Inheritance of resistance to Bt toxin crylac in a field-derived strain of pink bollworm (Lepidoptera: Gelechiidae)

Laboratory selection with Cry1Ac, the Bacillus thuringiensis (Bt) toxin in transgenic cotton, initially produced 300-fold resistance in a field-derived strain of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. After additional selection increased resistance to 3,100-fold, we tested the offspring of various crosses to determine the mode of inheritance of resistance to Cry1Ac. The progeny of reciprocal F1 crosses (resistant male x susceptible female and vice versa) responded alike in bioassays, indicating autosomal inheritance. Consistent with earlier findings, resistance was recessive at a high concentration of Cry1Ac. However, the dominance of resistance increased as the concentration of Cry1Ac decreased. Analysis of survival and growth of progeny from backcrosses (F1 x resistant strain) suggest that resistance was controlled primarily by one or a few major loci. The progression of resistance from 300- to 3,100-fold rules out the simplest model with one locus and two alleles. Overall the patterns observed can be explained by either a single resistance gene with three or more alleles or by more than one resistance gene. The pink bollworm resistance to Cry1Ac described here fits "mode 1" resistance, the most common type of resistance to Cry1A toxins in Lepidoptera.     

Effect of entomopathogenic nematodes on the fitness cost of resistance to Bt toxin crylac in pink bollworm (Lepidoptera: Gelechiidae)

The widespread use of crop plants genetically engineered to produce toxins from the bacterium Bacillus thuringiensis (Bt) imposes selection on insect populations to evolve resistance. The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major pest of cotton in the southwestern United States that is currently controlled with transgenic cotton that produces Bt toxin Cry1Ac. Previously reported theoretical work suggests that, in conjunction with a high dose/refuge strategy, fitness costs of Bt resistance can slow or prevent the evolution of resistance. We report here that the entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinernematidae) increased the fitness cost of resistance to Cry1Ac in P. gossypiella. Mortality of P. gossypiella from fourth instar to adult eclosion was significantly higher for a Bt-resistant strain than a susceptible strain in tests with two to 14 infective juveniles of S. riobrave per larva, but it did not differ between strains when nematodes were absent. Nematodes established in P. gossypiella larvae at all concentrations tested, and nematode reproduction in infected P. gossypiella larvae occurred at nematode concentrations of four to 14 infective juveniles per larva. Our results suggest that incorporation of entomopathogenic nematodes into an integrated resistance management strategy could help to delay pest resistance to Bt toxins.     

Effects of cotton cultivar on fitness costs associated with resistance of pink bollworm (Lepidoptera: Gelechiidae) to Bt cotton

Fitness costs associated with insect resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) reduce the fitness on non-Bt refuge plants of resistant individuals relative to susceptible individuals. Because costs may vary among host plants, choosing refuge cultivars that increase the dominance or magnitude of costs could help to delay resistance. Specifically, cultivars with high concentrations of toxic phytochemicals could magnify costs. To test this hypothesis, we compared life history traits of three independent sets of pink bollworm, Pectinophora gossypiella (Saunders), populations on two cotton cultivars that differed in antibiosis against this cotton pest. Each set had an unselected susceptible population, a resistant population derived by selection from the susceptible population, and the F1 progeny of the susceptible and resistant populations. Confirming previous findings with pink bollworm feeding on cotton, costs primarily affected survival and were recessive on both cultivars. The magnitude of the survival cost did not differ between cultivars. Although the experimental results did not reveal differences between cultivars in the magnitude or dominance of costs, modeling results suggest that differences between cultivars in pink bollworm survival could affect resistance evolution. Thus, knowledge of the interaction between host plants and fitness costs associated with resistance to Bt crops could be helpful in guiding the choice of refuge cultivars.     

Effects of refuge contamination by transgenes on Bt resistance in pink bollworm (Lepidoptera: Gelechiidae)

Refuges of non-Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum L., are used to delay Bt resistance in pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), a pest that eats cotton seeds. Contamination of refuges by Bt transgenes could reduce the efficacy of this strategy. Previously, three types of contamination were identified in refuges: 1) homozygous Bt cotton plants, with 100% of their seeds producing the Bt toxin Cry1Ac; 2) hemizygous Bt plants with 70-80% of their seeds producing Cry1Ac; and 3) non-Bt plants that outcrossed with Bt plants, resulting in bolls with Cry1Ac in 12-17% of their seeds. Here, we used laboratory bioassays to examine the effects of Bt contamination on feeding behavior and survival of pink bollworm that were resistant (rr), susceptible (ss), or heterozygous for resistance (rs) to Cry1Ac. In choice tests, rr and rs larvae did not differ from ss in preference for non-Bt versus Bt seeds. Survival of rr and rs also did not differ from ss on artificial outcrossed bolls (a mixture of 20% Bt and 80% non-Bt cotton seeds). On artificial hemizygous Bt bolls (70% Bt seeds) and homozygous Bt bolls (100% Bt seeds), rr had higher survival than ss, although rs and ss did not differ. In a simulation model, levels of refuge contamination observed in the field had negligible effects on resistance evolution in pink bollworm. However, in hypothetical simulations where contamination conferred a selective advantage to rs over ss individuals in refuges, resistance evolution was accelerated.     

