Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Universal Approach to Magnetic Second-Order Topological Insulator.

We propose a universal practical approach to realize magnetic second-order topological insulator (SOTI) materials, based on properly breaking the time reversal symmetry in conventional (first-order) topological insulators. The approach works for both three dimensions (3D) and two dimensions (2D), and is particularly suitable for 2D, where it can be achieved by coupling a quantum spin Hall insulator with a magnetic substrate. Using first-principles calculations, we predict bismuthene on EuO(111) surface as the first realistic system for a two-dimensional magnetic SOTI. We explicitly demonstrate the existence of the protected corner states. Benefitting from the large spin-orbit coupling and sizable magnetic proximity effect, these corner states are located in a boundary gap ∼83 meV, and hence can be readily probed in experiment. By controlling the magnetic phase transition, a topological phase transition between a first-order TI and a SOTI can be simultaneously achieved in the system. The effect of symmetry breaking, the connection with filling anomaly, and the experimental detection are discussed.

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control.

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP-47Aa, from an isolate of Pseudomonas mosselii. PIP-47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP-47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP-47Aa show significant protection from root damage by WCR. PIP-47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP-1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP-47Aa is a novel insecticidal protein for controlling the corn rootworm pests.

Quantum anomalous/valley Hall effect and tunable quantum state in hydrogenated arsenene decorated with a transition metal.

The quantum anomalous Hall (QAH) effect is superior to the quantum spin Hall (QSH) effect, which can avoid the inelastic scattering of two edge electrons located on one side of a topological nontrivial material, and thus it has attracted both theoretical and experimental interest. Here, we systematically investigate the lattice structures, and electronic and magnetic properties of hydrogenated arsenene decorated with certain transition metals (Cr, Mo and Cu) based on density-functional theory. A unique QAH effect in Mo@AsH is predicted, whose Chern number (C = 1) indicates only one chiral edge channel located on its one side. Then, we prove that this QAH effect realization is closely related with band inversion, which is the competitive result between its spin-orbit coupling (SOC) strength and exchange field. The quantum state of Mo@AsH can also be tuned by an external strain, similar to SOC, and it is noted that its increased topological gap of about 35 meV under 5.0% tensile strain, is large enough to realize the QAH effect at room-temperature. Additionally, the quantum valley Hall effect in Cu@AsH contributed by the inequality of AB sublattices is also found. Our results reveal the physical mechanism to realize the QAH effect in TM@AsH and provide a platform for electrically controllable topological states, which are highly desirable for nanoelectronics and spintronics.

Resistance of Trichoplusia ni populations selected by Bacillus thuringiensis sprays to cotton plants expressing pyramided Bacillus thuringiensis toxins Cry1Ac and Cry2Ab.

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.

Mis-spliced transcripts of nicotinic acetylcholine receptor alpha6 are associated with field evolved spinosad resistance in Plutella xylostella (L.).

The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR) and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxalpha6), at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxalpha6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxalpha6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species.

Structural journey of an insecticidal protein against western corn rootworm.

The broad adoption of transgenic crops has revolutionized agriculture. However, resistance to insecticidal proteins by agricultural pests poses a continuous challenge to maintaining crop productivity and new proteins are urgently needed to replace those utilized for existing transgenic traits. We identified an insecticidal membrane attack complex/perforin (MACPF) protein, Mpf2Ba1, with strong activity against the devastating coleopteran pest western corn rootworm (WCR) and a novel site of action. Using an integrative structural biology approach, we determined monomeric, pre-pore and pore structures, revealing changes between structural states at high resolution. We discovered an assembly inhibition mechanism, a molecular switch that activates pre-pore oligomerization upon gut fluid incubation and solved the highest resolution MACPF pore structure to-date. Our findings demonstrate not only the utility of Mpf2Ba1 in the development of biotechnology solutions for protecting maize from WCR to promote food security, but also uncover previously unknown mechanistic principles of bacterial MACPF assembly.

Initial detections and spread of invasive Spodoptera frugiperda in China and comparisons with other noctuid larvae in cornfields using molecular techniques.

