Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests. The main strategy proposed for delaying resistance to Bacillus thuringiensis (Bt) toxins in transgenic crops is the high-dose/refuge strategy. This strategy is based on the unverified assumption that resistance alleles are initially rare (<10(-3)). We used an F-2 screen on >1,200 isofemale lines of Ostrinia nubilalis Hifter (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999-2001. In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin. A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was <9.20 x 10(-4) with 95% probability, and a detection probability of >80%. In the northern US corn belt, the frequency of resistance to Bt maize was <4.23 x 10(-4) with 95% probability, and a detection probability of >90%. Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive. These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize.

Despite the potentially profound impact of genetically modified crops on agriculture and the environment, we know little about their long-term effects. Transgenic crops that produce toxins from Bacillus thuringiensis (Bt) to control insects are grown widely, but rapid evolution of resistance by pests could nullify their benefits. Here, we present theoretical analyses showing that long-term suppression of pest populations is governed by interactions among reproductive rate, dispersal propensity, and regional abundance of a Bt crop. Supporting this theory, a 10-year study in 15 regions across Arizona shows that Bt cotton suppressed a major pest, pink bollworm (Pectinophora gossypiella), independent of demographic effects of weather and variation among regions. Pink bollworm population density declined only in regions where Bt cotton was abundant. Such long-term suppression has not been observed with insecticide sprays, showing that transgenic crops open new avenues for pest control. The debate about putative benefits of Bt crops has focused primarily on short-term decreases in insecticide use. The present findings suggest that long-term regional pest suppression after deployment of Bt crops may also contribute to reducing the need for insecticide sprays.

The colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance has the potential use in an integrated pest management program for control of colorado potato beetle. During the 1998 and 1999 seasons, field studies were conducted to compare natural (leptine glycoalkaloids and glandular trichomes), engineered (Bt-cry3A and Bt-cry5 transgenic potato lines), and combined (Bt-cry5+glandular trichomes) plant resistance mechanisms of potato for control of colorado potato beetle. Nine different potato clones representing five different host plant resistance mechanisms were evaluated under natural colorado potato beetle infestation at the Montcalm Research Farm in Entrican, Michigan. The Bt-cry3A transgenic lines, the high leptine line (USDA8380-1), and the high foliar glycoalkaloid line (ND5873-15) were most effective for controlling defoliation by colorado potato beetle adults and larvae. The Bt-cry5 line (SPc5-G2) was not as effective as the Bt-cry3A transgenic lines ('Russet Burbank Newleaf,' RBN15, and YGa3.1). The glandular trichome (NYL235-4) and Bt-cry5+glandular trichome lines proved to be ineffective. Significant rank correlations for the potato lines between the two years were observed for egg masses, second and third instar, and fourth instar seasonal cumulative mean number of individuals per plant, and defoliation. Egg mass and first instar seasonal cumulative mean number of individuals per plant were not strong indicators of host plant resistance in contrast to second and third instars or adults. Based on these results, the Bt-cry3A transgenic lines, the high leptine line, and the high total glycoalkaloid line are effective host plant resistance mechanisms for control of colorado potato beetle.

Foliage from Solanum species found within or near potato fields in New Zealand was compared to potato (Solanum tuberosum) foliage for its ability to support growth of larvae of Phthorimaea operculella (potato tuber moth, PTM). Solanum species included S. laciniatum (poroporo), S. physalifolium (hairy nightshade), S. chenopodioides (velvety nightshade), S. nigrum (black nightshade), and S. dulcamara (bittersweet). In detached leaf bioassays larval growth was greater on velvety nightshade and poroporo than on foliage from the potato cultivar, 'Iwa', in the first year, but only larval growth on poroporo foliage was greater in the second year (P < 0.05). Larval growth on bittersweet foliage was similar to larvae on 'Iwa' foliage. No larvae survived on black nightshade beyond 5 days in both years. From a field survey, no PTM larval mines were found on any foliage of hairy, velvety, or black nightshade plants in or within 100 in of a field trial of transgenic PTM-resistant potato plants from a survey undertaken in the third summer. Over the entire survey, an average of 0.18 mines/plant were recorded on poroporo plants, all of which were <200 mm(2), compared to an average of 3.8 mines/non-transgenic potato plants (volunteer potato plants >100 m from the field trial and non-transgenic potato plants of the cultivars 'Iwa', 'Red Rascal', and breeding line 2390, used as experimental controls within the field trial). This study demonstrated that, under no-choice conditions, PTM larvae could grow on alternative Solanum species. However, PTM exhibited a clear preference for oviposition and development on potato plants in the field.

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 19992001 at 0.0037 (95% confidence interval = 0.00045-0.0080) 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.

Hybrid poplars (Populus tremula x Populus tremuloides) have been genetically engineered via Agrobacterium tumefaciens, to express a synthetic cry3Aa gene derived from the native Bacillus thuringiensis subsp. tenebrionis cry3Aa gene. The presence and the expression of the transgene have been verified in four transgenic poplar lines, using Southern, northern and western analyses. The transgenic poplar's toxicity towards the phytophagous beetle Chrysomela tremulae (Coleoptera, Chrysomelidae) has been assessed on six month-old greenhouse-grown selected plants in laboratory conditions. Laboratory experiments consisted of feeding tests of fresh detached leaves on C. tremulae at all developmental stages. Our results indicate that the transgenic poplar leaves, expressing a Cry3Aa protein amount in a range of 0.05-0.0025% of total soluble protein, were definitely deleterious for C. tremulae, regardless of the developmental stage.

Field and laboratory studies evaluated the influence of selected crop hosts on Helicoverpa zea (Boddie) population dynamics in relation to genetically engineered Bt (Bollgard) and non-Bt cottons. Host specific H. zea colonies were initiated with a colony originally collected from sweet corn. The colony was allowed to complete one generation on meridic diet then split into cohorts and allowed to complete one generation on field corn, grain sorghum, soybean, cotton, or meridic diet in individual 29.5 ml plastic cups. During the first part of the study, larval developmental times, pupal weights, and survival were measured. H. zea survival was higher on meridic diet and grain sorghum than on soybean and cotton. Development of H. zea larvae was faster on field corn than all other larval diets. Also, H. zea required a longer period of time to complete development on cotton than on the other hosts. Pupal weights were higher on meridic diet than the plant hosts. Pupal weights of H. zea that completed larval stadia on cotton were lower than on the other larval diets. Neonates (F-1) from each of the host specific colonies (200 per colony) were exposed to Bt and non-Bt cottons. Mortality of second generation H. zea on non-Bt and Bt cottons was measured at 96 h. H. Zea larvae from the cotton colony had higher mortality on non-Bt cotton than the other host specific colonies except the grain sorghum colony. On Bt cotton, larvae from the corn colony had a higher level of mortality than larvae from the soybean and grain sorghum colonies. These data provide valuable information for evaluating the contribution of cultivated hosts as additional, alternative refugia in Bt cotton resistance management plans.

Laboratory studies were performed to characterize the lepidopteran toxicity of cotton plants expressing two different toxin proteins from Bacillus thuringiensis (Bt), in order to assess insect resistance management implications of a commercial, two-toxin transgenic cotton. An independent and additive interactive effect of two Bt delta-endotoxins expressed by the transgenic cotton variety 15985 was demonstrated by examining the responses of Heliothis virescens (F.), Helicoverpa zea (Boddie), and Spodoptera frugiperda (J.E. Smith) larvae to field- or greenhouse-grown tissue from genetic near-isolines, which expressed Cry1A only, Cry2Ab only, or both toxins. In all cases, the Cry2Ab component was the larger contributor to total toxicity in the two-toxin isoline. Toxin-specific, quantitative enzyme-linked immunosorbent assay (ELISA) tests confirmed that the levels of each toxin in tissues of the two-toxin isoline were not statistically different (P > 0.05) from the levels found in the corresponding tissues of the respective single-toxin isoline. Resistance management considerations were discussed. Considering the additive interaction of toxins, a relatively simple insect resistance-monitoring procedure was proposed for the monitoring of commercial cotton varieties expressing both toxins.

If registered, transgenic corn, Zea mays L., with corn rootworm resistance will offer a viable alternative to insecticides for managing Diabrotica spp. corn rootworms. Resistance management to maintain susceptibility is in the interest of growers, the Environmental Protection Agency, and industry, but little is known about many aspects of corn rootworm biology required for an effective resistance management program. The extent of larval movement by the western corn rootworm, Diabrotica virgifera virgifera LeConte, that occurs from plant-to-plant or row-to-row after initial establishment was evaluated in 1998 and 1999 in a Central Missouri cornfield. Post-establishment movement by western corn rootworm larvae was clearly documented in two of four treatment combinations in 1999 where larvae moved up to three plants down the row and across a 0.46-m row. Larvae did not significantly cross a 0.91-m row after initial host establishment in 1998 or 1999, whether or not the soil had been compacted by a tractor and planter. In the current experiment, western corn rootworm, larvae moved from highly damaged, infested plants to nearby plants with little to no previous root damage. Our data do not provide significant insight into how larvae might disperse after initial establishment when all plants in an area are heavily damaged or when only moderate damage occurs on an infested plant. A similar situation might also occur if a seed mixture of transgenic and isoline plants were used and if transgenic plants with rootworm resistance are not repellent to corn rootworm larvae.

