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.     

Feeding behavior of bollworm and tobacco budworm (Lepidoptera: Noctuidae) larvae in mixed stands of nontransgenic and transgenic cotton expressing an insecticidal protein

Feeding behavior of third-instar bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), was observed in pure and mixed stands of nontransgenic and transgenic cotton (BTK), Gossypium hirsutum L., expressing an insecticidal protein CryIA(c) from a bacterium, Bacillus thuringiensis Berliner subsp. kurstaki. Five plant stands composed of BTK and non-BTK plants were evaluated; two pure stands and three mixed stands. Percentage ratios of BTK to non-BTK plants in the stands were 100:0, 75:25, 50:50, 25:75 and 0:100, respectively. In all stands with BTK plants, fewer bollworm and tobacco budworm larvae were found on BTK plants than non-BTK plants 24 h after infestation with third instars. At 48 h, significantly fewer tobacco budworm larvae, but not fewer bollworm larvae, were found on BTK plants. However, the number of larvae of either insect did not increase on non-BTK plants compared with the initial infestation density of three larvae per plant. The number of obacco budworm injured flower buds, and capsules was lower in all plant stands containing BTK plants compared with the pure stand of non-BTK at 48 h after infestation. Higher numbers of larvae on non-BTK plants were possibly the result of larval intoxication, reduced feeding, and increased plant abandonment and death on BTK plants rather than a classical feeding preference. Unexpectedly, the number of flower buds and capsules injured by bollworm and tobacco budworm when averaged per plant for all plants in a stand, differed little among the 75:25, 50:50 and 25:75 plant mixtures. These data suggest that larvae of both species frequently moved among plants, feeding indiscriminately on BTK and non-BTK plants.     

Overwintering cost associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae)

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance. We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton. Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter. Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived. This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains. Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent.     

Effects of cotton cultivar on fitness costs associated with resistance of pink bollworm (Lepidoptera: Gelechiidae) to Bt cotton

Fitness costs associated with insect resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) reduce the fitness on non-Bt refuge plants of resistant individuals relative to susceptible individuals. Because costs may vary among host plants, choosing refuge cultivars that increase the dominance or magnitude of costs could help to delay resistance. Specifically, cultivars with high concentrations of toxic phytochemicals could magnify costs. To test this hypothesis, we compared life history traits of three independent sets of pink bollworm, Pectinophora gossypiella (Saunders), populations on two cotton cultivars that differed in antibiosis against this cotton pest. Each set had an unselected susceptible population, a resistant population derived by selection from the susceptible population, and the F1 progeny of the susceptible and resistant populations. Confirming previous findings with pink bollworm feeding on cotton, costs primarily affected survival and were recessive on both cultivars. The magnitude of the survival cost did not differ between cultivars. Although the experimental results did not reveal differences between cultivars in the magnitude or dominance of costs, modeling results suggest that differences between cultivars in pink bollworm survival could affect resistance evolution. Thus, knowledge of the interaction between host plants and fitness costs associated with resistance to Bt crops could be helpful in guiding the choice of refuge cultivars.     

Behavior of bollworm (Lepidoptera: Noctuidae) larvae on genetically engineered cotton

Reports of bollworm, Helicoverpa zea (Boddie), larvae feeding in white flowers of Bollgard cotton have been relatively common since the commercialization of this technology in 1996. Field studies were conducted in Louisiana to determine if differences in bollworm larval behavior occuron non-Bollgard (cultivar 'Deltapine 5415') and Bacillus thuringiensis (Bt), Bollgard ('NuCOTN 33B') cottons. Larvae were placed on the terminal foliage of either single cotton plants or on all plants within 1-m row micro-plots. On preflowering cotton plants, significantly more bollworms moved from the site of infestation (terminal) on Bollgard plants compared with that on non-Bollgard plants. On individual flowering plants, the number of nodes larvae moved from the terminal and the number of infested bolls was greater on Bollgard cotton plants. Similar differences between Bollgard and non-Bollgard plants in the percentage of infested terminals and squares were observed at 48-h after infestation when 1-m rows were infested. These data will be used to refine scouting protocols for bollworm larvae on Bollgard cotton.     

