# Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens

Pierrick Labbé, Arnaud Berthomieu, Claire Berticat, Haoues Alout, Michel Raymond, Thomas Lenormand, Mylène Weill

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Pierrick Labbé, Arnaud Berthomieu, Claire Berticat, Haoues Alout, Michel Raymond, et al. Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens. Molecular Biology and Evolution, 2007, 24 (4), pp.1056-1067. 10.1093/mol-bev/msm025. halsde-00186494

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# Independent Duplications of the Acetylcholinesterase Gene Conferring Insecticide Resistance in the Mosquito *Culex pipiens*

Pierrick Labbé,*† Arnaud Berthomieu,* Claire Berticat,* Haoues Alout,* Michel Raymond,* Thomas Lenormand,† and Mylène Weill*

*Institut des Sciences de l'Evolution (UMR CNRS 5554), University Montpellier II, Montpellier, France; and †Centre d'Ecologie Fonctionnelle et Evolutive (UMR CNRS 5175), CNRS campus, Montpellier, France

Gene duplication is thought to be the main potential source of material for the evolution of new gene functions. Several models have been proposed for the evolution of new functions through duplication, most based on ancient events (Myr). We provide molecular evidence for the occurrence of several (at least 3) independent duplications of the *ace-1* locus in the mosquito *Culex pipiens*, selected in response to insecticide pressure that probably occurred very recently (<40 years ago). This locus encodes the main target of several insecticides, the acetylcholinesterase. The duplications described consist of 2 alleles of *ace-1*, 1 susceptible and 1 resistant to insecticide, located on the same chromosome. These events were detected in different parts of the world and probably resulted from distinct mechanisms. We propose that duplications were selected because they reduce the fitness cost associated with the resistant *ace-1* allele through the generation of persistent, advantageous heterozygosis. The rate of duplication of *ace-1* in *C. pipiens* is probably underestimated, but seems to be rather high.

## Introduction

Evolutionary potential is constrained by the number and type of genes present, but the nature of the constraints shaping the evolution of new functions remains a matter of debate. Gene duplication is thought to be a major feature of genome evolution and the main potential source of material for the origin of new evolutionary features, such as new gene functions (Haldane 1932; Ohno 1970). Two distinct phases can be distinguished in the evolution of a recent duplication: a polymorphic and a fixed period (Ohta 1988; Otto and Yong 2002). Most models of evolution following gene duplication concern the second phase—the fate of duplicated genes after fixation (for reviews see Zhang 2003; Lynch and Katju 2004), assuming that fixation is achieved by drift alone (Walsh 1995). However, several models have shown that selection can play a role in fixation (Ohta 1987; Clark 1994; Lynch et al. 2001), and increasing numbers of empirical studies have stressed the importance of selection in the early stages of duplication evolution (Hughes MK and Hughes AL 1993; Lynch and Conery 2000; Kondrashov et al. 2002). Focusing on the evolution of a new function by duplication, we can distinguish 4 scenarios with specific constraints (fig. 1; see also Otto and Yong 2002; Lynch and Katju 2004). In all these scenarios, selection favors the new function.

In the first scenario (Ohno 1970), no gene is available for the new function and no point mutation can solve this problem without altering existing (and presumably necessary) functions. In this case, only a redundant duplicated gene can accumulate the necessary mutations, with the original copy of the gene retaining its original function. An obvious constraint on this system is the number of deleterious mutations likely to disqualify the new duplicated gene before neofunctionalization (Walsh 1995; Lynch et al. 2001). Worse still, the duplicate is likely to be simply lost by drift before fixation, being initially neutral at best.

In the second scenario, an existing gene is able to perform, at least partially, different functions. After duplication, this “generalist” gene can then evolve by subfunctionalization (Hughes MK and Hughes AL 1993; Force et al. 1999), with each daughter copy retaining different subfunctions (although recent studies have suggested that subfunctionalization may be a transient mechanism on the road to neofunctionalization [He and Zhang 2005; Rastogi and Liberles 2005]). This process can evolve by the accumulation of mutations causing the loss (Force et al. 1999; Lynch and Force 2000; Ward and Durrett 2004) or improvement (Piatigorsky and Wistow 1991; Hughes 1994) of a subfunction of one of the duplicates (either by drift, in the case of subfunctionalization sensus stricto, or by selection, in the case of specialization). In cases of subfunction improvement, daughter copies specialize in a particular subfunction, removing the pleiotropic constraints that were presumably limiting the improvement of the generalist gene (e.g., evolution of crystallins [Piatigorsky and Wistow 1991]). Again, drift and deleterious mutations jeopardize the initial fixation and preservation of duplicates, although less crucially than in the previous scenario, as mutations advantageous for specialization are more likely than mutations generating an entirely new function. In both these scenarios, the new function emerges only after fixation, mostly by drift, of the initial duplication.

In the third scenario, gene duplications (or amplification) are first fixed in the population by selection, but for reasons other than the selection of a new function (e.g., an increase in protein production, as demonstrated for many adaptive gene amplifications [see Kondrashov et al. 2002, for a review]). Once the duplication is fixed, neo- or subfunctionalization can occur as above, with the additional constraint that these processes may conflict with selection for increased production of the original protein.

In the fourth scenario, a new function evolves by selection of a new allele (i.e., the new function is present before duplication). This allele is initially present in a heterozygous state in individuals able to perform both the original and the new function (overdominance). Duplication can then generate permanent heterozygosity, allowing the fixation of both alleles (Haldane 1954; Spofford 1969; Otto

Key words: insecticide resistance gene, duplication, mosquito, *Culex pipiens*, selection, *ace-1*.

E-mail: weill@isem.univ-montp2.fr.

Mol. Biol. Evol. 24(4):1056-1067. 2007
doi:10.1093/molbev/msm025
Advance Access publication February 5, 2007
<figure><img src="image_2.png" /><figcaption>FIG. 1.—Classical scenarios for the evolution of a new function through duplication. The different scenarios for the evolution of a new function through duplication proposed in the literature are illustrated. The new function is represented in gray. Short-term and long-term events are distinguished and the evolutionary force implied is indicated (in bold). Note that the case of subfunctionalization can be only considered to lead to the evolution of new functions as far as further improvement of at least one of the subfunction takes place.</figcaption></figure>

and Yong 2002). In this scenario, the duplication is less likely to be initially lost by drift (with a probability of ~1–2s, if *s* is the heterotic advantage, vs. ~1–1/2*N*<sub>e</sub> in the 2 first scenarios) and the main evolutionary constraint is the frequency of occurrence of the duplication itself. This type of duplication requires an unequal recombination between homologous chromosomes (presumably at meiosis) and may therefore occur less frequently than duplication of a gene on a single chromosome (i.e., replication slippage, which can occur at each round of DNA replication [Chen et al. 2005]).

In all these scenarios, 2 time scales should be consid-
ered (fig. 1): 1) an initial short period in which the critical
first mutation creating the new function appears, leading to
the preservation of these duplicates by selection and 2)
a longer time scale, in which new mutations may occur, re-
fining the new function and leading to further divergence of
the 2 copies. The initial period is longer for the first 3 sce-
narios (time for fixation plus time for appearance of the mu-
tation creating the new function) than for the fourth
scenario, in which it is instantaneous.

