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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an economically important pest of corn (maize) in North America and Europe. Current management practices for WCR involve transgenic expression of insecticidal proteins to minimize larval feeding damage to corn roots. The evolution of resistant WCR populations to transgenic corn expressing insecticidal proteins (e.g. Cry3Bb1, Gpp34Ab1/Tpp35Ab1) necessitates efforts to discover and deploy new modes of action for WCR control. Here, we tested the hypothesis that the addition of short peptides selected for binding to the WCR gut would restore insecticidal activity of Cry3Bb1 to resistant insects. Phage display technology coupled with deep sequencing was used to identify peptides selected for binding to WCR brush border membrane vesicles and to recombinant putative receptors aminopeptidase and cadherin. The binding and specificity of selected peptides was confirmed by ELISA and pull-down assays, and candidate gut surface binding partners were identified. Although production of 284 novel Cry3Bb1 variants with these peptides did not restore activity against resistant WCR in artificial diet bioassays, 112 variants were active against susceptible insects. These results provided insights for the mechanism of Cry3Bb1 activity and toward engineering a new mode-of-action via receptor re-targeting in the context of protein structure and function.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a significant pest of field corn, Zea mays L. (Poales: Poaceae), across the United States Corn Belt. Widespread adoption and continuous use of corn hybrids expressing the Cry3Bb1 protein to manage the western corn rootworm has resulted in greater than expected injury to Cry3Bb1-expressing hybrids in multiple areas of Nebraska. Single-plant bioassays were conducted on larval western corn rootworm populations to determine the level of resistance present in various Nebraska counties. The results confirmed a mosaic of susceptibility to Cry3Bb1 across Nebraska. Larval development metrics, including head capsule width and fresh weight, were measured to quantify the relationship between the level of resistance to Cry3Bb1 and larval developmental rate. Regression and correlation analyses indicate a significant positive relationship between Cry3Bb1 corrected survival and both larval development metrics. Results indicate that as the level of resistance to Cry3Bb1 within field populations increases, mean head capsule width and larval fresh weight also increase. This increases our understanding of western corn rootworm population dynamics and age structure variability present in the transgenic landscape that is part of the complex interaction of factors that drives resistance evolution. This collective variability and complexity within the landscape reinforces the importance of making corn rootworm management decisions based on information collected at the local level. [ABSTRACT FROM AUTHOR]

Perceived benefits of insecticidal transgenic crops include reduced usage of broad-based insecticides, and therefore lower risk to non-target organisms. Numerous studies have documented low or no direct toxicity of Bacillus thuringiensis (Bt)-derived toxins against non-target organisms, but there has been less research on (a) effects of secondary pest infestations on Bt expressing in crops and (b) behavioural responses by predators feeding on host arthropods from Bt crops - both topics are investigated in this study. We quantified predation by the obligate spider mite predator Phytoseiulus persimilis of carmine spider mites (Tetranychus cinnabarinus), reared on Bt or non-Bt corn (Zea mays). Both no-choice and two-choice studies were conducted. In addition, we quantified toxin levels in corn leaves with/without spider mite infestation. Under no-choice conditions, P. persimilis consumed non-Bt spider mites at a faster rate than Bt spider mites. Under two-choice conditions, P. persimilis spent more time in the vicinity of non-Bt spider mites than near Bt spider mites. Corn infested with spider mites exhibited lower toxin levels than non-infested plants. These results suggest potentially complex interactions among non-target herbivores, their natural enemies and Bt crops. © 2014 Association of Applied Biologists.

Following the discovery of western corn rootworm (WCR; Diabrotica virgifera virgifera) populations resistant to the Bacillus thuringiensis (Bt) protein Cry3Bb1, resistance was genetically mapped to a single locus on WCR chromosome 8 and linked SNP markers were shown to correlate with the frequency of resistance among field-collected populations from the US Corn Belt. The purpose of this paper is to further investigate the relationship between one of these resistance-linked markers and the causal resistance locus. Using data from laboratory bioassays and field experiments, we show that one allele of the resistance-linked marker increased in frequency in response to selection, but was not perfectly linked to the causal resistance allele. By coupling the response to selection data with a genetic model of the linkage between the marker and the causal allele, we developed a model that allowed marker allele frequencies to be mapped to causal allele frequencies. We then used this model to estimate the resistance allele frequency distribution in the US Corn Belt based on collections from 40 populations. These estimates suggest that chromosome 8 Cry3Bb1 resistance allele frequency was generally low (<10%) for 65% of the landscape, though an estimated 13% of landscape has relatively high (>25%) resistance allele frequency. [ABSTRACT FROM AUTHOR]

