• View in gallery

    Diagrammatic representation of the digestion profiles for Anopheles gambiae M, An. arabiensis, and An. gambiae S. The two arrows indicate the location of the forward and reverse polymerase chain reaction primers. The lines identify the relative locations of the two Mse I sites in An. arabiensis and An. gambiae M, with reference to the An. gambiae intergenic sequence (AF 470116). The size of each of the expected DNA fragments is reported above each fragment.

  • View in gallery

    Restriction digestion profiles. Lanes 1 and 10, DNA size standards (1-kb ladder; Gibco-BRL, Gaithersburg, MD); lane 2, An. gambiae M molecular form; lane 3, An. arabiensis; lane 4, An. gambiae S molecular form; lanes 5–9, pinned An. gambiae from 1970 (GA70), 1972 (GA72), and 1985 (GA1985). DNA extractions were from legs and abdomen (lane 5) and one leg (lanes 6–9). The GA70 and GA72 samples were from Banambani, Mali and the GA85 samples were from Ouagadougo, Burkina Faso.

  • 1

    Coluzzi M, Sabatini A, Petrarca V, di Deco MA, 1979. Chromosomal differentiation and adaptation to human environment in the Anopheles gambiae complex. Trans R Soc Trop Med Hyg 73 :483–497.

    • Search Google Scholar
    • Export Citation
  • 2

    Coluzzi M, Petrarca V, di Deco MA, 1985. Chromosomal inversion intergradation and incipient speciation in Anopheles gambiae.Boll Zool 52 :45–63.

    • Search Google Scholar
    • Export Citation
  • 3

    Coluzzi M, Sabatini A, della Torre A, Di Deco MA, Petrarca V, 2002. The polytene chromosome map of Anopheles gambiae: cytotaxonomic findings, patterns of rearrangements, evolutionary and malariological implications. Science 298 :1415–1418.

    • Search Google Scholar
    • Export Citation
  • 4

    Touré YT, Petrarca V, Traoré SF, Coulibaly A, Maiga HM, Sankaré O, Sow M, Di Deco MA, Coluzzi M, 1994. Ecological genetic studies in the chromosomal form Mopti of Anopheles gambiae s.s. in Mali, west Africa. Genetica 94 :213–223.

    • Search Google Scholar
    • Export Citation
  • 5

    Touré YT, Petrarca V, Traoré SF, Coulibaly A, Maiga HM, Sankaré O, Sow M, Di Deco MA, Coluzzi M, 1998. Distribution and inversion polymorphism of chromosomally recognized taxa of the Anopheles gambiae complex in Mali, west Africa. Parassitologia 40 :477–511.

    • Search Google Scholar
    • Export Citation
  • 6

    Powell JR, Petrarca V, della Torre A, Caccone A, Coluzzi M, 1999. Population structure, speciation, and introgression in the Anopheles gambiae complex.Parassitologia 41 :101–114.

    • Search Google Scholar
    • Export Citation
  • 7

    Bryan JH, Di Deco MA, Petrarca V, Coluzzi M, 1982. Inversion polymorphism and incipient speciation in Anopheles gambiae s. s. in The Gambia, west Africa. Genetica 59 :167–176.

    • Search Google Scholar
    • Export Citation
  • 8

    Mukabayire O, Touré YT, Coluzzi M, Besansky NJ, 2001. Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA, white and tryptophan oxygenase genes. Insect Mol Biol 10 :33–46.

    • Search Google Scholar
    • Export Citation
  • 9

    della Torre A, Fanello C, Akogbeto M, Dossouvovo J, Favia G, Petrarca V, Coluzzi. M, 2001. Cytogenetic and molecular evidence of incipient speciation within Anopheles gambiae s.s. in west Africa. Insect Mol Biol 10 :9–18.

    • Search Google Scholar
    • Export Citation
  • 10

    della Torre A, Costantini C, Besansky NJ, Caccone A, Petrarca V, Powell JR, Coluzzi M, 2002. Incipient speciation within Anopheles gambiae: the glass is half full. Science 298 :115–117.