Pairs of Helicoverpa zea (Boddie) larvae reared on diet-incorporated MON810 transgenic leaf tissue of field corn (Zea mays L.) were observed in the laboratory to characterize effects of sublethal levels of Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins on cannibalistic behavior and mortality. Feeding on sublethal levels of Bt corn reduced the frequency of cannibalistic behaviors exhibited by H. zea when uneven instars were paired together. Exposure to the Bt endotoxin had no significant effect on when cannibalistic mortality occurred or the level of mortality as a result of cannibalism. Assuming that H. zea larvae reared on nonBt corn tissue behaved in a similar way that resistant larvae would if feeding on Bt tissue, sublethal effects of Cry1Ab intoxication may reduce the chances of successful cannibalism by susceptible larvae and thus play a disproportionate role in the survival of multiple ear infestations. Furthermore, cannibalistic encounters could result in partially resistant larvae feeding on nontoxic food, thus temporarily providing an escape from exposure to the Bt endotoxin. These behavior alterations could increase the selective differential between susceptible individuals and those carrying resistance genes.

A 3-yr study (1996-1998) was conducted to evaluate the effects of MON810 Bt corn on Helicoverpa zea (Boddie) emergence and to determine whether delayed larval development as a result of Bt intoxication results in higher levels of diapause induction and pupal mortality. In the 1997 study, there was no difference in prepupal mortality between corn types, although significantly more prepupae from Bt plots than from non-Bt plots died in emergence buckets before constructing pupal chambers in 1998. In all years, significantly fewer moths emerged from prepupae collected from Bt plots, suggesting that effects of the expressed Cry1Ab extended to the prepupal and pupal stages. Late plantings of corn showed the greatest reductions in moth emergence from Bt corn because environmental conditions were more conducive to trigger diapause at the time H. zea was developing in these plantings. This was supported by a significantly greater proportion of diapausing pupae remaining in the ground in the late plantings of both Bt and non-Bt corn. For April and early May plantings, larval feeding on Bt corn delayed the time to pupation, although there was no significant difference in moth emergence between corn types for those larvae that successfully pupated. Although Bt expression had less impact on the proportion of moths emerging, the actual number of moths emerging from Bt corn was significantly reduced because fewer larvae reached pupation. Delays in adult emergence, along with significant reductions in adult emergence from MON810 Bt corn, should reduce the rates of colonization in soybean and other late host crops but may also result in asynchrony of mating between individuals emerging from Bt and non-Bt corn. This, in turn, may contribute to the evolution of resistance to Bt corn.

The phytophagous insects that damage crops are often polyphagous, feeding on several types of crop and on weeds. The refuges constituted by noncrop host plants may be useful in managing the evolution in pest species of resistance to the Bacillus thuringiensis toxins produced by transgenic crops. However, the benefits of these refuges may be limited because host-plant diversity may drive genetic divergence and possibly even host-plant-mediated sympatric speciation. The European corn borer, Ostrinia nubilatis Hubner (Lepidoptera: Crambidae), is the main pest of maize in Europe and North America, where it was introduced early in the 20th century. It has a wide host range but feeds principally on mugwort (Artemisia vulgaris L.) and maize (Zea mays L.). O. nubilalis is found on mugwort only in the northern part of France, whereas it is found on maize throughout France. The extent of genetic variation at allozyme markers was investigated in populations collected from the two host plants over the entire geographical distribution of the European corn borer on mugwort in France. Allelic differentiation between pairs of populations and hierarchical analyses of pools of samples from each host plant indicate that the group of populations feeding on maize differed from the group of populations feeding on mugwort. Our results suggest (1) host-plant-related divergent selection at the genomic region surrounding the Mpi locus and (2) limited gene flow between the populations feeding on mugwort and those infesting maize fields. These data indicate that adults emerging from mugwort would not be useful for managing the evolution of resistance to the B. thuringiensis toxins in European corn borer populations.

Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays. Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops. Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein. Larvae with two resistance alleles in-any combination were resistant, whereas those with one or none were susceptible to Cry1Ac. Together with previous evidence, the results reported here identify the cadherin genie as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests.

A simulation model is developed to examine the role of spatial processes in the evolution of resistance in Helcoverpa zea populations to Bt corn and Bt cotton. The model is developed from the stochastic spatially explicit Heliothis virescens model described by Peck et al. (1999), to accommodate a spatial mix of two host crops (corn and cotton), and to reflect the agronomic practices, as well as the spatial and temporal population dynamics of H.; ea, in eastern North Carolina. The model suggests that selection for resistance is more intense in Bt cotton fields than in Bt corn fields. It further suggests that local gene frequencies are highly dependent on local deployment levels of Bt crops despite the high mobility of the adult insects. Region-wide average gene frequencies depend on the region-wide level of Bt deployment, so incomplete technology adoption slows the rate of resistance evolution. However, on a local scale, H. zea populations in clusters of fields in which Bt use is high undergo far more rapid evolution than populations in neighboring clusters of fields in which Bt use is low. The model suggests that farm-level refuge requirements are important for managing tile risk of resistance. The model can be used as an aid in designing plans for monitoring for resistance by suggesting the appropriate distribution of monitoring locations, which should focus on areas of highest Bt crop deployment. The findings need to be placed in the context of the input parameters. many of which are uncertain or highly variable in nature, and therefore, a thorough sensitivity analysis is warranted.

The sensitivities of a model simulating the evolution of resistance in Helicoverpa zea to Bt toxins in transgenic crops were investigated by examining effects of each of the model parameters on the frequency of resistance alleles after 8 yr. The functional dominance of resistance alleles and the initial frequency of those alleles had a major impact on resistance evolution. The Survival of susceptible insects on the transgenic crops and the population dynamics of the insect, driven by winter survival and reproductive rates, were also important. In addition, agricultural practices including the proportion of the acreage planted to corn, and the larval threshold for spraying cotton fields affected the R-allele frequency. Many of these important parameters are inherently variable or cannot be measured with accuracy, so model output cannot be interpreted as being a forecast. However, this analysis is useful in focusing empirical research on those aspects of the insects' life system that have the largest effects on resistance development, and indicates ways in which to improve products and agricultural practices to increase the expected time to resistance. The model can thus be used as a scientific basis for devising a robust resistance management strategy for Bt crops.

The 'high-dose-refuge' (HDR) strategy is widely recommended by the biotechnology industry and regulatory authorities to delay pest adaptation to transgenic crops that produce Bacillus thuringiensis (Bt) toxins. This involves cultivating nontoxic plants (refuges) in close proximity to crops producing a high dose of Bt toxin. The principal cost associated with this strategy is due to yield losses suffered by farmers growing unprotected, refuge plants. Using a population genetic model of selection in a spatially heterogeneous environment, we show the existence of an optimal spatial configuration of refuges that could prevent the evolution of resistance whilst reducing the use of costly refuges. In particular, the sustainable control of pests is achievable with the use of more aggregated distributions of nontransgenic plants and transgenic plants producing lower doses of toxin. The HDR strategy is thus suboptimal within the context of sustainable agricultural development.

The potential of transferring herbicide resistance from transgenic rice (Oryza sativa L.) varieties to sexually compatible weeds is of paramount importance for development of effective weed control strategies. The objective of this research was to determine the genetic control and frequency of natural outcrossing between a transgenic, glufosinate-resistant rice line and a Louisiana biotype of red rice (Oryza sativa L.). Molecular and phenotypic data showed that outcrossing in field plots between a non-transgenic purple marker line and red rice did occur within one field season, but at a low rate of <1%. Similarly, molecular and phenotypic data demonstrated that glufosinate resistance was transferred from the transgenic line to the red rice biotype in the field within one year at a low frequency of 0.3%. Compared to parental lines, the transgenic-red rice hybrids were extremely late, tall, and never set seed during the normal field season. Genetic analyses in all F-2 populations showed glufosinate resistance behaved in a Mendelian fashion as a single, dominant gene. Presence of the bar gene for glufosinate resistance did not increase fitness or seed fecundity in hybrids or subsequent progeny. The genetic analyses and outcrossing results from this study suggest that an effective management program can be developed to prolong the usefulness of transgenic, glufosinate herbicide technology.

This study was undertaken to determine the effects of pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM, Plutella xylostella) resistant to one or the other protein. Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by sexual crosses of cry1Ac- and cty1C-transgenic plants. Plants containing both genes were selected by tests for resistance to kanamycin and hygromycin, and confirmed by PCR analysis for the Bt genes. Both cry1Ac and cry1C mRNAs were detected in the hybrid lines, and Cry1Ac and Cry1C proteins were stably produced at levels comparable to the parental plants. Plants producing both Cry1Ac and Cry1C, proteins caused rapid and complete mortality of DBM larvae resistant to Cry1A or Cry1C, and suffered little or no leaf damage. These plants, in combination with the resistant DBM populations available, will allow greenhouse or field studies of resistance management strategies involving gene pyramiding.

