Rhodnius (Stål, 1859) (Hemiptera, Triatominae) genus in Bolivian Amazonia: a risk for human populations?

BACKGROUND: Chagas disease, one of the most important neglected tropical diseases in the countries of Latin America, is considered to be a particularly important public health concern in the Amazon region due to increases in the number of outbreaks of acute Chagas disease and increased local transmission in the last 20 years. However, relative to other countries, in Bolivia there is little information available on its transmission in the Amazon region. The aim of this study was to investigate the infestation of palm trees, the main habitat of Triatominae in the region, in several localities, to evaluate the danger they represent to inhabitants. METHODS: Triatominae were collected using live bait traps left overnight in six localities in Pando and Beni Departments, Bolivia. DNA extraction and sequencing were used to establish the Triatominae species (Cytb, 16S and 28S-D2 gene fragments), and the blood meal sources (Cytb fragment). Trypanosoma sp. infection was analyzed by sequencing gene fragments (GPX, GPI, HMCOAR, LAP, PDH and COII) or by mini-exon multiplex PCR. RESULTS: A total of 325 Rhodnius were captured (97.3% of nymphs) from the 1200 traps placed in 238 palm trees and 32 burrows/ground holes. Sequence analyses on DNA extracted from 114 insects and phylogeny analysis identified two triatomine species: Rhodnius stali (17%) and Rhodnius montenegrensis (equated to Rhodnius robustus II, 83%). These were found in palm trees of the genera Attalea (69%), Astrocaryum (13%), Copernicia (12%), Euterpe (2%) and Acrocomia (1%). The infection rate was around 30% (165 analyzed insects), with 90% of analyzed insects infected by Trypanosoma cruzi (only the TcI discrete typing unit was detected), 3% infected by Trypanosoma rangeli (first time found in Bolivian Triatominae) and 7% infected by mixed T. cruzi (TcI)-T. rangeli. Rhodnius specimens fed on Didelphidae, rodents, gecko and humans. CONCLUSIONS: The results of this study highlight the epidemiological importance of Rhodnius in the Bolivian Amazon region. The huge geographical distribution of Rhodnius and their proximity to the human dwellings, high infection rate and frequent meals on the human population highlight a risk of transmission of Chagas disease in the region.

Evaluation of the Effectiveness of Chemical Control for Chagas Disease Vectors in Loja Province, Ecuador.

The objective of this study was to evaluate the effectiveness of selective and community-wide house insecticide spraying in controlling triatomines in the subtropical areas of Loja Province, Ecuador. We designed a quasi-experimental pre-post-test without a control group to compare entomological levels before and after spraying. The baseline study was conducted in 2008. Second, third, and fourth visits were conducted in 2010, 2011, and 2012 in three rural communities. Out of the 130 domestic units (DU) visited, 41 domestic units were examined in each of the four visits. Selective and community-wide insecticide interventions included spraying with 5% deltamethrin at 25 mg/m2 active ingredient. At each visit, a questionnaire was administered to identify the characteristics of households, and DUs were searched for triatomine bugs. In addition, parasitological analysis was carried out in life triatomines. One and two rounds of selective insecticide spraying decreased the probability of infestation by 62% (pairwise odds ratios [POR] 0.38, 95% confidence interval [CI] 0.17-0.89, p = 0.024) and 51% (POR 0.49, 95% CI 0.23-1.01, p = 0.054), respectively. A similar effect was observed after one round of community-wide insecticide application in Chaquizhca and Guara (POR 0.55, CI 0.24-1.25, p = 0.155) and Bellamaria (POR 0.62, CI 0.22-1.79, p = 0.379); however, it was not statistically significant. Trypanosoma cruzi infection in triatomines (n = 483) increased overtime, from 2008 (42.9% and 8.5% for Rhodnius ecuadoriensis and Panstrongylus chinai, respectively) to 2012 (79.5% and 100%). Neither of the two spraying methodologies was effective for triatomine control in this area and our results point to a high likelihood of reinfestation after insecticide application. This underscores the importance of the implementation of physical barriers that prevent invasion and colonization of triatomines in households, such as home improvement initiatives, accompanied by a concerted effort to address the underlying socioeconomic issues that keep this population at risk of developing Chagas disease.

