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Trypanosoma rangeli and Trypanosoma cruzi are generalist trypanosomes sharing a wide range of mammalian hosts; they are transmitted by triatomine bugs, and are the only trypanosomes infecting humans in the Neotropics. Their origins, phylogenetic relationships, and emergence as human parasites have long been subjects of interest. In the present study, taxon-rich analyses (20 trypanosome species from bats and terrestrial mammals) using ssrRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), heat shock protein-70 (HSP70) and Spliced Leader RNA sequences, and multilocus phylogenetic analyses using 11 single copy genes from 15 selected trypanosomes, provide increased resolution of relationships between species and clades, strongly supporting two main sister lineages: lineage Schizotrypanum, comprising T. cruzi and bat-restricted trypanosomes, and Tra[Tve-Tco] formed by T. rangeli, Trypanosoma vespertilionis and Trypanosoma conorhini clades. Tve comprises European T. vespertilionis and African T. vespertilionis-like of bats and bat cimicids characterised in the present study and Trypanosoma sp. Hoch reported in monkeys and herein detected in bats. Tco included the triatomine-transmitted tropicopolitan T. conorhini from rats and the African NanDoum1 trypanosome of civet (carnivore). Consistent with their very close relationships, Tra[Tve-Tco] species shared highly similar Spliced Leader RNA structures that were highly divergent from those of Schizotrypanum. In a plausible evolutionary scenario, a bat trypanosome transmitted by cimicids gave origin to the deeply rooted Tra[Tve-Tco] and Schizotrypanum lineages, and bat trypanosomes of diverse genetic backgrounds jumped to new hosts. A long and independent evolutionary history of T. rangeli more related to Old World trypanosomes from bats, rats, monkeys and civets than to Schizotrypanum spp., and the adaptation of these distantly related trypanosomes to different niches of shared mammals and vectors, is consistent with the marked differences in transmission routes, life-cycles and host-parasite interactions, resulting in T. cruzi (but not T. rangeli) being pathogenic to humans., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

The genus Phytomonas includes trypanosomatids transmitted to the fruits, latex, and phloem of vascular plants by hemipterans. We inferred the phylogenetic relationships of plant and insect isolates assigned to the previously defined genetic groups A-F and H of Phytomonas, particularly those from groups A, C and E comprising flagellates of Solanaceae fruits. Phylogenetic analyses using glycosomal Glyceraldehyde Phosphate Dehydrogenase (gGAPDH) and Small Subunit rRNA (SSU rRNA) genes strongly supported the monophyly of the genus Phytomonas and its division into seven main infrageneric phylogenetic lineages (Phy clades). Isolates from fruit or latex do not constitute monophyletic assemblages but disperse through more than one lineages. In this study, fruit flagellates were distributed in three clades: PhyA, formed by isolates from Solanaceae and phytophagous hemipterans; PhyC comprising flagellates from four plant families; and PhyE, which contains 15 fruit isolates from seven species of Solanaceae. The flagellates of PhyE are described as Phytomonas dolleti n. sp. according to their positioning in phylogenetic trees, complemented by data about their life cycle, and developmental and morphological characteristics in cultures, fruits of Solanum spp., and salivary glands of the vector, the phytophagous hemipteran Arvelius albopunctatus (Pentatomidae)., (Crown Copyright © 2016. Published by Elsevier GmbH. All rights reserved.)

