Ciliate diversity in rodrigo de freitas lagoon (Rio de Janeiro, Brazil) from an integrative standpoint.

The Rodrigo de Freitas Lagoon is a highly eutrophic lacustrine system and has one of the longest histories of exploration and anthropic alteration in Brazil. Despite its relevance, limited studies explored the diversity of micro-eukaryotes in the lagoon. Ciliates (Alveolata, Ciliophora) are overlooked in environmental microbiology, especially in tropical and subtropical ecosystems, resulting in limited knowledge about their diversity and functional relevance in South American habitats, particularly in coastal lagoons. To fill this gap, here we investigated the diversity of ciliates in a brackish coastal lagoon in an urban area of Rio de Janeiro, Brazil, applying and comparing the performance of morphological and metabarcoding approaches. The metabarcoding analysis, based on high-throughput sequencing of the hipervariable region V4 of the 18S rRNA genes detected 37 molecular operational taxonomic units (MOTUs) assigned to Ciliophora, representing only about a half (56.9%) of the diversity detected by microscopy, which counted 65 ciliate morphotypes. The most representative classes in both approaches were Spirotrichea and Oligohymenophorea. The metabarcoding analysis revealed that 35.3% of the ciliate MOTUs had less than 97% similarity to available sequences in the NCBI database, indicating that more than one-third of these MOTUs potentially represents still not represented or undescribed ciliate species in current databases. Our findings indicate that metabarcoding techniques can significantly enhance the comprehension of ciliate diversity in tropical environments, but the scarcity of reference sequences of brackish ciliates in molecular databases represents a challenge to the taxonomic assignment of the MOTUs. This study provides new insights into the diversity of ciliates in a threatened coastal lagoon, revealing a vast array of still unknown and rare ciliate taxonomic units in tropical environments.

Morphological and phylogenetic investigations shed light on evolutionary relationships of the enigmatic genus Copemetopus (Ciliophora, Alveolata), with the proposal of Copemetopus verae sp. nov.

Copemetopus Villeneuve-Brachon, 1940 is a rare, poorly known sapropelic ciliate genus composed of only two valid nominal species. Over time, Copemetopus was taxonomically assigned to Heterotrichea and Armophorea classes, but its phylogenetic affinities remained unknown. Until the present study, there were no molecular data available for Copemetopus representatives. Here, we present the 18S and 28S-rDNA sequences and the phylogenetic position of Copemetopus verae sp. nov., as well as its detailed morphological description based on live observations, protargol impregnation, and scanning electron microscopy. Transmission electron micrographs of the type species C. subsalsus Villeneuve-Brachon, 1940 reveal new morphological traits and a unique somatic ciliature pattern of Copemetopus, composed by short segments of dikinetids with one or two supplementary kinetosomes. The phylogenetic trees recovered Copemetopus as the sister group of the genus Protocruzia, both constituting early-divergent lineages that split first from a common ancestor of Intramacronucleta. Morphological and molecular evidence suggest that Copemetopus is neither a heterotrichean nor an armophorean ciliate, but a distinct clade related to Protocruzia.

Macronuclear Plasticity in Two South American Populations of Spirostomum (Ciliophora, Heterotrichea) Warns About its Use for Species Classification: Revision and New Insights.

Spirostomum is a widely distributed heterotrichean genus composed of well-known species with described ecology and phylogenetic affinities. The morphological classification of Spirostomum species is mostly based on the body size/shape, number of cortical granule rows and macronuclear characteristics. These features along with molecular phylogenies based on ribosomal genes divide the genus into two phylogroups, one including species with a compact macronucleus, and another including species with a moniliform macronucleus. Here, we present our observations on atypical Spirostomum specimens with unusually two distinct macronuclei and shortened adoral zone of membranelles. These atypical forms appeared in the cultures of S. minus and S. yagiui, sampled at different sites in South America (Chile and Brazil) and associated with unrelated substrate types. Morphological observations of living and stained cells, 18S rRNA gene analyses, and a thorough investigation of the literature suggest that the atypical phenotype may be a result of uncommon pathways during the conjugative process. Thus, we demonstrate that studies of ciliate natural populations and their morphological variations, especially from undersampled biogeographical regions, can reveal the boundaries of widely used morphological characters for Spirostomum taxonomy and species identification.