Antennal response of cotton bollworm (Heliocoverpa armigera) to volatiles in transgenic Bt cotton

Abstract:  Volatile compositions in transgenic Bt cotton variety 'GK-97' and its parental variety 'Simian No. 3' were analysed, and antennal response of cotton bollworm ( Heliocoverpa armigera ) to the volatiles were investigated. Volatile components in Bt cotton and regular cotton were almost same, except for absence of a sesquiterpene and a minor unknown compound in regular cotton. The results of gas chromatography-electroantennographic detection (GC-EAD) experiments showed that cotton bollworm antennae responded to seven compounds and two minor unknown compounds in Bt volatiles, among which concentrations of α -pinene and β -pinene in Bt cotton were relatively higher than those in regular cotton, and one minor unknown compound was absent in regular cotton.     

Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae)

In Australia, transgenic cotton plants expressing the cry1Ac gene from Bacillus thuringiensis Berliner variety kurstaki are less toxic to first-instar Helicoverpa armigera (Hübner) after the plant is producing fruit. We developed two bioassay methods (leaf mush, leaf disk) to test if the physiological state of the plants explained changes in toxicity and a third method (diet incorporation) was developed to quantify the toxicity of Bt leaves when mixed in chickpea diet. Cry1Ac protein was less toxic to H. armigera larvae when the protein was mixed with leaves from fruiting versus presquare conventional cotton. Differences in LC50 varied from 2.4- to 726-fold, depending on the source of toxin and conventional plant material. These results suggest that plant-toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein. The possible role of tannins in these changes is discussed.     

Mitochondrial genome of the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and comparison with other Lepidopterans

We present the complete sequence of the mitochondrial genome (mitogenome) of the cotton bollworm Helicoverpa armigera. The 15,347-bp mitogenome of H. armigera was arranged in the same order described for all other sequenced lepidopterans, which differs from the most common type found in insects, due to the movement of trnM to a position 5'-upstream of trnI. The gene overlap in the H. armigera mitogenome is totally 23 bp in six locations. The H. armigera mitogenome has a total of 175 bp of intergenic spacer sequences spread over 14 regions ranging in size from 1 to 45 bp. The nucleotide composition of the whole mitogenome of H. armigera is highly A+T biased, accounting for 80.97%, with a slightly positive AT skewness and negative GC skewness, indicating the occurrence of more A than T, C more than G. The protein-encoding genes have typical mitochondrial start codons, except for cox1, which contains the unusual CGA. The cox1, cox2, and nad4 genes have incomplete stop codons (T). The lrRNA and srRNA genes are 1395 and 794-bp long, respectively. All tRNAs have a typical cloverleaf structure of mitochondrial tRNAs, except for trnS1(AGN), the dihydrouridine arm of which could not form a stable stem-loop structure. The H. armigera A+T-rich region contains a conserved structure combining the motif ATAGA and a 19-bp poly-T stretch, but absence of the 9-bp poly-A element upstream of trnM.     

Fitness costs and maternal effects associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae).

Transgenic cotton producing a Bacillus thuringiensis (Bt) toxin is widely used for controlling the pink bollworm, Perctinophora gossypiella (Saunders). We compared performance of pink bollworm strains resistant to Bt cotton with performance of their susceptible counterparts on non-Bt cotton. We found fitness costs that reduced survival on non-Bt cotton by an average of 51.5% in two resistant strains relative to the susceptible strains. The survival cost was recessive in one set of crosses between a resistant strain and the susceptible strain from which it was derived. However, crosses involving an unrelated resistant and susceptible strain indicated that the survival cost could be dominant. Development time on non-Bt cotton did not differ between the two related resistant and susceptible strains. A slight recessive cost affecting development time was suggested by comparison of the unrelated resistant and susceptible strains. Maternal effects transmitted by parents that had eaten Bt-treated artificial diet as larvae had negative effects on embryogenesis, adult fertility, or both, and reduced the ability of neonates to enter cotton bolls. These results provide further evidence that fitness costs associated with the evolution of resistance to Bt cotton are substantial in the pink bollworm.     

