• View in gallery

    Genetic map of autosomal markers used to follow the fate of introgressed chromosomal regions between Anopheles gambiae and An. arabiensis. The genetic distances are based on GA × G backcross individuals. The X chromosome is not represented due to a lack of recombination on this chromosome in hybrids. Inversions in which markers are located are indicated in parentheses.

  • View in gallery View in gallery

    Frequency of Anopheles arabiensis alleles of microsatellite markers after introgression into An. gambiae in subsequent generations. The solid horizontal lines represent the expected frequencies of autosomal alleles under neutrality. Note that for the autosomes, the markers listed from left to right correspond to the 2R → 2L, and 3R → 3L.

  • View in gallery

    Frequency of Anopheles gambiae alleles of microsatellite markers after introgression into An. arabiensis in the F2 and F3 generation of the GA × A cross. Designations are as in Figure 2.

  • 1

    Coluzzi M, Sabatini A, Petrarca V, Di Deco MA, 1979. Chromosomal differentiation and adaptation to human environments 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

    Davidson G, 1962. The Anopheles gambiae complex. Nature 196 :907.

  • 4

    Davidson G, 1964. The five mating types of the Anopheles gambiae complex. Riv Malariol 13 :167–183.

  • 5

    Hunt RH, Coetzee M, Fettene M, 1998. The Anopheles gambiae complex: a new species from Ethiopia. Trans R Soc Trop Med Hyg 92 :231–235.

  • 6

    White GB, 1974. The Anopheles gambiae complex and disease transmission in Africa. Trans R Soc Trop Med Hyg 68 :278–302.

  • 7

    Coluzzi M, Sabatini A, 1967. Cytogenetic observations on species A and B of the Anopheles gambiae complex. Parassitologia 9 :73–88.

  • 8

    Coluzzi M, Sabatini A, 1968. Cytogenetic observations on species C of the Anopheles gambiae complex. Parassitologia 10 :155–166.

  • 9

    Coluzzi M, Sabatini A, 1969. Cytogenetic observations on the salt water species, Anopheles merus and Anopheles melas, of the gambiae complex. Parassitologia 11 :177–187.

    • Search Google Scholar
    • Export Citation
  • 10

    Bullini L, Coluzzi M, 1978. Applied and theoretical significance of electrophoretic studies in mosquitoes (Diptera: Culicidae). Parassitologia 20 :7–21.

    • Search Google Scholar
    • Export Citation
  • 11

    Miles SJ, 1978. Enzyme variation in the Anopheles gambiae Giles group of species (Diptera: Culicidae). Bull Entomol Res 68 :85–96.

  • 12

    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
  • 13

    Marchand RP, 1984. Field observations on swarming and mating in Anopheles gambiae mosquitoes in Tanzania. Neth J Zool 34 :367–387.

  • 14

    Curtis CJ, 1982. The mechanism of hybrid males sterility from crosses in the Anopheles gambiae and Glossina morsitans complexes. Steiner WM, ed. Recent Developments in the Genetics of Disease Vectors. Champaign, IL: Stripes Publishing Company, 290–312.

  • 15

    Slotman M, della Torre A, Powell JR, 2005. Female sterility in hybrids between An. gambiae and An. arabiensis and the causes of Haldane’s rule. Evolution 59 :1016–1026.

    • Search Google Scholar
    • Export Citation
  • 16

    Petrarca V, Beier JC, Onyango F, Koros J, Asiago C, Koech DK, Roberst CR, 1991. Species composition of the Anopheles gambiae complex (Diptera, Culicidae) at two sites in western Kenya. J Med Entomol 28 :307–313.

    • Search Google Scholar
    • Export Citation
  • 17

    Touré YT, Petrarca V, Traoré S, 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
  • 18

    Weill M, Chandre F, Brengues C, Manguin S, Akogbeto M, Pastuer 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
  • 19

    Coates CJ, Jasinskiene N, Miyashiro L, James AA, 1998. Mariner transposition and transformation of the yellow fever mosquito Aedes aegypti. Proc Natl Acad Sci USA 95 :3748–3751.

    • Search Google Scholar
    • Export Citation
  • 20

    Catteruccia F, Nolan T, Loukeris TG, Blass C, Savakis C, Kafatos FC, Crisanti A, 2000. Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature 405 :959–962.

    • Search Google Scholar
    • Export Citation
  • 21

    Brennan JD, Kent M, Dhar R, Fujioka HA, Kumar N, 2000. Anopheles gambiae salivary gland proteins as putative targets for blocking transmission of malaria parasites. Proc Natl Acad Sci USA 97 :13859–13864.

    • Search Google Scholar
    • Export Citation
  • 22

    Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C, Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA, Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Müller H-M, Osta MA, Paskewitz SM, Reichart J-M, Rzhetsky A, Troxler L, Vernick KD, Vlachou D, Volz J, von Mering C, Xu J, Zheng L, Bork P, Kafatos FC, 2002. Immunity-related genes and gene families in Anopheles gambiae. Science 298 :159–165.

    • Search Google Scholar
    • Export Citation
  • 23

    Dimopoulos G, Müller H-M, Levashina EA, Kafatos FC, 2001. Innate immune defense against malaria infection in the mosquito. Curr Opin Immunol 13 :79–88.

    • Search Google Scholar
    • Export Citation
  • 24

    Dimopoulos G, 2003. Insect immunity and its implication in mosquito-malaria interactions. Cell Microbiol 5 :3–14.

  • 25

    Coluzzi M, 1982. Spatial distribution of chromosomal inversions and speciation in anopheline mosquitoes. Barigozzi C, ed. Mechanisms of Speciation. New York: Alan R. Liss, 113–153.

  • 26

    Coluzzi M, Sabatini A, dellaTorre A, Di Deco MA, Petrarca V, 2002. A polytene chromosome analysis of the Anopheles gambiae species complex. Science 298 :1415–1418.

    • Search Google Scholar
    • Export Citation
  • 27

    Caccone A, Min GS, Powell JR, 1998. Multiple origins of cytologically identical chromosome inversions in the Anopheles gambiae complex. Genetics 150 :807–814.

    • Search Google Scholar
    • Export Citation
  • 28

    Garcia BA, Caccone A, Mathiopoulos KD, Powell JR, 1996. Inversion monophyly in African anopheline malaria vectors. Genetics 143 :1313–1320.

    • Search Google Scholar
    • Export Citation
  • 29

    della Torre A, Merzagora L, Powell JR, Coluzzi M, 1997. Selective introgression of paracentric inversions between two sibling species of the Anopheles gambiae complex. Genetics 146 :239–244.

    • Search Google Scholar
    • Export Citation
  • 30

    Mukabayire O, Caridi J, Wang X, Touré YT, Coluzzi M, Besansky NJ, 2001. Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA and single-copy loci. Insect Mol Biol 10 :33–46.

    • Search Google Scholar
    • Export Citation
  • 31

    Besansky NJ, Powell JR, Caccone A, Hamm DM, Scott JM, 1994. Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principle malaria vectors. Proc Natl Acad Sci USA 91 :6885–6888.