We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple-fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove-like forms are common in stationary-phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well-developed flagellar lamella, and many grooves in pumpkin-like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Background: Several Trypanosoma species transmitted by leeches infect marine and freshwater fish worldwide. To date, all South American fish trypanosome species identified have been based on unreliable morphological parameters. We recently isolated and cultured trypanosomes from the Brazilian armoured catfishes Hypostomus luetkeni and H. affinis. Here, we report the first phylogenetic analyses of South American (Brazilian) trypanosomes isolated from fish, and from leeches removed from these fish. We also analysed morphologically and morphometrically the different forms of fish, leech and cultured trypanosomes., Methods: V7V8 SSU rRNA and gGAPDH sequences were used for phylogenetic analysis of Brazilian fish and leech trypanosomes. Trypanosomes from cultures, fish blood and leech samples were also characterized morphologically and morphometrically by light and electron microscopy., Results: In blood smears from fish high trypanosome prevalence (90-100 %) and parasitemia (0.9-1.0x10(2)) were observed. Phylogenetic relationships using SSU rRNA and gGAPDH showed that, despite relevant sequence divergence, all Brazilian fish (and derived cultures) and leech trypanosomes clustered together into a single clade. The Brazilian clade clustered with European, North American and African fish trypanosomes. Based on sequence analysis, we uncovered a new species of Brazilian fish trypanosome, Trypanosoma abeli n. sp. Trypanosoma abeli cultures contained pleomorphic epimastigotes, small trypomastigotes and rare sphaeromastigotes. Ultrastructural features of T. abeli included a cytostome-cytopharynx complex in epi- and trypomastigotes, a compact rod-like kinetoplast, lysosome-related organelles (LROs) and multivesicular bodies. Trypanosomes found in fish blood smears and leech samples were highly pleomorphic, in agreement with sequence data suggesting that catfishes and leeches often have mixed trypanosome infections., Conclusions: Trypanosoma abeli n. sp. is the first trypanosome from South American fishes isolated in culture, positioned in phylogenetic trees and characterized at the ultrastructural level. Trypanosoma abeli n. sp. is highly prevalent in H. luetkeni and H. affinis armoured catfish from the Atlantic Forest biome, and in other catfish species from the Amazon and the Pantanal. Sequencing data suggested that Brazilian catfish often have mixed trypanosome infections, highlighting the importance of molecular characterization to identify trypanosome species in fishes and leeches.

Background: Proline racemase (PRAC) enzymes of Trypanosoma cruzi (TcPRAC), the agent of Chagas disease, and Trypanosoma vivax (TvPRAC), the agent of livestock trypanosomosis, have been implicated in the B-cells polyclonal activation contributing to immunosuppression and the evasion of host defences. The similarity to prokaryotic PRAC and the absence in Trypanosoma brucei and Trypanosoma congolense have raised many questions about the origin, evolution, and functions of trypanosome PRAC (TryPRAC) enzymes., Findings: We identified TryPRAC homologs as single copy genes per haploid genome in 12 of 15 Trypanosoma species, including T. cruzi and T. cruzi marinkellei, T. dionisii, T. erneyi, T. rangeli, T. conorhini and T. lewisi, all parasites of mammals. Polymorphisms in TcPRAC genes matched T. cruzi genotypes: TcI-TcIV and Tcbat have unique genes, while the hybrids TcV and TcVI contain TcPRACA and TcPRACB from parental TcII and TcIII, respectively. PRAC homologs were identified in trypanosomes from anurans, snakes, crocodiles, lizards, and birds. Most trypanosomes have intact PRAC genes. T. rangeli possesses only pseudogenes, maybe in the process of being lost. T. brucei, T. congolense and their allied species, except the more distantly related T. vivax, have completely lost PRAC genes., Conclusions: The genealogy of TryPRAC homologs supports an evolutionary history congruent with the Trypanosoma phylogeny. This finding, together with the synteny of PRAC loci, the relationships with prokaryotic PRAC inferred by taxon-rich phylogenetic analysis, and the absence in trypanosomatids of any other genera or in bodonids or euglenids suggest that a common ancestor of Trypanosoma gained PRAC gene by a single and ancient horizontal gene transfer (HGT) from a Firmicutes bacterium more closely related to Gemella and other species of Bacilli than to Clostridium as previously suggested. Our broad phylogenetic study allowed investigation of TryPRAC evolution over long and short timescales. TryPRAC genes diverged to become species-specific and genotype-specific for T. cruzi and T. rangeli, with resulting genealogies congruent with those obtained using vertically inherited genes. The inventory of TryPRAC genes described here is the first step toward the understanding of the roles of PRAC enzymes in trypanosomes differing in life cycles, virulence, and infection and immune evasion strategies.

It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellate's cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 2.4% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannamyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.