The fall armyworm, Spodoptera frugiperda, is a species native to the Americas and has spread to many countries in Africa and Asia in recent years. Proactive actions for potential invasion of S. frugiperda to China coordinated by government agencies and agricultural extension systems resulted in timely detection in January 2019 in Yunnan province neighboring onto Myanmar. The extensive monitoring in southern provinces of China since February 2019 resulted in dynamic tracking of S. frugiperda spreading to 13 provincial regions in China within 4 months by May 10, 2019, which is crucial for timely management actions in the fields. The first detections of S. frugiperda (corn strain) in China were confirmed using cytochrome oxidase subunit 1 (CO1) and triosephosphate isomerase (Tpi) genes molecular marker method. In addition to S. frugiperda, larvae of three other noctuid species with similar morphological appearance (S. litura, S. exigua and Mythimna separata) can occur simultaneously and cause similar damage in cornfields in southern China. Thus, we can use both morphological and molecular marker methods to compare larval stages of four noctuid species. Further, we discuss the risk of potential spread of invasive S. frugiperda to other regions and impact on corn production in China.

Genetic mapping of Bt-toxin binding proteins in a Cry1A-toxin resistant strain of diamondback moth Plutella xylostella.

A major mechanism of resistance to Bacillus thuringiensis (Bt) toxins in Lepidoptera is a reduction of toxin binding to sites in the midgut membrane. Genetic studies of three different species have shown that mutations in a candidate Bt receptor, a 12-cadherin-domain protein, confer Cry1A toxin resistance. Despite a similar resistance profile in a fourth lepidopteran species, Plutella xylostella, we have previously shown that the cadherin orthologue maps to a different linkage group (LG8) than Cry1Ac resistance (LG22). Here we tested the hypothesis that mutations in other genes encoding candidate Bt-binding targets could be responsible for Bt resistance, by mapping eight aminopeptidases, an alkaline phosphatase (ALP), an intestinal mucin, and a P252 glycoprotein with respect to the 29 AFLP marked linkage groups in a P. xylostella cross segregating for Cry1Ac resistance. A homologue of the Caenorhabditis elegans Bt resistance gene bre-2 was also mapped. None of the genes analysed were on the same chromosome containing the Cry1Ac resistance locus, eliminating them as candidate resistance genes in the parental resistant strain SC1. Although this finding excludes cis-acting mutations in these genes as causing resistance in this strain, one or more of the expressed proteins may still bind Cry1Ac toxin, and post-translational modifications could affect this binding and thereby exert a trans-acting effect on resistance.

Insect resistance management in GM crops: past, present and future.

Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were first commercialized in 1996 amid concern from some scientists, regulators and environmentalists that the widespread use of Bt crops would inevitably lead to resistance and the loss of a 'public good,' specifically, the susceptibility of insect pests to Bt proteins. Eight years later, Bt corn and cotton have been grown on a cumulative area >80 million ha worldwide. Despite dire predictions to the contrary, resistance to a Bt crop has yet to be documented, suggesting that resistance management strategies have been effective thus far. However, current strategies to delay resistance remain far from ideal. Eight years without resistance provides a timely opportunity for researchers, regulators and industry to reassess the risk of resistance and the most effective strategies to preserve Bt and other novel insect-resistant crops in development.

Susceptible and mCry3A resistant corn rootworm larvae killed by a non-hemolytic Bacillus thuringiensis Cyt1Aa mutant.

The western corn rootworm (WCR) Diabrotica virgifera virgifera causes substantial damage in corn. Genetically modified (GM) plants expressing some Bacillus thuringiensis (Bt) insecticidal Cry proteins efficiently controlled this pest. However, changes in WCR susceptibility to these Bt traits have evolved and identification of insecticidal proteins with different modes of action against WCR is necessary. We show here for the first time that Cyt1Aa from Bt exhibits toxicity against WCR besides to the dipteran Aedes aegypti larvae. Cyt1Aa is a pore-forming toxin that shows no cross-resistance with mosquitocidal Cry toxins. We characterized different mutations in helix α-A from Cyt1Aa. Two mutants (A61C and A59C) exhibited reduced or absent hemolytic activity but retained toxicity to A. aegypti larvae, suggesting that insecticidal and hemolytic activities of Cyt1Aa are independent activities. These mutants were still able to form oligomers in synthetic lipid vesicles and to synergize Cry11Aa toxicity. Remarkably, mutant A61C showed a five-fold increase insecticidal activity against mosquito and almost 11-fold higher activity against WCR. Cyt1Aa A61C mutant was as potent in killing WCR that were selected for resistance to mCry3A as it was against unselected WCR indicating that this toxin could be a useful resistance management option in the control of WCR.

Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution.

Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture. Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics. To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella. We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively. After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence. These results have important implications for the development and regulation of transgenic insecticidal plants.

An Alcaligenes strain emulates Bacillus thuringiensis producing a binary protein that kills corn rootworm through a mechanism similar to Cry34Ab1/Cry35Ab1.

Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.

Evaluation of insecticides and application methods against Contarinia nasturtii (Diptera: Cecidomyiidae), a new invasive insect pest in the United States.

The midge Contarinia nasturtii (Keiffer), a serious gall-forming insect pest of cruciferous plants in Europe and southwestern Asia, was first reported in the United States in summer 2004. It had not been recorded in North America until its discovery in Ontario, Canada, in 2000. Efficacy of 20 insecticides belonging to 12 different classes was evaluated by using a foliar spray, soil drench, or seed treatment method. The broccoli cultivar 'Packman' was used in all tests at the suitable stage of four to five true leaves. Results indicated that foliar sprays of lambda-cyhalothrin, acephate, acetamiprid, chlorpyrifos, and methomyl reduced C. nasturtii larval populations by 96.7-100%. Except for acetamiprid, the other four insecticides also were effective against adults and provided 100% mortality after 24 h. When applied by drench, acetamiprid, imidacloprid, and thiamethoxam provided 100% control of C. nasturtii larvae, and the duration of efficacy lasted at least 7 wk. When applied as seed treatment, clothianidin and thiamethoxam provided 100% control of larvae and did not significantly affect seed germination. Imidacloprid also provided 100% control but the percentage of germination after treatment was only 62% (96.9% in check). These results indicate that several insecticides may significantly reduce midge populations. The nicotinoid class of insecticides, which has strong systemic activity, is likely to be the first choice. It is necessary to explore and develop other control methods such as cultural control and host resistance to develop an effective integrated pest management system.

Onion maggot (Diptera: Anthomyiidae) resistance to chlorpyrifos in New York onion fields.

A larval immersion bioassay was developed to identify susceptibility of onion maggot, Delia antiqua (Meigen) (Diptera: Anthomyiidae), to chlorpyrifos and to determine whether this assay could be used to predict control in onion fields. Laboratory colonies were established from larvae collected in New York onion fields during 2003 and 2004, providing us with test insects to use in bioassays. The larval assay effectively determined susceptibility of D. antiqua to chlorpyrifos, and results were congruent with an adult bioassay. However, use of similar-aged larvae (4 d old) in the assays was critical because larvae became more tolerant to chlorpyrifos as they aged. In a field survey, six of the 13 populations had LC50 values above the recommended field rate of 3,600 ppm (range 4,031-6,869). Over two successive seasons in the same field, susceptibility of D. antiqua to chlorpyrifos decreased in two of three fields (by 45 and 42%) and remained the same in another field, indicating that resistance is not predictable from year to year. Based on the relationship between damage in the field and LC50 values from 11 of the populations mentioned above, all five populations that had LC50 values above the field rate caused unacceptable levels of damage, whereas five of six populations that had LC50 values below the field rate did not cause serious damage.

mCry3A-Selected Western Corn Rootworm (Coleoptera: Chrysomelidae) Colony Exhibits High Resistance and Has Reduced Binding of mCry3A to Midgut Tissue.

Several Bt maize events expressing various insecticidal Cry protein genes have been commercialized for management of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). We used high efficacy (>99.7%) experimental maize events that express mCry3A for selections under laboratory conditions to develop a western corn rootworm colony resistant to mCry3A at higher levels than published results. The resistance ratio (RR) to mCry3A was >97-fold based on LC 50 values in diet-based bioassays after six generations of selections when compared to that of an unselected Control colony. Using a sublethal seedling assay (SSA) method, we confirmed that the colony had no cross-resistance to maize event DAS-59122-7, which expresses Cry34/35Ab. Reciprocal crosses between the mCry3A-resistant colony and the susceptible colony were performed to test the inheritance of resistance. Larval survival and development evaluated by the SSA method indicated that resistance to mCry3A was inherited autosomally and was incompletely recessive (h = 0.23-0.25). Specific binding of mCry3A to brush border membrane vesicles of midgut tissue revealed reduced binding in the resistant colony when compared to binding in the susceptible colony. This is the first report where resistance in western corn rootworm has been shown to involve reduced binding of a Cry3-class protein in midgut tissue.