These scenarios are complicated by the potential dis-
ruption of gene dosage by multiple copies of the same (or
a similar) gene (Papp et al. 2003; Veitia 2005). Selection
may also favor duplication due to the masking of deleteri-
ous mutations. However, this effect has been shown to be
very weak and negligible in a first approximation (Clark
1994; Pàl and Hurst 2000; Otto and Yong 2002). The rel-
ative importance of the 4 evolutionary scenarios described
above is difficult to assess and may vary with population
size (Ohta 1987; Clark 1994; Walsh 1995; Force et al.
1999; Lynch and Force 2000; Lynch et al. 2001) or partic-
ular events (e.g., polyploidization [Otto and Whitton
2000]). Understanding how duplications are fixed in natural

populations is the first requirement—albeit a difficult em-
pirical challenge (Zhang 2003). The early stages of dupli-
cation are hard to follow and most studies have focused on
a posteriori analyses based on sequence data for fixed dupli-
cations (Long and Langly 1993; Hughes 1994; Ohta 1994;
Syvanen et al. 1996; Lynch and Conery 2000; Gu et al.
2002; Moore and Purugganan 2003; Zhang 2003). The
problem is that contemporary examples bearing witness
to the evolution of a new function are extremely rare.
The only case studied in detail concerns selection for insec-
ticide resistance in the mosquito *Culex pipiens* (common
house mosquito).

In *C. pipiens* populations exposed to organophosphate (OP) insecticides, at least one duplication—previously named *ace-1*<sup>RS</sup> but referred to here as *ace-1*<sup>D</sup>—combining resistant and susceptible alleles of the *ace-1* locus has recently appeared (Bourguet, Raymond, et al. 1996; Lenormand et al. 1998). This locus encodes acetylcholin-esterase (AChE1), the target of OP insecticides (Weill et al. 2002). The resistance allele, *ace-1*<sup>R</sup> is present world-wide and causes OP resistance in several mosquito species. It displays a single amino acid substitution, G119S, due to a mutation in the third exon of the *ace-1* gene, leading to the replacement of a glycine (GGC, susceptible alleles, *ace-1*<sup>S</sup>) by a serine (AGC [Weill, Lutfalla, et al. 2003; Weill et al. 2004]). This mutation is associated with reduced susceptibility to OP insecticide, modifications of the catalytic properties of AChE1, and a high fitness cost (for a review see Weill, Duron, et al. 2003). As no *ace-1* resistance alleles are detected in absence of OP insecticide, probably due to their high fitness cost, a duplication combining a resistant and a susceptible copies like *ace-1*<sup>D</sup> probably occurred very recently, that is, since OP insecticide treatments, less than 40 years ago anywhere in the world. The existence of *ace-1*<sup>D</sup> was inferred from enzymatic and genetic analyses, before cloning and sequencing of the *ace-1* gene.

Enzyme assays can be used to discriminate between
individuals expressing only the susceptible (AChE1S, phe-
notype [SS]), only the resistant (AChE1R, phenotype [RR]),
or both types (phenotype [RS]) of AChE1 (Test Propoxur
Propoxur [TPP] test [Bourguet, Pasteur, et al. 1996]). The
duplication of this gene was first suggested for Caribbean
strains of *C. pipiens* (from Martinique and Cuba), which
were mass selected in the laboratory and fixed with an
[RS] phenotype (Bourguet, Raymond, et al. 1996). A sim-
ilar duplication was next described in Southern France,
where some [RS] individuals had only [RS] progeny,
and an excess of the [RS] phenotype was observed in nat-
ural populations (Lenormand et al. 1998). The probable oc-
currence of this duplication in Southern France was traced
back to 1993—15 years after *ace-1*<sup>R</sup> was first detected in the
area. The duplication was shown to have gradually replaced
*ace-1*<sup>R</sup> in treated areas (Lenormand et al. 1998).

The gene ace-1 of *C. pipiens* has now been sequenced
(GenBank accession numbers AJ489456 and AJ515147
[Weill, Lutfalla, et al. 2003; Weill et al. 2004]), providing
molecular tools for more precise investigation of the gen-
eration and early evolution of ace-1 duplications. The pri-
mary aim of this study was to produce molecular evidence
for the existence of the ace-1<sup>D</sup> haplotype. The secondary
aim was to determine whether the Caribbean and South
<table><caption>**Table 1**
Nomenclature</caption><thead><tr><th rowspan="3">Genotype</th><th rowspan="3">Genotype Coding</th><th colspan="3">Phenotype Coding</th></tr><tr><th rowspan="2">Enzymatic Assay</th><th colspan="2">Molecular Identification</th></tr><tr><th>Position 119</th><th>Sequence</th></tr></thead><tbody><tr><td>ace-I<sup>R</sup> ace-I<sup>R</sup></td><td>(R/R)</td><td>[RR]</td><td>{R}</td><td>R</td></tr><tr><td>ace-I<sup>S</sup> ace-I<sup>S</sup></td><td>(S/S)</td><td>[SS]</td><td>{S}</td><td>S</td></tr><tr><td>ace-I<sup>R</sup> ace-I<sup>S</sup></td><td>(R/S)</td><td>[RS]</td><td>{S} and {R}</td><td>R, S</td></tr><tr><td>ace-I<sup>D</sup> ace-I<sup>D</sup></td><td>(D/D)</td><td>[RS]</td><td>{S} and {R}</td><td>D(R), D(S)</td></tr><tr><td>ace-I<sup>D</sup> ace-I<sup>R</sup></td><td>(D/R)</td><td>[RS]</td><td>{S} and {R}</td><td>D(R), D(S), R</td></tr><tr><td>ace-I<sup>D</sup> ace-I<sup>S</sup></td><td>(D/S)</td><td>[RS]</td><td>{S} and {R}</td><td>D(R), D(S), S</td></tr></tbody></table>

NOTE.—Diploid combinations of the 3 haplotypes—susceptible (ace-I<sup>S</sup>), resistant (ace-I<sup>R</sup>), and duplicated (ace-I<sup>D</sup>)—are given in the genotype column. For each genotype, the various phenotypes obtained with the different methods of identification are indicated. The enzymatic assay assesses the sensitivity or insensitivity to propoxur (an insecticide) of the AChE1 products (TPP test [Bourguet, Pasteur, et al. 1996]). Molecular identification is based on the presence ([R]) or absence ([S]) of a specific mutation in the first position of codon 119 (Weill et al. 2004), or the overall sequence of a large PCR fragment, allowing the attribution of {R} and [S] classes to specific alleles, ace-I<sup>R</sup> (R), ace-I<sup>S</sup> (S), or to specific copies of ace-I<sup>D</sup> haplotype, that is, susceptible (D(S)) or resistant (D(R)).

France duplications occurred independently or whether there was only one duplication that then spread worldwide. We also searched for ace-1 duplications in other populations. We then considered the mechanism by which the duplications occurred and spread and the likelihood of each of the possible scenarios for their evolution.

## Materials and Methods
Nomenclature

The precision of ace-1 genotyping in mosquitoes and the nomenclature depend on the technique used (enzymatic assay or molecular analysis) for characterization (table 1). The enzymatic assay (TPP test [Bourguet, Pasteur, et al. 1996]) measures the susceptibility of AChE1 to an insecticide (propoxur) and detects the presence of AChE1S and AChE1R, thus generating 3 phenotypes, [SS], [RS], and [RR] (table 1). This test is limited as the [RS] phenotype comprises the undistinguishable (ace-I<sup>S</sup>/ace-I<sup>R</sup>),

(ace-I<sup>D</sup>/ace-I<sup>D</sup>), (ace-I<sup>D</sup>/ace-I<sup>S</sup>), and (ace-I<sup>D</sup>/ace-I<sup>R</sup>) genotypes.

Molecular protocols generate 2 classes of fragments corresponding to susceptible or resistant copies. Fragments displaying the 119S mutation correspond either to ace-I<sup>R</sup> or to the resistant copy of ace-I<sup>D</sup>. These fragments, characteristic of resistant alleles, are collectively designated {R}. When additional sequence information is available, {R} fragments are attributed to an allele ace-I<sup>R</sup> (abbreviated to R) or to the resistant copy of an ace-I<sup>D</sup> haplotype (abbreviated to D(R)) (table 1). The second class of fragments generated by molecular protocols, displaying the 119G amino acid, corresponds to ace-I<sup>S</sup> or to susceptible copy of ace-I<sup>D</sup>. These fragments are collectively designated {S}. When additional sequence information is available, {S} fragments are attributed to an allele ace-I<sup>S</sup> (abbreviated to S) or to the susceptible copy of an ace-I<sup>D</sup> haplotype (abbreviated to D(S)) (table 1).

