The analysis of the relationship between domestic and silvatic populations of Rhodnius prolixus\robustus (Hemiptera : Reduviidae) in Venezuela by geometric morphometric and molecular methods

Despite four decades of control of Chagas disease in Venezuela, domestic infestations still persist and transmission of Trypanosoma cruzi may be increasing. This is in contrast to the Southern cone region where control has successfully eliminated domestic populations of the main vector Triatoma infestans over large areas. However unlike T. infestans, the main vector in Venezuela, Rhodnius prolixus, has a widespread silvatic distribution occurring primarily in palm trees, which are a ubiquitous feature of the Venezuelan landscape. The palm tree is an important part of campensino life and is maintained for fruit, shade and for use in house construction. Control failures may be due to reinvasion of houses by these prevalent silvatic populations. However, debate exists as to whether the silvatic populations are in fact Rhodnius robustus, a related species of minor epidemiological importance, and therefore no threat to control. With an estimated 800,000 people infected with T. cruzi in Venezuela and a further 3 million at risk of infection, an effective control programme is required, which necessitates that this relationship between silvatic and domestic populations is resolved. This study was undertaken in order to (1) confirm the identity of silvatic populations of Rhodnius in Venezuela and (2) determine if domestic and silvatic populations are isolated, thus to clarify the role of silvatic populations in maintaining house infestations. To achieve these aims field collected silvatic and domestic populations of Rhodnius from 5 States were analysed by genetic methods, direct sequencing (cytochrome b, D2) and microsatellites, and by geometric morphometric analysis. A total of 551 specimens from 31 localities in six Venezuelan states were analysed by direct sequencing of cytochrome b (cytb). Results confirmed the presence of R. prolixus in both silvatic and domestic ecotopes, dispelling the belief that all silvatic populations are R.robustus. Rhodnius robustus does however occur and was found in this study in the Andean State Trujillo. Here it was limited to the silvatic environment. This project found that silvatic and domestic populations of R. prolixus are not isolated, sharing 6 haplotypes, including four silvatic haplotypes also detected in domestic nymphs, indicating that silvatic specimens are capable of domestic colonisation. This was also confirmed from the analysis of adjacent domestic and silvatic populations in Portuguesa and Barinas State where population homogeneity was detected. Additionally cytb analysis identified an introgression event between Amazonian R. robustus and R prolixus, confirmed by incongruence of cytb and D2 nuclear characterisation. Phylogenetic analysis of specimens was also undertaken. To investigate further fine scale population heterogeneity a panel of microsatellite markers, hitherto unavailable, was developed for R prolixus, using an enrichment technique. A panel of 10 loci was available for analysis following PCR screening and linkage analysis. A total of 555 specimens were analysed from 33 populations . Microsatellite analysis also detected population homogeneity between ecotopes, including adjacent populations, indicating that silvatic populations are not isolated. Population heterogeneity was greater among localities in Portuguesa than Barinas, may be due to landscape variation. Differences between control programmes may also play a role. Geometric morphometries identified shape similarity between populations across all States. However, shape convergence by ecotope was detected and results indicated that morphometries might be of limited use for the analysis of populations of R prolixus, with the exception of post-control reinvasion\recrudescent studies. The three methods did not always concur precisely. However comparison was difficult due to detected introgression and shape convergence distorting, respectively, mitochondrial and morphometric analysis. Results indicated that a combined use of microsatellites and morphometries would be beneficial in the analysis of adjacent domestic and silvatic populations. A similar pattern of a lack of isolation between silvatic and domestic ecotopes was detected by both genetic methods, with limited morphometries overlap detected; broadly all three methods showed that populations from differing ecotopes are not isolated. This is also supported by a parallel project on risk-factor analysis. From this study it is clear that silvatic populations of R prolixus present an unquestionable threat to the successful control of Chagas disease in some endemic regions of Venezuela and, unlike the Southern cone, elimination of domestic infestations may not be possible in areas where silvatic R prolixus occur. A restructuring of the control programme in Venezuela is required to deal with this silvatic threat. This may include increased vigilance and methodical respraying and the incorporation of novel approaches to deal with silvatic invasion such as use of insecticide treated curtains; ultimately more investment in the improvement of the rural rancho is required.