• View in gallery

    Salivary glands of (A) Psammolestes tertius, (B) Rhodnius montenegrensis, and (C) Triatoma infestans. Note the red glands in (A) P. tertius and (B) R. montenegrensis (tribe Rhodniini). Bar = 10 μm. This figure appears in color at www.ajtmh.org.

  • 1.

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

  • 2.

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

  • 3.

    Justi S, Galvão C, 2017. The evolutionary origin of diversity in Chagas disease vectors. Trends Parasitol 33: 4252.

  • 4.

    Schofield CJ, 1988. The biosystematics of Triatominae. Service MW, ed. Biosystematics of Haematophagous Insects, Systematics Association Special. Oxford, United Kingdom: Clarenden Press, 284312.

    • Search Google Scholar
    • Export Citation
  • 5.

    Hellmann K, Hawkins RI, 1965. Prolixins-S and prolixin-G; two anticoagulants from Rhodnius prolixus Stal. Nature 207: 265267.

  • 6.

    Ribeiro JM, Hazzard JM, Nussenzveig RH, Champagne DE, Walker FA, 1993. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science 260: 539541.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lacombe D, 1999. Anatomia e histologia de glândulas salivares de triatomíneos. Mem Inst Oswaldo Cruz 94: 557564.

  • 8.

    Montandon CE, Barros E, Vidigal PM, Mendes MT, Anhê ANBM, Ramos HJO, Oliveira CJF, Mafra C, 2016. Comparative proteomic analysis of the saliva of the Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri triatomines reveals a high interspecific functional biodiversity. Insect Biochem Mol Biol 71: 8390.

    • Search Google Scholar
    • Export Citation
  • 9.

    Anhê AC, Azeredo-Oliveira MT, 2008. Cytochemical characterization of Triatoma infestans and Panstrongylus megistus salivary gland cells (Hemiptera, Reduviidae, Triatominae). Micron 39: 11261133.

    • Search Google Scholar
    • Export Citation
  • 10.

    Champagne DE, Nussenzveig RH, Ribeiro JMC, 1995. Purification, partial characterization and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus .J Biol Chem 270: 86918695.

    • Search Google Scholar
    • Export Citation
  • 11.

    Montfort WR, Weichsel A, Andersen JF, 2000. Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods. Biochim Biophys Acta 1482: 110118.

    • Search Google Scholar
    • Export Citation
  • 12.

    Soares RPP, Gontijo NF, Romanha AJ, Diotaiuti L, Pereira MH, 1998. Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae). Acta Trop 71: 285291.

    • Search Google Scholar
    • Export Citation
  • 13.

    Pacheco DE, 2014. Nitroforinas salivares de Rhodnius prolixus (Hemiptera: Reduviidae): Avaliação do RNAi parental e do papel da albumina do hospedeiro na sua atividade biológica. Available at: http://www.bibliotecadigital.ufmg.br/dspace/bitstream/handle/1843/BUOS-9KXJS7/dissertfinal.pdf?sequence=1. Accessed February 22, 2017.

    • Search Google Scholar
    • Export Citation
  • 14.

    Meirelles RM, Rodrigues IS, Steindel M, Soares MJ, 2003. Ultrastructure of the salivary glands of Rhodnius domesticus Neiva and Pinto, 1923 (Hemiptera: Reduviidae). J Submicrosc Cytol Pathol 35: 199207.

    • Search Google Scholar
    • Export Citation
  • 15.

    Bargues MD, Marcilla A, Ramsey J, Dujardin JP, Schofield CJ, Mas-Coma S, 2000. Nuclear rDNA-based molecular clock of the evolution of Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease. Mem Inst Oswaldo Cruz 95: 567573.

    • Search Google Scholar
    • Export Citation
  • 16.

    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
  • 17.

    Ribeiro JMC, Schneider M, Guimaraes JA, 1995. Purification and characterization of Prolixin S (Nitrophorin 2), the salivary anticoagulant of the blood sucking bug, Rhodnius prolixus .Biochem J 308: 243249.

