(27 ). In the beetle Tribolium castaneum,the three genes have a similar expressionpattern but pb expression is not diminishedin a Dfd mutant, suggesting that Dfd is notnecessary to activate pb in this species (28).This probably reflects a difference in pbregulatory sequences, as the Tribolium De-formed protein can activate Drosophila pbwhen it is expressed in Drosophila (29). Inthe bug Oncopeltus fasciatus, Dfd cannotactivate pb because pb is not even presentin the maxillae (30). Additionally, althoughpb and Scr are coexpressed in the labialappendages, RNA interference analysissuggests that Scr does not activate pb (31).Lastly, even in the Drosophila adult theregulatory hierarchy appears to be differentfrom that in the Drosophila embryo; Scrdoes not activate pb in adults, but rather pbis necessary to activate Scr (32). Thus, wehave three insect species and four differentregulatory systems to control the expres-sion of proboscipedia. Considering the mil-lions of different insect species, these re-sults suggest enormous diversity in the reg-ulation of this, and other, developmentalgenes.

The completed sequences of the Dro-

sophila and Anopheles genomes and theprospective sequencing of the Apis andAedes genomes will provide significant in-sights into the insects and their develop-ment, behavior, and evolution (Fig. 1). Butthese four species represent only the begin-ning of an analysis of the Insecta, much lessof the whole of the Arthropoda. Next wemight consider sequencing genomes of rep-resentatives from the Coleoptera and Lepi-doptera. These two insect orders containmany of our most serious agricultural pestsand, together with the Diptera and Hyme-noptera, comprise the “Big Four” insectorders that have evolved “complete” (holo-metabolous) development. The scientificcommunity is now blessed with a wealth ofsequencing capacity. Given the obvious im-portance of insects to our well-being andexistence, it is important that some of it beused to build a strong empirical foundationfor comparative insect genomics.

References1. R. A. Holt et al., Science 298, 129 (2002).2. M. D. Adams et al., Science 287, 2185 (2000).3. S. L. Hoffman, G. M. Subramanian, F. H. Collins, J. C.

Venter, Nature 415, 702 (2002).4. World Health Organization, The World Health Report

2000, Health Systems: Improving Performance (WorldHealth Organization, Geneva, 2000).

5. B. J. Beaty, Proc. Natl. Acad. Sci. U.S.A. 97, 10295(2000).

6. K. S. Aultman et al., Trends Parasitol. 17, 507 (2001).7. F. Catteruccia et al., Nature 405, 959 (2000).8. M. L. Allen et al., J. Med. Entomol. 38, 701 (2001).9. Z. N. Adelman et al., Mol. Biochem. Parasitol. 121, 1

(2002).10. J. Ito et al., Nature 417, 452 (2002).11. D. W. Severson et al., Annu. Rev. Entomol. 46, 183

(2001).12. K. S. Rai et al., Adv. Genet. 41, 1 (1999).13. I. V. Sharakhov et al., Science 298, 182 (2002).14. E. M. Zdobnov et al., Science 298, 149 (2002).15. J. R. Anderson et al., Mol. Phylogenet. Evol. 20, 316

(2001).16. D. W. Severson, personal communication.17. E. Pennisi, Science 296, 1589 (2002).18. G. E. Robinson, Trends Ecol. Evol. 14, 202 (1999).19. ���� , Science 297, 204 (2002).20. B. P. Berman et al., Proc. Natl. Acad. Sci. U.S.A. 99,

757 (2002).21. G. G. Loots et al., Science 288, 136 (2000).22. K. Ishihara et al., Genome Res. 10, 664 (2000).23. D. A. Papatsenko et al., Genome Res. 12, 470 (2002).24. R. C Brusca, G. J. Brusca, Invertebrates (Sinauer Asso-

ciates, Sunderland, MA, 1990).25. W. C. Wheeler et al., Cladistics 17, 113 (2001).26. E. Abouheif, G. A. Wray, Science 297, 249 (2002).27. D. B. Rusch, T. C. Kaufman, Genetics 156, 183 (2000).28. M. DeCamillis et al., Genetics 159, 1643 (2001).29. S. J. Brown et al., Dev. Genes Evol. 209, 389 (1999).30. B. T. Rogers et al., Evol. Dev. 4, 1 (2002).31. C. L. Hughes et al., Development 127, 3683 (2000).32. A. Abzhanov et al., Development 128, 2803 (2001).

