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Summary 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.

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.

Soil microbes yield insecticidal peptide The microbial peptide BT, derived from the bacterium Bacillus thuringiensis , is widely used to protect crops from insect pests. Schellenberger et al. identified another insecticidal peptide from a different soil-dwelling bacterium, Pseudomonas chlororaphis (see the Perspective by Tabashnik). Corn plants expressing the Pseudomonas peptide were protected from attack by western corn rootworm. Rootworms that were resistant to BT were susceptible to the Pseudomonas peptide. Addition of another insecticidal peptide diversifies the arsenal against insect pests, which may slow down the development of resistance. Science , this issue p. 634 ; see also p. 552

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 LC50 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.

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.

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.

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.

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.

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.

BACKGROUND: Developing scientifically valid, economically acceptable insecticide resistance management (IRM) programs is critical for sustainable insect management. The diamondback moth, Plutella xylostella (L.), has demonstrated an ability to developresistancetomanydifferentclassesofinsecticides,includingproteinsproducedbythebacteriumBacillusthuringiensis Berliner (Bt). Recently it has developed resistance to the novel compounds spinosad and indoxacarb. In greenhouse cage experiments, a laboratory-selected population of P. xylostella resistant to spinosad, indoxacarb and Bt was used to compare population growth and resistance evolution if these three insecticides were rotated or used in a mosaic fashion. RESULTS: The average population density through nine generations was lowest in the treatment in which the insecticide was rotated every generation (R-1) (x = 20.7 ± 3.20) compared with the treatment in which the insecticide was rotated every third generation (R-3) (x = 41.4 ± 17.6) or where the insecticides were applied as a mosaic (M) (x = 41.8 ± 6.53). After nine generations, the survival of resistant individuals increased for each insecticide (7.2‐73.5%) compared with the population without selection (CK) (0.73‐3.1%). Survival on spinosad was significantly lower (23.7%) in the single-generation rotation than for the other two treatments, both of which exceeded 72%. The calculated survival on all three insecticides treated simultaneously, according to the survival on each insecticide, was 0.26, 0.81 and 1.6% for R-1, R-3 and M treatments respectively. CONCLUSION: Results of both population density and resistance development indicated that insecticide rotation every generationwasbetterforIRMthaniftheinsecticidewasrotatedeverythirdgenerationorifthethreeinsecticideswereapplied as a mosaic. c ! 2010 Society of Chemical Industry

Advances in transgenic plants expressing Bacillus thuringiensis (Bt) insecticidal gene(s) offer a promising alternative to traditional insecticides for control of lepidopteran pests on important cruciferous vegetable crops such as cabbage and cauliflower. A public-private partnership, the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA), was formed in 2005 with the goal of developing dual-gene Bt cauliflower and cabbage, initially for India, to replace the use of broad spectrum, traditional insecticides. As a first step in this effort, the major lepidopteran pests of cruciferous vegetable crops [Plutella xylostella (L.), Pieris rapae (L.), Pieris brassicae (L.), Crocidolomia binotalis (L.), Hellula undalis (F.), Diacrisia obliqua Walker, Spodoptera litura F., and Helicoverpa armigera (Hubner)] were collected over a large geographic area (India, Indonesia, Taiwan, China, Australia, and the United States) and tested against purified Cry1Ba2 and Cry1Ca4 toxins, the toxins proposed to be expressed in the CIMBAA plants. Our results demonstrate that Cry1Ba2 and Cry1Ca4 were effective against the primary target of the CIMBAA plants, P. xylostella, regardless of geographic location, and had LC50 values

