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.     

Interaction of Cry1Ac toxin (Bacillus thuringiensis) and proteinase inhibitors on the growth, development, and midgut proteinase activities of the bollworm, Helicoverpa zea

Potential resistance development to Bt cotton in certain lepidopterans has prompted research to develop strategies that will preserve this environmental-friendly biotechnology. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against lepidopteran pests and for expanding the spectrum of control for other insects. Interactions of Bt toxin from Bacillus thuringiensis and proteinase inhibitors were investigated by monitoring growth, development, and gut proteinase activities of the bollworm, Helicoverpa zea. Several proteinase inhibitors were combined with Bt protoxin Cry1Ac in artificial diet and fed to newly molted 3rd-instar bollworm larvae to determine effects on larval body weight and length, pupation progress, and mortality rate. Major midgut proteinase activities, including caseinase, tryptic, and chymotrypsin activities, were examined after treatment. A concentration of Bt at a level causing minimal mortality (<10%), was mixed with the following proteinase inhibitors: benzamidine, phenylmethylsulfonyl fluoride (PMSF), and N-α-tosyl-l-lysine chloromethyl ketone (TLCK). When compared with controls, the synergistic effect of Bt toxin and proteinase inhibitors caused significant decreases in mean larval weight and length over time. Midgut samples tested against the substrates azocasein, α-benzoyl-dl-arginine-p-nitroanilide (BApNA), and N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA) showed significant decreases in the protease activity of larvae fed Bt plus inhibitor versus control. Interaction of Bt and proteinase inhibitors significantly retarded larval growth and resulted in developmental delay and up to 20% mortality.     

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.     

新疆地区棉铃虫自然种群对Bt棉的抗性频率监测

为监测新疆棉区棉铃虫Helicoverpa armigera(Hübner)田间种群对Bt棉的抗性频率,在2010年和2011年分别采集石河子和喀什地区莎车的棉铃虫单雌系,以Cry1Ac毒蛋白作为人工饲料,用单雌系F1/F2代法进行棉铃虫种群抗性个体检测。2010年筛选了123个石河子的棉铃虫单雌系,2 011年筛选了152个莎车的棉铃虫单雌系。两地的棉铃虫种群均没有筛选到相对平均发育级别≥0.8的抗Bt棉个体,估算出石河子和莎车的棉铃虫种群的抗性频率低于10-3。莎车F2单雌系与其对应的F1单雌系相对平均发育级别有明显差异。研究表明新疆石河子地区田间棉铃虫种群仍保持敏感状态,喀什地区田间棉铃虫种群对Bt棉的耐受性增高。     

Biochemical and physiological changes on Bacillus thuringiensis cotton after imidacloprid foliar spray

Bacillus thuringiensis cotton is a variety of cotton genetically modified to contain a gene derived from B. thuringiensis bacteria; which results in expression of toxin protein that confers resistance to bollworm complex (the most destructive pest of cotton). Introduction of Bt cotton lowered the need of insecticides, but still a number of insecticides are used for other insects like jassids, whitefly, aphids and tobacco caterpillar to which Bt gene does not provide effective control. Imidacloprid (tradename Imidacel 17.8 SL) is an insecticide designed for control of these major sucking/piercing insects that affect cotton. So in the present work we studied the post effect of imidacloprid insecticide on plant health of three Bt cotton hybrids (RCH-134, JKCH-1947, NCEH-6R) as there are reports of this insecticide causing growth and yield enhancements in absence of insect pests. Imidacloprid was first sprayed at recommended concentration (40 ml/acre) on 3 months old plants sown in randomly designed plots with three replications of each hybrid. The spray was repeated three times at 10 days interval. The level of B. thuringiensis gene expression, peroxidase activity and total phenols was measured on third day after every spray in leaves along with growth and yield of plants. The insecticide has shown to increase the level of B. thuringiensis protein, peroxidase enzyme activity, total phenols, height, number of bolls retained on plants and yield. These observations suggested that the imidacloprid treated plants showed better growth and development, thereby imidacloprid has growth enhancing effect on Bt cotton plants in addition to its insecticidal properties.     

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。     

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.     

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。     

Binding of three Cry1A toxins in resistant and susceptible strains of cotton bollworm (Helicoverpa armigera)

Evolution of resistance by pests is the greatest threat to the continuous success of theBacillus thuringiensis (Bt) toxins used in conventional sprays or in transgenic plants. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut. In this paper, binding experiments were performed with three ¹²⁵I-Cry1A toxins and the brush border membrane vesicles from Cry1Ac resistant or susceptible strains of Helicoverpa armigera. The homologous competition test showed that there was no significant difference in Cry1Ac-binding affinity, but the concentration of Cry1Ac-binding sites dramatically decreased in the resistant strain (R_t decreased from 5.87 ± 1.40 to 2.23 ± 0.80). The heterologous competition test showed that there were three Cry1Ac-binding sites in the susceptible strain. Among them, site 1 bound with all three Cry1A toxins, site 2 bound with both Cry1Ab and Cry1Ac, and site 3 only bound with Cry1Ac. In the Cry1Ac resistant strain, the binding capability of site 1 with Cry1Ab decreased and site 2 did not bind with Cry1Ac. It is suggested that the absence of one binding site is responsible for H. armigera resistance to Cry1Ac. This result also showed that the resistance fitted the “mode 1” pattern of Bt resistance described previously.     

