• 1.

    World Health Organization, 2015. Chagas disease (American trypanosomiasis). Wkly Epidemiol Rec 90: 3344.

  • 2.

    Alevi KCC, Moreira FFF, Jurberg J, Azeredo-Oliveira MTV, 2016. Description of diploid chromosome set of Triatoma pintodiasi (Hemiptera, Triatominae). Genet Mol Res 15: gmr15026343.

    • Search Google Scholar
    • Export Citation
  • 3.

    Galvão C, 2014. Vetores da Doença de Chagas no Brazil. Curitiba, Brazil: Sociedade Brasileira de Zoologia.

  • 4.

    Galvão C, Carcavallo RU, Rocha DS, Jurberg J, 2003. A checklist of the current valid species of the subfamily Triatominae Jeannel, 1919 (Hemiptera, Reduviidae) and their geographical distribution, with nomenclatural and taxonomic notes. Zootaxa 202: 136.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jurberg J, Cunha V, Cailleaux S, Raigorodschi R, Lima MS, Rocha DS, Moreira FFF, 2013. Triatoma pintodiasi sp. nov. do subcomplexo T. rubrovaria (Hemiptera, Reduviidae, Triatominae). Rev Pan-Amaz Saúde 4: 4356.

    • Search Google Scholar
    • Export Citation
  • 6.

    Motta FS, Moreira FFF, 2013. Description of the nymphs of Triatoma pintodiasi Jurberg, Cunha & Rocha, 2013 (Hemiptera: Reduviidae: Triatominae). Zootaxa 3947: 139145.

    • Search Google Scholar
    • Export Citation
  • 7.

    Simon C, Frati F, Bechenbach A, Crespi B, Liu H, Flook P, 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequence and compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87: 651701.

    • Search Google Scholar
    • Export Citation
  • 8.

    Thompson JD, Gibson TJ, Plewniak F, Jeamnougin F, Higgis DG, 1997. The CLUSTAL_X Windows Interface: Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools. Nucl Acids Res 25: 48764882.

    • Search Google Scholar
    • Export Citation
  • 9.

    Hall T, 1999. BioEdit: An important software for molecular biology. GERF Bull Biosci 2: 6061.

  • 10.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 27252729.

    • Search Google Scholar
    • Export Citation
  • 11.

    Justi SA, Russo CAM, Mallet JRS, Obara MT, Galvão C, 2014. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Paras Vect 7: 149.

    • Search Google Scholar
    • Export Citation
  • 12.

    Ceretti-Junior W, Vendrami DP, Gil JM, Barata JMS, Marrelli MT, 2008. Análise das relações taxonômicas e sistemáticas entre espécies de triatomíneos (Hemiptera, Reduviidae) de colônias mantidas pelo Serviço Especial de Saúde de Araraquara, inferida de seqüências do 16S rDNA mitochondrial. Rev Bras Entomol 52: 455462.

    • Search Google Scholar
    • Export Citation
  • 13.

    Alevi KCC, Reis YV, Guerra AL, Imperador CHL, Banho CA, Moreira FFF, Azeredo-Oliveira MTV, 2016. Would Nesotriatoma bruneri Usinger, 1944 be a valid species? Zootaxa 4103: 396400.

    • Search Google Scholar
    • Export Citation
  • 14.

    Justi SA, Galvão C, Schrago CG, 2016. Geological changes of the Americas and their influence on the diversification of the neotropical kissing bugs (Hemiptera: Reduviidae: Triatominae). PLoS Negl Trop Dis 10: e0004527.

    • Search Google Scholar
    • Export Citation
  • 15.

    Almeida CE, Marcet PL, Gumiel M, Takiya DM, Cardozo-de-Almeida M, Pacheco RS, Lopes CM, Dotson EM, Costa J, 2009. Phylogenetic and phenotypic relationships among Triatoma carcavalloi (Hemiptera: Reduviidae: Triatominae) and related species collected in domiciles in Rio Grande do Sul State, Brazil. J Vector Ecol 34: 164173.

    • Search Google Scholar
    • Export Citation
  • 16.

    Alevi KC, Oliveira J, Moreira FFF, Jurberg J, Rosa JA, Azeredo-Oliveira MTV, 2015. Chromosomal characteristics and distribution of constitutive heterochromatin in the Matogrossensis and Rubrovaria subcomplexes. Inf Gen Evol 33: 158162.

    • Search Google Scholar
    • Export Citation
  • 17.

