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Environmental Science and Pollution Research

, Volume 25, Issue 23, pp 22541–22551 | Cite as

Evaluation of larvicidal, adulticidal, and anticholinesterase activities of essential oils of Illicium verum Hook. f., Pimenta dioica (L.) Merr., and Myristica fragrans Houtt. against Zika virus vectors

  • Diego Gomes da Rocha Voris
  • Luciana dos Santos Dias
  • Josélia Alencar Lima
  • Keila dos Santos Cople Lima
  • José Bento Pereira Lima
  • Antônio Luís dos Santos Lima
Research Article

Abstract

Aedes aegypti is the vector responsible for transmitting pathogens that cause various infectious diseases, such as dengue, Zika, yellow fever, and chikungunya, worrying health authorities in the tropics. Due to resistance of mosquitoes to synthetic insecticides, the search for more effective insecticidal agents becomes crucial. The aim of this study was to verify the larvicidal, adulticidal, and anticholinesterase activities of the essential oils of the Illicium verum (EOIV), Pimenta dioica (EOPD), and Myristica fragrans (EOMF) against Ae. aegypti. The essential oils (EOs) were obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). The larvicidal and adulticidal activities of EOs were evaluated against third instar larvae and Ae. aegypti adult females, respectively, using the procedures of the World Health Organization (WHO) and the anticholinesterase activity of the EOs by the modified Ellman method. The following major components were identified: (E)-anethole (90.1%) for EOIV, methyl eugenol (55.0%) for EOPD, and sabinene (52.1%) for EOMF. All EOs exhibited larvicidal and adulticidal activity against Ae. aegypti. The highest larval mortality was observed in EOMF with LC50 = 28.2 μg mL−1. Adult mortality was observed after 1 (knockdown) and 24 h exposure, with the highest potential established by the EOIV, KC50 = 7.3 μg mg female−1 and LC50 = 10.3 μg mg female−1. EOIV (IC50 = 4800 μg mL−1), EOMF (IC50 = 4510 μg mL−1), and EOPD (IC50 = 1320 μg mL−1) inhibited AChE. EOMF (4130 μg mL−1) and EOPD (IC50 = 3340 μg mL−1) inhibited BChE whereas EOIV showed no inhibition. The EOs were toxic to larvae and adults of Ae. aegypti, as well as being less toxic to humans than the currently used insecticides, opening the possibility of elaboration of a natural, safe, and ecological bioinsecticide for vector control.

Keywords

Aedes aegypti Essential oils Larvicide Adulticide Anticholinesterase Bioinsecticide 

Notes

Acknowledgments

We acknowledge the members of the team of the Military Institute of Engineering (IME) and FIOCRUZ Entomology Laboratory located at the Institute of Biology of the Army (IBEX) for their kind cooperation and excellent assistance in this research project.

Funding information

This study received financial support from the Brazilian financial agencies, CNPq and CAPES. JAL is the recipiente of a CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) fellowship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

All procedures performed in animal studies were in accordance with the ethical standards of the institution or practice in which the studies were conducted and authorized by the Ethics Committee on Animal Use (LW- 20/14 March 31, 2014).

Supplementary material

11356_2018_2362_MOESM1_ESM.docx (4.2 mb)
ESM 1 (DOCX 4278 kb)