Novel polymorphic microsatellite markers developed in the cotton bollworm Helicoverpa armigera （Lepidoptera：Noctuidae）

A novel set of five polymorphic di- or trinucleofide microsatellite loci suitable for population genetic study were developed from an enriched genomic library for the pest insect cotton bollworm, Helicoverpa armigera, and cross-amplifiability of these and other published loci was tested in a closely related species, the tobacco budworm, H.assulta. The expected heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm. The observed allele numbers varies from 4 to 12 in the limited number of individuals tested. Although a large proportion of cloned microsatellite sequences are present in multi-copy in the cotton bollworm genome, the overwhelming majority of the finalized polymorphic diallelic loci are tri-nucleotide microsatellites - an unexpected outcome, which should facilitate subsequent genotyping analysis.     

转Bt基因抗虫棉对棉铃虫拒食作用及其机理研究

转Bt基因抗虫棉对 3 ,4,5龄棉铃虫幼虫的抗性表现形式为拒食作用 ,且随幼虫龄期的增加拒食作用明显降低 ,其中对 5龄幼虫的拒食作用很低。取食Bt棉后 ,3 ,4,5龄棉铃虫幼虫中肠消化酶比活力均较对照有所减退 ,且随幼虫龄期的增加减退率明显降低 ,其中对 5龄幼虫减退率最低。由此 ,解释了Bt棉对 3龄及 3龄以上棉铃虫幼虫抗性表现形式、抗性随不同幼虫龄期的差异性 ,及其抗性差异性的消化机理。     

Cytochrome P450 monooxygenases in the cotton bollworm (Lepidoptera: Noctuidae): tissue difference and induction

Differences in the microsomal P450 monooxygenase system and its inducibility by pentamethylbenzene (PMB) and naphathalene (NA) were investigated in midgut and fatbody tissues of the cotton bollworm, Helicoverpa armigera (Boddie), larvae. Orthogonal array design was used to establish the optimal conditions for measuring Aldrin epoxidation (AE). The optima for AE were similar for the midgut and the fatbody at a temperature of 30 degrees C, pH 7.4, and a time of 10 min. In comparison to fatbody, the midgut had higher levels of total cytochrome P450s, p-nitroanisole O-demethylation (ODM) and AE. In vivo administration of 0.2% PMB or 0.2% NA resulted in higher microsomal protein content and levels of total cytochrome P450 as well as the two examined monooxygenase activities. Total cytochrome P450 and ODM activity were induced to a greater degree in the fatbody. In the midgut, PMB was significantly more effective on ODM than NA. Differences existed in SDS-PAGE profiles between the midgut and the fatbody. The induction of the midgut with PMB and of the fatbody with NA and PMB resulted in marked intensification of the protein bands with molecular masses of 59,100, 53,400, 50,400 Da.     

棉铃虫对转Bt基因棉的抗性筛选及遗传方式的研究

通过室内筛选获得了棉铃虫Helicoverpa armigera (Hübner)对转Bt基因棉的抗性种群,并在此基础上进行了抗性遗传方式的研究。试验结果表明：经16代筛选,棉铃虫对转Bt基因棉的抗性倍数上升到43.3倍。筛选过程中,棉铃虫7日龄幼虫的体重和成虫羽化率变化明显,被选为确定筛选剂量的标准。敏感与抗性棉铃虫杂交,正交、反交的显性度都小于0、杂交过程中雌雄性比基本接近1∶1、假设抗性基因为单基因时的χ²值较低,因此,初步认为棉铃虫对转Bt基因棉的抗性是常染色体单基因控制的不完全隐性遗传。     

Diapause, migration and pyrethroid-resistance dynamics in the cotton bollworm, HeIicoverpa armigera (Lepidoptera: Noctuidae)

Abstract.

