• View in gallery
    Figure 1.

    Invasion map of Anopheles gambiae s.l. in Brazil. Arrows indicate the dispersal route of the mosquitoes towards the Brazilian mainland and the starting point of the invasion. Time progression is marked within the boxes. The maximum distribution range of An. gambiae s.l. in Brazil is depicted. Map redrawn from a previously published one.5

  • View in gallery
    Figure 2.

    Alignment of a portion of the produced IGS sequences together with the homologue sequences of Anopheles gambiae M (AF470112) and S (AF470116) molecular forms, and Anopheles arabiensis (AF470110). The diagnostic sites of the IGS fragment separating between the An. gambiae forms and between An. gambiae s.s. and An. arabiensis are identified by arrows. The nucleotide positions have been numbered based on the An. gambiae IGS sequence with accession number AF470116.

  • 1

    Breman JG, Egan A, Keusch GT, 2001. The intolerable burden of malaria: a new look at the numbers. Am J Trop Med Hyg 64 (Suppl): iv–vii.

    • Search Google Scholar
    • Export Citation
  • 2

    Kondrashin AV, Orlov VS, 1989. Migration and malaria. Service MW, ed. Demography and Vector Borne Diseases. Boca Raton, FL: CRC Press, 353–366.

  • 3

    Laird M, 1989 Vector–borne diseases introduced into new areas due to human movements: a historical perspective. Service MW, ed. Demography and Vector Borne Diseases. Boca Raton, FL: CRC Press, 17–33.

  • 4

    Lounibos LP, 2002. Invasion by insect vectors of human disease. Annu Rev Entomol 47 :233–266.

  • 5

    Soper FL, Wilson DB, 1943. Anopheles gambiae in Brazil 1930–1940. New York: Rockefeller Foundation.

  • 6

    White GB, 1974. Anopheles gambiae complex and disease transmission in Africa. Trans R Soc Trop Med Hyg 68 :278–298.

  • 7

    Levine R, Peterson AT, Benedict MQ, 2004. Geographic and ecologic distributions of the Anopheles gambiae complex predicted using a genetic algorithm. Am J Trop Med Hyg 70 :105–109.

    • Search Google Scholar
    • Export Citation
  • 8

    Santolamazza F, della Torre A, Caccone A, 2004. Short report: A new polymerase chain reaction–restriction fragment length polymorphism method to identify Anopheles arabiensis from An. gambiae and its two molecular forms from degraded DNA templates or museum samples. Am J Trop Med Hyg 70 :604–606.

    • Search Google Scholar
    • Export Citation
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Historical Analysis of a Near Disaster: Anopheles gambiae in Brazil

Aristeidis ParmakelisDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Michael A. RusselloDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Adalgisa CacconeDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Carlos Brisola MarcondesDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Jane CostaDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Oswaldo P. ForattiniDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Maria Anice Mureb SallumDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Richard C. WilkersonDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Jeffrey R. PowellDepartment of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, Florianópolis, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil; Division of Entomology Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC

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Attributed to human-mediated dispersal, a species of the Anopheles gambiae complex invaded northeastern Brazil in 1930. This event is considered unique among the intercontinental introductions of disease vectors and the most serious one: “Few threats to the future health of the Americas have equalled that inherent in the invasion of Brazil, in 1930, by Anopheles gambiae.” Because it was only in the 1960s that An. gambiae was recognized as a species complex now including seven species, the precise species identity of the Brazilian invader remains a mystery. Here we used historical DNA analysis of museum specimens, collected at the time of invasion from Brazil, and aimed at the identification of the Brazilian invader. Our results identify the arid-adapted Anopheles arabiensis as being the actual invading species. Establishing the identity of the species, in addition to being of intrinsic historical interest, can inform future threats of this sort especially in a changing environment. Furthermore, these results highlight the potential danger of human-mediated range expansions of insect disease vectors and the importance of museum collections in retrieving historical information.

The Anopheles gambiae species complex contains the most important vectors of the deadliest form of malaria in sub-Saharan Africa, where globally, ∼80% of malaria mortality and morbidity occurs.1 Incidents of Anopheles introduction have been reported worldwide but have been restricted.24 However, introductions coupled with suitable environmental conditions for Anopheles establishment are the most threatening. The Brazil invasion was one of these cases. On March 23, 1930, R. C. Shannon collected larvae of An. gambiae s.l., near Natal, Brazil (State of Rio Grande do Norte; Figure 1). The species expanded its range, and a serious increase in human malaria cases over 9 years followed with a 20–25% death rate in a largely immune-naïve population. The efforts of a Rockefeller Foundation–supported team led to the eradication of the invading species by 1940, averting a potential public health catastrophe.5