Ciliate Diversity From Aquatic Environments in the Brazilian Atlantic Forest as Revealed by High-Throughput DNA Sequencing.

Rainforest aquatic ecosystems include complex habitats with scarce information on their unicellular eukaryote diversity and community structure. We have investigated the diversity of ciliates in freshwater and brackish environments along the Brazilian Atlantic Forest, based on the hypervariable V4 region of the 18S-rDNA obtained by high-throughput DNA sequencing. Our analyses detected 409 ciliate taxonomic units (OTUs), mostly attributed to the classes Oligohymenophorea and Spirotrichea. A total of 11 classes, 12 subclasses, 112 genera, and 144 species were reported. We found the following: (a) the ciliate communities are more diverse in freshwater- than in Atlantic Forest-associated brackish environments; (b) the ciliate communities are composed by a small amount of highly abundant OTUs, but a high number of low-abundant or rare OTUs; (c) nearly one-third of the ciliate OTUs share less than 97% sequence identity to reference sequences and (d) phylogenetic inference supports the hypothesis that the V4 region of the Ciliophora 18S-rDNA is a suitable marker for accurate evolutionary inferences at class level. Our results showed that a considerable fraction of the HTS-detected diversity of ciliates from Brazilian Atlantic Forest is not represented in the currently available molecular databases.

Parablepharisma (Ciliophora) is not a Heterotrich: A Phylogenetic and Morphological Study with the Proposal of New Taxa.

The genus Parablepharisma Jankowski, 2007 at present includes five species, most of which have not been studied in detail, therefore phylogenetic affinities remained uninvestigated up to now. Parablepharisma is traditionally placed within Heterotrichea based on insufficient existing morphological data, and there are no available Parablepharisma gene sequences in molecular databases to support this placement. This work presents an 18S rDNA-based phylogeny of P. bacteriophora (Kahl, 1932) Jankowski, 2007 and P. brasiliensis sp. nov. We also provide a redescription of P. bacteriophora and P. chlamydophorum (Kahl, 1932) Jankowski, 2007 based on live morphological observations combined with silver impregnation and scanning and transmission electron microscopy. According to characters such as macro- and micronucleus number, the position of the ectosymbiotic bacteria, and the presence/absence of caudal cilia, two new species are described, i.e. P. granulata sp. nov. and P. brasiliensis sp. nov. In addition, we establish Kahlium gen. nov. to include P. chlamydophorum, which has a segmented anterior paroral portion and a twisted posterior paroral section as diagnostic features. To include Parablepharisma and Kahlium gen. nov., we propose Parablepharismidae fam. nov. According to our phylogenetic analyses, Parablepharisma belongs to SAL (Spirotrichea, Armophorea, Litostomatea), being a sister group of Cariacotrichea.

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.

Molecular phylogeny and comparative morphology indicate that odontostomatids (Alveolata, Ciliophora) form a distinct class-level taxon related to Armophorea.

The odontostomatids are among the least studied ciliates, possibly due to their small sizes, restriction to anaerobic environments and difficulty in culturing. Consequently, their phylogenetic affinities to other ciliate taxa are still poorly understood. In the present study, we analyzed newly obtained ribosomal gene sequences of the odontostomatids Discomorphella pedroeneasi and Saprodinium dentatum, together with sequences from the literature, including Epalxella antiquorum and a large assemblage of ciliate sequences representing the major recognized classes. The results show that D. pedroeneasi and S. dentatum form a deep-diverging branch related to metopid and clevelandellid armophoreans, corroborating the old literature. However E. antiquorum clustered with the morphologically discrepant plagiopylids, indicating that either the complex odontostomatid body architecture evolved convergently, or the positioning of E. antiquorum as a plagiopylid is artifactual. A new ciliate class, Odontostomatea n. cl., is proposed based on molecular analyses and comparative morphology of odontostomatids with related taxa.

