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.     

Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.     

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.     

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.     

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.     

The interaction of two-spotted spider mites, <Emphasis Type="Italic">Tetranychus urticae</Emphasis> Koch,with Cry protein production and predation by <Emphasis Type="Italic">Amblyseius andersoni</Emphasis> (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize

Crops producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), are an important tool for managing lepidopteran pests on cotton and maize. However, the effects of these Bt crops on non-target organisms, especially natural enemies that provide biological control services, are required to be addressed in an environmental risk assessment. Amblyseius andersoni (Acari: Phytoseiidae) is a cosmopolitan predator of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), a significant pest of cotton and maize. Tri-trophic studies were conducted to assess the potential effects of Cry1Ac/Cry2Ab cotton and Cry1F maize on life history parameters (survival rate, development time, fecundity and egg hatching rate) of A. andersoni. We confirmed that these Bt crops have no effects on the biology of T. urticae and, in turn, that there were no differences in any of the life history parameters of A. andersoni when it fed on T. urticae feeding on Cry1Ac/Cry2Ab or non-Bt cotton and Cry1F or non-Bt maize. Use of a susceptible insect assay demonstrated that T. urticae contained biologically active Cry proteins. Cry proteins concentrations declined greatly as they moved from plants to herbivores to predators and protein concentration did not appear to be related to mite density. Free-choice experiments revealed that A. andersoni had no preference for Cry1Ac/Cry2Ab cotton or Cry1F maize-reared T. urticae compared with those reared on non-Bt cotton or maize. Collectively these results provide strong evidence that these crops can complement other integrated pest management tactics including biological control.     

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.     

Effects of proportion and configuration of Bacillus thuringiensis cotton on pest abundance, damage, and yield

Bacillus thuringiensis (Bt) transgenic cotton, Gossypium hirsutum L., kills several economically important pests, reducing injury and increasing yields. Refuges of non-Bt cotton are currently planted with Bt cotton in different designs to slow pest resistance evolution. To compare the effects of differences in Bt/non-Bt plant heterogeneity found in different refuge designs on square (flower bud) damage, abscissions, sap-feeding herbivore densities, and yield in cotton, four types of 24-row cotton plots were planted in 2001 and 2002: 1) seed mixtures of Bt and non-Bt varieties, 2) 12-row strips of Bt and non-Bt, 3) solid Bt, and 4) solid non-Bt. For both years cotton bollworm, Helicoverpa zea (Boddie), damage was less in solid Bt plots than strips and mixtures and all were less than solid non-Bt plots. Cotton fleahopper, Pseudatomoscelis seriatus (Reuter), damage was affected by refuge, but only in 2002 when damage was greater in solid Bt plots than all other plots and greater in strips than solid non-Bt plots. Abscissions were least in solid non-Bt plots, and less in mixtures and strips than solid Bt plots. In 2001, western flower thrips, Frankliniella occidentalis (Pergande), density was greatest in mixtures, whereas sweetpotato whitefly, Bemisia tabaci (Gennadius), was greatest in solid Bt plots, and greater in mixtures than solid non-Bt plots. Yield also was affected by refuge, it was greater for solid Bt plots than for solid non-Bt plots and mixtures in 2001, but the reverse was true in 2002.     

Bt Crops Producing Cry1Ac,Cry2Ab and Cry1F Do Not Harm the Green Lacewing,Chrysoperla rufilabris

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.     

A Challenge for the Seed Mixture Refuge Strategy in Bt Maize: Impact of Cross-Pollination on an Ear-Feeding Pest,Corn Earworm

To counter the threat of insect resistance, Bacillus thuringiensis (Bt) maize growers in the U.S. are required to plant structured non-Bt maize refuges. Concerns with refuge compliance led to the introduction of seed mixtures, also called RIB (refuge-in-the-bag), as an alternative approach for implementing refuge for Bt maize products in the U.S. Maize Belt. A major concern in RIB is cross-pollination of maize hybrids that can cause Bt proteins to be present in refuge maize kernels and negatively affect refuge insects. Here we show that a mixed planting of 5% nonBt and 95% Bt maize containing the SmartStax traits expressing Cry1A.105, Cry2Ab2 and Cry1F did not provide an effective refuge for an important above-ground ear-feeding pest, the corn earworm, Helicoverpa zea (Boddie). Cross-pollination in RIB caused a majority (>90%) of refuge kernels to express ≥ one Bt protein. The contamination of Bt proteins in the refuge ears reduced neonate-to-adult survivorship of H. zea to only 4.6%, a reduction of 88.1% relative to larvae feeding on ears of pure non-Bt maize plantings. In addition, the limited survivors on refuge ears had lower pupal mass and took longer to develop to adults.     

