Cry1Ac Research Articles

Quantitative Study of Cry1Ac Protein and Bioassay for Helicoverpa armigera at Different Development Stages of Upland Cotton (Gossypium hirsutum L.)

Eight transgenic (Cry1Ac -endotoxin) genotypes and one non-GMO cotton (Gossypium hirsutum L.) variety were used in the investigation. Cry1Ac protein was expressed at various extents in several strains. In a greenhouse environment, sixty days after planting, the cry 1Ac gene's expression peaked. At 60 DAP, the variety Bt.CIM-598 (1.871 g/g) expressed the most toxin of any genotype. Throughout the cropping season, larvae of the Helicoverpa armigera were gathered from every section of the nation that produces cotton. H. armigera larvae that were captured in the field were raised on artificial feeds. There was a negative relationship between the DAP and the amount of Cry1Ac protein (-0.332). The greatest amount of death was seen when first-instar larvae consumed the leaves of 60 DAP plants. It was found that transgenic variants had a considerable effect on the percentage mortality of H. armigera. MNH-886 larvae showed the highest mortality percentage when fed on leaves that were harvested 30 DAP (days after planting). When larvae were fed on 60 and 90 DAP leaves on CIM-598 and IR-1524, the mortality rate was greater. However, AA-802 had the highest death rate when larvae were fed on 120 DAP leaves. The variety with the highest death rate was CIM-598. When H. armigera consumed 60 DAP leaves, the greatest overall mortality was noted. The plant-toxin interaction in cotton varied depending on a number of physiological changes in plants. Regardless of plant age or variety, the highest rate of mortality of H. armigera larvae was seen after feeding 48-hour of treatment or feeding.
 

Indian scenario on the occurrence of a dreaded insect pest Pink bollworm, Pectinophora gossypiella on Bt cotton-A review

The pink bollworm, Pectinophora gossypiella, has become a significant production constraint on Bt cotton in India. This problem is unique to India because the pest has developed multi-fold resistance to Cry toxins in many Indian populations but not in other countries. Most Indian populations have developed multifold resistance to Cry 1 Ac and Cry 1Ac + Cry 2 Ab toxins. Year-round cultivation of long-duration Bt cotton hybrids on a large scale has a pronounced impact on the incidence. Also discussed other factors responsible for the occurrence of pink bollworm on Bt cotton in India. Insecticide Resistance Management (IRM) strategies implemented by different cotton-growing countries globally; the USA, India, and China had a significant impact on the interaction of pink bollworm on Bt cotton. Huge selection pressure resulted in resistance to Cry toxins. Time-tested IPM, if implemented on a community basis focusing on pheromones technology and closed season, will help sustain the cotton cultivation in India in the future. Thus, this review aims to congregate exhaustive information on the history, biology, resistance to Bt cotton, and Integrated Management (IPM) options for the Indian scenario, which would help researchers in their future endeavors.

Field-Evolved Resistance to Cry 1ac and Cry2ab in Pink Bollworm, Pectinophora Gossiypiella Saunders from Andhra Pradesh, India

Field-Evolved Resistance to Cry 1ac and Cry2ab in Pink Bollworm, Pectinophora Gossiypiella Saunders from Andhra Pradesh, India

Convergence of Bar and Cry1Ac Mutant Genes in Soybean Confers Synergistic Resistance to Herbicide and Lepidopteran Insects.

