Cytolytic differences among lepidopteran cell lines exposed to toxins ofBacillus thuringiensis subst.Kurstaki. (HD-263) andAizawai (HD-112): Effect of aminosugars and N-glycosylation

Summary Comparison of lytic-dose response behavior of seven lepidopteran cell lines to the activated delta-endotoxin polypeptides ofBacillus thuringiensis subspecieskurstaki (HD-263) andaizawai (HD-112) indicated distinct differences among the lines. The lines derived fromSpodoptera speciesS. exigua (URC-SE-1A) andS. littoralis (UIV-SL-575) were more susceptible to lysis byaizawai towin (Bta) thankurstaki toxin (Btk) as were cells from theLymantria dispar line (IPLB-LD652Y). However, the concentrations of Bta required for lysis of 50% of URC-SE-1A and IPLB-LD652Y cells (LC₅₀) were 0.2 to 0.8 μg/ml compared to 5 to 9 μg/ml for UIV-SL-575 cells. In comparison, Btk LC₅₀ concentrations for the three lines were similar (14 to 19 μg/ml). Cells fromS. frugiperda (IPLB-SF-21AE) andTrichoplusia ni (TN368) were similar in their response to Bta (LC₅₀=2.5 to 3.7 μg/ml) and Btk (LC₅₀=1.0 to 2.8 μg/ml) whereas the lines derived fromHeliothis spp. were the least susceptible to both toxins. The LC₅₀ concentrations for Bta with theH. zea line (IPLB-HA-1075) andH. virescens line (BCIRL-HV-AM1) were >50 μg/ml and for Btk were >50 μg/ml and 42 to 50 μg/ml, respectively, yet for both lines Btk was the more cytolytic. Cytolysis of TN368 cells could be inhibited to varying extents by preincubation of the toxins with the aminosugars of galactose, mannose, and glucose and theirN-acetyl derivatives. The unsubstituted hexoses were not inhibitory. The order of decreasing inhibitory effectiveness was the same for both toxins regardless of the derivative species and followed the order galactose, mannose, and glucose. Also, inhibition of cytolysis could be achieved to varying extents by assaying cells grown in medium with tunicamycin. Lysis with Btk was inhibited 68 and 37% using treated cells of TN368 and IPLB-LD652Y, respectively; however, no inhibition was observed with URC-SE-1A cells. Further, no inhibition of Bta-mediated lysis was obtained with tunicamycin-grown cells of the three lines.     

Starch processing wastewater as a new medium for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL⁻¹ and higher entomotoxicity of 1,050 IU μL⁻¹ were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.     

受体介导的鳞翅目昆虫对Bt毒素抗性机制进展

苏云金芽孢杆菌作为一种对人畜安全、环境友好型绿色杀虫剂在全球被广泛使用。Bt毒素与昆虫中肠上特定毒素受体结合并发挥作用,形成毒素穿孔导致昆虫死亡是其重要的杀虫机制之一,靶标害虫对Bt毒素产生抗性是制约转Bt作物长期有效种植和Bt毒素持续使用的重要因素。文中从鳞翅目昆虫中肠细胞Bt毒素重要受体的研究阐述昆虫对Bt的抗性机制,为Bt抗性机制的深入研究和对害虫的防控与治理提供了一定的理论参考。     

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the Cry1A class of Bt toxins have been identified: an aminopeptidase N (APN-1) and a 270 kDa anionic glycoconjugate (BTR-270). Studies have shown that APN-1 has a relatively weak affinity and a very narrow specificity to Cry1Ac, the only Cry1A toxin that it binds. In contrast, BTR-270 binds all toxins that are active against L. dispar larvae, and the affinities for these toxins to BTR-270 correlate positively with their respective toxicities. In this study, an immunohistochemical approach was coupled with fluorescence microscopy to localize APN-1 and BTR-270 in paraffin embedded midgut sections of L. dispar larvae. The distribution of cadherin and alkaline phosphatase in the gut tissue was also examined. A strong reaction indicative of polyanionic material was detected with alcian blue staining over the entire epithelial brush border, suggesting the presence of acidic glycoconjugates in the microvillar matrix. The Cry1A toxin-binding sites were confined to the apical surface of the gut epithelial cells with intense labeling of the apical tips of the microvilli. APN-1, BTR-270, and alkaline phosphatase were found to be present exclusively along the brush border microvilli along the entire gut epithelium. In contrast, cadherin, detected only in older gypsy moth larvae, was present both in the apical brush border and in the basement membrane anchoring the midgut epithelial cells. The topographical relationship between the Bt Cry toxin-binding molecules BTR-270 and APN-1 and the Cry1A toxin-binding sites that were confined to the apical brush border of the midgut cells is consistent with findings implicating their involvement in the mechanism of the action of Bt Cry toxins.     

