We sequenced maxicircles fromT.cruzistrains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb.T.cruzimaxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional inT.cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomicallyT.cruziis a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist inT.cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present inT.cruzi.

Rhodnius montenegrensis (Triatominae), a potential vector of Chagas disease, was described after R. robustus-like bugs from southwestern Amazonia. Mitochondrial cytb sequence near-identity with sympatric R. robustus (genotype II) raised doubts about the taxonomic status of R. montenegrensis, but comparative studies have reported fairly clear morphological and genetic differences between R. montenegrensis and laboratory stocks identified as R. robustus. Here, we use a transcriptome-based approach to investigate this apparent paradox. We retrieved publicly-available transcriptome sequence-reads from R. montenegrensis and from the R. robustus stocks used as the taxonomic benchmark in comparative studies. We (i) aligned transcriptome sequence-reads to mitochondrial (cytb) and nuclear (ITS2, D2-28S and AmpG) query sequences (47 overall) from members of the R. prolixus–R. robustus cryptic–species complex and related taxa; (ii) computed breadth- and depth-coverage for the 259 consensus sequences generated by these alignments; and, for each locus, (iii) appraised query sequences and full-breadth-coverage consensus sequences in terms of nucleotide-sequence polymorphism and phylogenetic relations. We found evidence confirming that R. montenegrensis and R. robustus genotype II are genetically indistinguishable and, hence, implying that they are, in all likelihood, the same species. Furthermore, we found compelling genetic evidence that the benchmark ‘R. robustus’ stocks used in R. montenegrensis description and in later transcriptome-based comparisons are in fact R. prolixus, although likely mixed to some degree with R. robustus (probably genotype II, a.k.a. R. montenegrensis). We illustrate how public-domain genetic/transcriptomic data can help address challenging issues in disease-vector systematics. In our case-study, taxonomic confusion apparently stemmed from the misinterpretation of sequence-data analyses and misidentification of taxonomic-benchmark stocks. More generally, and together with previous reports of mixed and/or misidentified Rhodnius spp. laboratory colonies, our results call into question the conclusions of many studies (on morphology, genetics, physiology, behavior, bionomics or interactions with microorganisms including trypanosomes) based on non-genotyped ‘R. prolixus’ or ‘R. robustus’ stocks. Correct species identification is a prerequisite for investigating the factors that underlie the physiological, behavioral or ecological differences between primary domestic vectors of Chagas disease, such as R. prolixus, and their sylvatic, medically less-relevant relatives such as R. robustus (s.l.) including R. montenegrensis.

Triatoma sordida is a native South American species and the most frequently captured triatomine in artificial environments in Brazil. Although considered a secondary vector of Trypanosoma cruzi, it is typically associated with low infection rates. To investigate its role in an endemic area for Chagas disease in northern Minas Gerais, Brazil, we employed a multidimensional approach that combined triatomine capture data with quantitative and qualitative analyses of T. cruzi. A total of 1861 T. sordida specimens were captured, of which 1455 were examined and 210 (14.4%) were found to be infected with T. cruzi. The most prevalent discrete typing unit (DTU) was TcI (80%), followed by TcII (8%), TcV (5%), and TcIII (3%). Molecular techniques provided new insights into the ecology of T. sordida, revealing a higher infection rate than previously reported and a parasitic load lower than that observed in other quantified species. Chickens were confirmed as the primary food source, playing an epidemiological role in maintaining infected insects with four T. cruzi DTUs. The observed diversity of T. cruzi DTUs suggests a lack of environmental segregation, likely due to the extensive movement of various host species between wild and domestic habitats, resulting in overlapping transmission cycles.

We report the first known occurrence of Psammolestes tertius Lent & Jurberg, 1965 (Hemiptera, Reduviidae, Triatominae) in the state of Rio Grande do Norte, Brazil. In 2009 and 2017, 2 adult specimens, a female and a male respectively, were collected at the Seridó Ecological Station, which is located in the municipality of Serra Negra do Norte. These records broadens the geographical distribution of P. tertius in Brazil and the biodiversity of triatomines in Rio Grande do Norte.

Triatoma melanica is a sylvatic vector species in Brazil. In We aimed to characterize the Trypanosoma cruzi discrete typing units (DTUs), the parasitic loads, and the blood meal sources of insects collected in rocky outcrops in rural areas in the state of Minas Gerais. An optical microscope (OM) and kDNA-PCR were used to examine natural infection by T. cruzi, and positive samples were genotyped by conventional multilocus PCR. Quantification of the T. cruzi load was performed using qPCR, and the blood meal sources were identified by Sanger sequencing the 12S rRNA gene. A total of 141 T. melanica were captured. Of these, ~55% (61/111) and ~91% (63/69) were positive by OM and KDNA-PCR, respectively. We genotyped ~89% (56/63) of the T. cruzi-positive triatomines, with TcI (~55%, 31/56) being the most prevalent DTU, followed by TcIII (~20%, 11/56) and TcII (~7%, 4/56). Only TcI+TcIII mixed infections were detected in 10 (~18%) specimens. A wide range of variation in the parasitic loads of T. melanica was observed, with an overall median value of 10⁴ parasites/intestine, with females having higher T. cruzi loads than N2, N4, and N5. TcII showed lower parasitic loads compared to TcI and TcIII. The OM positive diagnosis odds ratio between T. cruzi infection when the parasite load is 10⁷ compared to 10³ was approximately 29.1. The most frequent blood meal source was Kerodon rupestris (~58%), followed by Thrichomys apereoides (~18%), Wiedomys cerradensis (~8%), Galactis cuja (~8%) and Gallus gallus (~8%). Our findings characterize biological and epidemiological aspects of the sylvatic population of T. melanica in the study area, highlighting the need to extend surveillance and control to this vector.

Background: A new triatomine species was discovered in the semiarid Caatinga region of Rio Grande do Norte, Brazil, where it coexists with Triatoma brasiliensis in both natural and artificial habitats. Methods: Triatomine specimens were captured in peridomestic and sylvatic environments in Rio Grande do Norte. Their identification was based on a combination of analyses, including morphology using dichotomous keys, head and hemelytron morphometry, and phylogeny using cytochrome b and internal transcribed spacer markers. Results: The new species exhibits morphological traits that are intermediate between those of Triatoma brasiliensis and Triatoma petrocchiae , but has distinct characteristics, leading to its designation as Triatoma chiarii sp. nov. Geometric morphometric analysis of its wings and head clearly distinguished T. chiarii sp. nov. from T. brasiliensis and T. petrocchiae , while phylogenetic reconstruction confirmed its placement within the T. brasiliensis species complex. Both approaches consistently supported T. chiarii sp. nov. as a species closely related to T. petrocchiae , but with sufficient phenotypic and genotypic divergence to warrant its recognition as a new taxonomic entity. Conclusions: Since T. chiarii sp. nov. was also found in the peridomestic environment, its possible role in the eco-epidemiology of Chagas disease warrants further investigation.