Frequency of Resistance to Bacillus thuringiensis Toxin Cry1Ab in ...

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Frequency of Resistance to Bacillus thuringiensis Toxin Cry1Ab in Greek and Spanish Population of Sesamia nonagrioides (Lepidoptera: Noctuidae) S. S. ANDREADIS,1,2 F. A´LVAREZ-ALFAGEME,2,3 I. SAŃCHEZ-RAMOS,3 T. J. STODOLA,4 D. A. ANDOW,4 P. G. MILONAS,1 M. SAVOPOULOU-SOULTANI,1,5 AND P. CASTAŃERA3

J. Econ. Entomol. 100(1): 195Ð201 (2007)

ABSTRACT The high-dose/refuge strategy is considered as the main strategy for delaying resistance in target pests to genetically modiÞed crops that produce insecticidal proteins derived from Bacillus thuringiensis Berliner. This strategy is based on a key assumption that resistance alleles are initially rare (⬍10⫺3). To test this assumption, we used an F2 screen on natural populations of Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) from Greece and Spain. In total, 75 lines from Greece and 85 lines from Spain were screened for survival of F2 larvae on Cry1Ab corn, Zea mays L., leaves. No major resistance alleles were found. The frequency of resistance alleles in the Greek population was ⬍9.7 ⫻ 10⫺3 with 95% probability, which was very similar to that of the Spanish population (⬍8.6 ⫻ 10⫺3 with 95% probability), and the expected frequencies were 3.2 ⫻ 10⫺3 (0 Ð 0.0097) and 2.9 ⫻ 10⫺3 (0 Ð 0.0086) in Greece and Spain (pooled 1.5 ⫻ 10⫺3). The experiment-wise detection probability of resistance was 94.0 and 97.5% for the Greek and the Spanish population, respectively. Evidence of alleles conferring partial resistance to Cry1Ab was found only for the Greek population. The frequency of alleles for partial resistance was estimated as 6.5 ⫻ 10⫺3 with a 95% credibility interval between 8 ⫻ 10⫺4 and 17.8 ⫻ 10⫺3 and a detection probability of 94%. Our results suggest that the frequency of alleles conferring resistance to Cry1Ab, regarding the population of S. nonagrioides, may be rare enough so that the high-dose/refuge strategy could be applied with success for resistance management. KEY WORDS high-dose/refuge strategy, Bacillus thuringiensis, Sesamia nonagrioides, transgenic maize, F2 screen resistance management

Corn, Zea mays L., plants expressing ␦-endotoxins from the bacterium Bacillus thuringiensis (Bt) have been used on 11.6 million ha in the United States (NASS 2005) and 1.6 million ha in Argentina (ArgenBio 2006),with these two countries growing the most Bt corn in the world. Most Bt corn commercially grown in the European Union is located in Spain with ⬇58,000 ha in 2004 (MAPA 2005). Bt corn, which expresses the insecticidal Cry1Ab toxin, provides economic control of Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) and the European corn borer, Ostrinia nubilalis (Hu¨ bner) (Lepidoptera: Crambidae), which are considered the main lepidopteran pests of corn in southern Europe (Commonwealth Institute of Entomology 1979). In Greece and Spain, S. nonagrioides is usually more abundant than O. nubi1 Laboratory of Applied Zoology and Parasitology, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece. 2 These authors equally contributed to this article. 3 Departamento de Biologia de Plantas, Centro de Investigaciones Biolo´ gicas, CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain. 4 Department of Entomology, University of Minnesota, 219 Hodson Hall, 1980 Folwell Ave., Saint Paul, MN 55108. 5 Corresponding author, e-mail: [email protected].