Several types of trap crops have been recommended for managing the diamondback moth, Plutella xylostella , including collards ( Brassica oleracea var. acephala ) and Indian mustard ( Brassica juncea L.). However, results have been variable perhaps because populations of P. xylostella develop on these trap crops and spill over to the cash crop. To overcome this problem, we sought to develop “dead-end” trap crops that were more attractive for oviposition than the cash crop but on which P. xylostella larvae cannot survive. We have produced Bacillus thuringiensis (Bt)-transgenic collard and Indian mustard lines with a cry1C gene that have the potential to be used as a “dead-end” trap crop for P. xylostella . Greenhouse and small cage studies confirmed the control of P. xylostella larvae on the Bt crops. Furthermore, Indian mustard was significantly preferred over cabbage and collards for oviposition, regardless of whether the Indian mustard was Bt or non-Bt. The use of Bt Indian mustard as a trap crop significantly reduced the number of larvae that appeared on a cabbage cash crop, compared with using a non-Bt Indian mustard trap crop. However, this reduction also occurred when using Bt collards as a trap crop, despite collards being less preferred for oviposition. In fact, despite the overall increase in oviposition caused by the presence of Indian mustard compared with collards, the use of either Bt Indian mustard or Bt collards provided the same level of protection to the cash crop. Both plants also resulted in significant suppression of a P. xylostella population over 3 generations in the greenhouse test and 2 generations in the small cage experiment, suggesting that in places where immigration may be limited some long-term population suppression may occur. We suggest that Bt trap crops may be useful tools in situations where the cash crop may not be suitable or desirable for genetic engineering.

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.

Swede midge (Contarinia nasturtii Keiffer) is a serious gall-forming insect pest of cruciferous plants in Europe and southwestern Asia. In North America, it was first identified in Ontario, Canada, in 2000 and in Niagara County, New York, US, in 2004. The insect is now rapidly spreading in Canada and the US. To date, infestation of C. nasturtii has been confirmed in 46 counties in Canada. The US Department of Agriculture has confirmed the presence of C. nasturtii in 6 counties in the US, although our surveys using pheromone traps have identified 19 counties in New York and one county in Massachusetts and New Jersey. Currently, hundreds of farms in the northeastern US that grow cruciferous vegetables are at risk for infestation of C. nasturtii through movement of vegetable seedlings and harvested produce from infested areas. To prevent the spread of C. nasturtii, the efficacy of acetamiprid, the first labeled insecticide in the US for C. nasturtii control, was evaluated by using foliar sprays on cauliflower seedlings. Results indicated the efficacy of acetamiprid on C. nasturtii was 99.52% 100% and 99.83% when cauliflower seedlings were sprayed before inoculation with C. nasturtii, 0 and 4 d after inoculation, respectively. The efficacy of acetamiprid was reduced to 69.89% when seedlings were sprayed 8 d after inoculation, and C. nasturtii larvae could successfully pupate and emerge after the spray. These results indicate that acetamiprid can effectively control C. nasturtii on cauliflower seedlings, especially in the early stage of insect occurrence. Based on these results, we suggest that seedlings be treated with acetamiprid as a foliar spray before shipment of seedlings from C. nasturtii infested areas.

The cabbage looper, Trichoplusia ni , is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni . This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut.

Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1 . Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori . The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis , the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper ( Trichoplusia ni ) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.

The thermoelasticity of rocksalt (RS) structure MgO is investigated by ab initio plane-wave pseudopotential density functional theory method and the quasi-harmonic Debye model. The obtained lattice constant, aggregate elastic moduli, pressure derivative of isothermal bulk modulus and elastic constants are well consistent with the experimental data and other theoretical results. Moreover, the pressure dependences of individual elastic modulus at 300 and 800 K are also discussed. It is shown that, at 300 K, the Debye temperatures from our obtained elastic constants are well consistent with other theoretical results. The Debye temperature increases nonlinearly with increasing pressure.

The cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), is an important cotton pest in northern China, especially in the seedling stage of cotton. After large scale commercial use of transgenic Bt cotton, cotton aphids became one of the most important cotton pests. A 2-year study was conducted to evaluate the role of four winter wheat varieties that were resistant or susceptible to wheat aphid, Sitobion avenae Fabricius (Homoptera: Aphididae), in conserving arthropod natural enemies and suppressing cotton aphids in a wheat-cotton relay intercropping system in northern China. The results indicated that wheat-cotton intercropping preserved and augmented natural enemies more than a monoculture of cotton. The density of natural enemies in cotton was significantly different among relay-intercropping fields with different wheat varieties. The highest density of natural enemies and low cotton aphid populations were found in the treatment of cotton in relay intercropped with the wheat variety Lovrin10, which is susceptible to wheat aphid. The lowest density of predators and parasitoids associated with high cotton aphid populations were found with the wheat variety KOK1679, which is resistant to wheat aphid. The results showed that wheat varieties that are susceptible or moderately resistant to wheat aphid might reduce cotton aphids more effectively than an aphid-resistant variety in the intercropping system by enhancing predators to suppress cotton aphids during the cotton seedling stage.