Oviposition on and mining in bolls of Bt and non-Bt cotton by resistant and susceptible pink bollworm (Lepidoptera: Gelechiidae)

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.     

Fitness costs and maternal effects associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae).

Transgenic cotton producing a Bacillus thuringiensis (Bt) toxin is widely used for controlling the pink bollworm, Perctinophora 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 an 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 and susceptible strain indicated that the survival cost could be dominant. Development time on non-Bt 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, or 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 Bt cotton are substantial in the pink bollworm.     

Overwintering cost associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae)

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.     

Effect of resistance to Bacillus thuringiensis cotton on pink bollworm (Lepidoptera: Gelechiidae) response to sex pheromone

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) could reduce male response to pheromone traps. Such costs would cause underestimation of resistance frequency if monitoring was based on analysis of males caught in pheromone traps. To develop a DNA-based resistance monitoring program for pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), we compared the response to pheromone traps of males with and without cadherin alleles associated with resistance to Bt cotton (Gossypium hirsutum L.). When irradiated males from two hybrid laboratory strains with an intermediate frequency of resistance alleles were released in large field cages, the probability of capture in pheromone traps was not lower for males with resistance alleles than for males without resistance alleles. These results suggest that analysis of trapped males would not underestimate the frequency of resistance. As the time males spent in traps in the field increased from 3 to 15 d, the success of DNA amplification declined from 100 to 30%. Thus, the efficiency of a DNA-based resistance monitoring program would be improved by analyzing males remaining in traps for 3 d or less.     

Effects of kaolin particle film on oviposition,larval mining,and infestation of cotton by pink bollworm (Lepidoptera: Gelechiidae)

We tested effects of kaolin particle film on oviposition, larval mining, and infestation of cotton by pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), in the laboratory, greenhouse, and field. In laboratory choice tests, females laid seven times more eggs on untreated bolls than on bolls treated with kaolin. When neonates were put on bolls in the laboratory, each boll with a treated and untreated half, larvae and mines were found 24 h later on the untreated half but not on the treated half. In oviposition choice tests with whole plants in the greenhouse, females laid four times more eggs on untreated plants than on treated plants and the number of eggs on bolls was five times higher for untreated plants than for treated plants. Kaolin treatments altered the distribution of eggs among plant parts, with untreated bolls receiving a higher percentage than treated bolls, whereas the opposite occurred for petioles. In field tests, treatment with kaolin alone reduced the proportion of bolls infested with pink bollworm, but a mixture of kaolin and the pyrethroid lambda-cyhalothrin was most effective. The results suggest that kaolin particle film may be useful against pink bollworm, particularly in conjunction with other control tactics.     

High-level resistance to Bacillus thuringiensis toxin crylac and cadherin genotype in pink bollworm

Resistance to transgenic cotton, Gossypium hirsutum L., producing Bacillus thuringiensis (Bt) toxin Cry1Ac is linked with three recessive alleles of a cadherin gene in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. Here, we analyzed a strain (MOV97-R) with a high frequency of cadherin resistance alleles, a high frequency of resistance to 10 microg of Cry1Ac per milliliter of diet, and an intermediate frequency of resistance to 1000 microg of Cry1Ac per ml of diet. We selected two strains for increased resistance by exposing larvae from MOV97-R to diet with 1000 microg of Cry1Ac per ml of diet. In both selected strains, two to three rounds of selection increased survival at 1000 microg of CrylAc per ml of diet to at least 76%, indicating genetic variation in survival at this high concentration and yielding >4300-fold resistance relative to a susceptible strain. Variation in cadherin genotype did not explain variation in survival at 1000 microg of Cry1Ac per ml of diet, implying that one or more other loci affected survival at this concentration. This conclusion was confirmed with results showing that when exposure to Cry1Ac stopped, survival at 1000 microg of Cry1Ac per ml of diet dropped substantially, but survival at 10 microg Cry1Ac per ml of diet remained close to 100% and all survivors had two cadherin resistance alleles. Although survival at 1000 microg of Cry1Ac per ml of diet is not required for resistance to Bt cotton, understanding how genes other than cadherin confer increased survival at this high concentration may reveal novel mechanisms of resistance.     

