Frequency of resistance alleles to Cry1Ac toxin from cotton bollworm, Helicoverpa armigera (Hübner) collected from Bt-cotton growing areas of Telangana state of India.

Transgenic cotton expressing Bacillus thuringiensis (Bt) cry1Ac and cry2Ab toxin genes is widely cultivated to manage bollworm complex in India. Cotton bollworm Helicoverpa armigera (Hübner) is one of the most serious of this complex. It is likely to evolve resistance to Cry toxins in view of continual selection pressure due to extensive cultivation of Bt cotton. Monitoring susceptibility of cotton bollworm using conventional bioassays is reported to have shown its increasing tolerance to Cry1Ac over the years. We report using an F2 screen Cry1Ac resistance allele frequencies of 0.050 (95% CI 0.022-0.076) and 0.056 (95% CI 0.035-0.075) in the insect populations collected from pigeon pea grown alongside Bt cotton in the respective years of 2016 and 2017 in the Telangana state of India. Compared to our earlier studies for 2013 and 2014, resistance allele frequency to Cry1Ac in the cotton bollworm in the following two years remains unchanged. The significance of these results is discussed in the context of non-Bt host crops acting as refuge for cotton bollworm for ensuring sustainable resistance management.

Development of an immunochromatographic assay for the specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Cry1Ab has been widely used in genetically modified (GM) crops and its amino acid sequence had high identity to Cry1Ac toxin. Existing nanogold immunochromatographic strips cannot distinguish Cry1Ab from Cry1Ac toxin. In this study, a rapid (5-6 min), qualitative nanogold immunochromatographic strip was successfully developed for the specific detection of Cry1Ab toxin. The assay was based on double antibody sandwich format with the visual detection limit (vLOD) of 0.1 µg mL-1. The results of immunochromatographic assay were all positive validated against the DAS-ELISA (recoveries between 109.6 and 111.8%). In addition, 10%, 5% and 0% error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1, 0.2, 0.5 and 1 µg mL-1, respectively, demonstrating the reproducibility of the test strip. Furthermore, the test strip could be stored for 3 months under dry conditions without significant loss of sensitivity. Furthermore, the practical sample analysis results showed that the test strip was able to detect the presence of Cry1Ab in GM materials containing as low as 0.5% MON 810 Bt maize which indicated the practical value of the test strip. To our knowledge, this is the first report on the detection of Cry1Ab by immunochromatographic assay without interference from Cry1Ac toxin.

Characterization of immune-related PGRP gene expression and phenoloxidase activity in Cry1Ac-susceptible and -resistant Plutella xylostella (L.).

Peptidoglycan recognition proteins (PGRPs) are important recognition receptors which play a critical role in signal identification and transmission in Toll or immune deficiency (IMD) pathways, particularly when pathogens evade and circumvent reactive oxygen species. Antimicrobial peptides (AMPs) synthesis can be activated by these signals to further eliminate pathogens. In this study, we cloned and characterized three different PGRP genes in Plutella xylostella strains, DBM1Ac-S, DBM1Ac-R and a field strain (DBMF). The results showed that PGRP1 belongs to the PGRP-SA family, PGRP2 to PGRP-LB, and PGRP3 to PGRP-LF. Moreover, PGRP1 expressed the highest transcript level, followed by PGRP3 and PGRP2, in two tissues including the gut and the larval carcass tissues of the DBM1Ac-S strain. Furthermore, altered expression levels of PGRP1-3 genes were detected in both gut and carcass tissues. Moreover, the DBM1Ac-R strain had the highest phenol oxidase (PO) activity among these three strains. The characterization of PGRP gene expression and PO activity in DBM1Ac-S, DBM1Ac-R and DBM-F provides insights into their important physiological roles in the immune system of P. xylostella exposed to Bt Cry1Ac toxin.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10-8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL-1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL-1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Identification of an alkaline phosphatase as a putative Cry1Ac binding protein in Ostrinia furnacalis (Guenée).