## Mosquito Collection
Recent Strains

We analyzed 8 strains from the laboratory, searching for the presence of duplications (table 2). These strains were BIFACE, from a population sampled in Ganges (Southern France, July 2002); MAURIN, from a population sampled in Maurin (Southern France, May 2005) (for precise location, see fig. 2 in Labbé et al. 2005); DUCOS, from a population sampled in Martinique in 2003 (Duron et al. 2005); PALAWAN and MANILLE, from populations sampled in the Philippines in 2003 (Duron et al. 2005); KUNU, from a population sampled in Crete in 2002 (Duron et al. 2005); and COTONOU, from a population sampled in Cotonou City in 2005 (Benin). All these strains contain individuals resistant to OP insecticides due to the modification of AChE1.

## Older Strains

Previous crossing experiments in several laboratory strains identified duplications. The mosquitoes concerned were preserved in liquid nitrogen for further analysis. These

<table><caption>**Table 2**
Mosquito Collection</caption><thead><tr><th>Strain</th><th>Haplotypes Present</th><th>Year of Collection</th><th>Origin</th><th>Subspecies</th><th>Original Field Sample</th><th>Reference</th></tr></thead><tbody><tr><td>BIFACE</td><td>ace-I<sup>R</sup>, ace-I<sup>D3</sup>, ace-I<sup>S</sup></td><td>2002</td><td>Southern France</td><td>C. p. pipiens</td><td>Ganges</td><td>This study</td></tr><tr><td>MAURIN</td><td>ace-I<sup>R</sup>, ace-I<sup>D2</sup>, ace-I<sup>D3</sup>, ace-I<sup>S</sup></td><td>2005</td><td>Southern France</td><td>C. p. pipiens</td><td>Maurin2</td><td>This study</td></tr><tr><td>DUMONT</td><td>ace-I<sup>D6</sup>, ace-I<sup>S</sup></td><td>1996</td><td>Southern France</td><td>C. p. pipiens</td><td>Maurin1</td><td>This study</td></tr><tr><td>DUCOS</td><td>ace-I<sup>D1</sup>, ace-I<sup>S</sup></td><td>2003</td><td>Martinique</td><td>C. p. quinque.</td><td>Ducos</td><td>Duron et al. 2005</td></tr><tr><td>MARTINIQUE</td><td>ace-I<sup>D1</sup></td><td>1994</td><td>Martinique</td><td>C. p. quinque.</td><td>—</td><td>Bourguet, Raymond, et al. 1996</td></tr><tr><td>M-RES</td><td>ace-I<sup>D5</sup></td><td>1986</td><td>Cuba</td><td>C. p. quinque.</td><td>—</td><td>Bourguet, Raymond, et al. 1996</td></tr><tr><td>PALAWAN</td><td>ace-I<sup>R</sup>, ace-I<sup>D4</sup>, ace-I<sup>S</sup></td><td>2003</td><td>Philippines</td><td>C. p. quinque.</td><td>Palawan</td><td>Duron et al. 2005</td></tr><tr><td>MANILLE</td><td>ace-I<sup>R</sup>, ace-I<sup>S</sup></td><td>2003</td><td>Philippines</td><td>C. p. quinque.</td><td>Manille</td><td>Duron et al. 2005</td></tr><tr><td>KUNU</td><td>ace-I<sup>R</sup>, ace-I<sup>S</sup></td><td>2002</td><td>Crete</td><td>C. p. pipiens</td><td>Kunu</td><td>Duron et al. 2005</td></tr><tr><td>SLAB</td><td>ace-I<sup>S</sup></td><td>1950</td><td>California</td><td>C. p. quinque.</td><td>—</td><td>Georghiou et al. 1966</td></tr><tr><td>COTONOU</td><td>ace-I<sup>R</sup>, ace-I<sup>S</sup></td><td>2005</td><td>Benin</td><td>C. p. quinque.</td><td>Cotonou</td><td>This study</td></tr></tbody></table>

NOTE.—Each strain used is indicated with the name of the field sample of origin (when available) and the year of collection. Taxonomic status (subspecies: *Culex pipiens pipiens* or *Culex pipiens quinquefasciatus*) is provided for each strain. The various ace-1 haplotypes present in the strain (considering the different susceptible alleles as a single class, ace-I<sup>S</sup>) are indicated.
<figure><img src="image_3.png" /><figcaption>FIG. 2.—Duplicated haplotype detection protocol. Females of the strain tested with an [RS] phenotype were crossed with SLAB males (genotype (S/S)). Their progenies were then selected individually, using propoxur insecticide at a concentration killing only susceptible individuals. If no mortality was detected in the progeny, the female was identified as bearing a duplicated *ace-1* gene and was thus analyzed. If some or all of the progeny died following exposure to insecticide, the corresponding female was eliminated.</figcaption></figure>

strains were analyzed to determine the stability of duplications over time.

MARTINIQUE and M-RES were derived from populations collected in Martinique in 1994 and in Cuba in 1987, respectively. These strains were assumed to be free of *ace-1*<sup>R</sup> and *ace-1*<sup>S</sup> alleles (Bourguet, Raymond, et al. 1996).

DUMONT was derived from a population sampled in Maurin in 1996 (Lenormand et al. 1998). This duplication-containing strain was backcrossed for 5 generations with the reference susceptible strain SLAB (Georghiou et al. 1966), with selection at each generation with propoxur concentrations giving 90% mortality. Individuals displaying only the duplicated haplotype *ace-1*<sup>D</sup> and the susceptible allele *ace-1*<sup>S</sup> of SLAB were present in the frozen DUMONT samples.

The taxonomic status of the mosquitoes of each strain was determined, using the molecular test discriminating between the *C. pipiens pipiens* and *C. pipiens quinquefasciatus* subspecies (Bourguet et al. 1998, table 2).

## Characterization of the Duplications

### Protocol for the Detection of Females Carrying *ace-1*<sup>D</sup>

No specific test (enzymatic or molecular) is currently available for detecting *ace-1* duplications. We overcame this problem by designing crosses and bioassays making possible to discard the confusing (R/S) genotype (fig. 2). Resistant females from each strain were crossed with (S/ S) males (strain SLAB). The progeny of each female was reared independently, and second instar larvae were exposed to $25 \times 10^{-6}$ M propoxur, which kills all (S/S) individuals. Mothers of progenies displaying no mortality were analyzed with TPP test (Bourguet, Pasteur, et al. 1996). All females with a [RS] phenotype correspond either to the (D/R) or to the (D/D) genotype (fig. 2). Their {S} copy of *ace-1* was therefore the D(S) sequences, and their {R} copies were either R or D(R) sequences.

For each female, there were 3 possible cases: 1) if the female was (D/D), with only one duplicated haplotype, 2 sequences were expected—1 susceptible, D(S), and 1 resis-

tant, D(R); 2) if 2 duplications were present in the same female, up to 2 susceptible, D(S)₁ and D(S)₂, and 2 resistant, D(R)₁ and D(R)₂ sequences were expected; and 3) if the female was (D/R), then up to 3 sequences were expected, one susceptible D(S), one resistant, R, and an additional resistant copy, D(R), if different from R.

## Female Progeny Analysis

Individuals from these progenies, carrying a chromosome inherited from the father ((S/S); SLAB) and a chromosome inherited from the mother (either (D/R) or (D/D)), were also sequenced. Two genotypes were possible, (D/ S) or (R/S). When the D(S) sequence was found in an individual (i.e., a {S} sequence different from that in SLAB), the associated {R} sequence was identified as D(R).

