Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that infects humans as well as domestic and sylvatic mammals. This disease can be transmitted by approximately 150 species of hemipteran insect vectors of which Triatoma infestans (Klug 1834) (Hemiptera: Reduviidae, Triatominae) is the most important in the southern countries of Latin America (Gurtler et al., 2008; Schofield et al., 2006). The most recent reports estimate that Chagas disease affects approximately 6 million people worldwide; approximately 70 million people are at risk of infection in 21 Latin American countries (WHO, 2015).

Domiciliary populations of T. infestans have been successfully controlled by spraying with pyrethroid insecticides in parts of southern South America (Schofield et al., 2006; Zerba, 1999); this contributed to the reduction in the geographical distribution of T. infestans from an estimated 6.28 million km² in the 1960s to less than 1 million km² today (Gorla, 2002; Schofield et al., 2006). Moreover, several countries and regions in South America, have been certified as having interrupted domestic vector transmission (Coura et al., 2014).

T. infestans has been found in sylvatic foci in several areas in the Gran Chaco and Andean regions, some reportedly invading houses, and are associated with T. cruzi transmission to humans (Breniere et al., 2017; Buitrago et al., 2013; Buitrago et al., 2016; Buitrago et al., 2010; Ceballos et al., 2009; Ceballos et al., 2011; Cortez et al., 2007; Noireau et al., 2000; Noireau et al., 2005; Rolón et al., 2011; Waleckx et al., 2012; Waleckx et al., 2011). Sylvatic populations are not targeted by vector control interventions and could re-invade houses treated with insecticide, especially if the population has resistant variants that can be selected under insecticidal pressure. Certain sylvatic populations from Bolivia have shown low susceptibility to pyrethroid insecticides (Roca-Acevedo et al., 2011; Depickère et al., 2012), and some also exhibited a lower sensitivity to fenitrothion (Santo-Orihuela et al., 2013). Furthermore, in the Andean region, post-spraying household reinfestation has been attributed partially to the dispersal of individuals from sylvatic populations (Noireau et al., 2005; Ceballos et al., 2011; Breniere et al., 2013; Waleckx et al., 2015) and domestic pyrethroid resistance has also been reported (Gomez et al., 2014).

Pyrethroid resistance in T. infestans was initially described in Argentina and Bolivia in the late 1990s. Increasing reports indicate that multiple natural populations are resistant to insecticides, and significant variability in susceptibility levels has been observed among them (Germano et al., 2010a; Mougabure-Cueto and Picollo, 2015; Pessoa et al., 2015; Picollo et al., 2005), at both the macro-geographic scale and even between ecotopes within the same household and peridomicile (Echeverria et al., 2018). Based on identification of a hybrid zone composed of Andean and non-Andean T. infestans from the Argentinean-Bolivian border, it has been proposed that the elimination of T. infestans by pyrethroid insecticides in Brazil, Chile, Uruguay and parts of Argentina and Paraguay indicated that resistance was less common in non-Andean regions and that Andean populations may have a particular genetic background that enabled the development of resistance more rapidly than non-Andean populations (Panzera et al., 2014). However, other studies have demonstrated that some of the highest resistance levels ever reported in T. infestans were in non-Andean individuals in Argentina (Picollo et al., 2005; Fronza et al., 2016); the evolutionary origins of pyrethroid resistance in T. infestans remains debated (Panzera et al., 2014). In these latter populations with extremely high pyrethroid resistance, which required 1000 times the amount of insecticide to kill the same proportion of susceptible individuals (Fronza et al., 2016), resistance was implicated as the cause of local control program failure. Similarly, in other parts of the Gran Chaco, rapid house re-infestation post-spraying and insecticide resistance are growing causes for concern (Cavallo et al., 2016; Cecere et al., 2006; Depickère et al., 2012; Gurevitz et al., 2011; Gurevitz et al., 2013; Lardeux et al., 2010; Piccinali et al., 2018; Cecere et al., 2019; Perez-Cascales et al., 2020).