Asian corn borer (ACB), Ostrinia furnacalis, is an important insect pest of maize susceptible to different Cry1A toxins. Based on amino acid sequence alignment of ALP sequences from lepidopteran larvae an alp gene was cloned from ACB, named ofalp. Pull dawn assays using biotinylated Cry1Ac and brush border membrane vesicles isolated from second instar ACB larvae showed that four proteins of 50, 65, 68 and 70kDa precipitated with the Cry1Ac. The 65kDa band cross-reacted with the anti-OfALP monoclonal antibody. GalNac was able to release the binding of Cry1Ac to the 65kDa OfALP in pull down assays. A 37kDa fragment from residues D173 to D473 of OfALP was cloned and expressed in Escherichia coli cells. We show that this ALP-fragment was able to bind Cry1Ac in ligand blot analysis. Our data also indicate that different ALP isoforms or variants may be also Cry1Ac binding proteins since more ALP enzymatic activity was pull down with Cry1Ac than with anti-OfALP antibody. We also analyzed the expression levels of ALP throughout the larval development by qPCR and ALP enzymatic activity. Our data indicated that ALP expression in ACB was observed preferentially in young instar larvae. Finally, we show that resistance in O. furnacalis ACB-AcR strain resistant to Cry1Ac did not correlate with changes in expression of this ALP protein since it shows similar gene expression of ofalp than the susceptible insect strain. Identification of Cry1Ac receptors will help to understand mechanism of action of Cry1Ac in O. furnacalis and to understand mechanism of Cry toxin resistance. Our data indicate that at least one ALP protein is involved in the binding interaction with Cry1Ac in O. furnacalis.

Contribution of the transcription factor SfGATAe to Bt Cry toxin resistance in Spodoptera frugiperda through reduction of ABCC2 expression.

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.

Reduced expression of the P-glycoprotein gene HaABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin but not Cry2Ab toxin in Helicoverpa armigera.

Rapid evolution of pest resistance to Bt insecticidal proteins presents a serious threat to the sustainable use of Bt crops. The cotton bollworm has been extensively exposed to Bt cotton worldwide and has evolved resistance in laboratory and field. Previous studies have highlighted the significant roles played by the ABC transporter proteins in Bt resistance. In this study, the ORF of HaABCB1 was cloned and analyzed. The expression of HaABCB1 was detected in all developmental stages and tissues, with the highest expression in third instar larvae stage and hindgut tissue. Compared with susceptible strain, a remarkable decrease of HaABCB1 expression in Cry1Ac resistant strain while no significant change in Cry2Ab resistant strain were found. The HaABCB1 expression reduced after susceptible larvae induced by Cry1Ac, but no obvious expression changes after Cry2Ab exposure. RNAi-mediated down-regulation of HaABCB1 could lead to a significant reduction in larval susceptibility to Cry1Ac, but not to Cry2Ab, in susceptible strain. Genetic linkage analysis confirmed that decreased expression of the HaABCB1 mediates resistance to Cry1Ac, but not Cry2Ab resistance. This knowledge contributes to better understanding of the complex molecular mechanisms underlying Bt resistance and provide theoretical foundation for the development of new strategies for pest resistance management.

Effects of Pb(II) and Zn(II) Contamination on Adsorption, Desorption and Degradation of Cry1Ac Toxin Identical to Bt Transgenic Poplar in Black Soil.

Bt transgenic white poplar has been commercially planted in China since 2002, and it showed obvious insect resistance in the field. However, the ecological risk of planting Bt transgenic poplar in a field contaminated with heavy metals has received little attention. The effects of Pb(II) and Zn(II) contamination on the adsorption, desorption and degradation of Bt toxin identical to Bt transgenic poplar in black soil were studied. The results showed that the adsorption of Bt toxin was enhanced and the desorption of Bt toxin was inhibited in black soil by Pb(II) and Zn(II) at concentrations between 0 and 1 mmol/L, and the effect of Pb(II) on Bt toxin was greater than that of Zn(II). In the presence of heavy metal ions, the Cry1Ac toxin molecules are oriented with domain I toward soil particles through the metal ion bridge. The promoting mechanism of Bt toxin adsorption by heavy metal ions in black soil is mainly attributed to cation-controlled electrostatic attraction (CCEA), which is different from patch-controlled electrostatic attraction (PCEA). With the increase in soil concentration from 1 to 4 mg/mL, the adsorption amount of Bt toxin showed a downward trend, and both Pb(II) and Zn(II) had the maximal promotion effect when the soil concentration was 2 mg/mL. The promoting effect of Zn(II) on the adsorption of Bt toxin increased with the increased temperature (5-45 °C), but the promoting effect of Pb(II) was maximal at 25 °C. Both Pb(II) and Zn(II) affected the degradation characteristics of Bt toxin in black soil. For the lead-contaminated black soil, the residual amount of Bt toxin increased in the early stage but decreased in the later stage compared to the control soil. For the zinc-contaminated black soil, the residual amount of Bt toxin decreased compared to the control soil except between the second and tenth days. In this study, it was observed that Bt toxin was degraded rapidly in the early stage, followed by a large amount of released Bt toxin and slow degradation in the middle and late stages.

