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|Does insecticide resistance alone account for the low genetic variability of asexually reproducing populations of the peach 29, F 35653 Le Rheu Cedex, France3Service Innovations, CETIOM, Thiverval Grignon 78850, FranceCorrespondence: T Guillemaud, Equipe Biologie des Populations en Interaction , UMR 1112, INRA. 400 route des Chappes BP 163, F 06903 Sophia Antipolis, France. Top of pageAbstractThe typical life cycle of aphids includes several parthenogenetic generations and a single sexual generation (cyclical parthenogenesis), but some species or populations are totally asexual (obligate parthenogenesis). Genetic variability is generally low in these asexually reproducing populations, that is, few genotypes are spread over large geographic areas. Both genetic drift and natural selection are often invoked to account for this low Moncler Down Jacket Men Down Vest Black Hood genetic variability. The peach potato aphid, Myzus persicae, which encompasses both cyclical and obligate parthenogens, has developed several insecticide resistance mechanisms as a consequence of intense insecticide use since the 1950s. We collected asexually reproducing M. persicae from oilseed rape and examined genetic variability at eight microsatellite loci and three insecticide resistance genes to determine whether their genetic structure was driven by drift and/or selection. We identified only 16 multilocus microsatellite genotypes among 255 individuals. One clone, which combined two insecticide resistance mechanisms, was frequently detected in all populations whatever their location over a large geographical area (the northern half of France). These unexpected findings suggest that drift is not the unique cause of this low variability. Instead, the intensification of both insecticide treatments and oilseed rape cultivation may have favored a few genotypes. Thus, we propose that selective pressures Moncler Shirt resulting from human activities have considerably modified the genetic structure of M. The annual life cycle of M. persicae includes cyclical parthenogenesis for several generations on herbaceous plants and one sexual generation on peach trees during the winter. By contrast, obligate parthenogenetic aphids have lost the ability to reproduce sexually and remain on herbaceous plants all year long, although they still retain the ability to produce some males (Blackman, 1972). By using hypervariable microsatellite markers or ITS fingerprints, several studies have shown that asexually reproducing populations of M. persicae sampled on herbaceous plants display a low genotypic variability, both within and among populations at a regional scale (Fenton et al, 1998; Terradot et al, 1999; Guillemaud et al, 2003b; Vorburger et al, 2003). These analyses of European and Australian populations also revealed that some clonal genotypes could be repeatedly found in geographically distant (more than 100 km apart) populations. What could be the causes of this low genetic variability? Does it result from selective factors favoring a few genotypes or from stochastic factors randomly acting on all genotypes during the asexual phase of the life cycle. Both drift and directional selection, in combination with clonal reproduction, could explain the coexistence of the limited number of clonal genotypes within populations (Balloux et al, 2003; Bengtsson, 2003). However, the low variability observed at a larger scale (within and among populations) is probably not the result of drift alone, because, being a random process, it would result in different genotypes becoming frequent in different populations (Wright, 1969; Bengtsson, 2003). Recent anthropogenic selection, such as insecticide treatment, is likely to explain the low genetic variability observed at a large scale. Guillemaud et al, 1996; Daborn et al, 2002). M. persicae has evolved at least four insecticide resistance mechanisms in the field: (1) The overproduction of detoxifying esterases, either E4 or FE4, and conferring resistance to organophosphates (OP) and pyrethroids. (2) The modification of the acetylcholinesterase (AChE), the target of OPs and carbamates, providing resistance against dimethyl carbamate insecticides (Moores and Devonshire, 2000). (3) The kdr mutation of the voltage gated sodium channel (Martinez Torres et al, 1999), which is the target of pyrethroids. (4) The rdl mutation of the GABA receptor (Anthony et al, 1998), which is the target of cyclodiene insecticides. The recent acquisition of insecticide resistance genes selected for in large geographical areas, combined with the absence or rarity of sexual recombination, could have led to the low genetic variability observed in M. persicae at a wide geographical scale. The comparison of populations from a large geographic area can be used to test this last hypothesis. Moreover, the analysis of the genetic variability of the putatively selected genes would directly address the selective hypothesis. We adopted this approach by studying the genetic variability of French populations of M. persicae collected on oilseed rape (Brassica napus, known as canola in the USA) using eight microsatellite markers and three insecticide resistance genes. Top of pageMaterials and methodsAphid samplesA total of 22 batches of 13 wingless parthenogenetic females of M. persicae were collected from oilseed rape in France between 19 October and 16 November 2001 (see Table 1 and Figure 1). The sampling period corresponds to the beginning of the oilseed rape cropping season in France. Only one aphid per plant was collected to avoid repeated sampling of aphids from the same colony, and between 13 and 54 plants