## Identification of the Different Copies Present

The simplest way to obtain R and S sequences from a strain is to sequence individuals displaying an [RR] or [SS] phenotype (i.e., in a genome without the *ace-1*<sup>D</sup> haplotype), identified with the TPP test (Bourguet, Pasteur, et al. 1996).

We amplified part of exon 3 of the *ace-1* gene from females displaying duplication and from their progeny. Polymerase chain reaction (PCR) products were then cloned (to separate the different copies present), using the TOPO® Cloning Kit (Invitrogen, Paisley, UK) according to the manufacturer's instructions. We expected a maximum of 2 {S} and 2 {R} clone types. A first screen was applied to discriminate {R} and {S} clones (PCR and AluI digestion, as described by Weill et al. 2004).

If 2 {S} clone types are present at the same frequency, the probability *P* of detecting both is $P = 1 - 1/2^{(n-1)}$, where *n* is the number of clones analyzed. Thus, to detect both types of clones with a risk of less than 5% (i.e., *P* > 0.95), a minimum of *n* = 6 clones should be analyzed. We followed the same reasoning for the resistant clones {R}. Thus, at least 6 clones were sequenced for each class ({S} or {R}), ensuring with a 95% probability that all the different copies present in an individual were detected. Finally, a minimum of 5 clones of each haplotype were analyzed to avoid *Taq* misincorporation errors.

## Susceptible Allele Variability

The S sequences were acquired from susceptible individuals (S/S) from the field samples identified as [SS] with the TPP test (table 2). PCR products were purified (Qiagen Purification Kit) and directly sequenced. For apparently heterozygous individuals (with 2 different S sequences), the PCR product was cloned and at least 6 clones were sequenced. Intron variability was assessed for the *C. p. quinquefasciatus* subspecies by analyzing the largest PCR fragment obtained from several susceptible individuals from the Ducos and Palawan field samples.

## Sequences of the Exon 3 of the *ace-1* Gene

DNA was extracted from single mosquitoes as described by Roger and Bendich (1988). Part of exon 3 of the *ace-1* gene, including position 119, was amplified using
<table><caption>**Table 3**
Duplicated Haplotype Sequences</caption><thead><tr><th rowspan="3">Copy</th><th rowspan="3">Strain</th><th colspan="12">Intron 2</th><th colspan="12">Exon 3</th></tr><tr><th colspan="12">Partial Exon 3 fragment</th></tr><tr><th>7</th><th>8</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>0</th><th>5</th><th>6</th><th>7</th><th>9</th><th>0</th><th>1</th><th>4</th><th>5</th><th>9</th><th>3</th><th>9</th><th>2</th><th>3</th><th>7</th><th>8</th><th>9</th><th>2</th><th>5</th><th>9</th><th>7</th><th>1</th><th>1</th><th>3</th><th>5</th><th>6</th><th>6</th><th>8</th><th>9</th><th>0</th><th>2</th><th>3</th></tr></thead><tbody><tr><td>D<sub>1</sub>(R)</td><td>MARTINIQUE DUCOS</td><td>T</td><td>A</td><td>G</td><td>G</td><td>G</td><td>C</td><td>T</td><td>T</td><td>T</td><td>T</td><td>C</td><td>A</td><td>T</td><td>T</td><td>G</td><td>C</td><td>A</td><td>C</td><td>C</td><td>C</td><td>A</td><td>T</td><td>C</td><td>G</td><td>A</td><td>T</td><td>A</td><td>G</td><td>G</td><td>C</td><td>C</td><td>C</td><td>C</td><td>A</td><td>C</td><td>C</td><td>C</td><td>C</td><td>C</td><td>A</td></tr><tr><td>D<sub>4</sub>(R)</td><td>PALAWAN</td><td>C</td><td>-</td><td>T</td><td>T</td><td>A</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>T</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>T</td><td>-</td><td>-</td><td>G</td><td>-</td><td>T</td><td>A</td><td>C</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>5</sub>(R)</td><td>M-RES</td><td>-</td><td>-</td><td>-</td><td>-</td><td>A</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>T</td><td>*</td><td>G</td><td>G</td><td>-</td><td>-</td><td>T</td><td>T</td><td>-</td><td>-</td><td>G</td><td>-</td><td>T</td><td>C</td><td>C</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>3</sub>(R)</td><td>BIFACE MAURIN</td><td>-</td><td>G</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>C</td><td>-</td><td>-</td><td>-</td><td>C</td><td>*</td><td>-</td><td>T</td><td>-</td><td>A</td><td>G</td><td>A</td><td>T</td><td>-</td><td>C</td><td>-</td><td>A</td><td>C</td><td>A</td><td>G</td><td>T</td><td>T</td><td>-</td><td>T</td><td>T</td><td>T</td><td>-</td><td>G</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>2</sub>(R)</td><td>MAURIN</td><td>-</td><td>G</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>C</td><td>-</td><td>-</td><td>-</td><td>C</td><td>*</td><td>-</td><td>T</td><td>-</td><td>A</td><td>G</td><td>A</td><td>T</td><td>-</td><td>C</td><td>-</td><td>A</td><td>C</td><td>A</td><td>G</td><td>T</td><td>T</td><td>-</td><td>T</td><td>T</td><td>T</td><td>-</td><td>G</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>6</sub>(R)</td><td>DUMONT</td><td>-</td><td>G</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>C</td><td>-</td><td>-</td><td>-</td><td>C</td><td>*</td><td>-</td><td>T</td><td>-</td><td>A</td><td>G</td><td>A</td><td>T</td><td>-</td><td>C</td><td>-</td><td>A</td><td>C</td><td>A</td><td>G</td><td>T</td><td>T</td><td>-</td><td>T</td><td>T</td><td>T</td><td>-</td><td>G</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>1</sub>(S)</td><td>MARTINIQUE DUCOS</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>4</sub>(S)</td><td>PALAWAN</td><td>-</td><td>-</td><td>-</td><td>-</td><td>A</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>T</td><td>*</td><td>G</td><td>G</td><td>-</td><td>-</td><td>T</td><td>T</td><td>-</td><td>-</td><td>G</td><td>-</td><td>T</td><td>C</td><td>C</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>5</sub>(S)</td><td>M-RES</td><td>-</td><td>-</td><td>-</td><td>-</td><td>A</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>T</td><td>*</td><td>G</td><td>G</td><td>-</td><td>-</td><td>T</td><td>T</td><td>-</td><td>-</td><td>G</td><td>-</td><td>T</td><td>C</td><td>C</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>3</sub>(S)</td><td>BIFACE MAURIN</td><td>-</td><td>G</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>C</td><td>-</td><td>-</td><td>-</td><td>C</td><td>*</td><td>G</td><td>T</td><td>-</td><td>A</td><td>G</td><td>A</td><td>T</td><td>-</td><td>C</td><td>-</td><td>C</td><td>C</td><td>-</td><td>-</td><td>T</td><td>G</td><td>A</td><td>T</td><td>-</td><td>T</td><td>G</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>2</sub>(S)</td><td>MAURIN</td><td>-</td><td>G</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>C</td><td>-</td><td>-</td><td>-</td><td>C</td><td>-</td><td>T</td><td>-</td><td>A</td><td>G</td><td>A</td><td>T</td><td>-</td><td>C</td><td>T</td><td>C</td><td>-</td><td>G</td><td>-</td><td>T</td><td>G</td><td>-</td><td>T</td><td>T</td><td>-</td><td>G</td><td>-</td><td>-</td><td>-</td></tr><tr><td>D<sub>6</sub>(S)</td><td>DUMONT</td><td>-</td><td>G</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td><td>C</td><td>-</td><td>-</td><td>C</td><td>*</td><td>-</td><td>T</td><td>-</td><td>A</td><td>G</td><td>A</td><td>T</td><td>-</td><td>C</td><td>T</td><td>C</td><td>-</td><td>G</td><td>-</td><td>T</td><td>G</td><td>-</td><td>T</td><td>T</td><td>-</td><td>G</td><td>-</td></tr></tbody></table>

NOTE.—Mutations are indicated for the D<sub>1</sub>(R) and D<sub>6</sub>(S) copies of each duplicated haplotype ace-1<sup>Di</sup>. The strains in which the duplicated haplotype was found are also indicated. D<sub>1</sub>(R) is the reference sequence. The dash (-) and the star (*) indicate identity or a deletion, respectively. The position from the first nucleotide of intron 2 or exon 3 is indicated at the top. The mutation at position 361 (boxed) is the only mutation conferring resistance (G119S, see Weill, Lutfalla, et al. 2003), discriminating between {R} copies (which possess an adenine at this position) and {S} forms (possessing a guanine at this position).