    • Search Google Scholar
    • Export Citation
  • 32

    Caccone A, Garcia BA, Powell JR, 1996. Evolution of the mitochondrial DNA control region in the Anopheles gambiae complex. Insect Mol Biol 5 :51–59.

    • Search Google Scholar
    • Export Citation
  • 33

    Besansky NJ, Krzywinski J, Lehmann T, Simard F, Kern M, Mukabayire O, Fontenille D, Touré Y, Sagnon N’F, 2003. Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: Evidence from multilocus DNA sequence variation. Proc Natl Acad Sci USA 100 :10818–10823.

    • Search Google Scholar
    • Export Citation
  • 34

    Slotman M, della Torre A, Powell JR, 2004. The genetics of inviability and male sterility in hybrids between Anopheles gambiae and An. arabiensis. Genetics 167 :275–287.

    • Search Google Scholar
    • Export Citation
  • 35

    Alstadt D, 1998 Populus version 4.3. Available at http://www.cbs.umn.edu/populus.

  • 36

    Zheng LB, Benedict MO, Cornel AJ, Collins FH, Kafatos FC, 1996. An integrated genetic map of the African human malaria vector mosquito Anopheles gambiae. Genetics 143 :941–952.

    • Search Google Scholar
    • Export Citation
  • 37

    Collins FH, Paskewitz SM, Finnerty V, 1989. Ribosomal RNA genes of the Anopheles gambiae complex. Harris KF ed. Advances in Disease Vector Research. New York: Springer-Verlag, 1–28.

  • 38

    Curtis CJ, Chalkey J, 1979. Lack of recombination between the X chromosomes of different members of the Anopheles gambiae complex. Heredity 42 :323–326.

    • Search Google Scholar
    • Export Citation
  • 39

    Schneider S, Roessli D, Excoffier L, 2000. ARLEQUIN, Version 2000: A Software for Population Genetic Data Analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva.

  • 40

    Guo SW, Thompson EA, 1992. A Monte-Carlo method for combined segregation and linkage analysis. Am J Hum Genet 51 :1111–1126.

  • 41

    Slatkin M, Excoffier L, 1996. Testing for linkage disequilibrium in genotypic data using the expectation-maximization algorithm. Heredity 76 :377–383.

    • Search Google Scholar
    • Export Citation
  • 42

    Rieseberg LH, Whitton J, Gardner K, 1999. Hybrid zones and the genetic architecture of a barrier to gene flow between two sunflower species. Genetics 152 :713–727.

    • Search Google Scholar
    • Export Citation
  • 43

    Noor MAF, Grams KL, Bertucci LA, Reiland J, 2001. Chromosomal inversions and the reproductive isolation of species. Proc Natl Acad Sci USA 98 :12084–12088.

    • Search Google Scholar
    • Export Citation
  • 44

    Wang RL, Wakeley J, Hey J, 1997. Gene flow and natural selection in the origin of Drosophila pseudoobscura and close relatives. Genetics 147 :1091–1106.

    • Search Google Scholar
    • Export Citation
  • 45

    Machado CA, Kliman RM, Markert JA, Hey J, 2002. Inferring the history of speciation from multilocus DNA sequence data: The case of Drosophila pseudoobscura and close relatives. Mol Biol Evol 19 :472–488.

    • Search Google Scholar
    • Export Citation
  • 46

    Wu C-I, 2001. The genic view of speciation. J Evol Biol 14 :851–865.

  • 47

    Ting CT, Tsaur SC, Wu CI, 2000. The phyglogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus. Proc Natl Acad SciUSA 97 :5313–5316.

    • Search Google Scholar
    • Export Citation
  • 48

    Krzywinski J, Besansky NJ, 2003. Molecular systematics of Anopheles: from subgenera to subpopulations. Annu Rev Entomol 48 :111–139.

  • 49

    Lanzaro GC, Touré YT, Carnahan J, Zheng LB, Dolo G, Traoré S, Petrarca V, Vernick KD, Taylor CE, 1998. Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by microsatellite DNA analysis. Proc Natl Acad Sci USA 95 :14260–14265.

    • Search Google Scholar
    • Export Citation
  • 50

    Taylor C, Touré YT, Carnahan J, Norris DE, Dolo G, Traoré SF, Edillo FE, Lanzaro GC, 2001. Gene flow among populations of the malaria vector, Anopheles gambiae, in Mali, west Africa. Genetics 157 :743–750.

    • Search Google Scholar
    • Export Citation
  • 51

    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
  • 52

    Wang R, Zheng L, Touré YT, Dandekar T, Kafatos FC, 2001. When genetic distance matters: measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species. Proc Natl Acad Sci USA 98 :10769–10774.

    • Search Google Scholar
    • Export Citation
  • 53

    Lehmann TM, Licht M, Elissa N, Maega BTA, Chimumbwa JM, Watsenga FT, Wondji CS, Simard F, Hawley WA, 2003. Population structure of Anopheles gambiae in Africa. J Hered 94 :133–147.

    • Search Google Scholar
    • Export Citation
  • 54

    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
  • 55

    Diabaté A, Baldet T, Chandre F, Dabire KR, Simard F, Ouedraogo JB, Guillet P, Hougard JM, 2004. First report of a kdr mutation in Anopheles arabiensis from Burkina Faso, west Africa. J Am Mosq Control Assoc 20 :195–196.

    • Search Google Scholar
    • Export Citation
  • 56

    Diabate A, Brengues C, Baldet T, Dabiré KR, Hougard JM, Akogbeto M, Kengne P, Simard F, Guillet P, Hemingway J, Chandre F, 2004. The spread of the Lue-Phe kdr mutation through Anopheles gambiae complex in Burkina Faso: genetic introgression and de novo phenomena. Trop Med Int Health 9 :1267–1273.

    • Search Google Scholar
    • Export Citation
  • 57

    Stump AD, Atieli FK, Vulule JM, Besansky NJ, 2004. Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide treated bed net trails. Am J Trop Med Hyg 70 :591–596.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

DIFFERENTIAL INTROGRESSION OF CHROMSOMAL REGIONS BETWEEN ANOPHELES GAMBIAE AND AN. ARABIENSIS

View More View Less
  • 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut; Istituto di Parassitologia, Fondazione Pastuer-Cenci Bolognetti, Università di Roma La Sapienza, Rome, Italy

Evidence for introgression between Anopheles gambiae and An. arabiensis has accumulated for some time. We examined the fate of introgressed DNA directly, using microsatellite markers located throughout the genome. Introgressed X chromosomes were removed within two generations. Furthermore, substantial differences in introgressive capacity between the two autosomes were found. After introgression from An. arabiensis into An. gambiae, most introgressed alleles at third chromosome markers, particularly those on 3R, decreased steadily, indicating selection against them. No such pattern was observed for 2L markers and several 2R markers. The frequency of introgressed alleles on 2L were close to the original frequency even after 19 generations, whereas only two 2R markers showed a modest decrease. Even though limited information was available on the reciprocal cross, the pattern appears to be identical. Although the decrease in frequency of the introgressed X chromosome can be attributed to the presence of sterility and inviability effects, the variation in introgressive capacity of the autosomes does not appear to be explained by the presence of inversion polymorphisms, or regions causing hybrid sterility and inviability. These results can have some important implications for the spread of insecticide resistance and the control of these vector populations via the release of transgenic mosquitoes.