Discovery of midgut genes for the RNA interference control of corn rootworm.

RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality.

A selective insecticidal protein from Pseudomonas for controlling corn rootworms.

The coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.

Evaluation of a chemically inducible promoter for developing a within-plant refuge for resistance management.

Chemically inducible production of Bacillus thuringiensis (Bt) toxins in transgenic plants may provide considerable benefits in preventing or delaying the evolution of insect resistance to Bt crops by creating within-plant temporal refuges. We examined the effect of inducible cry1Ab expression on survival of different genotypes (RR, RS, and SS) of diamondback moth, Plutella xylostella (L.), in transgenic broccoli, Brassica oleracea L., plants transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-s-methyl [= benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] (ASM) resulted in high levels of Bt toxin, and detached leaves from fully induced plants caused 100% mortality to all instars of P. xylostella SS and RS genotypes. When plants infested with larvae were treated with ASM, only a few larvae that were nearing completion of their development were able to survive the induction process. Signal transduction from ASM-treated leaves to new plant tissue also was evaluated using a larval assay. New foliage that emerged after plants were induced remained toxic to > or = 80% of RS larvae up to the fourth new leaf. In whole plant tests, however, induced plants remained protected from larval damage for > or = 3 wk. Uninduced PR-1a/cry1Ab plants seemed to produce low levels of Bt that were undetected by an enzyme-linked immunosorbent assay but that resulted in significant fitness costs for susceptible insects. The suitability of PR-1a/cry1Ab broccoli plants for insect resistance management and the requirements of an appropriate inducible promoter are discussed.

Selection and heritability of resistance to Bacillus thuringiensis subsp kurstaki and transgenic cotton in Helicoverpa armigera (Lepidoptera: Noctuidae).

Compared with an unselected susceptible population, a cotton bollworm, Helicoverpa armigera (Hübner), population selected for 22 generations with transgenic cotton leaves (modified Cry1A) in the laboratory developed 11.0-fold resistance to Cry1Ac (one single-protein product MVPII). Resistance to Bacillus thuringiensis Berliner subsp kurstaki (Btk) was selected for 22 generations with a 5.2-fold increase in LC50. The estimated realized heritabilities (h2) of resistance for transgenic-cotton- and Btk-selected populations were 0.1008 and 0.2341, respectively. This reflects the higher phenotypic variation in response to Cry1Ac in the transgenic-cotton-selected population. This variation may have been caused by differences in protein toxin levels expressed in different growth stages of the transgenic cotton. Because of the different slopes of the probit regression lines between Cry1Ac and Btk, the estimated realized h2 cannot be used visually to compare resistance development to Cry1Ac and Btk in H armigera. Thus, the response quotient (Q) of resistance was also estimated. The Q values of resistance for transgenic-cotton- and Btk-selected populations were 0.0763 and 0.0836, respectively. This showed that the rate of resistance development would be similar in both selection populations. This result indicates that the selection of resistance using transgenic cotton is different from that selected using the single toxin. Resistance risk to transgenic cotton and Btk in field populations was assessed assuming different pressures of selection by using the estimated h2. Assuming the h2 of resistance in a field population was half of the estimated h2, and the population received prolonged and uniform exposure to transgenic cotton or Btk causing >70% mortality in each generation, we predicted that resistance would increase 10-fold after <23 generations for Cry1Ac in transgenic cotton-selected-populations and after <21 generations for Btk in Btk-selected populations. Cross-resistance would be expected after <48 generations for Btk in transgenic-cotton-selected populations and after <21 generations for Cry1Ac in Btk-selected population. The results show that the potential to evolve resistance is similar in both transgenic-cotton- and Btk-selected populations, but that cross-resistance development to Btk is slower in transgenic-cotton-selected populations than cross-resistance development to Cry1Ac in Btk-selected populations.