Laurianne Leniaud

Philippe Audiot

Denis D Bourguet

Brigitte B Frérot

Gilles Genestier

Siu Fai Lee

Thibaut Malausa

Anne-Hélène Le Pallec

Marie-Claude Souqual

Sergine Ponsard
email: ponsard@cict.fr

Genetic structure of European and Mediterranean maize borer populations on several wild and cultivated host plants

Keywords: Lepidoptera, Crambidae, Noctuidae, Ostrinia nubilalis, Sesamia nonagrioides, transgenic insecticidal maize, host races, refuge crop, resistance evolution

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The European corn borer (ECB), Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), and the Mediterranean corn borer (MCB), Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae), are two major polyphagous maize (Zea mays L.) pests whose geographic ranges overlap in western Asia and in Europe south of the 45th parallel [START_REF] Hudon | Biology and population dynamics of the European corn borer (Ostrinia nubilalis) with special reference to sweet corn in Québec. I. Systematics, morphology, geographical distribution, host range, economic importance[END_REF][START_REF] Hudon | Seventy years of European corn borer (Ostrinia nubilalis) research in North America[END_REF][START_REF] Buès | Differences enzymatiques et écophysiologiques entre populations de Sesamia nonagrioides Lefèbvre (Lépidoptère: Noctuidae) originaires de l'Ouest du bassin méditérranéen[END_REF]. Both are targets of the Bacillus thuringiensis (Bt) toxin produced by transgenic Bt-maize varieties. A major concern about such varieties, especially if they are planted densely and over large acreages, is that pest populations may rapidly develop resistance to Bt toxins [START_REF] Bates | Insect resistance management in GM crops: past, present and future[END_REF]. A management strategy aimed at delaying resistance development has therefore been made mandatory in the USA (United States Environmental Protection Agency, 2001) and is under consideration in Europe: the so-called 'high-dose refuge' (HDR) strategy [START_REF] Alstad | Managing the evolution of insect resistance to transgenic plants[END_REF][START_REF] Gould | Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology[END_REF][START_REF] Bourguet | Regulating insect resistance management: the case of non-Bt corn refuges in the US[END_REF].

Essentially, the HDR strategy consists in setting aside Bttoxin-free 'refuge' areas, where alleles conferring susceptibility to the Bt toxin would be kept at high frequency in pest populations not exposed to the toxin's selection pressure. It is expected that, provided such refuges are set in close enough vicinity to Bt fields (<800 m; United States Environmental Protection Agency, 2001), random mating among adults will dilute resistance alleles within the global population. As these alleles are assumed to be rare and functionally recessive, it is further expected that most of them will be carried by heterozygotes and eliminated when the latter feed on Bt plants. Allozyme analyses of ECB populations feeding on maize showed a pattern compatible with random mating within fields and a high gene flow between fields across the whole France (Bourguet et al., 2000a), although other scenarios cannot be excluded [START_REF] Rousset | Spatially homogeneous dispersal: the island model and isolation by distance[END_REF]. In contrast, the possible effect of distance on genetic differentiation between MCB populations has been less extensively studied [START_REF] Buès | Differences enzymatiques et écophysiologiques entre populations de Sesamia nonagrioides Lefèbvre (Lépidoptère: Noctuidae) originaires de l'Ouest du bassin méditérranéen[END_REF], so that empirical bases are lacking to conjecture the efficiency of the HDR strategy in the case of this species.

Refuges can be Bt-toxin-free maize fields. However, in the absence of protection against pest attacks, yields may be expected to be low. Maize refuges thus appear economically unattractive in the short term for individual farmers, which jeopardizes the chances that they actually be implemented [START_REF] Bourguet | Regulating insect resistance management: the case of non-Bt corn refuges in the US[END_REF]. Alternatively or in addition to maize fields, refuges could also consist of areas planted with other plant species, provided they are good hosts for the target pests, and provided they host individuals that mate freely with adults emerging from maize [START_REF] Gould | Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology[END_REF][START_REF] Losey | Evaluation of noncorn host plants as a refuge in a resistance management program for European corn borer (Lepidoptera: Crambidae) on Bt-corn[END_REF].

In the northern half of Europe, where only ECB is present, mugwort (Artemisia vulgaris L.) and hop (Humulus lupulus L.), two common ECB host plants, appeared as potential candidates to serve as alternative refuges. However, they were found to host a particular race of the ECB, which interbreeds very little with the race feeding on maize (a finding which has also been discussed in the theoretical framework of sympatric speciation : Bourguet et al., 2000b;[START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF][START_REF] Thomas | Genetic isolation between host plant races of the European corn borer. I. Sex pheromone, moth emergence timing and parasitism[END_REF][START_REF] Malausa | Assortative mating in sympatric host races of the European corn borer[END_REF]. Moreover, the surface covered by hop and mugwort, compared with that devoted to maize, and the densities reached by the ECB on these three plants, make it unlikely that mugwort/hop refuges would provide the desired ratio of susceptible over resistant ECBs -according to the United States Environmental Protection Agency (2001), this ratio should be ca. 500 susceptible individuals for every resistant moth that might emerge from Bt-maize fields. Finally, the fact that not only ECB, but also MCB extensively infests maize in southern Europe makes it necessary to conceive a global resistance management strategy that takes both lepidopteran pest species into consideration. However, little is known about genetic isolation by distance and /or host plant between MCB populations [START_REF] Buès | Differences enzymatiques et écophysiologiques entre populations de Sesamia nonagrioides Lefèbvre (Lépidoptère: Noctuidae) originaires de l'Ouest du bassin méditérranéen[END_REF]. Especially, possible mating barriers between MCBs from populations developing on different host plants have, to our knowledge, never been examined.

The present study was aimed at identifying host plants that would be quantitatively and qualitatively suitable to serve as refuges for Bt-toxin susceptibility alleles in both the MCB and the ECB populations. For this purpose, we examined the densities and genetic variability of ECB and MCB populations collected as larvae in various locations of the French territory from maize and from five other species among their wild or cultivated host plants. We also examined the genetic variability of ECB populations collected on hop -a known host plant for a separate ECB race (Bourguet et al., 2000b;[START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF], as a control allowing to test for the presence of the hop host race among the ECB populations collected from the other plants under study. Finally, we identified the sexual pheromone of ECB females collected from two of the newly investigated host plants. The implications of our findings in terms of ECB host race distribution among host plants are discussed.

Materials and methods

Sampling sites

Samples for population genetic analysis. Samples of O. nubilalis and S. nonagrioides were taken from sites located in central, south-eastern, and south-western France and from one site located near Sevilla in southern Spain (Figure 1). Seven different host plants were considered: maize (Z. mays), pepper (Capsicum frutescens L.), sorghum (Sorghum spec.), sunflower (Helianthus annuus L.), cantaloupe (Cucumis melo L.), cocklebur (Xanthium spec.), and hop (H. lupulus). The cocklebur and hop stands were wild, whereas the five other plants were conventional crops -none of the fields sampled in this study was planted with transgenic varieties.

Individuals of both species were collected as diapausing larvae, their sex was determined, and they were directly frozen at -20 °C. On each host-plant individual, we did not collect more than a single larva per plant and the plants investigated for the presence of ECB and MCB were randomly chosen in order to reduce the risk of sampling genetically related individuals.

For O. nubilalis, we collected samples of 26 populations: nine from maize, three from hop, eight from pepper, two from sorghum, two from cocklebur, and two from sunflower (Table 1 andFigure 1A). For S. nonagrioides, we collected samples of 19 populations: 10 from maize, six from pepper, and one from cantaloupe, sorghum, and sunflower each (Table 1 and Figure 1B). Each sample consisted of 16-62 individuals. At least 100 plants were examined in each patch or field of the various host plants we examined. ECB and MCB densities were not formally measured. However, fields were roughly classified while sampling into one of three classes: <0.01 larva per plant, 0.01-0.1 larva per plant, and 0.1-5 larvae per plant.

Samples for sex pheromone analysis. Samples of O. nubilalis fifth-instar larvae were collected on pepper at Fongrave (44°53′N, 0°40′E, July 2004) and Orléans (47°55′N, 1°54′E, September 2004), and on cocklebur at Bourg St Andéol (44°23′N, 4°42′E, September 2004). Larvae were reared to adult stage either directly (if collected in July) or after completion of diapause at 4 °C (if collected in September). The sexual pheromones of nine, three, and 11 adult females could be determined for the samples collected at Fongrave, Orléans, and Bourg St Andéol, respectively.

Enzyme electrophoresis

Each ECB or MCB larva was homogenised in 150 µl of Tris-EDTA (pH 6.8). Horizontal starch gel electrophoreses of the homogenates were carried out using a Tris-Borate-EDTA (pH 8.6) buffer system [START_REF] Pasteur | Manuel Technique de Génétique par Electrophorèse des Protéines[END_REF]. Seven enzyme systems were revealed as described in Bourguet et al. (2000a) and [START_REF] Buès | Differences enzymatiques et écophysiologiques entre populations de Sesamia nonagrioides Lefèbvre (Lépidoptère: Noctuidae) originaires de l'Ouest du bassin méditérranéen[END_REF]. These systems were phosphoglucomutase (PGM, EC 5.4.4.2), mannose-6phosphate isomerase (MPI, EC 5.3.1.8), hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.30), glucose-phosphate isomerase (GPI, EC 5.3.1.9), aspartate aminotransferase (AAT, EC 2.6.1.1), triose-phosphate isomerase (TPI, EC 5.3.1.1), and creatine kinase (CK, EC 2.7.3.2). These seven systems were revealed for both species except for CK in O. nubilalis and except for MPI in S. nonagrioides. The Tpi locus of O. nubilalis is located on the Z chromosome [START_REF] Glover | Sex-linked control of sex pheromone behavioral responses in European corn-borer moths (Ostrinia nubilalis) confirmed with TPI marker gene[END_REF]. In Lepidoptera, females are the heterogametic (WZ) whereas males are the homogametic (ZZ) sex. Thus, at this locus, ECB females are hemiploid whereas ECB males are diploid.