Cry1Ac抗性亚洲玉米螟对四种Bt蛋白的交互抗性

种Bt蛋白Cry1Ac、Cry1Ab、Cry1Ah和Cry1Ie对敏感品系ACB-BtS和抗性品系ACB-AcR的毒力,结果显示,ACB-AcR对Cry1Ah的相对抗性倍数达14.9倍,有显著的交互抗性;对Cry1Ab的相对抗性倍数为4.3倍,交互抗性水平较低;对Cry1Ie的相对抗性倍数为0.9,即无交互抗性.4种蛋白对抗、感品系的EC50表明,ACB-AcR品系对Cry1Ac蛋白产生了显著的抗性,相对抗性达到32.6倍,对Cry1Ah和Cry1Ab有低水平的交互抗性,而对Cry1Ie没有交互抗性.     

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。     

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.     

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.     

Cry1Ac毒蛋白对亚洲玉米螟生长发育和种群增长的影响

以含不同浓度的Cry1Ac毒蛋白的人工饲料(不加毒蛋白为对照)饲喂亚洲玉米螟幼虫,采用实验种群生命表的方法,测定了Cry1Ac毒蛋白对亚洲玉米螟的控害作用。结果显示,含2 000.0ng/g Cry1Ac毒蛋白的人工饲料处理该虫第5天的幼虫存活率为0;200.0、20.0 ng/g CrylAc毒蛋白处理第10、15天的幼虫存活率、化蛹率、成蛾率、净增长率和种群增长指数均显著低于对照,且高浓度处理显著低于低浓度处理;200.0 ng/g CrylAc毒蛋白处理的世代平均周期和种群加倍时间显著长于其它处理;而200.0 ng/g CrylAc毒蛋白处理的周限增长率显著小于2.0、0.2 ng/g处理。表明微量的Cry1Ac毒蛋白对亚洲玉米螟的种群增长有显著影响,且随着浓度的增加控制作用增强。     

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.     

Bt稻Cry1Ab蛋白的表达和降解及对采后季节土表灰橄榄长角跳虫发生的影响

Bt水稻及其植株残体中的Bt杀虫蛋白对土壤跳虫具有潜在的生态风险。作者以Bt杂交稻东龙(DL)及非亲本对照汕优46(XY)和西农优1号(XN)为材料,采用ELISA法分析了DL各植株组织中Cry1Ab蛋白的田间表达和采后残留,并评价了采后季节DL植株秸秆还田对稻田土表跳虫发生量的影响。结果表明,在测定的各生长阶段,DL Cry1Ab在叶、茎和根系中的表达量为2.49~16.13μg/g FW;在采后季节,DL各秸秆还田植株残体中的Cry1Ab可残留较长时间,在试验第132天地表茎叶、稻桩和根系中的残留量分别为0.072、0.074和0.033μg/g DW;DL秸秆还田未对采后季节稻田土表发生量大的灰橄榄长角跳虫Entomobrya griseoolivata (Packard) 种群数量产生显著影响,该跳虫种群密度动态在DL与对照XY和XN稻田基本相似,多数调查日期发生量无显著差异。     

转基因玉米表达的Cry1Ab、PAT和EPSPS蛋白对日本通草蛉幼虫的安全风险评估

为评价我国自主研发的转基因玉米表达的Cry1Ab、PAT和EPSPS蛋白对日本通草蛉Chrysoperla nipponensis幼虫生长发育的安全风险,通过将外源蛋白混入日本通草蛉人工饲料中的方法,以加入砷酸二氢钾(KH_2AsO_4)的饲料为阳性对照,研究了日本通草蛉幼虫取食后的生长发育状况。结果表明:日本通草蛉取食含Cry1Ab和EPSPS蛋白饲料的幼虫发育历期、茧期、结茧率、羽化率及成虫体重等生物学参数与取食正常饲料处理相比均没有显著差异;而取食含PAT蛋白饲料的幼虫发育历期和结茧率分别为10.7 d和96.6%,与取食正常饲料的对照11.1 d和89.9%差异显著,即饲料中添加PAT蛋白显著提高了幼虫的存活率和发育速率;取食含KH_2AsO_4饲料的日本通草蛉幼虫不能存活到茧期,说明KH_2AsO_4具有显著的杀虫活性。ELISA检测结果表明,取食了分别添加有3种外源蛋白饲料的日本通草蛉幼虫体内可以检测到相应的Cry1Ab、EPSPS和PAT蛋白,含量分别为2 758.8~5 210.7、35 018.0~54 426.6、16.8~149.8 ng/g。表明转基因玉米所表达的Cry1Ab、EPSPS和PAT蛋白对日本通草蛉幼虫没有显著的不利影响。