Transcriptional Analysis of Cotton Bollworm Strains with Different Genetic Mechanisms of Resistance and Their Response to Bacillus thuringiensis Cry1Ac Toxin.

Transgenic crops producing Bacillus thuringiensis (Bt) insecticidal proteins are grown widely for pest control, but the evolution of resistance in target pests could reduce their efficacy. Mutations in genes encoding cadherin, ABC transporter or tetraspanin were linked with resistance to Cry1Ac in several lepidopteran insects, including the cotton bollworm (Helicoverpa armigera), a worldwide agricultural pest. However, the detailed molecular mechanisms by which these mutations confer insect resistance to Cry1Ac remain largely unknown. In this study, we analyzed the midgut transcriptomes of a susceptible SCD strain and three SCD-derived Cry1Ac-resistant strains of H. armigera (SCD-r1, with a naturally occurring deletion mutation of cadherin; SCD-KI, with a knock-in T92C point mutation in tetraspanin; and C2/3-KO, with both ABCC2 and ABCC3 knocked out). Evaluation of midgut transcript profiles of the four strains without Cry1Ac exposure identified many constitutively differentially expressed genes (DEGs) in the resistant SCD-r1 (n = 1355), SCD-KI (n = 1254) and C2/3-KO (n = 2055) strains. Analysis of DEGs in the midguts of each strain after Cry1Ac exposure revealed similar patterns of response to Cry1Ac in the SCD and SCD-r1 strains, but unique responses in the SCD-KI and C2/3-KO strains. Expression of midgut epithelium healing and defense-related genes was strongly induced by Cry1Ac intoxication in the SCD and SCD-r1 strains, while immune-related pattern recognition receptor and effector genes were highly expressed in the SCD-KI strain after Cry1Ac exposure. This study advances our knowledge of the transcriptomic basis for insect resistance to Bt toxins and provides a valuable resource for further molecular characterization of insect response to Cry1Ac toxin in H. armigera and other pest species.

Differential capability of Bacillus thuringiensis Cry1Ac protoxin and toxin to induce in vivo activation of dendritic cells and B lymphocytes.

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Study of the allergenic potential of Bacillus thuringiensis Cry1Ac toxin following intra-gastric administration in a murine model of food-allergy.

Cry1Ac toxin, from Bacillus thuringiensis, is widely used as a biopesticide and expressed in genetically modified (GM) plants used for human and animal consumption. Since Cry1Ac is also immunogenic and able to activate macrophages, it is crucial to thoroughly evaluate the immunological effects elicited after intra-gastric administration. The allergenic potential of purified Cry1Ac was assessed and compared with that induced in a murine model of food-allergy to ovalbumin (OVA), in which animals are sensitized with the adjuvant Cholera toxin (CT). Mice were weekly intragastrically administered with: i) vehicle phosphate-buffered saline (PBS), ii) OVA, iii) OVA plus CT iv) Cry1Ac or v) OVA plus Cry1Ac. Seven weeks after, mice were intragastrically challenged and allergic reactions along with diverse allergy related immunological parameters were evaluated at systemic and intestinal level. The groups immunized with, Cry1Ac, OVA/Cry1Ac or OVA/CT developed moderate allergic reactions, induced significant IgE response and increased frequencies of intestinal granulocytes, IgE+ eosinophils and IgE+ lymphocytes. These same groups also showed colonic lymphoid hyperplasia, notably in humans, this has been associated with food allergy and intestinal inflammation. Although the adjuvant and allergenic potential of CT were higher than the effects of Cry1Ac, the results show that applied intra-gastrically at 50 µg doses, Cry1Ac is immunogenic, moderately allergenic and able to provoke intestinal lymphoid hyperplasia. Moreover, Cry1Ac is also able to induce anaphylaxis, since when mice were intragastrically sensitized with increasing doses of Cry1Ac, with every dose tested, a significant drop in rectal temperature was recorded after intravenous challenge.

Combining the high-dose/refuge strategy and self-limiting transgenic insects in resistance management-A test in experimental mesocosms.