    Pita S, Lorite P, Nattero J, Galvão C, Alevi KC, Teves SC, Azeredo-Oliveira MTV, Panzera F, 2016. New arrangements on several species subcomplexes of Triatoma genus based on the chromosomal position of ribosomal genes (Hemiptera - Triatominae). Infect Genet Evol 43: 225231.

    • Search Google Scholar
    • Export Citation
  • 18.

    Gardim S, Rocha CS, Almeida CE, Takiya DM, Silva MT, Ambródio DL, Cicarelli RM, Rosa JA, 2013. Evolutionary relationships of the Triatoma matogrossensis subcomplex, the endemic Triatoma in central-western Brazil, based on mitochondrial DNA sequences. Am J Trop Med Hyg 89: 766774.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Mitochondrial Gene Confirms the Specific Status of Triatoma pintodiasi Jurberg, Cunha, and Rocha, 2013 (Hemiptera, Triatominae), an Endemic Species in Brazil

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  • 1 Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho”–São José do Rio Preto, São Paulo, Brazil.
  • 2 Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Instituto Oswaldo Cruz, LNIRTT/IOC/FIOCRUZ, Rio de Janeiro, Brazil.

Chagas disease is most frequently transmitted to humans through contact with feces of insects from the Triatominae subfamily. In Brazil, there are 65 species of triatomines distributed throughout the country's 27 states. Among the species in the state of Rio Grande do Sul, Triatoma rubrovaria, Triatoma oliveirai, Triatoma pintodiasi, Triatoma klugi, Triatoma carcavalloi, and Triatoma circummaculata (with the addition Triatoma limai, which is endemic to Argentina) form the T. rubrovaria subcomplex. The last species described and grouped into this subcomplex was T. pintodiasi. Thus, this study characterized the genetic distance between T. pintodiasi and of the other members of the T. rubrovaria subcomplex to evaluate the specific status of T. pintodiasi. The genetic distance observed between T. pintodiasi and the other species of the T. rubrovaria subcomplex was large, a finding which highlights the specific status of the species considered to be cryptic of T. circummaculata.

Chagas disease is a potentially life-threatening illness caused by the protozoan Trypanosoma cruzi Chagas, 1909. It is mainly distributed in endemic areas in 21 Latin American countries, where it is mostly vector borne and transmitted to humans through contact with feces of insects from the Triatominae subfamily. It is estimated that approximately 6–7 million people are infected worldwide, most of whom are located in Latin America where Chagas disease is endemic.1

There are currently 150 known species of triatomines distributed across 18 genera. All of the species are considered potential vectors of Chagas disease.2 In Brazil, there are 65 species distributed throughout the country's 27 states, and 64% of them are endemic species.3 In the state of Rio Grande do Sul, there are 12 species of triatomines: Triatoma rubrovaria, Triatoma platensis, Triatoma pintodiasi, Triatoma oliveirai, Triatoma klugi, Triatoma delpontei, Triatoma carcavalloi, Triatoma circummaculata, and Panstrongylus tupynambai, the latter of which has limited distribution in this Brazilian state.3

Among the species in Rio Grande do Sul, T. rubrovaria, T. oliveirai, T. pintodiasi, T. klugi, T. carcavalloi, and T. circummaculata (with the addition of Triatoma limai, which is endemic to Argentina)4 make up the T. rubrovaria subcomplex.5 The last species described and grouped into this subcomplex was T. pintodiasi.5 The description was based on chromatic, morphological, morphometric, and biochemical aspects of the species, which led the author to describe T. pintodiasi as a cryptic species of T. circummaculata.5 Recent studies on meiotic behavior2 and characterization of nymphs6 showed significant differences between T. pintodiasi and other species of the T. rubrovaria subcomplex. However, new comparative analyses were encouraged.2,5

Thus, this study characterized the genetic distance between the mitochondrial 16S rDNA gene of T. pintodiasi and that of the members of the T. rubrovaria subcomplex to evaluate the specific status of T. pintodiasi.

Three adult T. pintodiasi specimens were used in the study. The specimens were provided by the National and International Reference Laboratory of Taxonomy of Triatominae (LNIRTT) in Rio de Janeiro, Brazil. The triatomines were dissected, and genetic material was extracted from their legs using the DNeasy Blood and Tissue Kit (QIAGEN, São Paulo, Brazil) according to the manufacturer's instructions. Polymerase chain reactions (PCRs) were performed to amplify the 16S rDNA gene as described by Simon and others,7 with the forward sequence 5′-CCGGTTTGAACTCAGATCATGT-3′ and the reverse sequence 5′-CGCCTGTTTAACAAAAACAT-3′. The conditions for the amplification reactions of this gene were as follows: an initial cycle at 94°C for 2 minutes, followed by 35 cycles comprising denaturation (94°C, 30 seconds), annealing (50°C, 30 seconds), and extension (72°C, 1 minute), followed by a final single cycle at 72°C for 6 minutes. After electrophoresis, the amplified fragments were purified using the GFX PCR DNA and Gel Band Kit (GE Healthcare Life Sciences, São Paulo, Brazil) according to the manufacturer's instructions.