lalis and causes greater crop damage, resulting in severe yield losses (Stavrakis 1967, Castan˜ era 1986, Stamopoulos 1999). Large-scale cultivation of Bt crops will exert high selection pressure on target pest species; consequently, these pests may evolve resistance with repeated exposure (Gould 1998), thereby eliminating the beneÞt of these crops. The risk of target pests becoming resistant to Bt crops has led to the development of resistance management strategies to delay the evolution of resistance. Several strategies have been proposed regarding resistance management for transgenic crops (Gould 1998, Roush 1998, Shelton et al. 2002, Vacher et al. 2003, Zhao et al. 2005). Among these strategies, the most widely accepted strategy is the high-dose/refuge strategy (Alstad and Andow 1995). This strategy is expected to work best when 1) dose of toxin expressed in plant tissues is high enough so that resistance is rendered recessive, i.e., heterozygote (RS) survival is low and about equal to susceptible homozygote (SS) survival on the Bt crop; 2) resistance alleles are rare (P ⬍ 10⫺3), so that there will be few homozygous (RR) survivors (p2 ⬍⬍ 10⫺6) (Roush and Miller 1986); and 3) refuges of non-Bt plants are planted close to Bt plots, so that nearly all resistant survivors from the Bt

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Fig. 1. Geographic locations where S. nonagrioides was collected in Greece (f, Serres; Œ, Larissa; and F, Thessaloniki) and Spain (E, Erbo).

plots will mate with susceptible individuals from the non-Bt refuge plants to produce heterozygous progeny that cannot survive on Bt plants. So far, no control failures due to widespread resistance have been reported to Bt crops in the Þeld (Tabashnik et al. 2003). Nevertheless, alleles conferring resistance to Bt crops have been found in Þeld populations of several species, including Pectinophora gossypiella (Saunders) to Bt cotton, Gossypium hirsutum L., in the United States (Tabashnik et al. 2000), Chrysomela tremulae L. to Bt poplar (Populus spp.) in France (Ge´ nissel et al. 2003), Helicoverpa armigera (Hu¨ bner) to Bt cotton in Australia (Akhurst et al. 2003), and Diatraea saccharalis (F.) to Bt corn in the United States (Huang et al., 2007). None of the insect pests that have been selected for resistance under laboratory conditions (Tabashnik 1994, Huang et al. 1997, Bolin et al. 1999, Ferre´ and Van Rie 2002, Tabashnik et al. 2003, Farino´ s et al. 2004) have been resistant to a Bt crop, probably because laboratory colonies usually start with limited genetic variation and thus may not contain the rare resistant alleles present in Þeld populations. Farino´ s et al. (2004) performed mass selection with a laboratory strain of the S. nonagrioides for resistance to Cry1Ab toxin. After eight generations of selection, the selected strain had a 21-fold increase in tolerance to Cry1Ab compared with the unselected strain. However, no resistance to Bt corn was achieved. Only the diamondback moth, Plutella xylostella (L.), has exhibited control failures due to resistance under Þeld conditions, but this resistance is to Bt sprays (Tabashnik et al. 1990). Several techniques have been used to discover and estimate the frequency of rare resistance alleles in natural populations. When resistance alleles are thought to be rare and recessive, the most efÞcient method is an F2 screen (Andow and Ives 2002), which preserves genetic variation among isofemale lines and concentrates resistance alleles into homozygous genotypes of the F2 generation so that they can be detected. Mated females are the preferred stage for initiating an F2 screen (Andow and Alstad 1998), but many alternatives have been used (Bentur et al. 2000, Zhao et al. 2002, Stodola and Andow 2004, Stodola et al. 2006). So far, F2 screens have been used in O. nubilalis (Andow et al. 1998, 2000; Bourguet et al. 2003;

Stodola et al. 2006); P. xylostella (Zhao et al. 2002); yellow stem borer, Scirpophaga incertulas (Walker) (Bentur et al. 2000); H. armigera (Akhurst et al. 2003); C. tremulae (Ge´ nissel et al. 2003); and D. saccharalis (F.) (Huang et al., 2007). No resistance alleles have been recovered from populations of O. nubilalis or S. incertulas, but alleles have been recovered using F2 screens from natural populations of the other species. However, even in those cases where alleles have not been recovered, the F2 screen has allowed estimation of the frequency of resistance alleles in the natural population, which has been valuable for insect resistance management planning. F2 screens have never been developed or used for detecting resistance in S. nonagrioides. The objective of this research was to look for resistance alleles in natural populations and determine the initial frequency of resistance alleles to Bt corn expressing Cry1Ab by using the F2 screen on Þeld populations of S. nonagrioides from different geographical locations from Greece and Spain. Materials and Methods F2 Screen. Andow and Alstad (1998) described the F2 screen protocol for O. nubilalis, which is also applicable to many other insects that can be reared in the laboratory. This protocol was adapted to S. nonagrioides, by using wild mated females or pair-mating adults reared from late instars from natural populations to establish isofemale lines, rearing and sib-mating the F1 offspring in each isofemale line, and screening the F2 neonates on excised Bt plant leaf tissue. Knowing that each isofemale line carries at least four haplotypes (two from the female and two of her mate), each line allows us to characterize resistance on at least four haplotypes. After sib-mating the F1 generation, 1/16 of the F2 offspring are expected to be homozygous for the resistance allele if one of the P1 adults carried a resistance allele. Insect Collection. S. nonagrioides were collected from three different geographic locations from Greece (Serres, Larissa, and Thessaloniki) and one region from Spain (Ebro) during 2004 and 2005 (Fig. 1). Greece. S. nonagrioides adults were collected from the region of Serres in summer 2004 (15Ð30 August)

