Genes, genomes, and transcriptomes of ciliates and their prokaryotic endosymbionts

The topic of my PhD research was the study of symbiotic relationships between ciliated protozoa (phylum Ciliophora) and their bacterial symbionts, employing mainly molecular techniques with the goal of characterizing both partners in the symbiosis. My projects can be grouped into four categories, each corresponding to one of the parts of this thesis. In the first part I present the description of 6 bacterial species, providing information on their classification, relationship with the host, and phylogeny. The peculiar features of some of these prokaryotes, like the presence of flagella in representatives of lineages previously considered non-flagellated, allowed to propose evolutionary hypotheses of broader interest, including inferences on the characteristics of the mitochondria ancestor. The second part of the thesis contains the first genomic analysis of a bacterial symbiont, Polynucleobacter necessarius, harbored by a ciliate, Euplotes. The genomic structure and the gene content were comparatively studied with closely related free-living organisms in order to understand the physiological bases of the ciliate/symbiont interaction and study the process of genome reduction, that is seldom investigated outside of model arthropodan systems. In the third part of the thesis, I present systematic and multi-disciplinary surveys of symbiont-harboring ciliate genera, combining old and new data on many aspects of their diversity, including phylogeny and biogeography. The fourth part briefly reports the results of my most recent line of research, ciliate transcriptomics, and in particular the successful optimization of a single-cell RNA-seq protocol suitable for protists. In summary, this thesis provides characterization works on previously understudied organisms, hypotheses on the evolution of varied features, and the first results in two areas in their infancy: genomics of ciliate symbionts, and single-cell transcriptomics of protists. [ITA] L’argomento della mia ricerca di dottorato è stato lo studio delle relazioni simbiotiche tra protozoi ciliati (Phylum Ciliophora) e i loro endosimbionti batterici, utilizzando principalmente tecniche molecolari al fine di caratterizzare entrambi i partner della simbiosi. I miei progetti possono essere suddivisi in quattro categorie, corrispondenti ciascuna a una delle quattro parti di questa tesi. Nella prima parte presento la descrizione di 6 specie batteriche, fornisco informazioni sulla loro classificazione, rapporto con l’ospite, e filogenesi. Le caratteristiche più peculiari di alcuni di questi procarioti, come la presenza di flagelli in rappresentanti di gruppi precedentemente considerati non flagellati, ha consentito di proporre ipotesi evolutive di più ampio interesse, incluse ricostruzioni delle possibili caratteristiche dell’antenato dei mitocondri. La seconda parte di questa tesi contiene la prima analisi genomica su un simbionte batterico, Polynucleobacter necessarius, ospite di un ciliato, Euplotes. La struttura genomica e il contenuto genico sono stati studiati comparativamente rispetto a organismi a vita libera strettamente imparentati, per comprendere le basi fisiologiche dell’interazione ciliato/simbionte e studiare il processo di riduzione genomica, raramente investigato al di fuori dei sistemi modello negli artropodi. Nella terza parte della tesi presento indagini sistematiche e multidisciplinari su generi di ciliati che ospitano simbionti, integrando dati vecchi e recenti su molti aspetti della loro diversità, incluse filogenesi e biogeografia. La quarta parte riporta brevemente i risultati della mia più recente linea di ricerca, la trascrittomica dei ciliati, e in particolare il successo nell’ottimizzazione di un protocollo di RNA-seq su singola cellula efficace sui protisti. In sintesi, questa tesi fornisce caratterizzazioni di organismi precedentemente poco noti, ipotesi sull’evoluzione di diversi caratteri, e i primi risultati in due campi appena agli inizi: la genomica dei simbionti di ciliati, e la trascrittomica su singola cellula di protisti

Summer holidays as break-point in shaping a tannery sludge microbial community around a stable core microbiota

Recently, several investigations focused on the discovery of a bacterial consortium shared among different wastewater treatment plants (WWTPs). Nevertheless, the definition of a core microbiota over time represents the necessary counterpart in order to unravel the dynamics of bacterial communities in these environments. Here we performed a monthly survey on the bacterial community of a consortial industrial plant. Objectives of this study were: (1) to identify a core microbiota constant over time; (2) to evaluate the temporal dynamics of the community during one year. A conspicuous and diversified core microbiota is constituted by operational taxonomic units which are present throughout the year in the plant. Community composition data confirm that the presence and abundance of bacteria in WWTPs is highly consistent at high taxonomic level. Our results indicate however a difference in microbial community structure between two groups of samples, identifying the summer holiday period as the break-point. Changes in the structure of the microbial community occur otherwise gradually, one month after another. Further studies will clarify how the size and diversity of the core microbiota could affect the observed dynamics

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

Biogeography and Character Evolution of the Ciliate Genus Euplotes (Spirotrichea, Euplotia), with Description of Euplotes curdsi sp. nov.