were randomly chosen in each field. The aphids were stored at 80 until analysis. In three locations, samples were collected in October 2001 from both insecticide treated and untreated fields. In each case, the insecticide was alphamethrin, a pyrethroid insecticide (see Table 2 for details). persicae sampling sites. Boxed names are sites from which samples were used in the microsatellite analysis. When more than one field was sampled per locality, the first number in brackets corresponds to the number of fields sampled and the second number refers to the number of samples that were used in the microsatellite study. Resistance gene characterizationFor each of the 625 aphids sampled, three insecticide resistance mechanisms were analyzed: the class of esterase overproduction and the corresponding esterase allele, the presence of the kdr mutation and the modified AChE. The rdl mutation was not considered because cyclodiene insecticides are not commonly used against aphids in Europe. Single aphids were used both for DNA analysis and biochemical assays as described in Guillemaud et al (2003a). Aphids were homogenized in 100 l of 20 mM phosphate buffer pH 6.5, 0.1% Triton X 100 (PB triton) on ice. One third of the homogenate was used for DNA extraction (Sunnucks and Hales, 1996). The rest of the homogenate was centrifuged for 10 min at 10 000 g. The supernatant was carefully transferred to a new 1.5 ml tube and stored at 80 until biochemical analysis. Esterase overproduction The presence of the E4 and FE4 alleles at esterase genes in individual aphids was determined using a modified version (Guillemaud et al, 2003a) of the PCR RFLP method described by Field et al (1996). Genotypes could not be determined using this method because the esterase loci can be located at several different chromosomal sites (Blackman et al, 1995). The level of E4 and/or FE4 esterase overproduction was estimated for each aphid by using the microplate total esterase assay described by Devonshire et al (1992). This method can distinguish between three classes of aphids: susceptible, R1/R2 and R3 (Devonshire and Field, 1991). Ace genotype determination A fluorometric method was used to determine the genotype at the AChE locus, Ace (Moores and Devonshire, 2000; Guillemaud et al, 2003a). Kdr determination Pyrethroid resistance is correlated with the presence of the kdr mutation, that is, the presence of a T (kdr allele) instead of C (kds allele) at position 322 of the partial cDNA sequence of the para like gene of M. persicae (Martinez Torres et al, 1999). A PCR amplification of specific alleles (PASA) procedure (Sommer et al, 1992) was used to determine individual genotypes (kds/kds, kdr/kds and kdr/kdr) at this position as described by Guillemaud et al (2003a). In the following, the para like gene will be referred to as kdr gene. Microsatellite analysisNine of the 22 samples were subjected to microsatellite analysis to investigate the genotypic variability of M. persicae collected on oilseed rape over the geographic range considered for the insecticide resistance genes study: Le, Ba, Co, Fr, Pa, Su_1, Su_2, Su_3 and SG_2 (see Figure 1). Each aphid was genotyped using eight microsatellite loci: five (Myz25, M37, M40, M55 and M62) were isolated from M. percicae and three (S16b, S17b and S23) from Sitobion miscanthi (Takahashi) (Wilson et al, 2004). The microsatellites were amplified according to Sloane et al (2001) using the PMS1 PCR program. Microsatellite products were then loaded onto an 8% nondenaturing polyacrylamide gel and visualized by ethidium bromide staining as described by Vanlerberghe Masutti et al (1999). Each individual was described by its multilocus genotype (MLG): the allelic combination at the eight microsatellite loci. The observed MLGs were then carefully scored by using an automatic sequencer (ABI Prism 3100, Applied Biosystem) and reference DNAs from P. Sunnucks (La Trobe University, Australia). Analysis of genetic dataGenetic variability Allele frequencies, number of alleles and heterozygosity were calculated using the GENEPOP (ver. 3.3) software (Raymond and Rousset, 1995b). Moncler Kids Down Sleeveless Vest Pink Hardy (H W) equilibrium and linkage equilibrium are not expected if the samples display a clonal structure because of repeated parthenogenetic generations. We therefore tested for these equilibriums to evaluate the relative importance of the successive parthenogenetic generations and of the putative sexual reproduction in shaping the population genetic structure. Linkage disequilibrium (LD) between loci within each population and departure from H W equilibrium at each locus were tested using the pseudo exact test procedures in GENEPOP 3.3. Note that the LD test does not assume HW equilibrium. The spatial genetic structure revealed by the microsatellites was analyzed by estimating FST parameters (Weir and Cockerham, 1984) and by testing for MLG frequencies homogeneity using the Struc program in the GENEPOP 3.3 package. The use of FST to study a species with a mixture of cyclical and obligate parthenogens may be problematic. Indeed, FST can be hard to interpret in asexually reproducing populations where it does not necessarily reflect migration/drift equilibrium because of selection at loci which have not been scored. However, we chose this genetic distance parameter to enable comparisons with other published population genetics studies on aphids (eg Delmotte et al, 2002; Guillemaud et al, 2003b). The Isolde program from the GENEPOP package was used to test for an MLG isolation by distance model. This program performs Mantel tests of independence between the matrix of genetic distance and that of geographic distance.