Early detection of field-evolved resistance to Bt cotton in China: cotton bollworm and pink bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some major insect pests, but pests can evolve resistance and thereby reduce the effectiveness of such Bt crops. The main approach for slowing pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to cotton producing Bt toxin Cry1Ac, several countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. This strategy is designed for cotton bollworm (Helicoverpa armigera), which attacks many crops and is the primary target of Bt cotton in China, but it does not apply to pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we review evidence of field-evolved resistance to Cry1Ac by cotton bollworm in northern China and by pink bollworm in the Yangtze River Valley of China. For both pests, results of laboratory diet bioassays reveal significantly decreased susceptibility of field populations to Cry1Ac, yet field control failures of Bt cotton have not been reported. The early detection of resistance summarized here may spur countermeasures such as planting Bt cotton that produces two or more distinct toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

Predicting spring moth emergence in the pink bollworm (Lepidoptera: Gelechiidae): implications for managing resistance to transgenic cotton.

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.     

Effects of gossypol on fitness costs associated with resistance to Bt cotton in pink bollworm

Fitness costs associated with insect resistance to Bacillus thuringiensis (Bt) crops may help to delay or prevent the spread of resistance alleles, especially when refuges of non-Bt host plants are present. The potential for such delays increases as the magnitude and dominance of fitness costs increase. Here, we examined the idea that plant secondary chemicals affect expression of fitness costs associated with resistance to Bt cotton in Pectinophora gossypiella (Saunders). Specifically, we tested the hypotheses that gossypol affects the magnitude or dominance of fitness costs, by measuring performance of three independent sets of pink bollworm populations fed artificial diet with and without gossypol. Each set had an unselected susceptible population, a resistant population derived by selection from the susceptible population, and the F1 progeny of the susceptible and resistant populations. No individuals completed development on diets with gossypol in one set, suggesting that these individuals partially lost the ability to detoxify this chemical. In the other two sets, costs affecting survival did not support the hypotheses, but costs affecting pupal weight did. Adding gossypol to diet increased the magnitude and dominance of costs affecting pupal weight. In one of the two sets with survivors on diet with gossypol, costs affecting development time were less recessive when gossypol was present in diet. These results indicate that gossypol increased the magnitude and dominance of some fitness costs. Better understanding of the effects of natural plant defenses on fitness costs could improve our ability to design refuges for managing insect resistance to Bt crops.     

Association between resistance to Bt cotton and cadherin genotype in pink bollworm

Two strains of pink bollworm, Pectinophora gossypiella (Saunders), each derived in 1997 from a different field population, were selected for resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac in the laboratory. One strain (MOV97-R) originated from Mohave Valley in western Arizona; the other strain (SAF97-R) was from Safford in eastern Arizona. Relative to a susceptible laboratory strain, Cry1Ac resistance ratios were 1700 for MOV97-R and 520 for SAF97-R. For the two resistant strains, larval survival did not differ between non-Bt cotton and transgenic cotton producing CrylAc. In contrast, larval survival on Bt cotton was 0% for the two unselected parent strains from which the resistant strains were derived. Previously identified resistance (r) alleles of a cadherin gene (BtR) occurred in both resistant strains: r1 and r3 in MOV97-R, and r1 and r2 in SAF97-R. The frequency of individuals carrying two r alleles (rr) was 1.0 in the two resistant strains and 0.02 in each of the two unselected parent strains. Furthermore, in two hybrid strains with a mixture of susceptible (s) and r alleles at the BtR locus, all survivors on Bt cotton had two r alleles. The results show that resistance to Cry1Ac-producing Bt cotton is associated with recessive r alleles at the BtR locus in the strains of pink bollworm tested here. In conjunction with previous results from two other Bt-resistant strains of pink bollworm (APHIS-98R and AZP-R), results reported here identify the cadherin locus as the leading candidate for molecular monitoring of pink bollworm resistance to Bt cotton.     