Soybean is a globally important crop species, which is subject to pressure by insects and weeds causing severe substantially reduce yield and quality. Despite the success of transgenic soybean in terms of Bacillus thuringiensis (Bt) and herbicide tolerance, unforeseen mitigated performances have still been inspected due to climate changes that favor the emergence of insect resistance. Therefore, there is a need to develop a biotech soybean with elaborated gene stacking to improve insect and herbicide tolerance in the field. In this study, new gene stacking soybean events, such as bialaphos resistance (bar) and pesticidal crystal protein (cry)1Ac mutant 2 (M#2), are being developed in Vietnamese soybean under field condition. Five transgenic plants were extensively studied in the herbicide effects, gene expression patterns, and insect mortality across generations. The increase in the expression of the bar gene by 100% in the leaves of putative transgenic plants was a determinant of herbicide tolerance. In an insect bioassay, the cry1Ac-M#2 protein tested yielded higher than expected larval mortality (86%), reflecting larval weight gain and weight of leaf consumed were less in the T1 generation. Similarly, in the field tests, the expression of cry1Ac-M#2 in the transgenic soybean lines was relatively stable from T0 to T3 generations that corresponded to a large reduction in the rate of leaves and pods damage caused by Lamprosema indicata and Helicoverpa armigera. The transgenic lines converged two genes, producing a soybean phenotype that was resistant to herbicide and lepidopteran insects. Furthermore, the expression of cry1Ac-M#2 was dominant in the T1 generation leading to the exhibit of better phenotypic traits. These results underscored the great potential of combining bar and cry1Ac mutation genes in transgenic soybean as pursuant of ensuring resistance to herbicide and lepidopteran insects.

Susceptibility of the Egyptian Cotton Leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) to Entomocidal Crystal Proteins Cry1Ac and Cry 2Ab Baseline Responses

The present study maybe is the first attempt in Egypt to evaluate the effect of Entomocidal proteins (Cry toxins) from Bacillus thuringiensis (Bt)Cry 1Ac and Cry 2Ab againstthe Egyptian cotton leafworm Spodoptera littoralis. The cotton leafworm one of the important Lepidopteran species is the most important pests in cotton fields. It can be controlled using Genetically Modified (GM) crops expressing insecticidal Bt proteins like Bt cotton and Bt corn. The long-term success of this technology demands pest resistance management. Important information for the successful management of resistance is the baseline susceptibility of the different target pests to the different Bt proteins. The data on baseline susceptibility should enable risk assessors and managers to assess whether GM cotton produces a Bt protein in a high-dose to specific target organisms and resistance has evolved during the commercial cultivation of this GM cotton event. During this study, the toxicity of Cry 1Ac and Cry 2Ab from the bacterium Bacillus thuringiensis to larvae of S. littoralis was assessed using first instar larvae with diet incorporation bioassays. Larvae reared (4 generations) from 2 populations of S. littoralis collected from major cotton-growing regions of Sohag and Assuit Governorates were tested. The LC50 for the Assuit strain was 140.11 and 88.54 µg/ml forCry 1Ac and Cry 2Ab, respectively. While, the LC50 for the Sohag strain was 410.75 and 37.62 µg/ml for Cry 1Ac and Cry 2Ab, respectively. On the other hand, the laboratory strain was more sensitive for the same Cry’s (Cry 1Ac and Cry 2Ab) the LC50 was13.40 and 0.23 µg/ml, respectively. The lethal concentration (LC50) against S. littoralis for 2nd and 4th instars larvae when using the B. thuringiensis as spores (commercial product) was 1.16 and 2.49 for 2nd and 4th instars, respectively. This study is very important as a preliminary step and baseline ahead of the commercial cultivation and production of Genetically Modified cotton (Bt cotton).

New promising high yielding cotton Bt-Variety RH-647 adapted for specific agro-climatic zone.