2 specific primers: CpEx3dir 5'-CGA CTC GGA CCC ACT CGT-3' and CpEx3rev 5'-GAC TTG CGA CAC GGT ACT GCA-3', generating a 457 bp fragment. PCR was carried out with ~20 ng of genomic DNA, 10 pmol of each primer, 100 µM of each dNTP, 2.5 units of high fidelity Taq polymerase in 1 × reaction buffer (Tris-HCl [pH 9.0; 75 mM], (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> [20 mM], Tween 20 [0.1 g l<sup>-1</sup>], and MgCl<sub>2</sub> [1.25 mM]), in a final volume of 50 µl. The PCR mixture was subjected to 30 cycles of amplification (93 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min).

A larger part of ace-1 comprising the extreme end of exon 2, the following intron (intron 2) and exon 3 were amplified as described above, using primers Intron2dir 5'-GCG CGA GCA TAT CCA TAG CAC T-3' and CpEx3rev, generating a fragment 588-597 bp in size, depending on intron size. Fragments were sequenced with an ABI Prism 310 sequencer (BigDye Terminator Kit, Applied Biosystems, Foster City, CA).

## Sequence Analyses

Sequences were aligned with Multalin software (http://prodes.toulouse.inra.fr/multalin/multalin.html [Corpet 1988]). The similarity between the various sequences was assessed with ClustalW (Neighbor-Joining method, v1.83, http://www.ddbj.nig.ac.jp/search/clustalw-e.html [Thomopson et al. 1994]).

Nucleotide variability was analyzed with DnaSP, v. 4.10.3 (Rozas et al. 2003). We calculated the number of polymorphic sites and nucleotide diversity, which was estimated using Nei's π index (Nei 1987; Nei and Miller 1987).

Deduced amino acid sequences were obtained with ClustalW (Thomopson et al. 1994) to determine whether the mutations identified were synonymous or nonsynonymous.

When a nonsynonymous mutation was identified, the putative position of the mutated amino acid was sought on a 3-dimensional model of the *Torpedo californica* AChE (pdb 1EA5), using Swiss-PdbViewer v. 3.7 (Guex and Peitsch 1997), to determine whether this mutation was located at a site crucial for protein activity.

## Results

### Molecular Evidence for ace-1 Duplication

Females from several strains were analyzed using the "duplicated haplotype detection protocol" (fig. 2). This protocol allows identifying females of genotypes (D/R) or (D/D)—both display a [RS] phenotype and no mortality of their progeny in the presence of insecticide after mating with SLAB males (S/S). Such females were found in strains MAURIN, BIFACE, DUCOS, and PALAWAN, but not in strains MANILLE, KUNU, and COTONOU (see table 2 for strains description).

For each strain harboring duplication, a fragment of exon 3 of the ace-1 gene was cloned and sequenced for each female, to identify D(S) copies. The D(R) and R copies were identified by analyzing the sequences of the progeny of each female and of [RR] individuals respectively. Dupli-cated haplotypes were numbered in the order of their discovery and are summarized in table 3.

We analyzed 7 females carrying the duplication from the DUCOS strain collected in Martinique. All contained a single {S} (thus attributed to D(S)) and a single {R} copy, identical in all females. The D(R) sequence was identical to the single R sequence present in this strain and to the previously described ace-1<sup>R</sup> allele of *Culex p. quinquefasciatus* (Weill, Lutfalla, et al. 2003). This duplication was named ace-1<sup>Di</sup>. Note that D<sub>1</sub>(S) and D<sub>1</sub>(R) differed only
<figure><img src="image_4.png" /><figcaption>FIG. 3.—Diversity of the *ace-1* copies in *Culex pipiens pipiens* samples. Variability of the different resistant D<sub>f</sub>(R) and susceptible D<sub>l</sub>(S) copies of each duplicated haplotype *ace-1*<sup>D<sub>f</sub></sup> and the single copy alleles R and S found in the Ganges sample (G) are presented (Jukes–Cantor distance, ClustalW (1.83) software [Thomopson et al. 1994]), 1,000 bootstraps. The sequenced of *ace-1* considered is a part of the exon 3, for each copy. The G119S mutation, selected for resistance to OP, has been removed to consider only the neutral variation. Bootstraps are presented in percentage.</figcaption></figure>

for the G119S mutation (table 3). For the South France strain MAURIN, we analyzed 9 females with duplication. We found 2 {S} and 1 {R} copy. The first {S} copy was present in 8 females. Seven mutations differentiated the 2 {S} copies for this partial exon 3 fragment (table 3). The same {R} copy was found in all females. The D(R) sequence was identical to the single R sequence found in this strain and to the previously described *ace-1*<sup>R</sup> allele of *C. p. pipiens* (Weill, Lutfalla, et al. 2003). Duplications containing the first and second {S} copy were named *ace-1*<sup>D<sub>2</sub></sup> and *ace-1*<sup>D<sub>3</sub></sup>, respectively. Seven mutations differentiated D<sub>2</sub>(S) and D<sub>2</sub>(R) and 9 differentiated D<sub>3</sub>(S) and D<sub>3</sub>(R) (including G119S mutation; table 3). We analyzed 6 females with duplication from the South France strain BIFACE. Only one {S} and one {R} copy were found, identical in all females. The duplication in this strain was identical to the *ace-1*<sup>D<sub>3</sub></sup> duplication identified in MAURIN. For the PALAWAN strain from the Philippines, we tested 5 females and identified only one {S} and one {R} copy. The D(R) copy was identical to the single R in this strain, but different from the *ace-1*<sup>R</sup> allele described for *C. p. quinquefasciatus* (Weill, Lutfalla, et al. 2003). This duplication was named *ace-1*<sup>D<sub>4</sub></sup>. Three mutations differentiated D<sub>4</sub>(S) and D<sub>4</sub>(R) (including the G119S mutation; table 3).

All the sequences of the {R} or {S} copies were more
than 96% identical. D(S) sequences differed from each
other by at least 3 mutations (table 3), with a higher level
of divergence observed between than within subspecies.
We therefore identified 4 different duplicated haplotypes.
In all cases, the D(R) copy was identical to the single non-
duplicated {R} copy found in [RR] individuals from the
corresponding field sample.

We detected no recombinants in the progeny of any of
the crosses between strains harboring duplications carried
out in the laboratory (Lenormand T and Labbé P, Unpub-
lished data). The 2 *ace-1* copies therefore seem to be on the
same chromosome for all the duplications detected in this
study.

