Infection by Trypanosoma cruzi (Chagas, 1909) in a colony of Triatoma sherlocki Papa et al. 2002 (Reduviidae: Triatominae) present in a subsistence pig farm in Northeast Brazil.

Triatomines of the species Triatoma sherlocki are considered sylvatic; however, household invasion appears imminent, potentially carrying Trypanosoma cruzi, the causative agent of Chagas disease. The aim of this study was to report the first occurrence of a colony of T. sherlocki infected by T. cruzi in a subsistence pig farm. Triatomines collected underwent polymerase chain reaction (PCR) technique for T. cruzi detection and determination of blood meal source. The 19 triatomines collected in the pig farm were of the species T. sherlocki, comprising 26.3% nymphs (5/19), 52.6% males (10/19) and 21.1% females (4/19). PCR showed that 15.8% (3/19) of triatomines were infected by T. cruzi. The only detected blood meal source in triatomines (n = 11) was the domestic mammal Sus scrofa, commonly known as domestic pig, indicating that T. sherlocki is an opportunist, feeding on available vertebrates in the environment, including domestic animals such as pigs. These results highlight the possibility of domiciliation of the species T. sherlocki and its potential role in bridging the transmission of T. cruzi between sylvatic and domestic environments.

Trypanosoma cruzi infection reduces the population fitness of Mepraia spinolai, a Chagas disease vector.

The hematophagous insect Mepraia spinolai (Hemiptera: Reduviidae: Triatominae) is naturally infected with the protozoan parasite Trypanosoma cruzi, the agent of Chagas disease in humans. In this study, we compared the demographic parameters of M. spinolai with and without T. cruzi infection. We collected the immature life table data of 479 M. spinolai individuals of control cohort (reared on mice without T. cruzi infection) and 563 M. spinolai individuals of treatment cohort (reared on mice with T. cruzi infection). Nymphs were maintained in individual compartments inside a growth chamber (26°C; 65-75%) until adult emergence; moulting and survival were recorded daily. For the adult life table study of the control, we used 24 pairs of adults from the control cohort. For the adult life table study of T. cruzi-infected cohort, 25 infected females were paired with 25 males from the control cohort. Life table data were analysed using bootstrap-match technique based on the age-stage, two-sex life table. The preadult survival rate (0.5282) of the control cohort was significantly higher than that of the infected cohort (0.2913). However, the mean fecundity of reproductive females (Fr = 22.29 eggs/♀) and net reproductive rate of population (R0 = 5.07 offspring/individual) of the 0.5th percentile bootstrap-match control cohort were not significantly different from those of the infected cohort (Fr = 23.35 eggs/♀, R0 = 3.77 offspring/individual). Due to the shorter total preoviposition period and higher proportion of reproductive female, the intrinsic rate of increase (r = 0.0053 d-1 ) and finite rate of increase (λ = 1.0053 d-1 ) of control cohort of M. spinolai were significantly higher than those of the T. cruzi-infected cohort (r = 0.0035 d-1 , λ = 1.0035 d-1 ). These results suggest that T. cruzi infection reduces the population fitness of the Chagas disease vector M. spinolai.

Shelter selection in the kissing bugs Triatoma infestans and Rhodnius prolixus: Intra- and inter-specific interactions and quality evaluation.

Triatomines display most of their activities during the night. Before sunrise, they search, select and occupy adequate shelters to stay during the photophase, avoiding exposure to diurnal predators. In this work, we first explored the interactions between individuals of the same or different species in the shelter selection process of Triatoma infestans (Klug 1834) and Rhodnius prolixus (Stål 1859). When two groups of insects (either of different nutritional status, nymphal instar or species) were released together over an experimental arena containing two identical shelters, all nymphs were distributed randomly, suggesting the absence of intra- and inters-pecific interactions. Secondly, we analysed their preferences for particular features of shelters by releasing one group of insects (either T. infestans or R. prolixus) over an arena containing two different refuges. Nymphs exhibited preferences for darker shelters with a vertical orientation of its substrate and elevated from the ground, highlighting the importance of such features in a shelter selection context. We conclude that these species disregard the presence of other individuals but evaluate certain characteristics of the shelters to choose them. This information may contribute to understanding the colonization/recolonization dynamic processes of these Chagas disease vectors.