Evaluation of Selective Deltamethrin Application with Household and Community Awareness for the Control of Chagas Disease in Southern Ecuador.

Chagas disease is endemic in ~70% of Ecuador. Rhodnius ecuadoriensis and Triatoma carrioni (Hemiptera: Reduviidae) are the primary vectors of Chagas disease in Southern Ecuador. This study tested the effectiveness of selective deltamethrin application of Domiciliary Units (DUs) infested with triatomines, coupled with community education activities and a community-based surveillance system. Ten communities were selected in Loja Province, 466 DUs were examined, of these, 5.6% were infested with R. ecuadoriensis (Density [D] = 4 triatomines/DUs searched, Crowding [CR] = 71 triatomines/infested house, Colonization Index [CI] = 77% infested DUs with nymphs) and 8% with T. carrioni (D = 0.6, CR = 7, CI = 64%). Infested DUs were sprayed with deltamethrin. Subsequent visits were conducted at 6 and 12 mo after spraying. At each time point, new entomological searches were carried out in all DUs. All entomological indexes dropped significantly for the primary vector species one year after the initial intervention (R. ecuadoriensis: I = 2%, D = 0.1, CR = 7, CI = 100%; T. carrioni: I = 1.6%, D = 0.1, CR = 5.5, CI = 50%). Fifteen min educational talks were conducted in every DUs and workshops for schoolchildren were organized. Community-based surveillance system was established. However, there is a high risk of DUs reinfestation, possibly from sylvatic habitats (especially of R. ecuadoriensis) and reinforcing educational and surveillance activities are necessary.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae).

BACKGROUND: Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS: Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES: To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS: We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS: Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting.

Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches.

Human Blood Meals in Sylvatic Triatomines Challenges Domestic-Centered Strategies for Prevention of Trypanosoma cruzi Transmission in Ecuador.

Transmission risk of Chagas disease has been associated with human-vector contacts and triatomines colonizing dwellings, but alternative scenarios, independent of domestic colonization, are poorly documented. In the present work, we estimated the frequency of human blood meals in triatomines from domicile, peridomicile, and sylvatic environments in two endemic regions in Ecuador. Blood meal origins were identified by sequencing a cytb gene fragment. Human blood meals were detected in 42% of the triatomines among 416 analyzed, including 48% of sylvatic triatomines (both adults and nymphs). In triatomines from domicile and peridomicile, Trypanosoma cruzi infection rate was > 20%, and reached 48% in sylvatic triatomines. Human is a common source of blood for triatomines whether they live in or near dwellings in both regions, and the high rate of T. cruzi infection represents an important risk of transmission of Chagas disease. Consequently, control strategies should also take into account possible nondomestic transmission.

Triatomine Feeding Profiles and Trypanosoma cruzi Infection, Implications in Domestic and Sylvatic Transmission Cycles in Ecuador.