Carabids (Coleoptera: Carabidae) seem to be suitable bioindicators of the environmental impacts of novel agrotechnologies, including deployment of the genetically engineered (GE) crops. In this article, we describe our effort to employ carabids in the environmental risk assessment (ERA). GE maize MON88017, its near‐isogenic hybrid nontreated or treated with the soil insecticide chlorpyrifos, and two reference hybrids were used to compare three different ways how to utilize carabids in ERA. The analysis of abundance of all captured carabids or of the most abundant carabid species did not disclose any differences between the treatments. The analysis based on the categories of functional traits revealed distinct features of some treatments and proved suitable for ERA because it permitted field data transportability in spite of different species compositions. Our results indicate that GE maize has no detrimental environmental effect. On the other hand, we found significant trends toward lower abundance and lower species number (including analysis of all carabid species together) in plots treated with the insecticide, and some tendencies to higher abundance and higher species number in plots sown with the reference hybrid PR38N86. Using functional group indicators allows identification of unintended changes in ecological functions of agroecosystem and comparability across geographies. We recommend data evaluation at the level of the categories of functional traits in ERA of GE crops and other agricultural practices. [ABSTRACT FROM AUTHOR]

Abstract Maize MON 87411 was developed to confer resistance to corn rootworms (Diabrotica spp.) by the expression of a modified version of the Bacillus thuringiensis cry3Bb1 gene and a DvSnf7 dsRNA expression cassette, and tolerance to glyphosate‐containing herbicides by the expression of a CP4 5‐enolpyruvylshikimate‐3‐phosphate synthase (cp4 epsps) gene. The molecular characterisation data and bioinformatics analyses did not identify issues requiring assessment for food and feed safety. No statistically significant differences in the agronomic and phenotypic characteristics tested between maize MON 87411 and its conventional counterpart were identified. The compositional analysis of maize MON 87411 did not identify differences that required further assessment except for palmitic acid levels in grains from not treated maize MON 87411. The GMO Panel did not identify safety concerns regarding the toxicity and allergenicity of the Cry3Bb1 and CP4 EPSPS proteins, as expressed in maize MON 87411 and found no evidence that the genetic modification might significantly change the overall allergenicity of maize MON 87411. The nutritional impact of maize MON 87411‐derived food and feed is expected to be the same as those derived from the conventional counterpart and non‐GM commercial reference varieties. The GMO Panel concludes that maize MON 87411, as described in this application, is nutritionally equivalent to and as safe as the conventional counterpart and the non‐GM maize reference varieties tested, and no post‐market monitoring of food/feed is considered necessary. In the case of accidental release of viable maize MON 87411 grains into the environment, maize MON 87411 would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 87411. The GMO Panel concludes that maize MON 87411, as described in this application, is as safe as its conventional counterpart and the tested non‐GM maize reference varieties with respect to potential effects on human and animal health and the environment.

[Display omitted] • Risk assessment of GE crops is important in the EU. • Tetranychus urticae is a non-target pest species infesting maize. • No effects of GE maize on T. urticae compared to isogenic maize and insecticide. • Higher differences in T. urticae life history parameters among maize varieties. Genetically engineered (GE) maize expressing Bacillus thuringiensis Cry3Bb1 protein was developed to control Diabrotica spp. (Coleoptera: Chrysomelidae). However, Cry proteins have been reported to have effects on non-target arthropods. We therefore investigated whether the non-target pest Tetranychus urticae (Acari: Tetranychidae) was negatively affected by GE maize expressing the insecticidal Cry3Bb1 protein. Five treatments were used in the laboratory tests to determine the life history parameters of T. urticae on leaves of field-grown maize varieties: (1) GE maize MON 88017, (2) isogenic maize, (3) isogenic maize protected with the soil applied insecticide chlorpyrifos (Dursban 10G), and two unrelated varieties (4) Kipous and (5) PR38N86. Newly emerged T. urticae larvae were individually released on the upper surface of leaf discs placed on water saturated cotton wool. Immatures and adults survival, duration of developmental stages and female fecundity were recorded daily until T. urticae died. Age–stage, two-sex life table method and test for trends, revealed no significant differences in 13 of 18 studied parameters. The unrelated varieties Kipous and PR38N86 on one side and maize with the same genetic background, namely GE maize and isogenic maize with or without insecticide protection, on the other side, showed significant variations in male longevity, larval survival rate, preoviposition period, and fecundity. In addition to the differences between varieties, GE maize and insecticide-protected isogenic maize showed a substantial difference in age-specific fecundity, but not in the mean number of eggs laid by females. The obtained results do not indicate that consumption of Cry3Bb1 has negative effect on T. urticae and suggest that GE maize does not pose a risk with respect to the non-target mite pest T. urticae. The results may have implications for the approval and renewal of import and cultivation for GE crop in the European Union. [ABSTRACT FROM AUTHOR]

