Cry1Ab Protein Research Articles

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2-3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.

Fate of the Cry1Ab Protein from Bt-maize MON810 Silage in Biogas Production Facilities

Biogas plants fuelled with renewable sources of energy are a sustainable means for power generation. In areas with high infestation levels with the European corn borer, Ostrinia nubilalis (Hbn.), it is likely that transgenic Bt-maize will be fed into agricultural biogas plants. The fate of the entomotoxic protein Cry1Ab from MON810 maize was therefore investigated in silage and biogas production-related materials in the utilization chains of two farm-scale biogas plants. The Cry1Ab content in silage exhibited no clear-cut pattern of decrease over the experimental time of 4 months. Mean content for silage was 1878 +/- 713 ng Cry1Ab g(-1). After fermentation in the biogas plants, the Cry1Ab content declined to trace amounts of around 3.5 ng g(-1) in the effluents. The limit of detection of the employed ELISA test corresponded to 0.75 ng Cry1Ab g(-1) sample material. Assays with larvae of O. nubilalis showed no bioactivity of the reactor effluents. The utilization of this residual material as fertilizer in agriculture is therefore deemed to be ecotoxicologically harmless.

In situ studies on the time-dependent degradation of recombinant corn DNA and protein in the bovine rumen1

An in situ technique was adopted to investigate the time-dependent ruminal degradation of chloroplast compared with recombinant DNA of Bt176 corn using conventional and quantitative PCR assays. In parallel, the Cry1Ab protein content and fragment sizes were determined by ELISA and immunoblotting techniques. Triplicate nylon bags filled with 5 g of each substrate (whole-plant isogenic, whole-plant transgenic, ensiled isogenic, and ensiled transgenic corn) were positioned within the rumen of 5 rumen-cannulated, nonlactating cows and incubated for 2, 4, 8, 16, 24, and 48 h. To investigate the DNA degradation process, PCR assays were developed to detect fragments of the endogenous highly abundant rubisco gene (173, 896, 1,197, and 1,753 bp) and of the recombinant cry1Ab gene (211, 420, 727, and 1,423 bp). Short fragments of rubisco (<431 bp) and cry1Ab DNA (211 bp) were amplifiable in whole-plant and ensiled corn samples incubated in the rumen for 48 h, whereas the traceability of larger fragments depended on previous processing of the sample (whole-plant or ensiled corn), the length of the target sequence, and concomitantly on the length of time incubated in the rumen. Quantification of rubisco and cry1Ab gene fragments applying real-time PCR assays revealed degradation to <20% of initial 0-h values within 2 h and <0.5% after 48 h of ruminal incubation. Analysis of Cry1Ab protein in whole-plant corn using the ELISA technique revealed a decrease to 28.0% of the initial value within 2 h and to 2.6% within 48 h. The concentration of Cry1Ab protein of ensiled corn was only 10% that of whole-plant corn. Ensiled corn Cry1Ab protein decreased to 10% of initial values after 48 h of ruminal incubation. Using an immunoblotting technique, the full-size Cry1Ab protein was only detectable up to 8 h; thereafter, only fragments of approximately 17 and 34 kDa size were found. In conclusion, ruminal digestion decreased the presence of functional cry1Ab gene fragments. It is unlikely that full-size, functional Cry1Ab protein will be present after 8 h of incubation in the rumen. Therefore, results based on ELISA measurements should be interpreted carefully and verified by another detection method that discriminates between the full-size and fragmented Cry1Ab protein.

Environmental impact of conventional and Bt insecticidal cotton expressing one and two Cry genes in Australia