February 2007

ANDREADIS ET AL.: F2 SCREEN FOR GREEK AND SPANISH S. nonagrioides

with the use of a blacklight trap. Adults were collected before sunrise and placed in cages, males and females separated, and either shipped or carried to the laboratory within 4 h to avoid exposure to high temperatures. In the laboratory, each female was caged in a plastic cylinder (13 cm in diameter, 30 cm in height) with one corn plant and sugar water. In addition, late instars (fourth to Þfth) of S. nonagrioides were collected in the region of Larissa during fall 2004 (1Ð31 October) and 2005 (1Ð31 October) and in Thessaloniki in 2005 (20 SeptemberÐ20 October). Only one larva per corn plant was taken from plants at least 1.5 m apart to minimize the possibility that sibs were collected. Larvae were reared on an artiÞcial diet (Poitout and Bues 1970, Gonza´lez-Nun˜ ez et al. 2000) at 25 ⫾ 1⬚C, 70 ⫾ 5% RH, and a photoperiod of 16:8 (L:D) h. Pupae were sexed, and males and females were held separately. After adult emergence, males and females were paired to form the isofemale lines in similar plastic cylinders as for wild-collected adults. Spain. Larvae were collected by dissecting corn stalks in the region of Ebro (northwestern Spain) during the fall 2004 (19 Ð21 October) and 2005 (3Ð 6 October), by using similar methods as in Greece. Most larvae (⬇80%) were sixth instars. Field collected larvae were dipped in a 1% bleach solution for a few seconds to prevent pathogen contamination. Diapausing larvae were maintained in plastic cages containing vermiculite and meridic diet (Farino´ s et al. 2004). When required, diapause was broken by placing larvae at 28 ⫾ 0.3⬚C, 70 ⫾ 5% RH, and continuous light. Larvae were placed in plastic boxes (3Ð50 per box) and reared on an artiÞcial diet (Poitout and Bues 1970, Gonza´ lez-Nun˜ ez et al. 2000). Pupae were sexed and as adults emerged, males and females were paired in ventilated plastic cylinders (6 cm in diameter, 16 cm in height), containing a corn seedling, to form an isofemale line. All larvae, adults and eggs were held at 25 ⫾ 1⬚C, 70 ⫾ 5% RH, and a photoperiod of 16:8 (L:D) h. In both laboratories, F1 egg masses were collected daily, by removing corn leaves or entire plants with eggs, and the egg masses were placed in small plastic boxes (4.5 cm in diameter, 3 cm in height) with tightÞtting lids on moistened Þlter paper with propionic acid (1:1000 dilution) (Fantinou et al. 2003). Groups of 20 neonate larvae were placed in plastic petri dishes (9 cm in diameter, 1.5 cm in height). Every 3 d either new diet was added, or larvae were transferred to a new clean dish. Pupae from the same line were kept together. As adults emerged, males and females (F1 families) of each line were placed in large cages (30 by 30 by 30 cm) and were allowed to sib-mate at random (number of adults per line ⫾ SD: Serres, 44.9 ⫾ 21.7; Larissa, 34.1 ⫾ 17.0; Thessaloniki, 60.1 ⫾ 46.5; Ebro [2004], 26.9 ⫾ 10.5; Ebro [2005], 17.3 ⫾ 7.7). For each line, the number of F1 males and females that were sib-mated was recorded. Additionally, F1 cages were provided with several corn seedlings per cage, and F2 egg masses were collected daily. Neonates hatched from F2 egg masses were screened

