The Journal of Heredity 2001:92(4)
© 2001 The American Genetic Association 92:349-351
Brief Communication |
Recombination Between Two Amplified Esterase Alleles in Culex pipiens
From Team Genetics of Adaptation, Laboratoire Génétique et Environnement, Institut des Sciences de l'Evolution (UMR CNRS 5554), Université de Montpellier II (C.C. 065), F-34095 Montpellier cedex 05, France.
Address correspondence to Claire Berticat at the address above or e-mail: berticat{at}isem.univ-montp2.fr.
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Abstract |
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Esterase gene amplification at the Ester superlocus provides organophosphate resistance in the mosquito Culex pipiens (L.). In this study we explored the possibility of recombination between two amplified esterase alleles, thus generating a composite amplified allele. To do that, females heterozygous for two distinct amplified alleles (Ester2 and Ester4) were crossed with males homozygous for a third resistance allele (Ester8). Among analyzed offspring, one recombinant composite allele (Ester2–4) was detected, providing a rate of recombination of approximately 0.2%. This is the first report of a recombination between two distinct amplified esterase alleles. This phenomenon renders the predictability of allele evolution considerably more complex than was previously thought.
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Introduction |
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General models of population genetics that try to infer the outcome of adaptive genes are often based on simple approximations, like the existence of one or two loci with two alleles. Concerning resistance to organophosphorous (OP) insecticides in Culex pipiens mosquitoes, the adaptive system is more complex. The Ester superlocus, which is one of the main genome areas involved in this resistance (Lenormand et al. 1998), presents multiple resistance alleles. This superlocus is in fact composed of two loci on chromosome II, Est-3 and Est-2, separated by an intergenic DNA fragment of 2=n6 kb (Guillemaud et al. 1997; Heyse et al. 1996; Rooker et al. 1996) and both loci encode for detoxifying esterases A and B, respectively. The resistance conferred by Ester is due to an esterase overproduction that is the result of two nonexclusive mechanisms: gene amplification of one (Est-2) or both loci, or change in gene regulation (for a review, see Raymond et al. 1998). Six Ester alleles involved in resistance have been described: four correspond to the coamplification of both Est-2 and Est-3 loci (Ester2, Ester4, Ester5, and Ester8, encoding esterases A2-B2, A4-B4, A5-B5, and A8-B8, respectively), one corresponds to the exclusive amplification of Est-2 (EsterB1, encoding esterase B1), and one corresponds to an upregulation of Est-3 (Ester1, encoding esterase A1). Two other amplified esterase genes have been reported, but direct comparison with the above alleles is needed to confirm that they are distinct alleles (Vaughan et al. 1997; Xu et al. 1994). Est-3 and Est-2 loci have always been found in maximal linkage disequilibrium for alleles involved in resistance in field studies (see review in Chevillon et al. 1999; Raymond et al. 1998), which justifies the concept of Ester superlocus.
Recently molecular data have confirmed that the amplification level of amplified alleles is variable in natural populations (Callaghan et al. 1998; Weill et al. 2000). Moreover, level of resistance, esterase activity, and amplification covary so that the evolution of the amplification level can take place under selection (Guillemaud et al. 1999). The mechanism generating the amplification or the variability of copy number is currently unknown. We report here a laboratory experiment which shows that recombination between distinct amplified alleles is possible. To do that we crossed mosquitoes between three strains homozygous for different amplified alleles. We searched for recombinants characterized by the coexistence of the three overproduced esterases.
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Materials and Methods |
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Insects
Three laboratory mosquito strains were used: MAO from China (Qiao et al. 1998), SELAX from California (Raymond et al. 1987), and VIM from France (Poirié et al. 1992). They are homozygous for Ester8, Ester2, and Ester4 alleles, respectively. These strains display the overproduced esterases A8-B8, A2-B2, and A4-B4, respectively.
Crossing
Mass crossings between VIM females and SELAX males were performed in order to obtain F1 heterozygous mosquitoes Ester4/Ester2. The F1 females were then crossed with males of the MAO strain (Ester8/Ester8). The offspring of this cross were then reared to adult and deep frozen for further analyses.
Identification of Esterase Phenotypes
Esterase phenotypes were established by starch-gel electrophoresis in TME 7.4 buffer systems as described by Pasteur et al. 1988. Esterases A2-B2, A4-B4, and A8-B8 present clearly distinct electrophoregrams, that is, each of them has at least one of its two spots (esterase A and B) which has different electrophoretic mobility from the others. This allows us to easily detect individuals possessing all three.
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Results and Discussion |
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Of the 548 mosquitoes analyzed from the cross {female}(VIM*SELAX) * {male}MAO, 547 displayed an heterozygote phenotype, corresponding to Ester4/Ester8 and Ester2/Ester8 genotypes (in frequency, 0.46 and 0.54, respectively, not different from a 1:1 ratio, binomial exact test, P > .05). One mosquito displayed a phenotype with A2-B2 (Ester2), A4-B4 (Ester4), and A8-B8 (Ester8), as shown in Figure 1. It must be noted that this phenotype does not result from a mixture between the two flanking samples because the A8 and B8 bands are really less intense than would be expected from a mixture scenario. Thus this situation is easily interpreted by a recombination between Ester4 and Ester2 in the F1 females, generating a composite allele with both types of amplified alleles, designated Ester2–4 (Figure 2). This is the first evidence that there is recombination between two different amplified esterase alleles.