Genetically modified Bt cotton, expressing the Cry1Ac protein for specific insecticidal activity against economically significant lepidopteran pests, has been available commercially in Australia since 1996. This technology has been improved and superseded by the addition of a second gene, allowing new varieties to express both the Cry1Ac the Cry2Ab proteins. Bt cotton offers several advantages to the grower, mainly through reduced insecticide spray requirements. The environmental benefits of reduced insecticide usage are assessed in this paper using the environmental impact quotient (EIQ). The assessment included consideration of the impact of the expressed transgenic proteins Cry1Ac and Cry2Ab. EIQ values of the Cry1Ac and Cry2Ab proteins were calculated at 9.9 and 7.9, respectively. Bt protein expression, plant biomass, insecticide application records, constituent of active ingredient, and insecticide EIQ values were used to produce an environmental impact (EI) value for insecticide use (kg a.i./ha) for conventional non-GM and single- and 2-gene Bt cotton for the 1997–98 to 2003–04 seasons. Inclusion of the Cry proteins in the assessment increased the EI values for Bt cotton by only 2%. The average insecticide EI value, for 2002–03 and 2003–04 seasons, for conventional cotton was 135 kg a.i./ha, whereas for the 2-gene Bt variety it was only 28 kg a.i./ha. Results of the EI evaluation indicate that, due to changes in insecticidal choice and reduction in usage, there was a reduction of &gt;64% in EI from growing Bt cotton compared with conventional non-GM cotton in Australia.

Maize stemborer predator activity under ‘push – pull’ system and Bt-maize: A potential component in managing Bt resistance

Studies were conducted between 2002 and 2004 in experimental plots at two sites in western Kenya and one site in South Africa, to assess the potential role of the ‘push – pull’ strategy in stemborer resistance management in Bt-maize through its influence on the pests' oviposition and mortality from predation. Experiments in Kenya were conducted with non-Bt-maize while a combination of Bt-maize (expressing Cry1Ab proteins) and its non-Bt-maize isoline was used in South Africa. Treatments comprised of a maize monocrop and an intercrop of maize and desmodium, with Napier grass as a trap crop around the field in each site (‘push – pull’). A combination of natural and artificial infestation procedures were employed. Oviposition preference and predation rates of naturally infested stemborer eggs were assessed. Screen house-reared plants (non-Bt-maize) were infested with eggs, early-instar larvae, late-instar larvae and pupae of Busseola fusca and Chilo partellus in natural enemy exclusion studies. Significantly more egg batches were oviposited in the maize monocrop than in the ‘push – pull’ plots by C. partellus, but not B. fusca. Predation rates of the naturally oviposited C. partellus eggs were significantly higher in the ‘push – pull’ than in the maize monocrop plots, but not different with B. fusca eggs. Disappearance rates of C. partellus eggs on control plants (those exposed to predators), attributable to predatory activity, was significantly higher in control than exclusion plants and similarly higher in the ‘push – pull’ than in the maize monocrop plots. There were no differences in disappearance rates of B. fusca eggs between treatments. Recovery of early-instar larvae was generally low for both species, similar between control and exclusion plants, but significantly lower in the ‘push – pull’ plots in one of the sites. Recovery of late-instar larvae and pupae in control plants was similar between the two cropping systems for both species. The study revealed reduced oviposition by C. partellus and enhanced predatory activity on C. partellus eggs in the ‘push – pull’ system, pointing to the system becoming a component in Bt resistance management for the pest.

Cloning, Characterization, and Expression of a New cry2Ab Gene From Bacillus thuringiensis Strain 14-1

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1 + orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.

Degradation of transgenic Cry1Ab DNA and protein in Bt‐176 maize during the ensiling process

Maize silage is commonly used as feed for farm animals. The aim of this study was to monitor the time-dependent degradation of non-recombinant chloroplast DNA (exemplified by the rubisco gene) in comparison with the recombinant cry1Ab gene in the course of the ensiling process. In parallel, the Cry1Ab protein content and fragment sizes were determined. Fragments of the rubisco (173, 896, 1197, 1753 and 2521 bp) and of the cry1Ab gene (211, 420, 727 and 1,423 bp) were selected to investigate the DNA degradation process. The detection of the Cry1Ab protein was performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Rubisco gene fragments of 173 bp were still detectable after 61 days, while fragments of 1,197 and 2,521 bp were detectable up to 30 days and on the first day only respectively. Polymerase chain reaction (PCR) analyses revealed that fragments of the cry1Ab gene with sizes of 211 and 420 bp were detectable up to 61 days, fragments with sizes of 727 and 1,423 bp, 30 and 6 days respectively. The ELISA showed a decrease of the Cry1Ab protein in maize silage during the ensiling process. No marked degradation was observed during the first 43 h. Thereafter, a sharp decrease was measured. After 61 days, 23.6 +/- 0.9% of the initial Cry1Ab protein was still detectable. Immunoblotting confirmed the results of the ELISA showing a positive signal of approximately 60 kDa size for 8 days of ensiling; no further immunoactive fragments were detectable by immunoblotting. In conclusion, the ensiling process markedly decreases the presence of long functional cry1Ab gene fragments and full size Cry1Ab protein.

