Toxicity of chlorantraniliprole to Cry1Ac-susceptible and resistant strains of Helicoverpa armigera

Transgenic Bt cotton expressing Cry1Ac is important in controlling various agricultural pests, including Helicoverpa armigera. Especially for transgenic crops that are cultivated in large expanses, avoiding resistance development is a key for ensuring sustainability of Bt technologies. Integrated pest management, in which transgenic crops are strategically combined with rational pesticide use, may help to prevent H. armigera resistance acquisition in Bt cotton. In this study, we evaluated the toxicity of a novel insecticide (chlorantraniliprole) on Cry1Ac-susceptible and resistant individuals of H. armigera. More specifically, we assessed the effect of chlorantraniliprole on the activity of two enzymes and conducted laboratory bioassays to determine its toxicity on H. armigera larvae. Chlorantraniliprole increased esterase and glutathione-S-transferase activities in Cry1Ac susceptible and resistant populations of H. armigera. Cry1Ac resistant populations XJ-F (Cry1Ac resistance ratio 21.8-fold), XJ-10.0 (95.8-fold) and BTR (3536.5-fold) did not show cross-resistance to chlorantraniliprole, with LC₅₀ values of 0.0733 (μg/mL) in XJ-F, 0.0545 (μg/ml) in XJ-10.0 and 0.0731 (μg/mL) in BTR, which were close to that in the susceptible strain 96S (0.0954 μg/mL). Our work shows that chlorantraniliprole could be considered to be integrated in Bt cotton management schemes to delay the H. armigera resistance development.     

Cry1Ac的绿色荧光蛋白GFP标记及其在棉铃虫幼虫中肠的定位分布

为了明确Cry1Ac蛋白在棉铃虫体内与中肠组织的相互作用,采用重叠PCR方法将Bt-cry1Ac基因和绿色荧光蛋白GFP基因融合,构建含Cry1Ac毒蛋白和绿色荧光蛋白GFP原核表达载体,并在大肠杆菌大量表达。利用荧光显微镜观察发现,表达Cry1Ac-GFP融合蛋白的大肠杆菌在蓝光激发下发出绿色荧光。将含有融合蛋白的菌液拌入人工饲料饲喂3龄棉铃虫幼虫96h,取棉铃虫幼虫中肠做冰冻切片并在荧光显微镜下观察。结果显示,取食含有Cry1Ac-GFP融合蛋白饲料的棉铃虫幼虫中肠能够发出强烈荧光。比较Cry1Ac杀虫蛋白敏感和抗性棉铃虫幼虫中肠的发光部位,敏感棉铃虫幼虫的中肠围食膜已经消失,肠壁细胞发出强烈的荧光,而抗性棉铃虫的围食膜较健全并发出荧光。     

棉铃虫对Cry1Ac抗性的稳定性及其对适合度的影响

为明确棉铃虫Helicoverpa armigera（Hübner）对苏云金芽胞杆菌Bacillus thuringiensis（Bt）Cry1Ac毒蛋白抗性的稳定性及其适合度变化,利用生物测定的方法研究了Cry1Ac抗性品系棉铃虫转到正常饲料饲养后的抗性衰退及再次筛选后抗性的恢复情况,并比较了敏感、抗性和抗性衰退后各品系间的适合度差异。在失去选择压的情况下,高抗品系棉铃虫对Cry1Ac的抗性迅速衰退,经过4代后抗性水平由最初的3626.67倍下降到1436.67倍;到第12代时抗性水平已低于10倍,随后品系保持较稳定的低抗水平;当重新进行抗性再筛选时,其抗性水平可快速恢复,抗性倍数快速回升,5代后恢复到1123.33倍。与敏感品系相比,高抗棉铃虫品系的适合度明显降低,相对适合度仅为0.33,但转到正常饲料连续饲养14代后,棉铃虫适合度明显上升,相对适合度为0.87,主要表现为卵孵化率和幼虫存活率等显著提高。     

