New Finds on the Systematics of Cycloposthiid Ciliates (Ciliophora: Entodiniomorphida: Cycloposthiidae) Based on New 18S-rDNA Sequences from a Brazilian Capybara.

The family Cycloposthiidae (Entodiniomorphida) comprises ciliated protists that are symbionts of the gastrointestinal tract of several herbivore mammals, such as rodents, elephants, equids, primates, hippopotamus, marsupials, rhinoceros and tapir, where they contribute to the digestion of their host's plant-based diet. Despite the significance of these ciliates to the evolution of their hosts, many characters used in the taxonomy of the group are homoplastic and most of the valid species do not have molecular data available. For these reasons the systematics of this family is poorly understood. Here, we sequenced the 18S-rDNA of ten cycloposthiids, including nine Cycloposthium spp. and Monoposthium cynodontum, all of them isolated from the cecum of a Brazilian capybara. Our phylogenetic analyses indicate that the family Cycloposthiidae might be polyphyletic, while M. cynodontum and Cycloposthium spp. constitute a single monophyletic group. Given the great morphological and molecular similarities between members of M. cynodontum and Cycloposthium ciliates, it is possible that this species, although it has been described in the genus Monoposthium, is actually a Cycloposthium ciliate.

The Hidden World of Rickettsiales Symbionts: "Candidatus Spectririckettsia obscura," a Novel Bacterium Found in Brazilian and Indian Paramecium caudatum.

Symbioses between bacteria and eukaryotes are widespread and may have significant impact on the evolutionary history of symbiotic partners. The order Rickettsiales is a lineage of intracellular Alphaproteobacteria characterized by an obligate association with a wide range of eukaryotic hosts, including several unicellular organisms, such as ciliates and amoebas. In this work, we characterized the Rickettsiales symbionts associated with two different genotypes of the freshwater ciliate Paramecium caudatum originated from freshwater environments in distant geographical areas. Phylogenetic analyses based on 16S rRNA gene showed that the two symbionts are closely related to each other (99.4% identity), belong to the family Rickettsiaceae, but are far-related with respect to previously characterized Rickettsiales. Consequently, they were assigned to a new species of a novel genus, namely "Candidatus Spectririckettsia obscura." Screening on a database of short reads from 16S rRNA gene amplicon-based profiling studies confirmed that bacterial sequences related to the new symbiont are preferentially retrieved from freshwater environments, apparently with extremely scarce occurrence (< 0.1% positive samples). The present work provides new information on the still under-explored biodiversity of Rickettsiales, in particular those associated to ciliate host cells.

A House for Two--Double Bacterial Infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) Sampled in Southeastern Brazil.

Several ciliated protists form symbiotic associations with a diversity of microorganisms, leading to drastic impact on their ecology and evolution. In this work, two Euplotes spp. sampled in Rio de Janeiro, Brazil, were identified based on morphological and molecular features as Euplotes woodruffi strain Sq1 and E. encysticus strain Sq2 and investigated for the presence of endosymbionts. While E. woodruffi Sq1 stably hosts two bacterial populations, namely Polynucleobacter necessarius (Betaproteobacteria) and a new member of the family "Candidatus Midichloriaceae" (Alphaproteobacteria, Rickettsiales), here described as "Candidatus Bandiella woodruffii," branching with a broad host range bacterial group found in association with cnidarians, sponges, euglenoids, and some arthropods; in E. encysticus Sq2 no symbiotic bacterium could be detected. The dispersion ability of this novel bacterium was tested by co-incubating E. woodruffi Sq1 with three different ciliate species. Among the tested strains "Ca. B. woodruffii" could only be detected in association with E. encysticus Sq2 with a prevalence of 20 % after 1 week and 40 % after 2 weeks, maintaining this level for up to 6 months. Nevertheless, this apparent in vitro association was abolished when E. woodruffi Sq1 donor was removed from the microcosm, suggesting that this bacterium has the capacity for at least a short-term survival outside its natural host and the aptitude to ephemerally interact with other organisms. Together, these findings strongly suggest the need for more detailed investigations to evaluate the host range for "Ca. B. woodruffii" and any possible pathogenic effect of this bacterium on other organisms including humans.

Molecular Phylogeny of the Family Ophryscolecidae (Ciliophora, Litostomatea) Inferred from 18S rDNA Sequences.