Wild mammals involved in the transmission of Trypanosoma cruzi and food sources of Triatoma sherlocki in an endemic region of northeastern Brazil.

The present study, carried out in the municipality of Gentio do Ouro, Bahia, Brazil aimed to evaluate which wild mammals may be involved in the transmission of T. cruzi and which are the blood sources for triatomines collected in the study area. PCR analysis of 31 wild mammals captured revealed T. cruzi infection in 6.4% (2/31): one specimen of the opossum Didelphis albiventris (1/3) and one of the rodent Kerodon rupestris (1/5); despite being more frequent in the area, no specimen of the rodent Thrichomys sp. (0/23) was infected. A total of 169 triatomines were captured. The conclusive detection of food sources was possible only for Triatoma sherlocki Papa et al., 2002 (n = 56), with evidence for: K. rupestris (35.7%), Gallus (17.9%), D. albiventris (14.3%), Homo sapiens (14.3%), Tropidurus hispidus (7.1%), Leopardus geoffroyi (5.3%), Conepatus semistriatus (1.8%), Thrichomys inermis (1.8%) and Rattus norvegicus (1.8%). Triatomines of the species T. sherlocki showed food eclecticism, including feeding on humans, with some of them being captured at dwellings. These facts make this triatomine a potential link for the transmission of T. cruzi between wild and anthropic environments, highlighting a latent risk of the reemergence of Chagas disease outbreaks.

"Natural infections" with Trypanosoma cruzi via the skin of mice: size of mouthparts of vectors and numbers of invading parasites.

Investigating parameters influencing natural infections with Trypanosoma cruzi via the skin, the diameters of mouthparts of different stages of triatomines vectors were measured to determine the size of the channel accessible for T. cruzi during cutaneous infection. The mean diameters of the skin-penetrating mandibles of first to fifth instar nymphs of the vector Triatoma infestans increased from 18 to 65 µm. The mean diameter in fourth instar nymphs of Dipetalogaster maxima was 86 µm. Different numbers of isolated vector-derived metacyclic trypomastigotes (10-10,000) were injected intradermally into mice. Prepatent periods, parasitemia and mortality rates were compared with those of mice obtaining 10,000 metacyclic trypomastigotes that are usually present in the first drop of faeces onto the feeding wounds of fifth and fourth instar nymphs of T. infestans and D. maxima, respectively. After injection of 50-10,000 T. cruzi, in all 42 mice the infection developed. An injection of 10 parasites induced an infection in 8 out of 15 mice. With increasing doses of parasites, prepatent periods tended to decrease. The level of parasitemia was higher after injection of the lowest dose. Except for one mouse all infected mice died. After placement of 10,000 metacyclic trypomastigotes onto the feeding wound of fifth or fourth instar nymphs of T. infestans and D. maxima, respectively, the infection rates of the groups, prepatent periods and the levels of parasitemia of T. cruzi in mice indicated that about 10-1,000 metacyclic trypomastigotes entered the skin via this route. For the first time, the present data emphasise the risk of an infection by infectious excreta of triatomines deposited near the feeding wound and the low number of invading parasites.

Detection of Trypanosoma cruzi DNA in false negative samples of collected triatomines, xenodiagnosis material, and biopsies of experimentally infected animals.

Direct test over the gut material from triatomine vectors and xenodiagnosis over mammalian hosts are classical techniques for Trypanosoma cruzi parasitological diagnosis. Nevertheless, negative results can be a source of uncertainty. Experimental models have allowed evaluating the tissue invasion of different strains of T. cruzi, but conventional techniques for tissue biopsies involve time-consuming and elaborated procedures and have low sensitivity. Gut material of collected triatomines (microscopically negative) (n = 114), material of mammal xenodiagnoses (microscopically negative) (n = 138), and biopsy material (microscopically negative) from experimentally infected animals (n = 34) with isolates from endemic areas of Chagas' disease from Venezuela were used for DNA extraction and PCR for the amplification of kinetoplast DNA (kDNA) and satellite DNA (sDNA) of T. cruzi. Positive PCR was observed in 53.6% of collected triatomine material, 15.8% of parasitological negative xenodiagnosis material, and 70.6% in biopsies, revealing underestimation by the parasitological tests and the valour of this analysis with preserved material. Anzoátegui was the state with the highest percentage of infection, and the triatomine species Rhodnius prolixus and Panstrongylus geniculatus had the highest percentages of infection. Didelphis marsupialis and Canis familiaris were the most infected by T. cruzi revealed by PCR of xenodiagnosis material. In addition, the PCR technique allowed demonstrating the invasion of T. cruzi in all tissues analyzed, constituting a molecular marker of tissue invasion.