A Comparison of Soil Microbial Community Structure, Protozoa and Nematodes in Field Plots of Conventional and Genetically Modified Maize Expressing the Bacillus thuringiens is CryIAb Toxin

Field trials were established at three European sites (Denmark, Eastern France, South-West France) of genetically modified maize (Zea mays L.) expressing the CryIAb Bacillus thuringiensis toxin (Bt), the near-isogenic non-Bt cultivar, another conventional maize cultivar and grass. Soil from Denmark was sampled at sowing (May) and harvest (October) over two years (2002, 2003); from E France at harvest 2002, sowing and harvest 2003; and from SW France at sowing and harvest 2003. Samples were analysed for microbial community structure (2003 samples only) by community-level physiological-profiling (CLPP) and phospholipid fatty acid analysis (PLFA), and protozoa and nematodes in all samples. Individual differences within a site resulted from: greater nematode numbers under grass than maize on three occasions; different nematode populations under the conventional maize cultivars once; and two occasions when there was a reduced protozoan population under Bt maize compared to non-Bt maize. Microbial community structure within the sites only varied with grass compared to maize, with one occurrence of CLPP varying between maize cultivars (Bt versus a conventional cultivar). An overall comparison of Bt versus non-Bt maize across all three sites only revealed differences for nematodes, with a smaller population under the Bt maize. Nematode community structure was different at each site and the Bt effect was not confined to specific nematode taxa. The effect of the Bt maize was small and within the normal variation expected in these agricultural systems.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

苏云金芽胞杆菌Sigma54和CcpA共同调控的基因鉴定

【目的】通过综合分析苏云金芽胞杆菌(Bacillus thuringiensis)HD73菌株Sigma54缺失突变体的转录组数据和蜡样芽胞杆菌(Bacillus cereus)ATCC 14579菌株CcpA缺失突变体的转录组数据,并进行启动子与CcpA蛋白的体外结合验证,明确Bt HD73菌株中Sigma54和CcpA共同调控的基因,丰富了对微生物的代谢调控网络的认识。【方法】以转录组测序结果为基础,通过基因同源性的比对在Bt HD73菌株中寻找受Sigma54和CcpA共同调控的基因,在这些基因中找到具有cre序列的启动子,通过凝胶阻滞验证这些启动子与CcpA蛋白的结合。【结果】Bt HD73菌株中有31个基因受Sigma54和CcpA共同调控,其中14个基因的启动子序列包含cre序列,这些启动子都可以与CcpA蛋白发生体外结合。【结论】Bt HD73菌株中有14个基因直接受CcpA的调控,同时其转录受Sigma54的控制。     

Barley-based medium for the cost-effective production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Summary Studies conducted for the multiplication of Bacillus thuringiensis (Bt) using barley Hordeum vulgare as the carbon source led to the development of a protocol for the cost-effective, mass production of Bt. The production employs the simple shake flask method and can be easily adopted with a production potential of 1.5 kg Bt per day approximately at an overall production cost of Rs. 360 kg^-1 (8 US dollars). The protocol is suitable for promoting localized production of Bt at the village/district level. The product when tested as 0.1% (w/v) spray against the castor semilooper, Achoea janata proved highly effective, causing immediate feeding cessation of the larvae followed by 85% and 100% mortality by 48 and 72 h after treatment, respectively.     