Mpi haplotypes

For O. nubilalis larvae, polymerase chain reaction (PCR) amplifications of a fragment of the Mpi gene were performed on total DNA extracted as described by [START_REF] Estoup | Rapid one-tube DNA extraction for reliable PCR detection of fish polymorphic markers and transgenes[END_REF]. DNA extractions were performed on the homogenates used for enzyme electrophoresis. The PCR mixtures contained 1 × PCR buffer (Eurogentec, Seraing, Belgium), 0.1 m dTNPs, 1 unit Taq polymerase, and 1 µ of each primer (forward Mpi15: 5′-CACCGTTTGAGGCTCTCTGT-3′, and reverse Mpi16: 5′-TTGGTCTCCTTGGTCTTCTTG-3′). Cycling conditions were 95 °C for 1.5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C (annealing temperature) for 30 s, and a primer extension at 72 °C for 30 s. The Mpi fragment amplified by the combination of the Mpi15 and Mpi16 primers encompasses an intron displaying substantial length polymorphism (SF Lee, unpubl.). Four classes of alleles were defined according to the length polymorphism observed on more than 100 sequences of that fragment -these sequences were obtained for another purpose and will be reported elsewhere. The analysis of these sequences showed that the Mpi alleles differed either by point mutations or by substantial insertions or deletions (indels). Four discrete classes of alleles -with approximate sizes of 400, 450, 470, and 510 bp -were defined according to the size of this indel. Mpi fragments amplified by PCR were therefore assigned unambiguously to one of these four classes of alleles by estimating their length on 3% agarose gels.

Pheromone collection and identification

After emergence, females were placed in individual plastic containers with a water supply and were kept in a rearing room under 24 ± 2 °C, 70 ± 20% r.h., and a L16:D8 reversed photoperiod. Sex pheromone was collected individually from 2-to 5-day-old females sampled during the calling period (the last 3 h of darkness). The pheromone gland was extruded by gentle pressure on the abdomen and kept in this position with metallic forceps. The adsorbent part of an SPME fibre (CW/DVB, 65 µm, Supelco, Bellefonte, PA, USA) was gently rubbed on the gland surface [START_REF] Frérot | Solid-phase microextraction (SPME): a new tool in pheromone identification in Lepidoptera[END_REF]. The fibre was then desorbed on the injector (245 °C) of a Varian 3400 CX gas chromatograph (Varian Inc., Palo Alto, CA, USA) equipped with a polar column (Rtx Wax, 30 m, 0.32 id, 0.5 µm, Restek Corp., Bellefonte, PA, USA). The oven temperature was programmed as follows: hold initial temperature (50 °C) for 1 min, increase to 100 °C at 15 °C min -1 , hold for 1 min, and increase to 245 °C at 5 °C min -1 . The temperature of the FID detector was set at 260 °C. The carrier gas was helium (14 p.s.i.). Natural components were identified by comparison of their retention times with those of a hexane solution of synthetic Z and E isomers of the 11-14:OAc, the two known components of the ECB pheromone [START_REF] Klun | Insect sex pheromone: minor amount of the opposite geometrical isomer critical to attraction[END_REF]. The ratio of both components was calculated as the ratio of the areas of the corresponding peaks.

Analysis of data on population structure

For each sample, we estimated the allelic frequencies, the mean number of alleles (N all ), the observed heterozygosity (H o ) and the unbiased expected heterozygosity (H e ) as described by [START_REF] Nei | Estimation of average heterozygosity and genetic distance from a small number of individuals[END_REF], and f-values [i.e., F is estimates according to Weir & Cokerham (1984)] using GENETIX 4.0 [START_REF] Belkir | GENETIX 4.04, logiciel sous Windows TM pour la génétique des populations[END_REF]. We tested for deviations from Hardy-Weinberg expectations and calculated genotypic linkage disequilibria between loci, within each sample, with GENEPOP 3.2 [START_REF] Raymond | GENEPOP version 1.2: population genetic software for exact tests and ecumenicism[END_REF]. As Tpi is a sex-linked locus for O. nubilalis, H o and f-values for samples of this species were estimated and tests for deviations from Hardy-Weinberg expectations were carried out for males only. Finally, the frequency of null alleles at the Mpi locus was estimated using the EM algorithm [START_REF] Dempster | Maximum likelihood from incomplete data via the EM algorithm[END_REF] implemented in GENEPOP 3.2. The genetical structure between samples of a host-plant group (a host-plant group includes all samples collected on a given host plant) was analysed by testing for allelic differentiation using Fisher exact tests and calculating the 0 estimator of the F st as described by [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF], using GENEPOP 3.2. The 0-values calculated over all samples collected on hop and maize are referred to as 0 hop and 0 maize, respectively. We also carried out hierarchical analyses of population structure between host-plant groups [START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF], using TFPGA 1.3 software [START_REF] Miller | Tools for population genetic analysis (TFPGA 1.3): a Windows program for the analysis of allozyme and molecular population genetic data[END_REF]. Finally, isolation-by-distance patterns [START_REF] Slatkin | Isolation by distance in equilibrium and nonequilibrium populations[END_REF] were tested for samples of S. nonagrioides by assessing the independence of geographic and genetic distances for various geographical levels. The null hypothesis that geographic and genetic distances were independent was tested against the alternative hypothesis of a positive correlation expected under isolation by distance, estimated as Spearman's rank correlation coefficient. The calculated correlation coefficient was compared with the distribution of correlation coefficients obtained from Mantel-like permutations of the genetic [0/(1 -0)] and geographic [ln (geographical distance)] matrices as described by [START_REF] Rousset | Genetic differentiation and estimation of gene flow from F-statistics under isolation by distance[END_REF] and included in GENEPOP 3.2.

Results

Infestation levels of the various host plants

As shown in Table 1, the highest ECB densities were reached on maize (typically 0.1-5 larvae per plant), followed (at a much lower level) by sorghum, cocklebur, and sunflower (usually 0.01-0.1 larva per plant). Though economically damaging, densities on pepper and on cantaloupe were low (<0.01 larva per plant). MCBs were present at high and comparable densities in maize and sorghum (typically 0.1-5 larvae per plant), and at much lower densities on pepper, sunflower, and cantaloupe (0.01-0.1 larva per plant). It was not found on hop or cocklebur.

Population structure of ECB

All loci -Tpi, Pgm, Aat, Gpi, Hbdh, and Mpi -investigated in this study were polymorphic. However, the Hbdh locus displayed polymorphism in only eight (O-HO-1, O-MA-7, O-MA-8, O-PE-1, O-PE-2, O-PE-5, O-PE-6, and O-CO-2) out of the 26 samples. For most individuals, the staining of the MPI system was of insufficient quality for a reliable genotyping. Rather than merely omitting this locus from the analysis, we amplified a fragment of the Mpi locus encompassing an intron that displays four different sizes. This enabled us to genotype individuals according to PCR product sizes.

In agreement with our previous results on population genetic studies on ECB (Bourguet et al., 2000a,b;[START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF][START_REF] Bontemps | Managing the evolution of Bacillus thuringiensis resistance in natural populations of the European corn borer, Ostrinia nubilalis: host plant, host race and pherotype of adult males at aggregation sites[END_REF], we found no significant genotypic association between any pair of loci (data not shown). Allele frequencies at each locus and for each sample are given in Supplementary Appendix 1. Within samples, the mean number of alleles was 1.80-3.40 for the polymorphic loci tested, with apparently no particular trend distinguishing samples by host plant. The observed heterozygosity (H o ) was often lower than the expected heterozygosity (H e ). Accordingly, the f-values often indicated that heterozygosity tended to be lower than expected under Hardy-Weinberg equilibrium, the deviations from Hardy-Weinberg expectations for the six loci being significant in five of the 26 samples (O-MA-2, O-MA-6, O-MA-9, O-SU-1, and O-CO-2). Except for one sample (O-SU-1), these deviations remained significant even when a Bonferroni correction for multiple tests was applied [START_REF] Holm | A simple sequentially rejective multiple test procedure[END_REF]. This result is unexpected as all ECB samples investigated to date were found to be at Hardy-Weinberg equilibrium (Bourguet et al., 2000a,b;[START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF][START_REF] Bontemps | Managing the evolution of Bacillus thuringiensis resistance in natural populations of the European corn borer, Ostrinia nubilalis: host plant, host race and pherotype of adult males at aggregation sites[END_REF]. However, a closer examination of the data revealed that these heterozygote deficiencies were mainly due to the Mpi locus: indeed, f-values at this locus were always positive, and significant deviations from Hardy-Weinberg equilibrium were detected in 10 samples. When this locus was removed from the analysis, none of the samples displayed a significant deviation from Hardy-Weinberg expectations anymore. Hence, the most likely explanation for the deviation found on the complete data set is the presence of undetected 'null' alleles due to a lack of PCR amplifications of one or several Mpi haplotypes in these samples. Over all samples, the estimated mean frequency ± SE of this null allele equals 0.399 ± 0.174, ranging from 0.137 to 0.702.