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on Bt corn leaves (Compa CB, event 176, and Syngenta). F2 screens were performed in small rectangular boxes (10 by 5 by 5 cm). Approximately six to eight leaves of Bt corn tissue were put into the boxes over a moistened Þlter paper. Typically, 60 (and always ⬎20) S. nonagrioides neonates (⬍24 h) were transferred with a Þne brush into each box. Every 2 d, Bt corn leaves were replaced, and Þlter paper was remoistened. All dishes were examined for surviving larvae after 4 d. A fully positive line required many larvae feeding and growing to at least the second instar. To be considered partially positive, an isofemale line must produce leaf damage ⱖ2 on the Bolin scale (Bourguet et al. 2003). When needed, partially positive lines were rescreened during the F3 generation. Cost of F2 Screen. We estimated variable costs of the F2 screen including travel time for collecting females and late instars from natural populations, labor, and insect diet. Labor costs were estimated as a percentage of annual salary, including fringe beneÞts of all participants, including several research scientists. Fixed and capital costs, such as overhead, insect cages and dishes, and growth chambers, were not estimated in Greece, where these costs were low, but they were estimated as amortized costs in Spain, where growth chambers were used. Statistical Analysis. The two statistics needed to analyze results from a F2 screen are the expected resistance allele frequency (E[pR]) with its 95% credibility interval and the probability of a false negative (PNo) (i.e., probability we miss of Þnding a resistance allele that was present in a line). Bayesian analysis was used to estimate the expected frequency of resistance and credibility intervals, which enable us to make statistical inferences about the populations that we sampled (Andow and Alstad 1998, Andow and Alstad 1999, Schneider 1999). The statistical software Mathematica 5 (Wolfram Research 2003) was used to compute beta integrals. Probability of a false negative (PNo) depends on the number of F1 males (M) and F1 females (F) that contribute to the F2 generation, the number of F2 offspring screened per F1 female (J), and the nonscreen mortality of F2 larvae (␮). Analytical methods for calculating PNo are given in Stodola and Andow (2004). Results Greece. Serres. Of the 200 females collected, we established 194 (97.0%) isofemale lines. Of these 194 isofemale lines 140 (72.2%) produced fertile eggs to enable production of the sib-mated F1. The other lines were lost because P1 females did not produce enough eggs or F1 larva died before pupation. A similar result occurred for the other collecting sites. We were able to complete F2 screening on only 30 lines (15.46% of established isofemale lines; Table 1). Over these 30 isofemale lines, F1 family size was adequate with 24.4 ⫾ 12.0 males and 24.4 ⫾ 12.0 females. On average, 9.3 ⫾ 6.5 F2 neonates larvae per female (J) were tested for their susceptibility to Cry1Ab. Mortality on untreated diet (␮) was 51.3%. In no cases did a line

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Table 1.

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Number of lines progressing through the F2 screen experiment

