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Keywords:

  • Culex pipiens;
  • genetic drift;
  • global and local selection;
  • history;
  • insecticide resistance gene;
  • mosquito;
  • overproduced esterases

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Adaptation occurs by gene replacement (or transient balanced polymorphism). Replacement may be caused by selection (local or global) and/or genetic drift among alleles. In addition, historical events may blur the respective effects of selection and drift during the course of replacement. We address the relative importance of these processes in the evolution of insecticide resistance genes in the mosquito Culex pipiens. The resistance allele, Ester2, has a broad geographic distribution compared to the other resistance alleles. To distinguish between the different processes explaining this distribution, we reviewed the literature and analysed updated data from the Montpellier area of southern France. Overall, our data indicate that Ester2 prevails over other Ester resistance alleles in moderately treated areas. Such conditions are common and favour the hypothesis of selection acting at a local level. This places an emphasis on the importance of ecological conditions during the evolution of resistance. Finally, we highlight that historical events have contributed to its spread in some areas.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Adaptation occurs by gene replacement at one or several loci. In a large or structured population, several adaptive alleles may segregate simultaneously at a single locus. In the long run different outcomes are possible: (i) one of these alleles may prevail, i.e. the one that most likely confers the best adaptation (i.e. global selection); (ii) several adaptive alleles may coexist if they confer different phenotypes, which are favoured in different environments (i.e. local selection), or (iii) the different alleles may be adaptively equivalent and may coexist in a neutral way (i.e. genetic drift). Discriminating between these scenarios is relatively easy in the long run, when equilibrium has been reached. However, before equilibrium has been reached, due to historical events (i.e. initial conditions and contingency events), it is not easy to distinguish how these three scenarios influence the course of allele replacement (Fig. 1). Moreover, these processes are not mutually exclusive and can combine. Balanced polymorphism is also a possibility, although it will eventually be fixed (for example through duplication, Haldane, 1932). Sometimes it is possible to distinguish, at least partly, between selection (local or global), genetic drift, and history. For example, the G6PDA deficient allele, conferring malarial resistance in humans (i.e. it gives a selective advantage), is at higher frequencies than other deficient alleles of the G6PD gene. However, the frequency of another deficient allele, Med, providing less resistance than A, is as high as 70% in some populations, where apparently A has not spread yet. In this example, historical events are the most likely explanation for the prevalence of the Med allele (Tishkoff et al., 2001). A second example is the Adh gene in Drosophila melanogaster. Three different alleles are found: a wild one (named Standard or Low allele), an inverted allele (In(2L)t), most common in Africa, and a Fast allele, found essentially in Europe. In this example too, historical events and global selection are the most likely explanation for the distribution of alleles with the independent appearance of In(2L)t in Africa and Fast in Europe, and the subsequent spread of the Fast allele in Africa (Veuille et al., 1998). This spread is thought to occur because of a selective global advantage of the Fast allele. In these examples, the importance of historical events was inferred, although their roles remain uncertain due to the incomplete knowledge of the selective process at work.

image

Figure 1. Expected patterns under the different evolutionary hypotheses. The different shapes represent different environmental conditions. A, B, C are different adaptive alleles. Global selection: A is the fittest in each condition. Local selection: each allele is selected in a different environment. Genetic drift: the different adaptive alleles are neutral with respect to each other. To take account of historical contingency, expected patterns at equilibrium are indicated above and those occurring during gene replacement, and thus submitted to historical events, at the bottom. An absence of letters indicates the absence of an adaptive gene.

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The wide use of pesticides to control pests of agricultural and public-health importance has been a powerful and recent agent of selection in natural populations of many insect species. Here we took advantage of the knowledge on insecticide resistance genes in the mosquito Culex pipiens L. to investigate the relative importance of historical events, genetic drift between resistance alleles, and selection. This mosquito, common in temperate and tropical countries, is subjected to insecticide control in many places, particularly with organophosphate insecticides (OP), because it is a nuisance and a vector of human diseases (West Niles encephalitis, filariasis). These insecticides inhibit acetylcholinesterase (or AChE) in the central nervous system, causing death. This mosquito has rapidly developed various adaptations to this new and toxic component of its environment. This example of microevolution has been thoroughly investigated as an opportunity to characterize precisely both the new adapted phenotypes and the associated genetic changes. In addition, OP resistance in this mosquito was studied from its first appearance in one geographic area, and has been followed up in the context of a long-term project. In this species, only three loci have developed major OP resistance alleles: Est-2, Est-3 and ace-1. The first two loci, Est-2 and Est-3, code for detoxifying carboxylester hydrolases (or esterases). Resistance alleles correspond to an over-production of esterase (which binds or metabolizes the insecticide) relative to susceptible alleles. Several resistance alleles (each corresponding to a distinct over-produced allozyme) have been described at both loci (see for review Raymond et al., 2001). For most resistance alleles, the over-production of esterase is the result of gene amplification (i.e. several copies of the same gene are found in the same genome), affecting one or both Est loci. The latter situation, the co-amplification of two esterase loci that are adjacent in the genome, explains the tight statistical association of some electromorphs, e.g. A2–B2 (Guillemaud et al., 1996; Rooker et al., 1996). Although, strictly speaking, A2 is coded by an allele of the Est-3 locus, and B2 by an allele at the Est-2 locus, A2–B2 behave as if coded by an allele (named Ester2) of a single super locus (named Ester) due to the complete linkage in amplification of Est-2 and Est-3. Gene regulation is also present (i.e. esterase over-production is higher than expected by the amplification level), and is the major mechanism of over-production of A1 (Rooker et al., 1996). A third locus, ace-1, codes the insecticide target (acetylcholinesterase). The wild type and susceptible forms of this enzyme are inhibited by OP insecticides. Several resistance alleles at this locus have been described with reduced sensitivity towards OP, associated with their modified catalytic properties (Bourguet et al., 1997; Lenormand et al., 1998a; Weill et al., 2003,2004).