Bacillus thuringiensis Cry1Ab, but not Cry1Aa or Cry1Ac, disrupts liposomes

We evaluated the ability of Cry1Aa9, Cry1Ab4, and Cry1Ac1 insecticidal toxins from Bacillus thuringiensis to destroy liposomes. Cry1A toxins are thought to form pores in midgut apical cell membranes (BBMV), thereby disrupting midgut cells. Liposomes containing fluorescent calcein were prepared using phosphatidylcholine (PC) and phosphatidylserine (PS) (PC/PS-Liposomes) or PC alone (PC-Liposomes). Cry1Ab (1.4 μM), but not Cry1Aa or Cry1Ac, disrupted PC/PS-Liposomes and PC-Liposomes. PC/PS-Liposomes containing cholesterol and oligosaccharylceramide from Plutella xylostella midgut were damaged even more extensively by Cry1Ab, but the inclusion of either lipid alone had no effect. The initial velocity of Cry1Ab-mediated liposome disruption increased 17-fold when liposomes were prepared with Triton X-100-soluble proteins from Bombyx mori BBMV and PC (PC/Proteo-Liposomes), and Cry1Aa and Cry1Ac also caused slight disruption. These data suggest that Cry1Ab achieves higher penetration into PC/PS-Liposomes, PC-Liposomes, and PC/Proteo-Liposomes compared with Cry1Aa or Cry1Ac and that Cry1Ab may interact with membrane proteins.     

Cry1Ac和Cry2Ab蛋白对大草蛉生长发育及酶活力的影响

为明确转Cry1Ac和Cry2Ab基因棉花对大草蛉的影响,运用Bt蛋白与人工饲料混合的方法,以大于转基因棉花叶片中蛋白含量20倍的剂量饲喂大草蛉初孵幼虫,初步研究了Cry1Ac和Cry2Ab对大草蛉生物学参数和消化酶、解毒酶活性的影响。结果表明：取食含Bt蛋白饲料的大草蛉幼虫的发育历期、体重、蛹重、成虫体重、羽化率等生物学参数与对照相比均没有显著差异;在大草蛉幼虫体内可以检测到含量较高的Cry1Ac和Cry2Ab蛋白,分别为974.92~1 282.39 ng/g鲜重和5 592.62~6 082.92 ng/g鲜重,而在大草蛉成虫体内检测到的Cry1Ac和Cry2Ab蛋白含量非常低,分别为0.29~0.39 ng/g鲜重和50.34~56.71 ng/g鲜重;取食含Bt蛋白的饲料对大草蛉幼虫的类胰蛋白酶、类胰凝乳蛋白酶、氨肽酶和谷胱甘肽-S-转移酶活性有一定的影响,但对大草蛉成虫影响与对照差异不显著。表明大草蛉取食含Bt蛋白的人工饲料后,虽然体内可以检测到一定含量的Bt蛋白,但对大草蛉的生长发育并没有显著的直接不利影响。     

钙粘蛋白氨基酸点突变介导红铃虫对Cry1Ac的抗性

为明确室内筛选的红铃虫Pectinophora gossypiella抗性品系AQ-R对Cry1Ac的抗性机制,采用室内生物测定法明确该品系对Cry1Ac和Cry2Ab的敏感性,通过遗传杂交和基因克隆分析抗性基因的显隐性及突变位点,并进行细胞学试验分析突变蛋白的亚细胞定位。结果显示：红铃虫AQ-R抗性品系对Cry1Ac的抗性倍数为181.67倍,对Cry2Ab没有交互抗性;该品系携带了一种新型的隐性钙粘蛋白抗性等位基因PgCad1,其编码蛋白的钙粘蛋白重复区、前蛋白区和近膜区共发生了17个氨基酸替换。表达野生型PgCad1-s基因的Hi5细胞对Cry1Ac敏感,且钙粘蛋白定位于细胞膜;而表达抗性PgCad1-r基因的Hi5细胞则对Cry1Ac不敏感,且钙粘蛋白错误定位到内质网。表明钙粘蛋白氨基酸点突变能导致其定位错误,从而促成红铃虫AQ-R品系对Cry1Ac产生抗性。     

Does cotton bollworm show cross-resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab? A mini review