Paired P1 from Þeld-collected larvae

Location

P1 lines started

F1 eggs produced

F1 larvae produced

F1 adults produced

Screened

2004 2004 2005 2005 Total 2004 2005 Total

Serres Larissa Larissa Thessaloniki Greece Ebro Ebro Spain

194 90 16 183 483 141 254 395

140 44 5 76 265 133 205 338

96 20 4 37 157 64 81 145

45 11 4 30 90 53 58 111

30 11 4 30 75 53 32 85

produce any F2 larvae that survived ⬎4 d on Bt corn leaves. These results suggest that there were no major resistance alleles to Cry1Ab toxin among the isofemale lines we screened. The experiment-wise detection probability was 94.6%. Larissa. Regarding the F2 screening tests from Larissa, we pooled the results from both years 2004 and 2005, and of the initial 125 pairs, we managed to establish 106 (84.8%) isofemale lines. Of these 106 isofemale lines only 49 (46.2%) produced enough eggs to enable production of the sib-mated F1. We were able to perform F2 screen on only 15 lines (14.2% of established isofemale lines; Table 1). Over these lines, F1 family size was 17.9 ⫾ 3.3 males and 22.2 ⫾ 7.9 females. An average of 8.2 ⫾ 5.1 F2 neonates larvae per female (J) were tested on Bt corn leaves expressing Cry1Ab. Mortality on untreated diet (␮) was 42.8%. Although none of the 15 lines fed extensively on Bt corn, one line (#91) had survivors after 4 d and was scored as a potential resistant line. Untreated larvae of this line (#91) were fed on untreated diet and afterwards retested for Cry1Ab susceptibility at generation F3. None of the F3 larvae survived on Bt corn after 4 d of exposure. We did not identify any major or partial resistance alleles in the region of Larissa. The experiment-wise detection probability was 95.3%. Thessaloniki. Of the initial 216 pairs, we established 183 (84.7%) isofemale lines. Of these 183 isofemale lines only 76 (41.5%) lines laid fertile eggs to enable production of the sib-mated F1. We were able to perform F2 screen on 30 lines (16.4% of established isofemale lines; Table 1). Over these 30 isofemale lines, F1 family size was 25.7 ⫾ 21.6 males and 34.0 ⫾ 25.4 females. On average, 10.8 ⫾ 10.5 F2 neonates larvae per female (J) were tested on Bt corn leaves expressing Cry1Ab. Mortality on untreated diet (␮) was 42.8%. Although none of the 30 lines fed extensively on Bt corn, two lines (#151 and #154) had at least one larva still alive after 4 d and were considered potentially resistant lines. These lines (#151 and #154) were retested for Cry1Ab susceptibility at generation F3. None of the F3 larvae survived on Bt corn after 4 d of exposure. One line (#72) caused damage ⱖ2.0 on the Bolin scale over two generations (F2 and F3) testing, and was identiÞed as having partial resistance (Table 2). We did not identify any major resistance alleles in the region of Thessaloniki. The experimentwise detection probability was 92.7%.

Frequency of a Major Bt Resistance Allele. Overall, we observed S ⫽ 0 (number of isofemale lines with a major Bt resistance) over the three regions (Serres, Larissa, and Thessaloniki) from Greece that we screened, giving several estimates of major allele frequencies and their respective credibility intervals (CIs) (Table 3). If we combine the results from all years and all collecting sites together, S ⫽ 0 and N ⫽ 75 (total number of lines evaluated; 30 lines from Serres, 15 lines from Larissa and 30 lines from Thessaloniki), then the estimated R frequency for Greece is 0.0032 with a 95% CI between 0 and 0.0097 (Table 3). Thus, the frequency of resistance is ⬍9.7 ⫻ 10⫺3 with 95% probability. The cumulative probability of detecting a resistance allele in each of these isofemale lines for all sites is shown in Fig. 2A. The detection probability associated with this estimate is 94%. Frequency of a Partial Bt Resistance Allele. In 2004, we had no evidence for partial resistance alleles. However, in 2005 the extensive damage observed on Bt corn leaves over two generations for line #72 from the Thessaloniki region suggests that this line may have had an allele that conferred partial resistance to Cry1Ab. Assuming that the lines that were not rescreened did not have partial resistance and combining the results from Greece over years and sites, we observed that S ⫽ 1 and N ⫽ 75. Based on Bayesian statistics, the expected frequency of alleles conferring partial resistance is 0.0065 with a 95% CI between 0.0008 and 0.0178 (Table 3). Spain. Ebro. In 2004, we established 141 isofemale lines. Of these 141 isofemale lines 133 (94.3%) proTable 2. Results from F2 screen and subsequent rescreening of positive lines Yr Greece 2004 2005 Spain 2004 2005

Screen generation

Screen method

F2 F2 F3

Bt Event 176 corn leaves Bt Event 176 corn leaves Bt Event 176 corn leaves

0 1 1

0 0 0

F2 F2

Bt Event 176 corn leaves Bt Event 176 corn leaves

0 0

0 0

Partial Full positive positive

A full positive result required many larvae feeding and growing to at least the third instar on the screening media. A partial positive required a rating ⱖ2 on the Bolin Scale (corn), evidence of feeding and tunneling (diet), or larvae surviving to the second instar on either media.

February 2007 Table 3.


