Fernando A. Monteiro

Molecular Genetics Reveal That Silvatic Rhodnius prolixus Do Colonise Rural Houses

Background Rhodnius prolixus is the main vector of Chagas disease in Venezuela. Here, domestic infestations of poor quality rural housing have persisted despite four decades of vector control. This is in contrast to the Southern Cone region of South America, where the main vector, Triatoma infestans, has been eliminated over large areas. The repeated colonisation of houses by silvatic populations of R. prolixus potentially explains the control difficulties. However, controversy surrounds the existence of silvatic R. prolixus: it has been suggested that all silvatic populations are in fact Rhodnius robustus, a related species of minor epidemiological importance. Here we investigate, by direct sequencing (mtcytb, D2) and by microsatellite analysis, 1) the identity of silvatic Rhodnius and 2) whether silvatic populations of Rhodnius are isolated from domestic populations. Methods and Findings Direct sequencing confirmed the presence of R. prolixus in palms and that silvatic bugs can colonise houses, with house and palm specimens sharing seven cytb haplotypes. Additionally, mitochondrial introgression was detected between R. robustus and R. prolixus, indicating a previous hybridisation event. The use of ten polymorphic microsatellite loci revealed a lack of genetic structure between silvatic and domestic ecotopes (non-significant FST values), which is indicative of unrestricted gene flow. Conclusions Our analyses demonstrate that silvatic R. prolixus presents an unquestionable threat to the control of Chagas disease in Venezuela. The design of improved control strategies is essential for successful long term control and could include modified spraying and surveillance practices, together with housing improvements.

Ecology, evolution, and the long-term surveillance of vector-borne Chagas disease: a multi-scale appraisal of the tribe Rhodniini (Triatominae).

Chagas disease incidence has sharply declined over the last decade. Long-term disease control will, however, require extensive, longitudinal surveillance systems capable of detecting (and dealing with) reinvasion-reinfestation of insecticide-treated dwellings by non-domiciliated triatomines. Sound surveillance design calls for reliable data on vector ecology, and these data must cover different spatial scales. We conducted a multi-scale assessment of ecological and evolutionary trends in members of the tribe Rhodniini, including (i) a macroscale analysis of Rhodniini species richness and composition patterns across the Americas, and (ii) a detailed, mesoscale case-study of ecological and behavioural trends in Rhodnius neglectus and R. nasutus. Our macroscale overview provides some comprehensive insights about key mechanisms/processes probably underlying ecological and genetic diversification in the Rhodniini. These insights translate into a series of testable hypotheses about current species distributions and their likely causes. At the landscape scale, we used geometric morphometrics to identify dubious specimens as either R. neglectus or R. nasutus (two near-sibling species), and studied palm tree populations of these two vector taxa in five geographical areas. The data suggest that deforestation and the associated loss of habitat and host diversity might increase the frequency of vector-human contact (and perhaps Trypanosoma cruzi infection rates in vectors). Surveillance in central-northeastern Brazil should prioritise deforested landscapes where large palm trees (e.g., Attalea, Mauritia, Copernicia, Acrocomia or Syagrus) occur near houses. We anticipate that, by helping define the distribution patterns and ecological preferences of each species, multi-scale research will significantly strengthen vector surveillance systems across Latin America.

Biogeography and evolution of Amazonian triatomines (Heteroptera: Reduviidae): implications for Chagas disease surveillance in humid forest ecoregions.

An ecological-evolutionary classification of Amazonian triatomines is proposed based on a revision of their main contemporary biogeographical patterns. Truly Amazonian triatomines include the Rhodniini, the Cavernicolini, and perhaps Eratyrus and some Bolboderini. The tribe Rhodniini comprises two major lineages (pictipes and robustus). The former gave rise to trans-Andean (pallescens) and Amazonian (pictipes) species groups, while the latter diversified within Amazonia (robustus group) and radiated to neighbouring ecoregions (Orinoco, Cerrado-Caatinga-Chaco, and Atlantic Forest). Three widely distributed Panstrongylus species probably occupied Amazonia secondarily, while a few Triatoma species include Amazonian populations that occur only in the fringes of the region. T. maculata probably represents a vicariant subset isolated from its parental lineage in the Caatinga-Cerrado system when moist forests closed a dry trans-Amazonian corridor. These diverse Amazonian triatomines display different degrees of synanthropism, defining a behavioural gradient from household invasion by adult triatomines to the stable colonisation of artificial structures. Anthropogenic ecological disturbance (driven by deforestation) is probably crucial in the onset of the process, but the fact that only a small fraction of species effectively colonises artificial environments suggests a role for evolution at the end of the gradient. Domestic infestation foci are restricted to drier subregions within Amazonia; thus, populations adapted to extremely humid rainforest microclimates may have limited chances of successfully colonising the slightly drier artificial microenvironments. These observations suggest several research avenues, from the use of climate data to map risk areas to the assessment of the synanthropic potential of individual vector species.