The 18S rDNA was used to infer oral ciliature patterns of evolution within the family Ophryoscolecidae, with the addition of five new sequences of ciliates from the genus Ostracodinium. Our data confirmed the monophyly of the subfamilies Entodiniinae and Ophryoscolecinae, but more analysis would be required for the definition of the status of the subfamily Diplodiniinae. The oral infraciliature patterns reflect evolutionary divergence in the family Ophryscolecidae, observing monophyly on Entodinium-type, Diplodinium-type, Ostracodinium-type, Epidinium-type, and Ophryoscolex-type. The ancestral infraciliature of Entodinium-type cannot be proven, however, the position of Entodinium-type showed closer of Diplodinium-type than Ophryoscolex-type, corroborating previous studies using morphological characters. The observed inconsistencies reflect the need to increase the number of 18S rDNA sequences to family Ophryoscolecidae and investigate the evolution of this group using other molecular markers.

Toxicological impacts and likely protein targets of bisphenol a in Paramecium caudatum.

Bisphenol A (BPA) is a widely used plasticizer agent and a well-known ubiquitous endocrine disruptor, which is frequently associated with a series of reproductive, developmental, and transgenerational effects over wildlife, livestocks, and humans. Although extensive toxicological data is available for metazoans, the impact of BPA over unicellular eukaryotes, which represents a considerable proportion of eukaryotic diversity, remains largely overlooked. Here, we used acute end-point toxicological assay and an inverted virtual-screening (IVS) approach to evaluate cellular impairments infringed by BPA over the cosmopolitan ciliated protist, Paramecium caudatum. Our data indicate a clear time-dependent effect over P. caudatum survival, which seems to be a consequence of disruptions to multiple core cellular functions, such as DNA and cell replication, transcription, translation and signaling pathways. Finally, the use of this ciliate as a biosensor to monitor BPA within environments and the relevance of bioinformatic methods to leverage our current knowledge on the impacts of emerging contaminants to biological systems are discussed.

Molecular and Morphological Characterization of a Brazilian Lineage of Plasmodium ( Novyella) Unalis in Turdus Spp. (Passeriformes) of the Atlantic Forest, with Remarks on New Hosts and High Genetic Variation.

Plasmodium spp. are haemosporidian protozoans that alternate their live cycles between bloodsucking Culicidae dipterans and vertebrate hosts (mammals, reptiles, and birds). In birds, these parasites are the causative agents of the so-called avian malaria, a disease associated with considerable declines and extinctions in the avifauna in different geographical regions. In this work, we applied a multidisciplinary approach, light microscopy and cytochrome oxidase b (cyt b) gene sequence analysis, for characterization of Plasmodium spp. found in association with wild birds of the genus Turdus, collected in Atlantic forest fragments of southeastern Brazil. From the total 90 analyzed birds, 58 (47 Turdus rufiventris, 9 Turdus leucomelas, 1 Turdus albicollis, and 1 Turdus flavipes) were positively infected with Plasmodium unalis, a haemosporidian that was previously detected in Turdus fuscater in Colombia and in penguins in Brazil, but has never been found in association with these Turdus species of this present work. Moreover, all 7 new sequences of P. unalis cyt b gene clustered into a monophyletic clade with previously characterized P. unalis sequences with a mean genetic divergence of 1.6% and with a maximum divergence of 3.1%, indicating for a high degree of intraspecific polymorphism within this parasitic species. Together, our data highlight the existence a high degree of intraspecific variation within P. unalis and highlight the importance of integrative taxonomy to an accurate identification and characterization of avian haemosporidian parasites.

An Unbiased Genome-Wide View of the Mutation Rate and Spectrum of the Endosymbiotic Bacterium Teredinibacter turnerae.

Mutations contribute to genetic variation in all living systems. Thus, precise estimates of mutation rates and spectra across a diversity of organisms are required for a full comprehension of evolution. Here, a mutation-accumulation (MA) assay was carried out on the endosymbiotic bacterium Teredinibacter turnerae. After ∼3,025 generations, base-pair substitutions (BPSs) and insertion-deletion (indel) events were characterized by whole-genome sequencing analysis of 47 independent MA lines, yielding a BPS rate of 1.14 × 10-9 per site per generation and indel rate of 1.55 × 10-10 events per site per generation, which are among the highest within free-living and facultative intracellular bacteria. As in other endosymbionts, a significant bias of BPSs toward A/T and an excess of deletion mutations over insertion mutations are observed for these MA lines. However, even with a deletion bias, the genome remains relatively large (∼5.2 Mb) for an endosymbiotic bacterium. The estimate of the effective population size (Ne) in T. turnerae is quite high and comparable to free-living bacteria (∼4.5 × 107), suggesting that the heavy bottlenecking associated with many endosymbiotic relationships is not prevalent during the life of this endosymbiont. The efficiency of selection scales with increasing Ne and such strong selection may have been operating against the deletion bias, preventing genome erosion. The observed mutation rate in this endosymbiont is of the same order of magnitude of those with similar Ne, consistent with the idea that population size is a primary determinant of mutation-rate evolution within endosymbionts, and that not all endosymbionts have low Ne.