    • Search Google Scholar
    • Export Citation
  • 11

    Favia G, Dimopoulos G, della Torre A, Touré YT, Coluzzi M, Louis C, 1994. Polymorphisms detected by random PCR distinguishes between different chromosomal forms of Anopheles gambiae.Proc Natl Acad Sci USA 91 :10315–10319.

    • Search Google Scholar
    • Export Citation
  • 12

    Favia G, della Torre A, Bagayoko M, Lanfrancotti A, Sagnon N’F, Touré YT, Coluzzi M, 1997. Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation. Insect Mol Biol 6 :377–383.

    • Search Google Scholar
    • Export Citation
  • 13

    Favia G, Lanfrancotti A, Spanos L, Siden-Kiamos I, Louis C, 2001. Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s. Insect Mol Biol 10 :19–23.

    • Search Google Scholar
    • Export Citation
  • 14

    Gentile G, Slotman M, Ketmaier V, Powell JR, Caccone A, 2001. Attempts to molecularly distinguish cryptic taxa in Anopheles gambiae s.s. Insect Mol Biol 10 :25–32.

    • Search Google Scholar
    • Export Citation
  • 15

    Gentile G, della Torre A, Maegga B, Powell JR, Caccone A, 2002. Genetic differentiation in the African malaria vector, Anopheles gambiae s.s., and the problem of taxonomic status. Genetics 161: 1561–1578.

    • Search Google Scholar
    • Export Citation
  • 16

    Weill M, Chandre F, Brengues C, Manguin S, Akogbeto M, Pasteur N, Guillet P, Raymond M, 2000. The kdr mutation occurs in the Mopti form of Anopheles gambiae s.s. through introgression. Insect Mol Biol 9 :451–455.

    • Search Google Scholar
    • Export Citation
  • 17

    Gentile G, Santolamazza F, Fanello C, Petrarca V, Caccone A, della Torre A, 2004. Variation in an intron sequence of the voltage-gated sodium channel gene correlates with genetic differentiation between Anopheles gambiae s.s. molecular forms. Insect Mol Biol 13 : (in press).

    • Search Google Scholar
    • Export Citation
  • 18

    Edillo FE, Touré YT, Lanzaro GC, Dolo G, Taylor CE, 2002. Spatial and habitat distribution of Anopheles gambiae and Anopheles arabiensis (Diptera:Culicidae) in Banambani village, Mali. J Med Entomol 39 :70–77.

    • Search Google Scholar
    • Export Citation
  • 19

    Scott JA, Brogdon WG, Collins FH, 1993. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg 49 :520–529.

    • Search Google Scholar
    • Export Citation
  • 20

    Fanello C, Santolamazza F, della Torre A, 2002. Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP. Med Vet Entomol 16 :461–466.

    • Search Google Scholar
    • Export Citation
  • 21

    Coetzee M, Craig M, le Sueur D, 2000. Distribution of African malaria mosquitoes belonging to the Anopheles gambiae complex. Parasitol Today 16 :74–77.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 
 
 

 

 

 

 

 

 

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

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  • 1 Department of Ecology and Evolutionary Biology and Yale Institute for Biospheric Studies, Yale University, New Haven, Connecticut; Sezione di Parassitologia, Dipartimento di Scienze di Sanità Pubblica, Università di Roma La Sapienza, Rome, Italy

We present a polymerase chain reaction-restriction fragment length polymorphism method to simultaneously distinguish the two Anopheles gambiae M and S molecular forms and Anopheles arabiensis. This method uses different diagnostic sites than previously published methods, and it is based on the amplification of a smaller ribosomal DNA fragment. We have tested this protocol in a variety of samples from different geographic regions and various ages of preservation to ascertain the robustness of this protocol over a wide geographic window and on DNA templates of poor quality. This procedure is as efficient as previous ones in discriminating An. arabiensis from the two taxa in An gambiae s.s. However, it performs better than others on poor quality templates such as the ones from museum collections, and poorly stored field collected material. However, it must be noted that it does not allow the simultaneous discrimination of all the species in the An. gambiae complex.