The high-dose/refuge strategy has been the primary approach for resistance management in transgenic crops engineered with Bacillus thuringiensis toxins. However, there are continuing pressures from growers to reduce the size of Bt toxin-free refugia, which typically suffer higher damage from pests. One complementary approach is to release male transgenic insects with a female-specific self-limiting gene. This technology can reduce population sizes and slow the evolution of resistance by introgressing susceptible genes through males. Theory predicts that it could be used to facilitate smaller refugia or reverse the evolution of resistance. In this study, we used experimental evolution with caged insect populations to investigate the compatibility of the self-limiting system and the high-dose/refuge strategy in mitigating the evolution of resistance in diamondback moth, Plutella xylostella. The benefits of the self-limiting system were clearer at smaller refuge size, particularly when refugia were inadequate to prevent the evolution of resistance. We found that transgenic males in caged mesocosms could suppress population size and delay resistance development with 10% refugia and 4%-15% initial resistance allele frequency. Fitness costs in hemizygous transgenic insects are particularly important for introgressing susceptible alleles into target populations. Fitness costs of the self-limiting gene in this study (P. xylostella OX4139 line L) were incompletely dominant, and reduced fecundity and male mating competitiveness. The experimental evolution approach used here illustrates some of the benefits and pitfalls of combining mass release of self-limiting insects and the high-dose/refuge strategy, but does indicate that they can be complementary.

Does Bt maize expressing Cry1Ac protein have adverse effects on the parasitoid Macrocentrus cingulum (Hymenoptera: Braconidae)?

The potential effects of insect-resistant, genetically engineered (GE) crops on non-target organisms, especially on predators and parasitoids, must be evaluated before their commercial cultivation. The effects of GE maize that produces Cry1Ac toxin on the parasitoid Macrocentrus cingulum were assessed by direct bioassay and indirect bioassay. In the indirect bioassay, parasitism rate, cocoon weight and the number of M. cingulum progeny produced per host were significantly reduced when M. cingulum-parasitized Cry1Ac-susceptible Ostrinia furnacalis were fed a diet containing purified Cry1Ac; however, life-table parameters of M. cingulum were not adversely affected when the same assay was performed with Cry1Ac-resistant O. furnacalis. These results indicated that the detrimental effects detected with a Cry1Ac-susceptible host were mediated by poor host quality. In a direct bioassay, no difference in life-table parameters were detected when M. cingulum adults were directly fed a 20% honey solution with or without Cry1Ac; however, survival and longevity were significantly reduced when M. cingulum adults were fed a honey solution containing potassium arsenate, which was used as a positive control. The stability and bioactivity of Cry1Ac toxin in the food sources and Cry1Ac toxin uptake by the host insect and parasitoid were confirmed by enzyme-linked immunosorbent assay and sensitive-insect bioassays. Our results demonstrate that M. cingulum is not sensitive to Cry1Ac toxin at concentrations exceeding those encountered in Bacillus thuringiensis maize fields. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms and will be useful for assessing other non-target impacts.

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases.

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.

Mode of inheritance for biochemical traits in genetically engineered cotton under water stress.

Drought is an abiotic environmental stress that can significantly reduce crop productivity. We examined the mode of inheritance for different biochemical traits including total soluble proteins, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, total phenolic contents and enzymatic antioxidants (superoxide dismutase, peroxidase and catalase), and their relationship with Bacillus thuringiensis (Bt) toxin under control and drought conditions. Eight genetically diverse cotton genotypes were selfed for two generations to ensure homozygosity. Fifteen F1 hybrids were developed by crossing five non-Bt female lines with three Bt male testers. The F1 hybrids and eight parents were finally evaluated under control (100 % field capacity (FC)) and drought (50 % FC) conditions in 2013. The biochemical traits appeared to be controlled by non-additive gene action with low narrow sense heritability estimates. The estimates of general combining ability and specific combining ability for all biochemical traits were significant under control and drought conditions. The genotype-by-trait biplot analysis showed the better performance of Bt cotton hybrids when compared with their parental genotypes for various biochemical traits under control and drought conditions. The biplot and path coefficient analyses revealed the prevalence of different relationships between Cry1Ac toxin and biochemical traits in the control and drought conditions. In conclusion, biochemical traits could serve as potential biochemical markers for breeding Bt cotton genotypes without compromising the optimal level of Bt toxin.