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), and northern corn rootworm, Diabrotica barberi Smith and Lawrence (Coleoptera: Chrysomelidae), are important insect pests in corn, Zea mays L. For more than a decade, growers have been using transgenic plants expressing proteins from the bacterium Bacillus thuringiensis (Bt) to protect corn roots from feeding. In 2011, western corn rootworm populations were reported to have developed resistance to Bt hybrids expressing Cry3Bb1 and later found to be cross-resistant to hybrids expressing mCry3A and eCry3.1Ab. The identification of resistance to Cry3 (Cry3Bb1, mCry3A, and eCry3.1Ab) hybrids led to concerns about durability and efficacy of products with single traits and of products containing a pyramid of a Cry3 protein and the binary Bt proteins Cry34Ab1 and Cry35Ab1. From 2012 to 2014, 43 field trials were conducted across the central United States to estimate root protection provided by plants expressing Cry34Ab1/Cry35Ab1 alone (Herculex RW) or pyramided with Cry3Bb1 (SmartStax). These technologies were evaluated with and without soil-applied insecticides to determine if additional management measures provided benefit where Cry3 performance was reduced. Trials were categorized for analysis based on rootworm damage levels on Cry3-expressing hybrids and rootworm feeding pressure within each trial. Across scenarios, Cry34Ab1/Cry35Ab1 hybrids provided excellent root protection. Pyramided traits provided greater root and yield protection than non-Bt plus a soil-applied insecticide, and only in trials where larval feeding pressure exceeded two nodes of damage did Cry34Ab1/Cry35Ab1 single-trait hybrids and pyramided hybrids show greater root protection from the addition of soil-applied insecticides. [ABSTRACT FROM AUTHOR]

Volunteer corn (VC) has reemerged as a problematic weed in corn/soybean rotational cropping systems. This reemergence and increasing prevalence of volunteer corn has been correlated to an increased adoption of herbicide-resistant (HR) corn hybrids and the adoption of conservation tillage. Since the introduction of HR crops, control options, weed/crop competition, and other concerns (i.e., insect resistance management of Bt traits) have increased the amount of attention that volunteer corn is receiving. The objective of this review is to discuss what is known about VC prior to and after the introduction of HR crops, and to discuss new information about this important weed.

Staphylinid beetles are recommended bioindicators for the pre-market environmental risk assessment of genetically modified (GM) insect protected maize expressing the Cry3Bb1 toxin. Our multiannual study is a unique European analysis of a staphylinid community within a 14 ha maize field. GM maize, its near-isogenic hybrid (with or without insecticide treatment), and two other reference hybrids were each grown in five 0.5 ha plots. The opportunity for exposure to Cry toxin from plant residues ploughed into the soil was shown by the presence of saprophagous dipteran larvae that are common prey of predatory staphylinid species and hosts of the parasitoid species. 2587 individuals belonging to 77 staphylinid species were sampled using pitfall traps. Lesteva longoelytrata (31%), Oxypoda acuminata (12%), Aloconota sulcifrons (8%) and Anotylus rugosus (7%) were the most abundant beetles in the field. Bionomics, food specialization, temperature requirements and size group were assigned for 25 most common species. These traits determine the occurrence of staphylinid beetles in the field, the food sources they could utilize and thus also their likely contact with the Cry3Bb1 toxin. Statistical analysis of activity abundance, Rao indices and multivariate analysis of distribution of particular categories of functional traits in the field showed negligible effects of the experimental treatments, including the GM maize, upon the staphylinid community. Staphylinid beetles represent a considerably diverse part of epigeic field fauna with wide food specialization; these features render them suitable for the assessment of environmental safety of GM insect protected maize. However, the availability of prey and the presence of particular staphylinid species and their abundance are highly variable; this complicates the interpretation of the results. [ABSTRACT FROM AUTHOR]

BACKGROUND A SYBR Green I-based quantitative loop-mediated isothermal amplification ( LAMP) assay was developed for the rapid detection of genetically modified maize MON863. A set of primers was designed based on the integration region of the Cry3Bb1 and tahsp17 genes. RESULTS The qualitative and quantitative reaction conditions ( dNTPs, betaine, primers, Mg2+, Bst polymerase, temperature, reaction time) were optimized. The concentrations of Mg2+ and betaine were found to be important to the LAMP assay. The detection limits of both qualitative and quantitative LAMP for MON863 were as low as 4 haploid genomic DNA, and the LAMP reactions can be completed within 1 h at an isothermal temperature of 65 °C. CONCLUSION The results of this study demonstrate that this new SYBR Green I-based quantitative LAMP assay system is reliable, sensitive and accurate. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

