Wild populations of Triatoma infestans: Compilation of positive sites and comparison of their ecological niche with domestic population niche.

BACKGROUND: For several years, the wild populations of Triatoma infestans, main vector of Trypanosoma cruzi causing Chagas disease, have been considered or suspected of being a source of reinfestation of villages. The number of sites reported for the presence of wild T. infestans, often close to human habitats, has greatly increased, but these data are scattered in several publications, and others obtained by our team in Bolivia have not been published yet. METHODOLOGY/PRINCIPAL FINDINGS: Herein is compiled the largest number of wild sites explored for the presence of T. infestans collected with two methods The standardized methods aimed to determine the relationship between wild T. infestans and the ecoregion, and the directed method help to confirm the presence/absence of triatomines in the ecoregions. Entomological indices were compared between ecoregions and an environmental niche modelling approach, based on bioclimatic variables, was applied. The active search for wild T. infestans in Bolivia suggests a discontinuous distribution from the Andean valleys to the lowlands (Chaco), while the models used suggest a continuous distribution between the two regions and very large areas where wild populations remain to be discovered. The results compile the description of different habitats where these populations were found, and we demonstrate that the environmental niches of wild and domestic populations, defined by climatic variables, are similar but not equivalent, showing that during domestication, T. infestans has conquered new spaces with wider ranges of temperature and precipitation. CONCLUSIONS/SIGNIFICANCE: The great diversity of wild T. infestans habitats and the comparison of their ecological niches with that of domestic populations confirm the behavioural plasticity of the species that increase the possibility of contact with humans. The result of the geographical distribution model of the wild populations calls for more entomological vigilance in the corresponding areas in the Southern Cone countries and in Bolivia. The current presentation is the most comprehensive inventory of wild T. infestans-positive sites that can be used as a reference for further entomological vigilance in inhabited areas.

Molecular identification of wild triatomines of the genus Rhodnius in the Bolivian Amazon: Strategy and current difficulties.

The Amazon region has recently been considered as endemic in Latin America. In Bolivia, the vast Amazon region is undergoing considerable human migrations and substantial anthropization of the environment, potentially renewing the danger of establishing the transmission of Chagas disease. The cases of human oral contamination occurring in 2010 in the town of Guayaramerín provided reasons to intensify research. As a result, the goal of this study was to characterize the species of sylvatic triatomines circulating in the surroundings of Yucumo (Beni, Bolivia), a small Amazonian city at the foot of the Andes between the capital (La Paz) and Trinidad the largest city of Beni. The triatomine captures were performed with mice-baited adhesive traps mostly settled in palm trees in forest fragments and pastures. Species were identified by morphological observation, dissection of genitalia, and sequencing of three mitochondrial gene fragments and one nuclear fragment. Molecular analysis was based on (i) the identity score of the haplotypes with GenBank sequences through the BLAST algorithm and (ii) construction of phylogenetic trees. Thirty-four triatomines, all belonging to the Rhodnius genus, of which two were adult males, were captured in palm trees in forest fragments and pastures (overall infestation rate, 12.3%). The morphology of the phallic structures in the two males confirmed the R. stali species. For the other specimens, after molecular sequencing, only one specimen was identified with confidence as belonging to Rhodnius robustus, the others belonged to one of the species of the Rhodnius pictipes complex, probably Rhodnius stali. The two species, R. robustus and R. stali, had previously been reported in the Alto Beni region (edge of the Amazon region), but not yet in the Beni department situated in the Amazon region. Furthermore, the difficulties of molecular characterization of closely related species within the three complexes of the genus Rhodnius are highlighted and discussed.

Blood meal sources of wild and domestic Triatoma infestans (Hemiptera: Reduviidae) in Bolivia: connectivity between cycles of transmission of Trypanosoma cruzi.

