Identification of diverse RNA viruses in Obscuromonas flagellates (Euglenozoa: Trypanosomatidae: Blastocrithidiinae).

Trypanosomatids (Euglenozoa) are a diverse group of unicellular flagellates predominately infecting insects (monoxenous species) or circulating between insects and vertebrates or plants (dixenous species). Monoxenous trypanosomatids harbor a wide range of RNA viruses belonging to the families Narnaviridae, Totiviridae, Qinviridae, Leishbuviridae, and a putative group of tombus-like viruses. Here, we focus on the subfamily Blastocrithidiinae, a previously unexplored divergent group of monoxenous trypanosomatids comprising two related genera: Obscuromonas and Blastocrithidia. Members of the genus Blastocrithidia employ a unique genetic code, in which all three stop codons are repurposed to encode amino acids, with TAA also used to terminate translation. Obscuromonas isolates studied here bear viruses of three families: Narnaviridae, Qinviridae, and Mitoviridae. The latter viral group is documented in trypanosomatid flagellates for the first time. While other known mitoviruses replicate in the mitochondria, those of trypanosomatids appear to reside in the cytoplasm. Although no RNA viruses were detected in Blastocrithidia spp., we identified an endogenous viral element in the genome of B. triatomae indicating its past encounter(s) with tombus-like viruses.

Intricate balance of dually-localized catalase modulates infectivity of Leptomonas seymouri (Kinetoplastea: Trypanosomatidae).

Nearly all aerobic organisms are equipped with catalases, powerful enzymes scavenging hydrogen peroxide and facilitating defense against harmful reactive oxygen species. In trypanosomatids, this enzyme was not present in the common ancestor, yet it had been independently acquired by different lineages of monoxenous trypanosomatids from different bacteria at least three times. This observation posited an obvious question: why was catalase so "sought after" if many trypanosomatid groups do just fine without it? In this work, we analyzed subcellular localization and function of catalase in Leptomonas seymouri. We demonstrated that this enzyme is present in the cytoplasm and a subset of glycosomes, and that its cytoplasmic retention is H2O2-dependent. The ablation of catalase in this parasite is not detrimental in vivo, while its overexpression resulted in a substantially higher parasite load in the experimental infection of Dysdercus peruvianus. We propose that the capacity of studied flagellates to modulate the catalase activity in the midgut of its insect host facilitates their development and protects them from oxidative damage at elevated temperatures.

Phylogenetic framework to explore trait evolution in Trypanosomatidae.

The number of sequenced trypanosomatid genomes has reached a critical point so that they are now available for almost all genera and subgenera. Based on this, we inferred a phylogenomic tree and propose it as a framework to study trait evolution together with some examples of how to do it.

Parasites of firebugs in Austria with focus on the "micro"-diversity of the cosmopolitan trypanosomatid Leptomonas pyrrhocoris.

In this work, we investigated parasites of the firebug Pyrrhocoris apterus in Austria and demonstrated that in addition to the extensively studied Leptomonas pyrrhocoris, it can also be infected by Blastocrithidia sp. and by a mermithid, which for the first time has been characterized using molecular methods. This diversity can be explained by the gregarious lifestyle, as well as the coprophagous and cannibalistic behavior of the insect hosts that makes them susceptible to various parasites. In addition, we showed no tight association of the L. pyrrhocoris haplotypes and geographical locations (at least, considering the relatively small scale of locations in Austria) implying that the natural populations of L. pyrrhocoris are mixed due to the mobility of their firebug hosts.

The Mite Steatonyssus periblepharus Is a Novel Potential Vector of the Bat Parasite Trypanosoma dionisii.

Trypanosoma dionisii, for which only bat bugs (Cimicidae) had previously been demonstrated as vectors, was, for the first time, detected in the gamasine mite Steatonyssus periblepharus in Russia. The molecular phylogenetic analysis indicated that trypanosomes found in these mites belong to the "clade A" of T. dionisii, which, based on genetic distances, can be considered as a species separate from the sister clade B, and according to available data also has a distinct geographic distribution. The presence of developmental forms of T. dionisii resembling those previously described during the development of this trypanosome in cimicids suggests that S. periblepharus is a novel vector of the studied trypanosome.

