Acquisition of Cry1Ac Protein by Non-Target Arthropods in Bt Soybean Fields

Soybean tissue and arthropods were collected in Bt soybean fields in China at different times during the growing season to investigate the exposure of arthropods to the plant-produced Cry1Ac toxin and the transmission of the toxin within the food web. Samples from 52 arthropod species/taxa belonging to 42 families in 10 orders were analysed for their Cry1Ac content using enzyme-linked immunosorbent assay (ELISA). Among the 22 species/taxa for which three samples were analysed, toxin concentration was highest in the grasshopper Atractomorpha sinensis and represented about 50% of the concentration in soybean leaves. Other species/taxa did not contain detectable toxin or contained a concentration that was between 1 and 10% of that detected in leaves. These Cry1Ac-positive arthropods included a number of mesophyll-feeding Hemiptera, a cicadellid, a curculionid beetle and, among the predators, a thomisid spider and an unidentified predatory bug belonging to the Anthocoridae. Within an arthropod species/taxon, the Cry1Ac content sometimes varied between life stages (nymphs/larvae vs. adults) and sampling dates (before, during, and after flowering). Our study is the first to provide information on Cry1Ac-expression levels in soybean plants and Cry1Ac concentrations in non-target arthropods in Chinese soybean fields. The data will be useful for assessing the risk of non-target arthropod exposure to Cry1Ac in soybean.     

Bt Crops Producing Cry1Ac,Cry2Ab and Cry1F Do Not Harm the Green Lacewing,Chrysoperla rufilabris

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.     

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Evidence of Field-Evolved Resistance of Spodoptera frugiperda to Bt Corn Expressing Cry1F in Brazil That Is Still Sensitive to Modified Bt Toxins

Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.     

苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白及其分子设计

cry1Ac编码的杀虫晶体蛋白是苏云金芽孢杆菌(Bt)产生的多种杀虫晶体蛋白中对鳞翅目昆虫有很高毒性的蛋白.第一个Cry1Ac杀虫晶体蛋白最早在库斯塔克亚种HD73中以伴胞晶体形式分离获得,其编码区为3 534 bp,编码蛋白分子量为133 kD,含1 178个氨基酸,等电点为4.84.自此以来,Cry1Ac杀虫晶体蛋白结构、功能以及应用研究一直是Bt杀虫晶体蛋白研究的重要方向.本文介绍了苏云金芽孢杆菌中应用最广泛的Cry1Ac杀虫晶体蛋白家族的结构、功能及其基因分类,并进一步就基于苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白的基因工程研究做了分析,提出了持续利用BtCry1Ac杀虫晶体蛋白的一些见解.     

Movement of transgenic plant-expressed Bt Cry1Ac proteins through high trophic levels

The movement of Bacillus thuringiensis (Berliner) (Bt) Cry1Ac endotoxin through high trophic levels was assessed to help elucidate the effects of Bt toxin on non‐target insects. The diamondback moth (Plutella xylostella L., Lepidoptera: Plutellidae), the parasitic wasp (Cotesia vestalis Haliday, Hymenoptera: Braconidae) and the predatory green lacewing Chrysoperla carnea (Stephen) (Neuroptera: Chrysopidae) were used as a model system in this laboratory study. Bt‐resistant P. xylostella larvae fed Cry1Ac‐expressing transgenic oilseed rape (OSR, Brassica napus L., Cruciferae), before and after parasitization by C. vestalis, consumed Cry1Ac with the ingested plant material but only a proportion of Cry1Ac consumed was recovered from the bodies and faeces of P. xylostella larvae. Cry1Ac was not detected in newly emerged parasitoid larvae. In contrast, Cry1Ac was detected in C. carnea larvae fed on resistant P. xylostella larvae reared on Bt OSR. However, no Cry1Ac could be detected in C. carnea larvae when the lacewings were transferred to P. xylostella larvae reared on conventional OSR and tested 24–48 h. The metabolizing ability of Cry1Ac is discussed for the larvae of P. xylostella and C. carnea.     