Crispoic acid, a new compound from Laelia marginata (Orchidaceae), and biological evaluations against parasites, human cancer cell lines and Zika virus.

The phytochemical study of Laelia marginata (Lindl.) L. O. Williams (Orchidaceae) led to the isolation of a new natural product named crispoic acid (1), together with six other known compounds (2-7). The new natural product was identified as a dimer of eucomic acid and was structurally characterised based upon 1D and 2D NMR and HRMS data. Biological assays with plant crude extract, fractions and isolated compounds were performed against two human cancer cell lines (Hela and Siha), and the tropical parasites Trypanosoma cruzi and Leishmania (Leishmania) amazonensis. The phenantrenoid 9,10-dihydro-4-methoxyphenanthren-2,7-diol 2 was active against Hela and Siha cells (CC50 5.86 ± 0.19 and 20.78 ± 2.72 µg/mL, respectively). Sub-lethal concentrations of the flavone rhamnazin 4 were not able to rescue the viability of the Vero cells infected by Zika virus.

Coxiella symbionts are widespread into hard ticks.

Ticks are blood-feeding arthropods and can harbor several bacteria, including the worldwide zoonotic disease Q-fever agent Coxiella burnetii. Recent studies have reported a distinct group of Coxiella mostly associated with Ixodidae ticks, including the primary endosymbionts of Amblyomma americanum. In the present work, a screening for Coxiella infection was performed by 16S ribosomal DNA (rDNA) gene analyses in 293 tick samples of 15 different species sampled worldwide, including Brazil, Colombia, Kenya, and China. Different Coxiella phylotypes were identified, and these putative symbiotic bacteria were detected in ten different Amblyomma tick species. Approximately 61 % of Rhipicephalus sanguineus and ∼37 % of Rhipicephalus microplus DNA samples were positive for Coxiella. Sequence analysis and phylogenetic reconstruction grouped all the detected Coxiella with Coxiella-like symbionts from different Ixodidae ticks. This well-defined clade clearly excludes known phylotypes of C. burnetii pathogens and other Coxiella spp. detected in different environmental samples and other invertebrate hosts.

Rickettsia (Rickettsiales: Rickettsiaceae) Vector Biodiversity in High Altitude Atlantic Forest Fragments Within a Semiarid Climate: A New Endemic Area of Spotted-Fever in Brazil.

Rickettsioses are re-emerging vector-borne zoonoses with a global distribution. Recently, Rickettsia sp. strain Atlantic rainforest has been associated with new human spotted-fever (SF) cases in Brazil, featuring particular clinical signs: eschar formation and lymphadenopathy. These cases have been associated with the tick species, Amblyomma ovale From 2010 until 2015, the Brazilian Health Department confirmed 11 human SF cases in the Maciço de Baturité region, Ceará, Brazil. The present study reports the circulation of Rickettsia spp. in vectors from this entirely new endemic area for SF. A total of 1,727 ectoparasites were collected in this area from the environment, humans, and wild and domestic animals. Samples (n = 887) were screened by polymerase chain reaction (PCR), targeting the gltA and ompA rickettsial genes. Sequencing and phylogenetic analyses of gltA gene amplicons were carried out for 13 samples positive for both screening PCRs. Fragments of gltA and ompA from three samples were cloned, sequenced, and analyzed further. A. ovale and Rhipicephalus sanguineus specimens, collected from dogs, were found to be infected with Rickettsia sp. str. Atlantic rainforest, suggesting the importance of dogs in the epidemic cycle. Candidatus Rickettsia andeanae, Rickettsia felis, and Rickettsia bellii were also found infecting ticks and fleas in five municipalities, demonstrating the broad diversity of rickettsiae in circulation in the studied area. This study reports, for the first time, evidence of infection with Rickettsia sp. strain Atlantic rainforest in A. ovale and R. sanguineus in Ceará, and Ca. R. andeanae in an Atlantic rainforest environment of Brazil.

