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.

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.

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.

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.

Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite Leishmania.

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite Leishmania (Crithidia and Leptomonas), as well as plant-infecting PhytomonasLeptomonas pyrrhocoris was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as Leptomonas seymouri bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed "Leishbunyavirus" (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed "Leishbunyaviridae" Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus Leishmaniavirus (LRV1/2), implying that it was acquired at about the same time the Leishmania became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.

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.

Comparative genomics of Leishmania (Mundinia).

BACKGROUND: Trypanosomatids of the genus Leishmania are parasites of mammals or reptiles transmitted by bloodsucking dipterans. Many species of these flagellates cause important human diseases with clinical symptoms ranging from skin sores to life-threatening damage of visceral organs. The genus Leishmania contains four subgenera: Leishmania, Sauroleishmania, Viannia, and Mundinia. The last subgenus has been established recently and remains understudied, although Mundinia contains human-infecting species. In addition, it is interesting from the evolutionary viewpoint, representing the earliest branch within the genus and possibly with a different type of vector. Here we analyzed the genomes of L. (M.) martiniquensis, L. (M.) enriettii and L. (M.) macropodum to better understand the biology and evolution of these parasites. RESULTS: All three genomes analyzed were approximately of the same size (~ 30 Mb) and similar to that of L. (Sauroleishmania) tarentolae, but smaller than those of the members of subgenera Leishmania and Viannia, or the genus Endotrypanum (~ 32 Mb). This difference was explained by domination of gene losses over gains and contractions over expansions at the Mundinia node, although only a few of these genes could be identified. The analysis predicts significant changes in the Mundinia cell surface architecture, with the most important ones relating to losses of LPG-modifying side chain galactosyltransferases and arabinosyltransferases, as well as ß-amastins. Among other important changes were gene family contractions for the oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins. CONCLUSIONS: We suggest that adaptation of Mundinia to different vectors and hosts has led to alternative host-parasite relationships and, thereby, made some proteins redundant. Thus, the evolution of genomes in the genus Leishmania and, in particular, in the subgenus Mundinia was mainly shaped by host (or vector) switches.

Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution.

Unicellular flagellates of the family Trypanosomatidae are obligatory parasites of invertebrates, vertebrates and plants. Dixenous species are aetiological agents of a number of diseases in humans, domestic animals and plants. Their monoxenous relatives are restricted to insects. Because of the high biological diversity, adaptability to dramatically different environmental conditions, and omnipresence, these protists have major impact on all biotic communities that still needs to be fully elucidated. In addition, as these organisms represent a highly divergent evolutionary lineage, they are strikingly different from the common 'model system' eukaryotes, such as some mammals, plants or fungi. A number of excellent reviews, published over the past decade, were dedicated to specialized topics from the areas of trypanosomatid molecular and cell biology, biochemistry, host-parasite relationships or other aspects of these fascinating organisms. However, there is a need for a more comprehensive review that summarizing recent advances in the studies of trypanosomatids in the last 30 years, a task, which we tried to accomplish with the current paper.

The reduced genome of Candidatus Kinetoplastibacterium sorsogonicusi, the endosymbiont of Kentomonas sorsogonicus (Trypanosomatidae): loss of the haem-synthesis pathway.

Trypanosomatids of the genera Angomonas and Strigomonas (subfamily Strigomonadinae) have long been known to contain intracellular beta-proteobacteria, which provide them with many important nutrients such as haem, essential amino acids and vitamins. Recently, Kentomonas sorsogonicus, a divergent member of Strigomonadinae, has been described. Herein, we characterize the genome of its endosymbiont, Candidatus Kinetoplastibacterium sorsogonicusi. This genome is completely syntenic with those of other known Ca. Kinetoplastibacterium spp., but more reduced in size (~742 kb, compared with 810-833 kb, respectively). Gene losses are not concentrated in any hot-spots but are instead distributed throughout the genome. The most conspicuous loss is that of the haem-synthesis pathway. For long, removing haemin from the culture medium has been a standard procedure in cultivating trypanosomatids isolated from insects; continued growth was considered as an evidence of endosymbiont presence. However, we demonstrate that, despite bearing the endosymbiont, K. sorsogonicus cannot grow in culture without haem. Thus, the traditional test cannot be taken as a reliable criterion for the absence or presence of endosymbionts in trypanosomatid flagellates. It remains unclear why the ability to synthesize such an essential compound was lost in Ca. K. sorsogonicusi, whereas all other known bacterial endosymbionts of trypanosomatids retain them.

