Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC₅₀, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC₉₀, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC₉₀ level than at the LC₅₀ level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.     

Screening,Diversity and Partial Sequence Comparison of Vegetative Insecticidal Protein (<Emphasis Type="Italic">vip3A</Emphasis>) Genes in the Local Isolates of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Berliner

Characterization, direct sequencing of the PCR amplicon and phylogenetic relationship was done to discover a novel Vip protein genes of the Bt isolates, to improve the prospects for insect control, more Vip proteins should be sought out and researched to predict their insecticidal activity. Characterization was based on direct sequencing of PCR amplicon using primers specific to vip3A gene was presented here. 12 out of 18 isolates screened were positive for vip gene-specific primers. Homology search for the partial sequences using BLAST showed that 11 isolates had high similarity to vip3Aa gene and only one fragment with vip3Ae gene (25–100% at nucleotide and amino acid level). Phylogenetic analysis showed that the gene sequences were responsible for geographic separation for divergence within vip genes, consistent with the evaluation of distinct bacterial population. Despite the geographical distances, strains harbouring vip genes have originated from common ancestors may significantly contribute to control resistant insect pests. Some strains have evolved to be quite distinct and others remain as members of closely related groups. The reported method is a powerful tool to find novel Vip3A proteins from large-scale Bt strains which is effective in terms of time and cost. Further the Vip proteins produced by different strains of B. thuringiensis are unique in terms of the sequence divergence and hence may also differ in their insecticidal activities.     

Interaction between toxin crystals and vegetative insecticidal proteins of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in lepidopteran larvae

Insecticides based on crystalline toxins of Bacillus thuringiensis are very good biological plant protection products. However, the spectrum of activity of some toxins is narrow or resistance among insects has been developed. We tested the insecticidal activity of crystals of the B. thuringiensis MPU B9 strain alone and supplemented with Vip3Aa proteins against important pests: Spodoptera exigua Hübner (Lepidoptera: Noctuidae), Cydia pomonella L. (Lepidoptera: Tortricidae) and Dendrolimus pini L. (Lepidoptera: Lasiocampidae). The Cry toxins were more active for D. pini but less active against S. exigua and C. pomonella than Vip3Aa. Supplementation of Cry toxins by small amounts of vegetative insecticidal proteins demonstrated synergistic effect and significantly enhanced the toxicity of the insecticide. The results indicate the utility of Cry and Vip3Aa toxins mixtures to control populations of crops and forests insect pests.     

Genetic manipulation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> for strain improvement

Bacillus thuringiensis (Bt) has been used as a biopesticide in agriculture, forestry and mosquito control because of its advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms. The insecticidal properties of this bacterium are due to insecticidal proteins produced during sporulation. Despite these ecological benefits, the use of Bt biopesticides has lagged behind the synthetic chemicals. Genetic improvement of Bt natural strains, in particular Bt recombination, offers a promising means of improving efficacy and cost-effectiveness of Bt-based bioinsecticide products to develop new biotechnological applications.     

Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests

The insecticidal activities and specificities of the Vip3Aa proteins derived from different Bt strains are very different, although the similarities between these proteins are higher than 95%. In this study, we hypothesised that the differences in Vip3Aa11 and Vip3Aa39 C-terminal amino acids determine their differences in insecticidal activity against three Lepidoptera insects. To find the amino acid residues associated with insecticidal activity, nine different amino acid residues of Vip3Aa11 were substituted with the corresponding amino acid residues from Vip3Aa39 by site-directed mutagenesis. The toxicity of each protein was estimated by bioassays, and the results demonstrated that the mutant Y784N lost its insecticidal activity against three insects (Agrotis ipsilon, Helicoverpa armigera, and Spodoptera exigua). The insecticidal activity of S543N, I544L, and S686R against S. exigua increased 5-fold, 2.65-fold, and 8.98-fold, while the toxicity to H. armigera and A. ipsilon slightly decreased compared with that of the Vip3Aa11 protein. These findings indicate that the amino acid residues Ser⁵⁴³, Ile⁵⁴⁴, Thr⁶⁸⁵, Ser⁶⁸⁶, Arg⁷⁰⁴, Ile⁷⁸⁰, and Tyr⁷⁸⁴ may be insecticidal activity-related residues. Additionally, the trypsin activation of the four mutants indicated that all proteins can form a 62-kDa core fragment, except Y784N. A possible association between the insecticidal activity and trypsin sensitivity of Vip3A proteins is suggested.     

In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the ¹²⁵I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops.     

