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

https://doi.org/10.1016/j.meegid.2017.03.002Get rights and content

Highlights

  • Thirty four triatomines from palm trees were captured in Bolivian Amazon (Beni).

  • The overall infestation rate of palm trees in forest fragments and pastures was 12.3%.

  • Rhodnius stali species was identify by genitalia dissection in two males.

  • Sequencing identified one specimen of R. robustus and all others of R. pictipes complex, more probably R. stali species.

  • Phylogenetic trees were constructed showing some weakness for identification of Rhodnius genus species.

Abstract

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.

Introduction

Chagas disease, caused by Trypanosoma cruzi and mostly transmitted by blood-sucking bugs of the Triatominae subfamily, is a parasitic disease that may be fatal. According to World Health Organization (WHO) estimates, six to seven million people worldwide are infected by the parasite (WHO, 2016). In the Amazon region, Chagas disease can be considered as mostly enzootic because the parasite circulates among a wide number of wild mammal species, and foci of pre-adaptation or adaptation of triatomine vectors to the domicile or peridomicile are scarce, such as Triatoma maculata and Panstrongylus geniculatus in the Brazilian Amazon (Fe et al., 2009, Luitgards-Moura et al., 2005), Rhodnius stali in Alto Beni in Bolivia (on the edge of the Amazon Basin) (Justi et al., 2010) and Rhodnius prolixus in the Orinoco River of Venezuela (Aguilar et al., 2007). Some species of the genus Rhodnius, mainly found in forest habitat, have been implicated in T. cruzi transmission to humans, either attacking humans to take a blood meal, such as Rhodnius brethesi in the Brazilian state Amazonas (Coura et al., 1994) or flying to peridomestic and domestic areas where they can form small colonies, such as Rhodnius neglectus in Brazil (Garcia-Zapata et al., 1985). The latter process may be the first step in adapting to domestic habitat and could be the strategy used to increase their epidemiological role (Dujardin et al., 2000). R. stali is probably responsible for transmission of Chagas disease in indigenous communities in the Alto Beni region (Justi et al., 2010, Matias et al., 2003). Recently, at the foot of the Andes, on the edge of the Amazon Basin, P. geniculatus and Rhodnius robustus adults naturally infected with T. cruzi and collected from houses were described (Rojas-Cortez et al., 2016). In 2010, an outbreak by oral contamination was reported for the first time in Bolivia, (Guayaramerín, Beni department) (Santalla Vargas et al., 2011), showing that the Amazon has to be evaluated for the risk of Chagas disease transmission. In this context, a first exploration for the presence of triatomines was conducted in the surroundings of the small city of Yucumo, the largest Amazonian city on the road from La Paz (Andean capital) to Trinidad (centre of the Bolivian Amazon). The captures were done in forest fragments and pastures by positioning traps mostly in palm trees, and the species of the nymphs and adults were identified through morphology and genetic markers.

Section snippets

Biological material

The study was conducted near the small city of Yucumo in the province of General Jose Ballivian, Department of Beni (15° 08′43.3″S, 67° 02′03.01″W) in October 2012, by a group of researchers from the IRD, the FIOCRUZ and the local staff of the School of Pharmaceutical Sciences and Biochemistry (UMSA). The presence of triatomines was examined in six areas, two situated in forest fragments, two in pastures and two in peridomicile areas. Access to these forest and pasture lands was requested from

Collection of triatomines

A total of 26 triatomines were collected using mice-baited adhesive traps mostly positioned in palm trees (Table 1), and eight additional triatomines were collected during the dissection of one of the positive palm trees (with two positive traps out of four) previously sampled (S63) because the owner had decided to cut it. Fig. 1 shows some positive capture sites, and Table 1 details the geographical and habitat origins of the triatomines. Two principal landscapes were explored, fragment forest

Discussion

For many years, control of the transmission of Chagas disease in the Amazon was not an objective in itself; indeed this region, considered non-endemic, was excluded from the National Control Program of Chagas disease in Bolivia. Seventeen triatomine species were reported in Bolivia, and on the basis of these reports, predictive maps of the geographical distribution of each species were established (Rojas Cortez, 2007). In the department of Beni, which covers the widest part of the Bolivian

Conclusions

We conclude with three recommendations: (i) the sampling effort must be increased to determine the geographical distribution and the landscape categories where these species are present, and morphological and/or morphometric identifications must be continued, combined with molecular characterization which, used alone, can be unsatisfactory; (ii) for the molecular approach it is better to use several mitochondrial and nuclear genes in order to discover possible mitochondrial introgression

Acknowledgements

This study was funded by the IRD Institute, France (HC224R-2R224-MENG-07BO). We thank Dr Wilma Strauss for having received us in her laboratory as well as Dr Bernardo Torrico, director of the School of Pharmacy Sciences and Biochemistry, who authorized this study. We are also grateful to Dr Frédérique Cerqueira, from the genotyping platform in Montpellier, France, for sequencing the gene fragments, and Marcelo Claure (IRD member) for the assistance given during the fieldwork.

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