Understanding the blood meal patterns of insects that are vectors of diseases is fundamental in unveiling transmission dynamics and developing strategies to impede or decrease human-vector contact. Chagas disease has a complex transmission cycle that implies interactions between vectors, parasites and vertebrate hosts. In Ecuador, limited data on human infection are available; however, the presence of active transmission in endemic areas has been demonstrated. The aim of this study was to determine the diversity of hosts that serve as sources of blood for triatomines in domestic, peridomestic and sylvatic transmission cycles, in two endemic areas of Ecuador (central coastal and southern highland regions). Using conserved primers and DNA extracted from 507 intestinal content samples from five species of triatomines (60 Panstrongylus chinai, 17 Panstrongylus howardi, 1 Panstrongylus rufotuberculatus, 427 Rhodnius ecuadoriensis and 2 Triatoma carrioni) collected from 2006 to 2013, we amplified fragments of the cytb mitochondrial gene. After sequencing, blood meal sources were identified in 416 individuals (146 from central coastal and 270 from southern highland regions), achieving ≥ 95% identity with GenBank sequences (NCBI-BLAST tool). The results showed that humans are the main source of food for triatomines, indicating that human-vector contact is more frequent than previously thought. Although other groups of mammals, such as rodents, are also an available source of blood, birds (particularly chickens) might have a predominant role in the maintenance of triatomines in these areas. However, the diversity of sources of blood found might indicate a preference driven by triatomine species. Moreover, the presence of more than one source of blood in triatomines collected in the same place indicated that dispersal of vectors occurs regardless the availability of food. Dispersal capacity of triatomines needs to be evaluated to propose an effective strategy that limits human-vector contact and, in consequence, to decrease the risk of T. cruzi transmission.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae)

BACKGROUND Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity. METHODS Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples. OBJECTIVES To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons. FINDINGS We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%). MAIN CONCLUSIONS Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

Molecular data supports monophyly of Triatoma dispar complex within genus Triatoma.

The genus Triatoma contains numerous species, principal or secondary vectors of Chagas disease, which have been included in the three main lineages of Triatomini tribe based on morphological and biogeographical characteristics: North American, South American, and T. dispar complex. The three members of the T. dispar complex are distributed in Ecuador. This complex has been scarcely studied through molecular approaches, and the taxonomic position of this complex is not confirmed. In this study, we explored the phylogenetic relationships within the genus Triatoma, including five species from North and Central America, six from South America, and the three species belonging to the T. dispar complex. Partial sequences of four mitochondrial genes (Cyt b, COII, 16S-rRNA, 12S-rRNA) and two nuclear genes (18S-rRNA, ITS2) were obtained from 74 specimens. Phylogenetic trees were built with concatenated and single sequences through maximum likelihood (ML), maximum parsimony (MP), and Bayesian methods. The trees built using concatenated sequences showed three main branches (clusters) highly supported by significant bootstrap values; the T. dispar complex appeared as a monophyletic group separate from species of North and Central American origin and South American origin. On the contrary, for each gene tree, the three main clusters were not always significantly supported, mostly because genetic information is dramatically reduced when a single gene is considered. Consequently, concatenation of genes gives relevant results and is highly recommended for further in-depth examination of the relationships of several species and complexes of triatomines that remain unresolved. Moreover, our current molecular data fully revealed the division of genus Triatoma into at least three main genetic groups.

Would tropical climatic variations impact the genetic variability of triatomines: Rhodnius ecuadoriensis, principal vector of Chagas disease in Ecuador?

Rhodnius ecuadoriensis is one of the most important vector species of Chagas disease in Ecuador. This species is distributed in the Central coast region and in the south Andean region, and an incipient speciation process between these geographical populations was previously proposed. The current population genetics study only focused on the Central coast region and analyzed 96 sylvatic specimens of R. ecuadoriensis associated with Phytelephas aequatorialis palm trees. We used Cytb and 16S-rRNA sequences and a Cytb-16S-rRNA concatenated set to explore (i) the genetic variability, spatial structuring, and demographic history of R. ecuadoriensis, and to determine (ii) the relationship between the genetic and climatic variabilities. A particularly high genetic variability was observed without detectable general genetic structure; only some terminal genetic clusters were observed. We did not observe isolation by geographical distance (IBD), and it is likely that ancient expansion occurred, according to Fs index and mismatch distribution for Cytb-16S-rRNA concatenated sequences. Hierarchical clustering showed that the current locality origins of the bugs were grouped into four bioclimatic clusters. Genetic and bioclimatic distances were not correlated, but some genetic clusters were associated with bioclimatic ones. The results showed an ancient evolution of the species in the region with a possible old expansion. The absence of spatial genetic structure could be due to climatic conditions (possible selection of singular genotypes) and to passive transportation of palms tree materials where R. ecuadoriensis are living.

