Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts

The binding properties of Vip3A, a new family of Bacillus thuringiensis insecticidal toxins, have been examined in the major cotton pests, Heliothis virescens and Helicoverpa zea. Vip3A bound specifically to brush border membrane vesicles (BBMV) prepared from both insect larval midguts. In order to examine the cross-resistance potential of Vip3A to the commercially available Cry1Ac and Cry2Ab2 toxins, the membrane binding site relationship among these toxins was investigated. Competition binding assays demonstrated that Vip3A does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. BBMV protein blotting experiments showed that Vip3A does not bind to the known Cry1Ac receptors. These distinct binding properties and the unique protein sequence of Vip3A support its use as a novel insecticidal agent. This study indicates a very low cross-resistance potential between Vip3A and currently deployed Cry toxins and hence supports its use in an effective resistance management strategy in cotton.     

Survivorship of Helicoverpa zea and Heliothis virescens on cotton plant structures expressing a Bacillus thuringiensis vegetative insecticidal protein

A series of tests quantified bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), larval survival on plant structures of a nontransgenic cotton (Gossypium hirsutum L.), 'Coker 312', and two transgenic cottons expressing Vip3A protein or both Vip3A + CrylAb proteins (VipCot). Vegetative and reproductive structures including terminal leaves, flower bud (square) bracts, whole debracted squares, flower petals, flower anthers, and intact capsules (bolls) were harvested from plants in field plots. Each structure was infested with 2-d-old larvae from one of the two heliothine species. Larvae were allowed to feed for 96 h on fresh tissue. Survivorship at 96 h after infestation was significantly lower on all structures of Vip3A and VipCot cotton lines compared with similar structures of Coker 312. VipCot plant structures generally resulted in lower larval survivorship compared with similar structures of the Vip3A cotton line. H. zea survivorship ranged from 4 to 28% and from 1 to 18% on Vip3A and VipCot plant structures, respectively. H. virescens survivorship ranged from 10 to 43% and from 2 to 12% on Vip3A and VipCot plant structures, respectively. H. virescens survivorship was higher on VIP3A plant structures compared with that for H. zea on similar structures. These differences between species were not observed on plants from the cotton line expressing VipCot proteins. The results for these plant structures demonstrate that the combination of proteins expressed in VipCot cotton lines are more effective than Vip3A cotton lines against this heliothine complex.     

Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

A global approach to resistance monitoring

Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been grown in many parts of the world since 1996. In the United States, the Environmental Protection Agency (EPA) has required that industry submit insect resistance management (IRM) plans for each Bt corn and cotton product commercialized. A coalition of stakeholders including the EPA, USDA, academic scientists, industry, and grower organizations have cooperated in developing specific IRM strategies. Resistance monitoring (requiring submission of annual reports to the EPA), and a remedial action plan addressing any contingency if resistance should occur, are important elements of these strategies. At a global level, Monsanto conducts baseline susceptibility studies (prior to commercialization), followed by monitoring studies on target pest populations, for all of its commercialized Bt crop products. To date, Monsanto has conducted baseline/monitoring studies in Argentina, Australia, Brazil, Canada, China, Colombia, India, Mexico, the Philippines, South Africa, Spain, and the United States. Examples of pests on which resistance monitoring has been conducted include cotton bollworm, Helicoverpa zea, European corn borer, Ostrinia nubilalis, pink bollworm, Pectinophora gossypiella, Southwestern corn borer, Diatraea grandiosella, tobacco budworm, Heliothis virescens, and western corn rootworm, Diabrotica virgifera virgifera, in the United States, cotton bollworm, Helicoverpa armigera, in China, India and Australia, and H. virescens and H. zea in Mexico. No field-selected resistance to Bt crops has been documented.     

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.     

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.     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

Cross-resistance responses of CrylAc-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein vip3A