Cry1Ac toxicity enhancement towards lepidopteran pest Ephestia kuehniella through its protection against excessive proteolysis.

Bacillus thuringiensis has been extensively used in agroecosystems for four decades due to its high specific toxicity. Strategies based on B. thuringiensis proteins combinations for the improvement of its activity present an important focus for biopesticides development. However, the widespread use of B. thuringiensis δ-endotoxins has often been challenged by a lack of understanding of the target insect physiology as well as its midgut biochemistry. In the present investigation, we have evidenced and explained the toxicity improvement of Cry1Ac δ-endotoxins against Ephestia kuehniella larvae through in vivo combination with P20 helper protein. Tracking the fate of Cry1Ac in tested midgut larvae showed considerable differences between δ-endotoxins produced in the presence of P20 and those produced in its absence which could explain the obtained larvicidal activity enhancement. The P20 presence slightly increased Cry1Ac inclusions solubility in E. kuehniella midgut conditions. However, a protection against excessive degradation of protoxin and toxin forms of Cry1Ac was strongly decreased in the case of δ-endotoxins produced in the presence of P20 as compared to those from P20 lacking control. Thus, the P20 protective effect on Cry1Ac after larvae ingestion has been proven. This finding could be helpful to further understand the roles of P20 helper protein in toxicity enhancement of B. thuringiensis toxins.

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.

Comparative performance of modified full-length and truncated Bacillus thuringiensis-cry1Ac genes in transgenic tomato.

BACKGROUND: Bt-cry1Ac gene has been reputedly effective against Helicoverpa armigera a notorious lepidopteran pest. Reports on the expression of full-length and truncated cry1Ac genes in plants for effective resistance against Helicoverpa sp. have been documented however, their performance is still ambiguous. Moreover, the question remains to be addressed that truncation of 3' end of the native gene was documented and suggested for active insecticidal toxin production while the most successful transgenic event(s) of commercialized-cotton are based on full-length of the cry gene. Therefore, we performed a comparative study on the efficacy of the two versions of cry1Ac genes (full-length: 3,510 bp and truncated: 1,845 bp) in T0 and T1 transgenic tomato plants and analyzed the extent of protection against H. armigera and also compared the results with our previous findings related to a successful transgenic tomato line Ab25E, expressing cry1Ab gene. The integration of cry1Ac gene(s) in T0 transgenic plants and its inheritance in T1 progeny was observed by PCR, RT-PCR and Southern blot hybridization analysis while, the toxin integrity, expression and toxicity was monitored by Western immunoassay, DAS-ELISA and insect bioassay respectively. RESULTS: An average transformation frequency and Bt-Cry protein content of 16.93 ± 2.10 and 0.0020-0.0128% of total soluble protein (TSP) was obtained with pRD400 vector (Trcry1Ac) while, a much lower value of 9.30 ± 2.041 and 0.0001 - 0.0026% of TSP was observed with pNBRI-1 vector (Flcry1Ac), respectively. The promising Trcry1Ac T0 transgenic plants and their T1 progeny gave full protection from H. armigera. Although Flcry1Ac gene showed lower transformation frequency and lower expression, it showed higher toxicity to H. armigera when compared with truncated Trcry1Ac gene. CONCLUSIONS: The full-length cry1Ac gene can be redesigned for higher expression and performance in dicots or a hybrid gene could be designed having a blend of strong receptor binding and stable expression characteristics for enhanced efficacy and toxicity to the susceptible insects.

Owlet moths (Lepidoptera: Noctuoidea) associated with Bt and non- Bt soybean in the brazilian savanna / Noctuóides (Lepidoptera: Noctuoidea) associados a soja Bt e não-Bt no Cerrado brasileiro

Abstract The use of GMO expressing Bt toxin in soybean production has increased significantly in the last years in Brazil in order to manage the damage caused by lepidopteran pests. In this study, we compared the richness and abundance of owlet moths (Noctuoidea) associated with Bt and non-Bt soybean. We determined the temporal variations as a function of phenology, and correlated the population variations of the most common species with meteorological variables. The research was conducted at the experimental area of Embrapa Cerrados. The collection method used was differentiated being suppressive and absolute. A total of 13 species were collected, of which eight occurred on Bt soybeans. The most representative taxa were Chrysodeixis includens (72.87%), Anticarsia gemmatalis (18.17%) and Spodoptera spp (5.22%). The number of larvae belonging to species targeted by the Bt technology was 10 times lower on Bt than on non-Bt soybeans. Utetheisa ornatrix and Elaphria deltoides were recorded on soybean for the first time, observing larvae of both species in non-Bt soybean and those of U. ornatrix also in Bt soybean. Only A. gemmatalis larvae correlated (p <0.05) negatively with precipitation. This study provided field information on the abundance and species richness of owlet moths on non-Bt soybeans, associated with the effects of Bt soybean. When considering the different levels of infestation between cultivars as a criterion, larvae monitoring is of substantial importance in order to develop the lost control program.