Progress on the research and development of insect-resistant transgenic rice, especially expressing insecticidal proteins from Bacillus thuringiensis (Bt), in China has been rapid in recent years. A number of insect-resistant transgenic rice lines/varieties have passed restricted and enlarged field testing, and several have been approved for productive testing since 2002 in China, although none was approved for commercial use until 2006. Extensive laboratory and field trials have been conducted for evaluation of the efficiency of transgenic rice on target lepidoteran pests and potential ecological risks on non-target arthropods. The efficacy of a number of transgenic rice lines currently tested in China was excellent for control of the major target insect pests, the rice stem borers (Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens) and leaffolder (Cnaphalocrocis medinalis), and was better than most insecticides extensively used by millions of farmers at present in China. No significantly negative or unintended effects of transgenic rice on non-target arthropods were found compared with non-transgenic rice. In contrast, most of the current insecticides used for the control of rice stem borers and leaffolders proved harmful to natural enemies, and some insecticides may directly induce resurgence of rice planthoppers. Studies for developing a proactive insect resistance management of transgenic rice in the future are discussed to ensure the sustainable use of transgenic rice.

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 λ-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, clot...

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2-3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.

Microplitis mediator (Haliday) is an important endoparasitoid of the cotton bollworm, Helicoverpa armigera (Hubner) in northern China. Interactions among H. armigera, its larval parasitoid M. mediator, and insect–resistant transgenic cotton lines were evaluated under laboratory conditions. Two major transgenic cotton cultivars used in Hebei province of northern China, DP99B (Bollgard), carrying the cry1Ac gene, and SGK321, carrying both cry1A and CpTI (Cowpea trypsin inhibitor) genes, were used in the experiments. The results indicated that there was significant growth inhibition of the H. armigera larvae when they were fed a diet containing Bt transgenic cotton powder. The parasitoid offspring developed more slowly and pupal and adult weight was reduced significantly when the parasitized host larvae fed on the Bt cotton powder leaf diet compared with non-Bt treatment. With an increase of Bt cotton leaf powder concentration in the host larvae diet, the parasitism rate and adult emergence of the parasitoid decreased and the abnormal pupal rate increased. There was no evident difference in the effects on M. mediator between the transgenic single- and two-gene cotton cultivars; however, the parasitized host larval mortality was higher than that of unparasitized larvae in most treatments. The observed effects on M. mediator were probably host-quality mediated rather than direct effects of transgenic cotton because the H. armigera larvae which fed on diet with leaf powder of both transgenic cotton cultivars also experienced a significant decrease in weight, particularly when the host larvae were parasitized.

The transition phase of GaN from zincblende (ZB) structure to rocksalt structure (RS) is investigated by ab initio plane-wave pseudopotential density functional theory method, and the thermodynamic properties of the ZB and RS structures are obtained through the quasi-harmonic Debye model. We find that the transition phase from the ZB structure to the RS structure occurs at the pressure of 42.2 GPa, which is in good agreement with other calculated values. Moreover, the dependences of the relative volume V/V-0 on the pressure P, the Debye temperature Theta and heat capacity C-V on the pressure P, as well as the heat capacity C-V on the temperature T are also successfully obtained. (C) 2005 Elsevier Ltd. All rights reserved.

Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were grown on over 13 million ha in the United States and 22.4 million ha worldwide in 2004. Preventing or slowing the evolution of resistance by insects (“resistance management”) is critical for the sustainable use of Bt crops. Plants containing two dissimilar Bt toxin genes in the same plant (“pyramided”) have the potential to delay insect resistance. However, the advantage of pyramided Bt plants for resistance management may be compromised if they share similar toxins with single-gene plants that are deployed simultaneously. We tested this hypothesis using a unique model system composed of broccoli plants transformed to express different Cry toxins (Cry1Ac, Cry1C, or both) and a synthetic population of the diamondback moth ( Plutella xylostella ) carrying genes for resistance to Cry1Ac and Cry1C at frequencies of ≈0.10 and 0.34, respectively. After 24–26 generations of selection in the greenhouse, the concurrent use of one- and two-gene plants resulted in control failure of both types of Bt plants. When only two-gene plants were used in the selection, no or few insects survived on one- or two-gene Bt plants, indicating that concurrent use of transgenic plants expressing a single and two Bt genes will select for resistance to two-gene plants more rapidly than the use of two-gene plants alone. The results of this experiment agree with the predictions of a Mendelian deterministic simulation model and have important implications for the regulation and deployment of pyramided Bt plants.

