Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.     

High-throughput sequence-based analysis of the intestinal microbiota of weanling pigs fed genetically modified MON810 maize expressing Bacillus thuringiensis Cry1Ab (Bt maize) for 31 days

The objective of this study was to investigate if feeding genetically modified (GM) MON810 maize expressing the Bacillus thuringiensis insecticidal protein (Bt maize) had any effects on the porcine intestinal microbiota. Eighteen pigs were weaned at ~28 days and, following a 6-day acclimatization period, were assigned to diets containing either GM (Bt MON810) maize or non-GM isogenic parent line maize for 31 days (n = 9/treatment). Effects on the porcine intestinal microbiota were assessed through culture-dependent and -independent approaches. Fecal, cecal, and ileal counts of total anaerobes, Enterobacteriaceae, and Lactobacillus were not significantly different between pigs fed the isogenic or Bt maize-based diets. Furthermore, high-throughput 16S rRNA gene sequencing revealed few differences in the compositions of the cecal microbiotas. The only differences were that pigs fed the Bt maize diet had higher cecal abundance of Enterococcaceae (0.06 versus 0%; P < 0.05), Erysipelotrichaceae (1.28 versus 1.17%; P < 0.05), and Bifidobacterium (0.04 versus 0%; P < 0.05) and lower abundance of Blautia (0.23 versus 0.40%; P < 0.05) than pigs fed the isogenic maize diet. A lower enzyme-resistant starch content in the Bt maize, which is most likely a result of normal variation and not due to the genetic modification, may account for some of the differences observed within the cecal microbiotas. These results indicate that Bt maize is well tolerated by the porcine intestinal microbiota and provide additional data for safety assessment of Bt maize. Furthermore, these data can potentially be extrapolated to humans, considering the suitability of pigs as a human model.     

Effects of genetically modified maize events expressing Cry34Ab1, Cry35Ab1, Cry1F,and CP4 EPSPS proteins on arthropod complex food webs

Four genetically modified (GM) maize (Zea mays L.) hybrids (coleopteran resistant, coleopteran and lepidopteran resistant, lepidopteran resistant and herbicide tolerant, coleopteran and herbicide tolerant) and its non‐GM control maize stands were tested to compare the functional diversity of arthropods and to determine whether genetic modifications alter the structure of arthropods food webs. A total number of 399,239 arthropod individuals were used for analyses. The trophic groups’ number and the links between them indicated that neither the higher magnitude of Bt toxins (included resistance against insect, and against both insects and glyphosate) nor the extra glyphosate treatment changed the structure of food webs. However, differences in the average trophic links/trophic groups were detected between GM and non‐GM food webs for herbivore groups and plants. Also, differences in characteristic path lengths between GM and non‐GM food webs for herbivores were observed. Food webs parameterized based on 2‐year in‐field assessments, and their properties can be considered a useful and simple tool to evaluate the effects of Bt toxins on non‐target organisms.     

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.     

Fate of recombinant DNA and Cry1Ab protein after ingestion and dispersal of genetically modified maize in comparison to rapeseed by fallow deer (Dama dama)

The fate of recombinant DNA in fallow deer (Dama dama) was investigated by feeding a diet of isogenic or genetically modified (GM) maize expressing Cry1Ab protein against the European corn borer (Ostrinia nubilalis). To study the degradability of ingested DNA, polymerase chain reaction (PCR) assays were introduced to detect fragments of the endogenous, highly abundant chloroplast-specific rubisco gene, the maize-specific zein gene and the recombinant cry1Ab gene. PCR analysis revealed that small chloroplast- and maize-specific DNA fragments were detectable in contents of rumen, abomasums, jejunum, caecum and colon and occasionally in visceral tissues. In contrast, no fragments of the recombinant cry1Ab gene were detectable in gastrointestinal (GI) contents. The Cry1Ab protein was analysed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique. Neither ELISA nor immunoblotting yielded positive signals of immunoactive Cry1Ab protein in GI contents and tissues of fallow deer fed with GM maize. In conclusion, after uptake of GM maize, neither cry1Ab-specific gene fragments nor Cry1Ab protein were detected in the GI tract of fallow deer, indicating complete digestion of the GM maize. Additional investigations on the germination capacity of conventional rapeseed and maize seed after ingestion by fallow deer and faecal excretion (endozoochory) were performed to draw conclusions regarding a potential spreading of germinable GM crop seed by deer. Germination tests revealed that germinable rapeseed kernels were detectable in faeces; in contrast, no intact maize seeds were found in faeces.     