References

  1. Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–267CrossRefGoogle Scholar
  2. Abou-Elnaga ZS (2014) Insecticidal bioactivity of eco-friendly plant origin chemicals against Culex pipiens and Aedes aegypti (Diptera: Culicidae). J Entomol Zool Stud 2:340–347Google Scholar
  3. Affonso RS, Lima JÁ, Lessa B, Caetano JVO, Obara MT, Nóbrega A, Nepominova E, Musilek K, Kuca K, Slana GBCA, França TCC (2018) Quantification through TLC-Densitometric analysis, repellency and anticholinesterase activity of the homemade extract of Indian cloves. Biomed Chromatogr 32:e4096CrossRefGoogle Scholar
  4. Al-Malahmeh AJ, Al-Ajlouni AM, Wesseling S, Vervoort J, Rietjens IM (2017) Determination and risk assessment of naturally occurring genotoxic and carcinogenic alkenylbenzenes in basil-containing sauce of pesto. Toxicol Rep 4:1–8CrossRefGoogle Scholar
  5. Amer A, Mehlhorn H (2006) Repellency effect of forty-one essential oils against Aedes, Anopheles, and Culex mosquitoes. Parasitol Res 99:478–490CrossRefGoogle Scholar
  6. Ashokan AP, Paulpandi M, Dinesh D, Murugan K, Vadivalagan C, Benelli G (2017) Toxicity on dengue mosquito vectors through Myristica fragrans-synthesized zinc oxide nanorods, and their cytotoxic effects on liver cancer cells (HepG2). J Clust Sci 28:205–226CrossRefGoogle Scholar
  7. Atack JR, Yu QS, Soncrant TT, Brossi A, Rapoport SI (1989) Comparative inhibitory effects of various physostigmine analogs against acetyl-and butyrylcholinesterases. J Pharmacol Exp Ther 249:194–202Google Scholar
  8. Babushok VI, Linstrom PJ, Zenkevich IG (2011) Retention indices for frequently reported compounds of plant essential oils. J Phys Chem Ref Data 40:043101CrossRefGoogle Scholar
  9. Banumathi B, Vaseeharan B, Periyannan R, Prabhu NM, Ramasamy P, Murugan K, Canale A, Benelli G (2017) Exploitation of chemical, herbal and nanoformulated acaricides to control the cattle tick, Rhipicephalus (Boophilus) microplus—a review. Vet Parasitol 244:102–110CrossRefGoogle Scholar
  10. Basile K, Kok J, Dwyer DE (2017) Zika virus: what, where from and where to? Pathol 49:698–706CrossRefGoogle Scholar
  11. Beltrán-Silva SL, Chacón-Hernández SS, Moreno-Palacios E, Pereyra-Molina JA (2016) Clinical and differential diagnosis: dengue, chikungunya and Zika. Rev Med Hosp Gen (Mex).  https://doi.org/10.1016/j.hgmx.2016.09.011
  12. Benelli G (2016) Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol Res 115:23–34CrossRefGoogle Scholar
  13. Benelli G, Mehlhorn H (2016) Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control. Parasitol Res 115:1747–1754CrossRefGoogle Scholar
  14. Benelli G, Maggi F, Pavela R, Murugan K, Govindarajan M, Vaseeharan B, Petrelli R, Cappellacci L, Kumar S, Hofer A, Youssefi MR, Alarfaj AA, Hwang JS, Higuchi A (2017) Mosquito control with green nanopesticides: towards the one health approach? A review of non-target effects. Environ Sci Pollut Res 25:10184–10206.  https://doi.org/10.1007/s11356-017-9752-4 CrossRefGoogle Scholar
  15. Benelli G, Rajeswary M, Govindarajan M (2018) Towards green oviposition deterrents? Effectiveness of Syzygium lanceolatum (Myrtaceae) essential oil against six mosquito vectors and impact on four aquatic biological control agents. Environ Sci Pollut Res 25:10218–10227CrossRefGoogle Scholar
  16. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GRW, Simmons CP, Scott TW, Farrar JJ, Hay SI (2013) The global distribution and burden of dengue. Nature 496:504–507CrossRefGoogle Scholar
  17. Braga IA, Valle D (2007) Aedes aegypti: inseticidas, mecanismos de ação e resistência. Epidemiol Serv Saúde 16:179–293Google Scholar
  18. Brazil Ministry of Health (2017) Dengue, Chikungunya e Zika: Prevenção e Combate. http://portalms.saude.gov.br/saude-de-a-z/dengue/situacao-epidemiologica-dados. Accessed 7 Aug 2017
  19. Cardoso CW, Paploski IA, Kikuti M, Rodrigues MS, Silva MMO, Campos GS, Sardi SI, Kitron U, Reis MG, Ribeiro GS (2015) Outbreak of exanthematous illness associated with Zika, chikungunya, and dengue viruses, Salvador, Brazil. Emerg Infect Dis 21:2274–2276CrossRefGoogle Scholar
  20. Cavalcanti LPDG, Pontes RJS, Regazzi ACF, Júnior P, Frutuoso RL, Sousa EP, Dantas Filho FF, Lima JWDO (2007) Efficacy of fish as predators of Aedes aegypti larvae, under laboratory conditions. Rev Saude Publica 41:638–644CrossRefGoogle Scholar
