Field and laboratory evaluations of transgenic cottons expressing one or two Bacillus thuringiensis var. kurstaki Berliner proteins for management of noctuid (Lepidoptera) pests

Field studies were conducted from 1999 to 2001 to evaluate the efficacy of the transgenic cotton, Gossypium hirsutum (L.), genotype, Bollgard II (Monsanto 15985), which expresses two Bacillus thuringiensis Berliner (Bt) proteins (Cry1Ac + Cry2Ab) that are active against lepidopterous pests. Bollgard II was compared with Bollgard (DP50B), which expresses only one Bt protein (Cry1Ac), and, in all tests, the conventional variety, DP50, was used as a non-Bt control. Larval populations of the bollworm, Helicoverpa zea (Boddie), and the soybean looper, Pseudoplusia includens (Walker), were significantly lower in Bollgard II than in Bollgard and conventional cotton, and the proportion of fruit damaged by H. zea was also lower. Fall armyworm, Spodoptera frugiperda (J. E. Smith), populations were lower in Bollgard II than in Bollgard, although not significantly. Field tests were supplemented with laboratory bioassays in 2001 to compare mortality of S. frugiperda, and beet armyworms, Spodoptera exigua (Hübner), feeding on these genotypes. Mortality of both species was significantly greater on Bollgard II plant material than on either Bollgard or conventional cotton. This study demonstrated that the dual-toxin Bollgard II genotype is highly effective against lepidopterous pests that are not adequately controlled by the current single-toxin Bollgard varieties. If toxin expression in future Bollgard II varieties remains consistent with that of Monsanto 15985, supplemental insecticides will be reduced, and may be eliminated for lepidopterous pests in South Carolina.     

Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes.

Examination of commercial Cry1Ac transgenic Bacillus thuringiensis Berliner (Bt) cotton varieties (Bollgard, Monsanto, St. Louis, MO) and an experimental Cry1Ac + Cry2Ab transgenic Bt cotton variety (Bollgard II, Monsanto) for lepidopteran field efficacy was conducted during the 2000 growing season. In addition, a commercially available (Envirologix, Portland, ME) quantification assay (ELISA) was used to measure and profile the expression levels of Cry proteins in two of these varieties ['DP50B, Bollgard'; 'DP50BII, Bollgard II' (Delta & Pine Land, Scott, MS)]. Populations of beet army worms, Spodoptera exigua (Hübner), and soybean loopers, Pseudoplusia includens (Walker), were significantly lower (P < 0.05) in Bollgard II plots compared with Bollgard. Population numbers for fall army worms, Spodoptera frugiperda (J. E. Smith), and salt marsh caterpillars, Estigmene acrea (Drury), were lower in Bollgard II plots compared with Bollgard but means did not differ significantly (P > 0.05). Single and dual-toxin genotypes remained superior (P < 0.05) compared with conventional cotton against the tobacco budworm, Heliothis virescens (F.). The addition of Cry2Ab had no significant (P > 0.05) impact on Cry1Ac expression in Bollgard II compared with Cry1Ac expression in Bollgard. Furthermore, throughout the season Cry2Ab was present at much higher levels in the plant compared with Cry1Ac for Bollgard II plants. Possible species-specific reasons for increased efficacy of Bollgard II over Bollgard are discussed.     

Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.     

Partial characterization of cotton plants expressing two toxin proteins from Bacillus thuringiensis: relative toxin contribution, toxin interaction, and resistance management

Abstract:  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 δ -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.     

Binding of Bacillus thuringiensis toxin Cry1Ac to multiple sites of cadherin in pink bollworm

Toxins from Bacillus thuringiensis (Bt) are widely used for pest control. In particular, Bt toxin Cry1Ac produced by transgenic cotton kills some key lepidopteran pests. We found that Cry1Ac binds to recombinant peptides corresponding to extracellular regions of a cadherin protein (BtR) in a major cotton pest, pink bollworm (Pectinophora gossypiella) (PBW). In conjunction with previous results showing that PBW resistance to Cry1Ac is linked with mutations in the BtR gene, the results reported here support the hypothesis that BtR is a receptor for Cry1Ac in PBW. Similar to other lepidopteran cadherins that bind Bt toxins, BtR has at least two Cry1Ac-binding domains in cadherin-repeat regions 10 and 11, which are immediately adjacent to the membrane proximal region. However, unlike cadherins from Manduca sexta and Bombyx mori, toxin binding was not seen in regions more distal from the membrane proximal region. We also found that both the protoxin and activated toxin forms of Cry1Ac bound to recombinant BtR fragments, suggesting that Cry1Ac activation may occur either before or after receptor binding.     