Nutrient conditions determine the localization of Bacillus thuringiensis Vip3Aa protein in the mother cell compartment

Vip3Aa was first identified as a protein secreted during the vegetative growth phase of Bacillus thuringiensis (Bt) bacteria and which shows high insecticidal toxicity against lepidopteran insect pests (Estruch et al., 1996). Bt strains formulated as bio-insecticides only had low amounts of Vip3Aa secreted to the medium. Here, we report that Vip3Aa proteins produced by three different Bt strains, including an industrial strain, were indeed not secreted to the culture solution when grown in sporulation medium, but were retained in the mother cell compartment. In order to further investigate the Vip3Aa secretion and location, we grew the strains in rich medium. We found that in rich medium, a fraction of Vip3Aa was secreted, suggesting that Vip3Aa secretion is nutrient-dependent. Regardless of the growth conditions, we found that Vip3Aa retained in cell pellets exhibited high toxicity against Spodoptera frugiperda larvae. Hence, we speculate that the accumulation of Vip3Aa protein in the mother cell compartment under sporulation conditions could still be used as an efficient strategy for industrial production in commercial Bt strains.     

Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis

The potential of insecticidal Vip3Aa toxin peptide of Bacillus thuringiensis (Bt) as a resource for development of lepidopteran insect resistant transgenic crop plants has not yet been fully fathomed. The single mode of protection offered by the insecticidal Vip3Aa toxin against a broad spectrum of lepidopteran insect pests that invade crop field as secondary insect pests, carry definitive significance. However, lack of diversity amongst insecticidal Vip3A toxin towards toxicity for lepidopteran insects is often considered as disadvantage. In order to bring in improvement at this front, search for diversity and protein engineering of the toxin molecule for creation of diversity require to be undertaken in future. In that context, identification of the bioactive core component of Vip3BR toxin peptide of Bt an analogue of Vip3Aa toxin has been accomplished. The core component was found to contain enhanced potency of the insecticidal property 2–3 folds more than the native toxin against four major crop pests.     

Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins

Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.     

An evaluation of methods for assessing the impacts of Bt‐maize MON810 cultivation and pyrethroid insecticide use on Auchenorrhyncha (planthoppers and leafhoppers)

1 Auchenorrhyncha (Planthoppers and Leafhoppers) are not only pests of many crops, but they are also nontarget organisms with respect to Bt‐protein expressing genetically modified plants. As herbivorous arthropods, planthoppers and leafhoppers ingest Cry proteins depending on their feeding behaviour. Consequently, they are directly exposed to these entomotoxic proteins and can also serve as a source of Cry protein exposure to predatory arthropods. Therefore, it is reasonable to use Auchenorrhyncha in the risk assessment of genetically modified crops. 2 During a 2‐year field study, we evaluated four different methods in terms of their feasibility to assess the impacts of plant‐incorporated protectants from Bt‐maize and of insecticide use on this group of arthropods. Visual assessment of plants, sweep netting, yellow traps and custom made sticky traps were utilized in field plots of Bt‐maize MON810, untreated near‐isogenic maize and insecticide‐treated near‐isogenic maize and were compared with respect to their capability to reflect the diversity and abundance of Auchenorrhyncha species. 3 Zyginidia scutellaris (Herrich‐Schäffer) (Cicadomorpha: Cicadellidae) represented more than 94% of all captured individuals in both years. The analysis of Z. scutellaris data showed no consistent differences between Bt‐maize MON810 and the untreated near isogenic hybrid, demonstrating no negative impact of MON810 on this species. Insecticide treatment, on the other hand, was not equivalent to the isogenic maize in terms of Z. scutellaris densities. Based on the collected data and on practical considerations, we recommend the combined use of transect‐wise sweep netting and sticky traps for the sampling of Auchenorrhyncha in maize.     

Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation

Helicoverpa armigera is one of the most harmful pests in China. Although it had been successfully controlled by Cry1A toxins, some H. armigera populations are building up resistance to Cry1A toxins in the laboratory. Vip3A, secreted by Bacillus thuringiensis, is another potential toxin against H. armigera. Previous reports showed that activated Vip3A performs its function by inserting into the midgut brush border membrane vesicles (BBMV) of susceptible insects. To further investigate the binding of Vip3A to BBMV of H. armigera, the full-length Vip3Aa10 toxin expressed in Escherichia coli was digested by trypsin or midgut juice extract, respectively. Among the fragments of digested Vip3Aa10, only a 62 kDa fragment (Vip3Aa10-T) exhibited binding to BBMV of H. armigera and has insecticidal activity. Moreover, this interaction was specific and was not affected by the presence of Cry1Ab toxin. Binding of Vip3Aa10-T to BBMV resulted in the formation of an ion channel. Unlike Cry1A toxins, Vip3Aa10-T was just slightly associated with lipid rafts of BBMV. These data suggest that although activated Vip3Aa10 specifically interacts with BBMV of H. armigera and forms an ion channel, the mode of action of it may be different from that of Cry1A toxins.     