粘虫颗粒体病毒增效蛋白在苏云金芽胞杆菌中的表达及增效活性

【目的】在苏云金芽胞杆菌(Bacillus thuringiensis, Bt)中表达截短后的转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus-Ps, PuGV-Ps)增效蛋白,为构建增效Bt工程菌提供理论基础。【方法】通过对截短后增效蛋白的密码子进行优化,构建增效蛋白及其融合蛋白表达载体,分析不同启动子指导下增效蛋白表达量的变化,明确增效蛋白对Bt的增效活性。【结果】本研究构建了表达载体pHTP_cry1AcCoEn81、 pHTRHCoEn81和pHTNCCoEn81, SDS-PAGE结果显示pHTP_cry1AcCoEn81和pHTNCCoEn81分别可以产生81 kDa和134 kDa的重组蛋白。启动子Pcry1Ac和P_cry8E指导下的增效蛋白表达量和重组增效蛋白产量均无显著性差异。生物测定结果表明,重组增效蛋白可以显著增加Bt对小菜蛾的杀虫活性。【结论】研究结果表明,密码子优化的PuGV-Ps增效蛋白可以在Bt中表达并具有显著增效活性,为高效苏云金芽胞杆菌工程菌的构建及...     

Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

Bt新菌株资源的采集与鉴定

【目的】寻找对致倦库蚊高效的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)杀蚊菌株新资源。【方法】从福建省的武夷山自然保护区、建阳、建瓯、浦城等多个地区采集土壤样品,采用热处理法从土壤中分离Bt菌株,并测定其对致倦库蚊活性的效果。【结果】从125份土壤样品中分离出71株Bt菌株,经生物测定得到4株对致倦库蚊有效菌株(QQ13、QQ42、QQ66和QQ92)。其中,QQ66和QQ92有较高的毒性,均有几丁质酶基因,没有检测到cry1、cry1Ⅰ、cry2、cry4、cry5、cry6、cry7、cry8、cry9、cry10和cry11基因,在75～100 ku处各有一条杀虫晶体蛋白条带。【结论】采集和鉴定到的Bt新菌株资源将对致倦库蚊的生物防治起到促进作用。     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.     

Screening of <Emphasis Type="Italic">cry2</Emphasis> genes in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates from Argentina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identification of cry2 genes from Bacillus thuringiensis (Bt) was established. Strains from different sources of Argentina were analyzed to study the distribution of cry2 genes. The results showed that cry2Aa/cry2Ab profile was the most abundant irrespective of source and represented 56 of 59 Bt isolates (94.9%). Three different cry2 profiles were found in this collection, one of which was novel.     

Comparison of the activities of Bacillus thuringiensis mosquitocidal crystal proteins on mosquito cell lines (Diptera,Culicidae), using two colorimetric methods

Two colorimetric methods were compared for assaying the cell toxicity of soluble crystal proteins from five mosquitocidal serovarieties of Bacillus thuringiensis: israelensis, jegathesan, medellin, darmstadiensis and fukuokaensis. In mosquitoes cell lines from Culex quinquefasciatus, Aedes aegypti and Anopheles gambiae, all the crystal proteins were equally toxic. Both colorimetric methods MTT (tetrazolium salt) and Alamar Blue gave similar results, but the Alamar Blue method was simpler, faster, cheaper, and could be used to take readings from the same cell population at various time points. The most active crystal proteins were those of B. thuringiensis israelensis, followed by those of B. thuringiensis jegathesan, and B. thuringiensis medellin, which were much less active.     

AFLP fingerprinting and genotypic characterization of some serovars of Bacillus thuringiensis

Amplified fragment length polymorphism (AFLP)-based fingerprinting of 24 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 13 EcoR1 (+2) and Mse1 (+3) primer combinations for genotypic characterization. A high degree of polymorphism was established among the Bt serovars. A total of 1107 fragments ranging from 30–850 bp were generated out of which 1106 were polymorphic. Discrimination rates of different primer combinations at various band levels (1–5) among different Bt serovars were more than 90%. Cluster analysis revealed very low similarity values, ranging from 7–50%, among the Bt serovars indicating their remarkable genetic diversity. AFLP analysis establishes the molecular relatedness between the serovars and serotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Peritrophic membrane contribution to Bt Cry δ-endotoxin susceptibility in Lepidoptera and the effect of Calcofluor