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.     

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.     

Baseline susceptibility of tobacco budworm (Lepidoptera: Noctuidae) to Cry1F toxin from Bacillus thuringiensis

Transgenic cotton, Gossypium hirsutum L., lines expressing both Cry1F and Cry1Ac insecticidal proteins from Bacillus thuringiensis (Bt) have been commercially available in the United States since 2005. Both Bt proteins are highly effective against tobacco budworm, Heliothis virescens (F.), and other lepidopteran pests of cotton. Although CrylAc has been available in Bt cotton since 1996, the Cry1F component is relatively new. As part of the proactive resistance management program for Cry1F/Cry1Ac cotton, a susceptibility-monitoring program is being implemented. Baseline variation in the susceptibility to Cry1F in field populations of tobacco budworm was measured. There was a three-fold variation in the amount of Cry1F needed to kill 50% of the neonates from 15 different field populations from the southern and central United States. Future variation in susceptibility of tobacco budworm populations to Cry1F or even resistance evolution could be documented based on this baseline data. A candidate diagnostic concentration was determined that may be efficiently used to identify individuals that potentially carry major alleles conferring field-relevant resistance to Cry1F before such alleles spread through field populations.     

Similar genetic basis of resistance to Bt toxin Cry1Ac in Boll-selected and diet-selected strains of pink bollworm

Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F(1) progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona.     

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.     

Monitoring and management strategy for Helicoverpa armigera resistance to Bt cotton in China

The cotton bollworm, Helicoverpa armigera, is one of the most important insect pests in cotton growing regions of China. Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been deployed for combating cotton bollworm since 1997. Natural refugees derived from the mixed planting system consisting of cotton, corn, soybean, vegetables, peanut and others on single-family farms of a small scale were used for delaying the evolution of resistance to Bt cotton. Susceptibility of H. armigera field populations to the Bt insecticidal protein Cry1Ac was monitored from 1997 to 2006. The results indicate that the field populations are still susceptible to Cry1Ac, and monitoring indication no apparent shifts in susceptibility in field populations of this important pest.     

Selecting for efficacy of Bollgard cotton cultivars against various Lepidoptera using forward breeding techniques

Studies during the past 5 yr have shown that the overall level of protein (Cry1Ac) produced from the cry1Ac transgene (Monsanto Co., St. Louis, MO) differ among commercial Bollgard cotton, Gossypium hirsutum L., cultivars. These differences between cultivars are under genetic control and have been correlated with efficacy of certain lepidopteran pests. Previous studies have shown that the parental background (i.e., non-Cry1Ac conventional cultivar) has a significant influence on the amount of Cry1Ac protein in Bollgard cultivars. Unlike the backcross technique commonly used to acquire commercial Bollgard cultivars, we used forward breeding to obtain cultivars of Bollgard cotton that were selected for various levels of Cry1Ac. These differences in the amount of Cry1Ac were correlated with growth and survival of two lepidopteran pests of cotton. Implications for effective resistance management as well as relative ease of this procedure are discussed.     

Selective feeding of tobacco budworm and bollworm (Lepidoptera: Noctuidae) on meridic diet with different concentrations of Bacillus thuringiensis proteins