The overall differentiation between samples was highly significant (P<10 -5 ) but low (0 overall = 0.016) (Table 2). Similar to what has been previously found by [START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF] for ECB populations collected on mugwort, the group of samples collected on hop were significantly differentiated (0 hop = 0.049, P<10 -5 ) whereas the group of samples collected on maize were not (0 maize = -0.001, P = 0.134) (Table 2). The other groups of samples were weakly (pepper) or not (sorghum, sunflower, and cocklebur) differentiated (Table 2).

We carried out a hierarchical analysis of the distribution of the genetic variability in order to determine the amount of variance explained by differentiation between host-plant groups. As expected, this component of the total variance was significant between the maize and the hop group (0 host plant = 0.018, P<0.01). A significant differentiation between groups (0 host plant = 0.020, P<0.01) was also found when comparing the hop group with the group containing the samples collected on pepper, sorghum, sunflower, and cocklebur. When comparing this latter group with the maize group, the differentiation between host groups dropped to zero (0 host plant = -0.001, P<0.05). We analysed the genetic structure of each pair of samples more precisely. In agreement with the hierarchical analyses, most of the significant exact tests for allelic differentiation between samples involved one of the samples collected on hop (Figure 2). This result holds even when excluding the Mpi locus -which displayed heterozygote deficienciesfrom the analysis (details not shown).

Sex pheromone of ECB females from pepper and cocklebur

The pheromone glands of 23 females that emerged from larvae collected on pepper and cocklebur were analysed to study the pheromone compositions of the ECB infesting these two host plants. As previously reported in all studies on ECB pheromones (e.g., [START_REF] Klun | Insect sex pheromone: minor amount of the opposite geometrical isomer critical to attraction[END_REF][START_REF] Klun | Genetic basis of an insect chemical communication system: the European corn borer[END_REF], the studied components of the sexual pheromones were the E and Z isomers of 11-14:OAc. The experimental GC procedure produced a clear separation of these two isomers and mass spectra confirmed their identification and their purity.

All females either from pepper (n = 12) or from cocklebur (n = 11) displayed the Z11-14:OAc as the main component of their sexual pheromone. Indeed, the E11-14:OAc component represented only 1% on average. According to [START_REF] Klun | Genetic basis of an insect chemical communication system: the European corn borer[END_REF], Z and E females produce Z11-14:OAc and E11-14:OAc in ratios of 97 : 3 and 1 : 99, respectively. Consequently, the 20 females analysed in this study were clearly Z females.

Population structure of MCB Among the six loci examined, three -Aat, Gpi, and Pgmwere polymorphic in all samples. The Hbdh locus was monomorphic and the Tpi and Ck loci were polymorphic in only one (S-MA-5) and three (S-MA-1, S-MA-4, and S-MA-10) samples, respectively. Over all sites, we found no significant genotypic association between any pair of loci (results not shown). Allele frequencies at each locus and for each sample are given in Supplementary Appendix 2. Within samples, the mean number of alleles was 1.83-2.33 for the polymorphic loci tested. The observed heterozygosities (0.18-0.30) were slightly lower than the expected ones (0.23-0.32), and 10 out of the 19 samples displayed an f-value >0.1. These deviations from Hardy-Weinberg expectations over the loci were significant for five samples (S-CA-1, S-MA-3, S-MA-4, S-MA-5, and S-SO-1) after a Bonferroni correction for multiple tests was applied [START_REF] Holm | A simple sequentially rejective multiple test procedure[END_REF]. These deviations were not due to any particular locus, and a significant deficit in heterozygotes was detected over all sites for all three polymorphic loci: Aat (f = 0.139, P<10 -3 ), Gpi (f = 0.039, P<10 -3 ), and Pgm (f = 0.114, P<10 -5 ). The overall differentiation between samples was highly significant (P<10 -5 ) and the overall 0-value equalled 0.061, a value five times higher than that for O. nubilalis samples (Table 2). The Pgm locus displayed the highest 0-value (0 = 0.108) and, except at the Tpi locus, which was monomorphic in all samples but one, we found a significant differentiation at the four polymorphic loci (Table 2). The groups of samples collected on maize, on sorghum, or on the three other host plants (pepper, cantaloupe, and sunflower) were all differentiated (Table 2).

The hierarchical analysis of the distribution of the genetic variability showed that the maize group was differentiated both from the group of six samples collected on sorghum only (0 host plant = 0.010, P<0.01) and from the group of samples collected on all other, non-grass host plants (cantaloupe, pepper, and sunflower, 0 host plant = 0.010, P<0.01). Conversely, there was no differentiation between the sorghum group and the non-grass host-plant group (0 host plant = -0.008, P>0.05).

The genetic differentiation between the maize group and the group containing the samples collected on all the other host plants can be further investigated by comparing within-and between-group isolation by distance patterns [START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF]. We found no association between genetic and geographic distances within the group of samples collected on maize or between pairs of samples collected from different plants (maize vs. all other host plants) (Figure 3A; P>0.800 in both cases). Conversely, the level of differentiation within the group of samples collected on non-maize host plants was marginally correlated with distance (slope = 0.0158, R 2 = 0.44, P = 0.058) and was lower than within maize or between pairs of samples collected on different host-plant groups (Figure 3B).

Discussion

Apart from maize, we found both ECB and MCB larvae on pepper, sunflower, sorghum, and cantaloupe, although on the latter plant we found too few ECB larvae to analyse their genetical population structure. Only ECB was found on cocklebur and hop. This is consistent with previous reports of ECB feeding on a broader range of host plants than MCB: indeed, ECB has been recorded on species belonging to at least 42 different plant families (review in [START_REF] Ponsard | Carbon stable isotopes: a tool for studying the mating, oviposition, and spatial distribution of races of European corn borer, Ostrinia nubilalis, among host plants in the field[END_REF], whereas MCB is mainly known to feed on wild and cultivated monocotyledons (rice, sorghum, maize, and Typha spec. ;[START_REF] Hilal | Etude du développement de Sesamia nonagrioides et établissement de modèles pour la prévision de ses populations dans la nature[END_REF][START_REF] Buès | Differences enzymatiques et écophysiologiques entre populations de Sesamia nonagrioides Lefèbvre (Lépidoptère: Noctuidae) originaires de l'Ouest du bassin méditérranéen[END_REF]. Our results further draw attention to the fact that, within a given area, i.e., for a given global infestation level, not all host plants are infested at equal levels. Indeed, ECB densities were typically >10 times higher on maize than on any other plant we examined. Conversely, MCB was present at comparable densities on maize and on sorghum.

ECB samples showed no deviation from Hardy-Weinberg equilibrium (apart from what is probably an artefact due to the presence of null alleles at Mpi). A fraction (ca. 1 / 4 ) of the MCB samples did deviate, which might be due to recent demographic bottlenecks due to the high winter mortality in this species [START_REF] Galichet | Hibernation d'une population de Sesamia nonagrioides Lef. (Lep., Noctuidae) en France méridionale[END_REF][START_REF] Hassaine | La Noctuelle du maïs (Sesamia nonagrioides Lef.) résiste-t-elle au gel?[END_REF], although alternative explanations, such as a Wahlund effect due to the possible accidental presence in our samples of a few individuals of Sesamia cretica Led. (Lepidoptera: Noctuidae), cannot be ruled out. Indeed, this species is difficult to distinguish from S. nonagrioides at the larval stage and might be present, although at marginal densities, in our sampling area (JF Sylvain, pers. comm.).

The overall genetic differentiation between samples was higher within MCB (0 overall = 0.061, P<10 -5 ) than within ECB (0 overall = 0.016, P<10 -5 ) samples. As previously found [START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF], we detected no effect of geographic distance on the genetic differentiation between pairs of ECB samples. We also detected no significant effect of geographic distance on the genetic differentiation between pairs of MCB samples collected on maize. A small increase of genetic isolation with increased distance was, however, detected between pairs of MCB samples collected on other host plants. Under the reasonable assumption that both species reach comparable population densities, our results might indicate that MCB tends to be slightly less dispersive than ECB. However, this point would need to be confirmed by a more targeted study.

Our results further show evidence in both species for genetic differentiation between populations of larvae using different host plants. The MCB samples collected on maize showed a significant genetic differentiation from the samples collected on sorghum and from the samples collected on other host plants (the latter group could not be analysed separately for each plant due to limited sample size). Such differentiation may either be due to (1) different selection pressures exerted repeatedly on each MCB generation by both plant types, or (2) fixed genetic differences maintained from one generation to the next by mechanisms such as assortative mating and oviposition preferences, i.e., to the existence of MCB host races. Further investigation is needed in this respect. Indeed, if the genetic structure we observed results mainly from repeated selection, MCB populations feeding on sorghum could nevertheless contribute to diluting resistance alleles in populations feeding on maize. In this case, sorghum fields could be used to replace some of the 'refuges' -the Bt-toxin-free fields required by the HDR strategycurrently planted with non-Bt maize, or to increase the total amount of such refuges. Conversely, if the genetic structure results mainly from assortative mating, their efficiency in diluting resistance alleles would probably be more limited. In this case, sorghum would be of limited interest for managing the evolution of resistance in MCB populations.

