Cotton bollworm resistance to Bt transgenic cotton: A case analysis

Cotton bollworm (Helicoverpa armigera) is one of the most serious insect pests of cotton. Transgenic cotton expressing Cry toxins derived from a soil bacterium, Bacillus thuringiensis (Bt), has been produced to target this pest. Bt cotton has been widely planted around the world, and this has resulted in efficient control of bollworm populations with reduced use of synthetic insecticides. However, evolution of resistance by this pest threatens the continued success of Bt cotton. To date, no field populations of bollworm have evolved significant levels of resistance; however, several laboratory-selected Cry-resistant strains of H. armigera have been obtained, which suggests that bollworm has the capacity to evolve resistance to Bt. The development of resistance to Bt is of great concern, and there is a vast body of research in this area aimed at ensuring the continued success of Bt cotton. Here, we review studies on the evolution of Bt resistance in H. armigera, focusing on the biochemical and molecular basis of Bt resistance. We also discuss resistance management strategies, and monitoring programs implemented in China, Australia, and India.     

Tea: Biological control of insect and mite pests in China

Tea is one of the most economically important crops in China. To secure its production and quality, biological control measures within the context of integrated pest management (IPM) has been widely popularized in China. IMP programs also provide better control of arthropod pests on tea with less chemical insecticide usage and minimal impact on the environment. More than 1100 species of natural enemies including about 80 species of viruses, 40 species of fungi, 240 species of parasitoids and 600 species of predators, as well as several species of bacteria have been recorded in tea ecosystems in China. Biological and ecological characteristics of some dominant natural enemies have been well documented. Several viral, bacterial, and fungal insecticides have been commercially utilized at large scale in China. Progress in biological control methods in conjunction with other pest control approaches for tea insect pest management is reviewed in this article. Knowledge gaps and future directions for tea pest management are also discussed.     

湖北棉区转Bt基因棉对棉铃虫的控制作用

2000-2001年通过田间系统调查,表明转Bt基因棉（品种为GK19）在湖北江汉平原棉区对棉铃虫抗性稳定。试验设3个处理：转Bt基因棉化防田（使用化学农药控制害虫）、转Bt基因棉自控田（依靠天敌控制害虫）及常规棉对照田（利用综合防治措施控制害虫）。从棉铃虫的第2代到第5代整个发生期内,即使在不进行化学防治的情况下,棉铃虫在Bt棉田的发生量也保持在极低的水平(最高百株虫量为12头)。室内饲养结果表明,转Bt基因棉对棉铃虫的生长发育（幼虫体重、蛹重）有较为明显的影响,使6龄幼虫体重减少25.6%,蛹重减少18.2%。棉铃虫幼虫取食转Bt基因棉组织后,发育迟缓,相对于常规棉喂养的整个发育历期延长17 d,使棉铃虫在田间的危害减少至少一个世代。另外,接虫试验表明,棉铃虫幼虫在常规棉上的取食时间是转Bt基因棉株上的6.1倍,极大地减轻了棉铃虫的危害程度。     

麦套夏播转Bt基因棉R93-6对昆虫群落的影响

以转Bt(Bacillusthuringiensis)基因棉品系R93-6为试验材料,以中棉所16号为对照,研究了在麦套夏播条件下转基因棉对昆虫群落的影响。结果表明,转基因棉田昆虫群落、害虫和天敌亚群落的多样性指数和均匀度指数均低于常规棉田,而优势集中性则高于常规棉田,所以转基因棉田昆虫群落、害虫和天敌亚群落的稳定性不如常规棉田,某种害虫大发生的可能性较大。对季节性变化格局的研究表明,转基因棉田昆虫群落可以划分为前期(6月初至7月下旬)、中期(7月底至8月底)和后期(9月份以后)三个发展阶段,根据不同阶段害虫和天敌发生的特点, 提出了害虫综合治理的策略。即前期害虫的防治应以生物生态调控为主;中期以化学防治为主,以生物生态调控为辅,协调好生物防治和化学防治的矛盾;后期应以生物生态调控为主,并加强农业防治。     

Early detection of field-evolved resistance to Bt cotton in China: cotton bollworm and pink bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some major insect pests, but pests can evolve resistance and thereby reduce the effectiveness of such Bt crops. The main approach for slowing 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 cotton producing Bt toxin Cry1Ac, several 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. This strategy is designed for cotton bollworm (Helicoverpa armigera), which attacks many crops and is the primary target of Bt cotton in China, but it does not apply to pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we review evidence of field-evolved resistance to Cry1Ac by cotton bollworm in northern China and by pink bollworm in the Yangtze River Valley of China. For both pests, results of laboratory diet bioassays reveal significantly decreased susceptibility of field populations to Cry1Ac, yet field control failures of Bt cotton have not been reported. The early detection of resistance summarized here may spur countermeasures such as planting Bt cotton that produces two or more distinct toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