The potato tuber moth (Phthorimaea operculella Zeller) is the primary insect pest of cultivated potato (Solanum tuberosum L) in tropical and subtropical regions, causing both foliar and tuber damage. In contrast, the Colorado potato beetle (Leptinotarsa decemlineata Say) is the most important insect pest in the northern potato production latitudes. The codon-modified Bacillus thuringiensis Bt-cry5 gene (revised nomenclature cry1IaI), specifically toxic to Lepidoptera and Coleoptera, was transformed into cultivar Spunta using an Agrobacterium vector to provide resistance to both potato tuber moth and Colorado potato beetle. The Bt-cry5 gene was placed downstream from the constitutive CaMV35S promoter. Two transgenic 'Spunta' clones, G2 and G3, produced high levels of mortality in first instars of potato tuber moth in detached-leaf bioassays (80% to 83% mortality), laboratory tuber tests (100% mortality), and field trials in Egypt (99% to 100% undamaged tubers). Reduced feeding by Colorado potato beetle first instars was also observed in detached-leaf bioassays (80% to 90% reduction). Field trials in the United States demonstrated that the horticultural performance of the two transgenic lines was comparable to 'Spunta'. These Bt-cry5 transgenic potato plants with high potato tu er moth resistance have value in integrated pest management programs.

The existence of a continuous array of sympatric biotypes-from polymorphisms, through ecological or host races with increasing reproductive isolation, to good species-can provide strong evidence for a continuous route to sympatric speciation via natural selection. Host races in plant-feeding insects, in particular, have often been used as evidence for the probability of sympatric speciation. Here, we provide verifiable criteria to distinguish host races from other biotypes: in brief, host races are genetically differentiated, sympatric populations of parasites that use different hosts and between which there is appreciable gene flow. We recognize host races as kinds of species that regularly exchange genes with other species at a rate of more than ca. 1% per generation, rather than as fundamentally distinct taxa. Host races provide a convenient, although admittedly somewhat arbitrary intermediate stage along the speciation continuum. They are a heuristic device to aid in evaluating the probability of speciation by natural selection, particularly in sympatry. Speciation is thereby envisaged as having two phases: (i) the evolution of host races from within polymorphic, panmictic populations; and (ii) further reduction of gene flow between host races until the diverging populations can become generally accepted as species. We apply this criterion to 21 putative host race systems. Of these, only three are unambiguously classified as host races, but a further eight are strong candidates that merely lack accurate information on rates of hybridization or gene flow. Thus, over one-half of the cases that we review are probably or certainly host races, under our definition. Our review of the data favours the idea of sympatric speciation via host shift for three major reasons: (i) the evolution of assortative mating as a pleiotropic by- product of adaptation to a new host seems likely, even in cases where mating occurs away from the host; (ii) stable genetic differences in half of the cases attest to the power of natural selection to maintain multilocus polymorphisms with substantial linkage disequilibrium, in spite of probable gene flow; and (iii) this linkage disequilibrium should permit additional host adaptation, leading to further reproductive isolation via pleiotropy, and also provides conditions suitable for adaptive evolution of mate choice (reinforcement) to cause still further reductions in gene flow. Current data are too sparse to rule out a cryptic discontinuity in the apparently stable sympatric route from host-associated polymorphism to host-associated species, but such a hiatus seems unlikely on present evidence. Finally, we discuss applications of an understanding of host races in conservation and in managing adaptation by pests to control strategies, including those involving biological control or transgenic parasite-resistant plants.

Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments, have shown the high potential of other species to evolve resistance against Bt. We. have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect. Populations.

Data have been lacking on the proportion of Helicovera zea larvae that develop on noncotton host plants that can serve as a refuge from selection pressure for adaptation to transgenic cotton varieties that produce a toxin from the bacterium Bacillus thuringiensis. We found that individual H. zea moths that develop as larvae on cotton and other plants with C-3 physiology have a different ratio of C-13 to C-12 than moths that develop on plants with C-4 physiology, such as corn. We used this finding in determining the minimum percentage of moths that developed on noncotton hosts in two cotton-growing areas. Our results indicate that local corn can serve as a refuge for H. zea in midsummer. Our results contrast dramatically with the prevailing hypothesis that the large majority of late-season moths are produced from larvae feeding on cotton, soybean, and other C-3 plants. Typically, <50% of moths captured in August through October have isotope ratios indicative of larval feeding on C-3 plants. In one October sample, 100% of the moths originated from C-4 hosts even though C-4 crops were harvested at least 1 mo earlier, and no common wild C-4 hosts were available. These findings support other research indicating that many late-season H. zea moths captured in Louisiana and Texas are migrants whose larvae developed on corn in more northern locations. Our isotope data on moths collected in Texas early in the season indicate that the majority of overwintering H. zea do not originate from cotton- feeding larvae and may be migrants from Mexico. Non-Bt corn in Mexico and the U.S. corn belt appears to serve as an important refuge for H. zea.

The population dynamics and population genetics of two bivoltine species of corn borers are modeled in a hypothetical region of irrigated transgenic and nontransgenic corn. European corn borer, Ostrinia nubilalis (Hubner), adults were assumed to disperse throughout the landscape for both mating and oviposition. Southwestern corn borer, Diatraca grandiosella Dyar, adults were assumed to have very localized dispersal behaviors. Resistance developed quickly in both species when the allele for resistance to the transgenic toxin was dominant. When the allele for resistance was not dominant and few or none of the heterozygous larvae survived the toxin, the behaviors of adult insects determined the speed of resistance development. With block refuges of 10-40% the European corn borer developed resistance within 15-38 yr, but the southwestern corn borer never developed resistance within the 100 yr simulated. A row- strip refuge configuration (lid not change the time for resistance to develop in the European corn borer; however, row- strip refuges cannot be recommended for the southwestern corn borer. Uncertainty about adult behaviors in irrigated corn led us to examine the potential impact of behavior on resistance development, Adult behaviors influenced resistance development more than refuge size. For instance, if the first flight of moths exhibit random mating and uniform oviposition throughout the landscape and the second flight exhibits localized mating and oviposition, resistance developed at least five times faster in the southwestern corn borer population and three times slower in the European corn borer population compared with our standard assumptions. We discuss the implications of adult behavior, refuge configuration, refuge placement within the landscape and year-to-year relocation on resistance management plans.

A version of the Lotka-Volterra predator-prey model with logistic crop growth is modified to explore the rate of adaptation of a herbivore to a pest-resistant crop. This provides a phenotypic model for the evolution of resistance in a population comprising three different pest types each defined by differing parameter values for respiration rate and crop palatability. Expressions estimating the rates of increase of the fitter pest types are obtained as a function of the food qualities, and respiration and mortality rates. Potential strategies for delaying the rate of adaptation with regard to the expressions derived above, via the use of pest-susceptible refugia and natural enemies, are discussed. Although the model is formulated as one in which a single gene is the factor conferring resistance it can be interpreted and used independently of this. (C) 2002 Elsevier Science Limited. All rights reserved.

The Kansas Dipel-resistant and susceptible European corn borer, Ostrinia nubilalis(Hubner), were evaluated in the greenhouse on different Bt transgenic events expressed in corn hybrids. There were important differences in the resistance offered by the different Bt event corn hybrids. Hybrid comparison tests indicate that these Dipel-resistant first-instar European corn borer were not able to survive to adulthood on whorl-stage MON810, Bt11, or 176 Bt event corn plants. Third instars did not survive to adulthood on whorl-stage MON810 or Bt11. event corn plants but a small number of fifth instars were found on whorl-stage DBT418 plants infested with Dipel-resistant larvae. First and third instars of these Dipel-resistant European corn borers caused more leaf-feeding damage and more tunneling on whorl-stage Bt-corn plants than did the Dipel-susceptible European corn borers. However, in the single Bt corn hybrid test, there was no survival of the Dipel-resistant European corn borers on DK580BtX or MAX454 Bt plants 35 to 42 d after they had been infested with first instars. These results demonstrate that the current Kansas selection of Dipel- resistant European corn borer strain cannot establish reproducing populations in the tested Bt corn lines and hybrids.

New pesticidal crops are taking advantage of advances in genetic engineering. For example, corn has been engineered to express Bt proteins that are toxic to the European corn borer. These crops are effective pest management tools for United States growers. However, there is concern that pests will develop resistance to these crops resulting in the increased use of more hazardous pesticides. The purpose of this paper is to develop a stochastic dynamic bioeconomic simulation model to help guide regulatory policy designed to mitigate the threat of resistance to new pesticidal crops. The model is used to evaluate the insect resistance management guidelines mandated by the United States Environmental Protection Agency for the use of Bt corn in the Midwestern United States.

Widespread use of transgenic crops that express an insecticidal endotoxin from Bacillus thuringiensis increases the risk of evolution of resistance by the European corn borer and other insect pests. To delay resistance evolution, the high-dose refuge strategy is being implemented for Bt maize and Bt cotton. We develop a general modelling framework to understand the invasion and spread of alleles conferring resistance. We show that at least three processes are involved in explaining the effectiveness of the high-dose refuge strategy: the intensity of selection, assortative (non-random) mating due to spatial subdivision, and variation in male mating success also due to spatial subdivision. Understanding these processes leads to a greater range of possible resistance management tactics. For example, efforts to encourage adults to leave their natal fields may have the unwanted effect of speeding rather than slowing resistance evolution. Furthermore, when Bt maize causes high mortality to susceptible target pests, spraying insecticides in refuges to reduce pest populations may not greatly disrupt resistance management.

Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic of Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher (similar to 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt- transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.

When the application of pesticides places selective evolutionary pressure on pest populations it can be useful to plant refuge areas: crop areas intended to encourage the breeding of pests susceptible to the pesticide. Renewed interest in refuge areas has arisen with recent advances in biotechnology and genetically modified (GM) crops. This paper uses a simple model of evolution of pest population and pest resistance to characterize the socially optimal refuge strategy to manage pest resistance. We show both that the establishment of refuge areas might best be delayed until resistance becomes an important concern, and that the use of refuge areas in the long-run use will not be optimal if the fitness cost of resistance does not exceed the discount rate.