Direct sequencing was applied to the purified PCR products, and the samples were sent to the Human Genome and Stem Cell Research Center of the University of São Paulo in São Paulo, Brazil. The sequences generated were analyzed using the BioEdit software, version 7.0.5.8 A consensus sequence was obtained for each DNA segment, and the sequences were aligned using the ClustalW.9 Next, the MEGA 6.0 program10 for phylogenetic analysis was used using the maximum likelihood method as a distance criterion, and the bootstrapping resampling method was applied to assess support for the individual nodes (1,000 pseudoreplications). The Tamura 3-parameter model was used to calculate genetic distances pairwise between T. pintodiasi and the other species of the T. rubrovaria subcomplex in the MEGA 6.0 software,10 and the sequences used for the T. rubrovaria subcomplex species were deposited in GenBank (accession numbers: T. rubrovaria [KC249066]; T. klugi [KC249028]; T. carcavalloi [KC248991]; and T. circummaculata [KC248992]).

The genetic distance observed between T. pintodiasi and the other species of the T. rubrovaria subcomplex was large: 1.195 in the case of T. carcavalloi, 1.123 in the case of T. circummaculata, 1.184 in the case of T. klugi, and 1.194 in the case of T. rubrovaria. These results highlight the specific status of the species. They reflect the importance of the analysis used by Jurberg and others5 in the description of new species, particularly in the study of the phenomenon of cryptic speciation.

Although current phylogenetic analyses combine many different mitochondrial and nuclear genes,11 the 16S has been used alone to assess the phylogenetic relationships among triatomines. It is important to note the utility of the 16S gene as molecular marker among triatomine species and its importance in systematic and taxonomic issues.12 Furthermore, the extremely low genetic distance observed in this mitochondrial gene led to the suggestion that Nesotriatoma flavida and Nesotriatoma bruneri are the same species.13

Phylogenetic analyses have shown the T. rubrovaria subcomplex to be monophyletic11 (T. pintodiasi has never been included in these analyses due to its recent description). Studies have also determined that this subcomplex diversified from the other subcomplexes approximately 14 million years ago as a result of climatic changes demonstrated a consequence of the rapid Andean uplift.14 These triatomines share morphological15 and cytogenetic16 characteristics. Using chromosomal data, Pita and others17 recently proposed the grouping of Triatoma guasayana and Triatoma patagonica with the seven species of the T. rubrovaria subcomplex. This relationship was initially suggested by molecular data.11,18

Thus, we corroborate the specific status of T. pintodiasi. However, we suggest that different analyses should be combined to evaluate the evolutionary relationship of T. guasayana, T. patagonica, and T. pintodiasi with T. rubrovaria subcomplex.

  • 1.

    World Health Organization, 2015. Chagas disease (American trypanosomiasis). Wkly Epidemiol Rec 90: 3344.

  • 2.

    Alevi KCC, Moreira FFF, Jurberg J, Azeredo-Oliveira MTV, 2016. Description of diploid chromosome set of Triatoma pintodiasi (Hemiptera, Triatominae). Genet Mol Res 15: gmr15026343.

    • Search Google Scholar
    • Export Citation
  • 3.

    Galvão C, 2014. Vetores da Doença de Chagas no Brazil. Curitiba, Brazil: Sociedade Brasileira de Zoologia.

  • 4.

    Galvão C, Carcavallo RU, Rocha DS, Jurberg J, 2003. A checklist of the current valid species of the subfamily Triatominae Jeannel, 1919 (Hemiptera, Reduviidae) and their geographical distribution, with nomenclatural and taxonomic notes. Zootaxa 202: 136.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jurberg J, Cunha V, Cailleaux S, Raigorodschi R, Lima MS, Rocha DS, Moreira FFF, 2013. Triatoma pintodiasi sp. nov. do subcomplexo T. rubrovaria (Hemiptera, Reduviidae, Triatominae). Rev Pan-Amaz Saúde 4: 4356.

    • Search Google Scholar
    • Export Citation
  • 6.

    Motta FS, Moreira FFF, 2013. Description of the nymphs of Triatoma pintodiasi Jurberg, Cunha & Rocha, 2013 (Hemiptera: Reduviidae: Triatominae). Zootaxa 3947: 139145.