32 experimental plots were sown at Borovce (district Trnava) in Slovakia during 2008 with 8 maize hybrids, including coleopteran-protected MON 88017, the lepidopteran-protected MON 89034, the stacked product, MON 89034 × MON 88017, that same hybrids isoline that did not have the Bt trait or traits, and 4 commercial hybrids. Part of the fourth repetition was unexpectedly infested and damaged with the Western corn rootworm (WCR). High numbers of the WCR adults were found in the damaged plots on July 8. Adult WCR abundance remained concentrated in this area until August 12-19. The beetles apparently began to disperse during August 19-September 2, and after September 2. The trap captures increased in the uninfested areas up to September 16, no significant difference having been found in the adult WCR captures between the infested and uninfested plots, including the plots with MON 88017. Bt-maize hybrids containing MON 88017 strongly influenced the level of the WCR damage as assessed by lodging of the maize plants, and they also influenced the numbers of the WCR adults before maize flowering. We found that intensive movement of the WCR adults from the place of their origin started later in the vegetation season.

BACKGROUND Combining herbicide-resistant and Bacillus thuringiensis ( Bt) traits in corn ( Zea mays L.) hybrids may affect insect resistance management owing to volunteer corn. Some Bt toxins may be expressed at lower levels by nitrogen-deficient corn roots. Corn plants with sublethal levels of Bt expression could accelerate the evolution of Bt resistance in target insects. The present objective was to quantify the concentration of Bt ( Cry3Bb1) in corn root tissue with varying tissue nitrogen concentrations. RESULTS Expression of Cry3Bb1 toxin in root tissue was highly variable, but there were no differences in the overall concentration of Cry3Bb1 expressed between roots taken from Cry3Bb1-positive volunteer and hybrid corn plants. The nitrogen rate did affect Cry3Bb1 expression in the greenhouse, less nitrogen resulted in decreased Cry3Bb1 expression, yet this result was not documented in the field. CONCLUSION A positive linear relationship of plant nitrogen status on Cry3Bb1 toxin expression was documented. Also, high variability in Cry3Bb1 expression is potentially problematic from an insect resistance management perspective. This variability could create a mosaic of toxin doses in the field, which does not fit into the high-dose refuge strategy and could alter predictions about the speed of evolution of resistance to Cry3Bb1 in western corn rootworm Diabrotica virgifera virgifera LeConte. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: SmartStax® insect-protected corn (Zea mays L.) contains genes for six Bacillus thuringiensis (Bt) proteins controlling both lepidopteran pests and the corn rootworm complex (Diabrotica spp.). The properties of SmartStax, particularly the multiple effective modes of action (i.e., each Bt protein provides a high level of control of the target pests with a low probability of cross-resistance among the proteins), have provided the opportunity to add to previously approved structured refuge options by combining the non-Bt refuge seed with SmartStax seed in a seed mix. Seed mixes ensure that a refuge is present in every Bt field, remove concerns about grower compliance with refuge requirements, and provide grower convenience. However, seed mixes could increase the likelihood that larval insects move between Bt and non-Bt plants and vice versa. Assessing the insect resistance management (IRM) value of a seed mix refuge requires an assessment of the amount of larval movement, and the consequences of that movement, for the key target pests. The studies here present such data for control of corn rootworm by SmartStax corn, which contains the rootworm-active protein Cry3Bb1 and the binary protein Cry34Ab1/Cry35Ab1. In a growth chamber experiment, SmartStax was most effective against first instars and significantly effective against second instars, but did not control third instars. In a field study of movement from a heavily infested non-Bt plant onto surrounding plants, a larger percentage of insects successfully dispersed from the infested plant when the surrounding plants were non-Bt plants than when they were SmartStax plants. A paired-plant study showed that few larvae migrated from infested SmartStax plants and survived on nearby non-Bt plants; larvae that migrated from infested non-Bt plants had low survival if the adjacent plants were SmartStax. Replicated field studies of plant-to-plant movement indicated that the non-Bt plants in a 5% or 10% seed mix consistently supported large populations of susceptible insects and represented a productive refuge, whereas the SmartStax plants had few or no survivors. The timing of emergence from seed mix plots containing 5% or 10% non-Bt plants was more similar to that of the non-Bt plots than that of the SmartStax plots. Thus, the available growth chamber and field data indicate that a seed mix of 5% or more will provide an effective refuge for corn rootworm in SmartStax corn. [Copyright &y& Elsevier]