Molecular research and the control of Chagas disease vectors

Chagas disease control initiatives are yielding promising results. Molecular research has helped successful programs by identifying and characterizing introduced vector populations and by defining intervention targets accurately. However, researchers and health officials are facing new challenges throughout Latin America. Native vectors persistently reinfest insecticide-treated households, and sylvatic triatomines maintain disease transmission in humid forest regions (including Amazonia) without colonizing human dwellings. In these scenarios, fine-scale vector studies are essential to define epidemiological risk patterns and clarify the involvement of little-known triatomine taxa in disease transmission. These eco-epidemiological investigations, as well as the planning and monitoring of control interventions, rely by necessity on accurate taxonomic judgments. The problems of cryptic speciation and phenotypic plasticity illustrate this need--and how molecular systematics can provide the fitting answers. Molecular data analyses also illuminate basic aspects of vector evolution and adaptive trends. Here we review the applications of molecular markers (concentrating on allozymes and DNA sequencing) to the study of triatomines. We analyze the suitability, strengths and weaknesses of the various techniques for taxonomic, systematic and evolutionary investigations at different levels (populations, species, and higher taxonomic categories).

Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia

Rhodnius barretti , a new triatomine species, is described based on adult specimens collected in rainforest environments within the Napo ecoregion of western Amazonia (Colombia and Ecuador). R. barretti resembles Rhodnius robustus s.l. , but mitochondrial cytochrome b gene sequences reveal that it is a strongly divergent member of the “robustus lineage”, i.e., basal to the clade encompassing Rhodnius nasutus , Rhodnius neglectus , Rhodnius prolixus and five members of the R. robustus species complex. Morphometric analyses also reveal consistent divergence from R. robustus s.l. , including head and, as previously shown, wing shape and the length ratios of some anatomical structures. R. barretti occurs, often at high densities, in Attalea butyracea and Oenocarpus bataua palms. It is strikingly aggressive and adults may invade houses flying from peridomestic palms. R. barretti must therefore be regarded as a potential Trypanosoma cruzi vector in the Napo ecoregion, where Chagas disease is endemic.

A multiplex PCR assay that separates Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae)

Rhodnius prolixus is one of the most important primary vectors of human Chagas disease in Latin America. Its morphology is, however, identical to that of the members of the Rhodnius robustus cryptic species complex, which includes secondary vectors. The correct identification of these taxa with differential vector competence is, therefore, of great epidemiological relevance. We used the alignment of 26 mitochondrial cytochrome b haplotypes (663 bp) to select for PCR‐amplifiable species‐specific regions. We designed one forward primer on a region conserved across all haplotypes, and three reverse primers that anneal to species‐specific regions and amplify fragments of different lengths for R. prolixus (285 bp) and for members of the two major R. robustus lineages: group I (349 bp) and groups II–IV (239 bp). These fragments were easily identifiable on regular 1.5% agarose gels. This multiplex PCR assay was successfully tested on 81 specimens from six Latin American countries, and used to determine the phylogeographic boundaries for each species. It is a simple, objective, and cost‐effective assay. Its PCR‐based nature makes it applicable to any insect developmental stage, as well as to dried specimens, and insect remains. It should be particularly useful in areas where representatives of these Rhodnius species occur in sympatry.

Phylogeographic Pattern and Extensive Mitochondrial DNA Divergence Disclose a Species Complex within the Chagas Disease Vector Triatoma dimidiata

Background Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies. Methodology and Findings A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied. Conclusions We propose that Groups I–IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species – likely the basal lineage of the T. dimidiata complex, originated ∼8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I–IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.