    • Search Google Scholar
    • Export Citation
  • 18.

    Noeske-Jungblut C, Kratzschmar J, Haendler B, Alagon A, Possani L, Verhallen P, Donner P, Schleuning WD, 1994. An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis .J Biol Chem 269: 50505053.

    • Search Google Scholar
    • Export Citation
  • 19.

    Isawa H, Orito Y, Jingushi N, Iwanaga S, Morita A, Chinzei Y, Yuda M, 2007. Identification and characterization of plasma kallikrein-kinin system inhibitors from salivar glands of the blood-sucking insect Triatoma infestans .FEBS J 274: 42714286.

    • Search Google Scholar
    • Export Citation
  • 20.

    Ribeiro JMC, Assumpcao TC, Francischetti IMB, 2012. An insight into the sialomes of bloodsucking Heteroptera. Psyche (Stuttg) 2012: 16.

 
 
 

 

 

 

 

 

 

Study of the Salivary Glands in Triatominae (Hemiptera, Reduviidae, Triatominae): Their Color and Application to the Chagas Disease Vector Evolution

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  • 1 Laboratório de Parasitologia, Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCFAR/UNESP), Araraquara, Brazil;
  • | 2 Laboratório de Biologia Celular, Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho” (IBILCE/UNESP), São José do Rio Preto, Brazil;
  • | 3 Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz (LABE/IOC/FIOCRUZ), Rio de Janeiro, Brazil;
  • | 4 Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Instituto Oswaldo Cruz (LNIRTT/IOC/FIOCRUZ), Rio de Janeiro, Brazil

Chagas disease is caused by Trypanosoma cruzi and transmitted by feces of a triatomine that has the habit of defecating during blood feeding. The salivary glands of triatomines are important to hematophagy because their saliva is rich in anticoagulant and hemolytic proteins. The salivary glands of some Rhodnius species analyzed are reddish due to the presence of nitrophorins (antihemostatic activity). The present study aimed to analyze the color pattern of the salivary glands of 67 triatomine species to evaluate whether the presence of nitrophorins is a synapomorphy of Rhodnius or the tribe Rhodniini, or if it is shared with triatomines of the tribes Triatomini and Cavernicolini. Since only the species of the tribe Rhoniini present red glands, it is admitted that the presence of nitrophorin proteins is a synapomorphy of the tribe Rhodniini and that this tribe has derived more recently when compared with Triatomini and Cavernicolini.

Chagas disease is a vector-borne and potentially life-threatening illness caused by the protozoan Trypanosoma cruzi (Chagas, 1909). It occurs mainly in endemic areas in 21 Latin American countries, where it is transmitted to humans mostly by contact with feces of triatomines, known as “kissing bugs.” It is estimated that about 6–7 million people are infected worldwide, mostly in Latin America.1

Currently, there are 151 species of triatomines distributed in 18 genera and five tribes, all species being considered potential vectors of Chagas disease.2,3 As Chagas disease has no cure and treatment with benznidazole and nifurtimox is effective only in the acute phase of the disease (which is often asymptomatic), vector control is the most effective method of preventing this neglected disease.1 Thus, all knowledge about these hematophagous insects is important and can help and improve vector control programs.

Although the transmission of T. cruzi to the host occurs mostly through the feces of triatomines, hematophagous behavior is fundamental to the contamination with the protozoan, as such insects have the habit of defecating during blood feeding.2 It is believed that hematophagy was derived from ancestral generalist predators that initiated this behavior as opportunistic hematophagy, then it became facultative and finally evolved to mandatory hematophagy.4

The salivary glands of triatomines perform a fundamental role during hematophagy because their saliva is rich in proteins and anticoagulant and hemolytic enzymes.5,6 These structures have been studied anatomically,7 histologically,7 biochemically,5 molecularly,8 and cytogenetically9 based on a few triatomines of the genera Triatoma, Rhodnius, and Panstrongylus.

