The high triatomine infestation rates and densities of vectors found in this study confirm the white-naped squirrel (S. nebouxii) as an important natural food source for triatomines in the southern Andean region of Ecuador [3, 9]. Despite the extensive distribution of the vector R. ecuadoriensis, its presence in different habitats and its close association with other arboreal vertebrates, e.g. birds, rodents) , reaches its highest abundance in squirrel (S. nebouxii) nests [3, 9, 10, 12]. Also, other squirrel species of the genus Sciurus and the vector extensively overlap in both range and habitat, across the equatorial Pacific region of western Ecuador and northwestern Peru between 0–2000 masl [8, 15].
Our results reveal that ecological features of squirrel nests, such as tree height and tree richness in the surrounding forest, could favor the presence of triatomines. Although R. ecuadoriensis may not be specialized to S. nebouxii, ecological and environmental conditions might favor an overlap in geographical distribution of the two species [13, 16, 17], and thus, their interaction. Nevertheless, the high infestation rates could suggest that S. nebouxii favors R. ecuadoriensis population persistence in relation to other sympatric vertebrate hosts, where conditions are suitable. Furthermore, phenotypic variability in R. ecuadoriensis  could be a critical factor in the optimal exploitation of all available resources (i.e. host species) for successful vector colonization and multiplication .
Infestation of squirrel nest with triatomines
We detected a high infection rate (60%) and found only one species (R. ecuadoriensis) of the four species of triatomine reported for southern Ecuador. This finding is in accordance with other reports of R. ecuadoriensis (instars and adults) in sylvatic environments associated with squirrel nests. However, the infestation rate is higher than previously reported: 14%  and 12% .
In this study, triatomine population structure, with the presence of all nymphal stages and adults, revealed long-term colonization of triatomines. Considering that temperature influences the duration of triatomine life-cycle  and that R. ecuadoriensis requires around six months to reach the adult stage , the presence of nymphs could thus indicate stable conditions for triatomines in squirrel nests. The effectiveness of triatomines at colonizing nests of a particular host species could be primarily related to adult oviposition rate, offspring performance  and availability of at least one blood meal to molt . Survival under those conditions and within a seasonal environment, with a mortality risk during nymphal development, could be significantly maximized by biotic (e.g. protection against predation, intra- and interspecific competition) and abiotic (e.g. adequate temperature and humidity conditions) features of squirrel nest.
Trypanosome infection of triatomines
We detected T. cruzi and T. rangeli in 89% of the analyzed triatomines, of which more than half were nymphs (57%). The higher trypanosome prevalence in nymphs, along with nymph limited dispersal capacity [22, 23], suggests that they acquired the parasites from infected squirrels and/or the opportunistic rodents that use the abandoned nests. The effective parasite transmission could be related to the primary infection route through contact with triatomine feces (i.e. stercorarian transmission). However, the most effective pathway of infection for wildlife in sylvatic cycles is predation on infected bugs, as it happens in other natural hosts (e.g. raccoons, opossums) . Nevertheless, mammalian host immunity should be further explored in order to understand the influence of trypanosome parasitemia and the degree of tolerance to repeated triatomine exposure.
Selection of nesting sites by squirrels
Some species of tree squirrels, including the white-naped squirrel, build nests on trees. Nests are important resources, and they are used for sleeping, resting, and provide protection against weather conditions and predators, and serve as places to raise offspring [25, 26]. Simosciurus nebouxii nests are loosely constructed of woven sticks of about 30 cm in diameter . Nesting site selection depends on many factors (i.e. position, biome composition, the height of the tree) and can be a limiting factor for squirrel distribution . Our results demonstrated that tree height (TH) is an important variable for nesting site selection of S. nebouxii, together with the diameter at breast height (DBH), although to a lesser extent. These findings are in accordance with other studies of tree squirrels (i.e. Albert’s squirrel, Virginia northern flying squirrel) that reported TH and DBH as important variables for nesting site selection [27, 28].
In this analysis, tree species did not explain the absence or presence of squirrel nests. Only 30 species of trees (≥ 0.1 m of DBH) were identified. Of these, 22 species are higher than 800 cm up to almost 30 meters. Despite the diversity of tree species, only two species (Vachellia macracantha and Pisonia aculeata) accounted for 57% of trees and harbored the 48% of squirrels’ nests. The selection of a nesting site could explain this result is more related to the availability of trees than to a specific preference for tree species. In other squirrel species (e.g. Albert’s squirrel), tree size and access routes appear to be more important to the selection of nest sites than tree species .
Features of nesting habitat in relation to triatomine abundance and trypanosome presence
Wild and synanthropic rodents are important for T. cruzi transmission in several regions; however, their role varies with time and place . Biotic factors (i.e. nutritional status, age, stress conditions, abundance) are important in host-vector-parasite interaction  and the particular association of squirrel-triatomines-T. cruzi has been previously reported [3, 10, 12, 31, 32]. Nevertheless, little information is available about the ecological characteristics of this association.
Multi-model inference analysis revealed that tree height, nest occupancy and tree richness influence the presence of triatomines in squirrel nests. The importance of tree height has been demonstrated in previous studies that reported higher triatomine abundance in squirrel nests located five meters above ground level and close to human dwellings [3, 10]. At the same time, nest occupancy is an important factor due to the availability of blood, which is essential for triatomine development [20, 33]. In sylvatic environments as well as in environments near human dwellings, squirrels are not the only available host. Other arboreal rodents could, eventually, use the abandoned squirrel nest and serve as blood sources for triatomines. Within this context, the importance of tree richness might have an impact on vertebrate host diversity and the dynamics on rodents, which implies opportunistic behavior of other rodent species to use the squirrel nests and serve as an alternative blood source for triatomines.
Despite the role of other rodent species, it has been demonstrated that squirrels have a very important impact on triatomine abundance and distribution. Previous studies in sylvatic environment reported a triatomine infestation rate > 14% in squirrel nests, and much lower infestation rates in other habitats, such as bird and mouse/rat nests [3, 9]. Additionally, it has been shown that triatomines are associated with squirrels all year round and closely related to human activities such as cultivation of maize. Therefore, land use constitutes an important factor in the dispersal patterns of sylvatic triatomines, particularly of R. ecuadoriensis, which is temporally and spatially more closely related to squirrel dynamics than those of other available hosts . Moreover, land use impact might also be reflected in the presence of a pathogen within the host species, depending on the specific biology of the host-parasite relationship [34, 35]. Also, as important as the association of this squirrel species with triatomine abundance, it would be important to unveil the side effects of the use of abandoned nest by opportunistic rodents because of the generation of new habitats.