Transgenic cotton that produces insecticidal crystal protein Cry1Ac of Bacillus thuringiensis (Bt) has been effective in controlling pink bollworm, Pectinophora gossypiella (Saunders). We compared responses to bolls of Bt cotton and non-Bt cotton by adult females and neonates from susceptible and Cry1Ac- resistant strains of pink bollworm. In choice tests on caged cotton plants in the greenhouse, neither susceptible nor resistant females laid fewer eggs on Bt cotton bolls than on non-Bt cotton bolls, indicating that the Bt toxin did not deter oviposition. Multiple regression revealed that the number of eggs laid per boll was negatively associated with boll age and positively associated with boll diameter. Females also laid more eggs per boll on plants with more bolls. The distribution of eggs among bolls of Bt cotton and non-Bt cotton was clumped, indicating that boll quality rather than avoidance of previously laid eggs was a primary factor in oviposition preference. Parallel to the results from oviposition experiments, in laboratory no-choice tests with 10 neonates per boll, the number of entrance holes per boll did not differ between Bt cotton and non-Bt cotton for susceptible and resistant neonates. Also, like females, neonates preferred younger bolls and larger bolls. Thus, acceptance of bolls by females for oviposition and by neonates for mining was affected by boll age and diameter, but not by Bt toxin in bolls. The lack of discrimination between Bt and non-Bt cotton bolls by pink bollworm from susceptible and resistant strains indicates that oviposition and mining initiation are independent of susceptibility to Cry1Ac.

Genetic models have been used to examine the evolution of insecticide resistance in pest species subject to data and assumptions regarding genetic, biological, and operational parameters. We used time-series data on pyrethroid tolerance and simple genetic models to estimate underlying genetic and biological parameters associated with resistance evolution in tobacco budworm, Heliothis virescens (F.), and bollworm, Helicoverpa zea (Boddie), Louisiana field populations. Assuming pyrethroid resistance is conferred by one gene at one locus in both species, inheritance of pyrethroid resistance was partially dominant in the tobacco budworm and partially recessive in the bollworm. Relative fitness estimates indicated that fitness costs associated with resistance selected against resistance alleles in the absence of selection pressure in the tobacco budworm, but not in the bollworm. In addition, relative fitness estimates obtained using the indirect method outlined in this study were similar in magnitude to estimates obtained using traditional direct approaches.

The ability of non-crop plants to support complete development of insect pests is an important factor for determining the impact of those plants on resistance management programs for transgenic crops. We assessed the effect of one physical factor, plant stem diameter, on the ability of plants to support full development of the European corn borer (ECB), Ostrinia nubilalis Hubner (Lepidoptera: Crambidae), the target pest of transgenic Bt-corn. In the field, European corn borer larvae were significantly more likely to tunnel and survive in plants with larger stem diameters. Larvae were 40 x more likely to survive on corn, the largest plant tested, compared to many of the smaller plants. In the laboratory, larvae were more likely to survive in and less likely to abandon the largest diet-filled artificial stems that varied only in stem diameter. In conditions simulating those that an ECB larvae would encounter upon abandoning a host, larvae survived up to three weeks and were able to locate corn as a new host with a significantly higher frequency than would be expected if they were foraging randomly. These results indicate that the probability of ECB larval survival to maturity on a plant other than corn is relatively low and thus these smaller stemmed non-corn plants may not make a substantial contribution to the pool of susceptible adults. Conversely, since more mature larvae are not as susceptible as neonates, any larvae that partially develop on non-corn plants and subsequently colonize Bt-corn may not be exposed to a lethal dose of the toxin. Since some proportion of the individuals that survive could be partially resistant heterozygotes the presence of non-corn host plants could facilitate the development of resistant ECB populations.

We simulated the population dynamics and population genetics of two bivoltine species of corn borers, the European corn borer, Ostrinia nubilalis (Hubner), and the southwestern corn borer, Diatraea grandiosella Dyar, in a hypothetical region of irrigated transgenic and nontransgenic corn where insecticide was applied only to the nontransgenic refuge crop. Over the 100-yr time horizon, resistance developed quickly in both species and to both transgenic corn and the insecticide when the allele for resistance to the respective toxin was dominant. When the allele for transgenic resistance was not dominant and the refuge location was constant over the time horizon, spraying the refuge to control southwestern corn borer had no effect on how quickly resistance to the transgenic corn developed. In contrast, the European corn borer developed resistance to transgenic corn much sooner when the refuge was sprayed once per year, and the time to 3% resistance allele frequency decreased as efficacy of the insecticide increased. Only when the refuge was treated less than once every 5 yr (10 generations) did the frequency of application decline enough to permit resistance management for the European corn borer to approximate the effectiveness of an unsprayed refuge. A consistently sprayed refuge <40% of the corn acreage was an inadequate resistance management strategy for the European corn borer even when a low efficacy insecticide (70% mortality) was used. When assumptions about European corn borer adult behavior were changed and the adults behaved similarly to adult southwestern corn borer, the development of resistance to the transgenic crop was slowed significantly.

A major problem in insect pest and weed management is uncertainty. Managers are faced with three main types of uncertainty: uncertainty about biological and environmental processes, and observational uncertainty. Active adaptive management (AAM) is management with a deliberate plan for learning about the managed system, so that management can be improved in the face of uncertainty. We discuss the potential benefits of applying AAM to insect pest and weed control with reference to a number of examples. We first address the possible uses for AAM in biological control, with particular reference to agent selection and release. We also propose applying AAM methods to resistance management and to spatial strategies for pest control. We conclude with an overview of AAM, a discussion of some of the potential limitations to its use in pest management, and the possibilities for increased implementation of AAM in the future.

The density of Helicoverpa armigera (Hubner) populations on Bacillus thuringiensis Berliner (Bt) transgenic cotton, corn. peanut. and soybean: differences in its development on Bt cotton and common (nontransgenic) cotton and the potential for mating among populations from Bt cotton fields and other crop fields were investigated in the suburbs of Xinxiang City (Henan Province) and Langfang City (Hebei Province) in the southern and northern parts of north China, respectively, Although development of H. armigera on Bt cotton was much slower than on common cotton. there was a still high probability of mating between populations from Bt cotton and other sources due to the scattered emergence pattern of H. armigera adults, and overlap of the second and third generations. In a cotton and corn growing region. early and late planted Corn provided Suitable refugia for the third and fourth generations of H armigera, but not for the second generation. In a cotton and soybean/peanut mix system, noncotton crops provided a natural refugia from the second- to fourth-generation H, armigera, but function of the refuge would closely depend on the proportion of Bt cotton. Consequently. it may be necessary to compensate the original mixed cropping patterns in different areas for delaying resistance development of H. armigera to Bt cotton.

Sensitivities of Helicoverpa armigera (Hubner) field populations to Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac were monitored during 1998-2000 in China. A total of 41 strains was sampled, and most of them were collected from Bt cotton planting regions. The range of IC50 values (concentration producing 50% inhibition of larval development to third instar) among different populations in 1998, 1999, and 2000 were 0.020-0.105 mug/ml. 0.016-0.099 mug/ml, and 0.016- 0.080 mug/ml, respectively. Diagnostic concentration studies (IC99) showed that the percentage of individuals reaching third instar ranged from 0 to 4.35% with only eight of the 41 tested populations showing values above 0%. Also interesting was a trend over successive years in which fewer populations contained individuals that survived the diagnostic concentration by reaching third instar. Considering these data, it was determined that the field populations sampled during the 3-yr study were susceptible to Cry1Ac protein and that movement toward resistance among H. armigera populations was not apparent.

A synthetic laboratory population of the diamondback moth, Plutella xylostella (L.), was used to test the F-2 screen developed for detecting the frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bacillus thuringiensis (Bt). Of the 120 single-pair matings set up, 106 produced enough F-2 families for screening of Cry1Ac or Cry1C resistance alleles using both transgenic broccoli and an artificial diet overlay assay with a diagnostic dose. When using Bt broccoli plants as the F-2 screen method, only one F-2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C resistance using the diet assay. The survivors in the diagnostic diet assay were crossed with the resistant individuals to confirm their resistance genotypes. Four F-2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single-pairs and four other F-2 families contained an allele resistant to Cry1C. Our results suggest that using transgenic plants expressing a high level of a Bt toxin in an F-2 screen may underestimate the frequency of resistance alleles with high false negatives, or fail to detect true resistance alleles. The diagnostic diet assay was a better F-2 screen method to detect alleles, especially for the Cry1Ac resistance with monogenic inheritance in the diamondback moth. The estimated probabilities of false positives and false negatives were 33 and 1%, respectively, for detecting Cry1Ac resistance at the allele frequency of 0.012 using the diagnostic diet assay. Careful validation of the screening method for each insect-crop system is necessary before the F-2 screen can be used to detect rare Bt resistance alleles in field populations.