  21. Chaiyasit D, Choochote W, Rattanachanpichai E, Chaithong U, Chaiwong P, Jitpakdi A, Tippawangkosol P, Riyong D, Pitasawat B (2006) Essential oils as potential adulticides against two populations of Aedes aegypti, the laboratory and natural field strains, in Chiang Mai province, northern Thailand. Parasitol Res 99:715–721CrossRefGoogle Scholar
  22. Chang CL, Cho IK, Li QX (2009) Insecticidal activity of basil oil, trans-anethole, estragole, and linalool to adult fruit flies of Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae. J Econ Entomol 102:203–209CrossRefGoogle Scholar
  23. Chediak M, Pimenta Jr FG, Coelho GE, Braga IA, Lima JBP, Cavalcante KRL et al (2016) Spatial and temporal country-wide survey of temephos resistance in Brazilian populations of Aedes aegypti. Mem Inst Oswaldo Cruz 111:311–321CrossRefGoogle Scholar
  24. Cheng SS, Chang HT, Chang ST, Tsai KH, Chen WJ (2003) Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae. Bioresour Technol 89:99–102CrossRefGoogle Scholar
  25. Chung IM, Ro HM, Moon HI (2011) RETRACTED: major essential oils composition and immunotoxicity activity from leaves of Foeniculum vulgare against Aedes aegypti L. Immunopharmacol Immunotoxicol 33:450–453CrossRefGoogle Scholar
  26. Conti B, Flamini G, Cioni PL, Ceccarini L, Macchia M, Benelli G (2014) Mosquitocidal essential oils: are they safe against non-target aquatic organisms? Parasitol Res 113:251–259CrossRefGoogle Scholar
  27. Corbel V, Duchon S, Zaim M, Hougard JM (2004) Dinotefuran: a potential neonicotinoid insecticide against resistant mosquitoes. J Med Entomol 41:712–717CrossRefGoogle Scholar
  28. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB (2009) Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 360:2536–2543CrossRefGoogle Scholar
  29. Edwards FL, Tchounwou PB (2005) Environmental toxicology and health effects associated with methyl parathion exposure—a scientific review. Int J Environ Res Public Health 2:430–441CrossRefGoogle Scholar
  30. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88IN191–88I9095CrossRefGoogle Scholar
  31. Finney DJ (1971) Probit Analysis. Cambridge University Press, London. 68–72Google Scholar
  32. Fontoura NG, Bellinato DF, Valle D, Lima JBP (2012) The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem Inst Oswaldo Cruz 107:387–395CrossRefGoogle Scholar
  33. Freire JM (2008) Óleos essenciais de canela, manjerona e anis-estrelado: Caracterização química e atividade biológica sobre Staphylococcus aureus, Escherichia coli, Aspergillus flavus e Aspergillus parasiticus. Dissertation , Federal University of LarvasGoogle Scholar
  34. García-Fajardo J, Martínez-Sosa M, Estarrón-Espinosa M, Vilarem G, Gaset A, De Santos JM (1997) Comparative study of the oil and supercritical CO2 extract of Mexican pimento (Pimenta dioica Merrill). J Essent Oil Res 9:181–185CrossRefGoogle Scholar
  35. Govindarajan M, Benelli G (2016) α-Humulene and β-elemene from Syzygium zeylanicum (Myrtaceae) essential oil: highly effective and eco-friendly larvicides against Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus (Diptera: Culicidae). Parasitol Res 115:2771–2778CrossRefGoogle Scholar
  36. Govindarajan M, Mathivanan T, Elumalai K, Krishnappa K, Anandan A (2011) Ovicidal and repellent activities of botanical extracts against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi (Diptera: Culicidae). Asian Pac J Trop Biomed 1:43–48CrossRefGoogle Scholar
  37. Govindarajan M, Rajeswary M, Hoti SL, Benelli G (2016) Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus (Diptera: Culicidae). Res Vet Sci 104:77–82CrossRefGoogle Scholar
  38. Govindarajan M, Vaseeharan B, Alharbi NS, Kadaikunnan S, Khaled JM, Al-anbr MN, Alyahya SA, Maggi F, Benelli G (2018) High efficacy of (Z)-γ-bisabolene from the essential oil of Galinsoga parviflora (Asteraceae) as larvicide and oviposition deterrent against six mosquito vectors. Environ Sci Pollut Res 25:1–12Google Scholar
  39. Hari I, Mathew N (2018) Larvicidal activity of selected plant extracts and their combination against the mosquito vectors Culex quinquefasciatus and Aedes aegypti. Environ Sci Pollut Res 25:9176–9185CrossRefGoogle Scholar
