High-throughput analysis of the Trypanosoma cruzi minicirculome (mcDNA) unveils structural variation and functional diversity.

Trypanosoma cruzi causes Chagas disease and has a unique extranuclear genome enclosed in a structure called the kinetoplast, which contains circular genomes known as maxi- and minicircles. While the structure and function of maxicircles are well-understood, many aspects of minicircles remain to be discovered. Here, we performed a high-throughput analysis of the minicirculome (mcDNA) in 50 clones isolated from Colombia's diverse T. cruzi I populations. Results indicate that mcDNA comprises four diverse subpopulations with different structures, lengths, and numbers of interspersed semi-conserved (previously termed ultra-conserved regions mHCV) and hypervariable (mHVPs) regions. Analysis of mcDNA ancestry and inter-clone differentiation indicates the interbreeding of minicircle sequence classes is placed along diverse strains and hosts. These results support evidence of the multiclonal dynamics and random bi-parental segregation. Finally, we disclosed the guide RNA repertoire encoded by mcDNA at a clonal scale, and several attributes of its abundance and function are discussed.

Genome plasticity driven by aneuploidy and loss of heterozygosity in Trypanosoma cruzi.

Trypanosoma cruzi the causative agent of Chagas disease shows a marked genetic diversity and divided into at least six Discrete Typing Units (DTUs). High intra genetic variability has been observed in the TcI DTU, the most widely distributed DTU, where patterns of genomic diversity can provide information on ecological and evolutionary processes driving parasite population structure and genome organization. Chromosomal aneuploidies and rearrangements across multigene families represent an evidence of T. cruzi genome plasticity. We explored genomic diversity among 18 Colombian T. cruzi I clones and 15 T. cruzi I South American strains. Our results confirm high genomic variability, heterozygosity and presence of a clade compatible with the TcIdom genotype, described for strains from humans in Colombia and Venezuela. TcI showed high structural plasticity across the geographical region studied. Differential events of whole and segmental aneuploidy (SA) along chromosomes even between clones from the same strain were found and corroborated by the depth and allelic frequency. We detected loss of heterozygosity (LOH) events in different chromosomes, however, the size and location of segments under LOH varied between clones. Genes adjacent to breakpoints were evaluated, and retrotransposon hot spot genes flanked the beginning of segmental aneuploidies. Our results suggest that T. cruzi genomes, like those of Leishmania, may have a highly unstable structure and there is now an urgent need to design experiments to explore any potential adaptive role for the plasticity observed.

Genetic Diversity and Phylogenetic Relationships of Coevolving Symbiont-Harboring Insect Trypanosomatids, and Their Neotropical Dispersal by Invader African Blowflies (Calliphoridae).

This study is about the inter- and intra-specific genetic diversity of trypanosomatids of the genus Angomonas, and their association with Calliphoridae (blowflies) in Neotropical and Afrotropical regions. Microscopic examination of 3,900 flies of various families, mostly Calliphoridae, revealed that 31% of them harbored trypanosomatids. Small subunit rRNA (SSU rRNA) barcoding showed that Angomonas predominated (46%) over the other common trypanosomatids of blowflies of genera Herpetomonas and Wallacemonas. Among Angomonas spp., A. deanei was much more common than the two-other species, A. desouzai and A. ambiguus. Phylogenetic analyses based on SSU rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and internal transcribed spacer rDNA (ITS rDNA) sequences revealed a marked genetic diversity within A. deanei, which comprised four infraspecific genotypes (Dea1-Dea4), and four corresponding symbiont genotypes (Kcr1-Kcr4). Host and symbiont phylogenies were highly congruent corroborating their co-divergence, consistent with host-symbiont interdependent metabolism and symbiont reduced genomes shaped by a long coevolutionary history. We compared the diversity of Angomonas/symbionts from three genera of blowflies, Lucilia, Chrysomya and Cochliomyia. A. deanei, A. desouzai, and A. ambiguus were found in the three genera of blowflies in South America. In Africa, A. deanei and A. ambiguus were identified in Chrysomya. The absence of A. desouzai in Africa and its presence in Neotropical Cochliomyia and Lucilia suggests parasite spillback of A. desouzai into Chrysomya, which was most likely introduced four decades ago from Africa into the Neotropic. The absence of correlation between parasite diversity and geographic and genetic distances, with identical genotypes of A. deanei found in the Neotropic and Afrotropic, is consistent with disjunct distribution due to the recent human-mediated transoceanic dispersal of Angomonas by Chrysomya. This study provides the most comprehensive data gathered so far on the genetic repertoires of a genus of trypanosomatids found in flies from a wide geographical range.

Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit).

Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six discrete typing units (DTUs) designated as TcI-TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, multilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI-Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured populations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU.

New insights into the evolution of the Trypanosoma cruzi clade provided by a new trypanosome species tightly linked to Neotropical Pteronotus bats and related to an Australian lineage of trypanosomes.

BACKGROUND: Bat trypanosomes are implicated in the evolution of the T. cruzi clade, which harbours most African, European and American trypanosomes from bats and other trypanosomes from African, Australian and American terrestrial mammals, including T. cruzi and T. rangeli, the agents of the American human trypanosomiasis. The diversity of bat trypanosomes globally is still poorly understood, and the common ancestor, geographical origin, and evolution of species within the T. cruzi clade remain largely unresolved. METHODS: Trypanosome sequences were obtained from cultured parasites and from museum archived liver/blood samples of bats captured from Guatemala (Central America) to the Brazilian Atlantic Coast. Phylogenies were inferred using Small Subunit (SSU) rRNA, glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), and Spliced Leader (SL) RNA genes. RESULTS: Here, we described Trypanosoma wauwau n. sp. from Pteronotus bats (Mormoopidae) placed in the T. cruzi clade, then supporting the bat-seeding hypothesis whereby the common ancestor of this clade likely was a bat trypanosome. T. wauwau was sister to the clade T. spp-Neobats from phyllostomid bats forming an assemblage of trypanosome species exclusively of Noctilionoidea Neotropical bats, which was sister to an Australian clade of trypanosomes from indigenous marsupials and rodents, which possibly evolved from a bat trypanosome. T. wauwau was found in 26.5% of the Pteronotus bats examined, and phylogeographical analysis evidenced the wide geographical range of this species. To date, this species was not detected in other bats, including those that were sympatric or shared shelters with Pteronotus. T. wauwau did not develop within mammalian cells, and was not infective to Balb/c mice or to triatomine vectors of T. cruzi and T. rangeli. CONCLUSIONS: Trypanosoma wauwau n. sp. was linked to Pteronotus bats. The positioning of the clade T. wauwau/T.spp-Neobats as the most basal Neotropical bat trypanosomes and closely related to an Australian lineage of trypanosomes provides additional evidence that the T. cruzi clade trypanosomes likely evolved from bats, and were dispersed in bats within and between continents from ancient to unexpectedly recent times.

kDNA markers define two major Trypanosoma rangeli lineages in Latin-America.

Trypanosoma rangeli is a hemoflagellate parasite of man, domestic and wild animals in Central and South America. The genus Rhodnius is particularly susceptible to infection by T. rangeli and transmission by salivary inoculation has been demonstrated in 12 of 14 nominal species of naturally and experimentally infected insects. This report describes the molecular characterization of 37 strains of T. rangeli isolated from vertebrate and invertebrate hosts. Strains were analyzed by hybridization with kinetoplast DNA (kDNA) probes, polymerase chain reaction (PCR) amplification of kDNA minicircles and random amplification polymorphic DNA (RAPD). Strains isolated from Rhodnius prolixus present KP1, KP2 and KP3 minicircle amplification products but strains isolated from R. colombiensis or Panstrongylus megistus present amplification products derived only from KP2 and KP3 minicircles. The two T. rangeli groups defined as KP1(+) and KP1(-) present a high genetic divergence as they have probably been co-evolutioned with different adaptive radiated lines of the genus Rhodnius in Latin-America. The data obtained from insects with intestinal and salivary glands infections confirm that each Rhodnius species select the sub-population of T. rangeli KP1(+) or KP1(-) which is susceptible to transmit it by salivary inoculation to the vertebrate host.