"Candidatus Fokinia solitaria", a Novel "Stand-Alone" Symbiotic Lineage of Midichloriaceae (Rickettsiales).

Recently, the family Midichloriaceae has been described within the bacterial order Rickettsiales. It includes a variety of bacterial endosymbionts detected in different metazoan host species belonging to Placozoa, Cnidaria, Arthropoda and Vertebrata. Representatives of Midichloriaceae are also considered possible etiological agents of certain animal diseases. Midichloriaceae have been found also in protists like ciliates and amoebae. The present work describes a new bacterial endosymbiont, "Candidatus Fokinia solitaria", retrieved from three different strains of a novel Paramecium species isolated from a wastewater treatment plant in Rio de Janeiro (Brazil). Symbionts were characterized through the full-cycle rRNA approach: SSU rRNA gene sequencing and fluorescence in situ hybridization (FISH) with three species-specific oligonucleotide probes. In electron micrographs, the tiny rod-shaped endosymbionts (1.2 x 0.25-0.35 µm in size) were not surrounded by a symbiontophorous vacuole and were located in the peripheral host cytoplasm, stratified in the host cortex in between the trichocysts or just below them. Frequently, they occurred inside autolysosomes. Phylogenetic analyses of Midichloriaceae apparently show different evolutionary pathways within the family. Some genera, such as "Ca. Midichloria" and "Ca. Lariskella", have been retrieved frequently and independently in different hosts and environmental surveys. On the contrary, others, such as Lyticum, "Ca. Anadelfobacter", "Ca. Defluviella" and the presently described "Ca. Fokinia solitaria", have been found only occasionally and associated to specific host species. These last are the only representatives in their own branches thus far. Present data do not allow to infer whether these genera, which we named "stand-alone lineages", are an indication of poorly sampled organisms, thus underrepresented in GenBank, or represent fast evolving, highly adapted evolutionary lineages.

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.

Transgene expression in tick cells using Agrobacterium tumefaciens.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

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.

Turnerbactin, a novel triscatecholate siderophore from the shipworm endosymbiont Teredinibacter turnerae T7901.

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.

Identification of bacterial infection in neotropical primates.

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations.

A prevalent alpha-proteobacterium Paracoccus sp. in a population of the Cayenne ticks (Amblyomma cajennense) from Rio de Janeiro, Brazil.

As Rocky Mountain Spotted Fever is the most common tick-borne disease in South America, the presence of Rickettsia sp. in Amblyomma ticks is a possible indication of its endemicity in certain geographic regions. In the present work, bacterial DNA sequences related to Rickettsia amblyommii genes in A. dubitatum ticks, collected in the Brazilian state of Mato Grosso, were discovered. Simultaneously, Paracoccus sp. was detected in aproximately 77% of A. cajennense specimens collected in Rio de Janeiro, Brazil. This is the first report of Paracoccus sp. infection in a specific tick population, and raises the possibility of these bacteria being maintained and/or transmitted by ticks. Whether Paracoccus sp. represents another group of pathogenic Rhodobacteraceae or simply plays a role in A. cajennense physiology, is unknown. The data also demonstrate that the rickettsial 16S rRNA specific primers used forRickettsia spp. screening can also detect Paracoccus alpha-proteobacteria infection in biological samples. Hence, a PCR-RFLP strategy is presented to distinguish between these two groups of bacteria.

Coxiella symbionts in the Cayenne tick Amblyomma cajennense.

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.