V I E W P O I N T

Speciation Within Anopheles gambiae—the Glass Is Half Full

A. della Torre,1* C. Costantini,1* N. J. Besansky,3 A. Caccone,4 V. Petrarca,2 J. R. Powell,4 M. Coluzzi1

Restrictions to gene flow among molecular forms of the mosquito Anoph-eles gambiae sensu stricto reveal an ongoing speciation process affectingthe epidemiology of malaria in sub-Saharan Africa.

The most important vector of the malariaparasite in sub-Saharan Africa is the mosqui-to Anopheles gambiae sensu stricto (s.s.). Itbelongs to a group of sibling species—known as the A. gambiae complex—that aremorphologically indistinguishable but exhibitdistinct genetic and eco-ethological differ-ences reflected in their ability to transmitmalaria. Anopheles gambiae s.s. shows ex-treme genetic heterogeneity, revealed notonly by the traditional study of chromosomalinversions (1) but also by recent studies of

molecular markers such as X-linked ribosom-al DNA (rDNA). So far, extensive molecularanalyses have attempted to distinguish thenumber of isolated or semi-isolated geneticunits of A. gambiae s.s. that exist and whetherthese are evolving into separate species (spe-ciation). Elucidating the genetic populationstructure of the A. gambiae s.s. complex is aprerequisite for determining which geneticunits of the complex are the vectors of ma-laria, and unraveling the ecological and etho-logical differences that are relevant to diseasetransmission. Such knowledge will improveour understanding of malaria epidemiologyand will help in implementing appropriatevector control strategies.

Genotyping X-linked rDNA of A. gam-biae s.s. has led to the characterization of twomolecular forms (M and S) that differ in boththe transcribed and nontranscribed spacers inthe rDNA repeat unit (2–4). The relationshipbetween the M and S molecular forms and the

chromosomal forms—defined according tononrandom associations of inversions inchromosome 2 (1)—varies according to theirecological and geographic distribution (Fig.1). In some areas of West Africa (for ex-ample, Mali and Burkina Faso), there is aone-to-one correspondence between the Mmolecular form and the Mopti chromosom-al form. Similarly, the S molecular formalways corresponds to the Savanna or Ba-mako chromosomal form (5). In other areasof West Africa, this clear correspondencebreaks down (2). For example, in popula-tions inhabiting forests or humid savannas,both molecular forms are characterized byhigh frequencies of the standard arrange-ment in chromosome 2 indicative of theForest chromosomal form. Within the Sform, a small proportion show ambiguouscytological configurations, indicating thepresence of chromosome 2 arrangementstypical of chromosomal forms other thanSavanna and Bamako. Outside Mali andBurkina Faso, the M form may exhibitchromosomal arrangements typical of theBissau, Savanna, or Forest forms.

1Parasitology Unit, Department of Public Health, 2De-partment of Genetics and Molecular Biology, Univer-sity of Rome “La Sapienza,” P.le Aldo Moro 5, 00185Rome, Italy. 3Department of Biological Sciences, Uni-versity of Notre Dame, Notre Dame, IN 46556, USA.4Department of Ecology and Evolutionary Biology,Yale University, New Haven, CT 06520, USA.

*To whom correspondence should be addressed.E-mail: ale.dellatorre@uniroma1.it; carlo.costantini@uniroma1.it

