Diversity of RNA viruses in the cosmopolitan monoxenous trypanosomatid Leptomonas pyrrhocoris.

BACKGROUND: Trypanosomatids are parasitic flagellates well known because of some representatives infecting humans, domestic animals, and cultural plants. Many trypanosomatid species bear RNA viruses, which, in the case of human pathogens Leishmania spp., influence the course of the disease. One of the close relatives of leishmaniae, Leptomonas pyrrhocoris, has been previously shown to harbor viruses of the groups not documented in other trypanosomatids. At the same time, this species has a worldwide distribution and high prevalence in the natural populations of its cosmopolitan firebug host. It therefore represents an attractive model to study the diversity of RNA viruses. RESULTS: We surveyed 106 axenic cultures of L. pyrrhocoris and found that 64 (60%) of these displayed 2-12 double-stranded RNA fragments. The analysis of next-generation sequencing data revealed four viral groups with seven species, of which up to five were simultaneously detected in a single trypanosomatid isolate. Only two of these species, a tombus-like virus and an Ostravirus, were earlier documented in L. pyrrhocoris. In addition, there were four new species of Leishbuviridae, the family encompassing trypanosomatid-specific viruses, and a new species of Qinviridae, the family previously known only from metatranscriptomes of invertebrates. Currently, this is the only qinvirus with an unambiguously determined host. Our phylogenetic inferences suggest reassortment in the tombus-like virus owing to the interaction of different trypanosomatid strains. Two of the new Leishbuviridae members branch early on the phylogenetic tree of this family and display intermediate stages of genomic segment reduction between insect Phenuiviridae and crown Leishbuviridae. CONCLUSIONS: The unprecedented wide range of viruses in one protist species and the simultaneous presence of up to five viral species in a single Leptomonas pyrrhocoris isolate indicate the uniqueness of this flagellate. This is likely determined by the peculiarity of its firebug host, a highly abundant cosmopolitan species with several habits ensuring wide distribution and profuseness of L. pyrrhocoris, as well as its exposure to a wider spectrum of viruses compared to other trypanosomatids combined with a limited ability to transmit these viruses to its relatives. Thus, L. pyrrhocoris represents a suitable model to study the adoption of new viruses and their relationships with a protist host.

Vickermania gen. nov., trypanosomatids that use two joined flagella to resist midgut peristaltic flow within the fly host.

BACKGROUND: The family Trypanosomatidae encompasses parasitic flagellates, some of which cause serious vector-transmitted diseases of humans and domestic animals. However, insect-restricted parasites represent the ancestral and most diverse group within the family. They display a range of unusual features and their study can provide insights into the biology of human pathogens. Here we describe Vickermania, a new genus of fly midgut-dwelling parasites that bear two flagella in contrast to other trypanosomatids, which are unambiguously uniflagellate. RESULTS: Vickermania has an odd cell cycle, in which shortly after the division the uniflagellate cell starts growing a new flagellum attached to the old one and preserves their contact until the late cytokinesis. The flagella connect to each other throughout their whole length and carry a peculiar seizing structure with a paddle-like apex and two lateral extensions at their tip. In contrast to typical trypanosomatids, which attach to the insect host's intestinal wall, Vickermania is separated from it by a continuous peritrophic membrane and resides freely in the fly midgut lumen. CONCLUSIONS: We propose that Vickermania developed a survival strategy that relies on constant movement preventing discharge from the host gut due to intestinal peristalsis. Since these parasites cannot attach to the midgut wall, they were forced to shorten the period of impaired motility when two separate flagella in dividing cells interfere with each other. The connection between the flagella ensures their coordinate movement until the separation of the daughter cells. We propose that Trypanosoma brucei, a severe human pathogen, during its development in the tsetse fly midgut faces the same conditions and follows the same strategy as Vickermania by employing an analogous adaptation, the flagellar connector.

A Study of Oribatid Mites as Potential Intermediate Hosts of Anoplocephalid Tapeworms of Tatra chamois and Tatra marmots from the Tatra Mountains, Central Europe, and Report of a New Intermediate Host for Andrya cuniculi, the Parasite of Leporidae.