A particular OP resistance allele, Ester2, has a worldwide distribution. It has been shown by various molecular studies that this situation is the result of extensive migrations, and not due to independent origins (Raymond et al., 1991; Guillemaud et al., 1996; Callaghan et al., 1998). These migrations have been driven by human activity, as there is direct evidence of passive transportation of mosquitoes, including C. pipiens, by ships and planes (Asahina, 1970; Highton & van Someren, 1970; Curtis & White, 1984). The worldwide distribution of Ester2 has four possible and non-exclusive explanations. First Ester2 could be the first OP resistance gene to have occurred and spread in treated areas worldwide; this is the historical contingency hypothesis. Second and third, Ester2 could present some fitness advantage over other resistance genes, for example a lower cost or a better resistance level, its actual distribution thus being the result of a competitive advantage. This advantage may be local (i.e. only in some areas) or global (i.e. in all environments encountered); these are the local and globalselection hypotheses. Fourthly, Ester2 may be as fit as the other resistance alleles at the Ester locus, with its higher prevalence being explained only by chance; this is the drift hypothesis. In practice, different patterns of gene replacement are expected under these hypotheses (Fig. 1): local selection would cause Ester2 to be prevalent only in some type of environments, other resistance alleles being more frequent in others, whereas in the case of global selection, an intrinsic advantage would make Ester2 prevalent in all environmental conditions. Historical events would cause a less fit allele to be prevalent in environments where Ester2 would eventually prevail once introduced. With genetic drift among resistance alleles, no predictable pattern would emerge, different alleles prevailing independently of the type of selection or environmental variables.

In order to distinguish between these four hypotheses, we first reviewed all the locations where Ester2 has been reported, to establish if it is the only Ester resistance allele present and whether it occurred first or appeared later. Secondly, we present detailed studies from one particular area (Montpellier, southern France) where Ester2 was not the first Ester resistance allele to occur, in order to document its possible evolution at the population level.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Recorded data

To find all data available on Ester2, we performed literature searches in several databases for recently published studies, and then used cited literature to find older studies. As the nomenclature concerning resistance alleles at this locus has not been stable and consensual, all known designations were considered (Ester2, Est3A, A2–B2, estα21 and estβ21). Unpublished data, i.e. samples not formally and fully presented in a publication but available from the laboratory, were also used.

Mosquito sampling

Pupae and larvae were collected along a 50 km northwest-southeast transect crossing the treated-untreated boundary near the Montpellier area and studied previously (Guillemaud et al., 1998; Lenormand et al., 1999). This cline was sampled in July 1999 (n = 411), July 2001 (n = 736) and August 2002 (n = 521). Locations of samples are described in Fig. 2. Pupae and larvae were reared in the laboratory until emergence, and then adults were frozen and stored at −80 °C for further analyses. Data from the same transect, sampled in summer 1986 and 1991 (n = 354 and 217, respectively, Guillemaud et al., 1998), in summer 1995 (n = 1203, Lenormand et al., 1998b) and in summer 1996 (n =512, Lenormand & Raymond, 2000), were used for an overall comparison.

image

Figure 2. Sample site locations in the northwest southeast transect in the Montpellier area. Samples are indicated with black circle. The dashed line represents approximately the border between treated and untreated areas (modified from Lenormand et al., 1999). C. pipiens is present in the whole area.

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Identification of overproduced esterases

For each mosquito, the thorax and abdomen were used to detect overproduced esterases using starch-gel electrophoresis (Tris–Malate–EDTA 7.4 buffer, Pasteur et al., 1988) and to identify resistance alleles at the Ester locus. Heads were used to establish the phenotype at the ace-1 locus; these data will be reported elsewhere. Overproduced esterases are dominant over non-overproduced esterases under our electrophoretic conditions. Thus, this method does not allow complete genotype identification. See Lenormand et al. (1998b)) for the correspondence between each genotype and its corresponding phenotype detected by starch gel electrophoresis.