Biology of Chagas disease vectors: biological cycle and emergence rates of Rhodnius marabaensis Souza et al., 2016 (Hemiptera, Reduviidae, Triatominae) under laboratory conditions.

In Latin America, Chagas disease has been mostly transmitted to humans by contact with the feces or urine of triatomine species infected with the protozoan Trypanosoma cruzi. There are currently 156 species in the subfamily Triatominae, distributed in 18 genera and five tribes. The prolixus group of the genus Rhodnius is composed of 11 species. Rhodnius marabaensis was the last species described and considered in this grouping of vectors. Knowledge about the biology, ecology, and behavior of these vectors is of great epidemiological importance, and in order to expand the knowledge of the biology of R. marabaensis, this paper describes the biological cycle and emergence rates of the species under laboratory conditions. The experiment was carried out at temperatures ranging from 15.5 to 29 °C (average of 24 °C) and humidity ranging from 51.4 to 72.2 (average of 63). For each of the fifteen couples, the egg emergence rate was calculated throughout the oviposition period. The oviposition period lasted from February to September, and the emergence rate varied between 13.9 and 53.3%. R. marabaensis presented an emergence rate of 46.7% and a total biological cycle of 193 days (the mean time required for emergence (25.1 days), 1st nymphal instar (19.4 days), 2nd nymphal instar (22.1 days), 3rd nymphal instar (26.2 days), 4th nymphal instar (29.3 days), and 5th nymphal instar (70.9 days)). Based on the biological cycle of R. marabaensis and 14 other Rhodnius species already described in the literature, it was also possible to calculate the averages for the groups prolixus, pictipes, and pallescens and, mainly, for the genus Rhodnius, contributing to the knowledge of this important group of Chagas disease vectors.

Zuccagnia punctata Cav. Essential Oil into Poly(ε-caprolactone) Matrices as a Sustainable and Environmentally Friendly Strategy Biorepellent against Triatoma infestans (Klug) (Hemiptera, Reduviidae).

The main strategies against Triatoma infestans (primary vector responsible for the Chagas disease transmission) are the elimination or reduction of its abundance in homes through the application of insecticides or repellents with residual power, and environmental management through the improvement of housing. The use of plant-derived compounds as a source of therapeutic agents (i.e., essential oils from aromatic plants and their components) is a valuable alternative to conventional insecticides and repellents. Essential oil-based insect repellents are environmentally friendly and provide reliable personal protection against the bites of mosquitoes and other blood-sucking insects. This study investigates, for the first time to our knowledge, the potential repellent activity of Zuccagnia punctata essential oil (ZEO) and poly(ε-caprolactone) matrices loaded with ZEO (ZEOP) prepared by solvent casting. The analysis of its essential oil from aerial parts by GC-FID and GC-MS, MS allowed the identification of 25 constituents representing 99.5% of the composition. The main components of the oil were identified as (-)-5,6-dehydrocamphor (62.4%), alpha-pinene (9.1%), thuja-2, 4 (10)-diene (4.6%) and dihydroeugenol (4.5%). ZEOP matrices were homogeneous and opaque, with thickness of 800 ± 140 µm and encapsulation efficiency values above 98%. ZEO and ZEOP at the lowest dose (0.5% wt./wt., 96 h) showed a repellency of 33 and 73% respectively, while at the highest dose (1% wt./wt., 96 h) exhibited a repellent activity of 40 and 66 %, respectively. On the other hand, until 72 h, ZEO showed a strong repellent activity against T. infestans (88% repellency average; Class V) to both concentrations, compared with positive control N-N diethyl-3-methylbenzamide (DEET). The essential oils from the Andean flora have shown an excellent repellent activity, highlighting the repellent activity of Zuccagnia punctata. The effectiveness of ZEO was extended by its incorporation in polymeric systems and could have a potential home or peridomiciliary use, which might help prevent, or at least reduce, Chagas' disease transmission.

Triatomines of the Genus Rhodnius Do Not Mark Shelters with Feces.