• 1 A diapause induction and duration experiment was conducted in the laboratory on Indian Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) where 79% of individuals had extended pupal periods. At 22°C and 26°C respectively, 57% and 100% of the pupae had emerged 145 days after pupation.
• 2 A mathematical model was developed to investigate the interactions between diapause, migration and pyrethroid resistance frequency development in both eastern Australia and Andhra Pradesh, India.
• 3 The effect of diapause was different in the Australian and Indian cases, providing a mechanism to conserve resistance and susceptibility respectively.
• 4 For Australia, the model simulated discriminating dose data reasonably well and showed that resistance frequencies could rise prior to the pyrethroid window without invoking cross-resistance to non-pyrethroid insecticides applied to cotton.
• 5 The saw-toothed seasonal cycle of resistance development could be simulated in the Indian case without hypothesizing the existence of susceptible migrants.
• 6 The implications of‘refugia’populations for H.armigera insecticide resistance management programmes are discussed.

     

棉铃虫卵巢形态与卵子发生过程观察

害虫发生高峰期、 发生量的准确预测和田间防治适期的确定与种群雌虫卵巢结构及卵子发生过程密切相关。为了明确棉铃虫Helicoverpa armigera卵巢结构及卵子发生过程, 本研究利用光学体视显微镜和透射电子显微镜, 对棉铃虫成虫卵巢管和卵子的超微结构进行了研究, 并确定了发育级别划分标准。结果表明: 根据卵巢的形状、 卵的产生过程、 卵黄沉积情况等将棉铃虫卵巢发育程度分为6个级别, 即发育初期(0级)、 卵黄沉积前期(Ⅰ级)、 卵黄沉积期(Ⅱ级)、 成熟待产期(Ⅲ级)、 产卵盛期(Ⅳ级)和产卵末期(Ⅴ级)。根据卵子发生过程中滋养细胞、 卵母细胞的变化, 将卵子发生期分为3个阶段： 卵黄发生前期、 卵黄发生期和卵黄成熟期。本研究首次对棉铃虫的卵子发生进行电子显微观察, 并完善了棉铃虫卵巢发育的分级标准, 为进一步研究棉铃虫的生殖发育机理提供了理论参考, 对田间棉铃虫种群发生期和发生量的预测预报也有重要的实践参考价值。     

Gene-flow between populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) is highly variable between years

Both large and small scale migrations of Helicoverpa armigera Hübner in Australia were investigated using AMOVA analysis and genetic assignment tests. Five microsatellite loci were screened across 3142 individuals from 16 localities in eight major cotton and grain growing regions within Australia, over a 38-month period (November 1999 to January 2003). From November 1999 to March 2001 relatively low levels of migration were characterized between growing regions. Substantially higher than average gene-flow rates and limited differentiation between cropping regions characterized the period from April 2001 to March 2002. A reduced migration rate in the year from April 2002 to March 2003 resulted in significant genetic structuring between cropping regions. This differentiation was established within two or three generations. Genetic drift alone is unlikely to drive genetic differentiation over such a small number of generations, unless it is accompanied by extreme bottlenecks and/or selection. Helicoverpa armigera in Australia demonstrated isolation by distance, so immigration into cropping regions is more likely to come from nearby regions than from afar. This effect was most pronounced in years with limited migration. However, there is evidence of long distance dispersal events in periods of high migration (April 2001-March 2002). The implications of highly variable migration patterns for resistance management are considered.     

