been underestimated. In light of these considerations, the two 16S rRNA oligonucleotide probes here presented, specific for members of the Rickettsiaceae, can represent useful tools for further researches on the presence and the spread of these microorganisms in the natural environment.

Candidatus anadelfobacter veles and "Candidatus cyrtobacter comes," two new rickettsiales species hosted by the protist ciliate Euplotes harpa (Ciliophora, Spirotrichea).

ABSTRACT The order Rickettsiales (Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae, Anaplasmataceae, and Holosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “Candidatus Midichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “Candidatus Midichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity of Rickettsiales. In this paper, two new bacteria belonging to the “Candidatus Midichloria” clade, hosted by two different strains of the ciliate protist Euplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the family Rickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “Candidatus Midichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “Candidatus Midichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “Candidatus Anadelfobacter veles” and “Candidatus Cyrtobacter comes.”

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

Abstract 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.

Detection of a Novel Subspecies of Francisella noatunensis as Endosymbiont of the Ciliate Euplotes raikovi.

Francisella are facultative intracellular bacteria causing severe disease in a broad range of animals. Two species are notable: Francisella tularensis, the causative organism of tularemia and a putative warfare agent, and Francisella noatunensis, an emerging fish pathogen causing significant losses in wild and farmed fish. Although various aspects of Francisella biology have been intensively studied, their natural reservoir in periods between massive outbreaks remains mysterious. Protists have been suspected to serve as a disguised vector of Francisella and co-culturing attempts demonstrate that some species are able to survive and multiply within protozoan cells. Here, we report the first finding of a natural occurrence of Francisella sp. as a protist endosymbiont. By molecular and morphological approaches, we identified intracellular bacteria localized in a strain of the marine ciliate Euplotes raikovi, isolated from the coast of Adriatic Sea. Phylogenetic analysis placed these endosymbionts within the genus Francisella, in close but distinct association with F. noatunensis. We suggest the establishment of a novel subspecies within F. noatunensis and propose the cytoplasmatic endosymbiont of E. raikovi as “Candidatus F. noatunensis subsp. endociliophora” subsp. nov.

Feeding behaviour in ciliated protists

Abstract Ciliates are unicellular organisms characterized by the presence of cilia (at least in the juvenile stage), an oral cavity, nuclear dualism, conjugation, equatorial division plane in binary fission, and alveolar pellicular organization. Ciliates play an important role in the general economy of marine, fresh-water and soil environments, being essential links in the food chain. This article reviews the different feeding behaviours present in these protista together with the related digestive processes.

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.

Sulphide oxidation to elemental sulphur in a membrane bioreactor: performance and characterization of the selected microbial sulphur-oxidizing community.

In leather tanning industrial areas sulphide management represents a major problem. However, biological sulphide oxidation to sulphur represents a convenient solution to this problem. Elemental sulphur is easy to separate and the process is highly efficient in terms of energy consumption and effluent quality. As the oxidation process is performed by specialized bacteria, selection of an appropriate microbial community is fundamental for obtaining a good yield. Sulphur oxidizing bacteria (SOB) represent a wide-ranging and highly diversified group of microorganisms with the capability of oxidizing reduced sulphur compounds. Therefore, it is useful to select new microbes that are able to perform this process efficiently. For this purpose, an experimental membrane bioreactor for sulphide oxidation was set up, and the selected microbial community was characterized by constructing 16S rRNA gene libraries and subsequent screening of clones. Fluorescence in situ hybridization (FISH) was then used to assess the relative abundance of different bacterial groups. Sulphide oxidation to elemental sulphur proceeded in an efficient (up to 79% conversion) and stable way in the bioreactor. Both analysis of clone libraries and FISH experiments revealed that the dominant operational taxonomic unit (OTU) in the bioreactor was constituted by Gammaproteobacteria belonging to the Halothiobacillaceae family. FISH performed with the specifically designed probe tios_434 demonstrated that this OTU constituted 90.6+/-1.3% of the bacterial community. Smaller fractions were represented by bacteria belonging to the classes Betaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Clostridia, Mollicutes, Sphingobacteria, Bacteroidetes and Chlorobia. Phylogenetic analysis revealed that clone sequences from the dominant OTU formed a stable clade (here called the TIOS44 cluster), within the Halothiobacillaceae family, with sequences from many organisms that have not yet been validly described. The data indicated that bacteria belonging to the TIOS44 cluster were responsible for the oxidation process.

Bacterial communities in polluted seabed sediments: a molecular biology assay in Leghorn harbor

Seabed sediments of commercial ports are often characterized by high pollution levels. Differences in number and distribution of bacteria in such areas can be related to distribution of pollutants in the port and to sediment conditions. In this study, the bacterial communities of five sites from Leghorn Harbor seabed were characterized, and the main bacterial groups were identified. T-RFLP was used for all samples; two 16S rRNA libraries and in silico digestion of clones were used to identify fingerprint profiles. Library data, phylogenetic analysis, and T-RFLP coupled with in silico digestion of the obtained sequences evidenced the dominance of Proteobacteria and the high percentage of Bacteroidetes in all sites. The approach highlighted similar bacterial communities between samples coming from the five sites, suggesting a modest differentiation among bacterial communities of different harbor seabed sediments and hence the capacity of bacterial communities to adapt to different levels and types of pollution.

Resting cysts: A survival strategy in Protozoa Ciliophora

Abstract Encystment is a reversible cell differentiation process that also requires an antagonistic process, i.e. excystment. Both processes are genetically encoded. Encystment is of common occurrence among free-living ciliates and may serve several different purposes. In some ciliates, this process is part of the regular life cycle (reproductive cyst), while many other ciliates undergo encystment when environmental conditions become adverse (resting cyst). In this review, the different phases and aspects of the encystment process in ciliates, such as the ecological role, the cyst formation, the morphological characteristics of the cysts and their dynamic state during the time and the excysting process are reviewed on the basis on our own results and literature data.

In Situ Identification by Fluorescently Labeled Oligonucleotide Probes of Morphologically Similar, Closely Related Ciliate Species

Ciliate protozoa are important members of microbial communities in which they play specific ecological roles. The determination of single species distribution is fundamental for food web analysis, but species recognition, which is mainly based on morphological characters, is often difficult between closely related species. The use of species-specific, purposely designed, fluorescently labeled probes for in situ hybridization is here presented as an easy and fast identification method for three closely related species belonging to the widespread genus Euplotes, namely E. crassus, E. vannus, and E. minuta, that in spite of their remarkable morphological similarity have significant metabolic and ecological differences. These three species can be detected simultaneously, provided the probes employed are bound to different fluorescent dyes: in this way their relative abundance and their population dynamics in the natural environment can be evaluated. As more ciliate sequences become available in databases, species-specific probes can be designed for other ciliates, thus rendering the application of the method of more general importance. The probes used in this study may also provide a tool to prevent erroneous species identification in future studies.