Aggregation to volatile compounds emitted by feces has been demonstrated for several triatomine species. This signal guides the insects to suitable places that offer physical protection while providing contact with conspecifics. Though the use of aggregation marks has also been reported for Rhodnius spp., it is still unclear whether feces really cause these insects to aggregate inside shelters. In two-choice assays using artificial shelters, we found that refuges associated with a blend of synthetic compounds based on volatiles released by the feces of some triatomine species and reported to be attractive to several species, did not induce shelter choice in Rhodnius prolixus Stål, 1859. In addition, we show that refuges associated with feces of conspecifics did not induce shelter choice in R. prolixus, R. robustus Larrousse, 1927, R. neglectus Lent, 1954, and R. ecuadoriensis Lent and León, 1958. In contrast, as expected, control experiments with Triatoma infestans (Klug, 1834), another triatomine known to aggregate in shelters marked with feces, definitely showed preference for shelters containing feces of conspecifics. Our results clearly show that volatile signals associated with feces do not mediate shelter choice in Rhodnius spp. As a consequence, a paradigm shift will be necessary and, consequently, ab ovo investigations on the clues inducing aggregation behavior in these species.

Parasite × vector relationship in Chagas disease: does Trypanosoma cruzi (Chagas, 1909) infection affect the spermatogenesis of Triatoma infestans (Klug, 1834)?

The parasite-vector interaction of Chagas disease is still poorly understood and the understanding of this relationship can help in the development of new strategies to control Trypanosoma cruzi transmission, which is the etiological agent of this disease. Considering the need to know if T. cruzi can cause some pathology in the reproductive system of the Chagas disease vectors, we investigated the spermatogenesis of Triatoma infestans infected by T. cruzi through histological and cytogenetic analysis. Trypanosoma cruzi Bolivia strain infection was not pathogenic for the reproductive system of T. infestans, because all the analyzed males had normal spermatogenesis, with all phases (spermatocytogenesis, meiosis and spermiogenesis) happening without any change. Thus, we demonstrated that the presence of T. cruzi Bolivia strain does not have influence in the spermatogenesis of T. infestans and we suggest that the influences on reproductive system observed for other species were a result of the action of the parasite on gametogenesis of females.

Modeling the Spatial Spread of Chagas Disease.

The aim of this work is to understand the spatial spread of Chagas disease, which is primarily transmitted by triatomines. We propose a mathematical model using a system of partial differential reaction-diffusion equations to study and describe the spread of this disease in the human population. We consider the respective subclasses of infected and uninfected individuals within the human and triatomine populations. The dynamics of the infected human subpopulation considers two disease phases: acute and chronic. The human population is considered to be homogeneously distributed across a space to describe the local propagation of Chagas disease by triatomines during a short epidemic period. We determine the basic reproduction number that allows us to assess Chagas disease control measures, and we determine the speed of disease propagation by using traveling wave solutions for our model.

Ecological and physiological thermal niches to understand distribution of Chagas disease vectors in Latin America.

In order to assess how triatomines (Hemiptera, Reduviidae), Chagas disease vectors, are distributed through Latin America, we analysed the relationship between the ecological niche and the limits of the physiological thermal niche in seven species of triatomines. We combined two methodological approaches: species distribution models, and physiological tolerances. First, we modelled the ecological niche and identified the most important abiotic factor for their distribution. Then, thermal tolerance limits were analysed by measuring maximum and minimum critical temperatures, upper lethal temperature, and 'chill-coma recovery time'. Finally, we used phylogenetic independent contrasts to analyse the link between limiting factors and the thermal tolerance range for the assessment of ecological hypotheses that provide a different outlook for the geo-epidemiology of Chagas disease. In triatomines, thermo-tolerance range increases with increasing latitude mainly due to better cold tolerances, suggesting an effect of thermal selection. In turn, physiological analyses show that species reaching southernmost areas have a higher thermo-tolerance than those with tropical distributions, denoting that thermo-tolerance is limiting the southern distribution. Understanding the latitudinal range along its physiological limits of disease vectors may prove useful to test ecological hypotheses and improve strategies and efficiency of vector control at the local and regional levels.

Differences in competitive ability for the occupancy of shelters in triatomines.