INTRODUCTION

The two most important vectors of malaria in sub-Saharan Africa, Anopheles gambiae and An. arabiensis, belong to a group of closely related sibling species known as the An. gambiae complex.1,2 The members of this complex were originally considered a single species because they cannot be distinguished on the basis of their morphology. When crosses between several strains showed the presence of hybrid male sterility, it was realized that several reproductively isolated taxa exist.36 Their genetic distinctness was subsequently confirmed by polytene chromosome analysis,79 allozyme analysis,10,11 and the development of a species-specific diagnostic polymerase chain reaction (PCR).12 Currently, seven species with varying vectorial capacity are recognized within the complex.

Both An. gambiae and An. arabiensis are widespread in sub-Saharan Africa. They are both antropophilic, have a similar ecology, and are sympatric over much of their range. They even have been observed in the same mating swarms.13 Hybrid males are typically completely sterile, although a very small number of males with very low fertility can be found in hybrids produced by An. arabiensis females and An. gambiae males.14 Female F1 hybrids are fertile, but a substantial proportion of backcross females are partially or completely sterile.15 In addition to these types of post-mating isolation, strong pre-mating barriers exists, and hybrids are only found at very low frequency (< 0.1%).1,16,17 Therefore, the two species are separated by incomplete pre-mating and post-mating isolation mechanisms, allowing for the possibility of DNA exchange; i.e., introgression.

The possibility of introgression of genetic material between these two species has some important implications. Both species are a major threat to human health and economic potential in large areas of Africa, where they are subjected to continuous control programs. Currently, and in the conceivable future, insecticide use is an essential part of these programs, and the spread of insecticide resistance poses a serious threat to their success. At least one allele imparting insecticide resistance, knock down resistance (kdr), has been passed between different chromosomal forms of An. gambiae s.s. via introgression.18 Understanding the capacity of the various chromosomal regions to introgress between An. gambiae and An. arabiensis, is crucial for anticipating the spread of insecticide resistance genes such as kdr.

Additionally, much effort has been devoted to the development of transgenic mosquitoes,19,20 and identifying refractory genes.2124 The eventual goal of these efforts is the release of refractory transgenic mosquitoes as part of malaria control programs. Information about the introgressive ability of genomic regions in which genes are introduced will be needed for predicting their spread through An. gambiae and An. arabiensis populations.

Coluzzi and others have long advocated the importance of introgression in the evolutionary history of An. gambiae and An. arabiensis.1,25,26 They have shown that the frequency of two chromosomal inversions, the 2Rb and 2La, on the right and left arms of the second chromosome, respectively, is correlated with dry savanna environment. These investigators suggested that these inversions have originated in An. arabiensis and subsequently spread into An. gambiae through introgressive hybridization. This would have allowed an originally forest-adapted An. gambiae to extend its range into the drier savanna habitat. The effects on the transmission of human malaria by such a range expansion of the antropophilic An. gambiae presumably would have been large.1

The An. gambiae complex is characterized by the presence of numerous fixed and polymorphic chromosomal inversions. Coluzzi and others1,2 proposed a phylogeny based on the presence of shared inversions, assuming each inversion has a single origin; i.e., they are monophyletic. Anopheles gambiae and An. merus are both fixed for Xag, a large inversion covering two-thirds of the X chromosome, which suggests they are sister taxa. Although there is evidence that at least one inversion may have originated twice in this species complex,27 monophyly for the Xag inversion has been confirmed.28 However, An. gambiae and An. arabiensis share several second chromosomal inversions, which is hard to reconcile with the clustering based on the Xag inversion, unless these shared inversion polymorphisms are ancestral or introgression has occurred between these two species.1

Introgression of these shared second chromosome inversions has been demonstrated in a laboratory experiment.29 Inversions that are shared naturally between these species introgress readily in the laboratory, whereas non-shared inversions are removed after a few generations. Sequence data of genes inside the 2Rb have confirmed the introgression of this inversion in nature.30 Other evidence of introgression between these species is provided by molecular data from mitochondrial DNA31,32 and the 2La inversion.27 Furthermore, a recent study indicates that introgression may have occurred on the third chromosome.33

Here we present the results of a study into the capacity of different genomic regions to introgress between An. gambiae and An. arabiensis in a laboratory colony. The fate of 18 microsatellite markers distributed across the genome is followed, when introgressed from An. arabiensis into An. gambiae. This extends the work of della Torre and others29 to genomic regions outside of the inversions, as well as to all three chromosomes. Furthermore, we examine if the chromosomal regions containing hybrid sterility and inviability factors we identified previously34 pose a barrier for introgression.

MATERIALS AND METHODS

Crosses and strains.

An An. gambiae (Gasua) colony was started from females collected in Suakoko, Liberia in 1986. Originally, this colony was polymorphic for two chromosomal inversions: 2Rb and 2La. Later, a Gasua strain fixed for the 2La inversion and the standard 2R arrangement (2R+) was selected, and this strain was used in this study. The inversion karyotype for this strain can be described as Xag, 2R+, 2La, 3R+, 3L+. This strain belongs to the Mopti chromosomal form and the M molecular form.

The An. arabiensis (Armor) colony used in this study was started from adult females collected in Moribabougou, Mali in 1996. Armor, like all An. arabiensis, was fixed for the Xbcd and 2La inversions. In addition, it was polymorphic for one inversion on 3R, 3Ra, and three inversions on 2R. Two of these, 2Ra and 2Rb, were linked, creating a 2Rab arrangement. A third polymorphic inversion on 2R, 2Rc, was always linked to 2Rb. The inversion karyotype of Armor can therefore be described as Xbcd, 2Rab/c/+, 2La, 3Ra/+, 3L+.

Colonies were reared using standard methods. Larvae were reared in distilled water with 0.1% marine salt at 28° ± 1°C and fed on dried cat food pellets. Adults were kept at 26° ± 1°C and a relative humidity of 70 ± 5% in 0.125-meter3 cages and fed on a 1% sugar solution. Females were blood fed twice prior to oviposition on guinea pigs. Both larvae and adults were kept at a 12-hour photoperiod.

Gasua females were mated to Armor males to produce an F1 hybrid generation. Males of this generation are mostly or completely sterile, whereas females are generally fertile. The F1 females were mated to either Gasua or Armor males. The cross with Gasua males produced a GA × G backcross, here called the F2, which was used to start our GA × G introgression colony. In this colony, the fate of arabiensis DNA in a predominantly gambiae genetic background was followed. (GA × G)F2 males were mated to (GA × G)F2 females to produce an F3 generation. This process was repeated for subsequent generations until the F20 generation. The cross in the other direction proved to be less successful. Despite several attempts, we did not succeed in maintaining a GA × A introgression colony and for this cross data from an F2 and F3 generation reported previously were used.34

We used Populus35 to perform 1,000 simulations to determine the effective population size (Ne) required in our experimental design. With Ne too small, there would be a substantial probability that an allele is removed by chance. It was found that the critical Ne for this experiment was approximately 60. At this Ne, the probability that a neutral allele, with a starting frequency of 0.25, is removed from the population as the result of drift within 19 generations is 0.05. Since both F2 females and F2 males have significant hybrid sterility, we monitored the number of F2 and F3 females that contributed offspring to the next generation. In these generations, males and females were mated in cages containing approximately 200 individuals of both sexes. After blood feeding, approximately 130 gravid females were placed in single oviposition cups to determine the number of females producing offspring. Of these, 20 F2 females contributed to the F3 generation and 61 F3 females contributed to the F4. Following this generation, the colony was kept in two cages containing approximately 400 individuals each, and females were allowed to perform mass ovipositions.