Descriptive statistics

To test for the existence of frequency clines for each resistance allele along the transect and in order to compare frequency patterns among years, phenotypic data were fitted to a descriptive cline function. Frequency clines for each resistance allele i (i = 1, 2 or 3, respectively for Ester1, Ester2, and Ester4) at time j (j = 1986, 1991, 1995, 1996, 1999, 2001 and 2002) were simultaneously fitted to a scaled negative exponential

  • image

where x is the distance from the coast and hij, bij and aij are the estimated parameters (Lenormand & Raymond, 2000). bij and aij describe rates of decline in allele i frequency at time j with distance and with the square of distance from the coast, respectively. hij is the frequency of resistance allele i at time j and at x = 0 (i.e. at the coast). In order to test for trends in allelic replacements over the period 1986–2002, we supposed that hij values followed a logistic function of time

  • image

where t1j is the number of years after 1986 when the year of sampling is before 1986 + t* and is t* otherwise. t2j is the number of years after 1986 + t*. αi, βi, γi and t* are estimated parameters. Overall change in frequency over the 1986–2002 period is measured for each resistance allele i by αi and βi, which measure the rate of frequency change between 1986 and 1986 + t* and between 1986 + t* and 2002, respectively. We introduced t* to allow for changes in the rate of allele replacement due to the appearance of Ester2 allele. Parameter γi is related to the initial frequency hi0 of each allele i as: hi0= exp (γi)/[1+ exp (γi)]. The occurrence of allele replacement was tested by comparing models where αi and βi are estimated or set to zero. Changes in rates of allele replacement due to the presence of Ester2 allele were tested by comparing models where αi and βi are estimated independently or constrained such that αI = βi.

Expected phenotypic distributions were computed using allelic distributions and assuming each locus to be at Hardy–Weinberg equilibrium, at each location. The phenotype was considered to be a seven-state random variable. The log-likelihood of a sample was computed from the phenotypic multinomial distribution. Let nij and fij be the observed number and expected frequency of individuals having phenotype i in population j, respectively. The log likelihood L of observing all the data is proportional to

  • image

It was maximized for parameters joint estimation, using the Metropolis algorithm (see Lenormand et al., 1999,1998b; Lenormand & Raymond, 2000). Model simplification was performed using likelihood ratio tests corrected for over-dispersion (Lebreton et al., 1992; Anderson et al., 1994). Over-dispersion was computed from the full model as the ratio of residual deviance over residual d.f. We computed the percentage of total deviance explained by a model (%TD) as

  • image

where the maximal deviance (Dmax) is obtained by estimating the allele's average frequency among populations and the minimal deviance (Dmin) by estimating the allele's frequency independently in each population.

Departure from Hardy–Weinberg proportions F at the Ester locus was tested (and Bonferroni corrected) in each population (comparing models where F is either estimated or set to zero) and overall (comparing models where F is estimated for each population, averaged over populations or set to zero for all populations) by likelihood ratio tests. Testing for departure from Hardy–Weinberg proportion is possible at the Ester locus because seven phenotypes can be observed on electrophoresis gels for four alleles (all alleles are co-dominant markers except the susceptible allele, which is recessive, as for O in ABO blood groups).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Literature data

All data available on the distribution of Ester2 are summarized in Table 1. The first confirmed report of this gene dates from Liberia and Nigeria in 1977 (Villani et al., 1983; Urbanelli et al., 1985), although the earliest report could possibly be in 1970 in Japan (Yasutomi, 1970). Its present distribution encompasses Africa, Asia, Europe, North and South America, and French Polynesia (Fig. 3).

Table 1.  Geographic distribution of Ester2 within each geographic area.
Geographic areaCountryYear of first observationEster allelesReferences
  1. The country of first observation is underlined. Putative years of first observation are indicated with a question mark (see text). The presence of other resistance alleles of Ester locus is also indicated.