Triatomines (Hemiptera: Reduviidae: Triatominae) are nocturnal blood-sucking insects. During daylight hours they remain in an akinetic state inside their shelters, whereas at dusk they become active and move outside. When they are outside their shelters during the photophase, triatomines are vulnerable to diurnal predators and the period just before dawn is critical to their survival. This work analyses the existence of competitive interactions involved in the occupancy of shelters by triatomines. Behavioural assays were performed in which nymphs of different stages, nutritional status or species were released in an experimental arena containing a space-limited artificial shelter. The proportions of individuals occupying the shelter during the photophase were quantified to estimate the competitive abilities of each stage and species. Intraspecific comparisons showed higher levels of shelter occupancy for fourth over fifth instars and fed over unfed nymphs of Triatoma infestans. Interspecific comparisons showed higher rates of shelter occupancy for Triatoma sordida in comparison with T. infestans, and for T. infestans over Rhodnius prolixus. Arrival order was also relevant to determining shelter occupancy levels: early arrival was advantageous in comparison with later arrival. The study of intra- and interspecific competitive interactions for shelter occupancy provides relevant information about colonization and recolonization processes in the natural environments of triatomines.

Dicistroviridae: A new viral purification technique.

The family Dicistroviridae comprises three genera and about twenty species of RNA virus, most of them with health or agricultural importance. The Triatoma virus (TrV) is the only entomopathogenic virus identified in triatomine bugs up to the present. TrV replicates within the intestinal epithelial cells, causing high mortality rate and delayed development of the molt of these bugs. TrV has been proposed as a biological control agent for vectors of Chagas disease. Viral particles were purified from feces of 1, 5 and 10 insects from an experimental colony of Triatoma infestans infected with TrV. Viral concentration and infectivity were corroborated using polyacrylamide gels and RT-PCR, respectively. In this work we report a method of viral purification that allows to reduce necessary reagents and time, using a very small amount of fecal matter.

High-Resolution Molecular Typing of Trypanosoma cruzi in 2 Large Outbreaks of Acute Chagas Disease in Colombia.

Oral transmission of Trypanosoma cruzi has gained relevance because of its association with high morbidity and lethality rates. This transmission route is responsible for maintaining the infection of the parasite in sylvatic cycles, and human cases have been associated mainly with the consumption of food contaminated with triatomine feces or didelphid secretions. Several ecological changes allow the intrusion of sylvatic reservoirs and triatomines to the domestic environments with subsequent food contamination. Here, high-resolution molecular tools were used to detect and genotype T. cruzi across humans, reservoirs, and insect vectors in 2 acute outbreaks of presumptive oral transmission in eastern Colombia.

Invasion speeds of Triatoma dimidiata, vector of Chagas disease: An application of orthogonal polynomials method.

Demographic processes and spatial dispersal of Triatoma dimidiata, a triatomine species vector of Chagas disease, are modeled by integrodifference equations to estimate invasion capacity of this species under different ecological conditions. The application of the theory of orthogonal polynomials and the steepest descent method applied to these equations, allow a good approximation of the abundance of the adult female population and the invasion speed. We show that: (1) under the same mean conditions of demography and dispersal, periodic spatial dispersal results in an invasion speed 2.5 times larger than the invasion speed when spatial dispersal is continuous; (2) when the invasion speed of periodic spatial dispersal is correlated to adverse demographic conditions, it is 34.7% higher as compared to a periodic dispersal that is correlated to good demographic conditions. From our results we conclude, in terms of triatomine population control, that the invasive success of T. dimidiata may be most sensitive to the probability of transition from juvenile to adult stage. We discuss our main theoretical predictions in the light of observed data in different triatomines species found in the literature.

Origin, evolution and function of the hemipteran perimicrovillar membrane with emphasis on Reduviidae that transmit Chagas disease.

The peritrophic matrix is a chitin-protein structure that envelops the food bolus in the midgut of the majority of insects, but is absent in some groups which have, instead, an unusual extra-cellular lipoprotein membrane named the perimicrovillar membrane. The presence of the perimicrovillar membrane (PMM) allows these insects to exploit restricted ecological niches during all life stages. It is found only in some members of the superorder Paraneoptera and many of these species are of medical and economic importance. In this review we present an overview of the midgut and the digestive system of insects with an emphasis on the order Paraneoptera and differences found across phylogenetic groups. We discuss the importance of the PMM in Hemiptera and the apparent conservation of this structure among hemipteran groups, suggesting that the basic mechanism of PMM production is the same for different hemipteran species. We propose that the PMM is intimately involved in the interaction with parasites and as such should be a target for biological and chemical control of hemipteran insects of economic and medical importance.