Transgenic Cabbage Expressing Cry1Ac1 Does Not Affect the Survival and Growth of the Wolf Spider,Pardosa astrigera L. Koch (Araneae: Lycosidae)

Both herbivores that consume transgenic crops and their predators can be exposed to insecticidal proteins expressed in those crops. We conducted a tritrophic bioassay to evaluate the ecotoxicological impacts that Bt cabbage (Brassica oleracea var. capitata) expressing Cry1Ac1 protein might have on the wolf spider (Pardosa astrigera), a non-target generalist predator. Enzyme-Linked Immunosorbent Assays indicated that protein levels were 4.61 ng g^-1 dry weight in fruit flies (Drosophila melanogaster) fed with the transgenic cabbage and 1.86 ng g^-1 dry weight in the wolf spiders that preyed upon them. We also compared the life history traits of spiders collected from Bt versus non-Bt cabbage and found no significant differences in their growth, survival, and developmental rates. Because Bt cabbage did not affect the growth of fruit flies, we conclude that any indirect effects that this crop had on the wolf spider were probably not mediated by prey quality. Therefore, exposure to Cry1Ac1 protein when feeding upon prey containing that substance from transgenic cabbage has only a negligible influence on those non-target predatory spiders.     

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.     

转Bt基因稻谷对印度谷螟生长发育的影响

为建立仓储阶段转Bt水稻安全性评价中靶标害虫抗性汰选研究体系,配制了含不同比例(70%,50%,30%,10%)转Bt基因(Cry1Ab/Cry1Abc)明辉63水稻谷粉(简称Bt谷粉)的人工饲料饲喂印度谷螟Plodia interpunctella(Hübner),测定其对1～3龄幼虫在72h内的急性毒力,及对印度谷螟种群生长发育的影响,并采用ELISA法检测转基因稻谷和末龄幼虫体内Bt蛋白含量。结果发现:4种比例人工饲料对幼虫的毒力作用均发生在取食48h后,72h后剂量效应明显。含Bt水稻较高比例的饲料对印度谷螟发育的负面效应明显:幼虫死亡率高,发育历期延长。Bt蛋白在幼虫体内含量与对应饲料中的含量基本成正比。综合考虑,将Bt杀虫蛋白含量2.35μg/g作为转Bt基因稻谷对印度谷螟的亚致死剂量最为合适。     

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%–50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%–30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.     

Common, but Complex, Mode of Resistance of Plutella xylostella to Bacillus thuringiensis Toxins Cry1Ab and Cry1Ac

A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.     

Common Receptor for Bacillus thuringiensis Toxins Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua

Binding studies using ¹²⁵I-Cry1Ac and biotinylated Cry1Fa toxins indicate the occurrence of a common receptor for Cry1Ac, Cry1Fa, and Cry1Ja in Helicoverpa armigera, Helicoverpa zea, and Spodoptera exigua. Our results, along with previous binding data and the observed cases of cross-resistance, suggest that this pattern seems to be widespread among lepidopteran species.     

Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

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

Monitoring of resistance for the diamondback moth to Bacillus thuringiensis Cry1Ac and Cry1Ba toxins and a Bt commercial formulation

Abstract:  To monitor the resistance of field populations of the diamondback moth Plutella xylostella in China to the insecticidal protein Cry1Ac, Cry1Ba and commercial formulation Bacillus thuringiensis var. kurstaki (Btk), six representative populations of the diamondback moth were collected from Shanghai, Shandong, Hubei, Hunan, Zhejiang and Guangdong provinces of China where crucifer crop plants are intensively planted. Bioassay results showed that the populations of the diamondback moth from different locations exhibited different levels of resistance, compared with a susceptible laboratory population. The Guangdong field population was 56.15- and 21.90-fold resistant to Cry1Ac and Btk, respectively. Shanghai, Hunan, Shandong and Zhejiang populations were 37.85-, 17.24-, 10.24- and 9.41-fold resistant to Cry1Ac, respectively, but were not resistant to Btk. The Hubei population did not show resistance to Cry1Ac and Btk. Almost all tested populations were susceptible to Cry1Ba, but the Guangdong population showed some tolerance to Cry1Ba with a LC₅₀ of 0.69  μ g/ml which was 6.17-fold higher than that of the susceptible population. The results suggested that the complex resistance patterns of field populations of P. xylostella need to be considered for expression of Bt toxin genes in genetically-engineered crop plants and commercial formulations.     