  40. Her Z, Kam YW, Lin RT, Ng LF (2009) Chikungunya: a bending reality. Microbes Infect 11:1165–1176CrossRefGoogle Scholar
  41. Hoeveler M (2016) Produção de bioinseticida à base de Bacillus thuringiensis israelensis contra o Aedes aegypti. Dissertation, Federal University of Rio Grande do SulGoogle Scholar
  42. Huang Y, Ho SH, Lee HC, Yap YL (2002) Insecticidal properties of eugenol, isoeugenol and methyleugenol and their effects on nutrition of Sitophilus zeamais Motsch. (Coleoptera: Curculionidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). J Stored Prod Res 38:403–412CrossRefGoogle Scholar
  43. Jokanovic M, Prostran M (2009) Pyridinium oximes as cholinesterase reactivators. Structure-activity relationship and efficacy in the treatment of poisoning with organophosphorus compounds. Curr Med Chem 16:2177–2188CrossRefGoogle Scholar
  44. Kam YW, Ong EK, Rénia L, Tong JC, Ng LF (2009) Immuno-biology of chikungunya and implications for disease intervention. Microbes Infect 11:1186–1196CrossRefGoogle Scholar
  45. Kuno G (2010) Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains. J Med Entomol 47:957–971CrossRefGoogle Scholar
  46. Lemant J, Boisson V, Winer A, Thibault L, André H, Tixier F, Lemercier M, Antok E, Cresta MP, Grivard P, Besnard M, Rollot O, Favier F, Huerre M, Campinos JL, Michault A (2008) Serious acute chikungunya virus infection requiring intensive care during the Reunion Island outbreak in 2005–2006. Crit Care Med 36:2536–2541CrossRefGoogle Scholar
  47. Lima JA, Costa RS, Epifânio RA, Castro NG, Rocha MS, Pinto AC (2009) Geissospermum vellosii stembark: anticholinesterase activity and improvement of scopolamine-induced memory deficits. Pharmacol Biochem Behav 92:508–513CrossRefGoogle Scholar
  48. Montella IR, Martins AJ, Viana-Medeiros PF, Lima JBP, Braga IA, Valle D (2007) Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. Am J Trop Med Hyg 77:467–477CrossRefGoogle Scholar
  49. Naqqash MN, Gökçe A, Bakhsh A, Salim M (2016) Insecticide resistance and its molecular basis in urban insect pests. Parasitol Res 115:1363–1373CrossRefGoogle Scholar
  50. Norris EJ, Gross AD, Dunphy BM, Bessette S, Bartholomay L, Coats JR (2015) Comparison of the insecticidal characteristics of commercially available plant essential oils against Aedes aegypti and Anopheles gambiae (Diptera: Culicidae). J Med Entomol 52:993–1002CrossRefGoogle Scholar
  51. Pang EL, Loh HS (2017) Towards development of a universal dengue vaccine—how close are we? Asian Pac J Trop Med 10:220–228CrossRefGoogle Scholar
  52. Pavela R (2009) Larvicidal property of essential oils against Culex quinquefasciatus Say (Diptera: Culicidae). Ind Crop Prod 30:311–315CrossRefGoogle Scholar
  53. Pavela R (2014) Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna. J Asia Pac Entomol 17:287–293CrossRefGoogle Scholar
  54. Pavela R (2016) Encapsulation—a convenient way to extend the persistence of the effect of eco-friendly mosquito larvicides. Curr Org Chem 20:2674–2680CrossRefGoogle Scholar
  55. Pavela R, Benelli G (2016) Essential oils as ecofriendly biopesticides? Challenges and constraints. Trends Plant Sci 21:1000–1007CrossRefGoogle Scholar
  56. Pavela R, Maggi F, Lupidi G, Mbuntcha H, Woguem V, Womeni HM, Barboni L, Tapondjou LA, Benelli G (2018) Clausena anisata and Dysphania ambrosioides essential oils: from ethno-medicine to modern uses as effective insecticides. Environ Sci Pollut Res 25:10493–10503CrossRefGoogle Scholar
  57. Perumalsamy H, Kim NJ, Ahn YJ (2009) Larvicidal activity of compounds isolated from Asarum heterotropoides against Culex pipiens pallens, Aedes aegypti, and Ochlerotatus togoi (Diptera: Culicidae). J Med Entomol 46:1420–1423CrossRefGoogle Scholar
  58. Rajkumar S, Jebanesan A (2010) Chemical composition and larvicidal activity of leaf essential oil from Clausena dentata (Willd) M. Roam. (Rutaceae) against the chikungunya vector, Aedes aegypti Linn. (Diptera: Culicidae). J Asia Pac Entomol 13:107–109CrossRefGoogle Scholar
  59. Rocha DK, Matosc O, Novoa MT, Figueiredo AC, Delgado M, Moiteiro C (2015) Larvicidal activity against Aedes aegypti of Foeniculum vulgare essential oils from Portugal and Cape Verde. Nat Prod Commun 10:677–682Google Scholar