Europe
 WesternItaly, Greece, Spain, Portugal and France1984Ester4Ester1Ester5Bonning et al. (1991), Chevillon et al. (1995a), Chevillon et al. (1995b), Chevillon et al. (1995c), Eritja & Chevillon (1999), Gazave et al. (2001), Georghiou et al. (1988), Guillemaud et al. (1998), Rivet et al. (1993), Severini et al. (1994), Silvestrini et al. (1998), Villani & Hemingway (1987), and Unpublished data
 EasternCyprus1987Ester1Ester5Georghiou et al. (1987), Wirth & Georghiou (1996)
Asia
 Southern–westSri Lanka, Pakistan and India1978 Georghiou (1992), Georghiou et al. (1987), Peiris & Hemingway (1993), Villani et al. (1983) and Unpublished data
 Southern–eastThailand, South Korea, China and Vietnam1980? 1981EsterB1Ester8Ester9EsterB6EsterB7Callaghan et al. (1998), Georghiou et al. (1987), (1988), Jinfu (1999), Liu et al. (2000), Maruyama et al. (1984), Pasteur et al. (2001), Villani et al. (1983), Weill et al. (2001), and Xu et al. (1994)
 Japan1970? 1980 Georghiou (1992), Georghiou et al. (1987), Maruyama et al. (1984), Yasutomi (1970) and Yasutomi (1983)
 Middle EastSaudi Arabia and Israel1983Ester1Georghiou et al. (1987), Hemingway et al. (1990), (2000), Vaughan et al. (1997) and Villani et al. (1986)
Africa
 WesternLiberia, Nigeria, Burkina–Faso, Cape Verde, Niger, Senegal, Ivory Coast, Mali, Mauritania and Congo1977 Beyssat-Arnaouty et al. (1989), Callaghan et al. (1998), Chandre et al. (1997), Magnin (1986), Magnin et al. (1988), Majori et al. (1986), Urbanelli et al. (1985), Villani et al. (1983) and Unpublished data
 SouthernTanzania, Kenya, Zimbabwe and South Africa1979 Callaghan et al. (1998), Curtis & Pasteur (1981), Guillemaud et al. (1996), Vaughan et al. (1997), Villani et al. (1983) and Unpublished data
 NorthernEgypt, Tunisia and Algeria1983Ester4EsterA9Ester5Ben Cheikh et al. (1998), Ben Cheikh & Pasteur (1993), Villani et al. (1986) and Unpublished data
America
 North AmericaUSA (Louisiana, New–Mexico, Texas, California, New Jersey and Tennessee)1984EsterB1Georghiou et al., 1987, 1988; Pasteur & Georghiou, 1989; Pietrantonio et al. (2000), and Raymond et al. (1987)
 South AmericaBrazil, Venezuela and French Guyana1991Ester6EsterB1Gonzalez et al. (1999), Qiao & Raymond (1995) and Yébakima et al. (1995b))
 Caribbean islandsMost countries (Martinique)1988EsterB1Small et al. (1998), Yébakima et al. (1995a)) and Yébakima et al. (1995b))
Oceania
 PolynesiaFrench Polynesia (Society Is., Tuamotu Is., Australs Is.)1990EsterB1Pasteur et al. (1995)
image

Figure 3. Global distribution of Ester2. Stars (*) show the different places where this resistance gene has been reported (see references in Table 1).

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In some areas, Ester2 is the only resistance allele reported at the Ester locus, such as the sub-Saharan Africa. In other places, one or several resistant alleles are present, in addition to Ester2, e.g. China or in southern France. In the latter situations, different Ester alleles are competing, and allelic replacement among resistance alleles is possible. In Asia, North America, South America, Caribbean's and French Polynesia, it is most often found with EsterB1. In Mediterranean countries, Ester2 is present with different alleles: Ester4 mainly in occidental parts, Ester5 mainly in oriental ones, and Ester1 in all the Mediterranean Basin. Countries with the most diverse esterase-mediated insecticide resistance are in Asia, with five resistance alleles described in addition to Ester2: EsterB1, Ester8, Ester9, EsterB6 and EsterB7. There is several other resistance alleles not yet well characterized which are found together with Ester2: one in Tunisia (named A9 in Ben Cheikh et al., 1998) and one in Brazil and Venezuela (described as A6–B6 by Yébakima et al., 1995b; Gonzalez et al., 1999).

Thus, on a world scale, Ester2 is in competition with all the reported alleles of the Ester locus. In the majority of cases, Ester2 is found at relatively high frequencies (>0.2), and often being the most prevalent resistance gene. There is one exception in the western Mediterranean, where it is found at low frequencies (Severini et al., 1993; Guillemaud et al., 1998; Eritja & Chevillon, 1999).

Information on the process explaining the distribution of Ester2 and its evolutionary dynamics are limited. Indeed the new occurrence of Ester2 in a particular region is seldom documented except in France, where its occurrence is closely monitored. Rivet et al. (1993) showed that Ester2 was first detected in southern France in 1986, near the international airport and seaport of Marseille. Then it spread in southern France, and was first detected in the Montpellier area in 1990, where it remained at a low frequency (0.03 in 1995, <0.05 in 1997, Guillemaud et al., 1998).

Experimental data

In order to extend the monitoring (data from 1986 to 1996), the Montpellier area was sampled in summers 1999, 2001 and 2002 (Table 2). Cline models were fitted for resistance alleles frequencies. We first checked if there was no departure from Hardy–Weinberg proportions before using this assumption for estimating allelic clines.

Table 2.  Sampling results for Ester phenotypes.
SiteKilometers from the sea199920012002
n[0][1][2][4][12][14][24]n[0][1][2][4][12][14][24]n[0][1][2][4][12][14][24]
  1. For each year the numbers of individuals presenting each phenotype are indicated: for nomenclature, see Lenormand et al. (1998b)). Briefly, phenotype [i] corresponds to genotypes Esteri/Ester0 or Esteri/Esteri, and phenotype [ij] correspond to genotype Esteri/Esterj. n represents the total number of mosquitoes analysed for each year at each sampling site. (–) Indicates that the site was not sampled in the corresponding year. See Fig. 2 for details of the sampling location.

Pérols2.25635636042
Maurin55845336262784364318135832834137
Lattes5.258236311695843332367
Orstom1458253226020
Montferrier17125205000
Distill185816403403158152232142
StGely2160192133122
Cuculles265816532903258211135000
Viols30582911270005837101702158325219000
SML34583701190106929203503058353117011
NDL35584130120205828032302158271029001
Brissac4358431211001
StBauzille44512820190205836201901057251030010
Ganges49583521190016849411400058273223120
Total 4112041881632133737282322832533432521189211925161718
Hardy–Weinberg equilibrium

Hardy–Weinberg equilibrium was not rejected in any population. Only one population displayed a significant deficit in heterozygote before Bonferroni correction (Pérols population, 2001 transect, F = 0.707, inline image = 6.12, P < 0.05). Allelic frequencies for each population and each year are presented in Table 3. Each year, overall Hardy–Weinberg proportions were not rejected either (for each cline the best model is an average F equal to zero, F-test, P > 0.05 in each case).