In order to develop a resistance management strategy to control tropical pests based on the co-expression of different toxins, a fully modified Bacillus thuringiensis cr1B gene and the translational fusion cry1B-cry1Ab gene have been developed. Both constructs were cloned under the control of a maize ubiquitin-1 or a rice actin-1 promoter and linked to the bar gene driven by the CaMV 35S promoter. Immature embryos from the tropical lines CML72, CML216, and their hybrids, were used as the target for transformation by microprojectile bombardment. Twenty five percent of the transformed maize plants with cry1B expressed a protein that is active against southwestern corn borer and sugarcane borer. Ten percent of the transgenic maize expressed single fusion proteins from the translational fusion gene cry1B-1Ab and showed resistance to these two pests as well as to the fall armyworm. Transgenic maize plants that carried the cry1B gene in T1 to T3 progenies transmitted trangenes with expected Mendelian segregation and conferred resistance to the two target insects. Molecular analyses confirmed the cn, genes integration. the copy number, the size of protein(s) expressed in maize plants, the transmission, and the inheritance of the introduced cry gene. These new transgenic products will provide another recourse for reducing the build-up of resistance in pest populations

We produced 49 broccoli plants (Brassica oleracea L. ssp. italica) containing a Bacillus thuringiensis cry1Ab gene under control of the chemically inducible PR-1a promoter from tobacco. Most of them showed substantial or complete control of neonate diamondback moth larvae, regardless of whether the transgene was induced or not. Ten plants were selected for detailed study via northern and western analysis and insect bioassays. They expressed the cry1Ab gene and gave complete insect control when treated with the chemical inducers INA (2,6-dichloroiso-nicotinic acid) or BTH (1,2,3- benzothiadiazole-7-carbothioic acid S-methyl ester); however, leaves treated with water alone were also partially or completely protected from insect damage. Transgenic progeny plants showed greater inducibility than primary transformants at the molecular level. Two progeny lines produced cry1Ab mRNA and Cry1Ab protein and gave insect control only after induction, both when detached leaves and intact plants were tested. The relevance of these results to resistance management strategies is discussed.

The interaction of population dynamics and movement among two habitat types (toxic transgenic fields and nontoxic refuge fields) on the evolution of insecticide resistance was examined in two different simulation models. The two models were developed to test the hypothesis that increasing habitat grain from fine-grained to coarse-grained, and the resultant increase in nonrandom mating, would increase the rate of local adaptation, here the evolution of resistance. The first model, a complex, stochastic spatially explicit model, altered. habitat grain by varying adult dispersal rates between habitat patches. In contrast to the expectation that increasing patch isolation and increasing the coarseness of the habitats would increase the rate of resistance evolution, intermediate levels of dispersal actually delayed resistance by as much as fivefold over the range of dispersal levels observed. Source-sink dynamics related to ovipositional patterns and the related population dynamics appear to explain the results. A simple deterministic model was developed to abstract out the separate impacts of mating and ovipositional behaviors. This model showed qualitatively the same results, although under similar assumptions it predicted much longer delays in resistance evolution. In this model, nonrandom mating alone always increased the rate at which insects adapted to transgenic crops, but nonrandom mating in combination with nonrandom oviposition could significantly delay resistance evolution. Differences between the two models may be due to the population regulation incorporated in the spatially explicit model. The models clearly suggest that resistance management programs using untreated refuges should not over-emphasize random mating at the cost of making the habitat too fine-grained.

A major challenge for agriculture is management of insect resistance to toxins from Bacillus thuringiensis (Bt) produced by transgenic crops. Here we describe how a large-scale program is being developed in Arizona for management of resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), and other insect pests of cotton. Financial support from growers makes this program possible. Collaboration between the Arizona Cotton Research and Protection Council, the University of Arizona, and government agencies has led to development of resistance management guidelines, a remedial action plan, and tools for monitoring compliance with the proposed guidelines. Direct participation in development of resistance management policies is a strong incentive for growers to invest in resistance management research. However, more research, regularly updated regulations, and increased collaboration between stakeholders are urgently needed to maintain efficacy of Bt toxins in transgenic crops.

Transgenic cotton producing a Bacillus thuringiensis (Bt) toxin is widely used for controlling the pink bollworm. Peretinophora gossypiella (Saunders). We compared performance of pink bollworm strains resistant to Bt cotton with performance of their susceptible counterparts on non-Bt cotton. We found fitness costs that reduced survival on non-Bt cotton by all average of 51.5% in two resistant strains relative to the susceptible strains. The survival cost was recessive in one set of crosses between a resistant strain and the susceptible strain from which it was derived. However, crosses involving an unrelated resistant Laid susceptible strain indicated that the survival cost could be dominant. Development time oat non-BI cotton did not differ between the two related resistant and susceptible strains, A slight recessive cost affecting development time was suggested by comparison of the unrelated resistant and susceptible strains. Maternal effects transmitted by parents that had eaten Bt-treated artificial diet as larvae had negative effects on embryogenesis, adult fertility, nr both, and reduced the ability of neonates to enter cotton bolls. These results provide further evidence that fitness costs associated with the evolution of resistance to BL cotton are substantial in the pink bollworm.

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance. We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton. Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter. Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived. This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains. Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent.

Cultural control methods have been central in the southwestern United States for reducing pink bollworm, Pectinophora gossypiella (Saunders), damage to cotton. Nevertheless, it is not clear at present how such methods could be integrated within the novel pest management framework allowed by introduction of cotton producing a toxin from Bacillus thuringiensis (Bt) for pink bollworm control. Using, statewide pheromone trapping and climatic data in conjunction with deterministic simulation models, we investigated whether manipulation of cotton planting date and use of other cultural control methods could represent valuable tactics for control of the pink bollworm in Arizona. Accumulation of heat units from one January accurately predicted the rate of pink bollworm emergence from diapause in 15 cotton-producing regions. Significant variation in rate of emergence from diapause was present among regions, with earlier emergence at higher altitudes. Most adults emerge from diapause too early to reproduce successfully on cotton, a phenomenon known as suicidal emergence. A method for prediction of the fraction of suicidal emergence resulting from adoption of a given cotton planting date is presented. Results from simulation models suggest that manipulation of planting date and implementation of other control cultural methods, reduce the rate of application of insecticides and delay the evolution of resistance to Bt cotton in the pink bollworm.

The refuge-high-dose strategy for delaying insect adaptation to transgenic plants produces non-transgenic plants that enable survival of susceptible individuals. Previous theoretical work has suggested three requirements for success of the refuge- high-dose strategy: a low initial frequency of the resistance allele, extensive mating between resistant and susceptible adults and recessive inheritance of resistance. In order to understand an observed decrease in resistance frequency and improve the potential for managing resistance better, we used analytical and simulation models for exploring the conditions that prevent or reverse the evolution of resistance, even when resistance is not rare initially Assuming random mating and recessive or nearly recessive inheritance of resistance, the factors favouring reversal of resistance are non-recessive costs of resistance, low initial resistance allele frequency, large refuges, incomplete resistance and density-independent population growth in refuges.

Transgenic corn expressing the insecticidal toxin from Bacillus thuringiensis Berliner is gaining support as an effective control technology for use against lepidopteran pests, particularly European corn borer, Ostrinia nubilalis Hubner (Lepidoptera: Crambidae). However, there is concern that widespread adoption of transgenic plants will rapidly lead to B. thuringiensis toxin resistance, Thus, long-term selection of O. nubilalis populations with the Cry1Ab B. thuringiensis toxin has been undertaken in several laboratories in the United States and in Europe. We present results from two independent selection experiments performed in laboratories at the University of Nebraska and at the Institut National de In Recherche Agronomique in France. Although the protocols and methods used by the two laboratories were different, the results were comparable. The highest level of resistance occurred at generation 7 (14-fold), generation 9 (13-fold), and generation 9 (32-fold) for three different strains. For each strain, the level of resistance fluctuated from generation to generation, although there were consistently significant decreases in toxin susceptibility across generations for all selected strains. These results suggest that low levels of resistance are common among widely distributed O. nubilalis populations.

Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are widely used for pest control. Bt- resistant insect strains have been studied, but the molecular basis of resistance has remained elusive. Here, we show that disruption of a cadherin-superfamily gene by retrotransposon- mediated insertion was linked to high levels of resistance to the Et toxin Cry1Ac in the cotton pest Heliothis virescens. Monitoring the early phases of Bt resistance evolution in the field has been viewed as crucial but extremely difficult, especially when resistance is recessive. Our findings enable efficient DNA-based screening for resistant heterozygotes by directly detecting the recessive allele.

The long-term benefit of insecticidal products based on Cry toxins, either in sprays or as transgenic crops, is threatened by the development of resistance by target pests. The models used to predict evolution of resistance to Cry toxins most often are monogenic models in which two alleles are used. Moreover, the high-dose/refuge strategy recommended for implementation with transgenic crops relies on the assumption that the resistance allele is recessive. Using selection experiments, we demonstrated the occurrence in a laboratory colony of diamondback moth of two different genes (either allelic or nonallelic) that confer resistance to Cry1Ab. At the concentration tested, resistance was dominant in one selection line and partially recessive in the other. Resistant insects from the two selection lines also differed in their cross- resistance patterns. The diamondback moth colony was derived from a field population from the Philippines, which originally showed a different resistance phenotype. This is the first time that an insect population has been directly shown to carry more than one gene conferring resistance to the same Cry toxin.