Insecticidal toxins from Bacillus thuringiensis (Bt) are widely used to control pest insects, but evolution of resistance threatens their continued efficacy. The most common type of Bt resistance ('Mode 1') is characterized by recessive inheritance, > 500-fold resistance to at least one Cry1A toxin, negligible cross-resistance to Cry1C, and reduced binding of Bt toxins to midgut membrane target sites. Mutations affecting a Cry1A-binding midgut cadherin protein are linked to laboratory-selected Mode 1 resistance in Heliothis virescens and Pectinophora gossypiella. Here we show that field-evolved Mode 1 resistance in the diamondback moth, Plutella xylostella, has a different genetic basis, indicating that screening for resistance in the field should not be restricted to a previously proposed DNA-based search for cadherin mutations.

Interactions between the cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), its larval parasitoid Microplitis mediator (Haliday) (Hymenoptera: Braconidae), and the CrylAc toxin of Bacillus thuringiensis Berliner were evaluated under laboratory conditions. The growth of H. armigera larvae was delayed and its pupal rate and pupal weight decreased when they were fed on a diet containing CrylAc toxin. Due to the lowered growth rate of the host larvae, the time available for parasitization of H. armigera by M. mediator increased when the host larvae were reared on a diet containing CrylAc toxin at concentrations of 0.5, 1, 2, and 4 μg g -1 . The longevity of female and male parasitoids was not significantly affected when newly emerging wasps fed on honey solutions containing three different concentrations of CrylAc toxin (125, 250, and 500 μg ml -1 ). When female parasitoids were fed on honey solutions containing CrylAc, their offsprings' egg and larval development period, pupal weight, length of pupation, adult weight, and adult longevity did not change significantly in most of the treatments compared with controls. When the female parasitoids parasitized host larvae that had been fed on a diet containing 0.5, 1, 2, 4, and 8 μg g -1 CrylAc toxin, their offsprings' eggs and larvae were significantly delayed. Their pupal weight, adult weight, and adult longevity were also significantly less than controls.

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.

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F 1 reciprocal crosses and backcrosses between F 1 larvae and resistant (P R ) and susceptible (P S ) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F 1 crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F 1 larvae was slightly higher than that for P S , suggesting that resistance is partially recessive. The responses of both backcross progeny (F 1 × P R , F 1 × P S ) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F 1 × P R backcross but decreased the correspondence with the F 1 × P S backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.

Compared with an unselected susceptible population, a cotton bollworm, Helicoverpa armigera (Hubner), 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

Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) grew on >62 million ha worldwide from 1996 to 2002. Despite expectations that pests would rapidly evolve resistance to such Bt crops, increases in the frequency of resistance caused by exposure to Bt crops in the field have not yet been documented. In laboratory and greenhouse tests, however, at least seven resistant laboratory strains of three pests (Plutella xylostella [L.], Pectinophora gossypiella [Saunders], and Helicoverpa armigera [Hubner]) have completed development on Bt crops. In contrast, several other laboratory strains with 70- to 10,100-fold resistance to Bt toxins in diet did not survive on Bt crops. Monitoring of field populations in regions with high adoption of Bt crops has not yet detected increases in resistance frequency. Resistance monitoring examples include Ostrinia nubilalis (Hubner) in the United States (6 yr), P. gossypiella in Arizona (5 yr), H. armigera in northern China (...