Because of the level of shipping traffic between Brazil and Senegal in the 1920s/1930s, it has been assumed that the invader came from this African region, which is inhabited by An. gambiae s.s. (with its two molecular forms, M and S), Anopheles arabiensis, and Anopheles melas. Of these three taxa, An. melas is an unlikely candidate given its larval ecology (brackish water); both An gambiae s.s. and An. arabiensis have been speculated to be the invader.6,7

In this paper, we used museum specimens (Table 1), collected at the time of invasion from various localities in Brazil, and through DNA analysis, we aimed to identify the Brazilian invader. To avoid potential contaminations, DNA was extracted following strict ancient DNA (aDNA) protocols in an aDNA facility. DNA extractions (from entire mosquito or a few legs) were carried out using the EasyDNA kit (Invitrogen, Carlsbad, CA), using mussel glycogen and protein degrader according to the manufacturer’s protocol. For polymerase chain reaction (PCR) amplification, we targeted short regions (124–419 bp) of the ribosomal DNA (rDNA) intergenic spacer (IGS). This region contains one diagnostic site separating An. gambiae (both M and S forms) from An. arabiensis and one diagnostic site that allows the separation between the M and S An. gambiae molecular forms and between An. arabiensis and An. gambiae M molecular form.8 The legitimacy of the diagnostic sites is robust, having been assessed on a large database of IGS DNA sequences from An. gambiae s.l. samples collected from all over the African continent.8 The primers initially used in the PCR amplifications were IGS441 and IGS783,8 whereas additional primers targeting smaller and larger fragments were also designed and used when the initial IGS primers were not effective. The primers designed for this study were IGS565-forward, IGS581-forward, IGS659-forward, and IGS839-reverse (located in positions 565–584, 581–600, 659–679, and 839–858 of the An. gambiae IGS sequence with accession number AF470116, respectively). Primers were used either directly on the DNA extracts, or whenever that was feasible, in a nested PCR protocol. PCR products were visualized in ethidium bromide–stained agarose gels. The PCR products were either purified using the QIAquick PCR purification kit (Qiagen, German-town, MD) or excised from the gel and purified using the Freeze N’ Squeeze kit (Bio-Rad, Hercules, CA). Cycle sequencing of the purified PCR products was performed using Big Dye chemistry, and sequences were determined on an ABI 3730 automated sequencer.

IGS DNA sequences were successfully obtained (125–204 bp) from 11 of the 13 specimens. All 11 were An. arabiensis (Figure 2). This result is consistent with the prediction of An. arabiensis in Brazil6 based primarily on its ecology. An. arabiensis is the most arid-adapted member of the complex, and the area invaded is arid (Figure 1). Because An. arabiensis is arid-adapted, the humid rainforest surrounding the invaded area could well have been crucial in precluding its further spread in 1930s. With the increasing destruction of tropical forests in South America, more territory is converted into an ideal habitat for the previous invader and its equally dangerous sibling species, An. gambiae s.s.

Insect vectors of disease can spread to new regions, and it is critical to have surveillance capable of detecting new introductions early. The eradication of this fatal vector in Brazil in the 1930s was a success only because mosquito workers were in the region to combat yellow fever and, by chance, detected An. gambiae s.l. Our results point out the importance of museum collections in allowing retrieval of historical information as new understanding (e.g., taxonomy) and technology advances to take advantage of collections.

Table 1

Information on the A. gambiae s.l. museum specimens used

MuseumCollection year and localitySample codeAccession number
* Instituto Oswaldo Cruz, Coleção Entomológica Costa Lima, Rio de Janeiro, Brazil.
† Faculdade de Saúde Pública, Universidade de São Paulo, Brazil.
‡ National Museum of Natural History, Washington, DC.
1FIOCRUZ*1935, Santo Antonio (São Gonçalo), State of Rio Grande do Norte1073–1EU091298
2FIOCRUZ1935, Santo Antonio (São Gonçalo), State of Rio Grande do Norte1073–2
3FIOCRUZ1935, Santo Antonio (São Gonçalo), State of Rio Grande do Norte1074–1EU091299
4FIOCRUZ1935, Santo Antonio (São Gonçalo), State of Rio Grande do Norte1074–2EU091300
5FIOCRUZ1937, State of Ceará1419–1EU091301
6FIOCRUZ1937, State of Ceará1419–2EU091302
7FIOCRUZ1937, State of Ceará1419–3EU091303
8FIOCRUZ1937, State of Ceará1419–4EU091304
9FSP–USP†1939, Aracati, State of CearáFSP–5461EU091306
10FSP–USP1940, União Garça, State of CearáFSP–5468EU091307
11FSP–USP1932, State of Rio Grande do NorteFSP–60EU091305
12NMNH‡1940, State of CearáNMNH1
13NMNH1940, State of CearáNMNH2EU091308
Figure 1.
Figure 1.

Invasion map of Anopheles gambiae s.l. in Brazil. Arrows indicate the dispersal route of the mosquitoes towards the Brazilian mainland and the starting point of the invasion. Time progression is marked within the boxes. The maximum distribution range of An. gambiae s.l. in Brazil is depicted. Map redrawn from a previously published one.5

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.176

Figure 2.
Figure 2.