The European corn borer, Ostrinia nubilalis (Hubner), causes economic damage to corn, Zea mays L., throughout the Corn Belt. Because this insect has become the primary target of Bacillus thuringiensis Berliner (Bt) transgenic corn, current efforts addressing the management of O. nubilalis resistance to Bt corn require information on adult European corn borer dispersal and factors affecting its dispersal, In 1998 we conducted mark- release-recapture, release-recapture, and caged-mating studies to directly measure and compare local dispersal patterns of O. nubilalis adults within and proximal to irrigated and non- irrigated cornfields. Releases of marked adults were made corresponding to the first and second flight of O. nubilalis in eastern Nebraska. Adult dispersal was significantly different between irrigated and non-irrigated cornfields. Released adults tended to remain in and near irrigated cornfields, but dispersed out of and away from non-irrigated cornfields. When released at the edge of the cornfield, neither male nor unmated female O. nubilalis displayed an initial tendency to move out of irrigated corn and into the mixed smooth bromegrass (Bromus inermis Leyss) and broadleaf-weed field edge. Mating efficiency in a late-season cornfield was not significantly different than in dense foxtail (Setaria spp.). Generally, we found that adult O. nubilalis dispersal may vary depending on variables such as action-site availability and agronomic practices and their interaction with O. nubilalis life history.

We evaluated the effects of Bacillus thuringiensis (Bt) toxin Cry1Ac on survival and development of a susceptible strain and laboratory-selected resistant strains of pink bollworm, Pectinaphora gossypiella (Saunders). For susceptible and resistant strains tested on artificial diet, increases in Cry1Ac concentration reduced developmental rate and pupal weight. In greenhouse tests, survival of resistant larvae on transgenic cotton that produces Cry1Ac (Bt cotton) was 46% relative to their survival on non-Bt cotton. In contrast, Bt cotton killed all susceptible larvae tested. F, hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity. Recessive resistance to Bt cotton is consistent with one of the basic assumptions of the refuge strategy for delaying resistance to Bt cotton. Whereas slower development of resistant insects on Bt cotton could increase the probability of mating between resistant adults and accelerate resistance, negative effects of Bt cotton on the survival and development of resistant larvae could delay evolution of resistance.

Laboratory selection increased resistance of pink bollworm (Pectinophora gossypiella) to the Bacillus thuringiensis toxin Cry1Ac. Three selections with Cry1Ac in artificial diet increased resistance from a low level to >100-fold relative to a susceptible strain. We used artificial diet bioassays to test F-1 hybrid progeny from reciprocal crosses between resistant and susceptible strains. The similarity between F-1 progeny from the two reciprocal crosses indicates autosomal inheritance of resistance. The dominance of resistance to Cry1Ac depended on the concentration. Resistance was codominant at a low concentration of Cry1Ac, partially recessive at an intermediate concentration, and completely recessive at a high concentration. Comparison of the artificial diet results with previously reported results from greenhouse bioassays shows that the high concentration of Cry1Ac in bolls of transgenic cotton is essential for achieving functionally recessive inheritance of resistance.

Background: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. Results: We have determined the 2.2 Angstrom crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. Conclusions: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain- swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.

Survival and fecundity of Colorado potato beetle adults, Leptinotarsa decemlineata (Say), that had or had not fed previously on non-transgenic potato before exposure to transgenic potato containing the Bacillus thuringiensis subsp. tenebrionis Cry3A toxin (Bt) was investigated. In the laboratory, < 5% of first-generation adults survived after two weeks when restricted to Bt foliage since eclosion, but over 85% of adults that had fed initially on non-Bt potato survived exposure to Bt potato for two weeks. In field experiments, less than 0.5% of adults that were exclusively provided Bt potato plants survived overwinter, whereas 44% to 57% survived overwinter when fed non-Bt potato plants for two weeks before being provided Bt potato as a final pre-overwintering host. Survival through the winter increased as the duration of initial feeding on non-Bt potato increased and was similar for beetles provided either tubers or Bt potato plants as a final pre-overwintering host. Only overwintered beetles that fed initially on non-Bt potato before encountering either tubers or Bt potato as a final pre-overwintering host laid eggs the following spring. Survival and reproduction of potato beetle adults after colonizing Bt potato fields should not be adversely affected as long as they have had sufficient time to feed initially on non-Bt potato. Implications for how potato production practices in the Mid-Atlantic US may affect the utility of general resistance management plans for Bt potato are discussed.

A simulation model of the population dynamics and genetics of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was created for a landscape of corn, soybean, and other crops. Although, the model was created to study a 2-locus problem for beetles having genes for resistance to both crop rotation and transgenic cool, during this first phase of the project, the model was simulated to evaluate only resistance management plans for transgenic corn. Allele expression in the rootworm and toxin dose in the corn plant were the two most important factors affecting resistance development. A dominant resistance allele allowed quick evolution of resistance to transgenic corn, whereas a recessive allele delayed resistance >99 yr. With high dosages of toxin and additive expression, the time required to reach 3% resistance allele frequency ranged from 13 to >99 yr. With additive expression, lon er dosages permitted tire resistant allele frequency to reach 3% in 2-9 yr with refuges occupying 5-30% of the land. The results were sensitive to delays in emergence by susceptible adults and configuration of the refuge (row strips versus blocks).

Co-evolution between plants and their natural enemies is generally believed to have generated much of the Earth's biological diversity. A process analogous to co-evolution occurs in agricultural systems, in which natural enemies adapt to crop resistance introduced by breeding or genetic engineering. Because of this similarity, the investigation of resistance mechanisms in crops is helping to elucidate the workings of co-evolution in nature, while evolutionary principles, including those derived from investigation of co- evolution in nature, are being applied in the management of resistance in genetically engineered crops.

Field studies were conducted in 1992 and 1993 in Hermiston, Oregon, to evaluate the efficacy of transgenic Bt potato (Newleaf(R), which expresses the insecticidal protein Cry3Aa) and conventional insecticide spray programs against the important potato pest, Leptinotarsa decemlineata (Say), Colorado potato beetle (CPB), and their relative impact on non- target arthropods in potato ecosystems. Results from the two years of field trials demonstrated that Newleaf potato plants were highly effective in suppressing populations of CPB, and provided better CPB control than weekly sprays of a microbial Bt-based formulation containing Cry3Aa, bi-weekly applications of permethrin, or early- and mid-season applications of systemic insecticides (phorate and disulfoton). When compared with conventional potato plants not treated with any insecticides, the effective control of CPB by Newleaf potato plants or weekly sprays of a Bt-based formulation did not significantly impact the abundance of beneficial predators or secondary potato pests. In contrast to Newleaf potato plants or microbial Bt formulations, however, bi-weekly applications of permethrin significantly reduced the abundance of several major generalist predators such as spiders (Araneae), big-eyed bugs (Geocorus sp.), damsel bugs (Nabid sp.), and minute pirate bugs (Orius sp.), and resulted in significant increases in the abundance of green peach aphid (GPA), Myzus persicae (Sulzer) - vector of viral diseases, on the treated potato plots. While systemic insecticides appeared to have reduced the abundance of some plant sap-feeding insects such as GPA, lygus bugs, and leafhoppers, early and mid-season applications of these insecticides had no significant impact on populations of the major beneficial predators. Thus, transgenic Bt potato, Bt- based microbial formulations and systemic insecticides appeared to be compatible with the development of integrated pest management (IPM) against other potato pests such as GPA because these CPB control measures have little impact on major natural enemies. In contrast, the broad-spectrum pyrethroid insecticide (permethrin) is less compatible with IPM programs against GPA and the potato leafroll viral disease.

Planting of refuge crops and post-harvest cultivation of soil in winter are key elements of resistance management strategies (RMS) for Bt-transgenic (Ingard(lozenge)) and conventional cottons in southern Queensland and New South Wales. As part of a larger project to examine the feasibility of growing Ingard(lozenge) in central Queensland (CQ), field assessments were conducted during the 1996-97 growing season to examine the adequacy of the southern Bt-cotton RMS under local environmental conditions. The suitability of cotton and a number of other field crops as refuges for Bt-cotton was assessed in terms or their relative Helicoverpa pupal productivity. The practicality and potential effectiveness of post-harvest cultivation under CQ conditions were also assessed. Field assessments show that pigeon pea has the greatest potential as a refuge for Bt-cotton. Unsprayed cotton, sorghum and maize also produced substantially high pupal densities and hence are suitable refuge options, but they will require larger areas to be planted relative to pigeon pea. Post-harvest cultivation in cotton fields is largely ineffective for resistance management under CQ conditions. A Bt-cotton RMS for CQ is proposed. The CQ strategy includes refuge crop options contained in the southern strategy and the use of late season trap-crops of pigeon pea as an alternative to post-harvest cultivation.

Transgenic varieties of field corn that express the Cry1Ab B. thuringiensis (Bt) toxin in ear tissue present the potential of reducing ear feeding by the com earworm, Helicoverpa zea (Lepidoptera: Noctuidae), and for reducing the size of populations of the insect infesting other host crops. Life, history parameters of H. zea feeding on ears of conventional and Bt field corn varieties were measured in field plots in eastern North Carolina in 1997 and 1998. Transformation events investigated were Mon-810 and Bt-11. Bt corn was found to cause a steady mortality of larvae during development, but permitted approximate to 15-40% survival to the prepupal stage compared with non-Bt corn. Mortality of prepupae and pupae from Bt corn was also higher than from non-Bt com, reducing overall adult production by 65-95% The larvae that did survive grew more slowly on Bt than on non-Bt corn, and produced pupae that weighed 33% less. Pupation and adult eclosion were delayed by 6-10 d by feeding on Bt com ears. Com varieties, expressing Bt in ear tissue have the potential to reduce H. zea ear feeding by up to 80%, and the potential to reduce populations emerging from ear-stage corn fields to infest cotton, soybean and other crops by around 75%. To have a measurable effect on area-wide populations, Bt corn varieties would need to be planted in large proportions of corn fields, Extensive planting of varieties such as those tested here, having only moderate effects on H. zea, would raise concerns about rapid evolution of resistance.