Ciliates comprise a diverse and ecologically important phylum of unicellular protists. One of the most specious and best-defined genera is Euplotes, which constitutes more than 70 morphospecies, many of which have never been molecularly tested. The increasing number of described Euplotes taxa emphasizes the importance for detailed characterizations of new ones, requiring standardized morphological observations, sequencing of molecular markers and careful comparison with previous literature. Here we describe Euplotes curdsi sp. nov., distinguishable by the combination of the following features: 45±65 μm length, oval or elongated shape with both ends rounded, narrow peristome with 25±34 adoral membranelles, conspicuous paroral membrane, double-eurystomus dorsal argyrome type, 6±7 dorsolateral kineties and 10 frontoventral cirri. Three populations of the novel species have been found in brackish and marine samples in the Mediterranean and the White Sea. We provide the SSU rRNA gene sequences of these populations, and an updated phylogeny of the genus Euplotes. Using the molecular phylogenetic tree, we inferred aspects of the biogeographical history of the genus and the evolution of its most important taxonomic characters in order to provide a frame for future descriptions. Ultimately, these data reveal recurrent trends of freshwater invasion and highlight the dynamic, yet convergent, morphological evolution of Euplotes

Rediscovering the genus Lyticum, multiflagellated symbionts of the order Rickettsiales

Among the bacterial symbionts harbored by the model organism Paramecium, many still lack a recent investigation that includes a molecular characterization. The genus Lyticum consists of two species of large-sized bacteria displaying numerous flagella, despite their inability to move inside their hosts’ cytoplasm. We present a multidisciplinary redescription of both species, using the deposited type strains as well as newly collected material. On the basis of 16S rRNA gene sequences, we assigned Lyticum to the order Rickettsiales, that is intensely studied because of its pathogenic representatives and its position as the extant group most closely related to the mitochondrial ancestor. We provide conclusive proofs that at least some Rickettsiales possess actual flagella, a feature that has been recently predicted from genomic data but never confirmed. We give support to the hypothesis that the mitochondrial ancestor could have been flagellated, and provide the basis for further studies on these ciliate endosymbionts

Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria

We present the complete genomic sequence of the essential symbiont Polynucleobacter necessarius (Betaproteobacteria), which is a valuable case study for several reasons. First, it is hosted by a ciliated protist, Euplotes; bacterial symbionts of ciliates are still poorly known because of a lack of extensive molecular data. Second, the single species P. necessarius contains both symbiotic and free-living strains, allowing for a comparison between closely related organisms with different ecologies. Third, free-living P. necessarius strains are exceptional by themselves because of their small genome size, reduced metabolic flexibility, and high worldwide abundance in freshwater systems. We provide a comparative analysis of P. necessarius metabolism and explore the peculiar features of a genome reduction that occurred on an already streamlined genome. We compare this unusual system with current hypotheses for genome erosion in symbionts and free-living bacteria, propose modifications to the presently accepted model, and discuss the potential consequences of translesion DNA polymerase loss

Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic ‘joeniid’ Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp

Molecular characterization and phylogeny of four new species of the genus Trichonympha (Parabasalia, Trichonymphea) from lower termite hindguts

Members of the genus Trichonympha are among the most well-known, recognizable and widely distributed parabasalian symbionts of lower termites and the wood-eating cockroach species of the genus Cryptocercus. Nevertheless, the species diversity of this genus is largely unknown. Molecular data have shown that the superficial morphological similarities traditionally used to identify species are inadequate, and have challenged the view that the same species of the genus Trichonympha can occur in many different host species. Ambiguities in the literature, uncertainty in identification of both symbiont and host, and incomplete samplings are limiting our understanding of the systematics, ecology and evolution of this taxon. Here we describe four closely related novel species of the genus Trichonympha collected from South American and Australian lower termites: Trichonympha hueyi sp. nov. from Rugitermes laticollis, Trichonympha deweyi sp. nov. from Glyptotermes brevicornis, Trichonympha louiei sp. nov. from Calcaritermes temnocephalus and Trichonympha webbyae sp. nov. from Rugitermes bicolor. We provide molecular barcodes to identify both the symbionts and their hosts, and infer the phylogeny of the genus Trichonympha based on small subunit rRNA gene sequences. The analysis confirms the considerable divergence of symbionts of members of the genus Cryptocercus, and shows that the two clades of the genus Trichonympha harboured by termites reflect only in part the phylogeny of their hosts

Survey of Paramecium duboscqui using three markers and assessment of the molecular variability in the genus Paramecium.

The genus Paramecium (phylum Ciliophora) is one of the best-known among protozoa. Nevertheless, the knowledge on the diversity and distribution of species within this genus was remarkably scarce until recent times. In the last years a constantly growing amount of data has formed, especially on the distribution of species and the characterization of molecular markers. Much effort has been made on detecting clades inside each morphospecies, which could suggest the presence of sibling species complexes as in the famous case of Paramecium aurelia. In this work we present new data on Paramecium duboscqui, one of the morphospecies that have not yet been surveyed employing DNA sequences as markers. We obtained data from nine strains sampled around the world, using the three most commonly employed markers (18S rRNA gene, ITS1-5.8S-ITS2 and COI gene sequences). Moreover, we compared our results with those already available for other Paramecium species, and performed phylogenetic analyses for the entire genus. We also expanded the knowledge on the ITS2 secondary structure and its usefulness in studies on Paramecium. Our approach, that considers the data of all the species together, highlighted some characteristic patterns as well as some ambiguities that should be further investigated