First report of Rhodnius montenegrensis ( Hemiptera : Reduviidae : Triatominae ) infection by Trypanosoma rangeli

Introduction: This study reports for the fi rst time the infection of Rhodnius montenegrensis by Trypanosoma rangeli. Methods: The triatomines were manually collected in Attalea speciosa in the municipality of Buritis, Rondônia. The identifi cation of the trypanosomatid species was confi rmed by multiplex PCR. Results: All of the collected triatomines were R. montenegrensis. The analysis confi rmed that all of the adults were infected with the epimastigote form of T. rangeli. Conclusions: This report of a new vector of T. rangeli raises a warning for the State of Rondônia because the simultaneous presence of T. rangeli with T. cruzi in the same geographic region enables the occurrence of mixed infections in hosts and vectors, which complicates the differential diagnosis.

Trypanosoma rangeli is a heterogeneous hemoflagellate protozoan discovered in Venezuela in the intestinal contents of Rhodnius prolixus and, in addition to Trypanosoma cruzi, is the only trypanosome parasite of man found in Central and South America 1 .
From the earliest reports of human infection by T. rangeli in the Americas, more than 2,700 cases of human rangeliosis have been confirmed 6 .The only cases of human infection reported in Brazil have been in the States of Amazonas and Bahia 7,8 , but in contrast to T. cruzi, T. rangeli is not considered pathogenic to its vertebrate hosts 3 .
The transmission of T. rangeli to vertebrates is mainly related to species of triatomines of the genus Rhodnius, with 12 species reported as their natural vectors; of these 12 species, 6 are found in Brazil (Rhodnius neglectus, Rhodnius nasutus, Rhodnius neivai, Rhodnius domesticus, Rhodnius pictipes and Rhodnius robustus) 2,9,10 .The latter species may have been erroneously described in the State of Rondônia by some researchers 11,12 because of its similarity to R. montenegrensis, which had not been described at that time.
Rhodnius montenegrensis was first described in 2012 from specimens collected in the municipality of Monte Negro, Rondônia, Brazil.Initially, it was identified as R. robustus, but subsequent studies showed that it was a new species 13 .
The present study reports the first documented infection of Rhodnius montenegrensis (Hemiptera: Reduviidae: Triatominae) by Trypanosoma rangeli.
Monthly collections were performed with permission from the Brazilian Institute of Environment and Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis -IBAMA), permanent license No. 14934-1 from June to December of 2012 in the rural pasture area in the municipality of Buritis, Rondônia (Latitude 9º57'32.3"S-Longitude 64º08'05.2"W)(Figure 1).
Triatomines were collected from 14 specimens of Attalea speciosa (babassu) twice a month in the morning after downing a tree with a chainsaw and removing the bracts.Triatomines were manually collected through active searching.The A. speciosa were randomly selected, drawing any 1 from a group of 20.
The collected specimens were sent in a cooler at room temperature to the microscopy laboratory at the School of Education and Environment (Faculdade de Educação e Meio Ambiente -FAEMA) in the municipality of Ariquemes, Rondônia, where wet mount analysis and smear analysis of the adult triatomines' rectal contents were performed with an optical microscope at 1600X magnification after staining with triarylmethane (0.1%), xanthene (0.1%) and thiazine (0.1%) (Figure 2).
The identification of the trypanosomatid species was confirmed by multiplex chain reaction (PCR) in collaboration with the Department of Parasitology at the Institute of Biomedical Sciences, São Paulo University (ICB-USP), São Paulo (SP), Brazil.
Parasite deoxyribonucleic acid (DNA) was extracted from the triatomine rectal samples using a Qiagen DNA extraction kit.The multiplex PCR was performed according to Fernandes et al. 14 .This method amplifies a portion of the non-transcribed spacer of the mini-exon gene that varies between T. cruzi and T. rangeli species and between lines 1 and 2 of T. cruzi.The following primers were used: TC1, 5'-ACACTTTCTGGCGCTGATCG-3'; TC2, 250bp, 5'-TTGCTCGCACACTCGGCTGCAT-3'; Z3, 150bp, 5'-CCGCGCACAACCCCTATAAAAATG-3'; TR, 100bp, 5'-CCTATTGTGATCCCCATCTTCG-3' and EXON, 5'-TACCAATATAGTACAGAACTG-3'.The reaction mixture consisted of 100pmol of each primer and 150μM dNTPs in a buffer composed of 10mM Tris-HCl (pH 8.3), 1.5mM MgCl 2 , 25mM KCl, 0.1mg/ml bovine serum albumin, 2.5U of Taq DNA polymerase and 10ng of genomic DNA in a total volume of 50μL.The thermal cycling conditions were as follows: an initial step of 5min at 95°C, 34 cycles of 30sec at 94°C, 30sec at 55°C and 30sec at 72°C and a final extension of 10min at 72°C.The following reference strains were used as controls in each reaction: TC1, X10 Clone 1; TC2, Strain Y; Z3, Emerald Clone 1 and T. rangeli R1625.The amplified products were subjected to electrophoresis on a 2% agarose gel at 100V for 1h.After electrophoresis, the DNA was stained with ethidium bromide and visualized under ultraviolet light.A molecular marker of 50 base pairs was used as a size control for the amplified fragments.
The identification of the adult triatomine species was conducted based on the keys previously described by and Rosa et al. 12 .
A total of 120 triatomines were collected, providing an average of 8.6 specimens per Attalea speciosa, which is below the average reported by other studies in the state 9,11 .Of those triatomines, only 13 (10.8%)were adults, and all were R. montenegrensis.
The analysis confirmed that all of the adults were infected with trypanosomatids, with the T. rangeli in the epimastigote form.

FIGURE 1 -
FIGURE 1 -Geographical location of the municipality of Buritis, State of Rondônia, Brazil.