The potential ecological risks of Bacillus thurigiensis (Bt) insecticides and Bt-crops have caused increasing concern since their commercial release in the field, among which pests' resistance to Bt-crops is the major ecological risk. Refuge tactic, which can produce sensitive populations, has proved to be a key and sound resistance management strategy in USA and Australia; however, no tactics have been performed in China where Bt-cotton is mostly planted with other host crops of cotton bollworm. Genetic variation and gene flow among different host populations of the cotton bollworm Helicoverpa armigera were analyzed using PCR fingerprinting method. The results show that maize and castor-oil plant, as well as cotton can take effect as refuges to prevent resistance of cotton bollworm to Bt-cotton, while peanut and sesame are not as suitable for planting with Bt-cotton as refuges in the field as low gene flow was detected among populations on peanut, sesame and Bt cotton.

Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second tests, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1Cprotoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross- resistance patterns. We used 12 Bt protoxins for cross- resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (RR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene (s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin, The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.

Colorado potato beetle (CPB; Leptinotarsa decemlineata-Say) is a serious pest because it has developed resistance against insecticides. Three transgenic eggplant (Solanum melongena L,) lines bearing a mutagenized Bacillus thuringiensis; Berl, gene coding for the Cry3B toxin, and the nontransformed control DR2- line were tested in field trials to assess their insect resistance. The transgenic lines 3-2, 6-1, and 9-8 were tested at two different locations in a randomized complete-block design. Samples were taken biweekly to assess the level of CPB and the presence of other insects. At harvest, total yield and fruit number per plot were recorded. Two transgenic lines showed high levels of resistance at both locations, as measured by CPB abundance and yield, Fruit production was almost twice as great in the highly resistant lines (3-2 and 9-8) as in the nontransformed control. The 6-1 transgenic line showed an intermediate Level of resistance: it was similar to the control under heavy CPB pressure and was comparable to the other transgenic lines under milder infestations. Analysis by double antibody sandwich-enzyme linked immunosorbent assay (DAS- ELISA), performed on different tissues, revealed a fewer amount of Cry3B protein in the 6-1 transgenic line than in lines 3-2 and 9-8, No detrimental effects on nontarget arthropods (including the chrysomelid Altica) were evident. Field observations confirmed that Bt may be able to control CPB infestation in eggplant, representing a potential effective and environmentally safe means of pest control.

The resistance of vegetative, booting, and flowering stage plants of a variety of an aromatic rice, Oryza sativa L., transformed with a Bacillus thuringiensis Berliner cry1Ab gene under control of the maize phosphoenolpyruvate carboxylase (PEPC) promoter was evaluated against four lepidopterous lice pests-the stem borers Chilo suppressalis (Walker) (Lepidoptera: Crambidae) and Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae), and the foliage feeders Cnaphalocrocis medinalis Guenee (Lepidoptera: Pyralidae) and Naranga aenescens Moore (Lepidoptera: Noctuidae). Plants of the cry1Ab-transformed line (no. 827) were more resistant to young, larvae of S. incertulas. C. suppressalis, and C. medinalis than control plants at the vegetative stage but not at the flowering stage. Survival of 10-d-old stem borer larvae did not differ on cry1Ab plants and control plants at either the vegetative or flowering stage, but the development of 10-d-old C. suppressalis larvae was retarded on the vegetative stage cry1Ab plants. Immunological analysis also showed an apparent decline in Cry1Ab titer in leaf blades and leaf sheaths at the reproductive stage. In experiments comparing three fertilizer treatments (NPK, PK, and none), there was a significant interaction between fertilizer treatment and variety on larval survival only in whole-plant assays at booting stage with C. suppressalis. On cry1Ab plants, larval survival did not differ significantly among the three fertilizer levels, whereas on control plants survival was highest with the NPK treatment. cry1Ab plants tested at the sixth and seventh generations after transformation were more resistant than control plants to N. aenescens and C, suppressalis, respectively, suggesting that gene silencing will not occur in line 827. The results of the experiments are discussed in terms of resistance management for B. thuringiensis toxins in rice.

The refuge plus high-dose strategy for resistance management assumes that the frequency of resistance alleles is low. We used an F-2 screen to estimate the frequency of resistance to transgenic corn that produces Bacillus thuringiensis Berliner Cry1Ab toxin (Bt corn) in an Iowa population of European corn borer, Ostrinia nubilalis (Hubner). We also proposed a modification to the statistical analysis of the F-2 screen that extends its application for nonuniform prior distributions and for repeated sampling of a single population. Based on a sample of 188 isofemale lines derived from females caught at light traps during the 2nd flight of 1997, we show with 95% confidence that the frequency of resistance to Bt corn was <3.9 x 10(-3) in this Iowa population. These results provide weak evidence that the refuge plus high-dose strategy may be effective for managing resistance in O. nubilalis to Bt corn. Partial resistance to Cry1Ab toxin was found commonly. The 95% CI for the frequency of partial resistance were [8.2 x 10(-4), 9.4 x 10(-3)] for the Iowa population. Variable costs of the method were $14.90 per isofemale line. which was a reduction of 25% compared with our initial estimate.

Strategies proposed for delaying resistance to Bacillus thuringiensis toxins expressed by transgenic maize require intense gene flow between individuals that grew on transgenic and on normal (referred to as refuges) plants. To investigate gene flow in the European corn borer, Ostrinia nubilalis (Hubner), the genetic variability at 29 sampled sites from France was studied by comparing allozyme frequencies at six polymorphic loci. Almost no deviations from Hardy-Weinberg expectations occurred, and a high stability of allelic distribution was found among samples collected in the same site over two or three different generations, indicating a high stability of the genetic structure over time. The overall genetic differentiation was low at the region and whole country level, suggesting a high and homogeneous gene flow. These results are discussed in relation to the sustainability of transgenic insecticidal maize.

The strategies proposed for delaying the development of resistance to the Bacillus thuringiensis toxins produced by transgenic maize require high levels of gene flow between individuals feeding on transgenic and refuge plants. The European corn borer Ostrinia nubilalis (Hubner) may be found on several host plants, which may act as natural refuges. The genetic variability of samples collected on sagebrush (Artemisia sp.), hop (Humulus lupulus L.) and maize (Zea mays L.) was studied by comparing the allozyme frequencies for six polymorphic loci. We found a high level of gene flow within and between samples collected on the same host plant. The level of gene flow between the sagebrush and hop insect samples appeared to be sufficiently high for these populations to be considered a single genetic panmictic unit. Conversely the samples collected on maize were genetically different from those collected on sagebrush and hop. Three of the six loci considered displayed greater between-host-plant than within- host-plant differentiation in comparisons of the group of samples collected on sagebrush or hop with the group of samples collected on maize. This indicates that either there is genetic isolation of the insects feeding on maize or that there is host-plant divergent selection at these three loci or at linked loci. These results have important implications for the potential sustainability of transgenic insecticidal maize.

Dominance has been assessed in different ways in insecticide resistance studies, based on three phenotypic traits: the insecticide concentration required to give a particular mortality (D-LC) mortality at a particular insecticide dose (D- ML), and fitness in treated areas (D-WT). We propose a general formula for estimating dominance on a scale of 0 to 1 (0 = complete recessivity and 1 = complete dominance). D-LC, D-ML, and D-WT are not directly related and their values depend on genetic background and environmental conditions. We also show that pest management strategies can have die consequence to increase D-WT via the selection of dominance modifiers. Studies on resistance to Bacillus thuringiensis toxins provide the ultimate example of the complexity of the definition of the concept of dominance. Almost all studies have focused on calculation of D-LC, which provides little information about the efficiency of pest management programs. For instance, one assumption of the high dose/refuge strategy is that Bacillus thuringiensis resistance must be effectively recessive (i.e., D-ML must be close to zero). However, D-WT, rather than D-ML, is relevant to the resistance management strategy. Therefore, we strongly suggest that the time has come to focus on fitness dominance levels in the presence and absence of insecticide.

Simulations were conducted to guide development of resistance management strategies aimed at prolonging the usable life of B. thuringiensis (Bt) endotoxins in multiple cropping situations, where different crops expressing Bt endotoxins are host plants for a common pest. We used the New Zealand apple and clover model ecosystem to explore the relative impact on the rate of resistance development of varying levels of cross-resistance between different toxins expressed in these 2 potentially Bt- transformed crops. These 2 crops are hosts for a complex of leafrollers in New Zealand, including the lightbrown apple moth, used here as the model pest. Cross-resistance was varied between 0.0 and 0.5 (zero to partial cross-resistance) to allow for the case in which selection by one plant has a potential effect on resistance to the toxin in another plant. The largest factor affecting the evolution of resistance was the total habitat area occupied by transgenic orchards. The proportion of the clover habitat that was transformed was also an important. factor, even in the absence of cross-resistance. The effect of increasing the proportion of the second transformed crop (clover) acted on resistance evolution mainly by reducing the external refuge of susceptibility for the transgenic orchards. Hence, the ecological implications of reducing the available source of susceptible insects from clover, which can help to dow resistance development in the orchard ecosystem, had a more significant impact than the presence of cross-resistance. Partial cross-resistance between different toxins in the separate crops was overall of relatively minor importance. These simulations have implications for deployment decisions for individual transformed crops in multiple cropping systems, where there is the potential for the crops to serve as refuges for each other. These decisions may need to focus less on cross-resistance between toxins, than on economic trade-offs between the relative roles of individual crops as refugia maintaining susceptibility in the system as a whole.