Resistance traits are considered pre-adaptive, implying that there are a few resistant insects in each natural population, which would survive under pressure of insecticide exposure, selecting for the resistant genetic variants that would be inherited and in turn, become more frequent in subsequent generations. The dispersal rate of resistant traits will depend on pre-existing genetic variation in natural populations, selection intensity, fitness cost associated with expressing the resistant variant, local adaptation and levels of gene flow among populations. Such resistance dynamics have been reported in populations of other medically important vector species, particularly Aedes aegypti (Aguirre-Obando et al., 2015; Marcombe et al., 2013).

Elucidating population genetic dynamics through high resolution genetic markers in the context of well characterized insecticide resistance profiles could test these hypotheses and provide invaluable information for an integrated vector control approach. Establishing the role of sylvatic populations of T. infestans as potential sources of re-infestation and understanding the impact of insecticide resistance on triatomine population genetic diversity is crucial to safeguard the continued effectiveness of current control efforts. Previous studies using high resolution molecular markers demonstrated that genetic differentiation is detectable up to the capture site level (local populations) if sufficient numbers of bugs are collected per site to constitute a representative population for genetic analysis (Breniere et al., 2013; Marcet et al., 2008; Pérez de Rosas et al., 2008; Perez de Rosas et al., 2013).

The aim of this work was to establish and characterize the genetic profiles of four sylvatic and one domestic Andean populations of T. infestans from Bolivia, and to interpret their genetic relationships in the context of their differing levels of insecticide susceptibility. Moreover, an evaluation of the mechanisms likely responsible for pyrethroid resistance observed in these populations, including esterase enzymes and kdr mutations is discussed.

Through high resolution genotyping of five Andean T. infestans populations, we provide insights into natural population genetic structure, the relationship between genetic diversity of sylvatic versus domestic triatomine populations and their respective insecticide resistance profiles. All populations were genetically distinct, with no evidence of current gene flow among them, which correlates with previously described heterogeneity in insecticide susceptibility profiles (Roca-Acevedo et al., 2011). The pattern of inter-population differentiation was consistent with a significant isolation-by-distance (IBD) model, with geographically closer populations being more genetically-related, specifically adjacent sylvatic-domestic populations of Mataral. This observation was supported by both mitochondrial and nuclear microsatellite markers. The genetic pattern of allelic similarity reflected possible shared ancestry and/or secondary contact between domestic and sylvatic Mat populations. This observation further supports previous reports of dynamic gene flow between sylvatic and domestic populations in the Bolivian Andes (Breniere et al., 2013; Quisberth et al., 2011; Waleckx et al., 2011).

Both Mat populations were characterized by considerably higher genetic diversity compared to the other sylvatic populations evaluated in this work; and between the Mat populations, genetic diversity and substructure were higher for the sylvatic population. The combination of high levels of homozygosity, number of private alleles per locus, number of private mitochondrial haplotypes and divergence of mitochondrial sequences, suggest that Mat-S may be ancestral to the other sylvatic populations evaluated here. This observation is supported by the occurrence of two mitochondrial haplotypes within this population that correspond to haplotype E (Fig. 4), which has been previously reported as an ancient haplotype among other Andean T. infestans populations (Waleckx et al., 2011).

The populations 20oct, KM and Mat-S presented significant deviations from HW equilibrium, and all due to significant heterozygote deficits. As the samples were arbitrarily grouped per community, the significant Fis values and hierarchical AMOVA, revealed the majority of variation was present within individuals, which was likely attributable to these population being comprised of admixed individuals from multiple, independent non-panmictic founding populations (Wahlund effect). This would also account for the higher linkage disequilibrium detected in these populations. High standard deviations associated with D_AS values further supports the existence of intra-population sub-structuring. Alternatively, this genetic pattern could indicate that some members have undergone long-term inbreeding, following historical isolation. Both of these hypotheses would also be affected by our use of colony specimens (which may have undergone inbreeding during several generations of laboratory rearing) and not first-generation field captured individuals, a limitation of this study which must be acknowledged.

