Biological characteristics of black armyworm Spodoptera cosmioides on genetically modified soybean and corn crops that express insecticide Cry proteins.

This study aimed to evaluate the development and reproduction of the black armyworm, Spodoptera cosmioides when larvae fed on leaves of Bt-corn hybrids, expressing a single Cry1F and also Cry1F, Cry1A.105 and Cry2Ab2 in pyramided corn and their non-Bt-isoline (hybrid 2B688), as well as on leaves of two soybean isolines expressing the Cry1Ac protein and its non-Bt isoline (A5547-227). We also assessed the effect of these Bt and non-Bt plants on the leaf consumption rate of S. cosmioides larvae. This pest was unable to develop when fed on any of the corn isolines (Bt and non-Bt). When both 1st and 3rd instar larvae were fed on corn leaf, mortality was 100% in both Bt and non-Bt corn. In contrast, when corn leaves were offered to 5th instar larvae, there were survivors. Defoliation and leaf consumption was higher with non-Bt corn than with both of the Bt corn isolines. There was no negative effect of Bt soybean leaves on the development and reproduction of S. cosmioides with respect to all evaluated parameters. Our study indicates that both Bt and non-Bt corn adversely affect the development of S. cosmioides while Bt soybean did not affect its biology, suggesting that this lepidopteran has major potential to become an important pest in Bt soybean crops.

A theoretical model of the tridimensional structure of Bacillus thuringiensis subsp. medellin Cry 11Bb toxin deduced by homology modelling

Cry11Bb is an insecticidal crystal protein produced by Bacillus thuringiensis subsp. medellin during its stationary phase; this ¶-endotoxin is active against dipteran insects and has great potential for mosquito borne disease control. Here, we report the first theoretical model of the tridimensional structure of a Cry11 toxin. The tridimensional structure of the Cry11Bb toxin was obtained by homology modelling on the structures of the Cry1Aa and Cry3Aa toxins. In this work we give a brief description of our model and hypothesize the residues of the Cry11Bb toxin that could be important in receptor recognition and pore formation. This model will serve as a starting point for the design of mutagenesis experiments aimed to the improvement of toxicity, and to provide a new tool for the elucidation of the mechanism of action of these mosquitocidal proteins.

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bacillus thuringiensis var. israelensis SPS1: caracterização da região promotora de genes cry e efeito em larvas de Aedes aegypti (L.) (Diptera: Culicidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identificação, caracterização e teste de toxicidade contra nematóides de novos genes cry de Bacillus thuringiensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicidade e efeitos subletais de toxinas Cry de Bacillus thuringiensis Berliner em diferentes populações de Plutella xylostella (L.) (Lepidoptera: Plutellidae) em laboratório

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Induced-feeding bioassays for detection of Bacillus thuringiensis insecticidal activity against Epilachna paenulata (Coleoptera)

The objective of this work was to establish and test the induced-feeding bioassay in order to detect Bacillus thuringiensis insecticidal activity against Epilachna paenulata (Coleoptera: Coccinellidae). Larvae were induced to swallow high concentrations of spore-crystal suspensions of seven exotic and 30 Argentine B. thuringiensis strains. The great majority of strains showed no toxicity to E. paenulata larvae, and observed mortality was lower than 30%. Induced-feeding bioassay is feasible, and should be used for prospecting strains that produce right combinations of Cry proteins needed to an efficient pest control.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

Mode d’action moléculaire de la toxine anti-tumorale : PS1Aa2 du bacille de Thuringe

Les parasporines sont des toxines Cry du bacille de Thuringe actives contre des cellules tumorales. Ce travail montre que la parasporine PS1Aa2 (Cry31Aa2) forme des pores dans des membranes artificielles, comme de nombreuses toxines Cry. Ceux-ci ont plusieurs niveaux de conductance dont les plus fréquents étaient de 11, 16 et 21 pS dans une solution de 150 mM KCl. Nos résultats de microspectrofluorométrie avec la sonde Fura-2 montrent que la présence de la PS1Aa2 peut produire des augmentations du calcium intracellulaire, la plupart du temps sous la forme d’oscillations calciques et parfois des augmentations soutenues. Ces réponses ont été observées en présence et en absence de calcium extracellulaire, dans les lignées tumorales HeLa et HepG2 et dans la lignée non tumorale HEK 293. Bien que quelques études aient montré que le calcium semble intervenir dans leur mode d’action, de telles oscillations calciques n’ont jamais été décrites auparavant pour des toxines Cry. Les expériences ont dû être faites à des concentrations beaucoup plus élevées de toxine que prévues sur la base des résultats publiés de cytotoxicité. Malgré la présence des fragments identifiés auparavant comme actifs, sa faible efficacité semble liée à la présence d’ADN dans les préparations qui entraîne la précipitation de la protéine. Les travaux futurs sur cette toxine seraient donc grandement facilités par une amélioration de sa méthode de préparation.