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Expected frequencies of major and minor resistance alleles, with experiment-wise probability of a false negative

Location

Screened lines

E关pR兴 (95% Cl)

E关ppartial兴 (95% Cl)

Experiment-wise PNo

Serres Larissa Larissa Thessaloniki Greece (pooled) Ebro Ebro Spain (pooled)

30 11 4 30 75 53 32 85

0.0078 (0Ð0.0230) 0.0192 (0Ð0.0552) 0.0417 (0Ð0.1127) 0.0078 (0Ð0.0230) 0.0032 (0Ð0.0097) 0.0045 (0Ð0.0135) 0.0073 (0Ð0.0217) 0.0029 (0Ð0.0086)

0.0078 (0Ð0.0230) 0.0192 (0Ð0.0552) 0.0417 (0Ð0.1127) 0.0156 (0.0020Ð0.0418) 0.0065 (0.0008Ð0.0178) 0.0045 (0Ð0.0135) 0.0073 (0Ð0.0217) 0.0029 (0Ð0.0086)

0.054 0.059 0.014 0.073 0.060 0.01 0.05 0.025

duced enough offspring to enable production of the sib-mated F1. Finally, we were able to perform F2 screen on 53 lines (37.6% of established isofemale lines; Table 1). Over these isofemale lines, F1 family size was 24.7 ⫾ 9.2 males and 29.1 ⫾ 11.4 females. On average, 17.1 ⫾ 9.7 F2 neonates larvae per female (J) were tested on Bt corn leaves expressing Cry1Ab. Mortality on untreated diet (␮) was 28.5%. None of these 53 lines that we screened fed extensively on Bt corn leaves suggesting that there were no major resistance alleles to Cry1Ab toxin among the isofemale lines we screened (Table 2). The experiment-wise detection probability for 2004 was 99.0% (Table 3). In 2005, we established 254 isofemale lines. Of these 254 isofemale lines, 205 (80.7%) laid fertile eggs to enable production of the sib-mated F1. Overall, we were able to complete F2 screening only on 32 lines (12.6% of established isofemale lines; Table 1). Over these 32 isofemale lines, F1 family size was 16.0 ⫾ 5.9 males and 18.4 ⫾ 9.0 females. On average, 19.2 ⫾ 10.4 F2 neonates larvae per female (J) were tested on Bt corn leaves expressing Cry1Ab. Mortality on untreated diet (␮) was 27.8%. Larvae in none of these 32 lines that we screened fed extensively on Bt corn leaves, suggesting that there were no major resistance alleles to Cry1Ab toxin among the isofemale lines we screened (Table 2). The experiment-wise detection probability for 2005 was 95.0% (Table 3). Frequency of a Major Bt Resistance Allele. We observed S ⫽ 0 (number of isofemale lines with a major Bt resistance) over all samples that we screened from the region of Ebro during 2004 and 2005 (Table 2), giving several estimates of major allele frequencies and their respective CIs (Table 3). If we combine the results from all years together, S ⫽ 0 and N ⫽ 85 (total number of lines evaluated; 53 lines from 2004 and 32 lines from 2005), then the estimated R frequency for

Fig. 2. The cumulative probability density function (PDF) of detecting a resistance allele had one been present in a line, e.g., 1 ⫺ probability of a false negative (PNo). (A) Greece. (B) Spain.

Spain is 0.0029 with a 95% CI between 0 and 0.0086 (Table 3). Thus, the frequency of resistance is ⬍8.6 ⫻ 10⫺3 with 95% probability with a detection probability of 97.5% (Fig. 2B). Frequency of a Partial Bt Resistance Allele. From 2004 to 2005, we did not observe any extensive damage on Bt corn leaves by any line and thus found no evidence for partial resistance alleles (Table 2). So, the expected frequency of alleles conferring partial resistance is 0.0029 with a 95% CI between 0 and 0.0086 (Table 3). Cost of F2 Screen. The estimated variable cost of the F2 screen procedure for the Greek population was 287€ and 501€ per isofemale line when we used Þeld collected adults or late instars, respectively. The corresponding cost regarding the Spanish population was ⬇386€ per line. This included collecting late instars, egg collection from P1 adults, rearing and sibmating the F1 families, and Þnally screening the F2 offspring. The major part of the cost was for labor, and the high estimated costs reßect the salaries of research scientists devoting a considerable portion of time to learning the procedure. By using trained technicians and eliminating research time, we estimate this cost could be reduced by half. Discussion Detection of Resistance and Estimation of Resistance Frequencies. Many monitoring methods have been proposed so far that could be used in an adaptive resistance management plan, including in-Þeld screen (Venette et al. 2000), screening of Þeld-collected egg masses or larvae (Roush and Miller 1986), screening of laboratory selected stocks (Gould et al. 1997), and an F2 screen (Andow and Alstad 1998). The F2 screen is a genic screen and is particularly efÞcient for rare, recessive alleles. It allows us to detect any resistance allele that is present in the collected female, including those of her mates. We have made the Þrst effort to document the frequency of resistance alleles to the Cry1Ab of B. thuringiensis for populations of S. nonagrioides originating from Greece and Spain by using an F2 screen. The experiment-wise detection probability of resistance was 94.0 and 97.5% for the Greek and the Spanish population, respectively. The frequency of resistance alleles in the Greek population was ⬍9.7 ⫻ 10⫺3 with 95% probability, which was very similar to that of the Spanish population (⬍8.6 ⫻ 10⫺3 with 95% probabil-