Sustained susceptibility of pink bollworm to Bt cotton in the United States

Evolution of resistance by pests can reduce the benefits of transgenic crops that produce toxins from Bacillus thuringiensis (Bt) for insect control. One of the world's most important cotton pests, pink bollworm (Pectinophora gossypiella), has been targeted for control by transgenic cotton producing Bt toxin Cry1Ac in several countries for more than a decade. In China, the frequency of resistance to Cry1Ac has increased, but control failures have not been reported. In western India, pink bollworm resistance to Cry1Ac has caused widespread control failures of Bt cotton. By contrast, in the state of Arizona in the southwestern United States, monitoring data from bioassays and DNA screening demonstrate sustained susceptibility to Cry1Ac for 16 y. From 1996-2005, the main factors that delayed resistance in Arizona appear to be abundant refuges of non-Bt cotton, recessive inheritance of resistance, fitness costs associated with resistance and incomplete resistance. From 2006-2011, refuge abundance was greatly reduced in Arizona, while mass releases of sterile pink bollworm moths were made to delay resistance as part of a multi-tactic eradication program. Sustained susceptibility of pink bollworm to Bt cotton in Arizona has provided a cornerstone for the pink bollworm eradication program and for integrated pest management in cotton. Reduced insecticide use against pink bollworm and other cotton pests has yielded economic benefits for growers, as well as broad environmental and health benefits. We encourage increased efforts to combine Bt crops with other tactics in integrated pest management programs.     

Regulation of pheromone production by female pink bollworm moths Pectinophora gossypiella (Sanders) (Lepidoptera: Gelechiidae)

Abstract. We present in this study data which indicate that there is a diel periodicity in the pheromone production of the pink bollworm moth Pectinophora gossypiella (Sanders) (Lepidoptera: Gelechiidae) but that it is not well defined. Moreover the control mechanism of pheromone production differs somewhat from that reported for other moths. No pheromonotropic response was obtained when photophase females were injected with synthetic Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN). After decapitation for 24 h, Hez-PBAN did not induce pheromonotropic activity above control levels, which themselves remained relatively high. No effect on pheromone production was observed after treatment with the non-steroidal ecdysone agonist (RH5999). Decapitation for 72 h resulted in a significant drop in the control levels of pheromone titres. After decapitation for 72 h, stimulation by injections of Hez-PBAN and pink bollworm head extracts was observed. In addition, an enhancement of the PBAN stimulation was observed when combined with severance of the ventral nerve cord before injection. On the other hand, pink bollworm head extracts did not cross-react with Hez-PBAN antiserum in a radioimmunoassay, indicating that the pheromonotropic factor present is sufficiently different from Hez-PBAN and does not recognize the antigenic binding sites. In studies using isolated abdomen and pheromone gland cultures in vitro , no stimulation of de novo pheromone biosynthesis was observed but a 3-fold increase in the de novo fatty acid biosynthesis was detected in pheromone gland cultures.     

Selection for altered pheromone-component ratios in the pink bollworm moth,Pectinophora gossypiella (Lepidoptera: Gelechiidae)

Female pink bollworm moths, Pectinophora gossypiella(Saunders), were selected for altered component ratios of the long-distance sex pheromone, (Z, E)-and (Z, Z)-7,11- hexadecadienyl acetate. Selection for 12 generations increased the mean (± SD) percentage of the (Z, E)isomer from 42.9 ± 1.0 in the parental generation to 48.2 ± 1.2. Although statistically significant, a change of this magnitude may be transparent to males because of their relatively broad response spectrum. Selection for a lower percentage of the (Z, E)isomer yielded no change in the mean pheromone ratio. The total amount of pheromone produced declined in both selected lines. In the line selected for females producing a high percentage of the (Z, E)isomer, the duration of wing fanning by males to high (Z, E)blends was elevated.     

Isolation and characterization of polymorphic microsatellite loci from the pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae)

Thirteen polymorphic microsatellite markers were developed from an enriched partial genomic library for the pink bollworm, Pectinophora gossypiella (Saunders), one of the most important cotton pests in the world. The total number of alleles ranged from two to 12 for a sample of 50 individuals and the expected heterozygosity at these loci ranged from 0.042 to 0.830. Although the presence of null alleles in some loci is suspected, these polymorphic markers are likely to provide useful tools for the population genetic studies of this species.     

棉花无蜜腺性状抗棉铃虫及红铃虫效果研究

棉花无蜜腺性状抗棉铃虫及红铃虫效果研究郑冬官,方其英（安徽农业大学合肥２３００３３６）郑厚今（安徽省农业厅,合肥２３０００１）Ｒｅｓｉｓｔａｎｃｅｏｆｃｏｔｔｏｎｎｅｃｔａｒｉｌｅｓｓｃｈａｒａｃｔｅｒｔｏｂｏｌｌｗｏｒｍａｎｄｐｉｎｋｂｏｌｌｗｏｒ...     

Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.