Table 3.  Estimated frequencies for each population.
Site19951996199920012002
Ester1Ester2Ester4FisEster1Ester2Ester4FisEster1Ester2Ester4FisEster1Ester2Ester4FisEster1Ester2Ester4Fis
  1. For each year and each population the frequency of each Ester allele, and the estimated Fis is indicated. Bold numbers identify significant Fis (likelihood ratio tests, but see text). (–) Indicates that the site was not sampled in the corresponding year. See Fig. 2 for details of sampling location.

Brissac0.0090.0260.1090.000
Cournonteral0.1500.0160.3720.0000.000
Cuculles0.0720.0440.3560.2970.0090.0090.3720.000
Distill0.0620.0090.4110.0000.0610.0440.4090.000
Ganges0.0110.0000.1390.0000.0240.0060.1380.0000.0170.0170.1900.0250.0300.0070.1090.0000.0530.0260.2460.000
Lattes0.1400.0000.4810.0910.1520.5480.2950.1080.1170.5320.000
Maurin0.1090.0030.4760.0000.0000.1190.0630.5270.4380.0800.1370.5670.0000.0530.1530.5240.472
Montferrier0.0880.0000.2400.000
NDL0.0170.0060.1560.0030.0160.0000.1160.0000.0440.0000.1280.0000.0180.0360.2600.0000.0090.0090.3040.000
Orstom0.0440.0170.2810.348
Perols0.1410.0290.4270.0000.0000.1280.1140.5200.707
SML0.0290.0030.1680.0000.0080.0080.1970.8560.0090.0090.1900.0000.0370.0000.3270.0000.0350.0170.1790.000
St Bauzille0.0390.0040.1560.0510.0240.0000.1620.0000.0400.0000.2310.0000.0260.0000.1900.0000.0180.0000.3240.001
St Gely0.0790.0050.2460.6210.0870.0000.2930.0420.0340.3790.000
Triadou0.0660.0000.3890.000
Viols0.0430.0080.2030.0000.0090.0090.2700.0000.0260.0090.1880.0000.0440.0170.1810.000
Sumène0.0230.0000.145
Descriptive models

A decrease in allele frequency from the coast inland was observed for all resistance alleles (i.e. slope parameters a and/or b different from zero in the best model after model simplification, F-test, P < 0.05 in each case) and each year, except for the Ester2 allele, which presents a clinal pattern only since 1999. The majority of cline models were best fitted by an exp(−x) shape, with the exception of Ester4 cline of year 2001 and Ester1 cline of year 1995, which were best fitted by an exp(−x2) shape. The estimated parameters for allelic clines are consistent with those obtained in previous studies (see Table 4 and Guillemaud et al., 1998; Lenormand et al., 1998b; Lenormand & Raymond, 2000). Over-dispersion is low around fitted models, between 0.99 and 1.57. All fitted models explain more than 50% of the total observed deviance (see Table 4).

Table 4.  Parameters of the best-fitted clines.
YearAllele Ester1Allele Ester2Allele Ester4TD (%)
ha   ×  10−4b   ×  10−2hb   ×  10−4b   ×  10−2ha   ×  10−4b   ×  10−2
  1. The estimated value of parameters hij, aij and bij of the best model after likelihood ratio tests simplification procedure are given for each allele and each year. TD (%) is the part of total deviance explicated by the model (see text). (–) Means that this parameter is not significantly different from zero in the best model for the year considered, see text.

19950.12910.230.0080.5152.91570.7
19960.1914.8130.0020.4862.59159.9
19990.1274.5040.076.3390.5012.45150.6
20010.13.3010.1526.5730.5516.70273.4
20020.0872.8560.1957.610.5832.07260.9
Time variations of allelic frequencies

After model simplification, the best model describing frequency variations over the 1985–2002 period explains 62.05% of the total deviance with low over-dispersion (= 1.29). This model indicates that (i) Ester1 changes in frequency at a constant (α1 not different from β1, F1,327 = 3.52, P = 0.062) and negative (α1 = −0.1452, F1,327 = 61.38, P < 0.0001) rate, (ii) Ester2 changes in frequency at a constant (α2 not different from β2, F1,327 = 1.35, P = n.s.) and positive (α2 = 0.3835, F1,327 = 42.26, P < 0.0001) rate, (iii) Ester4 frequency changes at different rates before and after ∼1996 (t* =9.84, tmin = 9.43, tmax = 10.31, α4 different from β4, F1,327 = 19.11, P < 0.0001): it first increases (α4 =0.3016, F1,327 = 47.15, P < 0.0001) and then remains constant (β4 not different from zero, F1,327 = 1.02, P =n.s.). The fitted frequency variations are illustrated on Fig. 4.

image

Figure 4. Evolution of the insecticides resistant alleles in the Montpellier area since the beginning of the organophosphate treatments. The frequencies presented are the maximum frequencies, i.e. at the coast. The first allele that appeared was Ester1 (black squares), then replaced by Ester4 (white rounds) (Guillemaud et al., 1998). Recently, a third allele, Ester2, invaded the Montpellier area (white triangles). Dotted lines represent the fitted values according to the best logistic model (see material and methods).