The Anopheles gambiae complex presently includes seven species: gambiae (sensu stricto), arabiensis, quadriannulatus A and B, merus, melas, and bwambae. The complex is replete with chromosomal inversion polymorphisms within species and aspects of inversion polymorphism in the An. gambiae complex have been reviewed by several investigators.1–7 All taxa of this group are native to sub-Saharan Africa. The two most widespread and important species from an epidemiologic standpoint are An. gambiae and An. arabiensis. Both are closely associated with human habitats in Africa, readily bite humans, and are efficient vectors of malaria, Bancroftian filariasis, and some arboviruses.

A surprising finding is the indication that further subdivision of the species of the gambiae complex may exist. Based on gene arrangements of the 2R chromosomal arm (differing by inversions) five “chromosomal forms: have been recognized in An. gambiae s.s. (Mopti, Bamako, Forest, Savanna, and Bissau).2–7 Molecular studies of these “forms” have confirmed the existence of genetic discontinuity in this species. Diagnostic difference in the intergenic sequence (IGS) and internal transcribed sequence (ITS) regions of ribosomal DNA (rDNA) on the X chromosome distinguish two “molecular forms”, called M and S.8–15 Recently, nucleotide substitutions in intron I of the voltage-gated sodium channel gene located on the second chromosome have also been shown to segregate according to the M and S molecular forms.16,17 The correspondence between chromosomal and molecular forms is not completely resolved and it varies in different areas of the geographic range of the species.10,14,15 Understanding the spatial distribution of the molecular and chromosomal forms has important epidemiologic implications for the control of these most efficient vector species because ecologic and behavioral studies have shown that these forms may differ in their ecology and possibly transmission capacity.6,18

In this context, it is important to have easy and quick methods to diagnose the M and S forms. Three methods have been already published, all based on diagnostic size and site difference within the rDNA IGS region. The first12 includes a preliminary polymerase chain reaction (PCR) step to identify An. gambiae s.s.19 from the other species in the complex followed by the PCR amplification and restriction digestion of a 1.3-kb fragment of the IGS region. The second method combines the preliminary PCR step19 with a second PCR, and is based on primers designed on the diagnostic M/S site.13 The third method20 is a PCR-RFLP method that combines the protocol developed to distinguish the species within the An. gambiae complex19 and a restriction digestion using Hha I to distinguish M and S forms. The first method19 has been the most widely used, the second one is less reliable because of its sensitivity to PCR assay conditions, and the third one is the current method of choice, being recently and successfully adopted by several groups.

In this paper, we describe a new PCR-RFLP method that has been developed to work on DNA template of poor quality. These include old specimens from museum collections or poorly stored field-collected material. The procedure is based on a PCR amplification of a smaller fragment of the IGS region than other assays, followed by RFLP analysis of a different M/S diagnostic site from the one used before, located at position 647 in relation to the An gambiae sequence AF470116 (Figure 1), a position internal to the IGS fragment used for species identification.19 The method takes advantage of the fact that there are multiple variable sites in both the IGS and ITS regions that co-segregate with the site initially used for M/S identification.14,15 The primers IGS441 Forward (5′-TGG TCT GGG GAC CAC GTC GAC ACA GG-3′) and IGS783 Reverse (5′-CGT TTC TCA CAT CAA GAC AAT CAA GTC-3′), located in positions 441-466 and 781–754 of the An gambiae (GenBank sequence AF470116), amplifies a 288-basepair fragment of the An. gambiae and An. arabiensis IGS region (Figure 1). The PCRs are carried out in a volume of 15 μL using PCR buffer I (Applied Biosystems, Foster City, CA), 2.5 mM MgCl2, 0.5 mM of each dNTP, 6 pM of each primer, and 0.5 units of AmpliTaq Gold polymerase (Applied Biosystems). Cycling conditions include an initial 10-minute denaturation step at 94°C, followed by 30 cycles, each consisting of 30 seconds at 94°C, 30 seconds at 58°C, and 30 seconds at 72°C; the final products are extended for seven minutes at 72°C. After amplification, one unit of Mse I and 1× buffer 2 (New England Biolabs, Beverly, MA) ) is added to the PCR. Digestion is carried out for at least three hours at 37°C followed by separation of the bands by electrophoresis on a 1.5% agarose gel. The number and size of the digested fragments allow the simultaneous differentiation of An. arabiensis and An. gambiae M and S. The S form does not have any Mse I restriction site. The M form and An. arabiensis have Mse I sites located in different positions (Figure 1). Figure 2 shows the restriction profiles obtained for An. gambiae M and S, and for An. arabiensis.