Variability of Susceptible Copies

In order to compare the different duplicated haplo-
types and to elaborate a possible scenario for their occur-
rence, we measured the variability of a part of *ace-1* exon 3
in susceptible individuals from each field sample where
duplication was detected. For the Ducos field sample
(Martinique), we analyzed 10 [SS] individuals, and char-
acterized 7 S sequences, differing by 1–6 mutations, one
being identical to D₁(S) (supplementary table S1, Supple-
mentary Material online). For the Palawan field sample, 10
[SS] individuals were analyzed, and only 4 different S se-
quences were identified, differing from each other by 1 mu-
tation, 1 being identical to D₄(S) (supplementary table S1,
Supplementary Material online). For the South France sam-
ples, no susceptible individual was found in the Maurin2
field sample (intense insecticide treatment), so we se-
quenced [SS] individuals from the Ganges population (this
locality is less than 35 km North of Maurin). Sixteen [SS]
individuals were analyzed, leading to the description of 26
different S sequences, differing by 1–15 mutations, 1 being
identical to D₂(S) and 1 to D₃(S) (supplementary table S2,
Supplementary Material online and fig. 3). In all cases,
a {S} copy identical to the D(S) copy was found in [SS]
individuals from the corresponding field sample.
<figure><img src="image_5.png" /><figcaption>FIG. 4.—Diversity of the *ace-1* copies in *Culex pipiens quinquefasciatus* samples. Variability of the different resistant D₁(R) and susceptible D₁(S) copies of each duplicated haplotype *ace-1*<sup>D1</sup> and the single copy alleles R and S found in the Ducos (Du) and Palawan (Pa) samples are presented (Jukes–Cantor distance, ClustalW (1.83) software [Thomopson et al. 1994]), 1,000 bootstraps). The sequenced of *ace-1* considered encompassed the intron 2 and the exon 3, for each copy. The G119S mutation, selected for resistance to OP, has been removed to consider only the neutral variation. Bootstraps are presented in percentage. Considering only the part of the exon 3 available for every individual sequenced, this figure basically connects to figure 3 in a straightforward way, with 3 diagnostic mutations separating the sequences from the 2 subspecies.</figcaption></figure>

## Protein Sequence Variability

We compared the coding sequences of duplicated haplotypes, susceptible, and resistant single alleles (40 different sequences). Excluding the G119S mutation, 40 variable sites were identified on the partial exon 3 fragment, but no insertions/deletions (supplementary tables S1 and S2, Supplementary Material online). Nucleotide diversity was estimated at $\pi$ = 0.024. Almost all mutations were synonymous, with only 6 nonsynonymous mutations identified in susceptible individuals (3 in the Montpellier area, 1 in Martinique [supplementary tables S1 and S2, Supplementary Material online]). Protein modeling based on the structural model of AChE from *Torpedo californica* (Protein Data Bank [PDB] accession number 1EA5) indicated that these 4 mutations were located at some distance from the active site of AChE1 and were therefore unlikely to interfere with activity (data not shown).

## Intron Sequence Analysis

As the coding exon 3 partial sequence variability was low, especially in *C. p. quinquefasciatus* subspecies, we increased the power of the analysis by sequencing longer *ace-1* gene fragments, including the end of exon 2, intron 2, and almost all of exon 3.

We analyzed 10 [SS] individuals from the Ducos sample (Martinique), and described 7 different S sequences, differing by 2–11 mutations (supplementary table S1, Supplementary Material online and fig. 4). For the Palawan sample, we found 7 different S sequences, differing by 1–14 mutations, in the 5 [SS] individuals analyzed (supplementary table S1, Supplementary Material online and fig. 4). The *C. p. quinquefasciatus* populations appeared more structured, and only one susceptible allele of the Ducos sample (Ducos-S1) was found to be identical to D₄(R), the resistant copy of the Palawan duplicated allele (ignoring the G119S mutation, fig. 4). In each case, a single S and a single R copies identical to the D(S) and the D(R) copies

of the corresponding duplicated haplotype were found respectively.

The intron sequences of the duplicated haplotypes diverged considerably between the *C. p. pipiens* and *C. p. quinquefasciatus* subspecies (table 3), with an 8 bp insertion detected in the sequences of individuals from the DUCOS and PALAWAN strains (*C. p. quinquefasciatus*) but not in strains from the Montpellier area (*C. p. pipiens*). After this extended sequence analysis, for the *ace-1*<sup>D1</sup> haplotype, D₁(S) and D₁(R) still differed only for the G119S mutation. Similarly, D₂(R) and D₃(R) stayed strictly identical (table 3). However, the D₄(S) and D₄(R) intron sequences from PALAWAN differed considerably, by 6 mutations and 1 insertion (table 3).

## Stability of the Duplication Over Time

We analyzed individuals displaying duplication that had previously been collected from the same sample sites, to follow the evolution of duplicated haplotypes since their first detection.

In the Caribbean, duplications found in old strains from Martinique (MARTINIQUE, 1994) and Cuba (M-RES, 1987) were compared with the duplication found in recent samples from Martinique (DUCOS, 2003). In the MARTINIQUE strain, we found only one {S} and one {R} copy in 5 individuals. MARTINIQUE D(S) and D(R) sequences did not differ from DUCOS D₁(R) and D₁(S) sequences; these 2 strains therefore displayed the same haplotype, *ace-1*<sup>D1</sup> (table 3 and supplementary table S3, Supplementary Material online).

Two sequences were identified in the 5 M-RES individuals analyzed: a susceptible and a resistant sequence, attributed to D(S) and D(R), respectively. The MARTINIQUE and M-RES strain duplications were very different, particularly in terms of the intron 2 sequences (table 3 and fig. 4). The M-RES duplication was thus named *ace-1*<sup>D5</sup>. Note that D₅(S) and D₅(R) differed only for the G119S mutation. This analysis also showed that the D₅(S) was identical to the D₄(S) sequence found in PALAWAN (table 3 and fig. 4).

In the Montpellier area, the duplicated haplotype was analyzed in mosquitoes sampled in 1996 (DUMONT) and 2005 (MAURIN). As expected, 3 sequences, 2 {S} and 1 {R}, were identified from DUMONT (strain backcrossed on SLAB, composed of only (D/D) and (D/S) individuals) in the 5 individuals analyzed. One of the {S} sequences was identical to the SLAB sequence, and the other corresponded to D(S). The {R} sequence was attributed to D(R). D(R) in this strain was identical to the D₂(R) found in MAURIN. However, DUMONT D(S) was not identical to D₂(S), differing by one insertion in intron 2 and a synonymous mutation in exon 3 (table 3 and fig. 3 and supplementary table S3, Supplementary Material online). The DUMONT haplotype was thus named *ace-1*<sup>D6</sup>.

## Discussion

### Duplications Mechanisms and Independence of Events

We compared the duplicated haplotypes by sequencing a part of the *ace-1* gene of their D(S) and D(R) copies,
<figure><img src="image_6.png" /><figcaption>FIG. 5.—Origin of ace-1 duplications. Sensus stricto duplications (Type A event): scenario 3a unequal crossing-over or replication slippage in a resistant homozygote followed by reversion to susceptibility or recombination with a susceptible allele; scenario 3b unequal crossing-over or replication slippage in a susceptible homozygote followed by mutation toward resistance or recombination with a resistance allele; scenario 4 unequal crossing-over in an heterozygote individual. Examples of secondary recombination events that could modify the haplotype sequence are illustrated. Type B event: recombination in an individual carrying both a duplicated haplotype and a single copy allele, either a R allele (a) or a S allele (b). Type C event: crossing-over in an individual carrying 2 different duplicated haplotypes. Similar sequences are illustrated with the same color. Resistant copies are indicated by a bar representing the G119S mutation.</figcaption></figure>

and several nucleotide differences observed suggested different duplication events. The duplications described here are probably very recent: ace-1<sup>R</sup> allele is very costly for mosquitoes and cannot be detected in the absence of OP insecticides, and OP insecticides have been used against *C. pipiens* for only about 40 years in most parts of the world. Moreover, the probable occurrence of a duplication in Southern France was traced back to 1993—15 years after ace-1<sup>R</sup> was first detected in the area (Lenormand et al. 1998). This situation provides the first known contemporary example of ongoing evolution of a new function through duplication.