Conservation biological control and IPM practices in Brassica vegetable crops in China

Brassicas are major vegetable crops in China but the systems for growing the crops are complex. During the last 30 years, the area of vegetable crops has increased steadily, however, the control of insect pests on brassica vegetables has largely relied on the heavy use of chemical insecticides, resulting in high levels of resistance, insecticide residues hazardous to human health and other serious consequences. Nevertheless, efforts to develop practical and sustainable integrated pest management (IPM) strategies for brassica vegetables have been implemented. Here we first review the work on surveys of natural enemies of insect pests in brassicas and describe the biology and ecology of a few important parasitoids. We then introduce the progress of conservation biological control by reviewing studies on evaluation of natural enemies and selective insecticides, the work on the development of action thresholds and some successful examples of IPM field trials at the cropping system level. The successful examples of IPM practices in brassicas show the great potential of conservation biological control to reduce chemical pesticide input and improve vegetable production in the future.     

Sustained susceptibility of pink bollworm to Bt cotton in the United States

Evolution of resistance by pests can reduce the benefits of transgenic crops that produce toxins from Bacillus thuringiensis (Bt) for insect control. One of the world's most important cotton pests, pink bollworm (Pectinophora gossypiella), has been targeted for control by transgenic cotton producing Bt toxin Cry1Ac in several countries for more than a decade. In China, the frequency of resistance to Cry1Ac has increased, but control failures have not been reported. In western India, pink bollworm resistance to Cry1Ac has caused widespread control failures of Bt cotton. By contrast, in the state of Arizona in the southwestern United States, monitoring data from bioassays and DNA screening demonstrate sustained susceptibility to Cry1Ac for 16 y. From 1996-2005, the main factors that delayed resistance in Arizona appear to be abundant refuges of non-Bt cotton, recessive inheritance of resistance, fitness costs associated with resistance and incomplete resistance. From 2006-2011, refuge abundance was greatly reduced in Arizona, while mass releases of sterile pink bollworm moths were made to delay resistance as part of a multi-tactic eradication program. Sustained susceptibility of pink bollworm to Bt cotton in Arizona has provided a cornerstone for the pink bollworm eradication program and for integrated pest management in cotton. Reduced insecticide use against pink bollworm and other cotton pests has yielded economic benefits for growers, as well as broad environmental and health benefits. We encourage increased efforts to combine Bt crops with other tactics in integrated pest management programs.     

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.     

Diminishing Returns from Increased Percent Bt Cotton: The Case of Pink Bollworm

Regional suppression of pests by transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) has been reported in several cropping systems, but little is known about the functional relationship between the ultimate pest population density and the pervasiveness of Bt crops. Here we address this issue by analyzing 16 years of field data on pink bollworm (Pectinophora gossypiella) population density and percentage of Bt cotton in the Yangtze River Valley of China. In this region, the percentage of cotton hectares planted with Bt cotton increased from 9% in 2000 to 94% in 2009 and 2010. We find that as the percent Bt cotton increased over the years, the cross-year growth rate of pink bollworm from the last generation of one year to the first generation of the next year decreased. However, as the percent Bt cotton increased, the within-year growth rate of pink bollworm from the first to last generation of the same year increased, with a slope approximately opposite to that of the cross-year rates. As a result, we did not find a statistically significant decline in the annual growth rate of pink bollworm as the percent Bt cotton increased over time. Consistent with the data, our modeling analyses predict that the regional average density of pink bollworm declines as the percent Bt cotton increases, but the higher the percent Bt cotton, the slower the decline in pest density. Specifically, we find that 95% Bt cotton is predicted to cause only 3% more reduction in larval density than 80% Bt cotton. The results here suggest that density dependence can act against the decline in pest density and diminish the net effects of Bt cotton on suppression of pink bollworm in the study region. The findings call for more studies of the interactions between pest density-dependence and Bt crops.     