In an effort to determine a reason for differing susceptibilities to Bacillus thuringiensis (Bt) Cry δ-endotoxins amongst lepidopteran species, the peritrophic membrane (PM), and a number of agents that target the PM, were investigated to determine their effect on the efficacy of Bt toxins. In particular Calcofluor is able to disrupt the PM that acts as a barrier to microorganisms. Although Bt toxins have been shown to traverse the PM in some lepidopteran species, new data shows that toxins can bind the PM. Lepidopteran larval PMs also vary in thickness and composition that may determine the passage of Bt toxins. In non-susceptible insects the toxin associates with PM proteins and frass and is thought to be retained by the PM and then excreted before binding to the exposed target midgut membrane. However, the addition of Calcofluor to Bt toxins at an LC₅₀ for the recipient species did not result in an increase in the efficacy of the toxin. It is evident that Calcofluor does disrupt the PM but the toxin preferentially binds PM fragments and is excreted instead of binding the exposed target midgut membrane, therefore having little toxic effect. This study therefore concludes that Calcofluor is not as suitable as other toxin enhancing agents such as chitinase.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

Mechanisms for Bt Toxin Resistance and Increased Chemical Pesticide Susceptibility in Cry1Ac10-resistant Cultured Insect Cells

Cabbage looper moth (Trichoplusia ni) cell line BTI-Tn-5B1-4 (TnH5) has developed high-level resistance (>1000 fold) by the selection of Bt Cry1Ac10 toxin. In order to examine mechanisms of resistance to Cry1Ac10 toxin (biological pesticide), both general esterase activities and cell tolerance to osmotic lysis were compared between non-selected Cry1Ac10-susceptible Trichoplusia ni cell line TnH5-S and Cry1Ac10-resistant Trichoplusia ni cell line TnH5-R selected by Bt Cry1Ac10. The Cry1Ac10-resistant TnH5-R cells had lower general esterase activity than the non-selected TnH5-S cells, and the esterase isozyme bands for the Cry1Ac10-resistant TnH5-R cells were much weaker than that for the non-selected TnH5-S cells. Both activated Cry1Ac10 toxin and multi-toxin from Bacillus thuringiensis subsp. aizawai GC-91 (an engineering bacterium) could not inhibit the esterase activity both in the Cry1Ac10-susceptible and Cry1Ac10-resistant cells, but two chemical pesticides, chlopyrifos and methomyl, could greatly inhibit the esterase activities both in the TnH5-R and TnH5-S cells. On the other hand, cell tolerance to osmotic lysis caused by hypotonic solution for the Cry1Ac10-resistant TnH5-R cells was higher than that for the non-selected TnH5-S cells (2.5×). Based on these results, we made the following conclusions. The general esterase activities in the Cry1Ac10-resistant TnH5-R cells was not related to Bt Cry1Ac10 resistance, but the susceptibility to the two tested chemical pesticides increased in TnH5-R cells because of their lower esterase activity. The increase of cell tolerance to osmotic lysis for the Cry1Ac10-resistant TnH5-R cells may be one of the mechanisms for Bt toxin resistance because midgut cells of insects are also disrupted by an osmotic lysis caused by Bt toxin.     

Tritrophic Choice Experiments with Bt Plants,the Diamondback Moth (Plutella xylostella) and the parasitoid Cotesia plutellae

Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes. Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests. A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implementing measures for the conservation of parasitoid populations. It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations. The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins. However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P. xylostella. Cotesia plutellae is an important endoparasitoid of P. xylostella larvae. Although unable to survive in Bt-susceptible P. xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C. plutellae is able to complete its larval development in Bt-resistant P. xylostella larvae. Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C. plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts. This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage. Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P. xylostella larvae on Bt plants as on wildtype plants. In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant. The implications for integrated pest management and the evolution of resistance to Bt in P. xylostella are discussed.     

Reduction of resistance of Culex pipiens larvae to the binary toxin from Bacillus sphaericus by coexpression of cry4Ba from Bacillus thuringiensis subsp. israelensis with the binary toxin gene

The cry4Ba gene from Bacillus thuringiensis subsp. israelensis and the binary toxin gene from B. sphaericus C3-41 were cloned together into a shuttle vector and expressed in an acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. Transformed strain Bt-BW611, expressing both Cry4Ba protein and binary toxin protein, was more than 40-fold more toxic to Culex pipiens larvae resistant to B. sphaericus than the transformed strains expressing Cry4Ba protein or binary toxin protein independently. This result showed that the coexpression of cry4Ba of B. thuringiensis subsp. israelensis with B. sphaericus binary toxin gene partly suppressed more than 10,000-fold resistance of C. pipiens larvae to the binary toxin. It was suggested that production of Cry4Ba protein and binary toxin protein interacted synergistically, thereby increasing their mosquito-larvicidal toxicity.