Performance of Bt-susceptible and -heterozygous dual-gene resistant genotypes of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in seed blends of non-Bt and pyramided Bt maize

A seed blend refuge has been implemented in the U.S. Corn Belt for Bt maize resistance management. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a target pest of Bt maize in the Americas. The larvae of this pest are mobile, which may affect the efficacy of seed blend refuges. In this study, field and greenhouse trials were conducted to determine the performance of Bt-susceptible (aabb) and -heterozygous dual-gene-resistant (AaBb) genotypes of S. frugiperda in seed blends of non-Bt and pyramided Bt maize. Three field trials evaluated larval survival, larval growth, and plant injury with aabb in seed blends of Bt maize expressing Cry1A.105/Cry2Ab2/Vip3A with 0–30% non-Bt seeds. Greenhouse tests investigated the performance of aabb and AaBb in seed blends of Cry1A.105/Cry2Ab2 with 0–30% non-Bt seeds. In pure non-Bt maize plots, after 9–13 d of neonates being released on the plants, 0.39 and 0.65 larvae/plant survived with leaf injury ratings of 4.7 and 5.9 (Davis's 1–9 scale) in the field and greenhouse, respectively. In contrast, live larvae and plant injury were virtually not observed on Bt plants across all planting patterns. Larval occurrence and plant injury by aabb on non-Bt plants were similar between seed blends and pure non-Bt plantings, suggesting that the blended refuges could provide an equivalent susceptible population as structured refuge under the test conditions. In the greenhouse, the two insect genotypes in seed blends performed similarly, indicating that the seed blends did not provide more favorable conditions for AaBb over aabb. The information generated from this study should be useful in managing S. frugiperda and evaluating if send blends could be suitable refuge options for Bt resistance management in the regions where the insect is a primary target pest.     

Fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on transgenic cotton with reduced levels of Cry1Ac

The performance of Helicoverpa armigera (Hübner) on 15-wk-old cotton plants was compared for a susceptible strain, a near-isogenic laboratory-selected strain, and F1 progeny of the two strains. Glasshouse experiments were conducted to test the three insect types on conventional plants and transgenic plants that produced the Bacillus thuringiensis (Bt) toxin Cry1Ac. At the time of testing (15 wk), the Cry1Ac concentration in cotton leaves was 75% lower than at 4 wk. On these plants, < 10% of susceptible larvae reached the fifth instar, and none survived to pupation. In contrast, survival to adulthood on Cry1Ac cotton was 62% for resistant larvae and 39% for F1 larvae. These results show that inheritance of resistance to 15-wk-old Cry1Ac cotton is partially dominant, in contrast to results previously obtained on 4-wk-old Cry1Ac cotton. Growth and survival of resistant insects were similar on Cry1Ac cotton and on non-Bt cotton, but F1 insects developed more slowly on Cry1Ac cotton than on non-Bt cotton. Survival was lower and development was slower for resistant larvae than for susceptible and F1 larvae on non-Bt cotton. These results show recessive fitness costs are associated with resistance to Cry1Ac.     

Feeding and Dispersal Behavior of the Cotton Leafworm,Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), on Bt and Non-Bt Cotton: Implications for Evolution and Resistance Management

The host acceptance of neonate Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) larvae to Bt cotton plants exerts a strong influence on the potential risk that this pest will develop resistance to Bt cotton. This will also determine the efficiency of management strategies to prevent its resistance such as the “refuge-in-the-bag” strategy. In this study, we assessed the acceptance of neonate A. argillacea larvae to Bt and non-Bt cotton plants at different temperatures during the first 24 h after hatching. Two cotton cultivars were used in the study, one a Bt DP 404 BG (Bollgard) cultivar, and the other, an untransformed isoline, DP 4049 cultivar. There was a greater acceptance by live neonate A. argillacea larvae for the non-Bt cotton plants compared with the Bt cotton plants, especially in the time interval between 18 and 24 h. The percentages of neonate A. argillacea larvae found on Bt or non-Bt plants were lower when exposed to temperatures of 31 and 34°C. The low acceptance of A. argillacea larvae for Bt cotton plants at high temperatures stimulated the dispersion of A. argillacea larvae. Our results support the hypothesis that the dispersion and/or feeding behavior of neonate A. argillacea larvae is different between Bt and non-Bt cotton. The presence of the Cry1Ac toxin in Bt cotton plants, and its probable detection by the A. argillacea larvae tasting or eating it, increases the probability of dispersion from the plant where the larvae began. These findings may help to understand how the A. argillacea larvae detect the Cry1Ac toxin in Bt cotton and how the toxin affects the dispersion behavior of the larvae over time. Therefore, our results are extremely important for the management of resistance in populations of A. argillacea on Bt cotton.     