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The rate of recombination between amplified alleles observed here (1/548
0.2%; 95% confidence interval 0.0007–0.004) is probably an underestimation. This is because if the recombination occurs at a distal part of one of the amplified alleles involved, then the composite recombinant allele might produce an insufficient amount of one esterase type for detection by starch-gel electrophoresis. Thus with this technique the phenotype of individuals resulting from such distal recombination could be misinterpreted. Composite alleles, that is, different amplified esterase alleles within one amplification, have never been observed in field populations. This is probably not because intra-amplification recombination is rare, as the present estimate is relatively large. This absence of detection is best explained by the paucity of geographic areas where at least three different resistance alleles at Ester could be simultaneously detected in a single mosquito. Formation of a composite recombinant allele must occur in gametes of a heterozygote for two distinct amplified alleles. Its detection is then possible by starch-gel electrophoresis analysis of the offspring of this individual, provided that they carry a third distinct resistance allele, since susceptible esterases are not detectable in the presence of overproduced ones. Finally, the probability of detection of a composite allele would be maximized when the formation of both types of individuals described above (heterozygotes for two distinct amplified alleles and their offspring with a third distinct resistant one) is maximized, that is, for equifrequency of the three resistance alleles. A very low frequency of one of the three alleles could then compromise the detection of one recombinant.
Consequently no composite amplified allele could be detected in the Americas and Polynesia, where only Ester2 and EsterB1 were reported, in Africa, and in most parts of Asia where only Ester2 was reported (Georghiou 1992; Qiao and Raymond 1995; Raymond et al. 1991, 1998; Raymond and Pasteur 1996; Yébakima et al. 1995). Mediterranean countries and China are the only known areas where at least three resistance Ester alleles coexist (Chevillon et al. 1999; Raymond et al. 1998). In China, despite this favorable situation (Qiao et al. 1998; Xu et al. 1994), the rarity of population studies did not give a chance to detect any putative composite allele. In northern Italy there are three amplified alleles (Ester2, Ester4, and Ester5) plus the nonamplified resistant one (Ester1) (Silvestrini et al. 1998). If we assume that recombination occurs through unequal recombination between amplicons, then recombination involving Ester1 will be extremely rare because this allele is composed of a single unit, that is, one amplicon (the esterase overproduction of this allele is the result of a regulatory mutation; Rooker et al. 1996). Moreover, esterases encoded by Ester4 and Ester5 have a similar electrophoretic mobility (Poirié et al. 1992), such that a putative composite allele Ester4–5 could not be identified by starch-gel electrophoresis. Thus composite alleles that might be detected in Italy would be either Ester2–4 or Ester2–5 (indistinguishable from one another), provided that the other allele in the mosquito is Ester1. However, studies by Silvestrini et al. (1998) have shown that these three alleles coexisted only in two samples and that they were far from equifrequency with a very low frequency for Ester2. This explains why no Ester2–4/Ester1 (or Ester2–5/Ester1) heterozygote was detected. Ester1, Ester2, and Ester4 coexist in Mediterranean France too, especially around Montpellier, where field population studies have been intensively performed for 25 years. Among about 14,000 individuals characterized so far, the putative composite allele Ester2–4 (which might be detected here provided that the other allele in the mosquito is Ester1) has never been reported (Guillemaud et al. 1998; Lenormand et al. 1998; Lenormand and Raymond 2000). All three resistant alleles are now concentrated within the same costal populations, but Ester2 only recently appeared (1991) so the composite Ester2–4 allele could not be formed before the 1990s (Chevillon et al. 1995; Guillemaud et al. 1998; Lenormand et al. 1998). Since then, Ester4 frequency is greater than 0.4, and Ester2 and Ester1 maximal frequencies are less than 0.02 and 0.10, respectively, such that probability for detecting Ester2–4/Ester1 mosquitoes remains very low, as long as Ester2–4 remains at the frequency of formation.
The relatively low number of resistant alleles at Ester, recorded on a world scale for a pest species with large population sizes, indicates that advantageous mutations (e.g., amplification) at this locus could be limiting (Raymond et al. 1998). However, this paucity of resistance gene is compensated for by a variation of gene copy number for a given amplified allele (Guillemaud et al. 1999; Weill et al. 2000), and by the possible formation of composite alleles (this study). These two phenomena, which constantly generate new types of resistance alleles, cannot be ignored in field studies and could render the predictability of allele frequency evolution considerably more complex than was previously thought.
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Acknowledgments |
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We are very grateful to C. Bernard, and G. Pistre for technical assistance. This work was financed in part by the PNETOX program and the AIP "Génomes et Fonctions" 97/P00183 of INRA. Contribution 2001-051 of the Institut des Sciences de l'Evolution de Montpellier (UMR CNRS 5554).
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Footnotes |
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Corresponding Editor: Ross MacIntyre
Received August 12, 2000
Accepted March 31, 2001
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References |
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