Ex situ application of foliar-produced lepidoptericides from transgenic rice to control Pieris rapae and Cry1Ab stability in vivo

Insecticidal toxins produced by Bacillus thuringiensis (Bt toxins) have been widely used in plant protection in two different ways: spraying of bacterial Bt toxin formulations over plants and production of the toxins in planta by expressing the genes encoding for the proteins. In the second strategy, only the transgenic plants themselves are protected. We have now broadened the application of insect-resistant transgenics in a third scenario: protection of other crops that theoretically could be recalcitrant to DNA transformation. Using cabbage as a model crop well known to be susceptible to many pests, the Bt toxin Cry1Ab was first produced in transgenic rice ( Oryza sativa) plants and then was applied to the leaves of cabbage ( Brassica juncea) to test its toxicity against larvae of Pieris rapae. Seventy-eight to 100% larval mortality was observed for different instar larvae. Furthermore, no-choice feeding of the larvae on the Bt toxin-treated cabbage leaves had adverse effects on the oviposition rates and egg hatchability of surviving adult moths. Bt toxin-treated larvae had 30% and 46% lower moth emergence rates than their control counterparts. Female moths from the Bt toxin-treated larvae had significantly reduced egg production, 15.1 eggs per female versus the control 326.3 eggs, and lower hatching rates, 3.1% versus 33.8% in the control. Since long term stability is a key factor contributing to the potential of ex situ application of plant-made biocides, Cry1Ab protein stability was assessed in the detached rice leaf tissue. The toxin was found to be stable over a 5-month storage period, declining 31.7% and 20% from the initial content when stored at 4 and 37 °C, respectively.

Detection of transgenic maize Cry1Ab protein subjected to ruminal digestion

1. Pepsin Degradation of Cry1A(b) Protein Purified from Genetically Modified Maize (Zea mays) Luis de, María Lavilla, Lourdes Sánchez, Miguel Calvo, María Pérez Journal of Agricultural and Food Chemistry CrossRef

Impact of Transgenic VIP3A × Cry1Ab Lepidopteran-resistant Field Corn on the Nontarget Arthropod Community

A field experiment was conducted over 3 yr to assess the effects of transgenic field corn expressing stacked lepidopteran-active Bacillus thuringiensis (Bt)-derived VIP3A and Cry1Ab proteins on nontarget arthropods. Effects were compared with an isogenic conventional corn hybrid treated with a foliar insecticide and an isogenic control without insecticides. Plant inspections, pitfall traps, sticky cards, and emergence traps determined the abundance and diversity of plant, aerial, and surface-dwelling communities. More than 500,000 arthropods were enumerated, representing 203 taxonomic groups in 112 families and 13 orders. In terms of abundance, 70% of the arthropods were saprovores, 13% were herbivorous insects, 14% were predators, and 3% were parasitoids. Overall biodiversity and community-level responses were not significantly affected by expression of the stacked VIP3A and Cry1Ab proteins. Densities of most nontarget taxa exposed to the Bt hybrid showed no differences compared with the isogenic control. However, significant changes in certain taxa did occur in the Bt plots, which were indirectly related to plant-mediated factors, prey density responses, and the absence of plant injury. Arthropod communities in the insecticide-treated plots displayed both negative and positive changes in the abundances of individual taxa. Together, effects observed in the Bt plots were significantly less than the community disturbances caused by insecticide applications. Changes in nontarget communities in plots previously exposed to insecticides and the Bt hybrid did not carryover to the following growing season. Major arthropods as bioindicators, routes of exposures, and food web interactions relative to the observed effects are discussed.

