The Insecticidal Bacterial Toxins in Modern Agriculture

Increased awareness about environmental adverse effects of human activities has prompted the use of insecticides with low impact on systems associated to agriculture. Currently, the most successful biological products are based on protein toxins from the bacterial species Bacillus thuringiensis. Because of the remarkable properties of these proteins, their encoding genes were introduced into farming species (the so called Bt-crops), in such a way, that these plants are self-protected against some key insect pests. Despite the fact that a relatively large number of these toxins, with different toxicity ranges, have been described, it is still important to find new resources with novel capabilities to complement, or to replace in the future, the currently used ones. On another hand, it is important to continue studying their mode action in susceptible insects, and the changes occurred in resistant ones, to determine the most effective strategy for long lasting pest control. The focus of this Special Issue of Toxins is to provide updated information on the use of B. thuringiensis and their toxins on different field crops, the interactions of these toxins with other molecules, analyze the biochemical and molecular basis of emerging cases of resistance and, in general, to provide information which can contribute to an effective pest management with these toxins

Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)

<p>Abstract</p> <p>Background</p> <p>The accurate determination of the number of copies of a gene in the genome (gene dosage) is essential for a number of genetic analyses. Quantitative real time PCR (qPCR) with TaqMan detection has shown advantages over traditional Southern-blot and FISH techniques, however the high costs of the required labeled probes is an important limitation of this method. qPCR with SYBR Green I detection is a simple and inexpensive alternative, but it has never been applied to the determination of the copy number of low copy number genes in organisms with high allelic variability (as some insects), where a very small margin of error is essential.</p> <p>Findings</p> <p>We have tested the suitability of the qPCR with SYBR Green I detection methodology for the detection of low copy number genes in two insects: the genetically well characterized <it>Drosophila melanogaster </it>(Diptera) and the poor genetically characterized <it>Ostrinia nubilalis </it>(Lepidoptera). The system was applied to determine the copy number of: (1) the <it>O. nubilalis cadherin </it>gene, involved in the mode of action of <it>Bacillus thuringiensis </it>toxins, which showed indirect evidence of duplication, and (2) the <it>D. melanogaster BarH1 </it>and <it>BarH2 </it>genes, located within the <it>Bar </it>region of the X chromosome, to clearly determine whether they both are covered by the tandem duplication in the classical <it>Bar </it>(<it>B¹</it>) mutant. Our results showed that the <it>O. nubilalis cadherin </it>gene is an autosomal single copy gene and that <it>BarH1</it>, but not <it>BarH2</it>, is duplicated in the Drosophila <it>B¹</it>mutant.</p> <p>Conclusions</p> <p>This work shows that qPCR with SYBR Green I detection can be specific and accurate enough to distinguish between one and two gene copies per haploid genome of genes with high allelic variability. The technique is sensitive enough to give reliable results with a minimum amount of sample (DNA from individual thoraxes) and to detect gene duplications in tandem.</p

Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products

Study of the bacillus thuringiensis Cry1Ia protein oligomerization promoted by midgut brush border membrane vesicles of lepidopteran and coleopteran insects, or cultured insect cells

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities, the State Research Agency of Spain and the European FEDER founds (Refs. AGL2015-70584-C2 and RTI2018-095204-B-C21), and by the Generalitat Valenciana (GVPROMETEOII-2015-001). M. Domínguez received a predoctoral fellowship from the Universidad Pública de Navarra, Spain

The independent biological activity of Bacillus thuringiensis Cry23Aa protein against Cylas puncticollis

The Cry23Aa/Cry37Aa proteins fromBacillus thuringiensis(Bt) have been described toxic toCylas puncticollislarvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity towardC. puncticollislarvae has been assessed. The results showed that both proteins were toxic forC. puncticollislarvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity againstC. puncticollislarvae. Also, the binding behavior of Cry23Aa protein to midgut receptors ofC. puncticollislarvae has been determined. According to our results, Cry23Aa binds toC. puncticollisbrush border membrane vesicles (BBMV) specifically and independently of Cry37Aa. Due to the lack of common binding sites, Cry23Aa can be pyramided with Cry3Aa protein for better management ofC. puncticollis

Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae

Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case

Editorial for Special Issue: The Insecticidal Bacterial Toxins in Modern Agriculture

n/