Alignment of a portion of the produced IGS sequences together with the homologue sequences of Anopheles gambiae M (AF470112) and S (AF470116) molecular forms, and Anopheles arabiensis (AF470110). The diagnostic sites of the IGS fragment separating between the An. gambiae forms and between An. gambiae s.s. and An. arabiensis are identified by arrows. The nucleotide positions have been numbered based on the An. gambiae IGS sequence with accession number AF470116.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.176

*

Address correspondence to Aristeidis Parmakelis, 21 Sachem St., New Haven, CT 06520. E-mail: parmakel@nhmc.uoc.gr

Authors’ addresses: Aristeidis Parmakelis, Michael A. Russello, Adalgisa Caccone, and Jeffrey R. Powell, Department of Ecology and Evolutionary Biology, Yale University, 21 Sachem St., 06520, New Haven, CT, Telephone: 203–432–3886, Fax: 203–432–7394, E-mails: parmakel@nhmc.uoc.gr, adalgisa.caccone@yale.edu, jeffrey.powell@yale.edu. Aristeidis Parmakelis, present address: Department of Biology, University of Crete, Knossou Avenue, Irakleio, Crete GR-71409, Greece, Telephone: 2810–393282, E-mail: parmakel@nhmc.uoc.gr. Michael A. Russello, present address: Unit of Biology and Physical Geography, University of British Columbia Okanagan, 3333 University Way, SCI381, Kelowna, British Columbia V1V 1V7, Canada, Telephone: 250–807–8762, Fax: 250–807–8005, E-mail: michael.russello@ubc.ca. Carlos Brisola Marcondes, Department of Microbiology and Parasitology, Center of Biological Sciences, Universidade Federal de Santa Catarina, Trindade, 88040–900 Florianópolis, SC, Brazil, Telephone: 55–48–3721–5208, Fax: 55–48–3721–5208, E-mail: cbrisola@mbox1.ufsc.br. Jane Costa, Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil, Telephone: 55–21–2598–4401, Fax: 55–21–2573–7276, E-mail: jcosta@ioc.fiocruz.br. Maria Anice Mureb Sallum and Oswaldo P. Forattini, Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, Av. Dr. Arnaldo 715, CEP 01246–904, São Paulo, Brazil, Telephone: 55–11–30617731, Fax: 55–11–30812108, E-mails: masallum@usp.br, opforati@usp.br. Richard C. Wilkerson, Division of Entomology Walter Reed Army Institute of Research, Silver Spring, MD, and Department of Entomology, Smithsonian Institution, National Museum of Natural History, Washington, DC, Telephone: 301–238–1077, Fax: 301–238–3168, E-mail: wilkersonr@si.edu.

Acknowledgments: We thank M. Coluzzi and D. Fish for fruitful comments and discussion.

Financial support: This study was supported by NIH Grant RO1 AI046018 to JRP. AP was the beneficiary of a Marie Curie Outgoing International Fellowship (Contract MOIF-CT-2006-021357).

REFERENCES

  • 1

    Breman JG, Egan A, Keusch GT, 2001. The intolerable burden of malaria: a new look at the numbers. Am J Trop Med Hyg 64 (Suppl): iv–vii.

    • Search Google Scholar
    • Export Citation
  • 2

    Kondrashin AV, Orlov VS, 1989. Migration and malaria. Service MW, ed. Demography and Vector Borne Diseases. Boca Raton, FL: CRC Press, 353–366.

  • 3

    Laird M, 1989 Vector–borne diseases introduced into new areas due to human movements: a historical perspective. Service MW, ed. Demography and Vector Borne Diseases. Boca Raton, FL: CRC Press, 17–33.

  • 4

    Lounibos LP, 2002. Invasion by insect vectors of human disease. Annu Rev Entomol 47 :233–266.

  • 5

    Soper FL, Wilson DB, 1943. Anopheles gambiae in Brazil 1930–1940. New York: Rockefeller Foundation.

  • 6

    White GB, 1974. Anopheles gambiae complex and disease transmission in Africa. Trans R Soc Trop Med Hyg 68 :278–298.

  • 7

    Levine R, Peterson AT, Benedict MQ, 2004. Geographic and ecologic distributions of the Anopheles gambiae complex predicted using a genetic algorithm. Am J Trop Med Hyg 70 :105–109.

    • Search Google Scholar
    • Export Citation
  • 8

    Santolamazza F, della Torre A, Caccone A, 2004. Short report: A new polymerase chain reaction–restriction fragment length polymorphism method to identify Anopheles arabiensis from An. gambiae and its two molecular forms from degraded DNA templates or museum samples. Am J Trop Med Hyg 70 :604–606.

    • Search Google Scholar
    • Export Citation
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