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This overall pattern is consistent with previous studies of the decade 1985–1995, during which Ester2 was almost absent from Montpellier area, and has been detailed by Guillemaud et al. (1998). It corresponds to the replacement of Ester1 by Ester4 allele. However, in the most recent period (i.e. after ∼1996), Ester4 has not increased further in frequency, whereas Ester2 frequency increased rapidly. Ester2 was first reported in 1990 and remained at low frequency (h < 0.01) without a clear clinal pattern until 1999. Over the period 1999–2002, however, Ester2 increased sharply in frequency (α2 = 0.3835) and started to exhibit a clinal pattern (Table 4 and Fig. 5), almost reaching the frequency of 0.2 at the coast in 2002.

image

Figure 5. Recent evolution of Ester2-frequency clines in the Montpellier area. Clines of 5 years, over a period extending from 1995 to 2002, are presented: 1995, 1996, 1999, 2001 and 2002. See text for explanations.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Global distribution

Ester2 is widely distributed over the planet, as it has been reported in most places investigated. The actual geographic distribution probably corresponds to areas where OP insecticides are regularly applied. This is indicated by the disappearance of Ester2 whenever OP insecticides are no longer used. In Spain, OP insecticides were replaced by Bacillus sphaericus toxin the year after the first report of Ester2 (1993), and this allele was not found in later samples (Eritja & Chevillon, 1999). In Lucca, Italy, insecticide treatments were stopped in 1989, and Ester2, which was detected in 1988, no longer found in 1992. Across Italy, Ester2 was reported from places where OPs were continuously used, and was not detected in places where treatments have been stopped (Silvestrini et al., 1998). This suggests that a fitness cost is associated with Ester2, thus precluding the presence of Ester2 at high frequencies in non-treated areas.

Dynamics of Ester2 invasion

The current geographical distribution of Ester2 could be explained by four non-exclusive hypotheses. First, Ester2 could have been the first resistance gene to occur at the Ester locus, thus having an opportunity to spread widely, before other resistance alleles appeared (historical contingency hypothesis). Alternatively, Ester2 may possess a fitness advantage over the other resistant alleles, either locally (i.e. only in some environmental conditions) or globally (i.e. in all areas), and its present distribution would be the result of its competitive advantage (local or globalselection hypotheses). Third, it may be neutral with respect to other resistance genes, and chance alone explains this distribution (drift hypothesis). It is probably impossible to reject the historical contingency hypothesis, due to the lack of data during the 1960s and early 1970s, i.e. at a time where the first Ester resistance gene occurred. The pattern of replacement in resistance alleles and their global distribution do not suggest that genetic drift between resistance alleles (i.e. neutrality among resistance alleles) plays a predominant role: drift would generally create random fluctuations, which are not observed at a local scale (for example in the Montpellier area). Moreover, due to the large population sizes observed for this species, the time needed for a replacement between two resistance alleles (e.g. 7 years, Guillemaud et al., 1998) would be much longer than that observed if the process was driven by genetic drift only. Longitudinal studies are required to evaluate the selection hypotheses. To our knowledge, only eight cases are documented in the literature (Table 5), and five of them document the interaction of Ester2 with another resistance allele. In California, only EsterB1 was reported before 1979 (Pasteur & Georghiou, 1980). Then, Ester2 was also detected in the same populations in 1984, sometimes at a relatively high frequency (>0.5 at Long Beach in 1985, Raymond et al., 1987), suggesting that the replacement of EsterB1 by Ester2 was going on. Unfortunately no additional published data are available to document the evolution of this situation. In Cuba, the same process has apparently taken place: EsterB1 was first present, then Ester2 was detected, and its frequency rose and it became the most frequent resistance gene (Small, 1996). In Houston (Texas), Ester2 is also replacing EsterB1. In 1994, 20% of analysed mosquitoes were found carrying EsterB1, 40%EsterB1 and Ester2, and 40% only Ester2 (n = 104), whereas in 1998, the frequencies were respectively (n = 84) 11.5%, 11.9% and 68.7%, with 7.1% being susceptible mosquitoes (Pietrantonio et al., 2000). The authors explain the replacement by a decrease, since 1993, in malathion (an OP) use: both the number of areas treated and the number of applications decreased, to very low levels (even zero in some areas). In Martinique, the opposite situation is found: in 1990, Ester2 was at a high frequency (>37% in average, >66% in one population), while EsterB1 was very infrequent (<3%) and not found in three of seven populations sampled. Nine years later, EsterB1 had spread to all the sampled populations, with an overall frequency >30%, whereas the susceptible allele has decreased from >60 to <15%. In the same time, Ester2 decreased in some populations (e.g. from >60 to <30% in Carbet population), while increasing in few others (e.g. from <40 to >90% in Fort de France, alleles frequencies were computed using maximum likelihood from phenotype data provided in Yébakima et al., 2004). At the locations where EsterB1 has replaced Ester2, the authors explain this situation by a change of insecticide usage: during the increase of EsterB1 frequency, the intensity of insecticide treatment also increased (∼10-fold) and the authors show that EsterB1 confers a higher resistance to insecticides used in Martinique (Temephos) than Ester2. In Lucca (Italy), there is a possible replacement of Ester1 by Ester2 around 1985–1988 (Bonning et al., 1991; Villani & Hemingway, 1987) although published data are too scarce to firmly document it. However, Ester2 was present in 1985 in Lucca (Bonning et al., 1991), but it disappeared in 1992, probably because of the stoppage of insecticide treatments (Severini et al., 1994). Overall, this fragmentary dataset suggests that there is no overall trend, and that Ester2 could either replace or be replaced by another Ester allele. Thus, this dataset contradicts the global selection hypothesis, and is more consistent with the local selection hypothesis. Environmental conditions are probably involved in this interaction, such as the type or quantity of insecticide used. In Martinique, Ester2 decreased in frequency when treatments were increased, whereas in Houston its frequency increased when treatments were decreased. Both situations suggest that Ester2 is at a disadvantage when insecticide treatments are intensive. Its fitness advantage in California and Cuba, in conditions where insecticide doses are reduced, could be explained by lower fitness costs rather than better resistance. However, in most places, little information is available on pesticide application practices, precluding any firm conclusions. Nevertheless, this indicates that environmental conditions play a role during the course of adaptation at the local scale. There is an additional situation where a detailed longitudinal study has taken place, allowing a clearer understanding of the selection hypotheses.