Monsanto Company and Dow AgroSciences have used conventional breeding techniques to develop the combined-trait corn (Zea mays L.) product MON 89034 × TC1507 × MON 88017 × DAS-59122-7 (“SmartStax® corn”) that confers insect resistance against key lepidopteran pests and the corn rootworm complex (Diabrotica spp.), as well as herbicide tolerance. This product contains the Cry3Bb1, Cry34Ab1, and Cry35Ab1 proteins for corn rootworm (CRW) control. Replicated field trials were conducted in 2006 and 2007 to test the efficacy of Cry3Bb1, Cry34Ab1/Cry35Ab1, and SmartStax under both natural and controlled infestations. In both years, root damage and adult beetle emergence were significantly less for Bt-containing hybrids than for non-Bt controls. In trials with heavy insect pressure, adult beetle emergence (a measure of larval control) was significantly reduced for SmartStax hybrids compared with hybrids with the individual traits and with non-Bt controls. Similarly, strip-plot testing in 2012 in grower fields with high levels of root injury to non-Bt hybrids showed significantly lower feeding on SmartStax than on the non-Bt control or on either single-trait product. In grower fields where single-trait Cry3Bb1 products incurred heavy CRW damage in 2011, SmartStax provided consistent protection against CRW in 2012. The combination of these insecticidal proteins in a single plant provides better rootworm control than current single-trait Bt corn products and represents an effective approach for corn rootworm resistance management. [ABSTRACT FROM AUTHOR]

Abstract This Scientific Opinion reports on an evaluation of a risk assessment for placing on the market of genetically modified maize MON 88017 for cultivation. The EFSA GMO Panel considers that maize MON 88017 is unlikely to have any adverse effect on the environment, except for the possible resistance evolution to the Cry3Bb1 protein in coleopteran target pests; resistance evolution may lead to altered pest control practices that may cause adverse environmental effects. The cultivation management of maize MON 88017 could result in environmental harm. The EFSA GMO Panel therefore recommends managing the use of glyphosate on maize MON 88017 within diversified cropping regimes that have similar or reduced environmental impacts compared with conventional maize cultivation. The EFSA GMO Panel recommends the deployment of insect resistance management strategies and case‐specific monitoring to address (1) the possible resistance evolution to the Cry3Bb1 protein in coleopteran target pests, (2) changes in botanical diversity within fields due to novel herbicide regimes, and (3) resistance evolution to glyphosate in weeds due to novel herbicide regimes. The EFSA GMO Panel agrees with the general surveillance plan of the applicant, but requests that the proposals made to strengthen general surveillance are implemented. Whilst the scope of this application only covers the cultivation of maize MON 88017, this Scientific Opinion also updates the previous EFSA GMO Panel safety evaluation of the food and feed uses, import and processing of maize MON 88017 and derived products. The EFSA GMO Panel concludes that the information available for maize MON 88017 addresses the scientific comments raised by Member States and that maize MON 88017, as described in this application, is as safe as its conventional counterpart and commercial maize varieties with respect to potential adverse effects on human and animal health. If subjected to appropriate management measures, the cultivation management of maize MON 88017 is unlikely to raise safety concerns for the environment.

Der Westliche Maiswurzelbohrer ist einer der wirtschaftlich bedeutendsten Maisschädlinge weltweit. Eine Bekämpfungsmöglichkeit stellt der Anbau von B.t.-Mais dar. Ein großflächiger Anbau von B.t.-Mais erhöht jedoch die Wahrscheinlichkeit der Provozierung einer Resistenzentwicklung gegenüber den jeweils verwendeten B.t.-Toxinen. Um Testsysteme zu erstellen, die im Falle einer Resistenzentwicklung zur Aufklärung der Resistenzmechanismen herangezogen werden können, wurden verschiedene Parameter zur Verarbeitung von B.t.-Toxinen im Mitteldarm von L3 Larven des Westlichen Maiswurzelbohrers untersucht. Zur Identifizierung der im Darmsaft vorhandenen Proteasen und Quantifizierung ihrer Aktivitäten wurden photometrische Tests durchgeführt. Da der Darmsaft des Westlichen Maiswurzelbohrers mit einem durchschnittlichen pH-Wert von 5,75 leicht sauer ist, war anzunehmen, dass vor allem Proteasen mit saurem pH-Optimum aktiv wären, was die hohen Aktivitäten der Cystein-Endopeptidasen Cathepsin L mit Papain, gefolgt von Cathepsin B und Cathepsin H bestätigten. Die Aspartat-Endopeptidase Pepsin – eine Protease mit stark saurem pH-Optimum – wurde ebenfalls nachgewiesen. Aber auch andere Proteasen aus weiteren Klassen wurden identifiziert. Neben den Serin-Endopeptidasen Trypsin, Chymotrypsin und Elastase wurden die zur gleichen Klasse gehörigen Proteasen Cathepsin G, Plasmin und Thrombin nachgewiesen. Auch die Metallo-Endopeptidase Saccharolysin und die Exopeptidase Aminopeptidase sowie eine Omegapeptidase waren im Darmsaft des Westlichen Maiswurzelbohrers aktiv. Weiterhin wurde untersucht, ob wirksame B.t.-Toxine mit Darmsaft des Westlichen Maiswurzelbohrers abgebaut werden. Doch weder das 77 kDa Toxin Cry3Bb1 noch die binären Toxine Cry34Ab1 (14 kDa) und Cry35Ab1 (44 kDa) wurden mit Darmsaft abgebaut. Danach scheinen die Toxine – im Unterschied zu denen in B.t.-Pflanzenschutzmitteln – in einer aktiven Form vorzuliegen. Die Ligand-Blot Bindungsstudien mit den B.t.-Toxinen Cry3Bb1 und Cry34Ab1/Cry35Ab1 deuten auf verschiedene spezifische Rezeptoren in der Darmwand hin, die Molekulargewichte von ca. 30 kDa (Cry3Bb1), 110 kDa (Cry34Ab1) und 50 kDa (Cry35Ab1) aufweisen.