The bioassays performed in these populations demonstrated three different levels of altered susceptibility (Roca-Acevedo et al., 2011): high levels of decreased susceptibility in Mat-D and Mat-S, moderate levels of decreased susceptibility in KM and 20oct and limited altered susceptibility in Ili (Table 1). The resistance ratios (RRs) obtained for these populations are not as high as values from other areas that have presented difficulties for vector control (i.e. RR > 2000 in El Juramento, Guemes, Chaco, Argentina (Fronza et al., 2016), RR₅₀ = 541.6 in Tierras Nuevas, Tarija, Bolivia (Germano et al., 2010b) or RR = 818 in Taiguati in the Bolivian Chaco (Depickère et al., 2012)). However, in all but Ili, the RR values are above the level classified as resistant by PAHO guidelines, RR > 5 (fivefold higher than reference populations) (PAHO, 2005; Pessoa et al., 2015). Those guidelines also attribute altered susceptibility in field populations presenting RR < 5 to individual variability, which is not enough to cause resistance at the population level. As previously discussed (Depickère et al., 2012; Santo-Orihuela et al., 2013), elevated RR in a sylvatic population might result from at least three non-mutually exclusive hypotheses:

In the context of this study, the decreased susceptibility to deltamethrin observed in all populations may represent a shared ancestral trait, which has undergone further intensification in Mataral following insecticidal treatment, particularly for the domestic population, with possible gene flow between the two Mat populations. In this scenario, the intermediate RRs of 20oct and KM would have been maintained by moderate selection pressure, likely the indirect effect of agricultural spraying; the difference between them could be explained by intrinsic intra-species variability, consistent with the distinct genetic patterns observed. However, the minimal altered susceptibility observed in Ili would imply that this trait had been almost lost in this population in the absence of selection. This pattern would be consistent with a natural or ancestral tolerance trait of the species that evolved independently of insecticide application (Mougabure-Cueto and Picollo, 2015) but could have been selected subsequently under pressure.

Alternatively, insecticide resistance could have been acquired independently in all populations following different histories of insecticide exposure, with individual population RR directly corresponding to the intensity of selection each population has undergone. Recent modelling studies have also demonstrated the impact of key environmental factors, which may drive the evolution of insecticide resistance in T. infestans, particularly, temperature (proposed to influence triatomine physiological stress as well as insecticide toxicity levels), precipitation (found to be negatively correlated with residual insecticide efficacy) and village size (reflecting smaller effective population sizes that are more prone to stochastic effects, e.g. genetic drift or quicker fixation/loss of resistant alleles) (Fronza et al., 2019). Further characterization of the ecological context of the populations in these areas is necessary to evaluate these hypothesis or alternative explanations for the observed genetic patterns.

Insecticide-based interventions are expected to impact vector population structure and levels of genetic diversity. Selective bottlenecks imposed by insecticidal pressure are likely to result in both reductions in genetic variability and population size, such that a small proportion of survivors, possessing the requisite advantageous resistance genes, survive and reproduce (i.e. diversification that would be reflected in a genetic pattern as multiple independent “island effects”). In general, insecticide resistant populations are expected to be less genetically diverse than a wild-type population, as they have likely undergone a period of selective pressure (i.e. insecticide exposure). And within this context, domestic populations are assumed to have been the subject of greater selection pressures, compared to their sylvatic counterparts and therefore be less diverse. Recent studies of T. infestans populations in the Argentinean Chaco support some of these assumptions, with pyrethroid resistant individuals characterized by lower levels of genetic diversity than to their resistant counterparts (Piccinali et al., 2020).