One susceptible and three Cry1Ac-resistant strains of tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), were used in laboratory studies to determine the level of cross-resistance between the Bacillus thuringiensis (Berliner) toxins Cry1Ac and Vip3A by using concentration-mortality and leaf tissue experiments. Concentration-mortality data demonstrated that the three Cry1Ac-resistant H. virescens strains, YHD2, KCBhyb, and CxC, were at least 215- to 316-fold resistant to Cry1Ac compared with the susceptible strain, YDK. Results from Vip3A concentration-mortality tests indicated that mortality was similar among all four H. virescens strains. Relative larval growth on Cry1Ac reflected concentration-mortality test results, because YHD2 larval growth was mostly unaffected by the Cry1Ac concentrations tested. Growth ratios for KCBhyb and CXC indicated that they had a more moderate level of resistance to Cry1Ac than did YHD2. Relative larval growth on Vip3A was highly variable at lower concentrations, but it was more consistent on concentrations of Vip3A above 25 microg/ml. Differences in larval growth among strains on Vip3A were not as pronounced as seen in Cry1Ac experiments. Mortality and larval growth also was assessed in leaf tissue bioassays in which YDK, CxC, and KCBhyb neonates were placed onto leaf disks from non-Bt and Bt cotton, Gossypium hirsutum L., for 5 d. Three Bt lines were used in an initial bioassay and consisted of two Vip3A-containing lines, COT203 and COT102, and a Cry1Ac-producing line. Mortality of KCBhyb and CXC was lower than that of YDK larvae in the presence of leaf tissue from the Cry1Ac-producing line. Additionally, increased larval growth and leaf tissue consumption on Cry1Ac-containing leaf disks was observed for KCBhyb and CXC. Mortality and larval weights were similar among strains when larvae were fed leaf tissue of either non-Bt, COT203, or COT102. A subsequent leaf tissue bioassay was conducted that evaluated four cotton lines: non-Bt, Cry1Ab-expressing, Vip3A-expressing, and pyramided-toxin plants that produced both Cry1Ab and Vip3A. Mortality levels were similar among strains when fed non-Bt, Vip3A-expressing, or pyramided-toxin leaf tissues. Mortality was higher for YDK than for KCBhyb or CXC on Cry1Ab-expressing leaf tissues. No differences in larval weights were observed among strains for any genotype tested. Results of these experiments demonstrate that cross-resistance is nonexistent between CrylAc and Vip3A in H. virescens. Thus, the introduction of Vip3A-producing lines could delay Cry1Ac-resistance evolution in H. virescens, if these lines gain a significant share of the market.     

Vip3A resistance alleles exist at high levels in Australian targets before release of cotton expressing this toxin

Crops engineered to produce insecticidal crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) have revolutionised pest control in agriculture. However field-level resistance to Bt has developed in some targets. Utilising novel vegetative insecticidal proteins (Vips), also derived from Bt but genetically distinct from Cry toxins, is a possible solution that biotechnical companies intend to employ. Using data collected over two seasons we determined that, before deployment of Vip-expressing plants in Australia, resistance alleles exist in key targets as polymorphisms at frequencies of 0.027 (n = 273 lines, 95% CI = 0.019-0.038) in H. armigera and 0.008 (n = 248 lines, 0.004-0.015) in H. punctigera. These frequencies are above mutation rates normally encountered. Homozygous resistant neonates survived doses of Vip3A higher than those estimated in field-grown plants. Fortunately the resistance is largely, if not completely, recessive and does not confer resistance to the Bt toxins Cry1Ac or Cry2Ab already deployed in cotton crops. These later characteristics are favourable for resistance management; however the robustness of Vip3A inclusive varieties will depend on resistance frequencies to the Cry toxins when it is released (anticipated 2016) and the efficacy of Vip3A throughout the season. It is appropriate to pre-emptively screen key targets of Bt crops elsewhere, especially those such as H. zea in the USA, which is not only closely related to H. armigera but also will be exposed to Vip in several varieties of cotton and corn.     

The diversity of Bt resistance genes in species of Lepidoptera

Although the mode of action of Cry1A toxins produced by Bacillus thuringiensis is fairly well understood, knowledge of the molecular mechanisms by which lepidopteran species have evolved resistance to them is still in its infancy. The most common type of resistance has been called "Mode 1" and is characterized by recessive inheritance, >500-fold resistance to and reduced binding by at least one Cry1A toxin, and negligible cross-resistance to Cry1C. In three lepidopteran species, Heliothis virescens, Pectinophora gossypiella, and Helicoverpa armigera, Mode 1 resistance is caused by mutations in a toxin-binding 12-cadherin-domain protein expressed in the larval midgut. These mutations all interrupt the primary sequence of the protein and prevent its normal localization in the membrane, presumably removing a major toxic binding target of the Cry1A toxins. In Plutella xylostella, however, Mode 1 resistance appears to be caused by a different genetic mechanism, as Cry1A resistance is unlinked to the cadherin gene. Mapping studies in H. virescens have detected an additional major Cry1A resistance gene, which on the basis of comparative linkage mapping is distinct from the one in P. xylostella. An additional resistance mechanism supported by genetic data involves a protoxin-processing protease in Plodia interpunctella, and this is likely to be different from the genes mapped in Plutella and Heliothis. Thus, resistance to Cry1A toxins in species of Lepidoptera has a complex genetic basis, with at least four distinct, major resistance genes of which three are mapped in one or more species. The connection between resistance genes and the mechanisms they encode remains a challenging task to elucidate.     

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.     

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.     