The Bt-cotton RH-647 was developed by Cotton Research Institute CRI, Khanpur has been acknowledged for its possesses superior plant characteristics and potential to yield out under harsh agro-climatic conditions of cotton productive district of Rahimyar Khan in Bahawalpur Division and southern Punjab in 2016. RH- 647 for its novel plant structure and improved fiber quality heat and drought tolerant to withstand successfully sustain yield out in harsh, highly variable hot and dry climatic conditions of and harsh seasoned. RH-647 was developed through one-way hybridization of elite parental genotypes accompanied by pedigree selection method through gene pyramiding technique for incorporation of excellent combinations of fiber traits and CLCuV disease tolerance with higher yield potential right from F1 population. The superior plant combinations were selected in F2-F6 generations were entirely based on phenotypic plant traits and progeny yield potential in field, plant shape, number of bolls per plant, average boll weight (g) and fiber quality traits over standard varieties. The single plant progenies were selected 56 sister lines were tested for Bt-gene (Cry1 Ac) were evaluated for high yielding performance for this superior cross and finally RH-647 as superior breeding line was bulked in year 2010. The strain was evaluated in Randomized Complete Block Design in preliminary yield trials (PYT) and two years in Advance Yield Trials (AYT) trials and Zonal Varietal trials for two years. The superior line 647/10 was ensued for performance in variety attestation tests as RH-647. RH-647 performed best in two years varietal trials (NCVT and PCCT and DUS) conducted for two successive growing seasons (2014–2015 and 2015–2016). RH-647 yielded out significantly compared with standard varieties MNH-886, FH-142 and CIM 602. After completion of mandatory trials in year 2016, RH-647 was approved as new Bt. cotton variety “RH-647”. RH-647 is early in maturity with high yield potential and best suited for wheat-cotton cropping pattern. It has fluffy opening and is easy to pick, strongly tolerant to CLCuV disease, high Ginning out turn GOT% (40.2%) with improved fiber traits; staple length (28.3 mm), fiber strength (4.2ug/inch) is duly capable to fulfill all industrial requisitions.

Inseticidas nas sementes e a entomotoxina cry 1 ac protegem a soja contra a lagarta-do-velho-mundo

The Helicoverpa armigera caterpillar occurs from the emergence of the plants to the reproductive stage of the soybean crop, which can cause significant losses.As an initial pest, both neonate and more developed caterpillars can be found, the latter from the previous crop.The use of Bt cultivars and insecticides in seed treatment are options to pest managementat the beginning of soybean development, but there are doubtsabout the efficacy of these control methods. Thus, the objective was to evaluate the protective effect of insecticides applied to seeds and Cry 1Ac protein, in relation to the attack of the H. armigeracaterpillar, as an initial pest in soybean plants.Experiments were conducted in the laboratory, using a completely randomized design, with six treatments:four insecticides related to seeds and control (without insecticide), in conventional cultivar soybeans (Target BMX), and the Cry 1Ac protein in soybeans cultivate Bt (DM 5958 IPRO), without insecticide. Our results confirm that under laboratory conditions the insecticides cyantraniliprole (60 g ai / 100 kg of seeds) and chlorantraniliprole (62.5 g ai / 100 kg of seeds) protect the soybean against the injuries of H. armigera3º-4ºínstars, in cotyledons and unifoliolate and trifoliolate leaves, at 5 days after the emergenceof the plants. The protein Cry 1Acprotects the soybean plants from this type of injury caused byboth3rd-4thand 5th-6thínstarscaterpillars, at 5 and 10 days after emergence.

Expression and characterization of two chitinases with synergistic effect and antifungal activity from Xenorhabdus nematophila.

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65kDa and 78kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50°C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.

Control strategies for Chrysodeixis includens and Spodoptera eridania caterpillars (Lepidoptera: Noctuidae) and selection of resistant cultivars in soybean

Chrysodeixis includens and Spodoptera eridania caterpillars have been considered the most important pests for Glycine max in Brazil. This study evaluated different control strategies for these two important soybean caterpillars and the effects on crop productivity. The control strategies adopted were: BC, IPM, PUI and C (without pest control) and 2 cultivars (BMX Desafio - conventional and TEC Power IPRO – transgenic – Cry 1Ac). The experimental design was in randomized blocks in a factorial scheme (2 x 4) with four replications. The transgenic cultivar TEC Power IPRO presents efficiency in the control of Chrysodeixis includens and low efficiency for Spodoptera eridania. Harmonious practices such as IPM are the best alternative for soybean crops, to minimize Chrysodeixis includens and Spodoptera eridania infestations. The control strategy adopted did not influence soybean productivity.

Carotenoids moderate the effectiveness of a Bt gene against the European corn borer, Ostrinia nubilalis.