Vital Statistics of Triatominae (Hemiptera: Reduviidae) Under Laboratory Conditions: IV. Panstrongylus geniculatus.

A cohort of 100 eggs of Panstrongylus geniculatus (Latreille) was reared in the laboratory under constant conditions (temperature 26 ± 1°C, 60 ± 10% RH), with mortality and fecundity data recorded weekly. We calculated stage-specific development times, age-specific mortality and fecundity (18.4 eggs/♀/wk), and stage-specific and total preadult mortality (31.6%), and the weekly intrinsic rate of natural increase (r(o) = 0.096), the finite population growth rate (λ = 1.109), the net reproductive rate (R(0) = 60.45), and the generation time (T = 46.34 wk). Elasticity analysis showed that the dominant life-history trait determining λ was survival (particularly the adult female's survival). Adult females dominated the stage-specific reproductive value, and the egg stage dominated the stable stage distribution (SSD). The damping ratio (ρ = 1.096) suggests a relatively rapid period of recovery to a disturbed SSD. Results were compared with one previous study and conform relatively well, considering that environmental conditions were not the same. We estimated the colonizing ability of P. geniculatus, using as a criterion the ro/b index, and obtained the value of 0.74, an indicator of a good colonizer, and similar to well-known invasive species such as Rhodnius prolixus and Triatoma infestans. The life history traits and demographic parameters here presented for P. geniculatus are discussed in terms of their usefulness for evolutionary studies and vector control activities.

An entomological and seroepidemiological study of the vectorial-transmission risk of Chagas disease in the coast of northern Chile.

Four species of triatomines are known from Chile: Triatoma infestans Klug, Mepraia spinolai Porter, M. gajardoi Frías, Henry & González, and M. parapatrica Frías (Hemiptera: Reduviidae), the last three are endemic. The geographical distribution of M. gajardoi includes the coastal areas in the north of Chile between 18° and 21°S, an area with both a resident workforce and summer-season visitors. A study was developed to assess the risk of vectorial transmission of Chagas disease by M. gajardoi in hut settlements on the coast of the Tarapacá Region, in particular in Caleta San Marcos and Caleta Río Seco. The study comprised fingerstick sampling of 95 persons, venous samples from 29 domestic dogs and capture of 52 triatomines, from both fishing coves. The samples were analysed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) techniques. The results show that, of the total number of persons studied, 100% were negative for Trypanosoma cruzi Chagas (Trypanosomatida: Trypanosomatidae) antibodies, 10.34% of canids were positive for the antibody and 5.8% of M. gajardoi were infected to the PCR technique. The presence of this species in areas close to human settlements constitutes a risk to human populations established on the coast of northern Chile.

Biological parameters of interbreeding subspecies of Meccus phyllosomus (Hemiptera: Reduviidae: Triatominae) in western Mexico.

Understanding the biological parameters of some triatomine subspecies of Meccus phyllosomus (Burmeister) is a crucial first step in estimating the epidemiological importance of this group. Biological parameters related to egg eclosion, egg-to-adult development time, number of blood meals to moult, percentage of females at the end of the cycle, number of laid eggs, and the accumulative mortality for each instar of three M. phyllosomus subspecies [Meccus phyllosomus pallidipennis (Stål), Meccus phyllosomus longipennis (Usinger), and Meccus phyllosomus picturatus (Usinger)] as well as their laboratory hybrids were evaluated and compared. No significant differences (P > 0.05) were recorded among the experimental hybrids (M. p. longipennis × M. p. pallidipennis, M. p. longipennis × M. p. picturatus, M. p. pallidipennis × M. p. picturatus) and reciprocal cohorts. In five of the six studied parameters (egg eclosion, egg-to-adult development time, number of blood meals to moult, number of laid eggs and accumulative mortality), with the exception of the non-significant percentage of females obtained among all the studied cohorts, at least one of the parental cohorts in each set of crosses exhibited better fitness results than by those of their hybrid descendants. The lack of hybrid fitness in our study indicates the maintenance of reproductive isolation of parental genotypes. Moreover, the results lead us to propose that an incipient speciation process by distance is currently developing among the three studied subspecies, increasing the differences between them that modify the transmission efficiency of Trypanosoma cruzi to human beings in Mexico.