Resumo

Trypanosomatids are flagellated protozoa that comprise two distinct groups: (1) the monoxenic, which lives in one host, usually an insect, and (2) the heteroxenic, whose life cycle alternates between an invertebrate host and a second host (which may be a vertebrate or a plant). As there is no unanimously marker to taxonomic identification of protozoa in the literature yet, this study will evaluate the applicability of five molecular markers for identification of monoxenic trypanosomatids (76 species) deposited in the FIOCRUZ Protozoa Collection (COLPROT). Up to now, we used the mitochondrial gene cytochrome oxidase I (COI) and the nuclear target Alpha enolase. The sequencing was performed on the ABI 3730XL DNA sequencer (Applied Biosystems) at the PDTIS-FIOCRUZ DNA Sequencing facility. We have already obtained 16 sequences for each marker: 13 sequences from species belonging to the genera Angomonas, Crithidia, Herpetomonas, Leptomonas, Strigomonas, and three other sequences from isolates still awaiting identification: one obtained from the insect Zelus sp. (Reduviidae), another one obtained from Chamaesyce thymifolia (Euphorbiaceae) and one isolated from an HIV-positive patient. Sequences were aligned with the Bioedit v7 software, and a Neighbor-Joining tree (with 1000 bootstrap replicates), based on a Kimura-2-parameters distance matrix, was inferred with MEGA5. The two markers showed congruent results: Leptomonas samueli and L. lactosovorans have identical sequences, which may be an indication that they represent isolates of the same species. Similarly, Crithidia hutneri, C. luciliae termophila, as well as a yet unidentified Crithidia sp., isolated from Zelus sp., also presented very small genetic distances among them (range 0.2-0.7% for COI and 0.00-0.3% for Alpha-enolase). This may be an indicative that these are isolates of one single species. One organism isolated from Chamaesyce thymifolia and another from an HIV-positive patient showed very similar sequences for both genes (0.4-1,1% and 0-0.3%, respectively) in the same group that includes C. luciliae, C. ricardoe and C. fasciculata. The result suggests that these two isolates may belong to the Crithidia genus. These results obtained so far seem to indicate that COI and alpha-enolase genes are useful markers to discriminate monoxenic trypanosomatids, although larger studies including more samples, as well as other markers need to be performed to determine the exact taxonomic status of these species.