转Bt基因棉对非靶标害虫及害虫天敌种群动态的影响

2000～2001年,在湖北棉区系统研究了转Bt基因棉对棉田非靶标害虫及天敌种群动态的影响。试验设三个处理：转Bt化防田（使用化学农药控制害虫）、转Bt自控田（仅依靠田间自然天敌控制害虫）及常规对照棉田（利用综合防治措施控制害虫）。结果表明,在转Bt基因棉田中,除棉蓟马外,其它主要非靶标害虫（主要是刺吸性害虫）的种群发生数量呈明显的上升趋势。2000年棉蚜发生的总计值,化防田和自控田分别比常规对照田增加37.9%和71.4%,2001年则分别增加92.5%和134.9%;2000年朱砂叶螨发生的总计值,化防田和自控田分别比常规对照田增加181.1%和298.3%,2001年则分别增加69.9%和105.0%;转Bt基因棉对于斜纹夜蛾与烟粉虱这两种近几年来对棉花危害有加重趋势的害虫,在大田中没有表现出抗性。天敌的发生种类和数量也都远远高于常规对照棉田,几种主要天敌种群发生的总计值及高峰值都明显高于常规对照棉田。2000年蜘蛛类发生的总计值,化防田和自控田分别比常规对照田增加66.3%和112.1%,2001年则分别增加95.1%和111.7%;2000年龟纹瓢虫发生的总计值,化防田和自控田分别比常规对照田增加140.8%和135.4%,2001年则分别增加67.2%和109.5%;2000年在两块转Bt基因棉田中种群数量较大的大眼蝉长蝽在常规对照棉田的调查过程中没有发现,2001年在对照田中也仅记录到1头。试验表明,充分利用Bt棉田中天敌种类较丰富、种群数量较大的优势而加强生物防治,是湖北棉区转Bt基因棉田综防体系中的一个关键措施。     

Bt棉花害虫综合治理研究前沿

自1996年以来,全球Bt(Bacillus thuringiensis)棉花应用规模迅速增长,目前已占棉花种植总面积的60%左右,主要种植国家包括美国、澳大利亚、中国、印度和巴基斯坦等。大量研究表明,Bt棉花的大面积种植有效控制了多种靶标害虫的发生危害,从而大幅度减少了化学杀虫剂的使用量;化学杀虫剂的减少使用导致一些非靶标害虫的发生危害明显加重。针对Bt棉花生产中呈现出的害虫新问题,各国分别发展了由农业防治、生物防治、化学防治等不同措施构成的防控技术体系。     

Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm

Wide planting of transgenic Bt cotton in China since 1997 to control cotton bollworm (Helicoverpa armigera) has increased yields and decreased insecticide use, but the evolution of resistance to Bt cotton by H. armigera remains a challenge. Toward developing a new generation of insect-resistant transgenic crops, a chimeric protein of Vip3Aa1 and Vip3Ac1, named Vip3AcAa, having a broader insecticidal spectrum, was specifically created previously in our laboratory. In this study, we investigated cross resistance and interactions between Vip3AcAa and Cry1Ac with three H. armigera strains, one that is susceptible and two that are Cry1Ac-resistant, to determine if Vip3AcAa is a good candidate for development the pyramid cotton with Cry1Ac toxin. Our results showed that evolution of insect resistance to Cry1Ac toxin did not influence the sensitivity of Cry1Ac-resistant strains to Vip3AcAa. For the strains examined, observed mortality was equivalent to the expected mortality for all the combinations of Vip3AcAa and Cry1Ac tested, reflecting independent activity between these two toxins. When this chimeric vip3AcAa gene and the cry1Ac gene were introduced into cotton, mortality rates of Cry1Ac resistant H. armigera larvae strains that fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and cotton producing only Cry1Ac. These results suggest that the Vip3AcAa protein is an excellent option for a “pyramid” strategy for pest resistance management in China.     

Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.     

Biological control of insect pests in apple orchards in China

Apple is one of the most important fruits in China, and both yield and quality are greatly affected by insect pests. According to surveys, there are more than 200 species of natural enemies in apple orchards. Few, however, have been closely studied. Major natural enemies including parasitoids, predators and pathogens are briefly described in this review, especially focusing on two parasitoids of Trichogramma dendrolimi Matsumura and Aphelinus mali Haldeman, predatory mites and a pathogenic fungus of Beauveria bassiana (Balsamo) Vuillemin as case studies. Augmentation, one important strategy of biological control, supplements the natural control provided by the existing natural enemy community in apple orchards, and greatly increases their efficiency in controlling pests. Conservation biological control is also widely applied in four major apple-producing areas. Based on habitat manipulation, the ground cover planting system helps regulate the microclimate and enhance the biodiversity of apple orchards, effectively conserving the richness and diversity of beneficial insect species. Certain achievements have been made in the main biological control strategies including successful introduction of some exotic natural enemies such as A. mali and Typhlodromus occidentalis Nesbitt, augmentative production and application of biological control agents such as T. dendrolimi, B. bassiana and Bacillus thuringiensis, and further research in conservation of establishing adaptive ground cover planting patterns to local environment. Challenges, however, still exist. Biological control of insect pests in apple orchards is an important part of integrated pest management programs, requiring more research and application in China.     