Focusing on genera to improve species identification: revised systematics of the ciliate Spirostomum.

Although many papers dealing with the description of new ciliate taxa are published each year, species taxonomy and identification in most groups of the phylum Ciliophora remain confused. This is largely due to a scarcity of surveys on the systematics of immediately higher levels (genera and families) providing data for old and new species together. Spirostomum is a common and distinctive inhabitant of fresh- and brackish water environments, including artificial and eutrophic ones, and is a good model for applied ecology and symbiosis research. Despite this, only 3 of the numerous species are commonly cited, and no studies have yet confirmed their monophyly, with the consequence that reproducibility of the results may be flawed. In this paper we present morphological and molecular data for 30 Spirostomum populations representing 6 different morphospecies, some of which were collected in previously unreported countries. We performed a detailed revision of Spirostomum systematics combining literature surveys, new data on hundreds of organisms and statistical and phylogenetic analyses; our results provide insights on the evolution, ecology and distribution of known morphospecies and a novel one: Spirostomum subtilis sp. n. We also offer tools for quick species identification.

Genetic modification of Teredinibacter turnerae, an endosymbiont with biotechnological potential.

Teredinibacter turnerae belongs to a group of biotechnologically relevant bacteria. Gene transfer into T. turnerae was achieved by using pPROBE'-gfp[ASV] derived plasmids through conjugative mating with Escherichia coli DH5alpha pRK2073. Transferred plasmids were stably maintained and T. turnerae could also act as a donor to transfer these mobilizable plasmids. Constructs for both constitutive and IPTG-inducible gene expression were obtained, representing new tools for gene overexpression in T. turnerae.

Physiological traits of the symbiotic bacterium Teredinibacter turnerae isolated from the mangrove shipworm Neoteredo reynei.

Nutrition in the Teredinidae family of wood-boring mollusks is sustained by cellulolytic/nitrogen fixing symbiotic bacteria of the Teredinibacter clade. The mangrove Teredinidae Neoteredo reynei is popularly used in the treatment of infectious diseases in the north of Brazil. In the present work, the symbionts of N. reynei, which are strictly confined to the host's gills, were conclusively identified as Teredinibacter turnerae. Symbiont variants obtained in vitro were able to grow using casein as the sole carbon/nitrogen source and under reduced concentrations of NaCl. Furthermore, cellulose consumption in T. turnerae was clearly reduced under low salt concentrations. As a point of interest, we hereby report first hand that T. turnerae in fact exerts antibiotic activity. Furthermore, this activity was also affected by NaCl concentration. Finally, T. turnerae was able to inhibit the growth of Gram-negative and Gram-positive bacteria, this including strains of Sphingomonas sp., Stenotrophomonas maltophilia, Bacillus cereus and Staphylococcus sciuri. Our findings introduce new points of view on the ecology of T. turnerae, and suggest new biotechnological applications for this marine bacterium.

Physiological traits of the symbiotic bacterium Teredinibacter turnerae isolated from the mangrove shipworm Neoteredo reynei

Nutrition in the Teredinidae family of wood-boring mollusks is sustained by cellulolytic/nitrogen fixing symbiotic bacteria of the Teredinibacter clade. The mangrove Teredinidae Neoteredo reynei is popularly used in the treatment of infectious diseases in the north of Brazil. In the present work, the symbionts of N. reynei, which are strictly confined to the host's gills, were conclusively identified as Teredinibacter turnerae. Symbiont variants obtained in vitro were able to grow using casein as the sole carbon/nitrogen source and under reduced concentrations of NaCl. Furthermore, cellulose consumption in T. turnerae was clearly reduced under low salt concentrations. As a point of interest, we hereby report first hand that T. turnerae in fact exerts antibiotic activity. Furthermore, this activity was also affected by NaCl concentration. Finally, T. turnerae was able to inhibit the growth of Gram-negative and Gram-positive bacteria, this including strains of Sphingomonas sp., Stenotrophomonas maltophilia, Bacillus cereus and Staphylococcus sciuri. Our findings introduce new points of view on the ecology of T. turnerae, and suggest new biotechnological applications for this marine bacterium.