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The importance of chromosomal inver-sions in ecological adaptation has been wellestablished [reviewed in (6)], suggesting thatthe different chromosomal forms are indica-tors of adaptation to different ecological hab-itats. In contrast, the distinct M and S molec-ular forms reflect barriers to gene flow indic-ative of incipient speciation (2). Extensiveanalyses of DNA regions other than rDNAinitially failed to show consistent sequencedifferences corresponding to the M and Smolecular forms (4, 7). However, recentDNA-based data are emerging in support ofthe M and S distinctions. For example, al-though most microsatellite loci show similarallelic frequencies in M and S forms, differ-ences in allelic frequencies indicate restrictedgene flow in both Mali (8) and Cameroon (9).Two microsatellite loci near the centromericregion of the X chromosome exhibit verydistinct differences between M and S formsin Mali (10). The kdr allele in the parasodium channel gene, which confers resis-tance to pyrethroid insecticides, is found in Sform populations from several West Africancountries; it could not be detected in M formpopulations from the same locales (2, 11, 12),with the single exception of Benin (13).Sequence analysis of intron I upstream of the

kdr mutation has shown that S and M popu-lations across West Africa are consistentlydifferent at one nucleotide (14–16). Further-more, rDNA analysis in the closely relatedsibling species A. arabiensis would argueagainst the hypothesis that rDNA differencesbetween the molecular forms of A. gambiaeare due to unusual evolutionary dynamics(for example, very rapid concerted evolution)(17).

Although interbreeding between M andS forms yields fertile progeny, M-S hybridsare rarely observed in nature. Where theseforms overlap in time and space, the rate ofheterogamous insemination is �1% (18),clearly demonstrating the existence of apremating barrier, albeit an incomplete one.Thus, both indirect and direct genetic evi-dence indicates incomplete but substantialbarriers to gene flow between different A.gambiae s.s. molecular forms. Does ongo-ing gene flow signify a glass half empty, ordoes a premating barrier offer a glass halffull? The data suggest that we are observ-ing speciation at its very earliest stages,with the persistence of variation sharedbecause of recent common ancestry andwith low levels of gene flow continuing tohomogenize regions of the genome not di-

rectly involved in the speciationprocess. This may explain why arandom selection of nucleotide se-quences reveals no differentiation,in contrast to the recently emergedkdr allele and the more rapidlyevolving microsatellite markersand rDNA. In an attempt to recon-cile differences among data sets,Gentile et al. (17 ) proposed that Sand M forms “may have mosaicgenomes consisting of parts com-pletely differentiated betweenwhich gene flow is barred, where-as other parts of the genome arefree to pass between forms.” Ifcorrect, this suggests that a debateover taxonomic status hinging onabsolute levels of gene flow willlack biological relevance and dis-tract from the main issues: howrestrictions on gene flow affect theecology and behavior of the mo-lecular forms; to what extent andunder what circumstances theyhinder the circulation of traits suchas insecticide resistance or thepossible spread of transgenes; andwhether genetically engineeredmosquitoes should be used in vec-tor control measures (19, 20).

The vectorial potency of A. gam-biae s.s. stems from its strong associ-ation with humans, that is, its prefer-ence for biting humans exacerbatedby its capacity to exploit changes in

its natural habitat induced by Homo sapi-ens. There is evidence (21, 22) that inmarginal arid environments of BurkinaFaso—where the Savanna and Mopti chro-mosomal forms correspond to the S and Mmolecular forms, respectively— these twotaxa contrast significantly in the way theyexploit limiting resources, such as larvalbreeding and adult resting habitats. Eco-ethological differences regarding their de-gree of association with the human domes-tic environment when biting and resting areunder investigation. The M molecular formshows the closest association with the do-mestic environment and larval habitats cre-ated by human activities, whereas the Sform is more frequent in rain-dependenttemporary breeding sites (21, 22). Thisconfirms what was inferred from differen-tial microgeographic distributions for theMopti and Savanna chromosomal forms (1,23, 24 ).

These observations provide us with fur-ther clues to the nature and mechanisms ofthe speciation process. The occupation bythe M form of relatively recent ecologicalniches produced by human-made modifica-tions of the environment in marginal habi-tats has created new opportunities for spe-

Fig. 1. Geographical distribution of the molecular forms (S and M) of Anopheles gambiae sensu strictoand their relation to chromosomal forms. Chromosomal inversions characterizing each chromosomalform (1) are listed first, followed by less frequent arrangements listed in parentheses. Two-color circlesindicate the presence of one or both molecular forms (2, 4, 9, 31, 32) without reference to relativefrequencies; absence of one form refers only to lack of detection in the mosquito samples analyzed, notto its actual absence in the area. Chromosomal forms: FOR, Forest; SAV, Savanna; MOP, Mopti; BAM,Bamako; BIS, Bissau; n.d., karyotype not determined. African countries: 1, Angola; 2, Benin; 3, BurkinaFaso; 4, Cameroon; 5, Cote d’Ivoire; 6, Democratic Republic of the Congo; 7, Guinea; 8, Kenya; 9,Madagascar; 10, Malawi; 11, Mali; 12, Nigeria; 13, Senegal; 14, Tanzania; and 15, the Gambia.