Regarding ECB, our results confirm that populations are split into two genetically differentiated groups that differ by host-plant use, the maize race and the mugworthop race. Indeed, the groups of ECB samples collected on maize and hop showed the common and significantly different genetic patterns characterizing the ECB maize race and hop-mugwort race, respectively (Bourguet et al., 2000b;[START_REF] Martel | Host-plant associated genetic differentiation in northern French populations of the European corn borer[END_REF][START_REF] Thomas | Genetic isolation between host plant races of the European corn borer. I. Sex pheromone, moth emergence timing and parasitism[END_REF][START_REF] Bontemps | Managing the evolution of Bacillus thuringiensis resistance in natural populations of the European corn borer, Ostrinia nubilalis: host plant, host race and pherotype of adult males at aggregation sites[END_REF][START_REF] Malausa | Assortative mating in sympatric host races of the European corn borer[END_REF]. Furthermore, our results reveal no evidence suggesting the existence of any additional host race. Indeed, samples collected on pepper, sunflower, sorghum, and cocklebur are genetically distinct from the mugwort-hop race but not significantly different from the maize race: the most parsimonious conclusion is that they consisted mostly -if not entirely -of members of the maize race. This conclusion was reinforced by our results on the sex pheromone produced by females originating from pepper and cocklebur. Indeed, in France, the two ECB host races are known to differ, among other traits, by the sex pheromone produced by females and recognized by males. The maize race and the mugwort-hop race use the so-called Z and E pheromone type, respectively [START_REF] Thomas | Genetic isolation between host plant races of the European corn borer. I. Sex pheromone, moth emergence timing and parasitism[END_REF][START_REF] Bontemps | Managing the evolution of Bacillus thuringiensis resistance in natural populations of the European corn borer, Ostrinia nubilalis: host plant, host race and pherotype of adult males at aggregation sites[END_REF][START_REF] Pélozuelo | Host-plant sepcialization in pheromone strains of the European corn borer Ostrinia nubilalis in France[END_REF]. Hence, in agreement with the population genetic structure, all females emerging from pepper and cocklebur produced exclusively the Z pheromone type.

The absence or quasi absence of the mugwort-hop race in our samples collected on pepper, sunflower, sorghum, and cocklebur can result from one or a combination of three factors: (1) females of the mugwort-hop race are little attracted to these plant species for oviposition, (2) the mugwort-hop race is largely outnumbered by the maize race in our study areas, or (3) young instars of the mugwort-hop race have a lower survival on these plants than those of the maize race. There are currently no data that would allow us to favor one of these three hypotheses.

Our findings are also interesting in that they contrast somewhat with those of a previous cage experiment [START_REF] Bethenod | Genetic isolation between two sympatric host plant races of the European corn borer, Ostrinia nubilalis Hübner. II. Assortative mating and host-plant preferences for oviposition[END_REF] where ECB individuals were offered equal amounts of maize and mugwort to oviposit: the maize race almost exclusively oviposited on maize while the hop-mugwort race showed a less strong preference for mugwort. This result alone could be seen as an indication that the maize race is an oligophagous specialist, and that the wide-spectrum polyphagy of the ECB reported in the literature [START_REF] Caffrey | A progress report on the investigations of the European corn borer[END_REF][START_REF] Hudon | Seventy years of European corn borer (Ostrinia nubilalis) research in North America[END_REF] might mainly resort to observations of polyphagous members of the hop-mugwort race. Our present findings show that the maize race seems also able to oviposit -although to a much lower extent -on a broad range of plant species belonging not only to the Poaceae, such as sorghum and maize, but also to the Solanaceae (pepper) and Compositae (cocklebur).

Studies performed on American and former Soviet Union ECB strains have previously shown that the ECB is able to complete its entire life cycle on sunflower [START_REF] Legg | European corn borer (Lepidoptera: Pyralidae) oviposition preference and survival on sunflower and corn[END_REF], sorghum [START_REF] Atkins | Investigations of host plant resistance to the European corn borer in sorghum[END_REF][START_REF] Dyatlova | Egg laying and survival of the European corn borer, Ostrinia nubilalis, on sorghum vs. maize in two-generation zone of Russia[END_REF][START_REF] Dyatlova | Egg laying and survival of the European corn borer, Ostrinia nubilalis, on sorghum vs. maize in two-generation zone of Russia[END_REF]pepper (McLeod, 1981). Our results confirm that this is also possible, at least until the fifth instar (the stage at which our samples were collected), for French maize-race populations on pepper, sunflower, cocklebur, and sorghum. However, fitness (estimated as larval weight or developmental time) appeared lower for larvae feeding on sunflower [START_REF] Legg | European corn borer (Lepidoptera: Pyralidae) oviposition preference and survival on sunflower and corn[END_REF] or sorghum [START_REF] Dyatlova | Egg laying and survival of the European corn borer, Ostrinia nubilalis, on sorghum vs. maize in two-generation zone of Russia[END_REF] than for the corresponding control individuals feeding on maize. Oviposition was also higher on maize than on sunflower [START_REF] Legg | European corn borer (Lepidoptera: Pyralidae) oviposition preference and survival on sunflower and corn[END_REF]) and than on sorghum, at least at certain times of the season [START_REF] Dyatlova | Egg laying and survival of the European corn borer, Ostrinia nubilalis, on sorghum vs. maize in two-generation zone of Russia[END_REF].

Our data show that the net effect of possible differences in oviposition choices and larval development results in maize suffering much higher infestations than the other sympatric host plants. Pepper, sunflower, cocklebur, and sorghum thus probably function rather as sink habitats infested by more or less marginal oviposition of maizerace ECB females mainly attracted to nearby maize fields, than as 'true' hosts on which autonomous and dense ECB populations could develop over several generations.

In sum, from a genetic point of view, ECB adults emerged from any of the plants considered in this studyexcept hop -are probably able to mate with each other. However, sunflower, sorghum, cocklebur, and pepper are planted over smaller acreages and seem to host but low densities of ECB larvae compared with maize. Therefore, they may not replace non-Bt maize refuges in the framework of the HDR resistance-delaying strategy, but merely complement them.

Regarding MCB, the exact origin and strength of the genetic differentiation found between samples collected on maize and those found on sorghum, as well as the effect of geographic isolation on genetic distance between samples, call for closer examination. Indeed, the high MCB densities reached on sorghum could make it a valuable refuge crop for this species, provided neither reproductive nor geographic isolation prevent an efficient mixing between the Bt-maize field and the refuge populations.

Figure 2

 2 Figure 2 Pairwise exact tests of allelic differentiation for all loci between pairs of samples of Ostrinia nubilalis. Black squares indicate pairs of samples for which exact tests were significant (P<0.05). Labels O-SU, O-MA, O-PE, O-SO, O-CO, and O-HO correspond to samples collected on sunflower, maize, pepper, sorghum, cocklebur, and hop, respectively.

Figure 3

 3 Figure3Regressions of 0/(1 -0) against log-transformed geographical distances (km) for samples of Sesamia nonagrioides collected on maize (within maize) and between pairs of samples collected on (A) the two host-plant groups (between groups) and (B) on other host plants (within other host plants) to test for isolation by distance.

Table 1

 1 Characteristics of Ostrinia nubilalis and Sesamia nonagrioides samples: host plant, location and sample abbreviation, year of sampling, number (n) of individuals analysed, and density class (D): 1 = <0.01 larva per plant, 2 = 0.01-0.1 larva per plant, = 0.1-5 larvae per plant, NA = not available