BACKGROUND: Chagas disease is a major public health problem in Latin America. Its etiologic agent, Trypanosoma cruzi, is mainly transmitted through the contaminated faeces of blood-sucking insects called triatomines. Triatoma infestans is the main vector in various countries in South America and recently, several foci of wild populations of this species have been described in Bolivia and other countries. These wild populations are suspected of affecting the success of insecticide control campaigns being carried out in South America. To assess the risk that these T. infestans populations pose to human health, it is helpful to determine blood meal sources. METHODS: In the present work, blood meals were identified in various Bolivian wild T. infestans populations and in three specific areas, in both wild and intra-peridomestic populations to assess the links between wild and domestic cycles of T. cruzi transmission. PCR-HDA and sequencing of Cytb gene were used to identify these blood meal sources. RESULTS AND DISCUSSION: Fourteen vertebrate species were identified as wild blood meal sources. Of those, the most prevalent species were two Andean endemic rodents, Octodontomys gliroides (36%) and Galea musteloides (30%), while humans were the third most prevalent source (18.7%). Of 163 blood meals from peridomestic areas, more than half were chickens, and the others were generally domestic animals or humans. Interestingly, blood from wild animals was identified in triatomines captured in the peridomestic and domestic environment, and blood from domestic animals was found in triatomines captured in the wild, revealing links between wild and domestic cycles of T. cruzi transmission. CONCLUSION: The current study suggests that wild T. infestans attack humans in the wild, but is also able to bite humans in domestic settings before going back to its natural environment. These results support the risk to human health posed by wild populations of T. infestans.

Variations in time and space of an Andean wild population of T. infestans at a microgeographic scale.

BACKGROUND: Wild populations of Triatoma infestans are now believed to be the source of reinfestation of dwellings in some Andean areas and could impede the full achievement of vector control campaigns in this region. Given the poor knowledge of these populations in natural conditions, their basic biology traits, such as monthly demographic variations and movements of individuals, were explored. METHODS: A previously identified wild population of T. infestans in a field adjacent to a group of isolated houses in an Andean valley (department of La Paz, Bolivia) was explored using regular capture assays over 13 months in 50 sites selected at the beginning of the study. The capture-mark-recapture method was applied monthly using mouse-baited adhesive traps for captures and fingernail polish of different colors for the marking. RESULTS: The monthly capture assays did not show significant differences between rainy and dry seasons, showing evidence for a certain stability of the wild T. infestans population with only the nymph population tending to decline during the middle of the rainy season when rain is more intensive. Throughout the study, the monthly average number of bugs was 51.1 ± 25.3 per assay, 91.1% were nymphs, and they were found at 30 of the 50 sites (60%). The number of times a site was positive varied from one to 13. Site infestation was associated with the underground position of the traps, and rocks around and in the surroundings of the traps. The recaptures after marking were successful (138 recaptures over the study). The marking made it possible to detect for 14.5% of the recaptures significant movements of adults (up to 168 m) and nymphs (up to 34 m). Some bugs (nymphs and females) were recaptured after 5 months. For adults, recaptures (46 in total) mostly occurred between September and March. Females were recaptured twice as frequently as males. CONCLUSION: The Andean wild populations of T. infestans showed a strong spatial and temporal stability during the year-long study. Dispersal may occur mainly during the rainy season. The capture-mark-recapture method was successful and the longevity of the bugs and the distances covered by nymphs and adults were recorded.

Putative panmixia in restricted populations of Trypanosoma cruzi isolated from wild Triatoma infestans in Bolivia.

Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into six discrete typing units (DTUs; TcI-TcVI) of which TcI is ubiquitous and genetically highly variable. While clonality is the dominant mode of propagation, recombinant events play a significant evolutive role. Recently, foci of wild Triatoma infestans have been described in Bolivia, mainly infected by TcI. Hence, for the first time, we evaluated the level of genetic exchange within TcI natural potentially panmictic populations (single DTU, host, area and sampling time). Seventy-nine TcI stocks from wild T. infestans, belonging to six populations were characterized at eight microsatellite loci. For each population, Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), and presence of repeated multilocus genotypes (MLG) were analyzed by using a total of seven statistics, to test the null hypothesis of panmixia (H0). For three populations, none of the seven statistics allowed to rejecting H0; for another one the low size did not allow us to conclude, and for the two others the tests have given contradictory results. Interestingly, apparent panmixia was only observed in very restricted areas, and was not observed when grouping populations distant of only two kilometers or more. Nevertheless it is worth stressing that for the statistic tests of "HWE", in order to minimize the type I error (i. e. incorrect rejection of a true H0), we used the Bonferroni correction (BC) known to considerably increase the type II error ( i. e. failure to reject a false H0). For the other tests (LD and MLG), we did not use BC and the risk of type II error in these cases was acceptable. Thus, these results should be considered as a good indicator of the existence of panmixia in wild environment but this must be confirmed on larger samples to reduce the risk of type II error.

