In silico designing of insecticidal small interfering RNA (siRNA) for Helicoverpa armigera control

Helicoverpa armigera, a polyphagous lepidopteron insect pest causes severe yield loss in cotton, legumes, tomato, okra and other crops. Application of chemical pesticides although effective, has human health and environmental safety concerns. Moreover, development of resistance against most of the available pesticides is compelling to look for alternative strategies. Adoption of Bt transgenic crops have resulted in reduction in pesticide consumption and increasing crop productivity. However, sustainability of Bt transgenic crops is threatened by the emergence of insect resistance. In the present study potential insecticidal siRNA were identified in six H. armigera horrhonal pathway genes. Out of over 2000 computationally identified siRNA, 16 most promising siRNA were selected that address the biosafety concerns and have high potential of targeted gene silencing. These siRNA will be useful for chemical synthesis, in insect feeding assays and knockdown the target H. armigera hormone biosynthesis, consequently obstructing the completion of insect life cycle. The siRNA have a great potential of deployment to control H. annrmigera alone as well as with Bt for insect resistance management.     

Two-toxin strategies for management of insecticidal transgenic crops: can pyramiding succeed where pesticide mixtures have not?

Transgenic insect-resistant crops that express toxins from Bacillus thuringiensis (Bt) offer significant advantages to pest management, but are at risk of losing these advantages to the evolution of resistance in the targeted insect pests. All commercially available cultivars of these crops carry only a single Bt gene, and are particularly at risk where the targeted insect pests are not highly sensitive to the Bt toxin used. Under such circumstances, the most prudent method of avoiding resistance is to ensure that a large proportion of the pest population develops on non-transgenic ''refuge'' hosts, generally of the crop itself. This has generated recommendations that 20% or more of the cotton and maize in any given area should be non-transgenic. This may be costly in terms of yields and may encourage further reliance on and resistance to pesticides. The use of two or more toxins in the same variety (pyramiding) can reduce the amount of refuge required to delay resistance for an extended period. Cross-resistance among the toxins appears to have been overestimated as a potential risk to the use of pyramids (and pesticide mixtures) because cross-resistance is at least as important when toxicants are used independently. Far more critical is that there should be nearly 100% mortality of susceptible insects on the transgenic crops. The past failures of pesticide mixtures to manage resistance provide important lessons for the most efficacious deployment of multiple toxins in transgenic crops.     

Structure of Cry2Aa suggests an unexpected receptor binding epitope

BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.     

Regulation of Bt crops in Canada

The Canadian Food Inspection Agency (CFIA) regulates environmental releases of plants with novel traits, which include transgenic plants such as Bt crops. Bt crops are regulated in Canada because they express insect resistance novel to their species. Commercialization of crops with novel traits such as the production of insecticidal Bt proteins requires an approval for environmental release, as well as approvals for use as feed and food. Environmental factors such as potential impacts on non-target species are considered. Insect resistance management (IRM) may be imposed as a condition for environmental release of Bt crops to delay the development of resistance in the target insect. Bt potato and European corn borer-resistant Bt corn have been released with mandatory IRM. The CFIA imposes an IRM plan consisting of appropriate refugia, education of farmers and seed dealers, and monitoring and mitigation. Industry, regulators, government extension staff and public researchers provide expert advice on IRM.     

Sustainability of insect resistance management strategies for transgenic Bt corn

Increasing interest in the responsible management of technology in the industrial and agricultural sectors of the economy has been met thorough the development of broadly applicable tools to assess the "sustainability" of new technologies. An arena ripe for application of such analysis is the deployment of transgenic crops. The new transgenic pesticidal or plant-incorporated protectant (PIP) crops have seen widespread application in the United States based on the features of higher yield, lower applications of insecticides, and control of mycotoxin content. However, open rejection of these new crops in Europe and in other countries has been a surprising message and has limited their worldwide acceptance. The US Environmental Protection Agency's (USEPA) Office of Pesticide Programs (OPP) has worked on the development and analysis of insect resistance management (IRM) strategies and has mandated specific IRM requirements for Bacillus thuringiensis (Bt) crops since 1995 under the Food, Fungicide, Insecticide, and Rodenticide Act. Improvement of data quality and sustainability of IRM strategies have been targeted in an ongoing partnership between the USEPA Office of Research and Development and the Office of Pesticide Programs that will further enhance the agency's ability to develop sustainable insect resistance management strategies for transgenic field corn (Bt corn) producing B. thuringiensis (Bt) insecticidal proteins.     

Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

Insect resistance to Bt crops: evidence versus theory

Evolution of insect resistance threatens the continued success of transgenic crops producing Bacillus thuringiensis (Bt) toxins that kill pests. The approach used most widely to delay insect resistance to Bt crops is the refuge strategy, which requires refuges of host plants without Bt toxins near Bt crops to promote survival of susceptible pests. However, large-scale tests of the refuge strategy have been problematic. Analysis of more than a decade of global monitoring data reveals that the frequency of resistance alleles has increased substantially in some field populations of Helicoverpa zea, but not in five other major pests in Australia, China, Spain and the United States. The resistance of H. zea to Bt toxin Cry1Ac in transgenic cotton has not caused widespread crop failures, in part because other tactics augment control of this pest. The field outcomes documented with monitoring data are consistent with the theory underlying the refuge strategy, suggesting that refuges have helped to delay resistance.     

Capturing the interaction types of two Bt toxins Cry1Ac and Cry2Ab on suppressing the cotton bollworm by using multi‐exponential equations

Transgenic crops are increasingly promoted for their practical effects on suppressing certain insect pests, but all transgenic crops are not equally successful. The insect pests can easily develop resistance against single Bacillus thuringiensis (Bt) toxin transgenic crops. Therefore, transgenic crops including two or more mixed Bt‐toxins can solve this problem by delaying the resistance development and killing the majority of targeted pests before the evolution of resistance. It is important to test the controlling effects of transgenic crops including multiple mixed toxins on a particular insect pest. Previous research has checked the cross‐resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against one susceptible and four resistant strains of cotton bollworm. The results showed that independence was the main interaction type between two toxins for the susceptible strain, whereas synergism was the main interaction type for any one resistant strain. However, the optimal combinations of two toxins were not obtained. In the present study, we developed two multi‐exponential equations (namely bi‐ and tri‐exponential equations) to describe the combination effects of two Bt toxins. Importantly, the equations can provide predictions of combination effects of different continuous concentrations of two toxins. We compared these two multi‐exponential equations with the generalized linear model (GLM) in describing the combination effects, and found that the bi‐ and tri‐exponential equations are better than GLM. Moreover, the bi‐exponential equation can also provide the optimal dose combinations for two toxins.     

转Bt基因作物释放杀虫晶体蛋白对土壤生态安全的影响

随着转Bt基因抗虫作物的大面积推广种植,其环境安全性问题日益引起关注。转Bt基因作物在生长期内持续不断地向环境释放杀虫晶体蛋白,这些杀虫晶体蛋白积累一旦超过了昆虫的消耗及环境因子对其的钝化,就可能对非靶标昆虫或土壤微生物造成伤害。转Bt基因作物向土壤中释放杀虫晶体蛋白的途径主要有3种:根系分泌、花粉飘落和秸秆还田。释放到土壤中的Bt杀虫晶体蛋白能够迅速被土壤活性颗粒吸附,1～3 h就能达到吸附平衡。吸附态Bt杀虫晶体蛋白不易被土壤微生物或酶降解,导致杀虫活性持续时间显著延长。土壤微生物种群变化是衡量Bt作物对土壤生态影响的重要指标。研究表明,Bt作物根系分泌物或Bt生物体降解释放的杀虫晶体蛋白对于土壤蚯蚓、线虫、原生动物、细菌和真菌没有毒性,但可使丛枝菌根真菌(AMF)菌丝长度减小,不能形成侵染单元。Bt杀虫晶体蛋白对土壤酶活性的影响程度依这类蛋白的导入方式或Bt作物生育期的不同而呈现差异。土壤中Bt Cry1Ab蛋白能被部分后茬作物吸收,但不同的商品试剂盒检测结果存在差异。文章综述了Bt杀虫晶体蛋白在土壤中释放、吸附、残留特性及其对土壤动物、土壤微生物、土壤酶活性和后茬作物的影响,旨在为转Bt基因作物释放杀虫晶体蛋白的土壤生态安全评价提供参考依据。     