  60. Rosa CS, Veras KS, Silva PR, Neto JL, Cardoso HLM, Alves LPL, Brito MCA, Amaral FMM, Maia JGS, Monteiro OS, Moraes DFC (2016) Composição química e toxicidade frente Aedes aegypti L. e Artemia salina Leach do óleo essencial das folhas de Myrcia sylvatica (G. Mey.) DC. Rev Bras Pl Med 18:19–26CrossRefGoogle Scholar
  61. Rothman AL (2004) Dengue: defining protective versus pathologic immunity. J Clin Invest 113:946–951CrossRefGoogle Scholar
  62. Saavedra LM, Romanelli GP, Rozo CE, Duchowicz PR (2018) The quantitative structure–insecticidal activity relationships from plant derived compounds against chikungunya and zika Aedes aegypti (Diptera: Culicidae) vector. Sci Total Environ 610:937–943CrossRefGoogle Scholar
  63. Samarasekera R, Kalhari KS, Weerasinghe IS (2005) Mosquitocidal activity of leaf and bark essential oils of Ceylon Cinnamomum zeylanicum. J Essent Oil Res 17:301–303CrossRefGoogle Scholar
  64. Santos SR, Silva VB, Melo MA, Barbosa JD, Santos RL, De Sousa DP, Cavalcanti SC (2010) Toxic effects on and structure-toxicity relationships of phenylpropanoids, terpenes, and related compounds in Aedes aegypti larvae. Vector Borne Zoonotic Dis 10:1049–1054CrossRefGoogle Scholar
  65. Seo SM, Jung CS, Kang J, Lee HR, Kim SW, Hyun J, Park IK (2015) Larvicidal and acetylcholinesterase inhibitory activities of Apiaceae plant essential oils and their constituents against Aedes albopictus and formulation development. J Agric Food Chem 63:9977–9986CrossRefGoogle Scholar
  66. Shaalan EAS, Canyon DV (2018) Mosquito oviposition deterrents. Environ Sci Pollut Res 25:10207–10217CrossRefGoogle Scholar
  67. Siano F, Ghizzoni C, Gionfriddo F, Colombo E, Servillo L, Castaldo D (2003) Determination of estragole, safrole and eugenol methyl ether in food products. Food Chem 81:469–475CrossRefGoogle Scholar
  68. Tripp RA, Ross TM (2016) Development of a Zika vaccine. Expert Rev Vaccines 15:1083–1085CrossRefGoogle Scholar
  69. Valente VMM (2005) Caracterização de antifúngicos em óleo essencial de noz-moscada (Myristica fragans). Dissertation, Federal University of ViçosaGoogle Scholar
  70. Waliwitiya R, Kennedy CJ, Lowenberger CA (2009) Larvicidal and oviposition-altering activity of monoterpenoids, trans-anethole and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae). Pest Manag Sci 65:241–248CrossRefGoogle Scholar
  71. WHO G (1996) Report of the WHO informal consultation on the evaluation and testing of insecticides. World Health Organization GenevaGoogle Scholar
  72. WHO G (2005) Guidelines for laboratory and field testing of mosquitos larvicides. World Health Organization GenevaGoogle Scholar
  73. Wiesner J, Kříž Z, Kuča K, Jun D, Koča J (2007) Acetylcholinesterases—the structural similarities and differences. J Enzyme Inhib Med Chem 22:417–424CrossRefGoogle Scholar
  74. Yang YC, Park IK, Kim EH, Lee HS, Ahn YJ (2004) Larvicidal activity of medicinal plant extracts against Aedes aegypti, Ochlerotatus togoi, and Culex pipiens pallens (Diptera: Culicidae). J Asia Pac Entomol 7:227–232CrossRefGoogle Scholar
  75. Yang S, Fink D, Hulse A, Pratt RD (2017) Regulatory considerations in development of vaccines to prevent disease caused by chikungunya virus. Vaccine 35:4851–4858CrossRefGoogle Scholar
  76. Yeom HJ, Kang JS, Kim GH, Park IK (2012) Insecticidal and acetylcholine esterase inhibition activity of Apiaceae plant essential oils and their constituents against adults of German cockroach (Blattella germanica). J Agric Food Chem 60:7194–7203CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Diego Gomes da Rocha Voris
    • 1
  • Luciana dos Santos Dias
    • 2
    • 3
  • Josélia Alencar Lima
    • 1
  • Keila dos Santos Cople Lima
    • 1
  • José Bento Pereira Lima
    • 2
    • 3
  • Antônio Luís dos Santos Lima
    • 1
  1. 1.Section of Chemical Engineering, Chemical and Biological Defence LaboratoryMilitary Institute of EngineeringRio de JaneiroBrazil
  2. 2.Laboratory of Physiology and Control of Vector ArthropodsOswaldo Cruz Institute, FiocruzRio de JaneiroBrazil
  3. 3.Entomology LaboratoryInstitute of Biology of the ArmyRio de JaneiroBrazil

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