	Species	Host plant	Location	Sample	Latitude	Longitude	Year	n	D
	O. nubilalis	Hop	B ourg St Andéol	O-HO-1	44°23′N	4°42′E	2002		NA
			Beynes	O-HO-2	48°51′N	1°52′E	2002		NA
			Cft Arriège-Garonne	O-HO-3	43°31′N	1°27′E	2003		NA
		Maize	Vénerque	O-MA-1	43°26′N	1°27′E	2002		3
			Marmande	O-MA-2	44°30′N	0°10′E	2002		3
			Agen	O-MA-3	44°12′N	0°38′E	2002		3
			Fiac	O-MA-4	43°42′N	1°54′E	2002		3
			St Etienne de Tulmont	O-MA-5	44°03′N	1°28′E	2003		3
			Mollégès	O-MA-6	43°48′N	4°57′E	2003		3
			Côtes d'Arey	O-MA-7	45°28′N	4°51′E	2003		3
			Orléans	O-MA-8	47°55′N	1°54′E	2003		3
			Pierrelatte	O-MA-9	44°23′N	4°42′E	2003		3
		Pepper	Marmande site 1	O-PE-1	44°30′N	0°10′E	2002		2
			Marmande site 2	O-PE-2	44°30′N	0°10′E	2002		2
			Marmande site 3	O-PE-3	44°30′N	0°10′E	2002		2
			St Sylvestre sur Lot	O-PE-4	44°29′N	0°49′E	2002		2
			Montesquieu	O-PE-5	44°12′N	0°27′E	2002		2
			Petit Guérin	O-PE-6	44°12′N	0°27′E	2003		2
			Avignon	O-PE-7	43°53′N	4°49′E	2003		2
			Orléans	O-PE-8	47°55′N	1°54′E	2003		2
		Sorghum	St Etienne de Tulmont	O-SO-1	46°54′N	1°28′E	2003		2
			Côtes d'Arey	O-SO-2	45°28′N	4°51′E	2003		2
		Sunflower	Vénerque	O-SU-1	43°26′N	1°27′E	2002		2
			Nailloux	O-SU-2	43°22′N	1°38′E	2002		2
		Cocklebur	Bourg St Andéol	O-CO-1	44°23′N	4°42′E	2003		NA
			Muret	O-CO-2	43°28′N	1°21′E	2003		NA
	S. nonagroides	Cantaloupe	Saint Léon	S-CA-1	43°24′N	1°34′E	2002		1
		Maize	Pierrelatte	S-MA-1	44°23′N	4°42′E	2002		3
			Baziège	S-MA-2	43°27′N	1°37′E	2002		3
			Agen	S-MA-3	44°12′N	0°38′E	2002		3
			Vénerque	S-MA-4	43°26′N	1°27′E	2002		3
			Sevilla (Spain)	S-MA-5	37°22′N	5°59′W	2002		NA
			Dax	S-MA-6	43°43′N	1°03′W	2002		3
			Mollégès	S-MA-7	43°48′N	4°57′E	2002		3
			Grues	S-MA-8	46°23′N	1°18′W	2002		3
			Port Sainte Marie	S-MA-9	44°35′N	0°12′E	2002		3
			Saint Papoul	S-MA-10	44°20′N	2°02′E	2002		3
		Pepper	Marmande	S-PE-1	44°30′N	0°10′E	2002		1
		Sorghum	Vénerque site 1	S-SO-1	43°26′N	1°27′E	2002		3
			Montesquieu	S-SO-2	44°12′N	0°27′E	2002		3
			Vénerque site 2	S-SO-3	43°26′N	1°27′E	2002		3
			Fiac	S-SO-4	43°42′N	1°54′E	2002		3
			Pierrelatte	S-SO-5	44°23′N	4°42′E	2002		3
			Saint Hilaire	S-SO-6	43°25′N	1°16′E	2002		3
		Sunflower	Vénerque	S-SU-1	43°26′N	1°27′E	2002		2

Table 2

 2 Genetic differentiation within host-plant groups of Ostrinia nubilalis and Sesamia nonagrioides [0-values estimated according to[START_REF] Weir | Estimating F-statistics for the analysis of population structure[END_REF]] for the various loci and over all loci. NA = not available; NP = no polymorphism

			Locus
	Species	Group of samples	Tpi	Gpi	Aat	Pgm	Hbdh Mpi	Ck	All loci
	O. nubilalis	All host plants	0.056*** 0.005**	0.035*** 0.011	0.004 0.002* NA	0.016***
		Hop	0.181*** 0.054**	0.012	0.012*	0.021 -0.033 NA	0.049***
		Maize	-0.015	-0.003	0.012*	0.018*	0.002 -0.004 NA	-0.001
		Pepper	0.013	0.003	0.011*	-0.003	0.002 0.013 NA	0.019**
		Sorghum	-0.063	0.001	0.038*	0.003	NP 0.001 NA	0.005
		Sunflower	NA	-0.009	-0.006	-0.011	NP	NA NA	-0.009
		Cocklebur	-0.039	0.002	-0.013	NP	0.028 -0.031 NA	-0.013
	S. nonagrioides All host plants	0.003	0.028*** 0.019*** 0.108***	NP	NA 0.047*** 0.061***
		Maize	0.003	0.050*** 0.013**	0.147***	NP	NA 0.045*** 0.082***
		Sorghum	NP	0.007	0.025*	0.040***	NP	NA NP	0.027***
		Pepper + cantaloupe + sunflower	NP	0.006**	0.041*	0.058***	NP	NA NP	0.039***
	*P<0.05, **P<0.01, and ***P<10 -5 .

Acknowledgements

Warm thanks to all those who helped with laboratory work (C. Vauvarin, T. Ben Ari, M.-T. Bethenod, and M. Dépaulis) and sampling (N. Eychenne, L. Folcher, M. Delos, the staff of the different Services Régionaux de Protection des Végétaux, farmer families Biral, Bite, Cazonato, Cherron, Darqué, Dubourg, Gers, Grande, Guizet, Lamouroux, Marchi, Maurel, Pituello, Pressec and Villepin, H. Clerc (AIREL), F. Ortego and P. Castañera (CSIC, Madrid), and L. Pélozuelo. The financial support of this study was provided by the IFB -AO Biodiversité et Changement Global, the CNRS -AO Impact des Biotechnologies sur les Agrosystèmes, and the ACI-FNS 'Ecco' -Ecosphère Continentale : Processus et Modélisation. We finally thank the Comité de Biovigilance and the Direction Générale de l'Alimentation of the French Ministry of Agriculture for funding most of the sampling performed for this study.

Supplementary material

The following supplementary material is available for this article online at www.blackwellsynergy.com:


Anne Ge ´nissel

Sylvie Augustin

Claudine Courtin

Gilles Pilate

Philippe Lorme

Denis Bourguet

Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae

Keywords: Chrysomela tremulae, resistance-allele frequency, transgenic Bacillus thuringiensis poplar, high-dose plus refuge strategy, resistance management, F 2 screen

Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F 2 screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.000 45-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F 2 screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates.

INTRODUCTION

Genetically modified plants containing genes encoding toxins from the bacterium Bacillus thuringiensis (Bt plants) provide a safe and effective method for pest insect control [START_REF] Scott | Transgene risk is low[END_REF]. The estimated global area planted with transgenic plants of all types was 52.6 million hectares in 2001, with 12 million hectares (23%) planted with Bt plants [START_REF] James | Global review of commercialized transgenic crops[END_REF]. The increase in commercialization of these Bt plants has magnified the risk of targeted insect pest species rapidly adapting to this ecologically valuable class of toxin [START_REF] Gould | Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology[END_REF][START_REF] Wolfenbarger | Biotechnology and ecology: the ecological risks and benefits of genetically engineered plants[END_REF]. Indeed, Bt-resistant strains have been selected under laboratory conditions for several pest species (reviewed by [START_REF] Frutos | Managing insect resistance to plants producing Bacillus thuringiensis toxins[END_REF][START_REF] Sanchis | Biotechnological improvement of Bacillus thuringiensis for agricultural control of insect pests: benefits and ecological implications[END_REF], and field populations of Plutella xylostella have already been found to display substantial resistance to Bt toxins [START_REF] Tabashnik | Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae)[END_REF]). Therefore, one of the most important elements in the cultivation of Bt plants is the development of effective resistance-management plans, to delay the appearance of resistance to Bt toxins in the target pests [START_REF] Gould | Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology[END_REF]. With this aim in mind, governmental agencies, in collaboration with growers' associations and seed companies, have encouraged farmers to implement * Author for correspondence (bourguet@jouy.inra.fr). † Present address: University of California, Department of Ecology and Evolutionary Biology, 321 Steinhaus Hall, Irvine, CA 92697-2525, USA.

Ó the high-dose plus refuge strategy [START_REF] Georghiou | Operational influences in the evolution of insecticide resistance[END_REF][START_REF] Alstad | Managing the evolution of insect resistance to transgenic plants[END_REF], predicting that random mating between selected (in transgenic areas) and unselected (in refuges) insect populations can delay the evolution of resistance.

The high-dose plus refuge strategy may be very useful for delaying the evolution of Bt resistance. The degree to which this strategy can be expected to delay the evolution of widespread resistance increases as initial Bt resistanceallele frequency declines, as is true of other strategies. Evaluation of the initial frequency of resistance has been a challenging task in the last few years. Results from three landmark papers revealed that three lepidopteran pest species-Heliothis virescens, P. xylostella and Pectinophora gossypiella-display high initial frequencies of alleles for resistance to Bt crops, calling for a re-examination of the assumptions of resistance-management models [START_REF] Gould | Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens[END_REF][START_REF] Tabashnik | Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis[END_REF][START_REF] Tabashnik | Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm[END_REF]. The experiments described in these papers were, however, subject to a small flaw in that the frequencies of Bt resistance alleles were evaluated after the introduction of Bt plants and/or in geographical areas previously treated with biopesticide formulations containing Bt crystal proteins. Thus, the frequencies reported to date cannot rigorously be taken as actual initial allele frequencies-i.e. the frequencies at which the Bt resistance allele segregates in field populations before the introduction of artificial selection pressures resulting from pest management-and as such may not adequately reflect the probability of natural pest populations challenging the introduction of Bt plants.