Wild populations of Triatoma infestans are highly connected to intra-peridomestic conspecific populations in the Bolivian Andes.

Triatoma infestans, the major vector of Chagas disease south of the Amazon in South America, has a large distribution of wild populations, contrary to what has previously been stated. These populations have been suspected of being the source of reinfestation of human habitats and could impede the full success of vector control campaigns. This study examined gene flow between intra-peridomestic populations and wild populations collected in the surround areas in three Andean localities in Bolivia. The populations were defined according to temporal, ecological, and spatial criteria. After DNA extraction from the legs of each insect, the samples were analyzed using seven microsatellite markers. First, the analysis of molecular variance (AMOVA) detected an absence of differentiation between wild and intra-peridomestic populations, although strong structuring was observed between the populations within each environment. Then for some populations, the Bayesian method of assignment to inferred populations showed very similar assignment patterns of the members of wild or intra-peridomestic populations in each locality. Finally, the detection of the first-generation migrants within the different populations provided evidence of insect displacement from the wild to the intra-peridomestic environment. This result indicates that, after control campaigns in the Andes, controlling this new paradigm of vector transmission risk stemming from the invasion of human habitats by wild populations of T. infestans requires long-term maintenance of public monitoring to keep the risk at a minimal level. Since wild populations of T. infestans have also been detected elsewhere in Argentina, Paraguay, and Chile, there is an urgent need to take these populations into account in future monitoring of Chagas disease transmission.

Predominance of hybrid discrete typing units of Trypanosoma cruzi in domestic Triatoma infestans from the Bolivian Gran Chaco region.

In the Gran Chaco region the reinfestation by Triatoma infestans remains a major problem for control of Chagas disease. Trypanosoma cruzi the agent of the illness presents a broad genetic intraspecific variability which is poorly documented in the Bolivian Gran Chaco. This work presents the identification of the discrete typing units (DTUs) currently recognized for T. cruzi in T. infestans populations collected before and after residual insecticide spraying in four villages in this region. Before spraying, of 84 samples, the frequencies of the DTUs identified by using the multiplex PCR based on the non transcribed spacer of the mini-exon gene (MMPCR) were 0.21 for TcI, 0.70 for TcII/TcV/TcVI, and 0.17 for TcIII/TcIV and no significant difference was observed after spraying (76 samples). Moreover 13% of the total sample corresponds to T. infestans specimens with mixed infection of DTUs of which three were TcII/TcV/TcVI with TcIII/TcIV. The partial sequences of T. cruzi Gpi gene obtained from 14 PCR products agree the MMPCR DTU identification and allowed to precise the occurrence of TcIII, TcII and hybrid TcV/TcVI stocks which were not discriminated by the MMPCR. Given the high prevalence of hybrid stocks, the authors ask whether the recombination event at the origin of hybrids would have taken place in the Gran Chaco where the putative parents are also present.

Susceptibility and resistance to deltamethrin of wild and domestic populations of Triatoma infestans (Reduviidae: Triatominae) in Bolivia: new discoveries

Bolivia is a high-endemic country for Chagas disease, for which the principal vector is Triatoma infestans (Triatominae). This is a mainly domestic species that is also found in the wild environment. Recently, an increasing number of studies have shown the importance of Triatominae resistance to insecticides, especially in Bolivia. Data regarding the susceptibility/resistance of wild and domestic populations of T. infestans to deltamethrin are presented. For the first time, domestic populations of the department of Santa Cruz were tested, showing low resistance. Although most of the wild populations were found to be susceptible to deltamethrin, three populations from three departments showed a mortality rate of less than 100%. This result is emphasised here.