Since 1996, transgenic Bacillus thuringiensis(Bt) cotton has been commercially grown in numerous countries in an effort to stem the losses caused by key lepidopteran pests. However, the development of pest resistance to Bt toxins has jeopardized the continued utilization of Bt cotton. As a strategy designed to circumvent the development of resistance, Bt cotton varieties expressing two or more toxins targeting the same pest have been introduced. Nevertheless, from the perspective of long-term planting of Bt cotton, the potential risk of cross-resistance to these Bt toxins is a threat that cannot be ignored. In this paper, we review current research(including that based on the analysis of protein binding sites and resistance genes) on the resistance of cotton bollworm(Helicoverpa armigera) to the Bt toxins Cry1 Ac and Cry2 Ab and the interrelationship between these toxins. On the basis of existing evidence, we assume that the actions of Cry1 Ac and Cry2 Ab against cotton bollworm are not completely independent, and then propose the "resistance-associated gene mutation potential hypothesis". Although the mechanisms underlying the resistance of pests to Bt toxins are yet to be comprehensively elucidated, this hypothesis could undoubtedly have important implications for adopting "pyramid" strategy in the future. Further research is recommended to devise strategies to retard the development of H. armigera resistance to Bt cotton, either using different Bt toxins or their various combinations.     

我国棉铃虫对Bt蛋白Cry1Ac抗性现状及分子机制研究进展

棉铃虫Helicoverpa armigera是一种全球性的重要农业害虫,主要为害棉花、玉米和大豆等作物。长期种植单价Bt棉花（表达Cry1Ac蛋白）会使棉铃虫田间种群承受单一、持续的选择压力,必然会导致棉铃虫对Cry1Ac的抗性发生演化。该文概述我国棉铃虫田间种群对Cry1Ac的抗性现状、自然庇护所对棉铃虫Cry1Ac抗性演化的延缓作用以及棉铃虫对Cry1Ac抗性的遗传多样性,并对今后我国关于棉铃虫Bt抗性的治理对策进行了展望。     

四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化

为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。     

DNA-based screening for an intracellular cadherin mutation conferring non-recessive Cry1Ac resistance in field populations of Helicoverpa armigera

A number of cadherin mutants conferring resistance to Bt toxin Cry1Ac have been reported in three major lepidopteran pests, including Helicoverpa armigera. Unlike most of the cadherin mutants conferring recessive resistance, an allele (r₁₅) with a 55aa deletion in the intracellular domain of cadherin (HaCad) was previously identified to cause non-recessive resistance to Cry1Ac in H. armigera. In the present study, a DNA-based PCR method was developed to screen the r₁₅ allele from field populations of H. armigera collected from the main cotton planting areas of China in 2011 and 2012. Three heterozygous r₁₅ alleles were detected from 562 moths collected from northern China (with intensive Bt cotton planting), and r₁₅ allele frequency was estimated to be 0.0027. However, no r₁₅ allele was detected from 314 moths collected from Xinjiang (with limited Bt cotton use). Although all the r₁₅ alleles have the same deletion in the cDNA sequence, at least four different indels causing loss of exon 32 have been detected in the genomic DNA sequences flanking exon 32 of HaCad. Multiple origins of the r₁₅ alleles illustrate parallel genotypic adaption of H. armigera to the selection pressure of Bt cotton.     

Prohibitin,an essential protein for Colorado potato beetle larval viability,is relevant to Bacillus thuringiensis Cry3Aa toxicity

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30 kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828 pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5 days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.     

荧光增白剂对Cry1Ac抗感棉铃虫围食膜的影响及增效作用

为了提高Bt的杀虫活性,运用扫描电镜、SDS-PAGE电泳和生物测定技术,研究了荧光增白剂FB28对棉铃虫Cry1Ac抗性和敏感品系围食膜的影响及对Cry1Ac杀虫蛋白的增效作用。结果表明,正常棉铃虫围食膜表面光滑致密,没有空洞和缝隙。用含1%FB28的人工饲料饲喂刚蜕皮的5龄棉铃虫幼虫2 h后,抗感棉铃虫围食膜表面均出现许多空洞,且敏感棉铃虫的空洞明显大于抗性棉铃虫;随着处理时间的延长,围食膜上的空洞逐渐变小,13 h后恢复正常。用1%FB28离体或活体处理抗、感品系棉铃虫,均可造成围食膜蛋白被降解。用1%FB28与Cry1Ac杀虫蛋白混合饲喂抗、感棉铃虫初孵幼虫,可明显提高棉铃虫的死亡率,且对抗性品系的增效作用明显大于敏感品系,对敏感和抗性品系棉铃虫的增效比分别为2.23和9.34;1%FB28还可以明显增加对抗、感品系3龄棉铃虫幼虫的生长抑制作用。     