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%–50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%–30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.     

Determination of insecticidal Cry1Ab protein in soil collected in the final growing seasons of a nine-year field trial of Bt-maize MON810

Cultivation of genetically modified maize (Bt-maize; event MON810) producing recombinant δ-endotoxin Cry1Ab, leads to introduction of the insecticidal toxin into soil by way of root exudates and plant residues. This study investigated the fate of Cry1Ab in soil under long-term Bt-maize cultivation in an experimental field trial performed over nine growing seasons on four South German field sites cultivated with MON810 and its near isogenic non Bt-maize variety. Cry1Ab protein was quantified in soil (<2 mm size) using an in-house validated ELISA method. The assay was validated according to the criteria specified in European Commission Decision 2002/657/EC. The assay enabled quantification of Cry1Ab protein at a decision limit (CCα) of 2.0 ng Cry1Ab protein g⁻¹ soil with analytical recovery in the range 49.1–88.9%, which was strongly correlated with clay content. Cry1Ab protein was only detected on one field site at concentrations higher than the CCα, with 2.91 and 2.57 ng Cry1Ab protein g⁻¹ soil in top and lower soil samples collected 6 weeks after the eighth growing season. Cry1Ab protein was never detected in soil sampled in the spring before the next farming season at any of the four experimental sites. No experimental evidence for accumulation or persistence of Cry1Ab protein in different soils under long-term Bt-maize cultivation can be drawn from this field study.     

A proteomic-based approach to study underlying molecular responses of the small intestine of Wistar rats to genetically modified corn (MON810)

A genetically modified (GM) commercial corn variety, MON810, resistant to European corn borer, has been shown to be non-toxic to mammals in a number of rodent feeding studies carried out in accordance with OECD Guidelines. Insect resistance results from expression of the Cry1Ab gene encoding an insecticidal Bt protein that causes lysis and cell death in susceptible insect larvae by binding to midgut epithelial cells, which is a key determinant of Cry toxin species specificity. Whilst whole animal studies are still recognised as the ‘gold standard’ for safety assessment, they only provide indirect evidence for changes at the cellular/organ/tissue level. In contrast, omics-based technologies enable mechanistic understanding of toxicological or nutritional events at the cellular/receptor level. To address this important knowledge-gap and to gain insights into the underlying molecular responses in rat to MON810, differential gene expression in the epithelial cells of the small intestine of rats fed formulated diets containing MON810, its near isogenic line, two conventional corn varieties, and a commercial (Purina?) corn-based control diet were investigated using comparative proteomic profiling. Pairwise and five-way comparisons showed that the majority of proteins that were differentially expressed in the small intestine epithelial cells in response to consumption of the different diets in both 7-day and 28-day studies were related to lipid and carbohydrate metabolism and protein biosynthesis. Irrespective of the diet, a limited number of stress-related proteins were shown to be differentially expressed. However these stress-related proteins differed between diets. No adverse clinical or behavioural effects, or biomarkers of adverse health, were observed in rats fed GM corn compared to the other corn diets. These findings suggest that MON810 has negligible effects on the small intestine of rats at the cellular level compared with the well-documented toxicity observed in susceptible insects.

     

Effects of a diet containing genetically modified rice expressing the Cry1Ab/1Ac protein (Bacillus thuringiensis toxin) on broiler chickens

The aim of this study was to evaluate the effect of feeding Bacillus thuringiensis (Bt) rice expressing the Cry1Ab/1Ac protein on broiler chicken. The genetically modified (GM) Bt rice was compared with the corresponding non-GM rice regarding performance of feeding groups, their health status, relative organ weights, biochemical serum parameters and occurrence of Cry1Ab/1Ac gene fragments. One hundred and eighty day-old Arbor Acres female broilers with the same health condition were randomly allocated to the two treatments (6 replicate cages with 15 broilers in each cage per treatment). They received diets containing GM rice (GM group) or its parental non-GM rice (non-GM group) at 52–57% of the air-dried diet for 42 days. The results show that the transgenic rice had a similar nutrient composition as the non-GM rice and had no adverse effects on chicken growth, biochemical serum parameters and necropsy during the 42-day feeding period. In birds fed the GM rice, no transgenic gene fragments were detected in the samples of blood, liver, kidneys, spleen, jejunum, ileum, duodenum and muscle tissue. In conclusion, the results suggest that Bt rice expressing Cry1Ab/1Ac protein has no adverse effects on broiler chicken. Therefore, it can be considered as safe and used as feed source for broiler chicken.     

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.     

Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation

Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.     

Exposure of arthropod predators to Cry1Ab toxin in Bt maize fields

Abstract.  1. To assess the risks of an insect-resistant transgenic plant for non-target arthropods, it is important to investigate the exposure of non-target species to the transgene product. Exposure of predators in the field depends on the toxin levels in food sources, their feeding ecology and that of their prey.
2. To verify the transmission of Cry1Ab toxin through the food chain, and thus exposure of predators in the field, samples from different plant tissues, herbivores, and predators in Bt maize fields in Spain (Event 176) were collected at different periods over the season and the toxin content was measured using ELISA. Complementary laboratory studies were performed with the omnivorous predator Orius majusculus to assess the toxin uptake and persistence after feeding on variable Bt-containing food sources.
3. Field results revealed that toxin content in some herbivores was negligible (aphids, thrips, leafhoppers) compared with those in spider mites. The latter herbivore only occurred after pollen shed and contained three times greater toxin levels than Bt maize leaves.
4. Data confirmed that the Bt toxin can be transferred to predators, that is to say to Orius spp., Chrysoperla spp., and Stethorus sp. This only applied when Bt maize pollen or spider mites were available. The passage of Bt toxin to O. majusculus via these two food sources was also confirmed in the laboratory. Contrastingly, some predators in the field (hemerobiids, Nabis sp., Hippodamia sp., Demetrias sp.) contained no or negligible toxin levels even when pollen or spider mites were present.
5. Besides essential information for exposure assessment of numerous arthropod predators, this study provides an insight into the feeding ecology of different arthropods in the maize system.     

Extended monitoring of resistance to Bacillus thuringiensis Cry1Ab maize in Diatraea saccharalis (Lepidoptera: Crambidae)

The sugarcane borer, Diatraea saccharalis (F.), is a major target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid-Southern region of the United States. During 2007-2009, a total of 986 feral individuals of D. saccharalis were collected from maize fields in six locations of Louisiana and Mississippi and examined for resistance to Cry1Ab maize using F 1/F 2 screens. Major resistance alleles to Cry1Ab maize in the populations sampled from non-Bt maize plants during 2007 and 2008 in Louisiana and 2009 in Mississippi were rare. From a total of 487 individuals collected from three locations in Louisiana in 2007 and 2008, only one individual was identified with major resistance alleles. In addition, no major resistance alleles were detected in 242 individuals collected from three locations in Mississippi in 2009. The frequency of major resistance alleles was estimated to be 0.002 with a 95% CI of 0.00025-0.0057 for the Louisiana populations and < 0.0061, with 95% probability, for the Mississippi populations. The resistance frequency estimated for the Louisiana populations in 2007 and 2008 was not significantly different from those reported previously for populations sampled in 2004-2006. However, among 200 individuals sampled from non-Bt maize plants in 2009 in Louisiana, six individuals were identified to possess major resistance alleles. The estimated major resistance allele frequency for the populations sampled from non-Bt maize plants in 2009 in Louisiana was 0.0176 with a 95% CI of 0.0072 to 0.0328, which was significantly greater than those estimated for the populations collected in 2004-2008. Similarly, the frequency of minor resistance alleles to Cry1Ab maize for the Louisiana populations collected in 2009 was also significantly greater than those estimated for the populations sampled before. In addition, two out of 57 feral individuals collected from Bt maize plants in Louisiana in 2009 were identified to carry major resistance alleles to Cry1Ab maize. Since 2010, transgenic maize expressing pyramided Bt genes has been planted in the US mid-Southern region and by 2011, pyramided Bt maize has replaced Cry1Ab maize as the dominant Bt maize for managing lepidopteran pests including D. saccharalis. The timely switching from single-gene Cry1Ab maize to the pyramided Bt maize should prevent further increases in Cry1Ab resistance allele frequency and thus ensure the continued success of Bt maize for managing D. saccharalis in the region.     

Degradation of the Cry1Ab protein within transgenic Bacillus thuringiensis corn tissue in the field

Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem. Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues. Therefore, it is important to know how long the toxin persists in plant residues. In two field studies in the temperate corn-growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme-linked immunosorbent assay (ELISA). In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month. During the second month, Cry1Ab toxin concentrations decreased to approximately 20% of their initial values. During winter, there was no further degradation. When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June. In the second field trial, representing a no-tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil-incorporated Bt plants, to 38% of the initial concentration during the first 40 days. They then continued to decrease until the end of the trial after 200 days in June, when 0.3% of the initial amount of Cry1Ab toxin was detected. Our results suggest that extended pre- and post-commercial monitoring are necessary to assess the long-term impact of Bt toxin in transgenic plant residues on soil organisms.     

