Chagas disease is a complex anthropozoonosis with distinct domestic and sylvatic mammal species acting as potential reservoirs. The diversity of vector species and their habitats are among the factors that hinder the control of the disease. Control programs periodically monitor the prevalence of T. cruzi infection in insect bugs through microscopical observation of diluted feces. However, microscopy presents limited sensitivity in samples with low parasite numbers, difficulties in examining all evolutionary stages of the insect and may in turn be limited to differentiate T. cruzi from other morphologically similar trypanosomatids. Here, we report two highly sensitive and accurate methodologies to infer T. cruzi infection rates and to quantify parasite load in the gut of field-collected triatomines. Triatomines were manually collected in the period 2011–2012 and 2014–2015, in domestic, peridomestic or sylvatic habitats in rural areas of 26 municipalities, encompassing three distinct Brazilian biomes: Caatinga, Cerrado and Atlantic Rainforest. Following morphological and taxonomical identification, the search for flagellated protozoa was performed by optical microscopy. A conventional PCR targeting T. cruzi kDNA and a TaqMan qPCR directed to the parasite nuclear satellite DNA (SAT) were developed, both in multiplex, with the triatomine 12S subunit ribosomal RNA gene, used as internal amplification control. Both methods were used for detection (kDNA-PCR) and parasite load quantification (SAT-DNA-qPCR), to investigate T. cruzi infection in captured triatomines. The combined methods were assayed on a panel of 205 field-collected triatomine samples. Diagnostic analysis revealed 21% positivity for the kDNA-PCR, whereas microscopic examination enabled identification of T. cruzi in only 7.0% of the PCR-positive samples. Negative PCR results were confirmed by the absence of T. cruzi flagellates using microscopy. Caatinga biome yielded the highest T. cruzi infection rate (60%), followed by the Atlantic Rainforest and Cerrado with 7.1 and 6.1%, respectively. In addition, a wide range distribution of parasite load, varying from 8.05 × 10-2 to 6.31 × 1010 was observed with a median of 2.29 × 103 T. cruzi/intestine units. When parasite load was analyzed by triatomine species, a significantly higher median was found for Panstrongylus lutzi in comparison with Triatoma brasiliensis. Our results demonstrate highly sensitive PCR-based methodologies to monitor T. cruzi infection in triatomines. In addition, the qPCR assay offers the possibility of further evaluation parasite load, as a promising biomarker of the vectorial capacity of triatomines in Chagas disease endemic areas.

Several studies addressed changes on the insect vector behavior due to parasite infection, but little is known for triatomine bugs, vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. We assessed infection rates and metacyclogenesis of T. cruzi (TcVI) in fifth-instar nymphs of Triatoma rubrovaria comparing with the primary vector Triatoma infestans. Also, biological parameters related to feeding-excretion behavior were evaluated aiming to identify which variables are most influenced by T. cruzi infection. Fifth-instar nymphs of T. rubrovaria and T. infestans were fed on mice infected with T. cruzi (TcVI). We compared the presence and the number of parasite evolutive forms in excreta of both triatomine species at 30, 60 and 90 days post-infection (dpi) with traditional statistical analyses. Moreover, both species were analyzed through generalized linear models and multinomial logistic regression hypotheses for seven behavioral parameters related to host-seeking and feeding-excretion. Triatoma rubrovaria and T. infestans had similar overall infection and metacyclogenesis rates of T. cruzi TcVI in laboratory conditions. Regarding vector behavior, we confirmed that the triatomine’s tendency is to move away from the bite region after a blood meal, probably to avoid being noticed by the vertebrate host. Interspecific differences were observed on the volume of blood ingested and on the proportion of individuals that excreted after the blood meal, revealing the higher feeding efficiency and dejection rates of T. infestans. The amount of ingested blood and the bite behavior of T. rubrovaria seems to be influenced by TcVI infection. Infected specimens tended to ingest ~25% more blood and to bite more the head of the host. Noteworthy, in two occasions, kleptohematophagy and coprophagy behaviors were also observed in T. rubrovaria. Laboratory infections revealed similar rate of T. cruzi TcVI trypomatigotes in excreta of T. rubrovaria and T. infestans, one of the most epidemiological important vectors of T. cruzi. Therefore, TcVI DTU was able to complete its life cycle in T. rubrovaria under laboratory conditions, and this infection changed the feeding behavior of T. rubrovaria. Considering these results, T. rubrovaria must be kept under constant entomological surveillance in Rio Grande do Sul, Brazil. Chagas disease is caused by the etiological agent Trypanosoma cruzi and is mainly transmitted through the excreta of triatomine vectors. It is endemic in Latin America and the Caribbean, affecting ~8 million people. Control programs are based on the elimination of domestic vectors through insecticide-spraying indoors, since there is no vaccine or efficient treatment for chronic patients. However, this strategy is not sustainable where native triatomine species are capable of colonizing peridomestic structures and reinvading human dwellings. Since the ‘virtual’ elimination of Triatoma infestans in Brazil (residual foci remain in Rio Grande do Sul and Bahia), specimens of the native Triatoma rubrovaria have been constantly collected inside human dwellings and peridomiciliary ecotopes in Rio Grande do Sul, southern Brazil, which might represent a real risk for human infections. Here we analyzed a unique and large dataset through classical and modern statistical methods to evaluate T. cruzi infection in T. rubrovaria and to identify host-seeking, and also feeding/excretion behavioral traits that could be influenced by the parasite. Our results indicated that the parasite infection caused changes in T. rubrovaria feeding behavior that could increase T. cruzi TcVI transmission. Moreover, this vector species had similar infection rate to one of the main important Chagas disease vectors in South America, T. infestans. Therefore, T. rubrovaria must be kept under constant entomological surveillance in Rio Grande do Sul, Brazil.

The Triatoma rubrovaria subcomplex, comprising several triatomine species, plays a significant role in the transmission of Chagas disease in southern Brazil. Despite morphological distinctions among these species, their genetic differentiation remains poorly understood, particularly in sympatric regions. This study investigates the genetic diversity and phylogenetic relationships through DNA sequencing analysis of five sympatric species within the T. rubrovaria subcomplex (T. rubrovaria, T. carcavalloi, T. klugi, T. circummaculata, and T. pintodiasi), using a 542-bp fragment of the mitochondrial cytochrome b (mtCytb) gene. A total of 84 specimens were collected from six municipalities in Rio Grande do Sul, Brazil, and analyzed alongside laboratory-reared specimens and sequences from the GenBank. Bayesian phylogenetic reconstructions, haplotype networks, and population structure analyses revealed a lack of clear genetic differentiation among the five species, with overlapping intra- and interspecific divergences and shared haplotypes. These findings suggest either a single species exhibiting phenotypic plasticity or a group of incipient species with ongoing gene flow. This study highlights the need for a taxonomic revision and suggests that this group could serve as a valuable model for further genomic research to elucidate potential aspects of phenotypic plasticity and/or sympatric speciation in triatomines.