Tatra chamois (Rupicapra rupicapra tatrica (Blahout 1972)) and Tatra marmot (Marmota marmota latirostris (Kratochvíl 1961)) are significant endemic subspecies of the subalpine and alpine ranges of the Tatra Mountains in Central Europe. In four studied localities in the range of their typical biotopes in Slovakia and Poland, we investigated intestinal parasites of Tatra chamois and Tatra marmots, with an emphasis on anoplocephalid tapeworms. We also studied the occurrence, species diversity, and abundance of oribatid mites as intermediate hosts thereof, and the prevalence of cysticercoid larval stages of anoplocephalid tapeworms in collected oribatids using morphological and molecular methods. Coprological analyses revealed the average positivity of Moniezia spp. in chamois faeces at 23.5% and Ctenotaenia marmotae in marmot samples at 71.1%, with significant differences between the localities under study. Morphological analyses determined the presence of cysticercoids in five oribatid species: Ceratozetes gracilis, Edwardzetes edwardsi, Scheloribates laevigatus, Trichoribates novus, and Tectocepheus velatus sarekensis. This is the first record of T. v. sarekensis as an intermediate host of anoplocephalid tapeworms, as well as the first report of Andrya cuniculi occurrence in the territory of the Tatra Mountains, confirmed also by molecular methods.

Enhancing oral squamous cell carcinoma prediction: the prognostic power of the worst pattern of invasion and the limited impact of molecular resection margins.

Objective: Oral squamous cell carcinoma (OSCC) originates from the mucosal lining of the oral cavity. Almost half of newly diagnosed cases are classified as advanced stage IV disease, which makes resection difficult. In this study, we investigated the pathological features and mutation profiles of tumor margins in OSCC. Methods: We performed hierarchical clustering of principal components to identify distinct patterns of tumor growth and their association with patient prognosis. We also used next-generation sequencing to analyze somatic mutations in tumor and marginal tissue samples. Results: Our analyses uncovered that the grade of worst pattern of invasion (WPOI) is strongly associated with depth of invasion and patient survival in multivariable analysis. Mutations were primarily detected in the DNA isolated from tumors, but several mutations were also identified in marginal tissue. In total, we uncovered 29 mutated genes, mainly tumor suppressor genes involved in DNA repair including BRCA genes; however none of these mutations significantly correlated with a higher chance of relapse in our medium-size cohort. Some resection margins that appeared histologically normal harbored tumorigenic mutations in TP53 and CDKN2A genes. Conclusion: Even histologically normal margins may contain molecular alterations that are not detectable by conventional histopathological methods, but NCCN classification system still outperforms other methods in the prediction of the probability of disease relapse.

Comparative Analysis of Three Trypanosomatid Catalases of Different Origin.

Most trypanosomatid flagellates do not have catalase. In the evolution of this group, the gene encoding catalase has been independently acquired at least three times from three different bacterial groups. Here, we demonstrate that the catalase of Vickermania was obtained by horizontal gene transfer from Gammaproteobacteria, extending the list of known bacterial sources of this gene. Comparative biochemical analyses revealed that the enzymes of V. ingenoplastis, Leptomonas pyrrhocoris, and Blastocrithidia sp., representing the three independent catalase-bearing trypanosomatid lineages, have similar properties, except for the unique cyanide resistance in the catalase of the latter species.

Endosymbiont Capture, a Repeated Process of Endosymbiont Transfer with Replacement in Trypanosomatids Angomonas spp.

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of Angomonas ambiguus, which is sister to A. desouzai, but bears an endosymbiont genetically close to that of A. deanei. Based on phylogenetic inferences, it was proposed that the bacterium from A. deanei had been horizontally transferred to A. ambiguus. Here, we sequenced the bacterial genomes from two A. ambiguus isolates, including a new one from Papua New Guinea, and compared them with the published genome of the A. deanei endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of A. ambiguus were obtained from A. deanei and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.