Phylogenetic position of the pelobiont Mastigamoeba aspera and revision of the family Mastigamoebidae (Archamoebae, Pelobiontida).

In this study, we presented the results of our molecular phylogenetic analysis of Archamoebae using a newly obtained sequence of the 18S rRNA gene of Mastigamoeba aspera, the type species of the genus. In addition, we characterized the general organization of the tubulin cytoskeleton of M. aspera by immunofluorescent microscopy and TEM. Our findings allowed us to revise the family Mastigamoebidae and establish within it two subfamilies: Mastigamoebinae for the genus Mastigamoeba and Seraviniinae subfam. nov. for the genera Seravinia gen. nov., Paramastigamoeba gen. nov., Iodamoeba and Endolimax. The type genera Mastigamoeba and Seravinia are clearly distinguished by the structure of the basal apparatus of flagella. In addition, the tubulin cytoskeleton of several Mastigamoeba spp. contains a rim of microtubules around the nuclei.

RNA viruses of Crithidia bombi, a parasite of bumblebees.

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi - a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

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.

Shining the spotlight on the neglected: new high-quality genome assemblies as a gateway to understanding the evolution of Trypanosomatidae.

BACKGROUND: Protists of the family Trypanosomatidae (phylum Euglenozoa) have gained notoriety as parasites affecting humans, domestic animals, and agricultural plants. However, the true extent of the group's diversity spreads far beyond the medically and veterinary relevant species. We address several knowledge gaps in trypanosomatid research by undertaking sequencing, assembly, and analysis of genomes from previously overlooked representatives of this protistan group. RESULTS: We assembled genomes for twenty-one trypanosomatid species, with a primary focus on insect parasites and Trypanosoma spp. parasitizing non-human hosts. The assemblies exhibit sizes consistent with previously sequenced trypanosomatid genomes, ranging from approximately 18 Mb for Obscuromonas modryi to 35 Mb for Crithidia brevicula and Zelonia costaricensis. Despite being the smallest, the genome of O. modryi has the highest content of repetitive elements, contributing nearly half of its total size. Conversely, the highest proportion of unique DNA is found in the genomes of Wallacemonas spp., with repeats accounting for less than 8% of the assembly length. The majority of examined species exhibit varying degrees of aneuploidy, with trisomy being the most frequently observed condition after disomy. CONCLUSIONS: The genome of Obscuromonas modryi represents a very unusual, if not unique, example of evolution driven by two antidromous forces: i) increasing dependence on the host leading to genomic shrinkage and ii) expansion of repeats causing genome enlargement. The observed variation in somy within and between trypanosomatid genera suggests that these flagellates are largely predisposed to aneuploidy and, apparently, exploit it to gain a fitness advantage. High heterogeneity in the genome size, repeat content, and variation in chromosome copy numbers in the newly-sequenced species highlight the remarkable genome plasticity exhibited by trypanosomatid flagellates. These new genome assemblies are a robust foundation for future research on the genetic basis of life cycle changes and adaptation to different hosts in the family Trypanosomatidae.

Evolution of RNA viruses in trypanosomatids: new insights from the analysis of Sauroleishmania.

RNA viruses play an important role in Leishmania biology and virulence. Their presence was documented in three (out of four) Leishmania subgenera. Sauroleishmania of reptiles remained the only underinvestigated group. In this work, we analyzed the viral occurrence in Sauroleishmania spp. and detected RNA viruses in three out of seven isolates under study. These viruses were of two families-Narnaviridae and Totiviridae. Phylogenetic inferences demonstrated that totiviruses from L. adleri and L. tarentolae group together within a larger cluster of LRV2s, while a narnavirus of L. gymnodactyli appeared as a phylogenetic relative of narnaviruses of Blechomonas spp. Taken together, our work not only expanded the range of trypanosomatids that can host RNA viruses but also shed new light on the evolution and potential routes of viral transmission in these flagellates.

A neo-functionalized homolog of host transmembrane protein controls localization of bacterial endosymbionts in the trypanosomatid Novymonas esmeraldas.