Chagas vectors Panstrongylus chinai (Del Ponte, 1929) and Panstrongylus howardi (Neiva, 1911): chromatic forms or true species?

BACKGROUND: Chagas disease is a parasitic infection transmitted by "kissing bugs" (Hemiptera: Reduviidae: Triatominae) that has a huge economic impact in Latin American countries. The vector species with the upmost epidemiological importance in Ecuador are Rhodnius ecuadoriensis (Lent & Leon, 1958) and Triatoma dimidiata (Latreille, 1811). However, other species such as Panstrongylus howardi (Neiva, 1911) and Panstrongylus chinai (Del Ponte, 1929) act as secondary vectors due to their growing adaptation to domestic structures and their ability to transmit the parasite to humans. The latter two taxa are distributed in two different regions, they are allopatric and differ mainly by their general color. Their relative morphological similarity led some authors to suspect that P. chinai is a melanic form of P. howardi. METHODS: The present study explored this question using different approaches: antennal phenotype; geometric morphometrics of heads, wings and eggs; cytogenetics; molecular genetics; experimental crosses; and ecological niche modeling. RESULTS: The antennal morphology, geometric morphometrics of head and wing shape and cytogenetic analysis were unable to show distinct differences between the two taxa. However, geometric morphometrics of the eggs, molecular genetics, ecological niche modeling and experimental crosses including chromosomal analyses of the F1 hybrids, in addition to their coloration and current distribution support the hypothesis that P. chinai and P. howardi are separate species. CONCLUSIONS: Based on the evidence provided here, P. howardi and P. chinai should not be synonymized. They represent two valid, closely related species.

Determinants of intentions to prevent triatomine infestation based on the health belief model: An application in rural southern Ecuador.

INTRODUCTION: Control of triatomine infestation is a key strategy for the prevention of Chagas disease (CD). To promote this strategy, it is important to know which antecedents to behavioral change are the best to emphasize when promoting prevention. OBJECTIVE: The aim of this study was to determine predictors for intention to prevent home infestation based on the Health Belief Model (HBM), a commonly used health intervention planning theory. MATERIALS & METHODS: A cross-sectional study was conducted with 112 heads of household in six communities with endemic and high rates of triatomine infestation in Loja province, Ecuador. The data was collected by a questionnaire including perceived severity, susceptibility, benefits to action, barriers to action, and self-efficacy. These data were also used to predict actual infestation of homes. RESULTS: Community members reported strong intentions to prevent home infestation. HBM constructs predicted about 14% of the observed variance in intentions. Perceived susceptibility and severity did not predict behavioral intention well; perceived barriers to small-scale action that reduce likelihood of infestation and self-efficacy in participating in surveillance systems did. Self-efficacy and perception of barriers were equally powerful predictors. The HBM constructs, however, did not predict well actual infestation. CONCLUSION: The findings supported the HBM as a way to predict intentions to prevent infestation of the home by triatomine bugs. The findings highlight that messages emphasizing self-efficacy in participating in surveillance systems and overcoming barriers to small-scale action that reduce likelihood of infestation, rather than a focus on risk, should be central messages when designing and implementing educational interventions for CD. The gap between behavioral intention and actual infestation reveals the need to assess home practices and their actual efficacy to fully enact and apply the HBM.

Genetic data support speciation between Panstrongylus howardi and Panstrongylus chinai, vectors of Chagas disease in Ecuador.