An appetite for pests: Synanthropic insectivorous bats exploit cotton pest irruptions and consume various deleterious arthropods

Conservation biological control (CBC) seeks to minimize the deleterious effects of agricultural pests by enhancing the efficiency of natural enemies. Despite the documented potential of insectivorous bats to consume pests, many synanthropic bat species are still underappreciated as beneficial species. We investigated the diet of Kuhl's pipistrelle (Pipistrellus kuhlii), a common synanthropic insectivorous bat that forages in urban and agricultural areas, to determine whether it may function as a natural enemy in CBC. Faecal samples of P. kuhlii were collected throughout the cotton‐growing season from five roost sites near cotton fields located in a Mediterranean agroecosystem, Israel, and analyzed using DNA metabarcoding. Additionally, data on estimated abundance of major cotton pests were collected. We found that the diet of P. kuhlii significantly varied according to sites and dates and comprised 27 species of agricultural pests that were found in 77.2% of the samples, including pests of key economic concern. The dominant prey was the widespread cotton pest, the pink bollworm, Pectinophora gossypiella, found in 31% of the samples and in all the roosts. Pink bollworm abundance was positively correlated with its occurrence in the bat diet. Furthermore, the bats’ dietary breadth narrowed, while temporal dietary overlap increased, in relation to increasing frequencies of pink bollworms in the diet. This suggests that P. kuhlii exploits pink bollworm irruptions by opportunistic feeding. We suggest that synanthropic bats provide important pest suppression services, may function as CBC agents of cotton pests and potentially contribute to suppress additional deleterious arthropods found in their diet in high frequencies.     

Prospects for improved off-crop habitat management for pollen beetle control in oilseed rape

There is an urgent need to develop sustainable and environmentally benign integrated pest management (IPM) strategies for arable crops. The enhancement and manipulation of naturally occurring populations of the natural enemies of crop pests through habitat management for ‘conservation biological control’, as well as habitat management to manipulate populations of the pests themselves, have the potential to become major components of successful IPM strategies. We review the studies that have contributed to our current understanding of how the crop margin, local landscape, and regional landscape can influence pollen beetle Brassicogethes aeneus (syn. Meligethes aeneus) (Coleoptera: Nitidulidae) abundance and damage to oilseed rape crops (Brassica napus), and the efficacy of their natural enemies. We also discuss how habitat management across these multiple scales may improve pollen beetle control, reducing the need for insecticide use and contributing towards sustainable production of this important crop which is grown on increasing areas for both food and fuel.     

An overview on the natural enemies of Rhynchophorus palm weevils,with focus on R. ferrugineus

Rhynchophorus palm weevils are large insects belonging to the family Dryophthoridae. All Rhynchophorus species are polyphagous and have a similar life history but some are major pests because of the serious economic damage they cause, in particular to several species of the family Arecaceae. Here we review the natural enemies of Rhynchophorus species in both their native and introduced regions of the world, to assess the possibility of biological control of this taxon. Moreover, particular attention is paid to the well-studied and harmful species Rhynchophorus ferrugineus, about which more information is available, and to its natural enemies in the Mediterranean region, because the impact of this pest in this recently colonized area is particularly remarkable and also the recent trend in species management is looking for indigenous natural enemies.More than 50 natural enemies have been reported to attack Rhynchophorus species, even if most of them are associated to R. ferrugineus (Olivier), highlighting the lack of information on the other species of the genus. Pros and cons of all the biological control agents are then discussed: among the considered organisms, fungi are noteworthy to be considered for inclusion in integrated pest management programs.Overall, our overview underlines the need to increase knowledge on natural enemies of all the species of the genus Rhynchophorus, to isolate more virulent strains and to determine the optimum conditions for the actions of the biocontrol agents.     

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.     

Mutated Cadherin Alleles from a Field Population of Helicoverpa armigera Confer Resistance to Bacillus thuringiensis Toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r₁) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r₂ and r₃) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r₂ and r₃ alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.