Effects of entomopathogenic nematodes on evolution of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

The evolution of resistance by pests can reduce the efficacy of transgenic crops that produce insecticidal toxins from Bacillus thuringiensis (Bt). However, fitness costs may act to delay pest resistance to Bt toxins. Meta-analysis of results from four previous studies revealed that the entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinernematidae) imposed a 20% fitness cost for larvae of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), that were homozygous for resistance to Bt toxin Cry1Ac, but no significant fitness cost was detected for heterozygotes. We conducted greenhouse and laboratory selection experiments to determine whether S. riobrave would delay the evolution of pink bollworm resistance to Cry1Ac. We mimicked the high dose/refuge scenario in the greenhouse with Bt cotton (Gossypium hirsutum L.) plants and refuges of non-Bt cotton plants, and in the laboratory with diet containing Cry1Ac and refuges of untreated diet. In both experiments, half of the replicates were exposed to S. riobrave and half were not. In the greenhouse, S. riobrave did not delay resistance. In the laboratory, S. riobrave delayed resistance after two generations but not after four generations. Simulation modeling showed that an initial resistance allele frequency > 0.015 and population bottlenecks can diminish or eliminate the resistance-delaying effects of fitness costs. We hypothesize that these factors may have reduced the resistance-delaying effects of S. riobrave in the selection experiments. The experimental and modeling results suggest that entomopathogenic nematodes could slow the evolution of pest resistance to Bt crops, but only under some conditions.     

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.     

Relative fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton

Glasshouse and laboratory experiments were conducted to evaluate the relative fitness of Cry1A-susceptible and laboratory-selected resistant strains of Helicoverpa armigera (Hübner). Life history parameters of H. armigera larvae feeding on young cotton plants showed a significant developmental delay of up to 7 d for the resistant strain compared with the susceptible strain on non-Bacillus thuringiensis (Bt) cotton. This fitness cost was not evident on artificial diet. There was no developmental delay in the F1 hybrid progeny from the reciprocal backcross of the resistant and susceptible strains, indicating that the fitness cost is recessive. In two cohorts tested, survival to pupation of resistant larvae on Bt cotton expressing Cry1Ac was 54 and 51% lower than on non-Bt cotton, whereas all susceptible and F1 larvae tested on Cry1Ac cotton were killed. Mortality of susceptible larvae occurred in the first or second instar, whereas the F1 larvae were able to develop to later instars before dying, demonstrating that resistance is incompletely recessive. The intrinsic rate of increase was reduced by >50% in the resistant strain on Cry1Ac cotton compared with the susceptible strain on non-Bt cotton. There was a significant reduction in the survival of postdiapausal adults from the resistant strain and the F1 strains, indicating that there is a nonrecessive overwintering cost associated with Cry1A resistance in H. armigera.     

Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize

Although transgenic crops expressing either Cry1Ab or Cry1Ac, both derived from Bacillus thuringiensis (Bt), have been used commercially, the evolution of insects resistance to these CRY proteins has become a challenge. Thus, it has been proposed that co-expression of two Bt proteins with different modes of action may delay the development of resistance to Bt. However, few Bt proteins have been identified as having different modes of action from those of Cry1Ab or Cry1Ac. In this study, transgenic lines of maize over-expressing either Cry1Ie or Cry1Ac gene have been developed. Several independent transgenic lines with one copy of the foreign gene were identified by Southern blot analysis. Bioassays in the laboratory showed that the transgenic plants over-expressing Cry1Ie were highly toxic against the wild-type cotton bollworm (Heliothis armigera), producing mortality levels of 50 % after 6 days of exposure. However, the mortality caused by these plants was lower than that caused by the Cry1Ac transgenic plants (80 %) and MON810 plants expressing Cry1Ab (100 %), which both exhibited low toxicity toward the Cry1Ac-resistant cotton bollworm. In contrast, three transgenic maize lines expressing Cry1Ie induced higher mortality against this pest and were also highly toxic to the Asian corn borer (Ostrinia furnacalis) in the field. These results indicate that the Cry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins. Therefore, the use of transgenic plants expressing Cry1Ie might delay the development of Bt-resistant insects in the field.     