The genetically modified (GM) maize MON 88017 facilitates weed management owing to its tolerance to glyphosate, and resists western corn rootworm (WCR), Diabrotica virgifera virgifera, owing to the expression of Cry3Bb1 toxin. MON 88017 could therefore contribute to the solution of two major problems of European agriculture: continuous WCR spreading and high use of herbicides. To assess possible unwanted environmental impacts of MON 88017, we compared communities of spiders in plots planted in three successive years with this maize, its near isogenic non-GM cultivar treated or not treated with an insecticide and two unrelated maize cultivars. Each of the five treatments was applied on five 0.5 ha plots in a 14 ha field. Spiders were collected in five pitfall traps per plot five times per year. Upon reaching the waxy ripening stage, all plants of first-year cultivation were shredded to small pieces and ploughed into the soil in the respective plot, whereas in the 2nd and 3rd year the harvest was used for biogas production and only digestate was returned to the field. Out of 79 spider species, Pardosa agrestis, Pachygnatha degeeri and Oedothorax apicatus made up 28%, 25% and 23% of the total spider count in the 1st year of study; 2%, 8% and 84% in the 2nd; and 40%, 8% and 35% in the 3rd year. Statistical analysis did not reveal any influence of GM maize on the spider abundance and biodiversity. The abundance, and in two years also the species diversity, was insignificantly higher on the plots with GM maize than on plots with the insecticide-treated non-GM maize. The composition and size of spider community varied year to year, probably reflecting weather conditions and differences in field fertilization with organic matter. [ABSTRACT FROM AUTHOR]

Abstract: Transgenes in commercially available genetically modified plants are generally controlled by strong constitutive promoters to ensure a high level of expression at all stages of cultivation. Constitutive promoters however are influenced by a wide range of factors, and expression profiles of the transgenes in multiple genetic backgrounds have not yet been extensively studied. In this study a powerful expression profiling methodology for transgenic maize (Zea mays L.) is demonstrated on a large scale, analysing thousands of data points from three genotypes of herbicide and insect pest tolerant transgenic maize. Martonvásár inbred lines were crossed with LH244 maize line containing the MON 88017 events, and leaf tissue from the sixth backcross generation was sampled at four relevant phenological phases. Relative expression levels were determined using 18S rRNA as a reference and detailed statistical analysis performed. Expression levels of both transgenes are varied throughout plant development, and the interaction between the genetic background and phenophase are significant (p<0.05). Expression is present at a significant level throughout all the phenological stages. We found that the genetic background has a significant (p<0.01) effect on transgene expression levels in the case of the CP4epsps transgene, but not in the case of cry3Bb1, implying that the sensitivity of different constitutive promoter constructs to the effects of the genetic background is different. [Copyright &y& Elsevier]

Mortality of western corn rootworm ( Diabrotica virgifera virgifera LeConte) due to feeding on MON863 transgenic maize ( Zea mays L.) expressing the Cry3Bb1 protein was evaluated at three Missouri sites in both 2003 and 2004 and at one site each in South Dakota, Nebraska and Iowa in 2004. To do this, survivorship relative to survivorship on isoline maize (i.e. the same genetic background, but without Cry3Bb1) was evaluated. Comparisons were made using low (1650-2500 eggs/m) and high (3300-3500 eggs/m) western corn rootworm egg densities. Significantly fewer beetles were recovered from MON863 than from isoline maize. Emergence from MON863 as a percentage of viable eggs ranged from 0.02% to 0.10%, whereas percentage emergence from isoline maize ranged from 1.09% to 7.14%. Survivorship on MON863 relative to survivorship on isoline averaged 1.51% when averaged across all environments and both years, so mortality because of the Cry3Bb1 protein averaged 98.49%. The average time delay to 50% cumulative beetle emergence from MON863 was 18.3 days later than from isoline maize. Females comprised 56% and 71% of total beetles recovered from MON863 in 2003 and 2004, respectively. Results are discussed in relation to insect resistance management (IRM) of western corn rootworm. [ABSTRACT FROM AUTHOR]