Influence of the host plant on occluded virus production and lethal infectivity of a baculovirus

Intra- and inter-specific effects of cotton, soybean, and clover on the time until death of Helicoverpa zea (Boddie) and Heliothis virescens (F.) larvae lethally infected with H. zea nucleopolyhedrovirus (HzSNPV) were evaluated in the laboratory. In the first test, on second instar only, the time until death of lethally infected larvae of both species differed with the plant tissues (vegetative or reproductive) and plant species. The total viral activity produced per larva in LC(50) units (occluded viral bodies (OBs) per larva/LC(50) in OBs/mm(2) of diet surface) was greater from H. virescens larvae fed vegetative than reproductive tissues of all host plants, but from H. zea virus production was greater only when fed vegetative tissue of soybean. In a second test that compared second and fourth instar H. virescens on cotton, total viral activity from larvae treated in both instars was greater when fed vegetative than reproductive tissues. Results of these tests suggest that the ability of host plants to influence baculovirus disease is more complex than previously believed. When examining the epizootic potential of a baculovirus, more attention must be given to the effects of the host plant on the insect-virus interactions.     

A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae

Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.     

Monitoring Bacillus thuringiensis-susceptibility in insect pests that occur in large geographies: how to get the best information when two countries are involved

The adoption of cotton producing insecticidal proteins of Bacillus thuringiensis, commonly referred to as Bt cotton, around the world has proven to be beneficial for growers and the environment. The effectiveness of this important genetically-modified crop can be jeopardized by the development of resistance to Bt cotton by pests it is meant to control, with the possibility that this phenomenon could develop in one country and spread to another by means of insect migration. To preserve the effectiveness of this agricultural biotechnology, regulatory agencies have developed plans to mitigate the development of resistance, and research institutions constantly monitor for shifts in Bt-susceptibility in important pests. If Bt-resistance is detected, this finding needs to be corroborated by an independent laboratory according to current regulatory requirements; a process that presents numerous challenges. We investigated the biological activity of Bt-incorporated diet on Helicoverpa virescens L. after it was stored for several days at different temperatures. Diet stored up to nine days at different temperatures (-14 to 27 degrees C) produced the same biological effect on H. virescens as freshly-prepared diet. Elevating the temperature of Bt stock solution to 76 degrees C as compared to 26 degrees C yielded significantly higher reading of apparent Cry1Ac concentration from MVP II, but not enough to elicit a significant biological response when these stock solutions were incorporated into insect artificial diet. These findings are important particularly when the confirmation of resistance is done at a distant location, such as Mexico, or when diet is shared between laboratories, and must be stored for later use, as in the case of international collaboration.     

Monitoring and adaptive resistance management in Australia for Bt-cotton: current status and future challenges

In the mid-1990 s the Australian Cotton industry adopted an insect-resistant variety of cotton (Ingard) which expresses the Bt toxin Cry1Ac that is specific to a group of insects including the target Helicoverpa armigera. A conservative resistance management plan (RMP), that restricted the area planted to Ingard, was implemented to preserve the efficacy of Cry1Ac until two-gene transgenic cotton was available. In 2004/05 Bollgard II replaced Ingard as the transgenic cotton available in Australia. It improves on Ingard by incorporating an additional insecticidal protein (Cry2Ab). If an appropriate refuge is grown, there is no restriction on the area planted to Bollgard II. In 2004/05 and 2005/06 the Bollgard II acreage represented approximately 80 of the total area planted to cotton in Australia. The sensitivity of field-collected populations of H. armigera to Bt products was assayed before and subsequent to the widespread deployment of Ingard cotton. In 2002 screens against Cry2Ab were developed in preparation for replacement of Ingard with Bollgard II. There have been no reported field failures of Bollgard II due to resistance. However, while alleles that confer resistance to H. armigera in the field are rare for Cry1Ac, they are surprisingly common for Cry2Ab. We present an overview of the current approach adopted in Australia to monitor and adaptively manage resistance to Bt-cotton in field populations of H. armigera and discuss the implications of our findings to date. We also highlight future challenges for resistance management in Australia, many of which extend to other Bt-crop and pest systems.     

Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin

The use of combinations of Bacillus thuringiensis (Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species, Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.     

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.     

Helicoverpa armigera baseline susceptibility to Bacillus thuringiensis Cry toxins and resistance management for Bt cotton in India

Transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt), often referred to as Bt cotton, is widely grown in many countries. Bt cotton with a single cry1A gene and stacked also with cry2A gene has provided satisfactory protection against the damage by the lepidopteran bollworms, especially the cotton bollworm, Helicoverpa armigera (Hübner) which is considered as a key pest. The baseline susceptibility of the larvae of H. armigera to Cry1Ac and other toxins carried out in many countries has provided a basis for monitoring resistance. There is no evidence of development of field-level resistance in H. armigera leading to the failure of Bt cotton crop anywhere in the world, despite the fact that Bt cotton was grown on the largest ever area of 12.1 million hectares in 2006 and its cumulative cultivation over the last 11 years has surpassed the annual cotton area in the world. Nevertheless, the Bt resistance management has become a necessity to sustain Bt cotton and other transgenic crops in view of potential of the target insects to evolve Cry toxin resistance.     

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.