Different scenarios may account for the occurrence of these duplications (fig. 5). They correspond to scenarios 3 and 4 exposed in the introduction, as selection rather than drift seems necessary to explain the rapid emergence of the different duplications worldwide (mosquito population size is indeed certainly very large). The first scenario (3a) involves unequal crossing-over (or replication slippage [Chen et al. 2005]) in a resistant individual (i.e., of (R/R) genotype) followed by either a reversion (S119G) in one of the R copies or a supplemental recombination with an S allele. The second scenario (3b) involves unequal crossing-over (or replication slippage) in a susceptible individual (i.e., of (S/S) genotype) followed by mutation (G119S) in

one of the S copies or a supplemental recombination with an R allele. A mutation step will generate duplication with very similar D(S) and D(R) copies, whereas a recombination step will generate distinct D(S) and D(R) copies, whose divergence will depend on the diversity present in natural populations (fig. 5). Scenarios 3a and 3b are similar to the third scenario proposed in the introduction, the duplication being selected first and the divergence between copies being acquired later. The scenario 4 involves unequal crossing-over in a heterozygous individual (i.e., of (R/S) genotype), resulting immediately in a new ace-1<sup>D</sup> haplotype (fig. 5). This scenario corresponds to that proposed by Haldane (1954) as the functionally divergent copies are already present before the duplication. In such a scenario D(S) and D(R) copies should display distinct nucleotides sequences, whose divergence will also depend on the diversity of alleles present in natural populations (fig. 5). The likelihood of these scenarios differs, as scenarios 3a and 3b require an intermediate step. Also, scenarios 4 and 3a appear more probable than 3b as the corresponding duplications (RS and RR, respectively) would confer resistance to insecticides. Duplication with 2 R copies (scenario 3a) could be advantageous by restoring a part of the normal AChE1 activity (see paragraph “Advantages and Costs of Duplications for Insecticide Resistance”). By contrast,
the duplicated susceptible haplotype (SS duplication, scenario 3b) would not confer any resistance to insecticide and might even disrupt protein dosage. Unfortunately, if duplications involving 2 {S} or 2 {R} copies exist, they cannot be detected with our protocol and require more powerful molecular tools.

These 3 scenarios involve an unequal crossing-over (or replication slippage) in individual carrying only single copy alleles. We will call these events duplication sensus stricto or type A event. However, recombination in (D/S), (D/R), or (D/D) genotypes may also generate new duplicated haplotypes without adding a new gene (fig. 5). Because of these secondary events of recombination, no phylogenetic analysis of the haplotypes can be conducted based on the point mutations as the copies of a given haplotype may have different phylogenetic history. The type B event involves a crossing-over in individual carrying a single copy allele and a duplicated haplotype ((D/S) or (D/R)), and type C event involves a crossing-over between 2 different duplicated haplotypes ($D_x/D_y$). Type B and type C secondary events require a preliminary event of duplication, but result from a simple recombination and thus could be more frequent than type A events. A possible signature of B and C events would be the occurrence of different duplicated haplotypes sharing a similar copy (either D(S) or D(R)).

A first question is how many independent duplication events (i.e., type A rather than B or C events) can be detected within our data set? The comparison of sequences of susceptible and duplicated alleles from different geographic origins showed that the level of variability was low (as shown previously in Weill, Lutfalla, et al. 2003). Furthermore, the stability of the duplication from Martinique described in populations sampled in 1994 (MARTINIQUE) and 2003 (DUCOS), for intron 2 and exon 3 (corresponding to ~13% of total gene length, using *Anopheles gambiae* [GenBank accession number BN000066 and AJ515148] as a reference [Weill et al. 2002]) confirms that mutations in this part of the *ace-1* gene are rare. These observations increase the significance of any mutation found. First, the striking differences both of intron 2 and exon 3 between *C. p. pipiens* and *C. p. quinquefasciatus* sequences indicate that at least 2 types A events occurred, one in each subspecies (fig. 3 and 4).

In *C. p. pipiens* subspecies, 3 haplotypes (*ace-1*<sup>D2</sup>, *ace-1*<sup>D3</sup>, and *ace-1*<sup>D6</sup>) have been identified, sharing the same D(R) sequence but associated with different D(S) copies (fig. 3). These haplotypes may result from the same duplication sensus stricto event followed by recombination in D/S genotypes (type B (*b*) in fig. 5). Moreover, only 2 mutations were observed between duplicated haplotypes isolated from populations sampled in Maurin in 1996, *ace-1*<sup>D6</sup> (DUMONT) and in 2005, *ace-1*<sup>D2</sup> (MAURIN). This level of diversity is remarkably low. Thus, the duplicated haplotype *ace-1*<sup>D2</sup> may derive (by mutation) from *ace-1*<sup>D6</sup>. Thus, for *C. p. pipiens*, the minimum duplication event (type A) number is one.

In *C. p. quinquefasciatus* subspecies, 3 haplotypes have been identified. *ace-1*<sup>D4</sup> and *ace-1*<sup>D5</sup> display strictly identical D(S) copies and one of them may result from a type B (*a*) secondary event (fig. 5). However, *ace-1*<sup>D1</sup> from Martinique is highly divergent from *ace-1*<sup>D4</sup> and *ace-1*<sup>D5</sup>,

for both the resistant (D(R)) and the susceptible (D(S)) copies. This divergence suggests at least 2 independent sensus stricto duplications (fig. 4). Several arguments based on the distribution of alleles in the different populations corroborate this view: 1) in both Martinique and Palawan field samples, the local single R copy is identical to the local D(R) copy and these R (or D(R)) copies are different between the 2 populations. This differentiation on R and D(R) copies (they are private to each population) is a strong indication that these duplications (*ace-1*<sup>D1</sup> and *ace-1*<sup>D6</sup>) occurred independently in different places; 2) in both Martinique and Palawan field samples, there is a private S copy corresponding to the local D(S) copy. The possibility that duplicated genes can revert back to single S copies is very unlikely because pesticide treatments are still used in all populations studied; 3) all S alleles tend to globally cluster per population (see fig. 4), which reinforces the view that the populations are structured at *ace-1* locus so that the more likely explanation is again an independent origin of *ace-1*<sup>D1</sup> and *ace-1*<sup>D4</sup> in different places.

A process such as gene conversion between duplicated genes and alleles present in each population might blur the signal emerging from sequence data. However, this mechanism appears less likely than independent duplications for several reasons: 1) if gene conversion occurred, it should still be active and the duplicated $D_1(R)$ and $D_1(S)$ alleles sampled in Martinique in 2003 would not have been strictly identical to the ones sampled in 1994; 2) if gene conversion occurred at such a fast rate, no within-population divergence among alleles should be found; 3) gene conversion would also affect nonduplicated genes but previous data (Weill, Lutfalla et al. 2003) showed that all R alleles found in distinct *C. p. quinquefasciatus* populations (Africa, USA, China, and South America) are strictly identical, and homogenization with single S alleles present in natural populations was never detected; 4) gene conversion would not have discriminated between the {R} and {S} copies: in Martinique, $D_1(R)$ and $D_1(S)$ are identical to the local single R allele (except of course the codon conferring the insecticide insensitivity), suggesting that only the single R allele and not any single S allele could have been used for the conversion. In Palawan, however, $D_4(R)$ and $D_4(S)$ are identical to the local single R allele and to one local single S allele, respectively, suggesting that only the single R could have been used to convert $D_4(R)$, and only one single S to convert $D_4(S)$.

Thus, for *C. p. quinquefasciatus*, we conclude that the minimum number of duplication event (type A) is 2.

Overall, at least 3 duplications sensus stricto are required to explain the 6 observed duplicated haplotypes. In this minimum scenario, recombination (type B) does not appear to be more frequent than duplication sensus stricto.