Laboratory experiments were conducted to evaluate the behavior of bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), larvae on meridic diet with different concentrations of the Cry1Ac and Cry2Ab proteins from Bacillus thuringiensis subsp. kurstaki Berliner. The proteins used in these experiments are the ones in commercially available Bollgard and Bollgard II cotton. Both bollworms and tobacco budworms selectively fed on nontreated diet compared with diet treated with Cry1Ac. In addition, bollworms exhibited a concentration response with Cry1Ac. In general, bollworms selected diet with low concentrations of Cry1Ac compared with diet with higher concentrations of Cry1Ac. For Cry2Ab, the avoidance was not as prominent as that observed for Cry1Ac. Based on results from no-choice assays, the Cry1Ac and Cry2Ab concentrations used in choice assays represented a wide range of biological activity on both species. The lower concentrations provided low levels of mortality, whereas the higher concentrations provided high levels of mortality. Also, the developmental times of larvae were longer at higher concentrations of both proteins. These data provide important information about the behavioral response of key cotton pests to the B. thuringiensis proteins found in commercially available transgenic cotton. This information will be important to develop accurate scouting and management procedures for Bollgard and Bollgard II cotton.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.     

不同Bt蛋白对棉铃虫存活率和生长发育的影响

在我国Bt棉主要以Cry1Ab或Cry1Ac为主,其他新型Bt基因未被转入棉花中用来控制害虫,然而大面积种植单价Bt基因的棉花,将可能会大大增加靶标害虫对该类型Bt棉花抗性频率,因此研究其他新型Bt蛋白对靶标害虫的控制作用显得十分必要。采用蛋白混入人工饲料的生物测定方法,在室内测定了6种Bt蛋白对棉铃虫初孵幼虫的毒力,比较了浓度为1．0μg· g-1时不同Bt蛋白对棉铃虫幼虫生长发育的影响。毒力测定结果表明,不同Bt蛋白对棉铃虫初孵幼虫的毒力不同,LC50值由低到高依次为Cry1Ab 0．065μg· g-1、Cry1Ac 0．074μg· g-1、Cry2Ab 0．133μg· g-1、Cry2Aa 11．670μg· g-1、Cry1Ah 13．010μg· g-1和Cry1Ca＞20μg· g-1。生长发育测定结果表明,Cry1Ab和Cry1Ac对棉铃虫幼虫的生长发育影响最大,Cry2Ab次之;Cry1Ah和Cry2Aa对1龄幼虫的校正死亡率和体重抑制率差别不大,但对2龄幼虫的差异较大,Cry1Ah处理2龄幼虫后体重和生长发育参数与Cry2Ab接近,而Cry1Ca对棉铃虫幼虫生长发育几乎没影响。Cry1Ah、Cry2Aa和Cry2Ab的毒力不如Cry1Ac和Cry1Ab,但仍可以作为控制棉铃虫幼虫的替代策略。     

Disruption of a cadherin gene associated with resistance to Cry1Ac {delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera

A laboratory strain (GY) of Helicoverpa armigera (Hubner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to "mode 1," the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.     

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.     

Vip3Aa tolerance response of Helicoverpa armigera populations from a Cry1Ac cotton planting region

Transgenic cotton, Gossypium hirsutum L., that expresses the Bacillus thuringiensis (Bt) Cry1Ac toxin, holds great promise in controlling target insect pests. Evolution of resistance by target pests is the primary threat to the continued efficacy of Bt cotton. To thwart pest resistance evolution, a transgenic cotton culitvar that produces two different Bt toxins, cry1Ac and vip3A genes, was proposed as a successor of cry1Ac cotton. This article reports on levels of Vip3Aa tolerance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) populations from the Cry1Ac cotton planting region in China based on bioassays of the F1 generation of isofemale lines. In total, 80 isofemale families of H. armigera from Xiajin county of Shandong Province (an intensive Bt cotton planting area) and 93 families from Anci county of Hebei Province (a multiple-crop system including corn [Zea mays L.] , soybean [Glycine max (L.) Merr.], peanut (Arachis hypogaea L.), and Bt cotton) were screened with a discriminating concentration of both Cry1Ac- and Vip3A-containing diets in 2009. From data on the relative average development rates and percentage of larval weight inhibition of F1 full-sib families tested simultaneously on Cry1Ac and Vip3Aa, results indicate that responses to Cry1Ac and Vip3Aa were not genetically correlated in field population ofH. armigera. This indicates that the threat of cross-resistance between Cry1Ac and Vip3A is low in field populations of H. armigera. Thus, the introduction of Vip3Aa/Cry1Ac-producing lines could delay resistance evolution in H. armigera in Bt cotton planting area of China.