The stability of endosymbiotic associations between eukaryotes and bacteria depends on a reliable mechanism ensuring vertical inheritance of the latter. Here, we demonstrate that a host-encoded protein, located at the interface between the endoplasmic reticulum of the trypanosomatid Novymonas esmeraldas and its endosymbiotic bacterium Ca. Pandoraea novymonadis, regulates such a process. This protein, named TMP18e, is a product of duplication and neo-functionalization of the ubiquitous transmembrane protein 18 (TMEM18). Its expression level is increased at the proliferative stage of the host life cycle correlating with the confinement of bacteria to the nuclear vicinity. This is important for the proper segregation of bacteria into the daughter host cells as evidenced from the TMP18e ablation, which disrupts the nucleus-endosymbiont association and leads to greater variability of bacterial cell numbers, including an elevated proportion of aposymbiotic cells. Thus, we conclude that TMP18e is necessary for the reliable vertical inheritance of endosymbionts.

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.

The Roles of Mosquitoes in the Circulation of Monoxenous Trypanosomatids in Temperate Climates.

Monoxenous (insect-restricted) trypanosomatids are highly diverse and abundant in nature. While many papers focus on the taxonomy and distribution of these parasites, studies on their biology are still scarce. In particular, this concerns trypanosomatids inhabiting the ubiquitous mosquitoes. To shed light on the circulation of monoxenous trypanosomatids with the participation of mosquitoes, we performed a multifaceted study combining the examination of naturally- and experimentally-infected insects using light and electron microscopy and molecular identification of parasites. Our examination of overwintering mosquitoes (genera Culex and Culiseta) revealed that their guts contained living trypanosomatids, which can be spread during the next season. Experimental infections with Crithidia spp. demonstrated that imagines represent permissive hosts, while larvae are resistant to these parasites. We argue that for the parasites with wide specificity, mosquitoes act as facultative hosts. Other trypanosomatids may have specific adaptations for vertical transmission in these insects at the expense of their potential to infect a wider range of hosts and, consequently, abundance in nature.

Roe deer (Capreolus capreolus) are a novel potential reservoir for human visceral leishmaniasis in the Emilia-Romagna region of northeastern Italy.

Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum, responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region.

Elimination of LRVs Elicits Different Responses in Leishmania spp.

Leishmaniaviruses (LRVs) have been demonstrated to enhance progression of leishmaniasis, a vector-transmitted disease with a wide range of clinical manifestations that is caused by flagellates of the genus Leishmania. Here, we used two previously proposed strategies of the LRV ablation to shed light on the relationships of two Leishmania spp. with their respective viral species (L. guyanensis, LRV1 and L. major, LRV2) and demonstrated considerable difference between two studied systems. LRV1 could be easily eliminated by the expression of exogenous capsids regardless of their origin (the same or distantly related LRV1 strains, or even LRV2), while LRV2 was only partially depleted in the case of the native capsid overexpression. The striking differences were also observed in the effects of complete viral elimination with 2'C-methyladenosine (2-CMA) on the transcriptional profiles of these two Leishmania spp. While virtually no differentially expressed genes were detected after the LRV1 removal from L. guyanensis, the response of L. major after ablation of LRV2 involved 87 genes, the analysis of which suggested a considerable stress experienced even after several passages following the treatment. This effect on L. major was also reflected in a significant decrease of the proliferation rate, not documented in L. guyanensis and naturally virus-free strain of L. major. Our findings suggest that integration of L. major with LRV2 is deeper compared with that of L. guyanensis with LRV1. We presume this determines different effects of the viral presence on the Leishmania spp. infections. IMPORTANCE Leishmania spp. represent human pathogens that cause leishmaniasis, a widespread parasitic disease with mild to fatal clinical manifestations. Some strains of leishmaniae bear leishmaniaviruses (LRVs), and this has been shown to aggravate disease course. We investigated the relationships of two distally related Leishmania spp. with their respective LRVs using different strategies of virus removal. Our results suggest the South American L. guyanensis easily loses its virus with no important consequences for the parasite in the laboratory culture. Conversely, the Old-World L. major is refractory to virus removal and experiences a prominent stress if this removal is nonetheless completed. The drastically different levels of integration between the studied Leishmania spp. and their viruses suggest distinct effects of the viral presence on infections in these species of parasites.