However, our study results challenge these traditional paradigms and may instead be explained by an alternative hypothesis for genetic diversification. In a given location, a population may undergo a selection process from insecticide exposure independent of neighboring triatomine populations (Pessoa et al., 2014). A previous work comparing the genetic profiles of populations under different vector control pressure (Marcet et al., 2008) described populations within a community recovering from a recent massive spraying as much more genetically structured and diverse than populations that were not under systematic vector control pressure. The authors proposed that the distinct observed genetic pattern reflected the cumulative effect of migration among adjacent populations for several generations, which generated a pattern of genetic “homogenization” even among populations located geographically far apart. In contrast, populations recovering from a pool of survivors of insecticide spraying were observed as discrete genetic entities at the individual capture site (i.e. one household). Pooling together such individuals from these “genetically discrete” populations with bugs from other sites, for a random sampling of a given area, (i.e. at the community level, pooling individuals from different houses in the same sample set), would result in a highly genetically diverse meta-population, that is actually composed of several subpopulations, with genetic substructure and gene flow restrictions within. In this study, the elevated genetic diversity of both Mat populations could represent the assemblage of individuals from distinct genetic units, as they have been under higher selective pressure than the sylvatic populations from 20oct, KM and Ili, located further away from the domestic area subjected to vector control. High levels of genetic diversity have also been reported in several domestic non-Andean T. infestans populations from the Gran-Chaco area (Perez de Rosas et al., 2013; Piccinali et al., 2009), of which many had been subject to extensive vector control pressure.

More importantly, the existence of genetically-diverse populations displaying high levels of altered insecticide resistance has potential implications for longer-term efficacy of control interventions, such that the likelihood of individuals surviving future selection events is greater when the original population is composed of more unique genetic variants. It is noteworthy that three haplotypes found in the sylvatic populations analyzed here (A, C and E) were also identified in domestic populations in Cochabamba Department (Giordano et al., 2005), suggesting household colonization by sylvatic populations in the Bolivian Andes is a pervasive and contemporary process.

Regarding the resistance mechanism(s) underlying the altered susceptibility patterns in Mat, analyses of P450 monoxygenases and 7-CP esterases (Roca-Acevedo et al., 2011; Roca-Acevedo and Picollo, 2017) showed a lack of association between enzymatic activities and susceptibility ratios, indicating that these enzymes were not responsible for modified susceptibility in these sylvatic populations. In this work, we evaluated the presence of two kdr mutations and found one individual from Mat-S carrying the mutation L1014 (Fabro et al., 2012). Although most likely not responsible for the mechanism(s) altering insecticide susceptibility in these populations, the presence of this resistance mutation in this area represents a risk for selection under pressure and potential development of higher resistance intensities (Mougabure-Cueto and Picollo, 2015; Sierra et al., 2016). The absence of kdr target site mutations in our Bolivian populations contrasts with recent reports from the Argentinean Gran Chaco, where high frequencies of the L925I substitution were implicated in pyrethroid resistance (Fronza et al., 2020). Future work to characterize the underlying metabolic mechanisms of resistance in these populations is warranted, including screening for novel kdr mutations A934V and K964R, recently identified in Triatoma mazzottii and Triatoma longipennis, respectively (Davila-Barboza et al., 2018), as well as investigating a possible role for integumental detoxification in T. infestans pyrethroid resistance (Dulbecco et al., 2018).

Sylvatic populations of T. infestans represent a challenge for vector control. These populations are not targeted by control activities and could play a key role in post-spraying house re-infestation. The results presented here suggest that there might be an ancient trait(s) that confers resistance in T. infestans sylvatic populations that are capable of invading domiciles. These observations emphasize the need for stronger entomological surveillance in the region, including early detection of house invasion, particularly post-spraying, monitoring for resistance to pyrethroids and the design of integrative control actions that consider both sylvatic foci around domestic settings as well as bug dispersion dynamics.