This review paper explores whether the cultivation of the genetically modified Bt-maize transformation event MON 88017, expressing the insecticidal Cry3Bb1 protein against corn rootworms (Coleoptera: Chrysomelidae), causes adverse effects to non-target organisms (NTOs) and the ecological and anthropocentric functions they provide. Available data do not reveal adverse effects of Cry3Bb1 on various NTOs that are representative of potentially exposed taxonomic and functional groups, confirming that the insecticidal activity of the Cry3Bb1 protein is limited to species belonging to the coleopteran family of Chrysomelidae. The potential risk to non-target chrysomelid larvae ingesting maize MON 88017 pollen deposited on host plants is minimal, as their abundance in maize fields and the likelihood of encountering harmful amounts of pollen in and around maize MON 88017 fields are low. Non-target adult chrysomelids, which may occasionally feed on maize MON 88017 plants, are not expected to be affected due to the low activity of the Cry3Bb1 protein on adults. Impacts on NTOs caused by potential unintended changes in maize MON 88017 are not expected to occur, as no differences in composition, phenotypic characteristics and plant-NTO interactions were observed between maize MON 88017 and its near-isogenic line. [ABSTRACT FROM AUTHOR]

One of the concerns surrounding the commercial release of genetically modified ( GM) crops is the escape of transgenes into agricultural or semi-natural habitats through vertical gene flow, as this may cause environmental or economic problems. There is also the concern that GM crops may affect pollinators and the pollination services they provide. Despite the growing commercial interest of GM tomato ( Solanum lycopersicum), gene flow has been assessed only sparsely in tomato. To evaluate the likelihood of gene flow from GM tomato plants to sexually compatible plants, and to assess whether bumblebee activity is affected by GM tomato, three experiments were conducted under greenhouse conditions, using a Bt-tomato expressing the insecticidal Cry3Bb1 protein as model system: (a) artificial crosses between a GM tomato line, two wild tomato relatives ( Solanum hirsutum and Solanum nigrum) and a non- GM tomato variety; (b) bumblebee-mediated crosses between GM and non- GM tomato plants and (c) visual observations of bumblebees' feeding behaviour. No hybrids were obtained between the GM tomato line and S. hirsutum and S. nigrum. In an experimental design where non- GM receptor plants outnumbered GM plants by approximately 3:1, the bumblebee-mediated cross-fertilisation rate between GM and non- GM tomato plants was measured at 4.3 ± 5.47%. No significant differences in feeding behaviour of bumblebees foraging on GM and non- GM tomato plants were observed. Therefore, we conclude that: (a) the probability of transgene introgression between the GM tomato line used in this study and its wild relatives S. hirsutum and S. nigrum is negligible; (b) bumblebee activity can mediate cross-fertilisation between GM and non- GM tomato and (3) the Cry3Bb1-expressing tomato line tested does not adversely affect the feeding behaviour of bumblebees. [ABSTRACT FROM AUTHOR]

* Premise of the study: Insect-resistant Bacillus thuringiensis (Bt) maize is widely cultivated, yet few studies have examined the interaction of symbiotic arbuscular mycorrhizal fungi (AMF) with different lines of Bt maize. As obligate symbionts, AMF may be sensitive to genetic changes within a plant host. Previous evaluations of the impact of Bt crops on AMF have been inconsistent, and because most studies were conducted under disparate experimental conditions, the results are difficult to compare. * Methods: We evaluate AMF colonization in nine Bt maize lines, differing in number and type of engineered trait, and five corresponding near-isogenic parental (P) base hybrids in greenhouse microcosms. Plants were grown in 50% local agricultural soil with low levels of fertilization, and AMF colonization was evaluated at 60 and 100 d. Nontarget effects of Bt cultivation on AMF colonization were tested in a subsequently planted crop, Glycine max, which was seeded into soil that had been pre- conditioned for 60 d with Bt or P maize. * Key results: We found that Bt maize had lower levels of AMF colonization in their roots than did the non-Bt parental lines. However, reductions in AMF colonization were not related to the expression of a particular Bt protein. There was no difference in AMF colonization in G. max grown in the Bt- or P-preconditioned soil. * Conclusions: These findings are the first demonstration of a reduction in AMF colonization in multiple Bt maize lines grown under the same experimental conditions and contribute to the growing body of knowledge examining the unanticipated effects of Bt crop cultivation on nontarget soil organisms. [ABSTRACT FROM AUTHOR]