Recently, transgenic poplars producing high levels of the Bt Cry3A toxin have been produced [START_REF] Ge ´nissel | High tolerance against Chrysomela tremulae of transgenic poplar plants expressing a synthetic cry3aA gene from Bacillus thuringiensis ssp tenebrionis[END_REF]. The foliage of these transgenic poplars is highly toxic to Chrysomela tremulae (Coleoptera: Chrysomelidae), a polyvoltine oligophagous beetle responsible for massive attacks on native and introduced hybrid poplars [START_REF] Augustin | Life history of the poplar beetle Chrysomela tremulae in the central region of France[END_REF]. Bt poplars have not been disseminated and the Cry3A Bt toxin produced by these Bt poplars has never been used in French agricultural pestmanagement programmes. This situation provided us with a unique opportunity to evaluate the frequency of Bt resistance alleles in natural pest populations prior to the introduction of artificial positive selection pressures. Using the F 2 screen method proposed by Andow & Alstad (1998), we showed that, in a field population of C. tremulae surveyed over three consecutive years, the frequency of an allele conferring resistance to Bt poplars was 0.0037 (95% confidence interval (CI) = 0.000 45-0.0080).

MATERIAL AND METHODS

(a) F 2 screen

The F 2 screen method is conducted by: (i) sampling mated adult females from natural populations, and establishing isofemale lines in laboratory conditions; (ii) rearing and sib-mating F 1 progeny in each isofemale line; (iii) rearing eggs from the F 1 parents and screening F 2 neonates for Bt susceptibility; (iv) statistical analysis of the data; and (v) retesting of potential positive isofemale lines (Andow & Alstad 1998). As each female carries four haplotypes-two of her own and two from her mate-each isofemale line enables the characterization of four genomes.

(b) Sampling and sib-mating

Insects were sampled at a single site ('La Chesnaye', Vatan) located in the Centre region of France. At this site, three groups of adults were collected from young leaves and twigs of hybrid poplars (Populus deltoides ´P. trichocarpa and P. deltoides ´P. nigra) in August 1999, April 2000 and June 2001. To minimize the probability of collecting sib-related adults, adults were homogeneously sampled over the whole surface (ca. 1 ha) of the field. Adults sampled in 1999 corresponded to the first generation and those collected in 2000 and 2001 were from overwintering adults (C. tremulae being polyvoltine in the Centre region).

The sex of each insect was determined. The males were killed, and each female was isolated and kept in standard laboratory conditions (20 °C under a 16 L : 8 D photoperiod) in 12 cm ´12 cm ´7 cm boxes. The number of F 1 males and females that were sib-mated was recorded. Prior to the F 2 screen procedure, the F 0 and F 1 generations were fed on fresh leaves of a poplar hybrid clone (P. tremula ´P. tremuloides, Institut National de la Recherche Agronomique no. 353-38), grown in the field or in greenhouses.

(c) Screening procedure

Egg masses produced during the peak of egg production were collected from F 1 females for a few weeks, and incubated at 15 °C. F 2 neonates emerging from these masses were fed on leaf discs cut from fresh mature leaves of a transgenic Bt poplar line placed on moist filter paper to prevent them from drying out. This Bt poplar line produces an amount of Cry3A protein equivalent to ca. 0.05% of the total soluble protein in mature leaves [START_REF] Ge ´nissel | High tolerance against Chrysomela tremulae of transgenic poplar plants expressing a synthetic cry3aA gene from Bacillus thuringiensis ssp tenebrionis[END_REF]. The resulting concentration of Cry3A toxins is lethal to all susceptible C. tremulae neonates within 24 h of feeding, as shown by the results of bioassays performed on large samples of individuals from various susceptible laboratory strains [START_REF] Ge ´nissel | High tolerance against Chrysomela tremulae of transgenic poplar plants expressing a synthetic cry3aA gene from Bacillus thuringiensis ssp tenebrionis[END_REF]. After 72 h, surviving larvae that had fed actively on Bt poplar were classified as resistant larvae.

(d ) Expected proportions of resistant larvae

If one of the two F 0 parents giving rise to an isofemale line is heterozygous for a Bt resistance allele (R), then the expected number of heterozygotes (RS) in the F 1 generation is 50% of the total number of adults. If mating is random, the expected frequency of F 1 heterozygote by heterozygote matings (RS ´RS) is 0.25. Within egg masses produced from such matings, the number of resistant homozygotes (RR) is expected to be 25% of the total number of offspring. Therefore, 6.25% of the F 2 larvae should be homozygous and resistant (RR).

If the R allele confers recessive resistance to the amount of Cry3A toxin produced by Bt poplars then 6.25% of the F 2 neonates would be expected to survive on Bt poplar. From this expected frequency we can make two further predictions: (i) 25% of the egg masses produced by the F 1 females should give rise to resistant larvae (these egg masses are referred to as resistant egg masses); and (ii) the number of resistant larvae should be 25% of the total number of offspring emerging from resistant egg masses.

(e) Estimating the frequency of the resistance allele and experiment-wise probability

Expected allele frequencies were calculated using eqn 1 from Andow & Alstad (1998). From their later paper we calculated 95% CIs using eqn 5 if no resistant lines were detected, or eqn 7 if resistant lines were detected [START_REF] Andow | Credibility interval for rare resistance allele frequencies[END_REF]. Data for the three samples were pooled by assuming an uninformative beta prior distribution, Beta (u,v), with u = v = 1, appropriate when no prior data are available [START_REF] Andow | Frequency of resistance to Bacillus thuringiensis toxin CryIAb in an Iowa population of European corn borer (Lepidoptera: Crambidae)[END_REF]. For each line, detection probabilities were calculated using the algorithm described in Andow & Alstad (1998). This calculation gave the probability of detecting a resistance allele in an isofemale line if the line actually had a resistance allele. This probability is equal to [1 2 (probability of a false negative)] and is based on the probability that the resistance allele is lost prior to screening in the F 2 (Andow & Alstad 1998). An experiment-wise probability corresponding to the mean probability of not detecting a resistance allele was calculated for each sample and over the three samples.

RESULTS

(a) Allele frequency in the sample collected in 1999

Out of the 179 females collected, 128 (72%) produced enough offspring for the production of sib-mated F 1 . However, we were able to complete the F 2 screen for only 28 isofemale lines, with a mean ± s.d. of 171.2 ± 94.0 larvae tested per line. The number of F 1 females used to produce the F 2 of each line is indicated in figure 1a. Despite the small number of lines tested, one isofemale line (line 126) displayed resistant F 2 larvae.

For this positive line, 2.3% (20 out of 855) of the F 2 neonates tested survived on Bt poplar. This frequency was significantly lower (x 2 -test: x 2 1 = 21.65, p , 10 25 ) than the 6.25% of resistant larvae expected under the assumptions of the F 2 screen. When the proportion of resistant larvae was corrected by incorporating the mortality level on non-Bt poplar (14.4%, n = 125), the frequency was 2.7%, which is still significantly lower than the expected frequency (x 2 -test: x 2 1 = 14.86, p , 10 25 ). This lower frequency resulted from the proportion of resistant larvae per resistant egg mass being lower than expected. Indeed, the proportion of resistant egg masses was 33% (12 out of 36), consistent with the expected proportion of 25% (x 2test: x 2 1 = 0.93, p = 0.24). Conversely, the mean frequency (± s.e.) of resistant larvae per resistant egg mass, corrected according to the mortality level on non-Bt poplar, was 9.9 ± 1.8%, a value significantly lower (x 2 -test: x 2 1 = 37.61, p , 10 25 ) than the expected value of 25%. This may be caused by a fitness cost associated with the R allele. Alternatively, Bt poplar may produce enough toxin to kill a fraction of the RR individuals. Line 126 was retested over two more generations. Resistant larvae accounted for 2.1% (30 out of 1414) and 0.9% (13 out of 1425) of the F 3 and F 4 larvae tested, respectively. Isofemale line 126 was therefore considered to be resistant.

The most parsimonious explanation for these results is that one of the parents of isofemale line 126 was heterozygous for a Bt resistance allele. Based on Bayesian statistics, the expected frequency of this allele in the sampled population was 0.017 (95% CI = 0.0021-0.044; table 1). More than 80% of the lines had a detection probability of more than 95% (figure 2a) and the detection probability calculated over the 28 lines was 92.4% (table 1).

(b) Allele frequency in the sample collected in 2000

Out of the 51 isofemale lines collected, 39 (76.5%) produced enough fertile adults for the production of F 1 sibmated lines. Out of the 36 isofemale lines screened in the F 2 generation, we tested a mean ± s.d. of 393.1 ± 137.0 larvae per line on Bt poplar. The number of F 1 females used to produce the F 2 of each line is indicated in figure 1b. None of these lines produced F 2 resistant larvae. Bayesian statistics gave an estimated expected allele frequency of 0.0066 (95% CI = 0-0.016; table 1). In other words, the probability that the frequency was less than 0.016 was 95%. We calculated the cumulative probability of detecting a resistance allele and found that in more than 70% of the lines the probability of finding a resistance allele was greater than 95% (figure 2b). Over all the lines, the detection probability was 86.4% (table 1).

(c) Allele frequency in the sample collected in 2001

The last collection of 300 isofemale lines in 2001 generated 252 (84%) isofemale lines from which offspring could be produced by F 1 sib-mating, resulting in 206 isofemale lines that were tested in the F 2 generation (table 1). The number of F 1 females used to produce the F 2 of these lines is given in figure 1c. We screened a mean ± s.d. of 514.8 ± 255.2 larvae per line. Three isofemale lines (lines 15, 60 and 116) produced resistant larvae.

