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.

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.

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.

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.

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.

Genome Analysis of Endotrypanum and Porcisia spp., Closest Phylogenetic Relatives of Leishmania, Highlights the Role of Amastins in Shaping Pathogenicity.

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses.

Euglenozoa is a species-rich group of protists, which have extremely diverse lifestyles and a range of features that distinguish them from other eukaryotes. They are composed of free-living and parasitic kinetoplastids, mostly free-living diplonemids, heterotrophic and photosynthetic euglenids, as well as deep-sea symbiontids. Although they form a well-supported monophyletic group, these morphologically rather distinct groups are almost never treated together in a comparative manner, as attempted here. We present an updated taxonomy, complemented by photos of representative species, with notes on diversity, distribution and biology of euglenozoans. For kinetoplastids, we propose a significantly modified taxonomy that reflects the latest findings. Finally, we summarize what is known about viruses infecting euglenozoans, as well as their relationships with ecto- and endosymbiotic bacteria.

Development of Monoxenous Trypanosomatids and Phytomonads in Insects.

In this review, we summarize the current data on development of monoxenous trypanosomatids and phytomonads in various insects. Of these, Diptera and Hemiptera are the main host groups, and, consequently, most available information concerns their parasites. Within the insect body, the midgut and hindgut are the predominant colonization sites; in addition, some trypanosomatids can invade the foregut, Malpighian tubules, hemolymph, and/or salivary glands. Differences in the intestinal structure and biology of the host determine the variety of parasites' developmental and transmission strategies. Meanwhile, similar mechanisms are used by unrelated trypanosomatids, reflecting the limited range of options to achieve the same goal.

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.

The First Non-LRV RNA Virus in Leishmania.

In this work, we describe the first Leishmania-infecting leishbunyavirus-the first virus other than Leishmania RNA virus (LRV) found in trypanosomatid parasites. Its host is Leishmaniamartiniquensis, a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LmarLBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LmarLBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LmarLBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in L.martiniquensis poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other Leishmania species.

If host is refractory, insistent parasite goes berserk: Trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus.

Here we characterized the development of the trypanosomatid Blastocrithidia raabei in the dock bug Coreus marginatus using light and electron microscopy. This parasite has been previously reported to occur in the host hemolymph, which is rather typical for dixenous trypanosomatids transmitted to a plant or vertebrate with insect's saliva. In addition, C. marginatus has an unusual organization of the intestine, which makes it refractory to microbial infections: two impassable segments isolate the anterior midgut portion responsible for digestion and absorption from the posterior one containing symbiotic bacteria. Our results refuted the possibility of hemolymph infection, but revealed that the refractory nature of the host provokes very aggressive behavior of the parasite and makes its life cycle more complex, reminiscent of that in some dixenous trypanosomatids. In the pre-barrier midgut portion, the epimastigotes of B. raabei attach to the epithelium and multiply similarly to regular insect trypanosomatids. However, when facing the impassable constricted region, the parasites rampage and either fiercely break through the isolating segments or attack the intestinal epithelium in front of the barrier. The cells of the latter group pass to the basal lamina and accumulate there, causing degradation of the epitheliocytes and thus helping the epimastigotes of the former group to advance posteriorly. In the symbiont-containing post-barrier midgut segment, the parasites either attach to bacterial cells and produce cyst-like amastigotes (CLAs) or infect enterocytes. In the rectum, all epimastigotes attach either to the cuticular lining or to each other and form CLAs. We argue that in addition to the specialized life cycle B. raabei possesses functional cell enhancements important either for the successful passage through the intestinal barriers (enlarged rostrum and well-developed Golgi complex) or as food reserves (vacuoles in the posterior end).

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.

Development of Phytomonas lipae sp. n. (Kinetoplastea: Trypanosomatidae) in the true bug Coreus marginatus (Heteroptera: Coreidae) and insights into the evolution of life cycles in the genus Phytomonas.