Limited genetic data are currently available for three vectors of Chagas disease in Ecuador, Panstrongylus howardi, P. chinai, and P. rufotuberculatus. Previously regarded as mainly sylvatic, these species have been poorly studied. Recently, they have been more frequently reported in domiciles and peridomiciles and are now considered true secondary vectors of Chagas disease in a country where an estimated 200,000 people are infected by Trypanosoma cruzi, a causative agent of this disease. In order to fill this gap, we obtained DNA for sequencing from 53 insects belonging to these three species and mainly sampled from the two Ecuadorian provinces of Loja and Manabí. We used six mitochondrial loci (COI, COII, ND4, CytB, 16S, and 12S) and two nuclear ones (ITS2 and 18S). We interpreted the phylogenetic trees built with single and concatenated data through maximum likelihood, Bayesian Markov chain Monte Carlo, and maximum parsimony methods. We provide evidence that P. chinai and P. howardi are indeed two supported species closely related and derived from a common ancestor. Additionally, the phylogenetic position of P. rufotuberculatus was confirmed as being distant from P. chinai and P. howardi and clustered with Triatoma dimidiata, a species belonging to the Northern American Triatoma clade.

Life Cycle, Feeding, and Defecation Patterns of Triatoma carrioni (Hemiptera: Reduviidae), Under Laboratory Conditions.

Chagas disease is caused by Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). It is transmitted to humans primarily through contaminated feces of blood-sucking vectors of the subfamily Triatominae, known in Ecuador as 'chinchorros'. Some Triatominae species can adapt to domiciliary and peridomiciliary environments where T. cruzi can be transmitted to humans. Triatoma carrioni (Larrousse 1926) colonizes domestic and peridomestic habitats up to 2,242 m above sea level (masl) in southern Ecuador (Loja Province) and northern Peru. This study describes the life cycle, feeding, and defecation patterns of T. carrioni under controlled laboratory conditions using mice as hosts. Specimens were collected in Loja Province, Ecuador, and maintained in the laboratory. The life cycle was approximately 385.7 ± 110.6 d. There was a high mortality rate, 40.9% for first instars and 38.9% for fifth instars (NV). Feeding and defecation patterns for each life stage were examined by recording: insertion time of the proboscis into the host, total feeding time, time to first defecation, and weight of the bloodmeal. Total feeding time varied between 20.6 ± 11.4 min for first instars (NI) and 48.9 ± 19.0 min for adult females. The time to first defecation was variable but ranged from 9.8 ± 10.6 min for NI to 39.4 ± 24.7 min for NV during feeding. This suggests that T. carrioni has an annual life cycle and is a potential vector of T. cruzi in Loja Province. Improved knowledge of populations of T. carrioni in domestic and peridomestic environments of Ecuador can have a significant impact on the prevention and control of Chagas disease.

Triatominae: does the shape change of non-viable eggs compromise species recognition?

BACKGROUND: Eggs have epidemiological and taxonomic importance in the subfamily Triatominae, which contains Chagas disease vectors. The metric properties (size and shape) of eggs are useful for distinguishing between close species, or different geographical populations of the same species. METHODS: We examined the effects of egg viability on its metric properties, and the possible consequences on species recognition. Four species were considered: Panstrongylus chinai, P. howardi and Triatoma carrioni (tribe Triatomini), and Rhodnius ecuadoriensis (tribe Rhodniini). Digitization was performed on pictures taken when the viability of the egg could not clearly be predicted by visual inspection. We then followed development to separate viable from non-viable eggs, and the metric changes associated with viability status of the eggs were tested for species discrimination (interspecific difference). RESULTS: The shape of the complete contour of the egg provided satisfactory species classification (95% of correct assignments, on average), with improved scores (98%) when discarding non-viable eggs from the comparisons. Using only non-viable eggs, the scores dropped to 90%. The morphometric differences between viable and non-viable eggs were also explored (intraspecific comparison). A constant metric change observed was a larger variance of size and shape in non-viable eggs. For all species, larger eggs, or eggs with larger operculum, were more frequently non-viable. However, these differences did not allow for an accurate prediction regarding egg viability. CONCLUSIONS: The strong taxonomic signal present in egg morphology was affected by the level of viability of the eggs. The metric properties as modified in non-viable eggs presented some general trends which could suggest the existence of an optimum phenotype for size and for shape. Globally, viable eggs tended to have intermediate or small sizes, and presented a less globular shape in the Triatomini, or a relatively wider neck in Rhodnius ecuadoriensis.

