Trypanosoma cruzi infection by single and mixed DTUs occurs in several wild mammals and reduviid species in almost all natural environments in the Neotropical realm . Since most outbreaks of Chagas disease in humans originate in the wild ecotope, it is essential to understand the ecology of wild T. cruzi lineages, as well as the role of host species involved in the natural transmission cycles. In this sense, the discrimination of the BMS of triatomines provides data about the host species of the faunal community in the enzootic areas. The genetic detection of BMS is still little explored in Brazil, despite being a highly specific and sensitive methodology as well as a strategic instrument for understanding the ecology, at different levels, of American trypanosomiasis and other vector-borne infectious diseases. This is the first report of genetic BMS of triatomines in the Pantanal biome and the first in the country to use molecular cloning to achieve multi-host diversity per hematophagous vectors.
A unique molecular BMS study performed on T. sordida used four genetic markers for humans, dogs, cats, and birds, yielding generic results, and no other vertebrate could be detected as BMS . Triatoma sordida was historically indicated as the unique species of the genus with a BMS preference for birds . However, there are some BMS reports of other vertebrates, such as a variety of wild mammals including rodents, bats, and marsupials, a number of domestic animals, and even reptiles, detected by precipitin tests . Together with our finding that T. sordida can also feed on southern anteaters, the eclectic behavior of this triatomine species, rather than BMS preference, accepting a range of alternative sources is confirmed. As noted by Rabinovich et al. , the proximity and availability of a sort of host vertebrates may influence the opportunistic character of some triatomines.
Olifiers et al.  reported that South American coatis built arboreal nests with branches, leaves, and vines intertwined that were used for rest and reproduction. Additionally, de Lima et al.  showed that coatis’ nests can also be explored by other animals such as marsupials, rodents, and birds, based on precipitin BMS tests. These observations may explain the maintenance of triatomine colonies, including the non-volant nymphs, in nests abandoned by South American coatis . It was also observed that the coati nests may be visited by the spiny rat, Thrichomys fosteri, valid name T. pachyurus (Wagner, 1845) (Rodentia, Echimyidae), a scansorial species that has been reported with high frequency and parasitemia by T. cruzi (TcI) [4, 5, 15]. This caviomorph rodent species was also recorded inside South American coatis’ nests using camera traps to monitor it . In the present study, the results of genetic BMS identification of triatomines collected from a South American coati’s nest showed that these reduviid bugs fed on T. tetradactyla individuals, revealing that this habitat can also be occupied by this Pilosa species.
Given these shared occupations and the presence of infected triatomines, coatis’ nests can be considered an important core for maintaining different genotypes of T. cruzi. In fact, different DTUs including TcI, TcII, TcIII, and TcIV have been observed in hosts from the Pantanal biome, with the highest frequencies with both TcI and TcII, mainly in mixed infection [3, 16, 48]. In addition, Nasua nasua was indicated as the mammal responsible for retaining high parasitemia by both TcI and TcII in the biome, and mixed infections by different T. cruzi lineages have been recorded in the top predator species . Herein, TcI and TcII are confirmed in T. sordida that inhabit a South American coati nest, with a high frequency of TcI/TcII mixed infection and intra-DTU diversity, especially from TcI. The detection of haplotypes of classic reference strains (Sylvio X10, Y, and Esmeraldo) in the triatomine studied in this work showed that they are still circulating in the wild, together with other DTU variants. The heterogeneity of T. cruzi TcI was previously observed in wild mammals from diverse Brazilian biomes [49, 50], including from the Pantanal biome [49, 51], and in triatomine vectors , using high-resolution approaches, multilocus sequence typing (MLST) and multilocus microsatellite typing (MLMT). In this study, the mixed DTU infections per vector were detected due to the use of two molecular markers and the discriminatory capacity of the Barcoding DNA and molecular cloning methodologies. We highlighted that the cytb primers designed for this study were useful in complementing the T. cruzi diagnosis and genotyping based on 18S rDNA, and the target region was enough polymorphic and informative despite the small amplicon size generated, which also allowed some intra-DTU resolution, at least to TcI and TcII. We recommend the parasite diagnosis based on these two molecular markers, plus cloning, which is not frequently applied. It is possible that MLST and MLMT markers could reveal higher intra-TcI diversity in the triatomines examined, due to their remarkable resolution power. Therefore, mixed DTU and haplotype profiles must be more recurrent in this Pantanal subregion than those detected by conventional techniques, with consequences for the intricate T. cruzi transmission networks and for the fitness of the mammal hosts, which is still unknown.
The results of the present study suggest that TcI and TcII infection in the region may not be driven only by coatis, but apparently, southern tamandua, who also present high parasitemia in the studied area , seems to have a role when multiple DTU infections, and certainly reinfections, occur mediated by vectors during the shared coati nest occupation. Aside from southern anteaters being poorly studied, we are aware that the results of the present research should be corroborated by sampling more coati nests and in other subregions of the Pantanal biome. It is important to mention that nest colonization with triatomines is highly variable, with rates from 33.3% to 9.6%, even in the same landscape [9, 12]. This could be related to specific ecological characteristics of areas , and undoubtedly, to the BMS availability in the nests. However, the rate of T. cruzi infection of triatomines collected from coati nests can reach 70% per single nest .