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ity), and the expected frequencies were 3.2 ⫻ 10⫺3 and 2.9 ⫻ 10⫺3 in Greece and Spain. These results were comparable with that of other species (Andow et al. 2000, Bourguet et al. 2003, Stodola et al. 2006) and indicate that in each country the frequency of resistance approaches the value of 0.001 that would enable successful implementation of the high-dose refuge strategy for resistance management in Bt corn against S. nonagrioides. Recent studies have shown that S. nonagrioides may constitute a single large panmictic unit across all of southern Europe, because there were only small genetic differences between populations collected from Greece and Spain (De la Poza et al. 2006). Although more extensive studies should be carried on the differentiation of S. nonagrioides in southern Europe, under the assumption of a single panmictic unit, we can pool the data from Greece and Spain. In this case, S ⫽ 0 and N ⫽ 160, so the expected resistance allele frequency was 1.5 ⫻ 10⫺3 with a 95% CI of (0.0, 4.6 ⫻ 10⫺3), which is very near the value needed for effective resistance management. By similar reasoning, the frequency of partial resistance in S. nonagrioides in southern Europe was 3.1 ⫻ 10⫺3 with a 95% CI of (0.4 ⫻ 10⫺3, 8.5 ⫻ 10⫺3). Bourguet (2004) suggested that it is very common to recover partial resistance alleles to Bt toxins instead of major resistance if the founding population is small, possibly because the initial levels of genetic diversity are low. Regardless, identiÞcation of partial resistance alleles is not uncommon (Andow et al. 1998, Bourguet et al. 2003). Cost of F2 Screen. In the current study, the number of lines that were successfully screened was drastically less than the number originally established. From the initial 483 Greek and 395 Spanish parental lines, only 75 (15.5%) and 85 (21.5) lines were screened, respectively. Fewer of the Greek lines successfully produced F1 eggs than the Spanish lines, which may be due to population differences or differences in handling and rearing methods. In both countries many lines were lost between F1 egg production and the establishment of F1 larval populations. The reason for these high losses is not known. Once a line was established as a healthy F1 larval population, it was usually successfully screened. As a result of high line loss rates, the variable cost per isofemale line that was screened was much higher than for other species such as O. nubilalis (Andow et al. 1998, 2000). In Greece when we used Þeldcollected adults for the F2 screen, the cost per line was much lower in comparison with Þeld-collected larvae, because the time and money for rearing larvae to the adult stage was saved. Therefore, to improve the costeffectiveness of the F2 screen for S. nonagrioides, additional research should be conducted to improve and reduce the cost of rearing and handling methods. Molecular genetic methods could be developed to screen for resistance alleles. These would be genic methods similar to an F2 screen and would likely be more cost-effective than an F2 screen. However, before such molecular methods could be developed and veriÞed, it would be necessary to identify and recover a resistance allele. An F2 screen is the most cost-

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effective method for identifying and recovering rare, recessive resistance alleles. Thus, research to reduce per unit cost of an F2 screen for S. nonagrioides would be a valuable addition for resistance management. Acknowledgments We thank Syngenta Company for providing the Compa CB seeds. This work was funded by grants from the EU, Proposal No QLRT-2001-01969 (Protecting the BeneÞts of Bt-toxins from Insect Resistance Development by Monitoring and Management).

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