Leptomonas pyrrhocoris: Genomic insight into Parasite's Physiology.

BACKGROUND: Leptomonas pyrrhocoris is a parasite of the firebug Pyrrhocoris apterus. This flagellate has been recently proposed as a model species for studying different aspects of the biology of monoxenous trypanosomatids, including host - parasite interactions. During its life cycle L. pyrrhocoris never tightly attaches to the epithelium of the insect gut. In contrast, its dixenous relatives (Leishmania spp.) establish a stable infection via attachment to the intestinal walls of their insect hosts. MATERIAL AND METHODS: This process is mediated by chemical modifications of the cell surface lipophosphoglycans. In our study we tested whether the inability of L. pyrrhocoris to attach to the firebug's midgut is associated with the absence of these glycoconjugates. We also analyzed evolution of the proteins involved in proper lipophosphoglycan assembly, cell attachment and establishment of a stable infection in L. pyrrhocoris, L. seymouri, and Leishmania spp. Our comparative analysis demonstrated differences in SCG/L/R repertoire between the two parasite subgenera, Leishmania and Viannia, which may be related to distinct life strategies in various Leishmania spp. The genome of L. pyrrhocoris encodes 6 SCG genes, all of which are quite divergent from their orthologs in the genus Leishmania. Using direct probing with an antibody recognizing the ß-Gal side chains of lipophosphoglycans, we confirmed that these structures are not synthesized in L. pyrrhocoris. CONCLUSION: We conclude that either the SCG enzymes are not active in this species (similarly to SCG5/7 in L. major), or they possess a different biochemical activity.

RNA viruses in trypanosomatid parasites: a historical overview.

Viruses of trypanosomatids are now being extensively studied because of their diversity and the roles they play in flagellates' biology. Among the most prominent examples are leishmaniaviruses implicated in pathogenesis of Leishmania parasites. Here, we present a historical overview of this field, starting with early reports of virus-like particles on electron microphotographs, and culminating in detailed molecular descriptions of viruses obtained using modern next generation sequencing-based techniques. Because of their diversity, different life cycle strategies and host specificity, we believe that trypanosomatids are a fertile ground for further explorations to better understand viral evolution, routes of transitions, and molecular mechanisms of adaptation to different hosts.

Leptomonas seymouri: Adaptations to the Dixenous Life Cycle Analyzed by Genome Sequencing, Transcriptome Profiling and Co-infection with Leishmania donovani.

The co-infection cases involving dixenous Leishmania spp. (mostly of the L. donovani complex) and presumably monoxenous trypanosomatids in immunocompromised mammalian hosts including humans are well documented. The main opportunistic parasite has been identified as Leptomonas seymouri of the sub-family Leishmaniinae. The molecular mechanisms allowing a parasite of insects to withstand elevated temperature and substantially different conditions of vertebrate tissues are not understood. Here we demonstrate that L. seymouri is well adapted for the environment of the warm-blooded host. We sequenced the genome and compared the whole transcriptome profiles of this species cultivated at low and high temperatures (mimicking the vector and the vertebrate host, respectively) and identified genes and pathways differentially expressed under these experimental conditions. Moreover, Leptomonas seymouri was found to persist for several days in two species of Phlebotomus spp. implicated in Leishmania donovani transmission. Despite of all these adaptations, L. seymouri remains a predominantly monoxenous species not capable of infecting vertebrate cells under normal conditions.

Revised classification of the subfamily Leishmaniinae (Trypanosomatidae).

In the present study, we critically revised the recently proposed classification of the subfamily Leishmaniinae Maslov et Lukes in Jirku et al., 2012. Agreeing with erection of the genus Zelonia Shaw, Camargo et Teixeira in Espinosa et al., 2017 and the subgenus Mundinia Shaw, Camargo et Teixeira in Espinosa et al., 2017 within the genus Leishmania Ross, 1908, we argue that other changes are not well justified. We propose to: (i) raise Paraleishmania Cupolillo, Medina-Acosta, Noyes, Momen et Grimaldi, 2000 to generic rank; (ii) create a new genus Borovskyia gen. n. to accommodate the former Leptomonas barvae Maslov et Lukes, 2010 as its type and only species; (iii) leave the subfamily Leishmaniinae as originally defined, but establish two infrafamilies within it: Leishmaniatae infrafam. n. and Crithidiatae infrafam. n.