This study was undertaken to determine the effects of pyramiding two Bacillus thuringiensis (Bt) genes in the same plant on the production of Bt proteins and the control of diamondback moths (DBM, Plutella xylostella) resistant to one or the other protein. Broccoli lines carrying both cry1Ac and cry1C Bt genes were produced by sexual crosses of cry1Ac- and cry1C-transgenic plants. Plants containing both genes were selected by tests for resistance to kanamycin and hygromycin, and confirmed by PCR analysis for the Bt genes. Both cry1Ac and cry1C mRNAs were detected in the hybrid lines, and Cry1Ac and Cry1C proteins were stably produced at levels comparable to the parental plants. Plants producing both Cry1Ac and Cry1C proteins caused rapid and complete mortality of DBM larvae resistant to Cry1A or Cry1C, and suffered little or no leaf damage. These plants, in combination with the resistant DBM populations available, will allow greenhouse or field studies of resistance management strategies involving gene pyramiding.

A synthetic laboratory population of the diamondback moth, Plutella xylostella (L.), was used to test the F2 screen developed for detecting the frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bacillus thuringiensis (Bt). Of the 120 single-pair matings set up, 106 produced enough F2 families for screening of Cry1Ac or Cry1C resistance alleles using both transgenic broccoli and an artificial diet overlay assay with a diagnostic dose. When using Bt broccoli plants as the F2 screen method, only one F2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C resistance using the diet assay. The survivors in the diagnostic diet assay were crossed with the resistant individuals to confirm their resistance genotypes. Four F2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single-pairs and four other F2 families contained an allele resistant to Cry1C. Our results suggest that using transgenic plants expressing a high level of a Bt toxin in an F2 screen may underestimate the frequency of resistance alleles with high false negatives, or fail to detect true resistance alleles. The diagnostic diet assay was a better F2 screen method to detect alleles, especially for the Cry1Ac resistance with monogenic inheritance in the diamondback moth. The estimated probabilities of false positives and false negatives were 33 and 1%, respectively, for detecting Cry1Ac resistance at the allele frequency of 0.012 using the diagnostic diet assay. Careful validation of the screening method for each insect-crop system is necessary before the F2 screen can be used to detect rare Bt resistance alleles in field populations.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (RR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.

A resistant strain of Helicoverpa armigera selected by cyhalothrin was used to investigate the relative importance of knockdown resistance ( kdr ) for different levels of resistance to cyhalothrin and fenvalerate and the inheritance of kdr to fenvalerate. Pretreatment of piperonyl butoxide (PBO) decreased the resistance ratios from 30.6- to 5.0-fold for cyhalothrin and from 337.0- to 38.0-fold for fenvalerate. Electrophysiological studies showed that kdr ratios were 10.7- and 95.8-fold to cyhalothrin and fenvalerate, repectively, at EC 50 of burst discharge of neuromuscle preparation, and were 15.0- and 507.2-fold to cyhalothrin and fenvalerate, respectively, at EC 50 of blocked neuromuscular transmission. It was indicated that the difference in kdr to fenvalerate and cyhalothrin is the main reason for the difference in resistance level between the two pyrethroids in the resistant strain of H. armigera. Analysis of probit lines from F 1 reciprocal crosses indicated that kdr to fenvalerate was inherited autosomaly as an incompletely recessive factor. The degrees of dominance were −0.74 and −0.78, based on burst discharge and blocked transmission, respectively, with pooled F 1 generations. χ 2 analysis of knockdown responses of backcrossed offspring indicated that the resistance might be controlled by one major gene. Cross-resistance to aconitine was detected in resistant strain after applying PBO, indicating that sodium channels are different in the resistant and susceptible strains of H. armigera.

Topical application, leaf residue, and filter paper residue bioassay methods were used to assess the toxicity of permethrin and methomyl to larvae of the diamondback moth, Plutella xylostella (L.). The objective was to develop an on-farm insecticide resistance monitoring technique for diamondback moth that required only simple, inexpensive equipment and was easy to use. The results of tests using the three bioassay methods led to similar resistance ratios for three diamondback moth strains. Leaf residue and filter paper residue tests using discriminating concentrations could detect the relative susceptibility to permethrin and methomyl in three strains, indicating that both techniques were practical for on-farm resistance monitoring. Third instars were more suitable for the detection of resistance than fourth instars. A monitoring system incorporating concentrations f both the LC90 for susceptible diamondback moth and the recommended field concentration of insecticide is suggested to detect resistance at low levels and to help choose insecticides for the control of diamondback moth strains with higher resistance levels.