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cialization and the avoidance of intraspe-cific competition. This selective force ispresumably driving the speciation process.It has been proposed that co-adapted chro-mosomal inversions are crucial for estab-lishing populations in marginal habitatsthat could lead to the formation of newspecies, although the inversions per se arenot the cause of the evolution of subsequentbarriers to gene flow (25).

The taxonomic and genetic complexity of A.gambiae s.s. has serious consequences for ma-laria transmission. The ongoing speciation pro-cess leading to the M form has extended thetransmission potential of this vector in spaceand time (23, 24). In dry areas of West Africawhere malaria is hyper- to holoendemic (26),this taxon is able to exploit breeding opportu-nities due to human activities that would other-wise be available only to A. arabiensis; such isthe case in areas of Eastern Africa with a sim-ilar climate (like northern Sudan) where A.gambiae s.s. is absent and malaria is hypo- tomesoendemic (27). Moreover, in dry savannas,the ability of the M form to breed year-round inpermanent human-dependent larval habitats ex-tends the malaria transmission period well be-yond the rainy season, when the S form appar-ently disappears (28). Analogous situations areseen with other Afrotropical malaria mosquitovectors such as A. funestus, which has two WestAfrican chromosomal forms (Folonzo and Kiri-bina) that clearly differ in their degree of con-tact with humans and therefore have quite dif-ferent vectorial potentials (29). It is likely thatin both A. gambiae and A. funestus, chromo-somal inversions allow more specialized andtherefore more efficient exploitation of bothspatial and temporal environmental heterogene-ity. This is expected to have implications forsuch traits as the survival probability of indi-vidual mosquitoes and the stability of vectorpopulations, both important features of malariaepidemiology (30).

The complete genome sequence of A.gambiae will deepen our understanding of theprocess of adaptation and speciation of thisinsect vector. One immediate application ofthis information, already in progress, is thecloning of inversion breakpoints on chromo-some 2. Comparative analysis of the sequenc-es across and surrounding each breakpointwill allow us to identify and study the geneclusters protected by recombination and mayyield clues about the origin of inversions andtheir importance.

The concentration within four closely relat-ed species of the A. gambiae complex (A. gam-biae, A. arabiensis, A. melas, and A. merus) ofseveral inversions along the central and subte-lomeric sections of the 2R chromosomal arm isunlikely to be coincidental. These inversionsmay be associated with genome regions thatencode traits of ecological and behavioral im-portance. The availability of the entire A. gam-biae genome will facilitate polymerase chainreaction–based assays that will complement la-borious karyotyping of semigravid adult fe-males, providing new opportunities for fieldstudies on mosquito ecology and behavior. Along-term goal is gene discovery using a com-plete genome chip. The very recent divergenceof the A. gambiae s.s. molecular forms and thelikelihood that only a few genes are involved inreproductive isolation and ecological diversifi-cation means that the entire A. gambiae genomewill have to be screened in order to identifydifferences in gene sequence and coordinatedgene expression between incipient species.

A. gambiae provides us with an exceptionalopportunity to observe evolution in action, po-tentially operating over the time frame of thethousands of years since humans began to mod-ify the Afrotropical ecosystem (1, 6, 24). Thebuildup of barriers to gene flow during thespeciation process resulting in separation of themolecular forms of A. gambiae can be com-pared to a glass half full. Now, we must fully

elucidate the mechanisms and dynamics of evo-lutionary change in A. gambiae populations—information that will be essential if we are everto control this nefarious insect vector.