We assessed the effectiveness of a biofortified maize line (4BtxHC) which accumulates high levels of antioxidant carotenoids that also expressed the insecticidal Cry1Ac Bacillus thuringiensis (Bt) gene against the European corn borer Ostrinia nubilalis. This line had been previously engineered to accumulate carotenoids specifically in the seed endosperm, whereas the Bt gene was expressed constitutively. The concentrations of Bt toxin (Cry 1Ac) in the leaves of the 4Bt and 4BtxHC lines were not significantly different at 47±6 μg/g of fresh weight (FW); neither were they in the kernels of both lines (35±3 μg/g FW). The kernels and leaves were toxic to the larvae of O. nubilalis. However, the insecticidal activity was substantially lower (ca. 20%) than that of lines that expressed only Bt in spite that the two lines showed a quantity of toxin not significantly different in kernels or in leaves. Although the reduced effectiveness of Cry1Ac in kernels may not be entirely surprising, the observation of the same phenomenon in vegetative tissues was unexpected. When semi-artificial diets containing kernels from 4Bt supplemented with different levels of β-carotene were used in insect bioassays, the β-carotene moderated the effectiveness of the Bt similarly to the plant material with carotenoid enrichment. To elucidate the biochemical basis of the reduced effectiveness of Bt toxin in the carotenoid-enriched plants, we measured the activity of three enzymes known to be implicated in the detoxification defence, namely, catalase, superoxide dismutase and glutathione S-transferase. Whereas Cry1Ac expression significantly increased SOD and CAT enzymatic activity in the absence of carotenoids, carotenoids, either in 4BtxHC or in artificial diets enriched with β-carotene, significantly lowered CAT activity. Carotenoids can therefore moderate the susceptibility of the maize borer O. nubilalis to Cry1Ac, and we hypothesize that their role as antioxidants could explain this phenomenon via their scavenging of reactive oxygen species produced during Cry1Ac detoxification in the larvae. The involvement of this mechanism in the decreased mortality caused by Cry1Ac when carotenoids are present in the diet is discussed.

The current status and development of insect-resistant genetically engineered poplar in China

Poplar is one of the main afforestation tree species in China, and the use of a single, or only a few, clones with low genetic diversity in poplar plantations has led to increasing problems with insect pests. The use of genetic engineering to cultivate insect-resistant poplar varieties has become a hot topic. Over the past 20 years, there have been remarkable achievements in this area. To date, nearly 22 insect-resistant poplar varieties have been created and approved for small-scale field testing, environmental release, or pilot-scale production. Here, we comprehensively review the development of insect-resistant genetically modified (GM) poplars in China. This review mostly addresses issues surrounding the regulation and commercialization of Bt poplar in China, the various insecticidal genes used, the effects of transgenic poplars on insects, toxic protein expression, multigene transformation, the stability of insect resistance, and biosafety. The efficacy of GM poplars for pest control differed among different transgenic poplar clones, larval instars, and insect species. The Bt protein analysis revealed that the expression level of Cry3A was significantly higher than that of Cry1Ac. Temporal and spatial studies of Bt protein showed that its expression varied with the developmental stage and tissue. The inheritance and expression of the exogenous gene were reviewed in transgenic hybrid poplar progeny lines and grafted sections. Biosafety issues, in terms of transgene stability and the effects on soil microorganisms, natural enemies of insects, and arthropod communities are also discussed.

Efficacy ofBacillus thuringiensisSprays and Cotton Cultivars Expressing Cry Proteins in the Control ofEarias biplaga(Walker) (Lepidoptera: Noctuidae)

Earias biplaga (Walker) (Lepidoptera: Noctuidae) forms part of the bollworm pest complex of cotton in South Africa. Genetically modified Bt cotton that expresses Cry insecticidal proteins successfully controls the bollworm complex in South Africa. Although many insecticides are registered for use against Lepidoptera on this crop, Bt sprays are not registered for control of E. biplaga. The aim of this study was to determine the efficacy of Bacillus thuringiensis spray applications for the control of E. biplaga on non-Bt cotton and to compare the efficacy of sprays to that provided by Bt cotton. Bioassays were conducted in which cotton bolls of a non-Bt cotton cultivar (Delta Opal) were sprayed with different dosage rates of Dipel DF®, which contains the following insecticidal proteins: Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab. Bollgard® and Bollgard II® cotton which expresses Cry1Ac and both Cry 1Ac and Cry2Ab were used in the study. Neonate larvae were inoculated onto cotton bolls and survival observed 4, 7 and 10 days later. The Bt spray dosage rate recommended for control of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) on groundnut and sunflower in South Africa was used in this study and provided between 77 and 88%control of E. biplaga after 10 days. Bt cotton provided 100%control after 10 days. This study provides baseline data on the susceptibility of E. biplaga against Bt cotton which can also be used for future monitoring of resistance evolution.