Molecular markers.

Seventeen microsatellite loci distributed across the two autosomes, as well as two X-linked loci, were used as markers to follow the fate of introgressed chromosomal regions.36 Markers were tested on the two parental strains and only markers that did not share alleles were used. Locus 53 on the X chromosome is located within the Xag inversion. Locus 32J0, primers for which were kindly provided by L. Zheng (Department of Epidemiology and Public Health, Yale University, New Haven, CT), is located outside the Xag inversion. Locus 32J0 was used to analyze the F2. For the F3 and F4, a diagnostic PCR12 was used that distinguishes An gambiae and An. arabiensis ribosomal DNA (rDNA) located outside the Xag inversion.37 However, as reported previously,34,38 we did not detect any recombination between the gambiae and arabiensis X chromosomes. Therefore, for generations following the F4, a single microsatellite marker (53) was used to monitor the frequency of the arabiensis X chromosome (XA). Names of loci used follow those of Zheng and others36 with the prefix AGXH, AG2H, or AG3H removed. A genetic map of the markers is shown in Figure 1. This map was constructed using data from 800 (GA × G)F2 males and females from a previous study.34

Genotypes were obtained for 133–160 F2 males (average for all loci = 157). For the F3, genotypes were obtained for both males and females. The number of male genotypes for each locus ranged from 49 to 57 (average = 54), and the number of female genotypes ranged between 64 and 68 individuals (average = 67). Following the F3, only female genotypes were obtained. The sample size for each locus for all subsequent generations ranged from 108 to 126 (average = 122).

Molecular methods.

Extraction of DNA was conducted using the Easy DNA kit (Invitrogen, Carlsbad, CA). The PCRs were done using AmpliTaq Gold (Applied Biosystems, Foster City, CA) with the following program: 94°C for 12 minutes, 30 cycles at 94°C for 40 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes. One to four loci were amplified in a single PCR. Primers were labeled with fluorescent dye, and PCR products were sequenced on an ABI 373 automated sequencer (Applied Biosystems).

Analyses.

Extraction of genotypic data was performed using Genescan (Applied Biosystems) and Genotyper (Applied Biosystems). No distinction was made between different alleles within our strains, and alleles were coded based on the strain from which they were derived.

Arlequin39 was used to test for deviations from Hardy-Weinberg equilibrium and linkage disequilibrium. The procedure, followed by this program for testing Hardy-Weinberg equilibrium, is as outlined by Guo and Thompson.40 The number of steps in the Markov chain was 100,000 and the number of dememorization steps was 4,000. The presence of linkage disequilibrium was tested using a maximum likelihood estimation41 because the gametic phase of the individuals was not known. The number of permutations performed was 20,000 and the number of initial conditions was 100.

RESULTS

Introgression from An. arabiensis into An. gambiae.

We found marked differences between the three chromosomes in their ability to introgress. For autosomal loci, the expected frequency of arabiensis markers in the F2 was 0.25, whereas for the XA chromosome, the expected frequency was 0.33. For the F2, only males were analyzed. However, in the F2 of a separate, but identical cross, the frequencies of males and females carrying an arabiensis X chromosome (XA) were similar.34 Therefore, although genotypes were only obtained for F2 males in this experiment, the frequency of the XA in this generation was adjusted to represent that of a mixed population (Figure 2). The frequency of the XA is according to Mendelian expectation in the F2. In the F3, the XA frequency decreased dramatically, and only a single individual carrying an XA was found in the F4.

Although an excess of heterozygotes was found for several autosomal loci in the F2 of the GA × G cross, none of the differences were significant (P values ranged between 0.112 and 1 for all loci, by chi-square test). However, arabiensis alleles at several second chromosome loci, most notably 79 and 770, increased in frequency in the F3 and F4, only to decrease to lower frequencies in subsequent generations. These loci are both located on the right arm of the second chromosome (2R). Markers 175 and 79 are located in inversions 2Ra and 2Rb, respectively. These are polymorphic in our arabiensis strain and linked as 2Rab. Based on the result of della Torre and others,29 this inversion is expected to be removed rapidly from the population. However, although the frequency of introgressed alleles at marker 79 decreased in frequency until the F20, this was not the case for marker 175, which remained at the same frequency (~15%) between the F10 and the F20. Although marker 770 is located outside the inversion, the genetic distance between markers 79 and 770 and our linkage disequilibrium analysis indicated that these markers are significantly linked in all generations. However, marker 175, which is also located inside the 2Rab inversion, was at substantially higher frequency than marker 79, indicating that if there was selection against marker 79, it was not solely because of the presence of the 2Rab inversion.

The frequency of arabiensis alleles at loci on 2L appeared to be stable, and after 19 generations, none of the markers had appreciably decreased in frequency. This chromosome arm contains the large 2La inversion, which is fixed in An. arabiensis, but is polymorphic in An. gambiae in nature. Two of the markers on 2L are within the 2La inversion (marker 143 and 787) and both were at high frequencies up to the F20. Marker 675, which is close to the tip of 2L, was also at a high frequency. Linkage disequilibrium analysis indicates that marker 675 was not linked to marker 787 in the F10, although significant linkage was found in the F20. The genetic map (Figure 1) indicates a considerable genetic distance (~18.3 cM) between the two markers. Therefore, linkage disequilibrium due to physical linkage should have been mostly broken down by the F20. The linkage detected in the F20 may have been due to selection on the same alleles at epistatically interacting loci. Additionally, the section of the right arm of the second chromosome closest to the centromere (marker 786) was also at a high frequency throughout the experiment.

The frequencies of third chromosome markers were markedly different from those on the second chromosome. They decreased steadily starting in the F3. By the F20, marker 812 was completely removed, and all other loci on 3R were at low to very low frequencies. The frequency of 3L decreased less than 3R, with loci 127 and 758 more or less stable at frequencies of 9.2% and 9.1%, respectively, in the F20. However, marker 817 disappeared completely by the F20. On the third chromosome, marker 119 is located within the 3Ra inversion. Although we do not know the frequency of this inversion, it was polymorphic, and could not have been responsible for the almost complete disappearance of arabiensis alleles at several 3R markers.

Introgression from gambiae into arabiensis.