Based on the analysis of Rhodnius prolixus, it has been suggested that the salivary glands (principal glands) of the species of the genus Rhodnius are reddish.10 This characteristic results from the presence of nitrophorins,10 which are proteins with antihemostatic activity11 whose molecules contain a heme group responsible for that color. The relationship between red salivary glands and the presence of nitrophorins has recently been confirmed by Pacheco12 which inoculated nitrophorin inhibitors into Rhodnius eggs and the adult salivary glands were transparent.

Considering that the presence of nitrophorins is suggested for all species of the genus Rhodnius,13 and only R. prolixus,13 Rhodnius robustus,13 and Rhodnius domesticus14 have been studied, the present study aimed to analyze the color pattern of the salivary glands of 67 triatomine species, distributed in 10 different genera and grouped into three tribes, to evaluate whether the presence of nitrophorins is a synapomorphy of Rhodnius or the tribe Rhodniini, or if it is shared with triatomines of the tribes Triatomini and Cavernicolini.

At least two adult specimens of each species (males and/or females) were analyzed (tribe Cavernicolini: Cavernicola pilosa; tribe Rhodniini: Psammolestes tertius, Psammolestes coreodes, Psammolestes arthuri, Rhodnius brethesi, Rhodnius colombiensis, R. domesticus, Rhodnius ecuadoriensis, Rhodnius marabaensis, Rhodnius milesi, Rhodnius montenegrensis, Rhodnius nasutus, Rhodnius neglectus, Rhodnius neivai, Rhodnius pallescens, Rhodnius pictipes, R. prolixus, R. robustus, Rhodnius stali, tribe Triatomini: Dipetalogaster maxima, Eratyrus cuspidatus, Meccus pallidipennis, Meccus longipennis, Meccus picturata, Meccus phylossoma, Mepraia spinolai, Nesotriatoma bruneri sn Nesotriatoma flavida, Panstrongylus herreri sn Panstrongylus lignarius, Panstrongylus lignarius, Panstrongylus megistus, Panstrongylus lutzi, Triatoma arthurneivai, Triatoma bahiensis, Triatoma baratai, Triatoma brasiliensis, Triatoma brasiliensis macromelasoma, Triatoma carcavalloi, Triatoma circummaculata, Triatoma costalimai, Triatoma delpontei, Triatoma dimidiata, Triatoma garciabesi, Triatoma guasayana, Triatoma guazu, Triatoma infestans, Triatoma juazeirensis, Triatoma jurbergi, Triatoma klugi, Triatoma lectularia, Triatoma lenti, Triatoma maculata, Triatoma matogrossensis, Triatoma melanica, Triatoma melanocephala, Triatoma petrocchiae, Triatoma platensis, Triatoma protracta, Triatoma pseudomaculata, Triatoma pintodiasi, Triatoma rubrovaria, Triatoma sherlocki, Triatoma sordida, Triatoma tibiamaculata, Triatoma vandae, Triatoma vitticeps, Triatoma williami, Triatoma wygodzinskyi). The specimens were provided by the Triatominae Insectarium of Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil, and the Insectarium of the National and International Triatominae Taxonomy Reference Laboratory at Fiocruz, Rio de Janeiro, Brazil. The bugs were dissected and the salivary glands were then removed and examined by stereoscope microscope.

Through the analysis of the salivary glands, it was observed that the species of the tribe Rhodniini present red glands (represented by P. tertius [Figure 1A] and R. montenegrensis [Figure 1B]). On the other hand, all the other species analyzed exhibited transparent glands (represented by T. infestans [Figure 1C]).

Figure 1.
Figure 1.