Genetics of Heliothis and Helicoverpa resistance to chemical insecticides and to Bacillus thuringiensis

Genetic linkage maps of Heliothis virescens and Helicoverpa armigera are being used to identify and characterize resistance-conferring genes. The insensitive acetylcholinesterase conferring resistance to organophosphorus insecticides and the insensitive sodium channel conferring resistance to pyrethroids have both been mapped in H. virescens. The linkage mapping approach permits a genetic dissection of resistance, even when the mode of action and lethal target are not precisely known, such as for the insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). We have identified and mapped a major Bt-resistance locus in a strain of H. virescens exhibiting up to 10000-fold resistance to Cry1Ac toxin and are currently developing a linkage map for H. armigera with a set of ‘anchor’ loci to facilitate comparison with H. virescens. Both species are currently experiencing their first significant selective pressure in the field by transgenic cotton expressing Cry1Ac, and timely identification of resistance mechanisms and their underlying genetic basis will be essential in successfully managing the Bt resistance that will eventually appear. ©1997 SCI     

Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India

BACKGROUND: The pink bollworm is one of the most destructive pests of cotton. Transgenic cotton producing Bt toxin Cry1Ac or a combination of Cry1Ac and Cry2Ab2 has been used effectively against this pest. However, some other insects have evolved resistance to Bt toxins in the field. During the 2007–2008 and 2008–2009 seasons, pink bollworm populations in India were surveyed to evaluate their responses to Cry1Ac and seed powder containing Cry1Ac and Cry2Ab2. RESULTS: The results provide evidence that resistance to Cry1Ac had evolved by 2008 in a population sampled from non‐Bt cotton in the Amreli district of Gujarat in western India. The median lethal concentration of Cry1Ac for five‐day‐old larvae (LC₅₀) was significantly higher for insects derived in 2008 from Amreli than for any of the other field populations tested from four locations in India. For Cry1Ac, the mean LC₅₀ for the strain derived from Amreli in 2008 was 44 times higher than for the most susceptible population. However, for seed powder of Bollgard II containing primarily Cry2Ab2, the 2008 Amreli population was only slightly less susceptible than the most susceptible population. CONCLUSIONS: The data reported here constitute the first evidence of field‐evolved resistance of pink bollworm to Cry1Ac. This initial evidence spurred more extensive evaluations during the 2009–2010 growing season, which confirmed field‐evolved resistance to Cry1Ac in Amreli. The lack of cross‐resistance to Cry2Ab2 suggests that plants producing this toxin are likely to be more effective against resistant populations than plants producing only Cry1Ac. Copyright © 2011 Society of Chemical Industry     

Histochemical analysis of Bacillus thuringiensis CrylA toxin binding to midgut epithelial cells of Bombyx mori

We analyzed the binding of the Bacillus thuringiensis insecticidal toxins, CrylAa, CrylAb and CrylAc, to midgut tissue of the silkworm, Bombyx mori with ligand blot analysis and histochemical observations. CrylAa, CrylAb and CrylAc bound to unique sets of proteins in various subcellular fractions prepared by centrifugation. CrylAa bound to various proteins in all subcellular fractions, whereas CrylAb bound to a single protein of ∼180 kDa in all fractions as shown by Western blot analysis. Cry1Ac bound to proteins which were primarily ∼100-120 kDa in all fractions. CrylA toxins were labeled with fluorescent dye and Cy3-labeled CrylAa, CrylAb and CrylAc were shown to localize primarily to the apical membrane region. However, they also localized to basement or basolateral membranes. The distribution of a 252-kDa membrane protein (P252) of the B. mori midgut, which was recently identified as a plausible candidate for receptor of CrylA toxins were also examined with histochemical methods. Substantial signals of FITC-labeled antibody against P252, even though not all, were evident in the apical cells, and these were coincident with Cy3-CrylAa and Cy3-CrylAc signals.     

The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC₅₀ and LC_99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC₅₀ and LC_99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour.     