All the resistant larvae obtained in line 15 were detected in a single egg mass: nine resistant larvae out of 39 larvae tested. (A total of 90 egg masses (2124 larvae) were tested for line 15.). The survival of these larvae is probably not caused by a decrease in the toxin-expression level in the Bt poplar. First, the toxin gene is constitutively expressed in the plant, and preliminary bioassays have shown that it is lethal to susceptible larvae regardless of leaf age. Second, the same transgenic clone was always used to feed the larvae. Third, the F 2 screening procedures stimulated a very similar response in all experiments: the susceptible larvae were killed within 24-48 h as were the 30 larvae in the 'resistant' egg mass of line 15. Unfortunately, all the resistant larvae died before the adult stage. Moreover, we detected no further resistant larvae when this line was retested in the F 3 and F 4 generations from unselected F 2 larvae. Line 15 was therefore considered to be a false positive.

In line 60, resistant larvae accounted for 7.9% (171 out of 2157) of all the F 2 larvae tested. If corrected by incorporating the mortality level found on non-Bt poplar (11.4%, n = 44), the proportion of resistant larvae was 8.9%. This frequency is significantly higher (x 2 -test: x 2 1 = 23.28, p , 10 25 ) than the 6.25% expected under the assumptions of the F 2 screen. This was because the proportion of resistant egg masses was 42% (36 out of 85), a proportion significantly higher (x 2 -test: x 2 1 = 12.74, p = 0.0002) than the expected proportion of 25%. Nevertheless, the mean ± s.e. frequency of resistant larvae per resistant egg mass, once corrected by the mortality level found on non-Bt poplar, was 20.8 ± 1.6%. This value is significantly lower (x 2 -test: x 2 1 = 7.71, p = 4.8 ´10 23 ) than the expected value of 25%, as for the 1999 resistant line. We retested line 60 in the next generation and resistant larvae accounted for 2% (35 out of 1710) of all the F 3 larvae tested. We concluded that line 60 was a true resistant line. In the third positive line (line 116) resistant larvae accounted for only 1.9% (64 out of 3411) of all the F 2 larvae tested. When we took the mortality on non-Bt poplar (13.3%, n = 75) into account, the frequency of resistant larvae was 2.2%. This frequency is significantly lower (x 2test: x 2 1 = 83.53, p , 10 25 ) than the expected value. This was the result of the low proportion of resistant individuals recovered from each resistant egg mass, a tendency also observed for lines 126 (in 1999) and 60. Indeed, in resistant egg masses, the mean ± s.e. frequency of resistant larvae was, once corrected by the mortality level on non-Bt poplar, 9.7 ± 1.5%, whereas the expected proportion was 0.25 (x 2 -test: x 2 1 = 98.70, p , 10 25 ). Conversely, the proportion of resistant egg masses was 21% (24 out of 113), consistent with the expected proportion of 25% (x 2 -test:

x 2 1 = 0.66, p = 0.41). The F 3 generation was generated by pooling two resistant F 2 males with eight unselected virgin F 2 females. Almost 9% (206 out of 2309) of the F 3 neonates tested survived on Bt poplar. Based on these results, isofemale line 116 was also considered to be resistant.

We therefore concluded that one parent for each of the two lines 60 and 116 was heterozygous for a major Bt resistance allele. Based on Bayesian statistics, the expected frequency of this allele in the sampling population was 0.0036 (95% CI = 0.000 74-0.0085; table 1). The detection probability was greater than 95% for more than 80% of the lines (figure 2c) and the detection probability calculated over all the lines was 91.2%.

(d ) Global estimate

Results from the three samples were pooled to estimate the frequency of the Bt resistance allele that segregates in the population sampled in the Centre region of France. This estimation is based on the following assumptions: (i) all the resistant lines had the same Bt resistance allele; (ii) within each isofemale line, there was no divided paternity resulting from multiple matings; and (iii) the three samples were collected from the same site, as part of a single panmictic population [START_REF] Ge ´nissel | Population genetics of Chrysomela tremulae: a first step towards management of transgenic Bacillus thuringiensis poplars Populus tremula x tremuloides[END_REF]. As three resistant lines were detected in a total of 270 lines studied, the frequency of the allele conferring resistance to Bt poplar was 0.0037 (95% CI = 0.000 45-0.0080; table 1). The detection probability associated with this estimate is 90% (table 1).

(e) Life cycle of resistant larvae on Bt poplar

One of the three resistant lines (line 60) was further studied to determine whether or not resistant larvae were able to grow to adults when fed on Bt poplar only. Sixty resistant larvae were fed exclusively on Bt poplar. Twenty individuals (33%, 16 females and four males) reached the adult stage. These adults were pooled and gave birth to more than 25 fertile egg masses. The first three egg masses that were tested by the Bt poplar feeding test as for the F 2 screen procedure contained resistant larvae. We therefore concluded that the Bt resistance allele detected in line 60 confers a sufficient decrease in susceptibility to the Cry3A

Bt toxin that resistant beetles can complete their entire life cycle on Bt poplar.

DISCUSSION

Our results indicate that alleles enabling C. tremulae larvae to survive and reproduce on Bt poplar were segregating in a French poplar stand. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. The mean resistance-allele frequency for the period 1999-2001 was 0.0037 (95% CI = 0.000 45-0.0080), with a detection probability of 90%. This frequency is close to the highest values that are expected prior to the introduction of pesticide in the field, when considering fitness costs and typical mutation rates [START_REF] Roush | Ecological genetics of insecticide and acaricide resistance[END_REF].

High frequencies of alleles conferring resistance to Bt plants have already been detected in field populations of P. xylostella [START_REF] Tabashnik | Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis[END_REF], P. gossypiella [START_REF] Tabashnik | Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm[END_REF] and H. virescens [START_REF] Gould | Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens[END_REF]. However, these frequencies may have been artificially increased by man-made changes to the environment.

The R allele frequency of 0.12 reported for a susceptible strain of P. xylostella by [START_REF] Tabashnik | Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis[END_REF] was obtained in a sampling carried out on Hawaii, where cabbage, broccoli and watercress fields have been treated with Bt for many years [START_REF] Tabashnik | Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae)[END_REF]). Furthermore, even with stringent measures, [START_REF] Tabashnik | Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis[END_REF] recognized that a few individuals from Bt resistant strains may have occasionally contaminated this susceptible strain.

Cry1Ac-resistant larvae of P. gossypiella were recovered in populations captured in Arizona between 1997 and 1999 [START_REF] Tabashnik | Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm[END_REF]. However, in 1996, genetically modified cotton producing the Cry1Ac toxin was planted on 730 000 ha of US farm land [START_REF] Tabashnik | Global variation in the genetic and biochemical basis of diamondback moth resistance to Bacillus thuringiensis[END_REF], and Bt cotton accounted for more than half of the more than 100 000 ha of cotton in Arizona in [START_REF] Roush | Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management?[END_REF][START_REF] Gould | Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology[END_REF]and 1999[START_REF] Tabashnik | Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm[END_REF].

In H. virescens the frequency of the allele conferring resistance to Bt cotton was calculated for field-collected males captured in 1993 [START_REF] Gould | Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens[END_REF]. This frequency may therefore correspond to genuine initial conditions prior to the first commercial planting of transgenic cotton. However, Bt sprays were used in cotton fields in the mid-south of the US for a short period of time, and most of the samples screened came from this region (F. Gould, personal communication). Although local entomologists felt that these sprays were ineffective, they may have had sub-lethal effects on H. virescens populations, encouraging the development of resistance.

To date, Bt poplars have been planted only in a strictly protected insect-proof 20 m 2 greenhouse located ca. 100 km away from the sampling site, and French poplar plantations have never been treated with Bt sprays. Chrysomela tremulae feeds only on poplars (Augustin & Le ´vieux 1993) and could not have been exposed to Bt toxins from the treatment of other crops because formulations containing the Cry3A toxin-or any related toxin active against Chrysomelidae-have not yet been put on the market in France. Moreover, the migration of artificially selected Bt resistant genotypes originating in any bordering country is unlikely as there are neither Bt treated poplars nor Bt transgenic poplars within the poplar fields of these countries. Thus, our results provide the best evidence yet that alleles conferring resistance to Bt plants may be present at detectable frequencies in pest populations prior to any artificial selection resulting from pest management by humans.