    • Search Google Scholar
    • Export Citation
  • 7.

    Simon C, Frati F, Bechenbach A, Crespi B, Liu H, Flook P, 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequence and compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87: 651701.

    • Search Google Scholar
    • Export Citation
  • 8.

    Thompson JD, Gibson TJ, Plewniak F, Jeamnougin F, Higgis DG, 1997. The CLUSTAL_X Windows Interface: Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools. Nucl Acids Res 25: 48764882.

    • Search Google Scholar
    • Export Citation
  • 9.

    Hall T, 1999. BioEdit: An important software for molecular biology. GERF Bull Biosci 2: 6061.

  • 10.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 27252729.

    • Search Google Scholar
    • Export Citation
  • 11.

    Justi SA, Russo CAM, Mallet JRS, Obara MT, Galvão C, 2014. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Paras Vect 7: 149.

    • Search Google Scholar
    • Export Citation
  • 12.

    Ceretti-Junior W, Vendrami DP, Gil JM, Barata JMS, Marrelli MT, 2008. Análise das relações taxonômicas e sistemáticas entre espécies de triatomíneos (Hemiptera, Reduviidae) de colônias mantidas pelo Serviço Especial de Saúde de Araraquara, inferida de seqüências do 16S rDNA mitochondrial. Rev Bras Entomol 52: 455462.

    • Search Google Scholar
    • Export Citation
  • 13.

    Alevi KCC, Reis YV, Guerra AL, Imperador CHL, Banho CA, Moreira FFF, Azeredo-Oliveira MTV, 2016. Would Nesotriatoma bruneri Usinger, 1944 be a valid species? Zootaxa 4103: 396400.

    • Search Google Scholar
    • Export Citation
  • 14.

    Justi SA, Galvão C, Schrago CG, 2016. Geological changes of the Americas and their influence on the diversification of the neotropical kissing bugs (Hemiptera: Reduviidae: Triatominae). PLoS Negl Trop Dis 10: e0004527.

    • Search Google Scholar
    • Export Citation
  • 15.

    Almeida CE, Marcet PL, Gumiel M, Takiya DM, Cardozo-de-Almeida M, Pacheco RS, Lopes CM, Dotson EM, Costa J, 2009. Phylogenetic and phenotypic relationships among Triatoma carcavalloi (Hemiptera: Reduviidae: Triatominae) and related species collected in domiciles in Rio Grande do Sul State, Brazil. J Vector Ecol 34: 164173.

    • Search Google Scholar
    • Export Citation
  • 16.

    Alevi KC, Oliveira J, Moreira FFF, Jurberg J, Rosa JA, Azeredo-Oliveira MTV, 2015. Chromosomal characteristics and distribution of constitutive heterochromatin in the Matogrossensis and Rubrovaria subcomplexes. Inf Gen Evol 33: 158162.

    • Search Google Scholar
    • Export Citation
  • 17.

    Pita S, Lorite P, Nattero J, Galvão C, Alevi KC, Teves SC, Azeredo-Oliveira MTV, Panzera F, 2016. New arrangements on several species subcomplexes of Triatoma genus based on the chromosomal position of ribosomal genes (Hemiptera - Triatominae). Infect Genet Evol 43: 225231.

    • Search Google Scholar
    • Export Citation
  • 18.

    Gardim S, Rocha CS, Almeida CE, Takiya DM, Silva MT, Ambródio DL, Cicarelli RM, Rosa JA, 2013. Evolutionary relationships of the Triatoma matogrossensis subcomplex, the endemic Triatoma in central-western Brazil, based on mitochondrial DNA sequences. Am J Trop Med Hyg 89: 766774.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Kaio Cesar Chaboli Alevi, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo, 2265, Jardim Nazareth, CEP 15054-000, São José do Rio Preto, São Paulo, Brazil. E-mail: kaiochaboli@hotmail.com

Financial support: The study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (process no. 2013/19764-0, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil).

Authors' addresses: Kaio Cesar Chaboli Alevi, Ana Letícia Guerra, Carlos Henrique Lima Imperador, and Maria Tercília Vilela de Azeredo Oliveira, Departamento de Biologia, IBILCE, Universidade Estadual Paulista “Júlio de Mesquita Filho”–São José do Rio Preto, São Paulo, Brazil, E-mails: kaiochaboli@hotmail.com, analebio@yahoo.com.br, karlosimpe@gmail.com, and tercilia@ibilce.unesp.br. José Jurberg and Felipe Ferraz Figueiredo Moreira, Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil, E-mails: jjurberg@ioc.fiocruz.br and felipento@hotmail.com.

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