Expresión de la proteína recombinante Cry 1Ac en cultivos de células de papa en suspensión: Establecimiento del cultivo y optimización de la producción de la biomasa y la proteína mediante la adición de nitrógeno

Los cultivos in vitro de células vegetales en suspensión se han propuesto como plataformas alternativas de expresión de proteínas recombinantes con aplicación terapéutica por las ventajas que ofrecen sobre los sistemas tradicionales de expresión en células bacterianas y de mamíferos. En este trabajo se determinó un protocolo para el establecimiento de suspensiones de papa (S. tuberosum) genéticamente modificadas con el gen de la proteína Cry 1Ac y se caracterizaron las cinéticas de producción de la biomasa y la proteína recombinante. Los entrenudos y el medio MS suplementado con 2.0 mg L-1 de 2,4-D, mostraron los mejores porcentajes de formación de callo. La tasa máxima de crecimiento específico calculada para las suspensiones fue 0.12 d-1, con una concentración máxima de biomasa de 1.41 g L-1 al final de la fase exponencial, la cual logró aumentarse hasta 3.94 g L-1 duplicando la concentración de NO3- y NH4+ en el medio de cultivo.

Preparation of high stable core/shell magnetic nanoparticles and application in Bacillus thuringiensis Cry1Ac proteins detection

The silicon coated Magnetic nanoparticles (MNps) were prepared and applied to the genetically modified detection for Cry1Ac protein. TEM observations showed the bare MNps were in the size of 3–5nm and the silicon coated core/shell MNps were in the range of 30–60nm 1M HCl acidic solution was used to test the chemical stability of the resultant MNps and results showed the coated MNps were very stable in the acid environment even after 12h. Cy3 labelled IgG tests showed that the probe on the MNps was kept the bioactivity in the immunoassays. MNps based detection results showed that this analytical strategy had similar linear relation which R=0.995 with the commercial ELISA kit. The MNps based detections were reliable for the Cry1 Ac protein in the linear range from 0.1pg/L to 1.0mg/L, and showed better performance in the low concentration of analyte. This study provides a promising technique to detect the Cry1Ac protein with high sensitivity in the examination of the GM products which is of importance in food safety.

Impact of Bt-cotton on soil microbiological and biochemical attributes

Transgenic Bt-cotton produces Bt-toxins (Cry proteins) which may accumulate and persist in soil due to their binding ability on soil components. In the present study, the potential impacts of Bt- and non-Bt genotypes of cotton on soil microbial activity, substrate use efficiency, viable microbial population counts, and nutrient dynamics were studied. Two transgenic Bt-cotton genotypes (CIM-602 CIM-599) expressing cry1 Ac gene and two non-Bt cotton genotypes (CIM-573 and CIM-591) were used to evaluate their impact on biological and chemical properties of soil across the four locations in Punjab. Field trials were conducted at four locations (Central Cotton Research Institute-Multan, Naseer Pur, Kot Lal Shah, and Cotton Research Station-Bahawalpur) of different agro-ecological zones of Punjab. Rhizosphere soil samples were collected by following standard procedure from these selected locations. Results reveled that Bt-cotton had no adverse effect on microbial population (viable counts) and enzymatic activity of rhizosphere soil. Bacterial population was more in Bt-cotton rhizosphere than that of non-Bt cotton rhizosphere at all locations. Phosphatase, dehydrogenase, and oxidative metabolism of rhizosphere soil were more in Bt-cotton genotypes compared with non-Bt cotton genotypes. Cation exchange capacity, total nitrogen, extractable phosphorous, extractable potassium, active carbon, Fe and Zn contents were higher in rhizosphere of Bt-cotton genotypes compared with non-Bt cotton genotypes. It can be concluded from present study that the cultivation of Bt-cotton expressing cry1 Ac had apparently no negative effect on metabolic, microbiological activities, and nutrient dynamics of soils. Further work is needed to investigate the potential impacts of Bt-cotton on ecology of soil-dwelling insects and invertebrates before its recommendation for extensive cultivation.