The second question is which scenario leads to the different duplication events? Scenario 3 could be the more likely explanation for the Martinique (*ace-1*<sup>D1</sup>) and the Cuba (*ace-1*<sup>D5</sup>) haplotypes. This conclusion stems from the striking observation that, in these duplications, the D(R) and D(S) copies are exactly identical if we except for the G119S site characterizing {S} versus {R} copies. This observation is striking because, in each case/population,
many S alleles differing from the local R allele are segregating (see fig. 4). With scenario 4 (unequal crossing-over in a (R/ S) heterozygote), we do not expect D(R) and D(S) to be more similar than a R and S allele sampled randomly in the population and the exact D(R)–D(S) similarity seems therefore difficult to explain given the diversity of S alleles. On the contrary, with scenario 3, in particular 3a, where a RR duplication occurs first followed by a back (R to S) mutation at G119S site, we expect that R, D(R) and D(S) should be very similar compared with the divergence of S alleles in the population, which is in very close agree- ment with the data (this is particularly clear for Ducos al- leles, see fig. 4). Alternatively, gene conversion between the 2 copies of these duplicated haplotypes could be involved (Teshima and Innan 2004), although this seems unlikely as these duplications are very recent.

As the D(R) and the D(S) copies of the other haplo-
types differ by several mutations, they could result from
either scenario 3a involving additional recombination steps
or more simply from scenario 4 (fig. 5). Thus, duplications
described in this study probably occurred by different
mechanisms.

Advantages and Costs of Duplications for Insecticide
Resistance

Gene dosage has been shown to be important in many
cases and duplication may disrupt this balance (Kondrashov
et al. 2002; Papp et al. 2003; Veitia 2005). In the case of
*ace-1*, duplication could partly restore gene dosage, rather
than disrupting it. OP insecticides are lethal to mosquitoes
because they cause the accumulation of acetylcholine
(ACh) in synapses, due to the inhibition of AChE1, which
degrades ACh (Bourguet, Raymond, et al. 1997). The fit-
ness cost associated with *ace-1*<sup>R</sup> probably results from the
excess of ACh in synapses as the activity of the AChE1R is
more than 60% lower than that of the AChE1S (Bourguet,
Raymond, et al. 1996; Bourguet, Lenormand, et al. 1997).
Thus, a duplicated haplotype *ace-1*<sup>D</sup> could be advantageous
because it restores, at least in part, normal AChE1 activity.
It should be noted that a duplication with 2 R copies (sce-
nario 3a) could also restore a part of normal AChE1 activ-
ity, although at a lower level, and thus could be selected as
an intermediate step to *ace-1*<sup>D</sup> haplotypes. AChE1 activity
in *ace-1*<sup>D</sup> homozygotes has been shown to reach levels sim-
ilar to or larger than those in susceptible homozygotes
(Bourguet, Raymond, et al. 1996). The higher total AChE1
activity associated with *ace-1*<sup>D</sup> (15–30% higher than *ace-
1*<sup>S</sup>) may induce another type of fitness cost, resulting from
ACh deficit. However, excess or deficit of ACh may have
different fitness consequences so that the proximal reason
for which duplicated haplotypes could be advantaged over
*ace-1*<sup>R</sup> alleles in the field is still an open question. In addi-
tion, all duplicated haplotypes may not share the same level
or pattern of AChE1 activity (as for instance MARTINI-
QUE and M-RES haplotypes [Bourguet, Raymond, et al.
1996]) so that a detailed biochemical analysis in the differ-
ent haplotypes has to be performed, along with their fitness
consequences, to settle this issue.

Field surveys in Caribbean islands (Yébakima et al.
2004) and in the Montpellier area (Lenormand et al.

1998) have provided insight into the relative advantages
and costs of duplicated *ace-1*<sup>D</sup> haplotypes with respect
to *ace-1*<sup>R</sup> and *ace-1*<sup>S</sup>. In Martinique (Yébakima et al.
2004), resistance due to insensitive AChE1 was extremely
rare in 1990, whereas in 1999, half the population displayed
a resistant phenotype (i.e., [RS] or [RR]). Moreover, the
mean frequencies of each phenotype were 0.51, 0.49,
and less than 0.01 for [SS], [RS], and [RR], respectively,
with the frequency of [RS] reaching 0.76 in some popula-
tions. This corresponds to a very large departure from
Hardy-Weinberg equilibrium and is certainly due to the
high frequency of the duplicated haplotype (Lenormand
et al. 1998). The *ace-1*<sup>R</sup> allele was extremely rare in
Martinique (only one population displayed the [RR] pheno-
type, at a frequency of 0.03). As (D/D) individuals from
MARTINIQUE are less resistant to insecticides than (R/
R) individuals (Lenormand et al. 1998), the overall fitness
advantage of the duplicated haplotype may result from
a much lower fitness cost, but this hypothesis remains to
be tested.

In the Montpellier area, the adaptive sweep (i.e., adaptive replacement) by a duplicated haplotype (characterized in this study as 2 haplotypes, *ace-1*<sup>D2</sup> and *ace-1*<sup>D3</sup>) is consistent with the duplicated haplotypes being associated with a higher fitness than *ace-1*<sup>R</sup> (Lenormand et al. 1998). These duplicated haplotypes display a frequencycline across the treated and nontreated areas and seasonal oscillations in frequency, suggesting that they confer a lower fitness than *ace-1*<sup>S</sup> in the nontreated area.

Duplication Rate

Two studies estimated the number of duplications
through sequence analyses in 3 model organisms (Lynch
and Conery 2000; Gu et al. 2002). They reported mean rates
of gene duplications of 0.002, 0.01, and 0.02 per gene per
Myr for *Saccharomyces cerevisiae*, *Caenorhabditis ele-
gans*, and *Drosophila melanogaster*, respectively, although
slightly higher rates were reported for some genes, as
shown by variability in gene family size (Gu et al.
2002). These rates are probably underestimated, as only
fixed duplications were considered; whereas most duplica-
tions are likely to be lost by drift or selected against in the
early stages (Otto and Yong 2002).

It is not possible to estimate precisely the frequency of
duplication events in the mosquito *C. pipiens* as the genetic
protocol used to detect duplication requires crosses and
could not be applied extensively. Unfortunately, no con-
served feature was identified in the duplicated haplotypes,
precluding the design of a simple molecular detection test.
However, it is very remarkable that at least 3 *ace-1* duplic-
cations sensus stricto have appeared independently in such
a short lapse of time. Furthermore, this number is certainly
an underestimate because our geographic survey is limited.
This is clearly not representative of the entire genome of *C.
pipiens*, but demonstrates that some genes may have much
higher duplication rates than estimated by comparing se-
quences between species or within a single genome.

Our study demonstrates the importance of duplication
in the adaptive process and shows that selection may play an
important role in the occurrence of such events, as processes
driven by selection, rather than by drift, are more likely to
occur over such a short time scale. Insecticide resistance in
mosquitoes provides us with a unique opportunity to analyze
the dynamics of this process.

**Supplementary Material**

Supplementary tables S1–S3 are available at
*Molecular Biology and Evolution* online (http://www.mbe.
oxfordjournals.org/).

**Acknowledgments**

We would like to thank Janice Britton-Davidian, Mark Kirkpatrick, Thomas Galewski, Emmanuel Douzery, and Nicole Pasteur for helpful comments on the manuscript, C. Bernard, M. Marquine, and S. Unal for technical assistance, and V. Durand for the literature search. This work was funded in part by APR PNETOX 2001 (Ministère de l'Aménagement et du Territoire) and by ANR Morevol Sante-Environnement (Ministère délégué à la Recherche). Contribution 2007.017 of the Institut des Sciences de l'Evolution de Montpellier (UMR CNRS-UM2 5554).

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Manolo Gouy, Associate Editor

Accepted January 31, 2007