Dispersal of neonate European corn borers, Ostrinia nubilalis (Hubner), in seed mixtures of transgenic corn expressing Cry1Ab protein (Bt+) and nontransgenic corn (Bt-) was evaluated in a 2-yr field study. The main objective was to determine if larval dispersal limits the effectiveness of seed mixtures as a resistance management strategy. Mixtures evaluated included (1) all Bt+ plants, (2) every fifth plant Bt- with remaining plants Bt+, (3) every fifth plant Bt+ with remaining plants Bt-, and (4) all Bt- plants. The transformation events MON 802 (B73 BC1F2 X Mo17) and MON 810 (B73 BC1F1 X Mo17), which express the Cry1Ab endotoxin isolated from Bacillus thuringiensis subsp. kurstaki, were used as the sources of Bt+ seed in 1994 and 1995 respectively (YieldGard, Monsanto, St. Louis, MO). At corn growth stage V6-V8, subplots within each mixture (15-20 plants each) were infested so that every fifth plant in mixtures 1 and 4, every Bt- plant in mixture 2, and every Bt+ plant in mixture 3 received two egg masses. Larval sampling over a 21-d period indicated increased neonate dispersal off of Bt+ plants, reduced survival of larvae that dispersed from Bt+ plants to Bt- plants, and a low incidence of late-instar movement from Bt- plants to Bt+ plants. Computer simulations based on mortality and dispersal estimates from this study indicate that seed mixtures will delay the evolution of resistant European corn borer populations compared with uniform planting of transgenic corn. However, resistant European corn borer populations likely will develop faster in seed mixes compared with separate plantings of Bt and non-Bt corn.

Sowing seed mixtures of transgenic and nontransgenic plants is one approach to establishing refuges for resistance management of crops transformed with Bacillus thuringiensis (Bt) toxins. We studied larval dispersal and survival of two rice stern borers, Scirpophaga incertulas (Walker) and Chilo suppressalis (Walker), to evaluate the potential effectiveness of seed mixtures for resistance management of Bt rice, Oryza sativa L. Experiments were conducted with two cry1Ab-transformed rice varieties, 'IR58' and 'Tarom Molaii', and corresponding nontransgenic controls. During 0-24 and 24-48 h after egg hatch, the proportion of C. suppressalis larvae dispersing from the natal plant did not differ between transgenic and control plants for either rice variety Dispersal by S. incertulas did not differ between transgenic and control plants of IR58 during either 0-24 or 24-48 h, but a greater proportion of S. incertulas larvae dispersed from transgenic Tarom Molaii than from control plants 24-48 h after eclosion. Larvae that dispersed 0-24 h after eclosion on IR58 and 0-24 and 24-48 h after eclosion on Tarom Molaii were collected and transferred to stern pieces of control plants. No differences in survival were detected between larvae that dispersed from transgenic or control plants 24 (IR58) or 72 h (Tarom Molaii) after dispersal. Because both stem borer species move among plants during larval development, seed mixtures may not be the most effective approach to maintaining refuges for resistance management. However, additional experiments to compare the dispersal and fitness of Bt-resistant and -susceptible stem borer larvae are needed.

The present study evaluates, from a point of view of integrated plant protection, short-term and longterm crop protection effects found in a long-term trial with transgenic herbicide- resistant winter rape and maize. The trial started in 1996 and includes a 4-field crop rotation. Main points of consideration are the use of injury and control thresholds, control of volunteer rape, and an ecological evaluation of established and new herbicide strategies. Outcrossing of herbicide resistance is part of accompanying agroecological research. Use of LIBERTY (Glufosiant-ammonium) in rape and maize led to similar results as standard herbicides. There were no significant differences in yields. Systems with herbicide-resistant rape and maize have a smaller biological risk potential than conventional systems. Outcrossing rates in rape fields were clearly below 1%. Higher rates were observed only in directly neighbouring fields.

Transgenic head cabbage (Brassica oleracea var. capitata), resistant to diamondback moth (Plutella xylostella) larvae, was developed through Agrobacterium tumefaciens-mediated transformation with Bacillus thuringiensis (Bt) cry genes using a modified procedure. Factors important for transformation included cabbage cultivar; preculture: and coculture of explants on a callus initiation medium; use of appropriate amount; and delay in initial application of selective agents. A total of 15 independent transformed lines with over 100 plants were obtained from several transformation experiments, representing an overall transformation efficiency of similar to 1%. Cabbage plants transformed with a synthetic Bt gene, cry1Ab3, were all resistant to larvae of the diamondback moth, whereas all plants transgenic for cry11a3, a wild-type Bt gene, were susceptible. As a first step towards testing the hypothesis that reduced exposure of Bt to target insects would delay the evolution of insect resistance to Bt, cry1Ab3 expression was put under the transcriptional control of the soybean wound-inducible vspB promoter and transgenic cabbage was obtained. Insect bioassay showed that such plants were all resistant to diamondback moth even without induction for the expression of Bt.

Tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois), is a key pest of cotton in many states. Although this pest can damage cotton throughout most of the growing season, economic damage is most likely to occur during the period from first square through early bloom due to feeding on small squares and subsequent abscission of these squares. During this period, excessive damage by high TPB populations may result in reduced yields or delayed maturity. However, current research suggests that cotton can tolerate low levels of TPB damage without sustaining yield loss. Most states recommend monitoring both numbers of insects and percent square retention in order to obtain information on which to base TPB management decisions. The relative importance of TPB as a key pest of cotton is increasing due to a number of changes in cotton insect management systems. These include: development of insecticide resistance in TPB, boil weevil eradication, transgenic Bt cotton, and the development and availability of more target-specific foliar-applied insecticides.

In previous work, cabbage loopers (Trichoplusia ni) evolved 22- fold resistance to the single nucleocapsid nucleopolyhedrovirus of T. ni (TnSNPV) after 26 generations of selection with the virus. The goal of the present study was to determine if T. ni could evolve resistance to the recombinant Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV- AaIT) that; expresses an insect specific neurotoxin and to determine if it was influenced by prior development of resistance to TnSNPV. To answer these questions, the T. ni line that had been exposed to TnSNPV was divided into two sublines at generation 27. One of them was serially selected for resistance to AcRMNPV-AaIT (subline TnSNPV/AcMNPV-AaIT), while the other one was mock infected with distilled water (subline TnSNPV/H2O). The same was done with the line that was used as a control from generations 1 to 26 (subline H2O/AcMNPV-AaIT and subline H2O/H2O). After 17 generations of selection with AcMNPV-AaIT, T. ni that had not been previously exposed to TnSNPV evolved only twofold resistance to AcMNPV-AaIT, However, those that had been selected with TnSNPV evolved fourfold resistance to AcMNPV-AaIT. Exposure to AcMNPV-AaIT conferred cross-resistance to TnSNPV in only one subline, subline H2O/AcMNPV-AaIT. Resistance to AcMNPV-AaIT did not affect the developmental time, pupal weight, egg production, or percentage of egg hatch of T. ni. (C) 2000 Academic Press.

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic St crops approved by the EPA. Bf- transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a "refuge" composed of non-Bt plants to conserve susceptible alleles. We have used Bf-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a "separate refuge" will be more effective at conserving susceptible larvae than a "mixed refuge" and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.

We have attempted to review the potential and limitations of cultural approaches for control of Lygus populations in cotton, including management of alternate hosts and use of host plant resistance. Other than insecticides, cultural approaches seem the most likely of the currently available insect-management tactics to help in the management of Lygus populations. The use of transgenic or nectariless cotton cultivars that are resistant to Lygus could be easily implemented if they were available. Other kinds of cultural controls, such as management of alternate hosts, may not be adopted on a large scale unless they are part of an area-wide management program or until additional evidence of cost efficiency is demonstrated Adoption of cultural approaches will be largely influenced by the availability and efficacy of alternative management practices, including insecticides, and other socioeconomic factors. Cultural approaches are best suited in "soft" systems, with low use of disruptive insecticides, where natural enemies are relied upon most heavily to maintain cotton pests below economically damaging levels.

Strategies for delaying pest resistance to genetically modified crops that produce Bacillus thuringiensis (Bt) toxins are based primarily on theoretical models. One key assumption of such models is that genes conferring resistance are rare. Previous estimates for lepidopteran pests targeted by Pt crops seem to meet this assumption. We report here that the estimated frequency of a recessive allele conferring resistance to Pt toxin Cry1Ac was 0.16 (95% confidence interval = 0.05-0.26) in strains of pink bollworm (Pectinophora gossypiella) derived from 10 Arizona cotton fields during 1997. Unexpectedly, the estimated resistance allele frequency did not increase from 1997 to 1999 and Pt cotton remained extremely effective against pink bollworm. These results demonstrate that the assumptions and predictions of resistance management models must be reexamined.

We present a field-based approach to detect and monitor insects with resistance to insecticidal toxins produced ty transgenic plants. Out objective is to estimate the phenotypic frequency of resistance in a population by relating the densities of insects on genetically transformed plants to densities on nontransformed plants. We focus on European corn borer, Ostrinia nubilalis (Hubner), in sweet corn, Zea mays L., expressing Cry1Ab from Bacillus thuringiensis