Developing scientifically valid, economically acceptable insecticide resistance management (IRM) programs is critical for sustainable insect management. The diamondback moth, Plutella xylostella (L.), has demonstrated an ability to develop resistance to many different classes of insecticides, including proteins produced by the bacterium Bacillus thuringiensis Berliner (Bt). Recently it has developed resistance to the novel compounds spinosad and indoxacarb. In greenhouse cage experiments, a laboratory-selected population of P. xylostella resistant to spinosad, indoxacarb and Bt was used to compare population growth and resistance evolution if these three insecticides were rotated or used in a mosaic fashion.The average population density through nine generations was lowest in the treatment in which the insecticide was rotated every generation (R-1) (x = 20.7 ± 3.20) compared with the treatment in which the insecticide was rotated every third generation (R-3) (x = 41.4 ± 17.6) or where the insecticides were applied as a mosaic (M) (x = 41.8 ± 6.53). After nine generations, the survival of resistant individuals increased for each insecticide (7.2-73.5%) compared with the population without selection (CK) (0.73-3.1%). Survival on spinosad was significantly lower (23.7%) in the single-generation rotation than for the other two treatments, both of which exceeded 72%. The calculated survival on all three insecticides treated simultaneously, according to the survival on each insecticide, was 0.26, 0.81 and 1.6% for R-1, R-3 and M treatments respectively.Results of both population density and resistance development indicated that insecticide rotation every generation was better for IRM than if the insecticide was rotated every third generation or if the three insecticides were applied as a mosaic.

Transgenic cotton that has been engineered to produce insecticidal toxins from Bacillus thuringiensis (Bt) and so to resist the pest cotton bollworm ( Helicoverpa armigera ) has been widely planted in Asia. Analysis of the population dynamics of H. armigera from 1992 to 2007 in China indicated that a marked decrease in regional outbreaks of this pest in multiple crops was associated with the planting of Bt cotton. The study area included six provinces in northern China with an annual total of 3 million hectares of cotton and 22 million hectares of other crops (corn, peanuts, soybeans, and vegetables) grown by more than 10 million resource-poor farmers. Our data suggest that Bt cotton not only controls H. armigera on transgenic cotton designed to resist this pest but also may reduce its presence on other host crops and may decrease the need for insecticide sprays in general.

Transgenic brassica crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are being investigated as candidates for field release to control lepidopteran pests. Information on the potential impact of Bt brassica crops on pests and non-target natural enemies is needed as part of an environmental risk assessment prior to the commercial release. This first tier study provides insight into the tritrophic interactions among Bt broccoli plants, the herbivore Pieris rapae and its parasitoid Pteromalus puparum. We first evaluated the efficacy of three types of Bt broccoli plants, cry1Ac, cry1C and cry1Ac + cry1C, on different instars of P. rapae. Bt broccoli effectively controlled P. rapae larvae, although later instars were more tolerant. The efficacy of different Bt broccoli plants on P. rapae larvae was consistently cry1Ac > cry1Ac + cry1C > cry1C. When the parasitoid P. puparum developed in a P. rapae pupa (host) that had developed from Bt plant-fed older larvae, developmental time, total number and longevity of the P. puparum generated from the Bt plant-fed host were significantly affected compared with those generated from the non-Bt control plant-fed host. Simultaneously, negative effects on P. rapae pupae were found, i.e. pupal length, width and weight were significantly reduced after older P. rapae larvae fed on different Bt plants for 1 or 2 days. Cry1C toxin was detected using ELISA in P. rapae pupae after older larvae fed on cry1C broccoli. However, no Cry1C toxin was detected in newly emerged P. puparum adults developing in Bt-fed hosts. Only a trace amount of toxin was detected from entire P. puparum pupae dissected from the Bt plant-fed host. Moreover, no negative effect was found on the progeny of P. puparum developing from the Bt plant-fed host when subsequently supplied with a healthy host, P. rapae pupae. The reduced quality of the host appears to be the only reason for the observed deleterious effects on P. puparum. Our data suggest that the effects on P. puparum developing in Bt plant-fed P. rapae are mediated by host quality rather than by direct toxicity.