Salivary glands of (A) Psammolestes tertius, (B) Rhodnius montenegrensis, and (C) Triatoma infestans. Note the red glands in (A) P. tertius and (B) R. montenegrensis (tribe Rhodniini). Bar = 10 μm. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 97, 3; 10.4269/ajtmh.16-0814

It is estimated that the tribes Triatomini and Rhodniini diverged at 48.9–64.4 mya, when South America was already separated from Africa.15 Recently, it was suggested that the uplift of the Andes in South America and the variations in sea levels in North America are the events involved in the diversification of these tribes.16 Nitrophorin heme proteins could have appeared (and later they were positively selected) after the divergence of the tribes, more specifically in the common ancestor of the tribe Rhodniini.

Although there is no dating of the divergence of the tribe Cavernicolini, recently this tribe was presented as a brother group of Rhodniini.16 However, the absence of nitrophorins in the salivary glands of C. pilosa suggest that this tribe derived before Rhodniini. A possible conclusion is that the tribe Triatomini derived first, followed by Cavernicolini, and finally came the tribe Rhodniini. This highlights the need for studies using molecular clocks in Triatominae with representatives of all the tribes.

One of the antihemostatic activities of the nitrophorins is the storage and transport of nitric acid ligated into the center of ferric heme,6 which promotes vasodilation and inhibition of platelet aggregation when it is released in the microcirculation.17 This study demonstrates that the species of the tribes Triatomini and Cavernicolini do not have this heme protein in the composition of their salivary glands. In the few studies that characterize the salivary glands of other genera of the tribe Triatomini, the substances isolated were triabin and pallidipin in M. pallidipennis18; triafestins, triplatin, and trialysin in T. infestans19,20; procalin in T. protracta20; dipetalodipin in D. maxima20; and lipocalin in T. lectularia8 and P. herreri.8

Therefore, this study highlights the presence of nitrophorin proteins as a synapomorphy of the tribe Rhodniini and suggests that this tribe has derived more recently when compared with the tribes Triatomini and Cavernicolini, which contributes to understanding the evolutionary history of this important vector group.

REFERENCES

  • 1.

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

  • 2.

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

  • 3.

    Justi S, Galvão C, 2017. The evolutionary origin of diversity in Chagas disease vectors. Trends Parasitol 33: 4252.

  • 4.

    Schofield CJ, 1988. The biosystematics of Triatominae. Service MW, ed. Biosystematics of Haematophagous Insects, Systematics Association Special. Oxford, United Kingdom: Clarenden Press, 284312.

    • Search Google Scholar
    • Export Citation
  • 5.

    Hellmann K, Hawkins RI, 1965. Prolixins-S and prolixin-G; two anticoagulants from Rhodnius prolixus Stal. Nature 207: 265267.

  • 6.

    Ribeiro JM, Hazzard JM, Nussenzveig RH, Champagne DE, Walker FA, 1993. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science 260: 539541.

    • Search Google Scholar
    • Export Citation
  • 7.

    Lacombe D, 1999. Anatomia e histologia de glândulas salivares de triatomíneos. Mem Inst Oswaldo Cruz 94: 557564.

  • 8.

    Montandon CE, Barros E, Vidigal PM, Mendes MT, Anhê ANBM, Ramos HJO, Oliveira CJF, Mafra C, 2016. Comparative proteomic analysis of the saliva of the Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri triatomines reveals a high interspecific functional biodiversity. Insect Biochem Mol Biol 71: 8390.

    • Search Google Scholar
    • Export Citation
  • 9.

    Anhê AC, Azeredo-Oliveira MT, 2008. Cytochemical characterization of Triatoma infestans and Panstrongylus megistus salivary gland cells (Hemiptera, Reduviidae, Triatominae). Micron 39: 11261133.

    • Search Google Scholar
    • Export Citation
  • 10.

    Champagne DE, Nussenzveig RH, Ribeiro JMC, 1995. Purification, partial characterization and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus .J Biol Chem 270: 86918695.

    • Search Google Scholar
    • Export Citation
  • 11.

    Montfort WR, Weichsel A, Andersen JF, 2000. Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods. Biochim Biophys Acta 1482: 110118.

    • Search Google Scholar
    • Export Citation
  • 12.