Influence of ecological factors on the presence of a triatomine species associated with the arboreal habitat of a host of Trypanosoma cruzi.

BACKGROUND: The white-naped squirrel, Simosciurus nebouxii (previously known as Sciurus stramineus), has recently been identified as an important natural host for Trypanosoma cruzi in Ecuador. The nests of this species have been reported as having high infestation rates with the triatomine vector Rhodnius ecuadoriensis. The present study aims to determine the levels of nest infestation with R. ecuadoriensis, the ecological variables that are influencing the nest site selection, and the relationship between R. ecuadoriensis infestation and trypanosome infection. RESULTS: The study was carried out in transects in forest patches near two rural communities in southern Ecuador. We recorded ecological information of the trees that harbored squirrel nests and the trees within a 10 m radius. Manual examinations of each nest determined infestation with triatomines. We recorded 498 trees (n = 52 with nests and n = 446 without nests). Rhodnius ecuadoriensis was present in 59.5% of the nests and 60% presented infestation with nymphs (colonization). Moreover, we detected T. cruzi in 46% of the triatomines analyzed. CONCLUSIONS: We observed that tree height influences nest site selection, which is consistent with previous observations of squirrel species. Factors such as the diameter at breast height and the interaction between tree height and tree species were not sufficient to explain squirrel nest presence or absence. However, the nest occupancy and tree richness around the nest were significant predictors of the abundance of triatomines. Nevertheless, the variables of colonization and infection were not significant, and the data observed could be expected because of chance alone (under the null hypothesis). This study ratifies the hypothesis that the ecological features of the forest patches around rural communities in southern Ecuador favor the presence of nesting areas for S. nebouxii and an increase of the chances of having triatomines that maintain T. cruzi populations circulating in areas near human dwellings. Additionally, these results highlight the importance of including ecological studies to understand the dynamics of T. cruzi transmission due to the existence of similar ecological and land use features along the distribution of the dry forest of southern Ecuador and northern Peru, which implies similar challenges for Chagas disease control.

Distribution of triatomine species in domestic and peridomestic environments in central coastal Ecuador.

BACKGROUND: Although the central coast of the Ecuador is considered endemic for Chagas disease, few studies have focused on determining the risk of transmission in this region. In this study we describe the triatomine household infestation in Manabí province (Central Coast region), determine the rate of Trypanosoma cruzi infection and study the risk factors associated with infestation by Rhodnius ecuadoriensis. METHODOLOGY/PRINCIPAL FINDINGS: An entomological survey found three triatomine species (Rhodnius ecuadoriensis, Panstrongylus rufotuberculatus and P. howardi) infesting domiciles in 47.4% of the 78 communities visited (total infestation rate of 4.5%). Four percent of domiciles were infested, and nymphs were observed in 77% of those domiciles. The three species were found in altitudes below 500 masl and in all ecological zones except cloud forest. Within the domicile, we found the three species mostly in bedrooms. Rhodnius ecuadoriensis and P. rufotuberculatus were abundant in bird nests, including chicken coops and P. howardi associated with rats in piles of bricks, in the peridomicile. Triatomine infestation was characterized by high rates of colonization, especially in peridomicile. Flagelates infection was detected in only 12% of the samples by microscopy and Trypanosoma cruzi infection in 42% of the examined triatomines by PCR (n = 372). The most important risk factors for house infestation by R. ecuadoriensis were ecological zone (w = 0.99) and presence of chickens (w = 0.96). Determinants of secondary importance were reporting no insecticide applications over the last twelve months (w = 0.86) and dirt floor (w = 0.70). On the other hand, wood as wall material was a protective factor (w = 0.85). CONCLUSION/SIGNIFICANCE: According the results, approximately 571,000 people would be at high risk for T. cruzi infection in Manabí province. A multidisciplinary approximation and the adhesion to a periodic integrated vector management (IVM) program are essential to guarantee sustainable preventive and control strategies for Chagas disease in this region.