Despite repeated attempts, we were not successful in maintaining a GA × A colony. Therefore, our conclusions of introgression from gambiae into arabiensis are very limited, and are based on GA × A backcrosses and a (GA × A)F3 generation that were obtained for a separate study.34 The data for males and females were combined and are shown in Figure 3. Interestingly, the frequency of the autosomal markers is very similar to those in the GA × G introgression colony. In the F2, the frequency of introgressed gambiae alleles is higher than expected under neutrality. This was a backcross generation, and only arabiensis homozygotes or heterozygotes could be present. In the third generation, many of the second chromosome markers were still close to the expected frequency, but the frequency of gambiae alleles at loci 79 and 770 increased substantially. As is the case in the GA × G colony, the frequency of gambiae alleles of loci on the third chromosome decreased substantially in the F3. The XG chromosome also showed a pattern similar to the XA chromosome in the GA × G colony as well. It decreased dramatically in the F3. The lower than expected frequency of the XG in the F2 is due to a recessive inviability effect on the X chromosome that is expressed in a large proportion of these backcross males.34

Hardy-Weinberg equilibrium.

We tested for deviations from Hardy-Weinberg equilibrium to examine if preferential selection against heterozygotes or homozygotes, assortative mating, or a preferential transmission of alleles through one sex because of fertility effects could be detected. We did not observe a significant excess of heterozygotes in the F2 of the GA × G cross. Previously, we reported that this backcross shows highly significant excess of heterozygotes on the second, but not on the third chromosome.34 However, in the F3, several loci on the second chromosome have a significant excess of heterozygotes (Table 1). It is noteworthy that the third chromosome markers show no significant deviation from Hardy-Weinberg equilibrium. In all subsequent generations, only a few instances of either heterozygote or homozygote excess were found. In this cross, selection is acting against most arabiensis third chromosome markers in a gambiae background. The lack of deviation from Hardy-Weinberg equilibrium indicates that this selection is not primarily against either heterozygotes or homozygotes. In the F3 of the GA × A cross, all second and most third chromosome markers showed an excess of heterozygotes (Table 2). This corresponds with the observation that in the F2 of this cross both the second and third chromosomes showed significant heterosis.34

DISCUSSION

Marked differences exist between and within chromosomes in their capacity to introgress from An. arabiensis into An. arabiensis. The introgressed X chromosome is removed almost instantly. Most of the introgressed second chromosome remains at high frequency even after 19 generations, whereas much of the arabiensis third chromosome is mostly removed, albeit at a slower pace. Recently, four genes located on all three chromosomes were examined for evidence of introgression between the species of the An. gambiae complex.33 An X-linked gene examined in that study showed no evidence of introgression after the split of An. gambiae and An. arabiensis, whereas genes on both the second and third chromosomes indicated that introgression had occurred. Interestingly, the gene on the second chromosome provided a much lower Fst value than two genes on the third chromosome. This appears to coincide with our finding that introgression of second chromosome loci is more likely than that of third chromosome loci.

An experiment of this kind is critically dependent on Ne. Due to the high level of sterility in the F2, the number of males and females that contributed to the F3 was small. However, since we observed very similar patterns in the F3 of both backcrosses, we believe that it is unlikely that the immediate decrease in the frequency of foreign third chromosome alleles was due to the small Ne in the F2 generation. Additionally, there was clear trend of a decreasing frequency of these markers in subsequent generations in the GA × G colony. Considering that this colony consisted of approximately 800 individuals and that little hybrid sterility effects were present in subsequent generations, we are confident that this decrease was not an artifact of low Ne.

Inversions that are not shared naturally between An. gambiae and An. arabiensis do not introgress in the laboratory.29 In the present study, we found that the patterns of introgression of various chromosomal segments between An. gambiae and An. arabiensis are to a large extent independent of the presence of inversions. That is, although inversions undoubtedly play a role in determining whether chromosomal regions can introgress between these species, the larger pattern observed extends beyond the presence of inversions.

The accumulation of genetic differences between taxa during speciation results in incompatibilities between the evolving genomes, such that genes introduced into the genetic background of the other species cause low fitness. This could be in many different forms, e.g., sterility, inviability, hybrid courtship dysfunction, or maladaptation to a specific environment, but typically traits related to reproduction are among the first to be affected. In An. gambiae and An. arabiensis, several chromosomal regions have been identified that contribute to hybrid sterility.34 The expectation is that such genes pose a barrier for introgression of linked chromosomal regions.

Several studies have documented the effect of hybrid sterility genes on introgression. Rieseberg and others42 identified 26 chromosomal regions that have strong selection against introgression in hybrid zones of two species of sunflower. Sixteen of these regions were significantly associated with sterile pollen. In Drosophila pseudoobscura and D. persimilis, male hybrid sterility and female species preferences are almost exclusively associated with non-shared inversions.43 Little or no introgression of these inversions occurs between these species, whereas non-inverted regions do introgress.44,45

The X chromosomes of An. gambiae and An. arabiensis have a very large effect on hybrid male sterility.14,34 In almost all cases, backcross males carrying a foreign X chromosome are completely sterile. In addition, the X chromosome of both species plays a large role in hybrid female sterility,15 and inviability effects are associated with the XG.34 The very strong selection against the introgression of the X chromosomes we observed is therefore entirely expected.

We previously identified four quantitative trait loci (QTL) on the arabiensis autosomes that affect male hybrid fertility when heterozyogous in a gambiae background.34 These male sterility QTL are expected to affect the introgression of arabiensis alleles at linked marker loci; however, no clear pattern emerged from our experiment. Two of these male sterility QTL were linked to markers 79 and 787 on 2R and 2L, respectively. A third and fourth QTL were linked to markers 812 and 127, on the 3R and 3L, respectively. In the F20, the frequencies of arabiensis alleles at markers 79 and 812 were very low relative to the other markers on their respective chromosome, whereas the frequencies of arabiensis alleles at markers 787 and 127 were relatively high. This difference cannot be explained by the size of the effect of the QTL. The contribution of markers 79 and 812 to the hybrid male sterility was 4.6% and 4.2%, respectively, whereas that of markers 787 and 127 was 4.2% and 7.9%, respectively.34 Therefore, with the exception of the very large sterility and inviability effects of the X chromosome, the presence of hybrid male sterility loci does not appear to have a decisive effect on the capacity of linked regions to introgress between species, at least in the laboratory.

We also identified two female sterility QTL and two regions containing inviability loci, but these were mostly recessive and only had a significant effect when homozygous.15,34 These mostly recessive effects on female hybrid sterility and hybrid inviability are of less immediate consequence with respect to our experiment. Selection against such recessive genes will be weak, as long as they are at relatively low frequency. This is because at a low frequency, only a small proportion of these alleles will be in a homozygous state.

Other studies showing an association between sterility effects and selection against introgression involved natural populations.42,43 The small population size and limited number of generations in this experiment, compared with a natural population, makes selection relatively ineffective. It is likely that the effect of the autosomal hybrid sterility QTL was not sufficient to remove them from the colony within the duration of our experiment. Presumably, selection eventually prevents the introgression of hybrid sterility genes. However, this study shows that this may be a slow process, during which recombination can disassociate linked loci from the sterility genes. Unless genes are very closely linked to genes responsible for reproductive isolation, they may be able to introgress freely between populations whose reproductive isolation is incomplete.