Bt Cry1Ac毒素筛选的粉纹夜蛾离体抗性细胞与敏感细胞蛋白质组的差异分析

采用双向电泳技术和肽质量指纹图谱技术分析了Bt Cry1Ac毒素筛选的粉纹夜蛾Trichoplusia ni抗性BTI-Tn-5B1-4细胞与同源敏感细胞的蛋白质组的差。用Melanie ViewerⅡ软件在抗性细胞的双向电泳图谱上检测到的平均蛋白质点数为707±25个（n=3）,在敏感细胞的双向电泳图谱上检测到的平均蛋白质点数为637±19个（n=3）,其中分辨率高、重复性良好的显著差异点有10余个。对其中的一个敏感细胞特有的显著差异斑点进行了肽质量指纹图谱分析,经数据库 查询表明该蛋白质与胞质外周蛋白具有同源性。     

转（Bt Cry1Ac+CP4EPSPS）基因抗虫抗草甘膦棉花对草甘膦的耐受性研究

本研究比较了转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花与常规棉花在新疆棉区对草甘膦的耐受性差异。两年的研究结果表明,转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花对草甘膦有较好的耐受性,而对照棉花中棉所49对草甘膦耐受性较差。苗期喷施草甘膦后转基因抗棉铃虫抗草甘膦棉花生长发育没有受到影响,而对照棉花中棉所49喷施草甘膦后生长发育受到了明显的影响,个别植株甚至死亡。转Bt Cry1Ac+CP_4EPSPS基因抗棉铃虫抗草甘膦棉花株高、真叶数、蕾数、产量等指标与对照相比差异显著。转Bt Cry1Ac+CP_4EPSPS基因棉花对草甘膦的耐受程度显著高于非转基因棉花。草甘膦对转基因抗草甘膦棉花无负面影响。     

Bt Cry1Ac抗性粉纹夜蛾离体细胞与敏感细胞mRNA的差异显示

为了研究昆虫对Bt的抗性机制 ,以苏云金芽孢杆菌Cry1Ac活性毒素对粉纹夜蛾离体细胞连续筛选 80代 ,获得了高抗性细胞。采用DDRT PCR技术比较了该抗性细胞与非选择敏感细胞mRNA的差异 ,经反向RNA斑点印迹杂交确证了 5个片段为两种细胞的显著差异表达序列标签(ESTs)。序列分析结果表明 ,敏感细胞特有的三种ESTs都位于cDNA的非编码区 ,两种ESTs(GenBank注册号 :S1 ,CF32 2 4 1 5 ;S3,CF32 2 4 1 7)与数据库中EST没有显著同源性 ,另一种S2(GenBank注册号 :CF32 2 4 1 6)与已登录的某些昆虫的ESTs具有一定的同源性。从抗性细胞特有的R1 (GenBank注册号 :CF32 2 4 1 3)推导出的氨基酸序列与磷酸烯醇式丙酮酸羧激酶有 60 %～63%的同源性 ,从抗性细胞特有的R2 (GenBank注册号 :CF32 2 4 1 4)推导出的氨基酸序列与黏蛋白类有约 30 %的同源性。这些差异表达基因可能与抗性的形成相关。     

Bt毒蛋白Cry1Ac在人造土壤中对赤子爱胜蚓毒理及生化影响

Bt毒素能通过转基因作物的花粉、根和残株进入土壤.为评估转基因作物对土壤动物的影响,本文模拟转基因棉的Bt毒素进入土壤的发生程度,用含不同浓度Bt毒蛋白Cry1Ac的人造土壤处理蚯蚓,测定蚯蚓存活率、重量变化及体内总蛋白含量和过氧化氢酶(CAT)、乙酰胆碱酯酶(AchE)、谷胱甘肽-S-转移酶(GST)和纤维素酶活性.结果表明,Bt毒蛋白对蚯蚓的生物量和生理水平影响均不明显,不存在急毒性和亚致死毒性影响,对蚯蚓比较安全.