To be of diagnostic value, this protocol needs to be as reliable as previous methods over a wide geographic scale.14,15 To be preferred over other protocols, it needs to be perform better in a given set of circumstances than other methods. To evaluate the first point, we used this protocol in parallel with the method of Fanello and others.20 On 123 samples of An. arabiensis and An. gambiae M and S from 13 sub-Saharan countries: Angola, Benin, Burkina Faso, Cameroon, Congo, Ghana, Côte d’Ivoire, Mali, Nigeria, Senegal, Tanzania, The Gambia, and Sao Tomé (a list of specimens and form identification are available from the first author upon request). This screening was important because this new assay is based on a different diagnostic site than the previous ones (position 540 in AF470116); thus, we needed to test if it holds over the same large geographic window. For all 123 samples tested, the new method gave the same result as the protocol of Fanello and others,20 correctly identifying the An. gambiae M and S forms, the An. arabiensis samples, and one M/arabiensis hybrid.

To evaluate if the new method was more successful than the protocol of Fanello and others20 when using DNA of poor quality, we performed the two assays on DNA extracted from single legs of museum specimens. The analysis was carried out on 16 pinned specimens from 1970, 1972, and 1985 from Banambani, Mali and Ouagadougou, Burkina Faso. Isolations of DNA were carried out using the Easy DNA Kit (Invitrogen, Carlsbad, CA) after homogenization in liquid nitrogen. In our experience, grinding specimens in liquid nitrogen gives better DNA yields than standard homogenization procedures, when dealing with dry field-collected, museum-preserved material, and also larval collections. Conversely, the Invitrogen kit DNA extraction protocol gives only marginally better yields than standard non-kit-based DNA extraction procedures. The new PCR-RFLP method described here allowed M/S diagnosis for all the museum samples, while the method of Fanello and others20 on the same DNA extractions failed. We did not compare this new method with the other available protocols because they are based on larger PCR fragments than the method of Fanello and others,20 and thus are unlikely to perform better than this method on degraded templates.

If one uses the same protocol, it is possible to amplify the same-sized PCR product for all the species within the An. gambiae complex. The An. merus and An. bwambae PCR fragments have a Mse I site that produces a species-specific restriction profile with two bands each (An. merus: 77 and 213 basepairs, An. bwambae: 74 and 214 basepairs). Thus, if these fragments were subjected to electrophoresis on 2–3% agarose gels rather than the standard 1.5% gel we used for this assay, it will be possible to distinguish An gambiae S specimens from An. merus and An. bwambae. However, it must be pointed out that this assay cannot distinguish An. quadriannulatus and An. melas from An. gambiae S, since no Mse I site occurs in the PCR fragment. This limits the use of this method for species identification to areas where An. melas and An. quadriannulatus do not co-occur with the other taxa. However, given the range overlap of these species with the other members of the An. gambiae complex, this method is still useful for samples collected in most of sub-Saharan Africa.21

In conclusion, this method seems as robust as previous ones in identifying the M and S molecular forms within An. gambiae s.s. and A. arabiensis. The results suggest that it is also better suited than previously published protocols to deal with DNA templates of low quality such as the ones from old museum collections, poorly preserved field-collected material, or degraded DNA templates.

Figure 1.
Figure 1.

Diagrammatic representation of the digestion profiles for Anopheles gambiae M, An. arabiensis, and An. gambiae S. The two arrows indicate the location of the forward and reverse polymerase chain reaction primers. The lines identify the relative locations of the two Mse I sites in An. arabiensis and An. gambiae M, with reference to the An. gambiae intergenic sequence (AF 470116). The size of each of the expected DNA fragments is reported above each fragment.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 6; 10.4269/ajtmh.2004.70.604

Figure 2.
Figure 2.