Genetic characterization of Trypanosoma cruzi DTUs in wild Triatoma infestans from Bolivia: predominance of TcI.

BACKGROUND: The current persistence of Triatoma infestans (one of the main vectors of Chagas disease) in some domestic areas could be related to re-colonization by wild populations which are increasingly reported. However, the infection rate and the genetic characterization of the Trypanosoma cruzi strains infecting these populations are very limited. METHODOLOGY/PRINCIPAL FINDINGS: Of 333 wild Triatoma infestans specimens collected from north to south of a Chagas disease endemic area in Bolivia, we characterized 234 stocks of Trypanosoma cruzi using mini-exon multiplex PCR (MMPCR) and sequencing the glucose phosphate isomerase (Gpi) gene. Of the six genetic lineages ("discrete typing units"; DTU) (TcI-VI) presently recognized in T. cruzi, TcI (99.1%) was overdominant on TcIII (0.9%) in wild Andean T. infestans, which presented a 71.7% infection rate as evaluated by microscopy. In the lowlands (Bolivian Chaco), 17 "dark morph" T. infestans were analyzed. None of them were positive for parasites after microscopic examination, although one TcI stock and one TcII stock were identified using MMPCR and sequencing. CONCLUSIONS/SIGNIFICANCE: By exploring large-scale DTUs that infect the wild populations of T. infestans, this study opens the discussion on the origin of TcI and TcV DTUs that are predominant in domestic Bolivian cycles.

New discoveries of sylvatic Triatoma infestans (Hemiptera: Reduviidae) throughout the Bolivian Chaco.

Sylvatic populations of Triatoma infestans might be involved in the recolonization of human dwellings. We report here the discoveries of new T. infestans sylvatic foci in the Bolivian Chaco. Eighty-one triatomines were caught, 38 of which were identified as T. infestans. Triatoma sordida and Panstrongylus geniculatus were the other species collected. One T. infestans and one T. sordida were infected with Trypanosoma cruzi TcI; one T. infestans was infected with TcII. These discoveries add to the debate on the geographic distribution of sylvatic T. infestans populations, the geographic origin of the species, and the epidemiological role of these populations.

Combination of cytochrome b heteroduplex-assay and sequencing for identification of triatomine blood meals.

The identification of blood meals in vectors contributes greatly to the understanding of interactions between vectors, microorganisms and hosts. The aim of the current work was to complement the validation of cytochrome b (Cytb) heteroduplex assay (HDA) previously described, and to add the sequencing of the Cytb gene of some samples for the identification of blood meals in triatomines. Experimental feedings of reared triatomines helped to clarify the sensitivity of the HDA. Moreover, the sequencing coupled with the HDA, allowed the assessment of the technique's taxonomic level of discrimination. The primers used to produce DNA fragments of Cytb genes for HDA had a very high sensitivity for vertebrate DNAs, rather similar for mammals, birds and reptiles. However, the formation of heteroduplex depended on blood meal's quality rather than its quantity; a correlation was observed between blood meals' color and the positivity of HDA. HDA electrophoresis profiles were reproducible, and allowed the discrimination of blood origins at the species level. However, in some cases, intraspecific variability of Cytb gene generated different HDA profiles. The HDA based on comparison of electrophoresis profiles is a very useful tool for screening large samples to determine blood origins; the subsequent sequencing of PCR products of Cytb corresponding to different HDA profiles allowed the identification of species whatever the biotope in which the vectors were captured.

Susceptibility and resistance to deltamethrin of wild and domestic populations of Triatoma infestans (Reduviidae: Triatominae) in Bolivia: new discoveries.