Several investigators42,43,46 have advocated a view of speciation in which emerging reproductive isolation is a property of small chromosomal regions, rather than the entire genome. For example, in D. mauritiana and D. similans, genes as little as 2 kb removed from a sterility factor show no evidence of reproductive isolation.47 Krzywinski and Besansky48 and Besansky and others33 have argued that the data available for the An. gambiae complex so far match this view.

Coluzzi and others suggested that the adaptation of second chromosome inversions to environmental conditions and their introgression from An. arabiensis into An. gambiae allowed the expansion of An. gambiae into drier savanna habitat.1,2 Introgression of these inversions in the past, accompanied by selective sweeps, may have led to the exchange of other parts of the second chromosome. However, regardless of the role of inversions, it seems likely that the capacity of the second chromosome to introgress between An. gambiae and An. arabiensis is due to introgression of this chromosome in the past. This would have allowed the divergence of other chromosomes, while sharing genetic variation on the second chromosome. Under this scenario, the capacity of different regions to introgress between these species should be similar in both directions. This view is consistent with the data from our GA × A cross. Although these data are limited, they suggest that the pattern of introgression is the same in both directions, at least in the first two generations.

An important question is whether introgression between An. gambiae and An. arabiensis is an ongoing phenomenon. Evidence for introgression between An. gambiae and An. arabiensis is plentiful,1,27,29,30,33 but is mostly based on analysis of shared variation in natural populations. Using these data, it is difficult to establish if introgression is ongoing or an event in the past. Estimates of gene flow between An. gambiae and An. arabiensis based on Fst values obtained from microsatellite analysis have provided Nem estimates ranging from 0.97 for the X chromosome to 1.7 for the second and third chromosomes.49,50 These values are almost certainly too high. Although the lowest gene flow estimate reported was for the X chromosome, our results, as well as the previous observation of della Torre and others,29 indicate that any gene flow of X-linked loci between An. gambiae and An. arabiensis is extremely unlikely. The reported Fst values may be due to factors other than gene flow. For example, as pointed out in both studies, it is possible that the populations of An. gambiae and An. arabiensis have not yet reached a state of equilibrium between drift and migration.49,50 Therefore, the reported Nem for the second and third chromosomes between these two species should also be interpreted with caution. Regardless, substantial Nem values could also be indicative of historic, rather than ongoing, gene flow.

Our results suggest that current introgression on the second chromosome is quite possible, and that selection is acting against introgression on much of the third chromosome. Because removal of third chromosome introgressions may not occur immediately, recombination may disassociate third chromosome loci and allow their introgression. However, based on our results, loci on most of the third chromosome are much less likely to introgress between An. gambiae and An. arabiensis than loci on the second chromosome.

Anopheles gambiae s.s. is now thought to be subdivided into several, at least partially reproductively isolated populations. Several so-called chromosomal forms have been defined, based on the distribution of 2R inversions,17 the most important of which are Mopti, Savanna, and Bamako. An alternative classification of M and S molecular forms exists based on fixed differences in the X-linked rDNA.51 Studies of gene flow between molecular forms have found differentiation between the molecular forms in the area linked to the rDNA,52,53 as well as on the second chromosome.49 However, little differentiation was found between the molecular or chromosomal elsewhere in the genome.30,52,54 Our gambiae strain belongs to the M molecular form, which poses the question to what extend our results can be extrapolated to the S molecular form. Considering the lack of differentiation found between the M and S form in much of the genome, we expect that the S form will show a similar pattern of introgression with An. arabiensis for the X, 2L, and third chromosomes. However, as shown by della Torre and others, introgression of 2R is likely strongly influenced by which specific inversions are present.29

Our experiment was conducted using laboratory strains. Laboratory rearing conditions are different from conditions in nature and selection pressures will not be identical. However, the laboratory strains that we have used have been kept in the laboratory for several years under identical conditions. Both strains have therefore been adapted to similar ecologic parameters. That is, the situation in the laboratory is less complex than that in nature. The pattern of introgression we have identified is therefore likely due to interactions between the two different genomes that affect the constitution of the mosquito in some way. Any such interactions would be very strongly selected against in the natural population as well, and the pattern of introgression observed in this experiment is therefore expected to hold in nature.

Depending on their position in the genome, the introgression of autosomal genes that provide an adaptive advantage when transferred between these species, such as insecticide-resistance genes, may be possible. This could have important implications for the control of these mosquitoes. For example, in An. gambiae, the widespread kdr gene confers resistance against pyrethroids and has been mapped to the left arm of the second chromosome. Our results imply that introgression of kdr into An. arabiensis is not hampered by selection against the chromosomal region in which it is contained, and based on our results introgression of the kdr into An. arabiensis is expected. Recently a single specimen of An. arabiensis carrying kdr was discovered in Burkina Faso,55 but it was subsequently determined that this kdr allele likely arose de novo.56 The kdr allele was also recently found in a single specimen of An. arabiensis in western Kenya.57 It is not known if this kdr allele was acquired from An. gambiae through introgression, or whether it arose independently.

The priority of much current research involving An. gambiae focuses on the development of refractory transgenic mosquitoes with the goal of their future release as part of malaria control programs. Predicting the spread of novel genes introduced into a mosquito population will be crucial for the success of this effort. Understanding the capacity of introgression of different chromosomal regions between these vectors of malaria may assist in the design of a strategy for introducing novel genes.

Table 1

Significance of excess/shortage of heterozygotes in the GA × G population*

MarkerF3F4F6F10F20
* P values were calculated according to Guo and Thompson.40 − = P > 0.05.
(+) = heterozygote excess; (−)= heterozygote shortage.
P < 0.05.
P < 0.01.
§ P < 0.001.
X
53-----
Second chromosome
    417(+)†----
    175(+)‡---(−)‡
    79-----
    770-----
    786(+)‡(+)‡---
    143(+)†----
    787(+)§----
    675(+)†----
Third chromosome
    776-----
    812-----
    249--(−)†--
    119-----
    311-(+)†---
    127-----
    758-----
    102-----
    817-----
Table 2

Significance of excess/shortage of heterozygotes in the GA × G population*

MarkerF3
* P values were calculated according to Guo and Thompson.40 – = P > 0.05.
(+) = heterozygote excess; (−) = heterozygote shortage.
P < 0.05.
P < 0.01.
§ P < 0.001.
X
53
Second chromosome
    417(+)†
    175(+)‡
    79(+)§
    770(+)§
    786(+)§
    143(+)§
    787(+)‡
    675(+)§
Third chromosome
    776(+)†
    812(+)§
    249(+)‡
    119(+)‡
    311(+)‡
    127(−)†
    758
    102(+)†
    817(+)†
Figure 1.
Figure 1.

Genetic map of autosomal markers used to follow the fate of introgressed chromosomal regions between Anopheles gambiae and An. arabiensis. The genetic distances are based on GA × G backcross individuals. The X chromosome is not represented due to a lack of recombination on this chromosome in hybrids. Inversions in which markers are located are indicated in parentheses.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 2; 10.4269/ajtmh.2005.73.326

Figure 2.
Figure 2.
Figure 2.