Restriction digestion profiles. Lanes 1 and 10, DNA size standards (1-kb ladder; Gibco-BRL, Gaithersburg, MD); lane 2, An. gambiae M molecular form; lane 3, An. arabiensis; lane 4, An. gambiae S molecular form; lanes 5–9, pinned An. gambiae from 1970 (GA70), 1972 (GA72), and 1985 (GA1985). DNA extractions were from legs and abdomen (lane 5) and one leg (lanes 6–9). The GA70 and GA72 samples were from Banambani, Mali and the GA85 samples were from Ouagadougo, Burkina Faso.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 6; 10.4269/ajtmh.2004.70.604

Authors’ addresses: Federica Santolamazza and Alessandra della Torre, Sezione di Parassitologia, Dipartimento di Scienze di Sanità Pubblica, Università di Roma La Sapienza, Piazzale Aldo Moro, 5 00185 Rome, Italy, Telephone: 39-06-4991-4932, Fax: 39-06-4991-4653, E-mails: Federica.Santolamazza@uniroma1.it and ale.dellatorre@uniroma1.it. Adalgisa Caccone Yale Institute for Biospheric Studies-Ecosave Molecular Systematics and Conservation Genetics Laboratory, Yale University, 21 Sachem Street, New Haven, CT 06520, Telephone: 203-432-5259 Fax: 203-432-7394, E-mail: adalgisa.caccone@yale.edu.

Acknowledgments: We thank all the scientists who made this study possible by providing us with field samples, Melissa Garren for laboratory assistance, Mario Coluzzi for stimulating the development of the method, and Jeffrey Powell for financial advice and support. We also thank two anonymous reviewers for their useful comments.

Financial support: Federica Santolamazza and Alessandra Della Torre were supported by the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (TDR) and by MIUR/COFIN grants. Federica Santolamazza was supported by RO1AI-46018 to Jeffrey Powell while at Yale Univesity.

REFERENCES

  • 1

    Coluzzi M, Sabatini A, Petrarca V, di Deco MA, 1979. Chromosomal differentiation and adaptation to human environment in the Anopheles gambiae complex. Trans R Soc Trop Med Hyg 73 :483–497.

    • Search Google Scholar
    • Export Citation
  • 2

    Coluzzi M, Petrarca V, di Deco MA, 1985. Chromosomal inversion intergradation and incipient speciation in Anopheles gambiae.Boll Zool 52 :45–63.

    • Search Google Scholar
    • Export Citation
  • 3

    Coluzzi M, Sabatini A, della Torre A, Di Deco MA, Petrarca V, 2002. The polytene chromosome map of Anopheles gambiae: cytotaxonomic findings, patterns of rearrangements, evolutionary and malariological implications. Science 298 :1415–1418.

    • Search Google Scholar
    • Export Citation
  • 4

    Touré YT, Petrarca V, Traoré SF, Coulibaly A, Maiga HM, Sankaré O, Sow M, Di Deco MA, Coluzzi M, 1994. Ecological genetic studies in the chromosomal form Mopti of Anopheles gambiae s.s. in Mali, west Africa. Genetica 94 :213–223.

    • Search Google Scholar
    • Export Citation
  • 5

    Touré YT, Petrarca V, Traoré SF, Coulibaly A, Maiga HM, Sankaré O, Sow M, Di Deco MA, Coluzzi M, 1998. Distribution and inversion polymorphism of chromosomally recognized taxa of the Anopheles gambiae complex in Mali, west Africa. Parassitologia 40 :477–511.

    • Search Google Scholar
    • Export Citation
  • 6

    Powell JR, Petrarca V, della Torre A, Caccone A, Coluzzi M, 1999. Population structure, speciation, and introgression in the Anopheles gambiae complex.Parassitologia 41 :101–114.