Table 5.  Literature data on allele replacement.
AreaAllele replacementObservations
  1. For Montpellier different replacements occurred through time and are all indicated separately, see text for explanation and references.

CaliforniaEster2 taking over EsterB1No change in insecticide
CubaEster2 taking over EsterB1No change in insecticide
TexasEster2 taking over EsterB1OP quantities decreased
MartiniqueEsterB1 taking over Ester2OP quantities increased
ItalyEster0 taking over Ester2OP insecticides removed
BarcelonaEster0 taking over Ester2OP insecticides removed
MontpellierEster1 taking over Ester0OP introduced
MontpellierEster4 taking over Ester1Temephos partially replacing chlorpyrifos OP quantities decreased
MontpellierEster2 taking over Ester1 and Ester4No change in insecticide

Montpellier area

The Montpellier area is the subject of a long-term longitudinal study for the evolution of the resistance genes since 1972 (Pasteur & Sinègre, 1975; Chevillon et al., 1995c; Guillemaud et al., 1998; Lenormand et al., 1999). Since the 1960s, OP treatments have been used on an area extending from the coast to approximately 20 km. Resistance to these insecticides is conferred in this area by different Ester alleles. The first to appear was Ester1, in 1972, followed by Ester4 in 1984 and then, Ester2, in 1990 (Guillemaud et al., 1998). The Ester1 and Ester4 alleles are known to harbour deleterious effects in the absence of OP insecticides (the so-called ‘cost’ of resistance), because they decline in frequency along transects from treated to non-treated areas (Guillemaud et al., 1998; Lenormand et al., 1999) and they alter fitness-related life history traits (e.g. Berticat et al., 2002a,2004; Agnew et al., 2004). In this study, we show that Ester2 also is costly in the absence of insecticides, as this allele declines in frequency from the treated coastal area to untreated inland areas (Fig. 5). During the nineties, Ester4 has replaced Ester1 (Fig. 4, Guillemaud et al., 1998). This increase in the frequency of Ester4 also corresponds to an environmental change, as the quantities of OP decrease dramatically from about 2700 L applied per year in the Languedoc–Roussillon region, to about 300 L/year from 1987 to 1992, and to a change from chlorpyrifos to temephos (EID, 1992). As Ester4 is known to confer a slightly lower OP resistance level, its advantage over Ester1 could possibly be due to lower costs (Guillemaud et al., 1998).

Although the appearance of Ester2 in southern France was documented in 1986, it was detected in the Montpellier area for the first time in 1990, at a low frequency (Rivet et al., 1993). It remained at a low frequency (<0.1) during the following years (Guillemaud et al., 1998; Gazave et al., 2001). We report here that it increased in frequency since 1996, reaching a frequency of 0.2 in treated areas. Since the increase in frequency of Ester2, the frequency of Ester1 has continued to decrease at the same rate. During the same period, the frequency of Ester4 stabilized, after a sharp increase during the period 1986–1996. Although the data seems to indicate a slow increase, we did not detect a significant variation over the 1996–2002 period and strongly rejected the idea of a slower but continuous rise of Ester4. Moreover, the higher frequency in 1995 is not an artifact as more than 1200 mosquitoes were analysed for that particular year. Thus, overall, the replacement of Ester1 by Ester4 started in 1986 and stopped in 1996, with Ester2 now replacing both Ester1 and Ester4. This pattern of replacement clearly indicates that Ester2 enjoys a competitive advantage, at least locally, over previously prevalent resistance alleles. This change in the course of allele replacement is, however, not correlated with any noticeable change in insecticide treatment practices since 1996. This result confirms that selection plays a major role on the evolution of Ester resistance alleles and strengthens the local selection hypothesis. It also emphasizes the role of treatment practices in this evolutionary dynamic, at least in the replacement of Ester1 by Ester4.