Here we described a new trypanosomatid species, Phytomonas lipae, parasitizing the dock bug Coreus marginatus based on axenic culture and in vivo material. Using light and electron microscopy we characterized the development of this flagellate in the intestine, hemolymph and salivary glands of its insect host. The intestinal promastigotes of Phytomonas lipae do not divide and occur only in the anterior part of the midgut. From there they pass into hemolymph, increasing in size, and then to salivary glands, where they actively proliferate without attachment to the host's epithelium and form infective endomastigotes. We conducted molecular phylogenetic analyses based on 18s rRNA, gGAPDH and HSP83 gene sequences, of which the third marker performed the best in terms of resolving phylogenetic relationships within the genus Phytomonas. Our inference demonstrated rather early origin of the lineage comprising the new species, right after that of P. oxycareni, which represents the earliest known branch within the Phytomonas clade. This allowed us to compare the development of P. lipae and three other Phytomonas spp. in their insect hosts and reconstruct the vectorial part of the life cycle of their common ancestor.

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.

Correction: Obligate development of Blastocrithidia papi (Trypanosomatidae) in the Malpighian tubules of Pyrrhocoris apterus (Hemiptera) and coordination of host-parasite life cycles.

[This corrects the article DOI: 10.1371/journal.pone.0204467.].

RNA Viruses in Blechomonas (Trypanosomatidae) and Evolution of Leishmaniavirus.

In this work, we analyzed viral prevalence in trypanosomatid parasites (Blechomonas spp.) infecting Siphonaptera and discovered nine species of viruses from three different groups (leishbunyaviruses, narnaviruses, and leishmaniaviruses). Most of the flagellate isolates bore two or three viral types (mixed infections). Although no new viral groups were documented in Blechomonas spp., our findings are important for the comprehension of viral evolution. The discovery of bunyaviruses in blechomonads was anticipated, since these viruses have envelopes facilitating their interspecific transmission and have already been found in various trypanosomatids and metatranscriptomes with trypanosomatid signatures. In this work, we also provided evidence that even representatives of the family Narnaviridae are capable of host switching and evidently have accomplished switches multiple times in the course of their evolution. The most unexpected finding was the presence of leishmaniaviruses, a group previously solely confined to the human pathogens Leishmania spp. From phylogenetic inferences and analyses of the life cycles of Leishmania and Blechomonas, we concluded that a common ancestor of leishmaniaviruses most likely infected Leishmania first and was acquired by Blechomonas by horizontal transfer. Our findings demonstrate that evolution of leishmaniaviruses is more complex than previously thought and includes occasional host switching.IMPORTANCE Flagellates belonging to the genus Leishmania are important human parasites. Some strains of different Leishmania species harbor viruses (leishmaniaviruses), which facilitate metastatic spread of the parasites, thus aggravating the disease. Up until now, these viruses were known to be hosted only by Leishmania Here, we analyzed viral distribution in Blechomonas, a related group of flagellates parasitizing fleas, and revealed that they also bear leishmaniaviruses. Our findings shed light on the entangled evolution of these viruses. In addition, we documented that Blechomonas can be also infected by leishbunyaviruses and narnaviruses, viral groups known from other insects' flagellates.

Obligate development of Blastocrithidia papi (Trypanosomatidae) in the Malpighian tubules of Pyrrhocoris apterus (Hemiptera) and coordination of host-parasite life cycles.

Blastocrithidia papi is a unique trypanosomatid in that its life cycle is synchronized with that of its host, and includes an obligate stage of development in Malpighian tubules (MTs). This occurs in firebugs, which exited the winter diapause. In the short period, preceding the mating of overwintered insects, the flagellates penetrate MTs of the host, multiply attached to the epithelial surface with their flagella, and start forming cyst-like amastigotes (CLAs) in large agglomerates. By the moment of oviposition, a large number of CLAs are already available in the rectum. They are discharged on the eggs' surface with feces, used for transmission of bugs' symbiotic bacteria, which are compulsorily engulfed by the newly hatched nymphs along with the CLAs. The obligate development of B. papi in MTs is definitely linked to the life cycle synchronization. The absence of peristalsis allow the trypanosomatids to accumulate and form dense CLA-forming subpopulations, whereas the lack of peritrophic structures facilitates the extensive discharge of CLAs directly into the hindgut lumen. The massive release of CLAs associated with oviposition is indispensable for maximization of the infection efficiency at the most favorable time point.

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.

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.