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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.2–4 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
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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.
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). 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.
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Received May 4, 2007. Accepted for publication August 18, 2007.
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).
* Address correspondence to Aristeidis Parmakelis, 21 Sachem St., New Haven, CT 06520. E-mail: parmakel{at}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{at}nhmc.uoc.gr, adalgisa.caccone{at}yale.edu, jeffrey.powell{at}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{at}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{at}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{at}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{at}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{at}usp.br, opforati{at}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{at}si.edu.
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