Minnesota field population of western corn rootworm (Coleoptera: Chrysomelidae) shows incomplete resistance to Cry34Ab1/Cry35Ab1 and Cry3Bb1

In the United States of America, the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is commonly managed with transgenic corn (Zea mays L.) expressing insecticidal proteins from the bacteria Bacillus thuringiensis Berliner (Bt). Colonies of this pest have been selected in the laboratory on each commercially available transformation event and several resistant field populations have also been identified; some field populations are also resistant. In this study, progeny of a western corn rootworm population collected from a Minnesota corn field planted to SmartStax^® corn were evaluated for resistance to corn hybrids expressing Cry3Bb1 (event MON88017) or Cry34/35Ab1 (event DAS‐59122‐7) and to the individual constituent proteins in diet‐overlay bioassays. Results from these assays suggest that this population is resistant to Cry3Bb1 and is incompletely resistant to Cry34/35Ab1. In diet toxicity assays, larvae of the Minnesota (MN) population had resistance ratios of 4.71 and >13.22 for Cry34/35Ab1 and Cry3Bb1 proteins, respectively, compared with the control colonies. In all on‐plant assays, the relative survival of the MN population on the DAS‐59122‐7 and MON88017 hybrids was significantly greater than the control colonies. Larvae of the MN population had inhibited development when reared on DAS‐59122‐7 compared with larvae reared on the non‐Bt hybrid, indicating resistance was incomplete. Overall, these results document resistance to Cry3Bb1 and an incomplete resistance to Cry34/35Ab1 in a population of WCR from a SmartStax^® performance problem field.     

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 < 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 (<10‐fold), the ACB apparently is susceptible to Cry1Ab across its range within China.     

Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications

Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% × (0.5 ± 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N × GM wt% × (0.5 ± 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.     

Cross-resistance studies of Cry1Ac-resistant strains of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry2Ab

Bioassays and binding tests between Cry toxins (CrylAa, CrylAb, CrylAc, and Cry2Ab) and brush-border membrane vesicles (BBMVs) from larvae of a Bacillus thurningiensis (Bt)-susceptible (96S) and two CrylAc-resistant strains (BtR and LFR10) were conducted for investigating cross-resistance of CrylAc-resistant strains to Cry2Ab in Helicoverpa armigera (Hiibner) (Lepidoptera: Noctuidae). The resistance ratio (RR) values of the BtR and LFR10 strains to CrylAc and Cry2Ab were 2,971- and 1.1-fold and 253- and 1.0-fold, respectively, indicating that there was no cross-resistance to Cry2Ab. The binding experiments between Cry toxins and BBMVs from BtR, LFR10, and 96S larvae showed that all of the toxins could bind with these BBMVs, but the Cry2Ab could not displace 125I labeled CrylAc and CrylAb. The same results were observed in reciprocal binding tests, demonstrating that CrylA and Cry2Ab had different binding sites in H. armigera and providing a potential mechanism for the lack of cross-resistance between CrylA and Cry2Ab toxins. These results suggest that the transgenic cotton, Gossypium hirsutum L., expressing CrylAc and Cry2Ab genes may be deployed for management of CrylAc resistant H. armigera.     

Occurrence of aflatoxin B1 in dairy cow feed over 10 years in Portugal (1995-2004)

In Portugal, there is rather little information about the natural occurrence of aflatoxin in feedstuffs. The aim of this work was to report the results of screening the natural incidence of aflatoxin B1 (AFB1) in samples of cattle feed collected from seven dairy cow's farms from Portugal distributed in several locations of the country. One thousand and one samples were taken from 1995 to 2004. High performance liquid chromatography (HPLC) was used for separation, identification and quantification of the compound. Detection limit was 1 microg/kg. Aflatoxin B1 was detected in 374 (37.4%) of the samples. The incidence and mean content of AFB1 was generally low. Levels of aflatoxin B1 above the maximum limit established in Portugal (5 microg/kg) for dairy cattle feed samples were observed in 62 samples (6.2%) with levels ranging from 5.1 to 74 microg/kg. Out of those 62 samples, 3.7% had levels between 5.1 to 10 (mean 7.8); 1.8% had a contamination level of 10.1 to 20 (mean 12.0), and 0.7% exceeded 20.1 microg/kg (mean 50.4). On the last two years (2003-04) none of the samples exceeded the maximum permissible level of the toxin.