Diversity of Trypanosomatids in Cockroaches and the Description of Herpetomonas tarakana sp. n.

In this study, we surveyed six species of cockroaches, two synanthropic (i.e. ecologically associated with humans) and four wild, for intestinal trypanosomatid infections. Only the wild cockroach species were found to be infected, with flagellates of the genus Herpetomonas. Two distinct genotypes were documented, one of which was described as a new species, Herpetomonas tarakana sp. n. We also propose a revision of the genus Herpetomonas and creation of a new subfamily, Phytomonadinae, to include Herpetomonas, Phytomonas, and a newly described genus Lafontella n. gen. (type species Lafontella mariadeanei comb. n.), which can be distinguished from others by morphological and molecular traits.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era.

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Host specificity, pathogenicity, and mixed infections of trypanoplasms from freshwater fishes.

This work summarizes the results of the 8-year study focused on Trypanoplasma sp. parasitizing freshwater fishes in the vicinity of Kyiv, Ukraine. Out of 570 fish specimens of 2 different species analyzed, 440 individuals were found to be infected. The prevalence of infection ranged from 24 % in Abramis brama Linnaeus (freshwater bream) to 100 % in Cobitis taenia Linnaeus (spined loach). The level of parasitemia also varied between moderate in freshwater bream and very high in spined loach. Interestingly, no clinical manifestations of trypanoplasmosis were observed even in extremely heavily infected C. taenia. We hypothesize that different species may differ in evolutionary timing allowing for reciprocal adaptation of the members of the "host-parasite" system. Molecular analysis of the 18S rRNA sequences revealed that several specimens were simultaneously infected with at least two different trypanoplasm species. To the best of our knowledge, this is the first report of the mixed infection with fish trypanoplasms.

Reisolation and redescription of Balantidium duodeni Stein, 1867 (Litostomatea, Trichostomatia).

In this work, we present reisolation and redescription of Balantidium duodeni Stein, 1867 from the European common brown frog Rana temporaria Linnaeus, 1758 using light and electron microscopy. This species has a unique morphological feature--its cells are flattened along the dorsoventral axis. Because of its unique morphology and localization (duodenum) in the gastrointestinal tract of the host, it has been proposed to recognize B. duodeni as a member of separate genus, Balantidiopsis Penard, 1922. Molecular phylogenetic analysis demonstrates it to be close to the type species Balantidium entozoon (Ehrenberg, 1838). We argue that its placement into separate genus is not substantiated. We also propose to reinstate the genus Balantioides Alexeieff, 1931 with the type species Paramecium coli (Malmstein, 1857). The recently proposed generic name for this taxon, Neobalantidium Pomajbíková et al., 2013, is a junior synonym of the previously recognized name.

High prevalence of trypanosome co-infections in freshwater fishes.

One thousand three hundred seventy three fish specimens of eight different species from the vicinity of Kyiv, Ukraine, were examined for the presence of trypanosomes and 921 individuals were found to be infected. The prevalence of infection ranged from 24% in freshwater bream, Abramis brama (Linnaeus), to 100% in spined loach, Cobitis 'taenia' Linnaeus. The level of parasitaemia also varied significantly between generally mild infections in pikeperch, Sander lucioperca (Linnaeus), and heavy ones in C. 'taenia'. In most cases the infections with trypanosomes were asymptomatic. Cases of co-infection with species of Trypanoplasma Laveran et Mesnil, 1901 were documented for five out of eight examined host species. Molecular analysis of the 18S rDNA sequences revealed that four hosts, namely northern pike, Esox lucius Linnaeus, freshwater bream, spined loach and European perch, Perca fluviatilis Linnaeus, were simultaneously infected with two different trypanosome species. Our findings advocate the view that to avoid the risk posed by mixed infections, subsequent molecular taxonomic studies should be performed on clonal lines derived from laboratory cultures of fish trypanosomes.