Effects of insect-resistant transgenic cotton on ground-dwelling beetle assemblages (Coleoptera)

AbstractWe compared the ground-dwelling beetle assemblages under four scenarios in which transgenic Bt (Cry 1Ac) cotton (33B), transgenic Bt (Cry 1Ac)+CpTI cotton (SGK321), conventional cotton (33), conventional cotton (Shiyuan 321) in North China. During the survey in two years (2009–2010), 24 ground beetle species were captured with pitfall traps in 20 plots which included five replicates for each cotton type. No significant difference was observed in the number of ground beetle species captured, activity density, evenness and Shannon-Wiener diversity among the four cotton varieties. Chlaenius posticalis was less abundant in transgenic Bt+CpTI cotton (SGK321) fields than its conventional cotton (Shiyuan 321), but more abundant in transgenic Bt cotton (33B) fields compared with its conventional cotton (33). There was no significant difference for other abundant species between in transgenic cotton and in conventional cotton fields. Based on non-metric multidimensional scaling (NMDS) analysis, ground-dwelling beetle assemblages were similar in transgenic and conventional cotton over the two years, but the ground-dwelling beetle assemblages in transgenic cotton 33B significantly differed from that in the conventional cotton (strain 33) in 2010. No strong evidence that the transgenic cotton effect on ground-dwelling beetle assemblages was found in this study.

Selection and characterization of Bacillus thuringiensis isolates with a high insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae)

Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) affects diverse crops of great economic interest, for instance, it can cause severe yield losses in maize, rice and sorghum. In this study, a selection and characterization of Bacillus thuringiensis (BERLINER, 1911) isolates with a high insecticidal activity against S. frugiperda was performed. Fifty-two crystal-forming B. thuringiensis isolates that were identified from 3384 Bacillus -like colonies were examined and screened by PCR for the presence cry genes ( cry 1, cry 1Aa, cry 1Ab, cry 1Ac, cry 1D, cry 2 and cry 2Ab). Four isolates that showed high toxicity towards S. frugiperda were shown to harbor cry 2 genes. The crystals were analyzed by electron microscopy and showed bipyramidal and cuboidal shapes. Furthermore, these four isolates had lethal concentration (LC 50 ) values of 44.5 ng/cm 2 (SUFT01), 74.0 ng/cm 2 (SUFT02), 89.0 ng/cm 2 (SUFT03) and 108 ng/cm 2 (SUFT 04) to neonate S. frugiperda larvae. An ultrastructural analysis of midgut cells from S. frugiperda incubated with the SUFT01 spore-crystal complex showed disruptions in cellular integrity and in the microvilli of the midgut columnar cells. The isolates characterized in this work are good candidates for the control of S. frugiperda , and could be used for the formulation of new bioinsecticides.

Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant--Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation.

In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt) Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o.) or with a single intraperitoneal (i.p.) injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed). This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.

A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants.

Effect of Bt Soybean on Larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) causes economically significant damage to soybeans Glycine max (L.) Merrill (Fabaceae: Phaseoleae). The genetically modified soybean expressing the Cry1Ac protein of Bacillus thuringiensis Berliner, which is toxic to lepidopterans, is a potential alternative tool to manage this pest. Bioassays with H. armigera larval instars were conducted with Bt (Cry1Ac) and non-Bt soybean plants in order to evaluate the efficacy of control with the modified cultivar. Bt soybean affected the mean time of mortality in each larval instar, but its efficacy was not affected by the larval stage. Mortality was 100% in all six instars, indicating that the Bt soybean expressing the Cry 1Ac protein is an efficient control tactic for this pest.