Astylus atromaculatus (Coleoptera: Melyridae): abundance and role in pollen dispersal in Bt and non-Bt cotton in South Africa

In South Africa, modified Bt (Cry1 Ac) cotton cultivars and organic ones coexist. This raises the question of the risk of dissemination of genetically modified (GM) pollen to non-GM crops by visiting insects. We inventoried the flower-visiting insects in Bt and non-Bt cotton fields of the South African Highveld region and investigated their role in pollen dispersal. Their diversity and abundance varied slightly among sites, with Astylus atromaculatus as the predominant insect on both Bt and non-Bt cotton flowers. The other major flower-visiting species were Apis mellifera and solitary Apidae. No differences were found in the abundance of each taxum between Bt and non-Bt cotton except for Scoliidae and Nitidulidae, which were scarce overall (<0.5%) but more abundant on the non-Bt flowers in the central area of the field at one site. The pollen load on A. atromaculatus was as high as on Apis mellifera. Cage tests showed that A. atromaculatus can pollinate female cotton plants by transferring pollen from male donor plants. In the field, the flight range of this insect was generally short (25 m), but it can occasionally reach up to 200 m or even more. This study therefore highlights that A. atromaculatus, commonly regarded as a pest, could be an unexpected but efficient pollinator. Because its population density can be high, this species could mediate unwanted cotton pollen flow when distances between coexiting fields are not sufficient.     

Increasing tolerance to Cry1Ac cotton from cotton bollworm, Helicoverpa armigera, was confirmed in Bt cotton farming area of China

Abstract.  1. Changes in the frequency of Cry1Ac resistance genes and shifts in tolerance of cotton bollworm, Helicoverpa armigera , to the Cry1Ac toxin were assessed using bioassays of F₁ and F₂ offspring of isofemale lines from Anci County of Hebei Province (a multiple-crop system including corn, soybean, peanut, and Bt cotton) and Xiajin County of Shandong Province (an intensive Bt cotton planting area) in Northern China during 2002–2005.
2. A conservative analysis of the overall results indicated that there was a small increase in the frequency of major, non-recessive resistance genes over time.
3. The relative average development ratings [RADR – growth rate of a line on a Bt diet in proportion to the growth rate on a non-Bt (NBT) diet] of the bollworm larvae in F₁ tests increased significantly from year to year, indicating a gradual trend towards higher tolerance to Cry1Ac in the field populations.
4. There were also significant positive correlations between RADR of the lines in the F₁ generation and the RADR of their F₂ offspring, indicating that the tolerance was genetically based.
5. Quantitative genetic simulation analysis showed that resistance of H . armigera to Bt cotton in Xiajin could evolve to a high level in 11–15 years if no effective resistance management measures are carried out.     

Dispersal behavior of neonate European corn borer (Lepidoptera: Crambidae) on Bt corn

European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), has historically been a significant economically important insect pest of corn (Zea mays L.) in the United States and Canada. The development in the 1990s of genetically modified corn expressing genes derived from Bacillus thuringiensis (Bt) that encodes insecticidal crystalline (Cry) proteins has proven to be effective in controlling this insect as well as other corn pests. The purpose of this study was to assess the movement and dispersal behavior of neonate European corn borer on Bt corn. We examined differences in neonate European corn borer dispersal behavior for the first 4 h after eclosion in the field among a stacked pyramid (Cry1F X Cry1Ab X Cry34/35Ab1) Bt corn, a Cry1F Bt corn, and a non-Bt sweet corn; and in the laboratory among a Bt corn hybrid containing Cry1F, a hybrid containing Cry1Ab, a pyramid combining these two hybrids (Cry1F X Cry1Ab), and a non-Bt near isoline corn. In field experiments, we found that dispersal was significantly higher on Bt corn compared with sweet corn. In laboratory experiments, dispersal was significantly higher on Cry1Ab Bt corn and Cry1F X Cry1Ab Bt corn than on non-Bt near isoline corn. Results indicated that neonate dispersal may be significantly greater in Bt cornfields compared with non-Bt cornfields. The findings on dispersal behavior in this study will be useful in evaluating the efficacy of a blended seed refuge system for managing European corn borer resistance in Bt corn.