The impact of secondary pests on Bacillus thuringiensis (Bt) crops

The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to pesticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered (GE) insect resistant crops could mitigate many of the negative side effects of pesticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro-ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if non-susceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers’ disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible longterm ecological trophic interactions of employing this technology.

Caracterização de novos isolados de Bacillus thuringiensis para o controle de importantes insetos-praga da agricultura

A bactéria Bacillus thuringiensis Berliner produz um corpo de inclusão paraesporal (cristal) de natureza proteica, formado durante a esporulação, que atua de forma eficiente no controle de insetos-praga de culturas economicamente importantes. Esse cristal é constituído de proteínas Cry, que são codificadas pelos genes cry; um isolado pode ser caracterizado pelo conteúdo de genes cry que apresenta. Visando caracterizar novos isolados no combate de insetos-praga pertencentes às ordens Lepidoptera e Coleoptera, 76 isolados bacterianos foram analisados molecularmente e tiveram seu potencial de controle avaliado por meio de bioensaios com larvas de Spodoptera frugiperda (J.E. Smith), Sphenophorus levis Vaurie e Tenebrio molitor Linnaeus. As análises moleculares indicaram 11 isolados (14,5% da coleção), contendo genes lepidóptero-específicos e 17 (22,37%) com genes coleóptero-específicos. As análises de patogenicidade revelaram dois isolados com alto potencial de controle para lagartas de S. frugiperda, um para larvas de S. levis e seis prejudiciais ao desenvolvimento das larvas de T. molitor. Esses isolados de B. thuringiensis podem ser promissores no controle biológico das referidas pragas.

cry1 genes from Bacillus thuringiensis: specificity determination and implications for primer design

Some pest management programs employ PCR to identify cry1 genes from Bacillus thuringiensis to predict bacterial toxicity towards different insect pests. However, due to changes on the mode of action of the Cry proteins, new primers had to be designed to detect the new genes. Therefore, an 'in-silico' study of genetic sequences from five cry1 subclasses was carried out and characterized by molecular tools. The design of new primers allows for more precise selection of B. thuringiensis isolates, helping to better direct the programs employing biological control.

Bacillus Thuringiensis mutant increase activity against Apodoptera Frugiperda larvae

Bacillus thuringiensis is a Gram-positive bacterium which main characteristic is the production of Cry proteins, that is toxic to some insects. These proteins, when ingested by susceptible insects, become active causing their death. In nature, it is possible to found B. thuringiensis strains which produce these proteins, but they differ in productivity (some of these isolates are more productive then others), and as to the toxicity levels of the produced proteins. Two B. thuringiensis strains that were highly effective against Spodoptera frugiperda larvae were chosen to verifying genetic mutation implication on Cry proteins productivity. One strain with a prolific spores production, while the other one only produced small amounts of spores. A genomic mutant library of these two isolates was, separately, constructed by genome Tn-5 transposon random insertion. Data analysis showed that mutation had a direct effect on the spores production, inducing an increase as well as a decrease in the production, according to the different strain observed. These results indicate, for the first time, that it is possible to use the described technique with B. thuringiensis, as well as the possibility to genetically breeding this bacteria. Another possibility introduced here is the possibility to do functional genetic studies mediated by mutagenesis in this bacterium.

Expression of a new Bacillus thuringiensis Cry1Ia gene in Escherichia coli with strong activity against cotton pests

The control of cotton pests may be accomplished using Bacillus thuringiensis Cry proteins. For this purpose, the objective of this work was to evaluate the insecticidal activity of a new Cry1Ia protein against neonatal larvae of Spodoptera frugiperda and Anthonomus grandis. The complete cry1Ia gene, previously obtained by PCR with oligonucleotide primers based on the sequenced gene, was cloned into the vector pET28a(+), introduced into Escherichia coli BL21(DE3) and expressed by induction with IPTG. The expression of the Cry1Ia protein was confirmed with molecular weight of approximately 81 kDa. The results demonstrated the efficiency of the bacterial system for the expression of B. thuringiensis Cry1Ia protein, which was subsequently used in quantitative bioassays against S. frugiperda and A. grandis larvae, resulting in an extremely toxic protein for both species. This characteristic is exceptionally important for obtaining transgenic cotton plants resistant to these pests.

Proteínas Cry1 e Vip3A de Bacillus thuringiensis: sinergismo e efeito sub-letal no controle de Heliothis virescens

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)