Microbial control of cotton pests. Part I: Use of the naturally occurring entomopathogenic fungus Aspergillus sp. (BC 639) in the management of Creontiades dilutus (Stal) (Hemiptera: Miridae) and beneficial insects on transgenic cotton crops

The development and adoption of transgenic (Bt) crops that express the Bacillus thuringiensis (Bt) toxin has reduced the use of synthetic insecticide on transgenic crops to target Helicoverpa spp., the major insect pest of cotton in Australia. However, it has also increased the threat posed by sucking pests, particularly Creontiades dilutus (green mirid), which are unaffected by the Bt toxins in transgenic cotton crops. Here we report the efficacy of the entomopathogenic fungus Aspergillus sp. (BC 639) in controlling the infestation of transgenic cotton crops by C. dilutus and promoting interactions of transgenic cotton with beneficial insects. The results showed that the number of C. dilutus adults and nymphs recorded on plots treated with 1000, 750, 500, 250 ml/ha BC 639 fungus formulation were the same as on plots treated with the recommended concentration of the commercial insecticide Fipronil. The fungus was found to have minimal effect on predatory insects compared with Fipronil and was most effective against C. dilutus when applied at the rate of 500 ml/ha (equivalent to 50 g spores/ha). At this rate, the fungus was as effective as Fipronil for controlling C. dilutus populations and ensured the survival of predatory beetles, lacewings and spiders compared with Fipronil treatment. The yield from fungus-treated plots was 5.24 bales per acre compared with 5.40 and 3.88 bales per acre for Fipronil-treated and unsprayed plots, respectively. The ability of the BC 639 strain to control C. dilutus infestations of transgenic cotton crops while conserving beneficial insect populations suggests its potential for supplementing integrated pest management programs to reduce the use of synthetic insecticides for transgenic cropping systems.     

Detection and monitoring of insect resistance to transgenic Bt crops

Transgenic crops expressing Bacillus thuringiensis （Bt） endotoxins havebecome one of the most important tools for managing corn and cotton insect pests in the US and other countries. The widespread adoption of transgenic Bt crops could place a high degree of selection pressure on the target insect populations and accelerate development of resistance, raising concerns about the long-term durability of Bt plants as an effective pest management tool. Conservation of Bt susceptibility in insects has become one of the most active research areas in modern agriculture. One of the key factors for a successful Bt resistance management plan is to have a cost-effective monitoring system that can provide information on. （i） the initial Bt resistance allele frequencies at low levels in field insect populations; and （ii） early shifts in Bt resistance allele frequencies so that proactive measures for managing resistance can be deployed well before field control failures. Developing such a monitoring program has been difficult because： （i） resistance traits that occur at very low frequencies are hard to detect; （ii） many factors affect the sensitivity and accuracy of a Bt resistance monitoring program; and （iii） monitoring resistance is costly. Several novel methods for detecting Bt resistance alleles developed during the last decade have made a cost-effective monitoring system possible. Future studies should focus on how to improve and standardize the methodologies for insect sampling and Bt resistance detection.     

基于微生物源的抗虫基因发掘

植物转基因抗虫技术在害虫控制方面取得了巨大成功。商业化运用的抗虫基因目前全部来源于苏云金杆菌（Bacillus thuringiensis,Bt）的杀虫晶体蛋白基因,存在抗虫谱较窄及害虫逐渐产生抗性等问题,表明新型抗虫基因的筛选尤为重要。已有的文献研究表明,除了继续发掘Bt来源的新型杀虫蛋白基因以外,非Bt杀虫细菌及杀虫真菌也具有重要的发掘价值。     

昆虫对Bt作物抗性监测技术

Bt棉花等转抗虫基因作物已在许多国家商业化种植 ,靶标昆虫的潜在抗性是广泛关注的重要问题。为了准确地监测昆虫的抗性动态 ,近年来在传统的基于标准生物测定确定抗性指数的基础上先后发展了诊断剂量、单对杂交、F2 代检测和分子生物学检测抗性等位基因等抗性监测方法。该文综述了相关的研究进展。     