Abstract: Transgenic maize expressing Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins may contribute to a decreased need for chemical pesticides, providing benefits for human health and the environment. However, there is public concern regarding the environmental safety of transgenic crops, which relates also to potential adverse effects on plant-microbe interactions by affecting the plant-associated, endophytic microflora. Thus, we compared the endophytic bacterial communities in plants of the three transgenic Bt maize lines MON89034 (cry3Bb1), MON88017 (cry1A.105 and cry2Ab2) and the stacked event MON88017xMON89034 (cry1A.105 and cry2Ab2, cry3Bb1), with those of the respective near-isogenic line, and with those in plants of three additional, conventional maize lines. The maize plants were grown in a containment system on two different soils which were commonly used for maize cultivation in Lower Austria. We obtained 700 bacterial endophytes and characterized them regarding their phylogenetic diversity and specific plant growth promoting functions, including the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and indole-3-acetic acid (IAA). Both the soil environment and the plant cultivar had an effect on the phylogenetic diversity of the endophytic communities, but there were no specific effects of the transgenic varieties. Diversity measures of endophytic isolates were not different in Bt- versus non-Bt maize varieties. A variable number of isolates showed ACC deaminase activity irrespective of the soil and plant cultivar. Generally, the number of high IAA producers appeared to be affected by the soil environment but not by the plant cultivar. Cultivation-independent profiling via terminal restriction fragment polymorphism (T-RFLP) analysis of the 16S rRNA gene revealed that maize root endophytes were differentially composed dependent on soil type and plant cultivar, but they were not discriminated with respect to Bt- versus non-Bt varieties. In conclusion, maize endophyte communities studied were highly diverse and showed a high potential for plant growth promotion, emphasizing their importance in modern agricultural systems irrespective of the maize cultivar, including Bt-modifications. [Copyright &y& Elsevier]

Abstract: Before pest-resistant genetically modified maize can be grown commercially, the risks for soil-beneficial, non-target organisms must be determined. Here, a tiered approach was used to assess the risk to free-living soil nematodes posed by maize genetically modified to express the insecticidal Cry3Bb1 protein (event Mon88017), which confers resistance towards western corn rootworm (Diabrotica virgifera; Coleoptera). The toxicity of purified Cry3Bb1 for the nematode Caenorhabditis elegans was determined using a bioassay and gene expression analysis. In addition, a soil toxicity test was used to assess the effects on C. elegans of rhizosphere soil obtained from plots of an experimental field grown with Mon88017, the near-isogenic cultivar, or either of two conventional cultivars. Finally, the indigenous nematode communities from the experimental field site with Mon88017 and from the control cultivars were analyzed. The results showed a dose-dependent inhibitory effect of Cry3Bb1 on the growth and reproduction of C. elegans, with EC50 values of 22.3mgl−1 and 7.9mgl−1, respectively. Moreover, Cry-protein-specific defense genes were found to be up-regulated in the presence of either Cry1Ab or Cry3Bb1. However, C. elegans was not affected by rhizosphere soils from Mon88017 compared to the control plots, due to the very low Cry3Bb1 concentrations, as indicated by quantitative analyses (<1ngg−1 soil). Nematode abundance and diversity were essentially the same between the various maize cultivars. At the last sampling date, nematode genus composition in Bt-maize plots differed significantly from that in two of the three non-Bt cultivars, including the near-isogenic maize, but the shift in genus composition did not influence the composition of functional guilds within the nematode communities. In conclusion, the risk to free-living soil nematodes posed by Mon88017 cultivation can be regarded as low, as long as Cry3Bb1 concentrations in soil remain four orders of magnitude below the toxicity threshold. [Copyright &y& Elsevier]

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Abstract: Some GM crops including MON863 corn and stack varieties contain Cry3Bb1 protein. Cry3Bb1 is very important from the standpoint of assessing the safety of GM crops. In this study Cry3Bb1 was assessed from the standpoint of possible binding to IgE from allergy patients. First, an ELISA that was improved in our laboratory was used to test serum samples from 13 corn allergy patients in the United States with recombinant Cry3Bb1 expressed in Escherichia coli, and serum samples from 55 patients in Japan with various food allergies were also assayed. Two samples from the Japanese allergy patients were suspected of being positive, but Western blotting analysis with purified Cry3Bb1 indicated that the binding between IgE and Cry3Bb1 was nonspecific. Ultimately, no specific binding between IgE and recombinant Cry3Bb1 was detected. Next, all proteins extracted from MON863 corn and non-GM corn were probed with IgE antibodies in serum samples from the corn allergy patients by Western blotting, but the staining patterns of MON863 and non-GM corn were similar, meaning that unintended allergic reactions to MON863 are unlikely to occur. Our study provides additional information that confirms the predicted lack of IgE binding to Cry3Bb1 in people with existing food allergies. [Copyright &y& Elsevier]