2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador.

BACKGROUND: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. METHODOLOGY/PRINCIPAL FINDINGS: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment.

Pioneer study of population genetics of Rhodnius ecuadoriensis (Hemiptera: Reduviidae) from the central coastand southern Andean regions of Ecuador.

Effective control of Chagas disease vector populations requires a good understanding of the epidemiological components, including a reliable analysis of the genetic structure of vector populations. Rhodnius ecuadoriensis is the most widespread vector of Chagas disease in Ecuador, occupying domestic, peridomestic and sylvatic habitats. It is widely distributed in the central coast and southern highlands regions of Ecuador, two very different regions in terms of bio-geographical characteristics. To evaluate the genetic relationship among R. ecuadoriensis populations in these two regions, we analyzed genetic variability at two microsatellite loci for 326 specimens (n=122 in Manabí and n=204 in Loja) and the mitochondrial cytochrome b gene (Cyt b) sequences for 174 individuals collected in the two provinces (n=73 and=101 in Manabí and Loja respectively). The individual samples were grouped in populations according to their community of origin. A few populations presented positive FIS, possible due to Wahlund effect. Significant pairwise differentiation was detected between populations within each province for both genetic markers, and the isolation by distance model was significant for these populations. Microsatellite markers showed significant genetic differentiation between the populations of the two provinces. The partial sequences of the Cyt b gene (578bp) identified a total of 34 haplotypes among 174 specimens sequenced, which translated into high haplotype diversity (Hd=0.929). The haplotype distribution differed among provinces (significant Fisher's exact test). Overall, the genetic differentiation of R. ecuadoriensis between provinces detected in this study is consistent with the biological and phenotypic differences previously observed between Manabí and Loja populations. The current phylogenetic analysis evidenced the monophyly of the populations of R. ecuadoriensis within the R. pallescens species complex; R. pallescens and R. colombiensis were more closely related than they were to R. ecuadoriensis.

The modern morphometric approach to identify eggs of Triatominae.

BACKGROUND: Egg morphometrics in the Triatominae has proved to be informative for distinguishing tribes or genera, and has been based generally on traditional morphometrics. However, more resolution is required, allowing species or even population recognition, because the presence of eggs in the domicile could be related to the species ability to colonize human dwellings, suggesting its importance as a vector. RESULTS: We explored the resolution of modern morphometric methods to distinguish not only tribes and genera, but also species or geographic populations in some important Triatominae. Four species were considered, representing two tribes and three genera: Panstrongylus chinai and P. howardi, Triatoma carrioni and Rhodnius ecuadoriensis. Within R. ecuadoriensis, two geographical populations of Ecuador were compared. For these comparisons, we selected the most suitable day of egg development, as well as the possible best position of the egg for data capture. The shape of the eggs in the Triatominae does not offer true anatomical landmarks as the ones used in landmark-based morphometrics, except for the egg cap, especially in eggs with an evident "neck", such as those of the Rhodniini. To capture the operculum shape variation, we used the landmark- and semilandmark-based method. The results obtained from the metric properties of the operculum were compared with the ones provided by the simple contour of the whole egg, as analyzed by the Elliptic Fourier Analysis. Clear differences could be disclosed between the genera, between the species - among which two very close species (P. chinai and P. howardi), as well as between two allopatric, conspecific populations. The whole egg contour (including the operculum) produced reclassification scores much more satisfactory than the ones obtained using the operculum only. CONCLUSIONS: We propose the outline-based approach as the most convenient characterization tool to identify unknown eggs at the species or population levels.