Determination of baseline susceptibility to Cry1Ab protein for Asian corn borer (Lep., Crambidae)

Abstract: Although transgenic Bacillus thuringiensis (Bt) corn can provide a new tool for control of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), concern has been raised regarding the possibility of the target insect evolving resistance to the Bt protein under intensive selection pressure from Bt corn. Therefore, it is necessary to establish baseline data to enable detection of changes in susceptibility in field populations after prolonged exposure to Bt corn. Susceptibility to purified Cry1Ab protein from Bt was determined for 10 populations of ACB from the major corn‐growing regions of China, ranging geographically from Heilongjiang Province in the northeast to Shaanxi Province in the east‐central part. Neonate ACB were exposed to semi‐artificial diet incorporated with increasing Cry1Ab protein concentrations, and mortality and growth inhibition were evaluated after 7 days. The range of LC50 (50% lethal concentration) among the populations was 0.10 to 0.81 μg/g (Cry1Ab protein/diet). Differences (P &lt; 0.05) in susceptibility among the populations were significant. LC50s generated from the Huanghuaihai Summer Corn Region were higher than those from the Spring Corn Regions. Bt was one of the significant natural biomortality factors of overwintering generation ACB. There was a significant correlation between percentage of the larvae infected with Bt and their LC50 values to Cry1Ab protein in geographic distinct populations (r = 0.7350*, d.f. = 8, r0.05 = 0.632). Based on the background of Bt formulations used for corn insect pests control in these areas, these differences were not caused by prior exposure to Bt insecticides. Instead, the small differences likely reflect natural Bt selection pressure. Because the variation in susceptibility to Cry1Ab was small (&lt;10‐fold), the ACB apparently is susceptible to Cry1Ab across its range within China.

Can Bacillus thuringiensis ( Bt) corn residues and Bt-corn plants affect life-history traits in the earthworm Aporrectodea caliginosa?

Bt-corn is genetically engineered to express proteins from the common soil bacterium Bacillus thuringiensis which presents specific toxicity to targeted insect pests. One type of Bt-corn expresses the Cry1Ab protein that provides protection against certain lepidopteran pests, mainly the European corn borer ( Ostrinia nubilalis) and the Mediterranean stalk borer ( Sesamia nonagroides). Vast areas of agricultural soils worldwide are grown with transgenic Bt-corn. With such widespread use of Bt-corn, it is important to evaluate the potential risks of Bt-protein to non-target organisms in agro-ecosystems such as earthworms. In this study, we investigated the effects of Bt-corn on important life-history traits (survival, reproduction and growth) of the earthworm Aporrectodea caliginosa under various experimental conditions. Finely ground Bt-corn leaves added to soil had no deleterious effects on survival, growth, development or reproduction in A. caliginosa, even in high concentrations that could be considered as a worst-case scenario. Also, growth of juvenile A. caliginosa was unaffected when worms were kept in pots with a growing Bt-corn plant. Only when considering cocoon hatchability did we see a slight, but statistically significant, negative effect of Bt-corn residues. The implications of these results for risk assessment of Bt-corn are discussed.

Cry1Ab Protein Does Not Persist in Soil After 3 Years of Sustained Bt Corn Use

The purpose of this study was to assess the persistence and accumulation of the Cry1Ab protein in soil as a result of sustained planting of genetically modified Bacillus thuringiensis (Bt) corn hybrids. Soil samples were collected from agricultural fields in five corn-growing regions of the United States where Bt corn hybrids (MON 810 or Bt11) had been planted for at least 3 consecutive yr. At each site, soil samples were collected during the corn-growing period (postanthesis) and again within 6 wk after harvest. Multiple soil specimens from matched Bt cornfields and nearby, non-Bt control fields were analyzed by diet-incorporation insect bioassay, using growth inhibition (GI) of the European corn borer (Ostrinia nubilalis) as the toxicity endpoint. Positive control soil samples containing Cry1Ab protein at the GI50 level (0.05 μg/g soil) were analyzed in tandem with test and control samples to verify that the bioassay was able to detect low levels of Cry1Ab protein. The limit of detection for Cry1Ab protein in soil was 0.03 μg/g soil. The presence of Cry1Ab protein in soil was assessed by statistical comparison of the insect toxicity (GI) of soils collected from Bt and non-Bt (control) cornfields. Only one soil sample, collected postanthesis in a Bt cornfield that had also been treated with carbofuran insecticide, showed insect toxicity. This toxicity was below the GI50 level, and no toxicity was detected in the soil collected from the same plot shortly after harvest. Therefore, there is no evidence of persistence or accumulation of Cry1Ab protein in soils from fields planted for at least three consecutive growing seasons with Bt corn hybrids.

Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay

AbstractLaboratory bioassays were conducted to evaluate the response of first instar larvae of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Danaidae), a non‐target species, to pollen from corn, Zea mays L. (Commelinales: Poaceae), from two new corn hybrids genetically modified to express different types of insecticidal proteins derived from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). One hybrid expresses both Cry1Ab and Cry2Ab2 proteins (MON 810 × MON 84006), active against lepidopteran pests, and the other expresses Cry3Bb1 protein (MON 863), targeted against coleopteran pests. First instar larvae were placed on milkweed leaves (Asclepias syriaca L.) (Gentianales: Asclepiadaceae) dusted with doses of either Bt pollen or its nonexpressing (isoline) pollen counterpart ranging from 50 to 3200 grains cm−2 of milkweed leaves, or no pollen at all. Larvae were exposed to pollen for 4 days, then moved to pollen‐free leaves and observed for another 6 days. Survival was observed after 2, 4, and 10 days. Weight gain was estimated after 4 and 10 days, leaf consumption after 2 and 4 days, and larval development after 10 days. Exposure to pollen of the Cry1Ab/Cry2Ab2‐Bt expressing hybrid reduced larval survival approximately 7.5–23.5% at the dose ranges tested relative to a no pollen control. Larval weight gain and consumption were reduced for larvae exposed to pollen of this hybrid and a small minority of larvae (3.1%) never developed past the third instar after 10 days of observation. Exposure to pollen of the Cry3Bb1‐Bt expressing hybrid had no negative effects on larval mortality, weight gain, consumption, or development relative to the consumption of Bt‐free corn pollen. The relevance of these findings to the risk that these Bt corn hybrids pose to monarch populations is discussed.

Field studies on the environmental fate of the Cry1Ab Bt‐toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere

Field studies were done to assess how much of the transgenic, insecticidal protein, Cry1Ab, encoded by a truncated cry1Ab gene from Bacillus thuringiensis (Bt), was released from Bt-maize MON810 into soil and whether bacterial communities inhabiting the rhizosphere of MON810 maize were different from those of the rhizosphere of nontransgenic maize cultivars. Bacterial community structure was investigated by SSCP (single-strand conformation polymorphism) of PCR-amplified 16S rRNA genes from community DNA. Using an improved extraction and detection protocol based on a commercially available ELISA, it was possible to detect Cry1Ab protein extracted from soils to a threshold concentration of 0.07 ng/g soil. From 100 ng of purified Cry1Ab protein added per gram of soil, only an average of 37% was extractable. At both field sites investigated, the amount of Cry1Ab protein in bulk soil of MON810 field plots was always lower than in the rhizosphere, the latter ranging from 0.1 to 10 ng/g soil. Immunoreactive Cry1Ab protein was also detected at 0.21 ng/g bulk soil 7 months after harvesting, i.e. in April of the following year. At this time, however, higher values were found in residues of leaves (21 ng/g) and of roots (183 ng/g), the latter corresponding to 12% of the Cry1Ab protein present in intact roots. A sampling 2 months later indicated further degradation of the protein. Despite the detection of Cry1Ab protein in the rhizosphere of MON810 maize, the bacterial community structure was less affected by the Cry1Ab protein than by other environmental factors, i.e. the age of the plants or field heterogeneities. The persistence of Cry1Ab protein emphasizes the importance of considering post-harvest effects on nontarget organisms.

Modeling effects of environment, insect damage, and Bt genotypes on fumonisin accumulation in maize in Argentina and the Philippines