Engineering of Bacillus thuringiensis insecticidal proteins

Bacillus thuringiensis (Bt) has been used as sprayable pesticides for many decades. Bt strains utilized in these products produce multiple insecticidal proteins to complement a narrow insect specificity of each protein. In the late 1990s, genes encoding Bt insecticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry1Ac to control Heliothis virescens (tobacco budworm). Cry1Ab was applied to corn to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry1Ac and Cry1Ab, new Bt proteins are required to overcome the resistance. In order to protect corn furthermore, it is desired to control Diabrotica virgifera (Western corn rootworm), Helicoverpa zea (corn earworm) and Spodoptera frugiperda (fall armyworm). Recently, many new Bt insecticidal proteins have been discovered, but most of them require protein engineering to meet the high activity standard for commercialization. The engineering process for higher activity necessary for Bt crops is called optimization. The seed industry has been optimizing Bt insecticidal proteins to improve their insecticidal activity. In this review, several optimization projects, which have led to substantial activity increases of Bt insecticidal proteins, are described.     

Bt-maize effects on biological parameters of the non-target aphid Sitobion avenae (Homoptera: Aphididae) and Cry1Ab toxin detection

Bt-maize crop is increasingly used worldwide and the study of ecological side effects is a major subject in this domain. Under laboratory conditions, we determined Bt-maize effects on the non-target aphid Sitobion avenae (Fabricius) (Homoptera: Aphididae). We found no significant differences between S. avenae on MON810 and the near-isogenic line when alate offspring production, apterous survivorship, longevity, intrinsic rates of natural increase (r_m), finite rates of increase and doubling times were compared. No significant differences were found between treatments for apterous pre-reproductive and reproductive periods. Additionally, we used immunological tests (ELISA) to detect Cry1Ab protein in maize leaves and S. avenae nymphs. Results showed that Bt-maize leaves expressed 0.203 (±0.05) μg Cry1Ab/g leaf tissue (Mean ± SEM). No Cry1Ab protein was present in S. avenae nymphs developing on Bt or conventional maize. We conclude that Bt-maize does not affect the development of the non-target aphid S. avenae and that Cry1Ab toxin quantities in these aphids are nil, suggesting an inconsequential risk for natural enemies of this aphid species.     

转cry1Ac玉米对亚洲玉米螟的抗性评价

目标性状的有效性评价是转Bt基因抗虫玉米研发和安全性评价研究的重要内容之一。采用酶联免疫吸附剂测定（ELISA）、室内生测和田间人工接虫鉴定研究了转cry1Ac玉米纯合群体BT-X和分离群体BT-38、BT-181、BT-43、BT-105等转化事件Cry1Ac蛋白的表达量以及对亚洲玉米螟Ostrinia furnacalis（Guenée）的室内杀虫和田间抗虫效果。转cry1Ac玉米鲜组织中Cry1Ac蛋白含量在44.07～438.00 ng/g之间,不同转化事件及不同组织之间差异显著,其中BT-X鲜花丝中Bt蛋白表达量显著低于未展开心叶。室内生测试验中除BT-43与对照没有显著差异外,取食其它转基因玉米心叶的亚洲玉米螟初孵幼虫6或7天的存活率为0～15.3%,显著低于非转基因对照;取食夏播BT-X鲜花丝6天的幼虫存活率为62.5%,显著低于Bt11及非转基因对照。田间心叶期接虫鉴定显示BT-X高抗亚洲玉米螟。说明转cry1Ac玉米BT-X、BT-181、BT-38和BT-105对亚洲玉米螟有很高的杀虫作用,BT-X田间抗虫效果良好。     

粉纹夜蛾离体细胞对Bt Cry1Ac毒素的抗性发展及其交互抗性

以CrylAc活性毒素逐代筛选抗性粉纹夜蛾离体细胞BTI-TN-5B1抗性发展速度呈S形曲线，经56代选择后，抗性比上升至1280倍。在去除选择压力后，抗性比逐渐下降。低水平抗性细胞抗性衰退较快，而高水平抗性细胞抗性衰退较缓慢。抗性比衰退至1．5倍的细胞在复筛后抗性比恢复较快。抗性细胞对苏云金杆菌工程菌GC-91(BtGC-91)、苏云金杆菌鲇泽亚种(B.t.atoazai)和苏云金杆菌库斯塔克亚种(B．t．kurstaki)的复合毒素晶体的活性毒素都具有较高的交互抗性，但对农药无交互抗性，对杆状病毒的敏感性也没发生变化。