Tamandua tetradactyla is an autochthonous South American mammal species, occurring in all Brazilian biomes . It is primarily arboreal, using branches for moving and foraging, but can also move, feed, and rest on the ground. It has a predominantly nocturnal habit, and trees, burrows of armadillos, or other natural cavities are indicated as the preferable spaces to rest . Tamandua tetradactyla feeds on insects, mostly nests of ants or termites, from the ground or in trees .
A T. cruzi infection in the southern tamandua has already been reported. In 1942, the first report in the Amazon basin was on three animals by parasitological direct tests of fresh blood and xenodiagnosis . Also, in the Amazon rainforest, de Araújoet al.  revealed mixed infection by Trypanosoma rangeli, Leishmania infantum, and T. cruzi TcI, using mini-exon gene analysis. Recently, Santos et al.  reported for the first time T. cruzi infection in T. tetradactyla from the Pantanal wetland, the site of the present study. The TcI lineage was isolated from hemocultures, indicating high parasitemias. In the present work, anteaters are the unique BMS from T. sordida specimens infected by TcI, TcII, and mixed TcI/TcII.
Regarding the BMS methodology, it was possible to detect three different T. tetradactyla haplotypes using the cytb marker and two haplotypes based on 12S rDNA. Since the cytb marker is the most variable target , it was expected that more cytb haplotypes would be identified than the 12S rDNA target. Furthermore, the vertebrate cytb sequence dataset available in GenBank is richer than the 12S rDNA dataset. It is possible that the two closely related cytb haplotypes HapA and HapC harbor the same 12S rDNA haplotype. The limited genetic variability of 12S rDNA and the lack of taxonomic definition in the discrimination of some species of Caprinae and Didelphinae were observed . However, due to the small amplicon size, it is efficient in revealing BMS in degraded DNA , and as demonstrated in the present study, in previously negative samples by using the cytb marker (Table 1). The pros and cons of these molecular markers make the use of both in parallel the best methodological strategy in the detection and identification of vertebrate species acting as BMS.
Three southern tamandua individuals were the BMS of T. sordida collected from the coati nest. Medri et al.  reported that the parental care of a single offspring usually lasts 1 year; it is carried on the mother's back or left in “a nest” when it is going to feed. Since anteaters have an individualist nonsocial behavior, the three individuals identified in the present study occupied the nest at different times. Another possibility is that two individuals corresponding to a mother and her single offspring, potentially the closely related animals HapA and HapC, and the other individual, possibly the genetically distant anteater HapB, visited the nest at different times. However, as maternally related animals may have the same mitochondrial DNA (mtDNA) haplotype, it is plausible that more than three different individuals of T. tetradactyla visited the coati nest.
We could also hypothesize that the massive frequency of animal HapA could represent a long-lasting exploration of the coati nest, while the other animals could be punctual events or old previous visits, reflected by low BMS DNA detection. However, an initial massive feeding by fasting triatomines in anteater individual HapA, and after their feeding on individuals HapB and Hap C, could not be ruled out.
In this regard, the absence of coati DNA detection as BMS of the bugs collected from their nest suggests the persisting exploration of anteaters in the coati nest, and the total abandonment by the “constructors.” Abandoned coati nests have been reported in the literature [4, 9, 13]. Recently, it was demonstrated that BMS can be detected in triatomines until 12 weeks after the last feeding, using the same molecular method applied in this study, under experimental conditions . Proportionally speaking, we are inclined to suggest that the coatis abandoned this nest for months, which was later used only by anteaters. The direction of the transmission, from triatomines to anteaters or vice versa, is not possible to determine, but the coati nest acting as a hub of the T. cruzi transmission is clear.
The analysis of BMS from the triatomine vector is a relevant and non-faunal invasive approach for gathering information on which wild mammals participate in a local transmission network. The data presented herein implicate the T. tetradactyla participation in the T. cruzi enzootic scenario in this Pantanal subregion, not only for this niche occupation but also due to its ecological peculiarities in exploring the arboreal and terrestrial strata.
Desbiez and Kluyber (2013) reported that T. tetradactyla was the most frequent vertebrate using burrows of giant armadillo, “the ecosystem engineers,” from other 24 species documented in the Brazilian Pantanal . Tamandua tetradactyla was found as the only feeding source by molecular BMS analysis in 14/31 Rhodnius robustus collected in an Attalea phalerata palm tree crown from the Brazilian Amazon region, and was suggested as an important reservoir for T. rangeli . Since T. tetradactyla has been found in arboreal coati nests, giant armadillo burrows, and palm tree crows, this anteater species may be connecting different T. cruzi transmission cycles that would be occurring in the canopy and on the ground.
As bioaccumulator of T. cruzi DTUs, South American coatis were proposed as a transmission hub linking different sylvatic cycles [15, 17]. However, would the South American coatis themselves or what they build be considered as hubs of T. cruzi cycle transmission? In the strict sense, it would not be them, but their nests. South American coatis build the structure and modify the environment, so they are responsible for their origin, but even within the species, there is no exclusivity of use for the animals that build them. Indeed, based on our results and previous data on serological tests and camera traps [3, 9], there is no use restricted to the South American coati species. As highlighted earlier, arboreal nests represent an example of the richness of possibilities of encounters between mammals and reduviid species for T. cruzi transmission . Therefore, these nests have intra- and interspecific communal use for those who explore the arboreal stratum and consequently act as T. cruzi transmission hubs, available to all the competent hosts that potentially could frequent them.