Differential proteomic analysis of Trichoplusia ni cells after continuous selection with activated Cry1Ac toxin

Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.     

Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

A range of crops have been transformed with delta-endotoxin genes from Bacillus thuringiensis (Bt) to produce transgenic plants with high levels of resistance to lepidopteran pests. Parasitoids are important natural enemies of lepidopteran larvae and the effects of Bt plants on these non-target insects have to be investigated to avoid unnecessary disruption of biological control. This study investigated the effects of Cry1Ac-expressing transgenic oilseed rape (Brassica napus) on the solitary braconid endoparasitoid Cotesia plutellae in small-scale laboratory experiments. C. plutellae is an important natural enemy of the diamondback moth (Plutella xylostella), the most important pest of brassica crops world-wide. Bt oilseed rape caused 100% mortality of a Bt-susceptible P. xylostella strain but no mortality of the Bt-resistant P. xylostella strain NO-QA. C. plutellae eggs laid in Bt-susceptible hosts feeding on Bt leaves hatched but premature host mortality did not allow C. plutellae larvae to complete their development. In contrast, C. plutellae developed to maturity in Bt-resistant hosts fed on Bt oilseed rape leaves and there was no effect of Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence and percentage adult emergence from cocoons. Weights of female progeny after development in Bt-resistant hosts did not differ between plant types but male progeny was significantly heavier on wildtype plants in one of two experiments. The proportion of female progeny was significantly higher on Bt plants in the first experiment with Bt-resistant hosts but this effect was not observed again when the experiment was repeated.     

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.     

Bacillus thuringiensis: a biotechnology model

This paper is on the different biotechnological approaches that have been used to improve Bacillus thuringiensis (Bt) for the control of agricultural insect pests and have contributed to the successful use of this biological control agent; it describes how a better knowledge of the high diversity of Bt strains and toxins genes together with the development of efficient host-vector systems has made it possible to overcome a number of the problems associated with Bt based insect control measures. First we present an overview of the biology of Bt and of the mode of action of its insecticidal toxins. We then describe some of the progress that has been made in furthering our knowledge of the genetics of Bt and of its insecticidal toxin genes and in the understanding of their regulation. The paper then deals with the use of recombinant DNA technology to develop new Bt strains for more effective pest control or to introduce the genes encoding partial-endotoxins directly into plants to produce insect-resistant trangenic plants. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt or their persistence in the field by protecting them against UV degradation are presented and discussed. Finally, based on our knowledge of the mechanism of transposition of the Bt transposon Tn4430, we describe the construction of a new generation of recombinant strains of Bt, from which antibiotic resistance genes and other non-Bt DNA sequences were selectively eliminated, using a new generation of site-specific recombination vectors. In the future, continuing improvement of first generation products and research into new sources of resistance is essential to ensure the long-term control of insect pests. Chimeric toxins could also be produced so as to increase toxin activity or direct resistance towards a particular type of insect. The search for new insecticidal toxins, in Bt or other microorganisms, may also provide new weapons for the fight against insect damage.     

Assessment of fitness costs in Cry3Bb1‐resistant and susceptible western corn rootworm (Coleoptera: Chrysomelidae) laboratory colonies

Abstract Maize production in the United States is dominated by plants genetically modified with transgenes from Bacillus thuringiensis (Bt). Cry3Bb delta endotoxins expressed by Bt maize specifically target corn rootworms (genus Diabrotica) and have proven highly efficacious. However, development of resistance to Bt maize, especially among western corn rootworm (Diabrotica virgifera virgifera) populations, poses a significant threat to the future viability of this pest control biotechnology. The structured refuge insect resistance management (IRM) strategy implemented in the United States for Bt maize adopts a conservative approach to managing resistance by assuming no fitness costs of Bt resistance, even though these trade‐offs strongly influence the dynamics of Bt resistance within numerous agricultural pest species. To investigate the effects of Bt resistance on fitness components of western corn rootworm, we compared survivorship, fecundity and viability of five Bt‐resistant laboratory lines reared on MON863 (YieldGard Rootworm), a Bt maize product that expresses Cry3Bb1 delta endotoxin, and on its non‐transgenic isoline. Analysis of performance on the isoline maize demonstrated no fitness costs associated with Bt resistance. In fact, resistant lines emerged approximately 2–3 days earlier than control lines when reared on both MON863 and the isoline, indicating that selection for Bt resistance resulted in a general increase in the rate of larval development. In addition, resistant lines reared on Bt maize displayed higher fecundity than those reared on the isoline, which may have significant management implications. These data will be valuable for formulating improved IRM strategies for a principal agricultural pest of maize.     

Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.     

害虫Bt抗性机制研究新方向：昆虫体液免疫系统

作为最成功的生物农药,苏云金芽孢杆菌Bacillus thuringiensis (Bt)杀虫剂已在农业生产中应用了约80年。Bt由于其特异性强、安全高效的特点而得到广泛、成功的应用,极大减少了化学农药的用量,为环境保护作出了巨大贡献。然而,由于长期使用,一些靶标害虫逐渐对Bt产生抗性。本文对昆虫体液免疫及昆虫Bt抗性机制的研究成果进行了总结,已有研究认为害虫对Bt产生抗性的主要原因是毒素激活受阻及(或)毒素受体突变或减少。然而近年越来越多的研究表明,昆虫的Bt抗性还与其免疫系统,特别是与Toll, IMD和proPO-AS等体液免疫通路有关。由此,本文对昆虫体液免疫系统参与昆虫Bt抗性形成的主要通路进行了归纳和推论。IMD免疫通路可能通过MAPK信号通路参与调节昆虫Bt抗性,或可能通过多种免疫反应对抗因中肠组织被Bt破坏而引起的败血症,并通过JNK信号通路促使中肠组织愈合,进而提高其对Bt的抗性。从体液免疫系统切入研究,可能成为深入探索昆虫Bt抗性机制的新方向。     

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

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

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

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

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

Sublethal effects of Bt toxin and chlorpyrifos on various Spodoptera exigua populations

Bt cotton (Cry1Ac) has been commercially grown in China since 1997, saving China's cotton production from attack by Bt‐target pests and also tremendously reducing pesticide usage. In recent years, however, Bt cotton, with 4.2 million ha of cultivation, has suffered from a secondary target pest, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In China, growers have even had to re‐adopt conventional pesticides to control the pest, and this practice has already caused serious pesticide residue. In order to clarify the sublethal effects of chemical pesticide, the responses of a Bt‐susceptible and a Bt‐tolerant (Bt10) S. exigua strain to three treatment combinations were examined, including Bt toxin, sublethal chlorpyrifos, and Bt + sublethal chlorpyrifos. The susceptible and the Bt10 strain responded differently to dual pressure. Bt toxin + sublethal chlorpyrifos treatment lowered larval mortality and stimulated population increase of the susceptible S. exigua, whereas it delayed growth and development of the Bt10 strain. Under dual pressure, although larvae of the Bt10 strain developed faster than larvae of the susceptible strain, the Bt10 population experienced higher larval mortality, prolonged pupal duration, decreased pupal weight, decreased emergence rate, and shortened adult longevity. Compared with the susceptible strain, the Bt10 strain was deleteriously affected by sublethal chlorpyrifos. The Bt‐tolerant/resistant S. exigua population was more vulnerable to chemical pesticides like chlorpyrifos regardless of whether it was exposed to Bt toxin or not. Our study provides a reference for increasing the efficacy of control of S. exigua in Bt‐cotton planting areas.     

盐碱土壤转Bt基因棉花外源蛋白表达量时空变化及对抗虫性的影响

盐碱地是潜在的可利用耕地资源,但土壤盐碱化严重制约了农业生产的可持续发展。基于棉花机械化程度低、劳动力成本和生产资料投入剧增、比较效益下降和实施粮食生产安全战略等因素影响,我国长江流域和黄河流域棉花面积锐减,种植区域向内陆盐碱旱地或滨海盐碱地转移,但目前针对盐碱地转Bt基因棉种植可能带来的生态安全性问题研究甚少,正成为国内外研究的焦点和热点。伴随着棉花向盐碱地大面积转移种植趋势,检测盐胁迫是否影响转基因抗虫棉抗虫性,明确其影响程度,直接关系到转基因抗虫棉种植的安全性,也是目前抗虫棉扩大生产中迫切需要解决的问题。以非转基因棉花为对照,分别在低盐、中盐和高盐土壤种植的棉花的苗期、蕾期和花铃期采样,室内测定了转Bt基因棉花叶片对棉铃虫幼虫校正死亡率和外源蛋白表达量。研究结果发现盐分胁迫下转Bt基因棉花苗期叶片对棉铃虫幼虫校正死亡率下降了9.22%—47.46%,蕾期下降了31.61%—45.42%,花铃期下降了3.59%—18.52%;土壤盐分显著降低了转Bt基因棉花叶片中外源蛋白的表达量,苗期功能叶外源蛋白表达量下降了7.66%—29.86%;蕾期下降了3.77%—36.85%;花铃期下降了18.13%—41.02%;相关性分析表明,盐分胁迫条件下转Bt基因棉花叶片中外源蛋白表达量与其对棉铃虫抗性程度存在正相关关系。结果表明,盐碱土壤显著降低了转Bt基因棉花叶片外源杀虫蛋白表达量,从而导致转Bt基因棉花叶片对棉铃虫的抗虫性下降。研究土壤盐分对转Bt基因棉花对棉铃虫的影响及其作用机制,可为建立盐碱地转Bt基因棉花田害虫综合防控技术体系、转Bt基因棉花环境安全评价及转Bt基因棉安全管理提供依据。     