Reduced Mythimna separata infestation on Bt corn could benefit aphids

Use of genetically engineered plants that express insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) have been proven efficacious for managing lepidopteran pests. However, in some cases herbivores that are not targeted by the Bt trait have increased in importance. It has been suggested that reduced caterpillar damage to Bt crops could lead to decreased levels of induced plant defensive compounds which might benefit other non-target herbivores. Here we investigated the potential effect of reduced damage by larvae of Mythimna separata on aphid populations in Bt corn. We compared the performance of Rhopalosiphum maidis feeding on non-Bt corn plants that had been infested by M. separata larvae or were uninfested. The results showed that caterpillar-infested corn plants significantly reduced the fitness of R. maidis leading to a prolonged nymphal development time, reduced adult longevity and fecundity compared to uninfested plants. Consequently, the population growth rate of corn aphids feeding on caterpillar-infested corn plants was significantly lower than on uninfested plants. As expected, the aphids performed significantly better on Lepidoptera-resistant Bt corn than on non-Bt corn when plants were infested with M. separata, since the caterpillars caused very little damage to the Bt plants. The current findings indicate that reduced M. separata infestation could benefit aphid development in Bt corn. Bt corn has the potential to be commercialized in China in the near future and aphids and other non-target pests should be monitored in the farming fields.     

Toxicological Study and Oxidative Stress Evaluation for Safety Assessment of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Vip3Aa16 Toxin in Adult Mice

Vegetative insecticidal proteins were produced by some Bacillus thuringiensis strains and were successfully used in biological control against different agricultural pests such as Lepidoptera. To assess the safety of Vip3Aa16 toxins in mammalian organisms, we evaluated their toxicity using histological, hematological, and oxidative stress parameters on albino Swiss mice. The animals were orally treated with 2500, 5000, 7500 milligrams (mg) of the toxin/kilogram (kg) of body weight for 14 days. Then samples of blood, kidney and hepatic tissues were collected at the end of the treatment. Hematological parameters were monitored by RBC, WBC, hemoglobin, hematocrit, MCV, MCH, and MCHC. Liver and kidney MDA, SOD, vitamin C and H₂O₂ were analyzed to assess oxidative damage. Hepatotoxicity was monitored by analysis of the plasma enzymes ALT and AST and bilirubin levels. Renal toxicity was tested by urea, uric acid and creatinine evaluation. The histopathology of kidney and liver tissues was also investigated. The results of the toxicological study revealed that the Vip3AaA16 has no lethal effect since no mortality was observed at any dose. Moreover, body weight, hematological, histological, biochemical and oxidative findings showed no significant differences between treated and control groups. All these findings confirmed that this toxin is highly safe and doesn’t represent any risk on animal health and subsequently, Vip3Aa16 toxin can be safely used in biological programs to control Lepidopteran pests attacking crops around the world.     

Bt蛋白Vip3Aa19、Cry1Ab和Cry1Ah对玉米蚜的效应评价

[目的] 探讨转基因玉米表达的3种Bt蛋白对非靶标害虫玉米蚜的影响效应,为农田生态系统中转基因玉米的环境安全评价提供依据。[方法] 在玉米蚜全纯人工饲料中分别添加Bt蛋白Vip3Aa19、Cry1Ab和Cry1Ah饲养玉米蚜,并以PBS缓冲液或Na₂CO₃溶液为阴性对照,添加酪蛋白（casein,CS）为中性对照,添加印楝素（neem oil）为阳性对照,比较分析Bt蛋白等各处理对玉米蚜存活率、发育历期、有翅蚜率及繁殖力的影响。[结果] 低浓度印楝素（Neem-L）处理后玉米蚜半数个体生存时间（ST₅₀）为3.2~4.0 d,高浓度印楝素（Neem-H）处理后,玉米蚜在第4天全部死亡,这2个处理均没有子代若蚜产生。添加Bt蛋白和CS对玉米蚜的生存时间没有显著影响,ST₅₀在8.3~9.6 d之间。与阴性对照相比,3个Bt蛋白和CS处理的若蚜期显著短1.0~2.9 d,产出的下一代若蚜数显著增多。Vip3Aa19、Cry1Ab以及CS处理后,有翅蚜比例显著高于其阴性对照。[结论] 饲料中分别添加3种Bt蛋白Vip3Aa19、Cry1Ab和Cry1Ah对玉米蚜的存活率没有显著影响,但具有与添加CS等同提高饲料营养质量的效果;与阴性对照相比,添加3种Bt蛋白对玉米蚜的生长发育和繁殖具有显著的促进效应。     