Frequency of Anopheles arabiensis alleles of microsatellite markers after introgression into An. gambiae in subsequent generations. The solid horizontal lines represent the expected frequencies of autosomal alleles under neutrality. Note that for the autosomes, the markers listed from left to right correspond to the 2R → 2L, and 3R → 3L.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 2; 10.4269/ajtmh.2005.73.326

Figure 3.
Figure 3.

Frequency of Anopheles gambiae alleles of microsatellite markers after introgression into An. arabiensis in the F2 and F3 generation of the GA × A cross. Designations are as in Figure 2.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 2; 10.4269/ajtmh.2005.73.326

*

Address correspondence to Dr. Michel A. Slotman, Department of Entomology, University of California, 1 Shields Avenue, Davis, CA 95616. E-mail: maslotman@ucdavis.edu

Authors’ addresses; Michel A. Slotman, Department of Entomology, University of California, 1 Shields Avenue, Davis CA 95616, Telephone: 530-752-7333, Fax: 530-752-1537, E-mail: maslotman@ucdavis.edu. Alessandra della Torre and Maria Calzetta, Istituto di Parassitologia, Fondazione Pasteur-Cenci Bolognetti, Università di Roma La Sapienza, P.le A. Moro 5, 00185 Rome, Italy. Jeffrey R. Powell, Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect Street, New Haven, CT 06511.

Acknowledgments: We thank M. Coluzzi for general support of the work, and G. Petrangeli for instrumental technical support. We also thank two anonymous reviewers, F. Tripet, and D. A. Elnaiem for constructive comments on the manuscript.

Financial support: This study was supported by National Institutes of Health grant R01 46018. Michel A. Slotman was also supported by the Centers for Disease Control and Prevention Fellowship Training Program inVector-Borne Infectious Disease (T01/CCT122306). Alessandra della Torre was supported by the United Nations Development Program for Research and Training in Tropical Diseases (Tropical Disease Research) and the Ministro dell’Istruzione, Dell’ Università e della Ricerca/COFIN.

REFERENCES

  • 1

    Coluzzi M, Sabatini A, Petrarca V, Di Deco MA, 1979. Chromosomal differentiation and adaptation to human environments 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

    Davidson G, 1962. The Anopheles gambiae complex. Nature 196 :907.

  • 4

    Davidson G, 1964. The five mating types of the Anopheles gambiae complex. Riv Malariol 13 :167–183.

  • 5

    Hunt RH, Coetzee M, Fettene M, 1998. The Anopheles gambiae complex: a new species from Ethiopia. Trans R Soc Trop Med Hyg 92 :231–235.

  • 6

    White GB, 1974. The Anopheles gambiae complex and disease transmission in Africa. Trans R Soc Trop Med Hyg 68 :278–302.

  • 7

    Coluzzi M, Sabatini A, 1967. Cytogenetic observations on species A and B of the Anopheles gambiae complex. Parassitologia 9 :73–88.

  • 8

    Coluzzi M, Sabatini A, 1968. Cytogenetic observations on species C of the Anopheles gambiae complex. Parassitologia 10 :155–166.

  • 9

    Coluzzi M, Sabatini A, 1969. Cytogenetic observations on the salt water species, Anopheles merus and Anopheles melas, of the gambiae complex. Parassitologia 11 :177–187.

    • Search Google Scholar
    • Export Citation
  • 10

    Bullini L, Coluzzi M, 1978. Applied and theoretical significance of electrophoretic studies in mosquitoes (Diptera: Culicidae). Parassitologia 20 :7–21.

    • Search Google Scholar
    • Export Citation
  • 11

    Miles SJ, 1978. Enzyme variation in the Anopheles gambiae Giles group of species (Diptera: Culicidae). Bull Entomol Res 68 :85–96.

  • 12

    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
  • 13

    Marchand RP, 1984. Field observations on swarming and mating in Anopheles gambiae mosquitoes in Tanzania. Neth J Zool 34 :367–387.

  • 14

    Curtis CJ, 1982. The mechanism of hybrid males sterility from crosses in the Anopheles gambiae and Glossina morsitans complexes. Steiner WM, ed. Recent Developments in the Genetics of Disease Vectors. Champaign, IL: Stripes Publishing Company, 290–312.

  • 15

    Slotman M, della Torre A, Powell JR, 2005. Female sterility in hybrids between An. gambiae and An. arabiensis and the causes of Haldane’s rule. Evolution 59 :1016–1026.

    • Search Google Scholar
    • Export Citation
  • 16

    Petrarca V, Beier JC, Onyango F, Koros J, Asiago C, Koech DK, Roberst CR, 1991. Species composition of the Anopheles gambiae complex (Diptera, Culicidae) at two sites in western Kenya. J Med Entomol 28 :307–313.

    • Search Google Scholar
    • Export Citation
  • 17

    Touré YT, Petrarca V, Traoré S, 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
  • 18

    Weill M, Chandre F, Brengues C, Manguin S, Akogbeto M, Pastuer 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
  • 19

    Coates CJ, Jasinskiene N, Miyashiro L, James AA, 1998. Mariner transposition and transformation of the yellow fever mosquito Aedes aegypti. Proc Natl Acad Sci USA 95 :3748–3751.

    • Search Google Scholar
    • Export Citation
  • 20

    Catteruccia F, Nolan T, Loukeris TG, Blass C, Savakis C, Kafatos FC, Crisanti A, 2000. Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature 405 :959–962.

    • Search Google Scholar
    • Export Citation
  • 21

    Brennan JD, Kent M, Dhar R, Fujioka HA, Kumar N, 2000. Anopheles gambiae salivary gland proteins as putative targets for blocking transmission of malaria parasites. Proc Natl Acad Sci USA 97 :13859–13864.

    • Search Google Scholar
    • Export Citation
  • 22

    Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C, Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA, Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Müller H-M, Osta MA, Paskewitz SM, Reichart J-M, Rzhetsky A, Troxler L, Vernick KD, Vlachou D, Volz J, von Mering C, Xu J, Zheng L, Bork P, Kafatos FC, 2002. Immunity-related genes and gene families in Anopheles gambiae. Science 298 :159–165.

    • Search Google Scholar
    • Export Citation
  • 23

    Dimopoulos G, Müller H-M, Levashina EA, Kafatos FC, 2001. Innate immune defense against malaria infection in the mosquito. Curr Opin Immunol 13 :79–88.

    • Search Google Scholar
    • Export Citation
  • 24

    Dimopoulos G, 2003. Insect immunity and its implication in mosquito-malaria interactions. Cell Microbiol 5 :3–14.

  • 25

    Coluzzi M, 1982. Spatial distribution of chromosomal inversions and speciation in anopheline mosquitoes. Barigozzi C, ed. Mechanisms of Speciation. New York: Alan R. Liss, 113–153.

  • 26

    Coluzzi M, Sabatini A, dellaTorre A, Di Deco MA, Petrarca V, 2002. A polytene chromosome analysis of the Anopheles gambiae species complex. Science 298 :1415–1418.

    • Search Google Scholar
    • Export Citation
  • 27

    Caccone A, Min GS, Powell JR, 1998. Multiple origins of cytologically identical chromosome inversions in the Anopheles gambiae complex. Genetics 150 :807–814.