    Soares RPP, Gontijo NF, Romanha AJ, Diotaiuti L, Pereira MH, 1998. Salivary heme proteins distinguish Rhodnius prolixus from Rhodnius robustus (Hemiptera: Reduviidae: Triatominae). Acta Trop 71: 285291.

    • Search Google Scholar
    • Export Citation
  • 13.

    Pacheco DE, 2014. Nitroforinas salivares de Rhodnius prolixus (Hemiptera: Reduviidae): Avaliação do RNAi parental e do papel da albumina do hospedeiro na sua atividade biológica. Available at: http://www.bibliotecadigital.ufmg.br/dspace/bitstream/handle/1843/BUOS-9KXJS7/dissertfinal.pdf?sequence=1. Accessed February 22, 2017.

    • Search Google Scholar
    • Export Citation
  • 14.

    Meirelles RM, Rodrigues IS, Steindel M, Soares MJ, 2003. Ultrastructure of the salivary glands of Rhodnius domesticus Neiva and Pinto, 1923 (Hemiptera: Reduviidae). J Submicrosc Cytol Pathol 35: 199207.

    • Search Google Scholar
    • Export Citation
  • 15.

    Bargues MD, Marcilla A, Ramsey J, Dujardin JP, Schofield CJ, Mas-Coma S, 2000. Nuclear rDNA-based molecular clock of the evolution of Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease. Mem Inst Oswaldo Cruz 95: 567573.

    • Search Google Scholar
    • Export Citation
  • 16.

    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
  • 17.

    Ribeiro JMC, Schneider M, Guimaraes JA, 1995. Purification and characterization of Prolixin S (Nitrophorin 2), the salivary anticoagulant of the blood sucking bug, Rhodnius prolixus .Biochem J 308: 243249.

    • Search Google Scholar
    • Export Citation
  • 18.

    Noeske-Jungblut C, Kratzschmar J, Haendler B, Alagon A, Possani L, Verhallen P, Donner P, Schleuning WD, 1994. An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis .J Biol Chem 269: 50505053.

    • Search Google Scholar
    • Export Citation
  • 19.

    Isawa H, Orito Y, Jingushi N, Iwanaga S, Morita A, Chinzei Y, Yuda M, 2007. Identification and characterization of plasma kallikrein-kinin system inhibitors from salivar glands of the blood-sucking insect Triatoma infestans .FEBS J 274: 42714286.

    • Search Google Scholar
    • Export Citation
  • 20.

    Ribeiro JMC, Assumpcao TC, Francischetti IMB, 2012. An insight into the sialomes of bloodsucking Heteroptera. Psyche (Stuttg) 2012: 16.

Author Notes

Address correspondence to Jader de Oliveira, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista (FCFAR/UNESP), Rodovia Araraquara-Jaú km 1, 14801-902, Araraquara, Sao Paulo, Brazil. E-mail: jdr.oliveira@hotmail.com

Financial support: The study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Process numbers 2013/19764-0, 2013/08826-5, and 2015/11372-1, FAPESP, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil process: 142284/2015-7).

Authors’ addresses: Jader de Oliveira, Eder dos Santos Souza, and João Aristeu da Rosa, Laboratório de Parasitologia, Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCFAR/UNESP), Araraquara, Brazil, E-mails: jdr.oliveira@hotmail.com, ederss1@hotmail.com, and rosaja@fcfar.unesp.br. Amanda Ravazi and Kaio Cesar Chaboli Alevi, Laboratório de Biologia Celular, Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Júlio de Mesquita Filho” (IBILCE/UNESP), São José do Rio Preto, Brazil, E-mails: amandaravazi95@gmail.com and kaiochaboli@hotmail.com. Felipe Ferraz Figueiredo Moreira, Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz Fundacao Oswaldo Cruz, (LBE/IOC/FIOCRUZ), Rio de Janeiro, Brazil, E-mail: felipento@hotmail.com. Cleber Galvão, Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, E-mail: clebergalvao@gmail.com.

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