Development of two species of the Trypanosoma theileri complex in tabanids.

BACKGROUND: Trypanosoma theileri species complex includes parasites of Bovidae (cattle, sheep, goat, etc.) and Cervidae (deer) transmitted mainly by Tabanidae (horse flies and deerflies) and keds (Hippoboscidae). While morphological discrimination of species is challenging, two big clades, TthI and TthII, each containing parasites isolated from bovids and cervids, have been identified phylogenetically. To date, the development in the vector has been studied in detail only for the ked-transmitted sheep parasite T. melophagium (TthII), while the fate of trypanosomes in tabanids was described only briefly by light microscopy. METHODS: We collected infected tabanids of various species and identified trypanosomes by molecular phylogenetic analysis. The morphology and development of trypanosomes was studied using the combination of statistical analyses as well as light and electron microscopy. RESULTS: Two trypanosome species belonging to both TthI and TthII clades of the T. theileri complex were identified. The phylogenetic position of these two trypanosomes suggests that they parasitize deer. Both species were indiscernible by morphology in the vector and showed the same development in its intestine. In contrast to the previously described development of T. melophagium, both trypanosomes of tabanids only transiently infected midgut and settled mainly in the ileum, while pylorus and rectum were neglected. Meanwhile, the flagellates developing in the tabanid ileum (pyriform epimastigotes and metacyclic trypomastigotes) showed similarities to the corresponding stages in T. melophagium by morphology, mode of attachment to the host cuticle and formation of the fibrillar matrix surrounding the mass of developing parasites. In addition, for the first time to our knowledge we documented extraintestinal stages in these trypanosomes, located in the space between the epithelium and circular muscles. CONCLUSIONS: The development of different species of flagellates of the T. theileri complex in their insect vectors shows many similarities, which can be explained not only by their common origin, but also the same transmission mode, i.e. contamination of the oral mucosa with the gut content released after squashing the insect either by tongue or teeth. The observed differences (concerning primarily the distribution of developmental stages in the intestine) are associated rather with the identity of vectors than the phylogenetic position of parasites.

Analyses of Leishmania-LRV Co-Phylogenetic Patterns and Evolutionary Variability of Viral Proteins.

Leishmania spp. are important pathogens causing a vector-borne disease with a broad range of clinical manifestations from self-healing ulcers to the life-threatening visceral forms. Presence of Leishmania RNA virus (LRV) confers survival advantage to these parasites by suppressing anti-leishmanial immunity in the vertebrate host. The two viral species, LRV1 and LRV2 infect species of the subgenera Viannia and Leishmania, respectively. In this work we investigated co-phylogenetic patterns of leishmaniae and their viruses on a small scale (LRV2 in L. major) and demonstrated their predominant coevolution, occasionally broken by intraspecific host switches. Our analysis of the two viral genes, encoding the capsid and RNA-dependent RNA polymerase (RDRP), revealed them to be under the pressure of purifying selection, which was considerably stronger for the former gene across the whole tree. The selective pressure also differs between the LRV clades and correlates with the frequency of interspecific host switches. In addition, using experimental (capsid) and predicted (RDRP) models we demonstrated that the evolutionary variability across the structure is strikingly different in these two viral proteins.

Genomics of Trypanosomatidae: Where We Stand and What Needs to Be Done?

Trypanosomatids are easy to cultivate and they are (in many cases) amenable to genetic manipulation. Genome sequencing has become a standard tool routinely used in the study of these flagellates. In this review, we summarize the current state of the field and our vision of what needs to be done in order to achieve a more comprehensive picture of trypanosomatid evolution. This will also help to illuminate the lineage-specific proteins and pathways, which can be used as potential targets in treating diseases caused by these parasites.

A New Model Trypanosomatid, Novymonas esmeraldas: Genomic Perception of Its "Candidatus Pandoraea novymonadis" Endosymbiont.

The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.