References and Notes1. M. Coluzzi et al., Science, in press; published online 3

October 2002 (10.1126/science.1077769).2. A. della Torre et al., Insect Mol. Biol. 10, 9 (2001).3. G. Favia et al., Insect Mol. Biol. 10, 19 (2001).4. G. Gentile et al., Insect Mol. Biol. 10, 25 (2001).5. G. Favia et al., Insect Mol. Biol. 6, 377 (1997).6. J. R. Powell et al., Parassitologia 41, 101 (1999).7. O. Mukabayire et al., Insect Mol. Biol. 10, 33 (2001).8. C. Taylor et al., Genetics 157, 743 (2001).9. C. Wondji et al., Insect Mol. Biol. 11, 11 (2002).

10. R. Wang et al., Proc. Natl. Acad. Sci. U.S.A. 98, 10769(2001).

11. F. Chandre et al., Parassitologia 41, 319 (1999).12. C. Fanello et al., in preparation.13. C. Fanello et al., Trans. R. Soc. Trop. Med. Hyg. 94,

132 (2000).14. M. Weill et al., Insect Mol. Biol. 9, 451 (2000).15. F. Santolamazza et al., Parassitologia 44, 165 (2002).16. In Benin, both the kdr allele and the “S-type” intron

I were acquired by M from S.17. G. Gentile et al., Genetics 161, 1561 (2002).18. F. Tripet et al., Mol. Ecol. 10, 1725 (2001).19. T. W. Scott, W. Takken, B. G. J. Knols, C. Boete,

Science 298, 117 (2002).20. L. Alphey et al., Science 298, 119 (2002).21. N. F. Sagnon et al., Parassitologia 42, 157 (2000).22. C. Costantini et al., unpublished data.23. Y. T. Toure et al., Genetica 94, 213 (1994).24. Y. T. Toure et al., Parassitologia 40, 477 (1998).25. M. Coluzzi, in Mechanisms of Speciation, C. Barigozzi,

Ed. (Liss, New York, 1982), pp. 143–153.26. The degree of malaria endemicity in an area is clas-

sified according to the proportion of children with apalpably enlarged spleen. Increasing levels of ende-micity are expressed as hypoendemic (0 to 10%),mesoendemic (11 to 50%), hyperendemic (�50%),or holoendemic (�75%).

27. A. R. Zahar, Vector Bionomics in the Epidemiology andControl of Malaria. Part I. The WHO African Region &the Southern WHO Eastern Mediterranean Region.Section III: Vector Bionomics, Malaria Epidemiologyand Control by Geographical Areas. (D) East Africa;(E) Eastern Outer Islands; (F) Southwestern ArabiaVBC/85.3 MAP/85.3 (World Health Organization, Ge-neva, 1985).

28. Y. T. Toure et al., Med. Vet. Entomol. 10, 197 (1996).29. C. Costantini et al., Parassitologia 41, 595 (1999).30. M. Coluzzi, Bull. WHO 62, 107 (1984).31. G. C. Carrara et al., Parassitologia 44, 43 (2002).32. T. Lehmann, unpublished data.

V I E W P O I N T

The Ecology of Genetically ModifiedMosquitoes

Thomas W. Scott,1* Willem Takken,2 Bart G. J. Knols,2 Christophe Boete3

Ecological and population biology issues constitute serious challenges tothe application of genetically modified mosquitos (GMM) for diseasecontrol.

Optimism that mosquito-borne diseases suchas malaria, dengue, and filariasis can be ef-fectively controlled or even eradicated withinexpensive drugs, vaccines, or insecticideshas been sorely tested (1). The impact ofdrugs is debatable, vaccine development is

slow, and mosquitoes are becoming resistantto insecticides, including those used to treatbed nets (2). Such shortfalls have been usedto justify research on mosquito populationreplacement—that is, the release into naturalmosquito populations of genetically modified

mosquitoes (GMM) rendered refractory topathogen infection—to reduce or eliminatedisease transmission (3).

A big hurdle to battling vector-borne diseas-es is our incomplete understanding of parasitetransmission ecology, which is hamperingGMM efforts in particular and public healthinitiatives in general. The GMM strategyshould serve as a case study for ways to im-prove overall disease prevention, because the

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