棉铃虫漆酶2基因的分子鉴定

【目的】黑化反应在昆虫表皮骨化以及免疫防御过程中起着重要作用, 酚氧化酶是黑化反应中的关键酶类, 漆酶2 (laccase2, LAC2)是酚氧化酶的一种, 在昆虫变态发育和免疫系统中起着重要的作用。本研究旨在探讨LAC2在棉铃虫Helicoverpa armigera表皮骨化中表达模式及激素调控作用。 【方法】采用PCR及RACE的方法, 从棉铃虫5龄幼虫中得到了lac2 cDNA全序列。利用荧光定量PCR、 激素处理及RNA干扰方法, 对LAC2的表达模式差异和激素调控作用进行分析。【结果】序列分析表明, lac2 cDNA全长3 221 bp, 编码框长度为2 268 bp, 编码756个氨基酸残基。发育时序表达分析发现, lac2在幼虫各龄期表达规律相似, 均在蜕皮期高水平表达, 在5龄96 h转录水平达到最高峰。组织表达结果分析, lac2基因在幼虫表皮和成虫卵巢以及触角表达量较高。激素处理实验发现, 保幼激素类似物（methoprene）对lac2基因转录有抑制作用; 蜕皮激素（20-hydroxyecdysone）则促进其表达。进一步利用RNA干扰蜕皮激素受体EcR （ecdysone receptor）和USP （ultraspiracle isoform）基因发现, 干扰后蜕皮激素受体的表达明显受到抑制, 同时lac2基因的表达也显著受到抑制, 表明蜕皮激素调控lac2基因转录。【结论】这些结果为进一步研究漆酶在昆虫表皮的骨化以及免疫防御等方面不同的生理功能提供理论依据。     

Female moths of cotton bollworm (Lepidoptera: Noctuidae) captured by waterbasin traps baited with synthetic female sex pheromone

Cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is one of the most important pest insects in cotton fields in China. Female moths were captured by waterbasin traps with a synthetic female sex pheromone blend in cotton fields over three years. The blend contained (Z)‐11‐hexadecenal and (Z)‐9‐hexadecenal with a ratio of 97:3. Each pheromone dispenser was impregnated with 2.0 mg of pheromone blend and 0.2 mg of antioxidant dissolved with 0.1 mL of hexane, and there was a control dispenser with a similar amount of antioxidant and solvent only. Waterbasin traps were deployed in three configurations in the fields. ‘A’ was pheromone traps only, ‘B’ was both pheromone and control traps, ‘C’ was control traps only. (i) In four plots of ‘A’, the average weekly female catch was 1.5, and more females were captured by centrally located pheromone traps, (ii) In three plots of ‘Brsquo;, control traps also captured female as well as male moths, but average weekly female catches of control traps was significantly lower than that in pheromone‐baited traps. (iii) There were significant linear relationships between the average weekly female catch and the corresponding layer in pheromone‐baited traps in both ‘A’ and ‘B’ plots, and in quadratic equations in control in ‘B’ plots. (iv) With the increase of the interval of traps, average weekly female catches per trap increased but average weekly female catches per hectare decreased. (v) Among the female moths captured by pheromone traps, 88.3% were mated female moths which each containing 1.46 spermatophores, while in control traps 86.9% of the mated female moths had 0.90 spermatophores. There was a significant difference between the average numbers of spermatophores of mated females in pheromone traps and in controls.     

A review on biological interactions and management of the cotton bollworm,Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is one of the most damaging insect pests globally, causing estimated global economic losses of over 3 billion US dollars annually. Crops most affected include cotton, tomato, soybean, grain crops such as corn and sorghum, chickpea and other pulses. Adults of this species possess strong migratory abilities (>2000 km), high fecundity and rapid reproductive rates; completing 4–6 generations per year in most cropping regions. Furthermore, the larvae are polyphagous, with a wide and diverse host range and possess the ability to enter diapause in order to survive adverse climatic conditions. At present, it is distributed across most of Oceania, Asia, Africa and southern Europe and has recently spread to South America. Various control measures have been trialled or proposed for the treatment of this pest, including synthetic insecticides, phytopesticides, microbial pesticides, macro-biocontrol agents (both parasitoids and predators) and the development of genetically modified crops (e.g. Bt cotton). Successful control necessitates the use of an integrated pest management (IPM) approach, wherein biological, chemical and physical control measures are combined for the greatest control efficacy.