New insights on the spread of Triatoma infestans from Bolivia-Implications for Chagas disease emergence in the Southern Cone

Triatoma infestans, now eliminated from most of South America by control campaigns, has been and still is the main Chagas disease vector due to its ability to colonize rural dwellings. The traditional hypothesis put forth to explain T. infestans adaptation to the synanthropic environment rests on the domestication of wild guinea pigs, one of its natural hosts, by Andean tribes about 5000 BC. Here we present two new hypotheses, based on organized human social activities. The first involves maize production, storage and distribution during the Inca period. Maize granaries could host wild rodent populations that would attract sylvatic T. infestans that were later dispersed during maize distribution. The second hypothesis is associated with the contemporary Urkupiña Virgin festival, near Cochabamba, where thousands of pilgrims gather for rituals in an area that is part of a sylvatic T. infestans focus, thus favoring the contact with the insects and leading to their passive dispersal.

Systematics of Mepraia (Hemiptera-Reduviidae): cytogenetic and molecular variation.

The haematophagous insects of the subfamily Triatominae (Hemiptera-Reduviidae) have great epidemiological importance as vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Mepraia was originally described as a monotypic genus comprised of Mepraia spinolai, distributed along coastal areas of northern Chile (from Region I to the Metropolitan Region). Recently, some M. spinolai populations have been ranked as a new species named Mepraia gajardoi. Several populations along the distribution range of the genus were sampled, and genetic differentiation was studied based upon the analysis of three molecular markers: cytogenetics (karyotype and chromosome behaviour during meiosis using the C-banding technique), mitochondrial DNA (a cytochrome oxidase I gene fragment), and nuclear ribosomal DNA (intergenic region including the two internal transcribed spacers ITS-1 and ITS-2 and the 5.8S rRNA gene). The data here presented indicate that populations within the Mepraia genus (excluding Region II specimens) can be divided into two separate lineages. One lineage is comprised of specimens from the northernmost Region I and represents M. gajardoi. The other includes samples from the southern III, IV and the Metropolitan Regions, and represents M. spinolai. Region II individuals deserve particular attention as their relationship to the two identified lineages is not clear-cut. While they appear to belong to M. spinolai based on cytogenetics and rDNA markers, COI results indicate a closer relationship to M. gajardoi. This disagreement can be due to mitochondrial DNA introgression or the retention of ancestral polymorphisms.

On palms, bugs, and Chagas disease in the Americas

Palms are ubiquitous across Neotropical landscapes, from pristine forests or savannahs to large cities. Although palms provide useful ecosystem services, they also offer suitable habitat for triatomines and for Trypanosoma cruzi mammalian hosts. Wild triatomines often invade houses by flying from nearby palms, potentially leading to new cases of human Chagas disease. Understanding and predicting triatomine-palm associations and palm infestation probabilities is important for enhancing Chagas disease prevention in areas where palm-associated vectors transmit T. cruzi. We present a comprehensive overview of palm infestation by triatomines in the Americas, combining a thorough reanalysis of our published and unpublished records with an in-depth review of the literature. We use site-occupancy modeling (SOM) to examine infestation in 3590 palms sampled with non-destructive methods, and standard statistics to describe and compare infestation in 2940 palms sampled by felling-and-dissection. Thirty-eight palm species (18 genera) have been reported to be infested by ∼39 triatomine species (10 genera) from the USA to Argentina. Overall infestation varied from 49.1-55.3% (SOM) to 62.6-66.1% (dissection), with important heterogeneities among sub-regions and particularly among palm species. Large palms with complex crowns (e.g., Attalea butyracea, Acrocomia aculeata) and some medium-crowned palms (e.g., Copernicia, Butia) are often infested; in slender, small-crowned palms (e.g., Euterpe) triatomines associate with vertebrate nests. Palm infestation tends to be higher in rural settings, but urban palms can also be infested. Most Rhodnius species are probably true palm specialists, whereas Psammolestes, Eratyrus, Cavernicola, Panstrongylus, Triatoma, Alberprosenia, and some Bolboderini seem to use palms opportunistically. Palms provide extensive habitat for enzootic T. cruzi cycles and a critical link between wild cycles and transmission to humans. Unless effective means to reduce contact between people and palm-living triatomines are devised, palms will contribute to maintaining long-term and widespread, albeit possibly low-intensity, transmission of human Chagas disease.