Fumonisins are common contaminants of maize (Zea mays L.) grain products, especially in countries where maize is a major constituent of the diet and are harmful to human and animal health. There is a need to better define environmental conditions that favor fumonisin accumulation in the grain of maize. The impacts of biotic and abiotic factors, and hybrids containing the Cry1Ab protein from Bacillus thuringiensis (Bt), were associated with fumonisin accumulation in the grain of maize across contrasting environments in Argentina and the Philippines between 2000 and 2002. Average fumonisin concentrations in grain samples varied from 0.5 to 12 microg g(-1) across field locations in Argentina, and from 0.3 to 1.8 microg g(-1) across locations in the Philippines. The ratio of fumonisin B1 to fumonisin B2 was <3.0 in four of nine locations in Argentina, which proved to be due to a higher prevalence of Fusarium proliferatum in those locations. Most of the variability of total fumonisins among maize grain samples was explained by location or weather (47%), followed by insect damage severity in mature ears (17%), hybrid (14%), and with the use of Bt hybrids (11%). In Argentina, where conditions were more favorable for accumulation of fumonisin in the years considered, fumonisin concentrations were lower in Bt hybrids compared to their genetic isolines by an average of 40%. A model was developed to predict fumonisin concentration using insect damage to ears and weather variables as predictors in the model. Four periods of weather around silking were identified as critical for fumonisin concentrations at harvest. The model accounted for 82% of the variability of total fumonisin across all locations in 2 years of the study.

Decomposition processes under Bt ( Bacillus thuringiensis) maize: Results of a multi-site experiment

The effects of maize expressing the Bacillus thuringiensis Cry1Ab protein ( Bt maize) on decomposition processes under three different European climatic conditions were assessed in the field. Farming practices using Bt maize were compared with conventional farming practices using near-isogenic non- Bt maize lines under realistic agricultural practices. The litter-bag method was used to study litter decomposition and nitrogen mineralization dynamics of wheat straw. After 4 months incubation in the field, decomposition and mineralization were mainly influenced by climatic conditions with no negative effect of the Bt toxin on decomposition processes.

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.

Abundance of non-target pests in transgenic Bt-maize: A farm scale study

The impact of transgenic Bt-maize, expressing the Cry1Ab protein, on aphids, leafhoppers, cutworms and wireworms was evaluated at the farm scale by comparing their abundance on Bt-plots and those sown with the isogenic variety over three consecu- tive growing seasons. The impact of Bt-maize was different on each of the three-herbivore groups. There were significantly more aphids on the Bt-maize but in terms of aphid species, the difference was only statistically significant for Sitobion avenae and not for the other three most abundant species (Metopolophium dirhodum, Rhopalosiphum padi and Macrosiphum euphorbiae). The analysis of aphid age structure showed consistently more individuals on Bt-plots; differences were significant for alates, apterous adults and young nymphs of R. padi, apterous adults and apterous fourth instar nymphs of S. avenae, alates, apterous adults and apterous fourth instar nymphs of M. dirhodum. Leafhoppers (Zyginidia scutellaris), particularly mature nymphs, were also more abundant on the Bt- maize. In spite of this there was no difference in leafhopper damage to Bt and non Bt-maize. The reasons for this are unclear but may be due to changes in Bt-maize that favoured aphids and leafhoppers. Differences in aphid and leafhopper densities were not high enough to affect yield. However, they may have affected the availability of prey for polyphagous predators such as Orius sp. The Bt- maize did not affect the incidence of cutworms (Agrotis segetum) or wireworms (Agriotes lineatus). These results indicate that Bt- maize does not have a negative impact on the non-target maize biocenosis at the farm scale.

Detection of Genetically Modified Corn (Bt176) in Spiked Cow Blood Samples by Polymerase Chain Reaction and Immunoassay Methods

The fate of DNA and protein transgenic sequences in products derived from animals fed transgenic crops has recently raised public interest. Sensitive molecular tests targeting the Bt176 genetic construct and the transgenic Cry1Ab protein were developed to determine whether plant sequences, especially transgenic sequences, are present in animal products. A protocol for total DNA extraction and purification from cow whole blood samples was first drawn up and assessed by spiking with known amounts of DNA from Bt176 maize. The limit of detection for transgenic sequences (35S promoter and Bt176-specific junction sequence) was determined by both the polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and the 5'-nuclease PCR assay. Four additional PCR systems were built to substantiate the results. The first detects a mono-copy maize-specific sequence (ADH promoter). Two others target multi-copy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The last one, used as a positive control, targets a mono-copy animal sequence (alpha(s1)-casein gene). Both methods detected a minimum spiking at 25 copies of Bt176 maize/mL in 10 mL whole blood samples. The sandwich ELISA kit used detected down to 1 ng transgenic Cry1Ab protein/mL spiked whole blood.