    • Search Google Scholar
    • Export Citation
  • 7

    Bryan JH, Di Deco MA, Petrarca V, Coluzzi M, 1982. Inversion polymorphism and incipient speciation in Anopheles gambiae s. s. in The Gambia, west Africa. Genetica 59 :167–176.

    • Search Google Scholar
    • Export Citation
  • 8

    Mukabayire O, Touré YT, Coluzzi M, Besansky NJ, 2001. Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA, white and tryptophan oxygenase genes. Insect Mol Biol 10 :33–46.

    • Search Google Scholar
    • Export Citation
  • 9

    della Torre A, Fanello C, Akogbeto M, Dossouvovo J, Favia G, Petrarca V, Coluzzi. M, 2001. Cytogenetic and molecular evidence of incipient speciation within Anopheles gambiae s.s. in west Africa. Insect Mol Biol 10 :9–18.

    • Search Google Scholar
    • Export Citation
  • 10

    della Torre A, Costantini C, Besansky NJ, Caccone A, Petrarca V, Powell JR, Coluzzi M, 2002. Incipient speciation within Anopheles gambiae: the glass is half full. Science 298 :115–117.

    • Search Google Scholar
    • Export Citation
  • 11

    Favia G, Dimopoulos G, della Torre A, Touré YT, Coluzzi M, Louis C, 1994. Polymorphisms detected by random PCR distinguishes between different chromosomal forms of Anopheles gambiae.Proc Natl Acad Sci USA 91 :10315–10319.

    • Search Google Scholar
    • Export Citation
  • 12

    Favia G, della Torre A, Bagayoko M, Lanfrancotti A, Sagnon N’F, Touré YT, Coluzzi M, 1997. Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation. Insect Mol Biol 6 :377–383.

    • Search Google Scholar
    • Export Citation
  • 13

    Favia G, Lanfrancotti A, Spanos L, Siden-Kiamos I, Louis C, 2001. Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s. Insect Mol Biol 10 :19–23.

    • Search Google Scholar
    • Export Citation
  • 14

    Gentile G, Slotman M, Ketmaier V, Powell JR, Caccone A, 2001. Attempts to molecularly distinguish cryptic taxa in Anopheles gambiae s.s. Insect Mol Biol 10 :25–32.

    • Search Google Scholar
    • Export Citation
  • 15

    Gentile G, della Torre A, Maegga B, Powell JR, Caccone A, 2002. Genetic differentiation in the African malaria vector, Anopheles gambiae s.s., and the problem of taxonomic status. Genetics 161: 1561–1578.

    • Search Google Scholar
    • Export Citation
  • 16

    Weill M, Chandre F, Brengues C, Manguin S, Akogbeto M, Pasteur N, Guillet P, Raymond M, 2000. The kdr mutation occurs in the Mopti form of Anopheles gambiae s.s. through introgression. Insect Mol Biol 9 :451–455.

    • Search Google Scholar
    • Export Citation
  • 17

    Gentile G, Santolamazza F, Fanello C, Petrarca V, Caccone A, della Torre A, 2004. Variation in an intron sequence of the voltage-gated sodium channel gene correlates with genetic differentiation between Anopheles gambiae s.s. molecular forms. Insect Mol Biol 13 : (in press).

    • Search Google Scholar
    • Export Citation
  • 18

    Edillo FE, Touré YT, Lanzaro GC, Dolo G, Taylor CE, 2002. Spatial and habitat distribution of Anopheles gambiae and Anopheles arabiensis (Diptera:Culicidae) in Banambani village, Mali. J Med Entomol 39 :70–77.

    • Search Google Scholar
    • Export Citation
  • 19

    Scott JA, Brogdon WG, Collins FH, 1993. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg 49 :520–529.

    • Search Google Scholar
    • Export Citation
  • 20

    Fanello C, Santolamazza F, della Torre A, 2002. Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP. Med Vet Entomol 16 :461–466.

    • Search Google Scholar
    • Export Citation
  • 21

    Coetzee M, Craig M, le Sueur D, 2000. Distribution of African malaria mosquitoes belonging to the Anopheles gambiae complex. Parasitol Today 16 :74–77.

    • Search Google Scholar
    • Export Citation
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