The compatibility of a nucleopolyhedrosis virus control with resistance management for Bacillus thuringiensis: co-infection and cross-resistance studies with the diamondback moth, Plutella xylostella

The use of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins can lead to the reduction in application of broad-spectrum pesticides and an increased opportunity for supplementary biological control. Bt microbial sprays are also used by organic growers or as part of integrated pest management programs that rely on the use of natural enemies. In both applications the evolution of resistance to Bt toxins is a potential problem. Natural enemies (pathogens or insects) acting in combination with toxins can accelerate or decelerate the evolution of resistance to Bt. In the present study we investigated whether the use of a nucleopolyhedrovirus (AcMNPV) could potentially affect the evolution of resistance to the Bt toxin Cry1Ac in Plutella xylostella. At low toxin doses there was evidence for antagonistic interactions between AcMNPV and Cry1Ac resistant and susceptible insects. However, this antagonism was much stronger and more widespread for susceptible larvae; interactions were generally not distinguishable from additive for resistant larvae. Selection for resistance to Cry1Ac in two populations of P. xylostella with differing resistance mechanisms did not produce any correlated changes in resistance to AcMNPV. Stronger antagonistic interactions between Bt and AcMNPV on susceptible rather than resistant larvae can decrease the relative fitness between Bt-resistant and susceptible larvae. These interactions and the lack of cross-resistance between virus and toxin suggest that the use of NPV is compatible with resistance management to Bt products.     

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program. This revised version was published online in November 2006 with corrections to the Cover Date.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

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.     

Zero effect of Bt rice on expression of genes coding for digestion,detoxification and immune responses and developmental performances of Brown Planthopper Nilaparvata lugens (Stål)

Transgenic Cry1Ac, Cry2Aa and Cry1Ca (Bt toxins) rice lines are well developed to manage lepidopteron pests in China. The impact of transgenic Bt rice on the non-target Brown Planthopper (BPH) has become an essential part of environmental risk assessment, however, scanty evidence is found addressing on developmental and molecular responses of BPH to the ingestion of Bt protein from transgenic rice. The focus of the current study is to examine the developmental characteristics and the expression profiles of gene in relation to digestion, detoxification and immune responses were examined. Our study strongly revealed that the tested Bt rice strains have no unfavorable effect on fecundity, survival and growth of BPH. Furthermore, each of the tested genes did not exhibit distinct expression pattern responding to non Bt parental cultivar, thus, it could be concluded that Bt rice have no detrimental effects on the physiological processes of digestion, detoxification and immune responses of BPH.     

苏云金杆菌发酵后处理技术与产量关系研究

本文采集并分析了苏云金杆菌Bacillus thuringiensis发酵后处理技术,揭示了Bt发酵后处理生产膜过滤数据分析、喷雾干燥数据分析和产量的关系,以期对Bt实际生产过程进行高效控制。     

不同添加物对苏云金芽孢杆菌杀虫活性的增效作用研究进展

苏云金芽孢杆菌（Bacillius thuringiensis,Bt）制剂是当前应用最广、最有效的生物杀虫剂之一,因其对多种昆虫具有特异性杀虫活性,而被广泛用于农林业和公共卫生等领域的害虫防治,但田间施用后,其速效性差、持效期短和防效不稳定等弊端限制了其进一步的推广。将Bt制剂与增效物质（剂）、因子混合使用以提高其杀虫活性和田间防效稳定性,是最快速、有效的途径之一,因而国内外对此开展了广泛而深入的研究。主要介绍了化学添加剂、化学杀虫剂和生物杀虫剂等添加物对Bt制剂杀虫活性的增效作用研究进展,并探讨了增效物质（剂）、因子的开发和应用前景,以期为开发安全、高效的Bt制剂的增效物质（剂）、因子提供一定的参考。