Extrapolating non-target risk of Bt crops from laboratory to field

The tiered approach to assessing ecological risk of insect-resistant transgenic crops assumes that lower tier laboratory studies, which expose surrogate non-target organisms to high doses of insecticidal proteins, can detect harmful effects that might be manifested in the field. To test this assumption, we performed meta-analyses comparing results for non-target invertebrates exposed to Bacillus thuringiensis (Bt) Cry proteins in laboratory studies with results derived from independent field studies examining effects on the abundance of non-target invertebrates. For Lepidopteran-active Cry proteins, laboratory studies correctly predicted the reduced field abundance of non-target Lepidoptera. However, laboratory studies incorporating tri-trophic interactions of Bt plants, herbivores and parasitoids were better correlated with the decreased field abundance of parasitoids than were direct-exposure assays. For predators, laboratory tri-trophic studies predicted reduced abundances that were not realized in field studies and thus overestimated ecological risk. Exposure to Coleopteran-active Cry proteins did not significantly reduce the laboratory survival or field abundance of any functional group examined. Our findings support the assumption that laboratory studies of transgenic insecticidal crops show effects that are either consistent with, or more conservative than, those found in field studies, with the important caveat that laboratory studies should explore all ecologically relevant routes of exposure.     

Ser-Substituted Mutations of Cys Residues in Bacillus thuringiensis Vip3Aa7 Exert a Negative Effect on its Insecticidal Activity

Vegetative insecticidal proteins (VIPs), which were produced by Bacillus thuringiensis during its vegetative growth stage, display a broad insecticidal spectrum to Lepidoptera larvae. Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7 protein by site-directed mutagenesis. Bioassays using the third instar larvae of Plutella xylostella showed that the toxicity of C401S and C507S mutants were completely abolished. To find out the inactivity reason of mutants, three mutants and the wild-type Vip3Aa7 were treated with trypsin. The results indicated that the C507S mutant was rapidly cleaved and resulted in decrease of the 62?kDa toxic core fragment. These results indicated that the replacement of the Cys⁵⁰⁷ with a Ser⁵⁰⁷ caused decrease in C507S resistance against trypsin degradation. It is suggesting a possible association between insecticidal activity and trypsin sensitivity of Vip3A proteins. This study serves a guideline for the study of Vip3A protein structure and active mechanism.     

Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis

The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. The Egyptian cotton leaf worm (Spodoptera littoralis) is an important agricultural pest that is susceptible to the Vip3Aa16 protein of Bacillus thuringiensis kurstaki strain BUPM95. The midgut histopathology of Vip3Aa fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration. Biotinylated Vip3Aa toxin bound proteins of 55- and 100-kDa on blots of S. littoralis brush border membrane preparations. These binding proteins differ in molecular size from those recognized by Cry1C, one of the very few Cry proteins active against the polyphagous S. littoralis. This result supports the use of Vip3Aa16 proteins as insecticidal agent, especially in case of Cry-resistance management.     

Co-expression and Synergism Analysis of Vip3Aa29 and Cyt2Aa3 Insecticidal Proteins from Bacillus thuringiensis

Vegetative insecticidal protein (Vip3) from Bacillus thuringiensis shows high activity against lepidopteran insects. Cytolytic δ-endotoxin (Cyt) also has high toxicity to dipteran larvae and synergism with other crystal proteins (Cry), but synergism between Cyt and Vip3 proteins has not been tested. We analyzed for synergism between Cyt2Aa3 and Vip3Aa29. Both cyt2Aa3 and vip3Aa29 genes were co-expressed in Escherichia coli strain BL21 carried on vector pCOLADuet-1. Vip3Aa29 showed insecticidal activity against Chilo suppressalis and Spodoptera exigua, with 50% lethal concentration (LC(50)) at 24.0 and 36.6 μg ml(-1), respectively. It could also inhibit Helicoverpa armigera growth, with 50% inhibition concentration at 22.6 μg ml(-1). While Cyt2Aa3 was toxic to Culex quinquefasciatus (LC(50): 0.53 μg ml(-1)) and Chironomus tepperi (LC(50): 36 μg ml(-1)), it did not inhibit C. suppressalis, S. exigua, and H. armigera. However, the co-expression of Cyt2Aa3 and Vip3Aa29 showed synergistic effect on C. suppressalis and S. exigua, and the individual activities were strengthened 3.35- and 4.34-fold, respectively. The co-expression had no synergism against C. tepperi and H. armigera, but exerted some antagonistic effect on Cx. quinquefasciatus. The synergism between Cyt2Aa and Vip3Aa was thus discovered for the first time, which confirmed that Cyt toxin can enhance the toxicity of other toxins against some non-target insects. By synergism analysis, the effectiveness of microbial insecticides can be verified.