转Bt基因抗虫作物对非靶标害虫的影响

转基因作物的长期大面积种植, 在为农业生产带来惠益的同时, 对农业生态系统的健康和稳定可能会产生潜在的影响。转基因作物表达的Bt蛋白对靶标害虫起到较好的控制效果, 而对Bt蛋白不敏感的非靶标害虫种群可能会迅速发展起来, 对作物造成为害。随着抗虫转基因作物的连续多年种植, 科学家们对于田间杀虫剂施用量的增减看法不尽一致。通过总结已有的研究报道, 本文以Bt玉米和Bt棉花为例, 分析了大田中非靶标害虫暴发的现状, 以及暴发的主要原因(如杀虫剂的使用、害虫天敌减少和物种替代)。在生产实践中, 抗虫作物的长期大面积释放导致广谱杀虫剂施用量减少, 田间非靶标害虫数量上升。因此今后需要继续开展更多的研究来综合评估种植转Bt基因作物产生的长期潜在影响, 优化害虫防治措施, 避免非靶标害虫暴发。     

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.     

Molecular approaches for identification and construction of novel insecticidal genes for crop protection

Summary The insecticidal cry (crystal) genes from Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. Discovery of new insecticidal genes is of importance for delaying the development of resistance in target insects. The diversity of Bt strains facilitates isolation of new types of cry and vip (vegetative insecticidal protein) genes. PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities. PCR together with other methods of analysis such as RFLP, gene sequence determination, electrophoretic, immunological and chromatographic analysis of Cry proteins and insect bioassays for evaluation of toxicity have been employed for identification of new insecticidal proteins. Some other new approaches have also been devised. Many Bt strains with novel insecticidal genes have been found. A desired combination of Cry proteins can be assembled via site-specific recombination vectors into a recipient Bt strain to create a genetically improved biopesticide. For better pest control, the cry genes have been transferred to plants. Stacking of more than one insecticidal gene is required for resistance management in transgenic crops. Modification of Cry proteins through protein engineering for increasing the toxicity and/or the insecticidal spectrum is also a promising approach, but requires detailed understanding of the structure and function of these proteins and analysis of toxin-receptor interactions. More research into this area will provide useful insights for the design of toxins for management of insect resistance. Insecticidal genes from other bacteria and plants are also being examined for their potential for deployment in transgenic crops. Stringent implementation of resistance management is needed for maintaining the efficacy of Bt transgenic crops and deriving maximum economic and environmental benefit.     

转Bt基因玉米的生态安全性研究进展

随着转基因作物的应用和推广 ,转 Bt基因作物释放后对生态环境及其它方面产生的潜在影响越来越受到重视。分别从生物活性杀虫晶体蛋白在土壤中的残留特性、杀虫晶体蛋白对土壤中非目标生物的影响、转 Bt基因玉米植株体成分的变化、转Bt基因玉米花粉中杀虫晶体蛋白的表达特性及其在田间和马力筋叶片上的散积状况、花粉中表达的杀虫晶体蛋白对君主斑蝶的毒性、君主斑蝶幼虫暴露在 Bt花粉中的概率及综合风险评价估算等方面对转 Bt基因玉米产生的杀虫晶体蛋白与土壤生态环境的相互作用、花粉对非目标生物影响的研究现状进行了综述。通过对转 Bt基因作物生态安全性的科学评价和广泛宣传 ,以确保生物技术的健康发展。     

A meta-analysis of effects of Bt crops on honey bees (Hymenoptera: Apidae)

Background

Honey bees (Apis mellifera L.) are the most important pollinators of many agricultural crops worldwide and are a key test species used in the tiered safety assessment of genetically engineered insect-resistant crops. There is concern that widespread planting of these transgenic crops could harm honey bee populations.

Methodology/Principal Findings

We conducted a meta-analysis of 25 studies that independently assessed potential effects of Bt Cry proteins on honey bee survival (or mortality). Our results show that Bt Cry proteins used in genetically modified crops commercialized for control of lepidopteran and coleopteran pests do not negatively affect the survival of either honey bee larvae or adults in laboratory settings.

Conclusions/Significance

Although the additional stresses that honey bees face in the field could, in principle, modify their susceptibility to Cry proteins or lead to indirect effects, our findings support safety assessments that have not detected any direct negative effects of Bt crops for this vital insect pollinator.