    • Search Google Scholar
    • Export Citation
  • 28

    Garcia BA, Caccone A, Mathiopoulos KD, Powell JR, 1996. Inversion monophyly in African anopheline malaria vectors. Genetics 143 :1313–1320.

    • Search Google Scholar
    • Export Citation
  • 29

    della Torre A, Merzagora L, Powell JR, Coluzzi M, 1997. Selective introgression of paracentric inversions between two sibling species of the Anopheles gambiae complex. Genetics 146 :239–244.

    • Search Google Scholar
    • Export Citation
  • 30

    Mukabayire O, Caridi J, Wang X, Touré YT, Coluzzi M, Besansky NJ, 2001. Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA and single-copy loci. Insect Mol Biol 10 :33–46.

    • Search Google Scholar
    • Export Citation
  • 31

    Besansky NJ, Powell JR, Caccone A, Hamm DM, Scott JM, 1994. Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principle malaria vectors. Proc Natl Acad Sci USA 91 :6885–6888.

    • Search Google Scholar
    • Export Citation
  • 32

    Caccone A, Garcia BA, Powell JR, 1996. Evolution of the mitochondrial DNA control region in the Anopheles gambiae complex. Insect Mol Biol 5 :51–59.

    • Search Google Scholar
    • Export Citation
  • 33

    Besansky NJ, Krzywinski J, Lehmann T, Simard F, Kern M, Mukabayire O, Fontenille D, Touré Y, Sagnon N’F, 2003. Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: Evidence from multilocus DNA sequence variation. Proc Natl Acad Sci USA 100 :10818–10823.

    • Search Google Scholar
    • Export Citation
  • 34

    Slotman M, della Torre A, Powell JR, 2004. The genetics of inviability and male sterility in hybrids between Anopheles gambiae and An. arabiensis. Genetics 167 :275–287.

    • Search Google Scholar
    • Export Citation
  • 35

    Alstadt D, 1998 Populus version 4.3. Available at http://www.cbs.umn.edu/populus.

  • 36

    Zheng LB, Benedict MO, Cornel AJ, Collins FH, Kafatos FC, 1996. An integrated genetic map of the African human malaria vector mosquito Anopheles gambiae. Genetics 143 :941–952.

    • Search Google Scholar
    • Export Citation
  • 37

    Collins FH, Paskewitz SM, Finnerty V, 1989. Ribosomal RNA genes of the Anopheles gambiae complex. Harris KF ed. Advances in Disease Vector Research. New York: Springer-Verlag, 1–28.

  • 38

    Curtis CJ, Chalkey J, 1979. Lack of recombination between the X chromosomes of different members of the Anopheles gambiae complex. Heredity 42 :323–326.

    • Search Google Scholar
    • Export Citation
  • 39

    Schneider S, Roessli D, Excoffier L, 2000. ARLEQUIN, Version 2000: A Software for Population Genetic Data Analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva.

  • 40

    Guo SW, Thompson EA, 1992. A Monte-Carlo method for combined segregation and linkage analysis. Am J Hum Genet 51 :1111–1126.

  • 41

    Slatkin M, Excoffier L, 1996. Testing for linkage disequilibrium in genotypic data using the expectation-maximization algorithm. Heredity 76 :377–383.

    • Search Google Scholar
    • Export Citation
  • 42

    Rieseberg LH, Whitton J, Gardner K, 1999. Hybrid zones and the genetic architecture of a barrier to gene flow between two sunflower species. Genetics 152 :713–727.

    • Search Google Scholar
    • Export Citation
  • 43

    Noor MAF, Grams KL, Bertucci LA, Reiland J, 2001. Chromosomal inversions and the reproductive isolation of species. Proc Natl Acad Sci USA 98 :12084–12088.

    • Search Google Scholar
    • Export Citation
  • 44

    Wang RL, Wakeley J, Hey J, 1997. Gene flow and natural selection in the origin of Drosophila pseudoobscura and close relatives. Genetics 147 :1091–1106.

    • Search Google Scholar
    • Export Citation
  • 45

    Machado CA, Kliman RM, Markert JA, Hey J, 2002. Inferring the history of speciation from multilocus DNA sequence data: The case of Drosophila pseudoobscura and close relatives. Mol Biol Evol 19 :472–488.

    • Search Google Scholar
    • Export Citation
  • 46

    Wu C-I, 2001. The genic view of speciation. J Evol Biol 14 :851–865.

  • 47

    Ting CT, Tsaur SC, Wu CI, 2000. The phyglogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus. Proc Natl Acad SciUSA 97 :5313–5316.

    • Search Google Scholar
    • Export Citation
  • 48

    Krzywinski J, Besansky NJ, 2003. Molecular systematics of Anopheles: from subgenera to subpopulations. Annu Rev Entomol 48 :111–139.

  • 49

    Lanzaro GC, Touré YT, Carnahan J, Zheng LB, Dolo G, Traoré S, Petrarca V, Vernick KD, Taylor CE, 1998. Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by microsatellite DNA analysis. Proc Natl Acad Sci USA 95 :14260–14265.

    • Search Google Scholar
    • Export Citation
  • 50

    Taylor C, Touré YT, Carnahan J, Norris DE, Dolo G, Traoré SF, Edillo FE, Lanzaro GC, 2001. Gene flow among populations of the malaria vector, Anopheles gambiae, in Mali, west Africa. Genetics 157 :743–750.

    • Search Google Scholar
    • Export Citation
  • 51

    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
  • 52

    Wang R, Zheng L, Touré YT, Dandekar T, Kafatos FC, 2001. When genetic distance matters: measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species. Proc Natl Acad Sci USA 98 :10769–10774.

    • Search Google Scholar
    • Export Citation
  • 53

    Lehmann TM, Licht M, Elissa N, Maega BTA, Chimumbwa JM, Watsenga FT, Wondji CS, Simard F, Hawley WA, 2003. Population structure of Anopheles gambiae in Africa. J Hered 94 :133–147.

    • Search Google Scholar
    • Export Citation
  • 54

    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
  • 55

    Diabaté A, Baldet T, Chandre F, Dabire KR, Simard F, Ouedraogo JB, Guillet P, Hougard JM, 2004. First report of a kdr mutation in Anopheles arabiensis from Burkina Faso, west Africa. J Am Mosq Control Assoc 20 :195–196.

    • Search Google Scholar
    • Export Citation
  • 56

    Diabate A, Brengues C, Baldet T, Dabiré KR, Hougard JM, Akogbeto M, Kengne P, Simard F, Guillet P, Hemingway J, Chandre F, 2004. The spread of the Lue-Phe kdr mutation through Anopheles gambiae complex in Burkina Faso: genetic introgression and de novo phenomena. Trop Med Int Health 9 :1267–1273.

    • Search Google Scholar
    • Export Citation
  • 57

    Stump AD, Atieli FK, Vulule JM, Besansky NJ, 2004. Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide treated bed net trails. Am J Trop Med Hyg 70 :591–596.

    • Search Google Scholar
    • Export Citation
Save