Bolivia is a high-endemic country for Chagas disease, for which the principal vector is Triatoma infestans (Triatominae). This is a mainly domestic species that is also found in the wild environment. Recently, an increasing number of studies have shown the importance of Triatominae resistance to insecticides, especially in Bolivia. Data regarding the susceptibility/resistance of wild and domestic populations of T. infestans to deltamethrin are presented. For the first time, domestic populations of the department of Santa Cruz were tested, showing low resistance. Although most of the wild populations were found to be susceptible to deltamethrin, three populations from three departments showed a mortality rate of less than 100%. This result is emphasised here.

New insights on the Chagas disease main vector Triatoma infestans (Reduviidae, Triatominae) brought by the genetic analysis of Bolivian sylvatic populations.

Triatoma infestans is the main and most widespread vector of Chagas disease in South America. For the first time, a large sample of sylvatic populations of T. infestans was analyzed by ITS-2 and mtCytB sequencing. ITS-2 showed a low level of polymorphism but revealed a dichotomy between the Andean and non-Andean sylvatic populations. On the contrary, mtCytB sequences showed a high polymorphism (19 haplotypes determined by 35 variable sites) revealing a strong structuring between most of the sylvatic populations and possible ancient isolation and bottleneck in the Northern Andes. The dichotomy Andean vs. non-Andean populations was not observed with this marker. Moreover, mtCytB haplotype genealogies showed that the non-Andean haplotypes would have derived from the Andean ones, supporting somewhat an Andean origin of the species. Nevertheless, a non-Andean origin could not be discarded because a remarkable genetic diversity was found in the non-Andean sample. The comparison of the sylvatic haplotypes with the domestic ones from GenBank suggested multiple events of T. infestans domestication in Andean and non-Andean areas, instead of a major and unique domestication event in the Bolivian Andes, as previously proposed.

PCR-RFLP of ribosomal internal transcribed spacers highlights inter and intra-species variation among Leishmania strains native to La Paz, Bolivia.

Human leishmaniasis is highly endemic in Bolivia and shows a growing incidence. This report reveals the genetic variability of 35 isolates mainly belonging to Leishmania braziliensis and Leishmania amazonensis species. Among them, 31 were from human patients with different clinical presentations, 3 strains from Lutzomya nuneztovari anglesi (the proven vector of L. amazonensis) and 1 strain of a mammal (Conepatus chinga). The isolates were analyzed by isoenzyme electrophoresis (MLEE) and PCR-RFLP of ITS rRNA genes, a genetic marker highly polymorphic and better adapted to sub-structuring of populations. MLEE and RFLP-ITS were in agreement to discriminate the species, 12 belong to L. (V.) braziliensis, 21 to L. (L.) amazonensis, 1 to Leishmania (V.) lainsoni and 1 to Leishmania (L.) chagasi. Among L. (V.) braziliensis the RFLP-ITS only highlights variability. Ten isolates from either cutaneous or mucocutaneous clinical forms, were grouped together (bootstrap value of 99.8%) apart from two others, one from a mammal (C. chinga), the other from a patient with a cutaneous form. Among L. (L.) amazonensis both markers detect variability but no significant sub-division was identified including isolates from different clinical forms. Moreover, the high frequency of several isolates from cutaneous forms occurred during an outbreak, with putative hybrid character (multiloci heterozygous patterns depicted by MLEE) could be linked to better fitness of these parasites. However, in the absence of observation of hypothetical parents, their hybrid status remains a question.

First report of widespread wild populations of Triatoma infestans (Reduviidae, Triatominae) in the valleys of La Paz, Bolivia.

Wild populations of Triatoma infestans, the main vector of Chagas disease in the Southern Cone countries, may be involved in reinfestation of human dwellings, limiting the success of vector-control campaigns in Bolivia. Knowledge of the distribution of these populations remains incomplete. We report here the detection of T. infestans wild populations in large areas in the department of La Paz, Bolivia. Among 18 sylvatic areas investigated, 17 were positive with T. infestans specimens. The infection rate of captured T. infestans with Trypanosoma cruzi was 85.7% in adult specimens. These results expand the geographical distribution of wild populations of T. infestans; it may be distributed throughout the Inter-Andean Dry Forest eco-region of Bolivia. The current information allows us to propose the hypothesis that a sylvatic origin of the reinfestation is located in the valleys of La Paz.