Alleles conferring pesticide resistance may be part of the existing genetic variation prior to pesticide treatment, and may be generated by means of recurrent mutations [START_REF] Ffrench-Constant | The molecular and population genetics of cyclodiene insecticide resistance[END_REF][START_REF] Andreev | Multiple origins of cyclodiene insecticide resistance in Tribolium castaneum (Coleoptera: Tenebrionidae)[END_REF]) and/or migration events [START_REF] Raymond | Worldwide migration of amplified insecticide resistance genes in mosquitoes[END_REF][START_REF] Guillemaud | Testing the unique amplification event and the worldwide migration hypothesis of insecticide resistance genes with sequence data[END_REF]. As individuals carrying these alleles often pay a fitness cost in the absence of pesticide [START_REF] Roush | Ecological genetics of insecticide and acaricide resistance[END_REF][START_REF] Coustau | Resistance to xenobiotics and parasites: can we count the cost?[END_REF], the alleles are expected to segregate at a mutation-selection balance prior to selection. This frequency is ca. u/hs, with u being the mutation rate, s the fitness cost and h the dominance of this cost [START_REF] Hartl | Principles of population genetics[END_REF]. In such conditions, how is it possible for a Bt resistance allele to be present at a frequency of greater than 10 23 ? One possibility is a combination of a high mutation rate (e.g. u = 10 25 ) with a low (e.g. s = 0.01) and/or recessive (e.g. h = 0.1) fitness cost. It is also possible that resistance to Bt is naturally selected in field populations of C. tremulae. The ecological characteristics of Bt are largely unknown but this bacterium has been shown to be pathogenic to insects: although rare, natural epizootics do occur in field conditions [START_REF] Damgaard | Natural occurrence and dispersal of Bacillus thuringiensis in the environment[END_REF]. This bacterium is present not only in dead insects, but also in soils (e.g. [START_REF] Chilcott | Isolation and toxicity of Bacillus thuringiensis from soil and insect habitats in New Zealand[END_REF][START_REF] Chaufaux | Recherche de souches naturelles du Bacillus thuringiensis dans diffe ´rents biotopes a `travers le monde[END_REF], water (e.g. [START_REF] Iriarte | Isolation and characterization of Bacillus thuringiensis strains from aquatic environments in Spain[END_REF] and the phylloplane of many plants (e.g. [START_REF] Smith | The phylloplane as a source of Bacillus thuringiensis variants[END_REF][START_REF] Mizuki | Ubiquity of Bacillus thuringiensis on phylloplanes of arboreous and herbaceous plants in Japan[END_REF]. Bt strains producing the Cry3A toxin have not yet been shown to be present in poplar plantations or in dead insects of C. tremulae, but if such strains were shown to be present, they provide a natural source of selection for the Bt resistance allele recovered in this study.

As alleles conferring resistance to insecticidal proteins are not necessarily rare, it is essential to evaluate their frequencies before deploying transgenic plants producing these insecticidal proteins. The discriminative-dose assay approach (consisting of a selection at high Bt dose able to discriminate resistant phenotypes (e.g. [START_REF] Roush | Considerations for design of insecticide resistance monitoring programs[END_REF][START_REF] Sims | Field evaluation of transgenic corn containing a Bacillus thuringiensis Berliner insecticidal protein gene against Helicoverpa zea (Lepidoptera: Noctuidae)[END_REF]) is not sensitive enough because resistance to Bt toxins is often recessive [START_REF] Bourguet | Insecticide resistance and dominance levels[END_REF][START_REF] Ferre | Biochemistry and genetics of insect resistance to Bacillus thuringiensis[END_REF]. This is apparently the case in C. tremulae: the proportion of resistant larvae recovered in the three resistant lines suggests that resistance to the doses of Cry3A produced by Bt poplar is recessive (preliminary results of crosses between the resistant three lines and a susceptible line also indicate that resistance to Bt poplar is recessive). With recessive resistance and genotypic frequencies at Hardy-Weinberg equilibrium, the frequency of resistant individuals in a population equals the square of the allele frequency. Based on our estimates, we would therefore need to assay ca. 100 000 C. tremulae larvae to find one resistant individual; this number is probably greater than the number of individuals in the population in the poplar stand that has been considered here. In fact, the discriminative-dose assay approach is relevant only for detecting or monitoring alleles that are already at a very high frequency, as in natural populations of P. gossypiella, in which the frequency of the recessive Cry1Ac resistance allele may be as high as 0.16 [START_REF] Tabashnik | Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm[END_REF], or that confer dominant resistance to Bt toxins.

A more sensitive technique for estimating the initial frequency of Bt resistance was developed by [START_REF] Gould | Initial frequency of alleles for resistance to Bacillus thuringiensis toxins in field populations of Heliothis virescens[END_REF]. Field-collected males of H. virescens were mated with virgin homozygous resistant females from a resistant laboratory strain. The genotypes of the offspring were determined by discriminating-dose assay, so that the number of male parents that carried the resistance allele could be inferred. However, this single-pair mating design is effective only if a resistance allele has already been identified and fixed in a resistant strain, a condition that may not be easily satisfied when considering the initial situation before intervention. Moreover, this technique cannot take multiple resistance genes into account (Andow & Alstad 1998).

In this study, we used the F 2 screen developed by Andow & Alstad (1998) and refined by [START_REF] Andow | Credibility interval for rare resistance allele frequencies[END_REF]. This screening procedure increases the likelihood of detecting recessive and rare resistance alleles over the other two screening procedures cited. In particular, the F 2 screen can be used to estimate the frequency of any resistance allele sampled from the natural population and is suitable for estimating the statistical robustness of any experiment (see Andow & Alstad (1998) and [START_REF] Venette | An in-field screen for early detection and monitoring of insect resistance to Bacillus thuringiensis in transgenic crops[END_REF][START_REF] Venette | Strategies and statistics of sampling for rare individuals[END_REF] for a more detailed comparison of the various screening methods). The feasibility of this method has been demonstrated and it has been used to estimate the frequencies of Bt resistance alleles in field populations of the European corn borer, Ostrinia nubilalis (Andow et al. 1998(Andow et al. , 2000;;[START_REF] Bourguet | Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of the European corn borer, Ostrinia nubilalis[END_REF], and the rice stem borer, Scirpophaga incertulas [START_REF] Bentur | Frequency of alleles conferring resistance to a Bacillus thuringiensis toxin in a Philippine population of Scirpophaga incertulas (Lepidoptera: Pyralidae)[END_REF]. Although partial Bt resistance alleles, conferring a level of resistance not sufficiently high for survival on Bt plants producing large amounts of toxin, have been identified in populations of these pest species, major Bt resistance alleles have not been detected. These 'negative' results and those reported by [START_REF] Zhao | Examination of the F 2 screen for rare resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae)[END_REF] shed some doubts on the ability of the F 2 screen to detect low frequencies of R alleles. The US Environmental Protection Agency therefore waited for data to emerge before using this screening procedure in pest-management programmes (Environmental Protection Agency 2001). Our study demonstrates the usefulness of the F 2 screen for recovering major resistance alleles from pest populations, and validates this technique for monitoring the evolution of recessive Bt resistance alleles.

Genetic models have indicated that an allele conferring a recessive resistance at a frequency similar to that reported here for C. tremulae could lead to the rapid evolution of resistant populations in the absence of refuges for susceptible individuals (e.g. [START_REF] Mallet | Preventing insect adaptation to insect-resistant crops: are seed mixtures or refugia the best strategy?[END_REF][START_REF] Alstad | Managing the evolution of insect resistance to transgenic plants[END_REF]. Conversely, if refuges are planted over 50% of the cultivated area, the evolution of Bt resistance can be delayed by up to 20 generations [START_REF] Alstad | Managing the evolution of insect resistance to transgenic plants[END_REF]. If the resistance is completely recessive, a refuge as low as 5% might still delay the evolution of resistance for at least 50 generations [START_REF] Roush | Bt-transgenic crops: just another pretty insecticide or a chance for a new start in resistance management?[END_REF]). Moreover, our results suggest that resistant beetles may not be as fit as susceptible beetles. During the F 2 screen, the proportion of resistant individuals was lower than expected and only 33% of the resistant beetles completed their life cycle when feeding exclusively on Bt poplar. These data suggest that the resistance allele induced a fitness cost and/or conferred an incomplete resistance to Bt poplar. Carrie `re & [START_REF] Carrie `re | Reversing insect adaptation to transgenic insecticidal plants[END_REF] have shown that these two factors may prevent and even reverse the evolution of resistance, even if the initial frequency of Bt resistance is 0.1. Therefore, rather than challenging the high-dose plus refuge strategy, the results found in the present study provide empirical support for theoretical models using 0.001 and values slightly larger or smaller for the initial resistance-allele frequency.

Figure 1 .

 1 Figure 1. Number of F 1 females per F 0 during F 2 screen experiments in (a)1999 (28 F 0 ); (b) 2000 (36 F 0 ); and (c) 2001 (206 F 0 ). The mean ± s.d. number of F 1 females was 14.46 ±3.65, 11.83 ± 2.38 and 14.92 ± 2.50 in 1999, 2000 and 2001, respectively.

Figure 2 .

 2 Figure 2. Cumulative probabilities of detecting a resistance allele {S N i = 1 N i /N } (N = total number of lines) in isofemale lines tested in (a) 1999; (b) 2000; and (c) 2001.

Table 1 .

 1 Estimated expected frequency (E[p]) of the allele conferring resistance into Bt poplar C. tremulae.

			number of lines		estimated R allele frequency
	year	F 0	F 1	F 2	n a	E[p]	95% CI	detection probability (%)
	1999	179	128	28	1	0.017	(0.0021-0.044)	92.4
	2000	51	39	36	0	0.0066	(0-0.016)	86.4
	2001	300	252	206	2	0.0036	(0.000 74-0.0085)	91.2
	total	530	419	270	3	0.0037	(0.000 45-0.0080)	90.0

a Number of F 2 resistant lines.

We thank G. Magnanon, O. Denux and V. Dubois for assistance in screening the isofemale lines, and D. A. Andow, F. Gould, A. D. Long, A. McLysaght and M. Tenaillon for helpful comments on the manuscript. We also thank T. Stodola for providing the revised and unpublished version of Calculvar. This work was supported by a grant from the French Ministe `re de l'Education Nationale, de la Recherche et de la Technologie (Action Organise ´e 'Impact des Organismes Ge ´ne ´tiquement Modifie ´s').