Four advantages could exist for Ester2. First, it could provide better protection against the insecticide used, as the levels of resistance to OP insecticides used in the Montpellier area (temephos and chlorpyrifos) are higher for Ester2 than for Ester1 or Ester4 (Raymond et al., 1986; Wirth et al., 1990; Poiriéet al., 1992). Second, Ester2 may confer lower fitness costs than other Ester resistance alleles. Comparisons of life history traits have revealed that mosquitoes bearing Ester2 fare better than those with other resistance alleles, in particular for larval mortality (Duron et al. unpublished data), and endure a lower density of endosymbiotic bacteria (Wolbachia, Berticat et al., 2002b). These trends are confirmed by population cage experiments in the absence of insecticide, showing that the fitness costs associated with Ester2 are lower than that of Ester1, Ester4 or EsterB1 (Berticat, 2001; Berticat et al., 2004). The nature of the cost displayed by the various resistance alleles is currently unknown, thus the reduced costs associated with Ester2 are also enigmatic. Third, as proposed by Guillemaud (1997) and Berticat (2001) for Ester4 when replacing Ester1, Ester2, having a greater gene copy number, may recombine less than the other alleles with the locus ace-1 (same linkage group), whose resistance allele (ace-1R) confers a much higher level of insecticide resistance. Thus, Ester2 would be favoured by hitchhiking with ace-1R resistance gene. This hypothesis is currently under consideration. Lastly, the advantage of Ester2 could be indirect, and due to another gene located in its amplicon, coding for an aldehyde-oxydase (Hemingway et al., 2000). According to these authors, this gene is thought to provide resistance against a carbamate insecticide (aldicarb), although this effect has not yet been shown. The effect of a co-amplified gene remains unclear for any advantage it may confer, although it may enhance fitness costs, through biochemical perturbation due to over-expression. Further comparative data between the various Ester resistance alleles could settle this issue.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

The allele Ester2 is found globally and its current distribution probably reflects the areas where OP insecticides are regularly used. There are four possible explanations for this prevalence: the historical contingency, the drift and the global and localselection hypotheses. The data available in the literature, although not refuting the historical contingency hypothesis, show that selection is also at work, as in five cases Ester2 has replaced one or several previous Ester resistance alleles. Drift between resistance alleles does not appear to be a major mechanism in this process. A long-term longitudinal study the Montpellier area has found evidence that Ester2 is taking over previously prevalent resistance alleles (Ester1 and Ester4). So, at least in southern France, this allele confers a sufficient fitness advantage to be selected for in conditions where other resistant alleles were already present at high frequencies, offering a unique opportunity to distinguish between selection, drift and historical events at the gene level. Different types of advantages could explain the selection of Ester2, although the data are currently inconclusive, and it is not excluded that several coexist. However, in Martinique another allele is currently replacing Ester2, showing that the selective advantages of Ester2 do not appear to be constitutive and are probably dependent on environmental conditions, weakening the global selection hypothesis. The data suggest that Ester2 may be the best allele so far, but only in moderately treated areas. This conclusion is consistent with the local selection hypothesis. The global distribution of this allele would then simply reflect the fact that most treated areas are now moderately treated. The traditional way of using insecticides is a high dose strategy, which favours alleles conferring strong resistance despite a high cost (Denholm & Rowland, 1992). Modern practices are more refined, thus decreasing insecticide quantities, with the consequences that an alternative resistance allele becomes fitter. This must be taken into account in insecticide treatment strategies. The importance of environmental conditions should also be considered when studying the evolution of such adaptations to anticipate and predict the long-term outcomes. In the case of insecticide resistance, an increased knowledge of pesticide applications across the planet is required to improve our understanding of the process of allele replacement at local and global scales.

Allele replacement is not an instantaneous process: it took approximately 7 years for Ester4 to become prevalent over Ester1 (Fig. 4) and Ester1 is still present at a low frequency 14 years later (Guillemaud et al., 1998; this study). So, in countries where Ester2 was the only allele described during the 1980s (e.g. Sub-Saharan Africa and southern-western Asia, see Table 1), allele replacement is probably not yet complete. In these regions, Ester2 was probably the first allele to appear and spread and, there, this historical event contributed to its present prevalence.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

We are very grateful to P. Agnew, C. Berticat, N. Pasteur, A. Porter and M. Weill for helpful comments on the manuscript, C. Bernard, A. Berthomieu, C. Berticat, M. Marquine, 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). Contribution 2005–015 of the Institut des Sciences de l'Evolution de Montpellier (UMR CNRS-UM2 5554).

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  4. Material and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
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