Resumo O uso de OGM que expressam toxina Bt na produção de soja tem aumentado significativamente nos últimos anos no Brasil e são utilizados para conter os danos causados ​​pelos lepidópteros pragas. Neste estudo comparamos a riqueza e a abundância de Noctuoides (Noctuoidea) associados à soja Bt e não-Bt. Determinamos as variações temporais em função da fenologia e correlacionamos às variações populacionais das espécies mais comuns com variáveis ​​meteorológicas. A pesquisa foi conduzida na área experimental da Embrapa Cerrados. O método de coleta utilizado foi diferenciado sendo supressivo e absoluto. Um total de 13 espécies foram coletadas, das quais oito ocorreram em soja Bt. Os taxa mais representativos foram Chrysodeixis includens, Anticarsia gemmatalis e Spodoptera spp. O número de larvas pertencentes às espécies alvo da tecnologia Bt foram 10 vezes menores na soja Bt do que em soja não-Bt . Utetheisa ornatrix e Elaphria deltoides foram registradas na soja pela primeira vez, observando-se larvas de ambas espécies na soja não-Bt e as de U. ornatrix também na soja Bt. Somente as larvas de A. gemmatalis se correlacionaram (p <0,05) negativamente com a precipitação. Este estudo forneceu informações em campo sobre a abundância e riqueza de espécies na soja não- Bt, associada aos efeitos da soja Bt. A importância do monitoramento das lagartas é substancial, a fim de tomar a melhor decisão de controle, considerando-se os diferentes níveis de infestação entre cultivares como critério.

Owlet moths (Lepidoptera: Noctuoidea) associated with Bt and non- Bt soybean in the brazilian savanna

Abstract The use of GMO expressing Bt toxin in soybean production has increased significantly in the last years in Brazil in order to manage the damage caused by lepidopteran pests. In this study, we compared the richness and abundance of owlet moths (Noctuoidea) associated with Bt and non-Bt soybean. We determined the temporal variations as a function of phenology, and correlated the population variations of the most common species with meteorological variables. The research was conducted at the experimental area of Embrapa Cerrados. The collection method used was differentiated being suppressive and absolute. A total of 13 species were collected, of which eight occurred on Bt soybeans. The most representative taxa were Chrysodeixis includens (72.87%), Anticarsia gemmatalis (18.17%) and Spodoptera spp (5.22%). The number of larvae belonging to species targeted by the Bt technology was 10 times lower on Bt than on non-Bt soybeans. Utetheisa ornatrix and Elaphria deltoides were recorded on soybean for the first time, observing larvae of both species in non-Bt soybean and those of U. ornatrix also in Bt soybean. Only A. gemmatalis larvae correlated (p 0.05) negatively with precipitation. This study provided field information on the abundance and species richness of owlet moths on non-Bt soybeans, associated with the effects of Bt soybean. When considering the different levels of infestation between cultivars as a criterion, larvae monitoring is of substantial importance in order to develop the lost control program.

Resumo O uso de OGM que expressam toxina Bt na produção de soja tem aumentado significativamente nos últimos anos no Brasil e são utilizados para conter os danos causados pelos lepidópteros pragas. Neste estudo comparamos a riqueza e a abundância de Noctuoides (Noctuoidea) associados à soja Bt e não-Bt. Determinamos as variações temporais em função da fenologia e correlacionamos às variações populacionais das espécies mais comuns com variáveis meteorológicas. A pesquisa foi conduzida na área experimental da Embrapa Cerrados. O método de coleta utilizado foi diferenciado sendo supressivo e absoluto. Um total de 13 espécies foram coletadas, das quais oito ocorreram em soja Bt. Os taxa mais representativos foram Chrysodeixis includens, Anticarsia gemmatalis e Spodoptera spp. O número de larvas pertencentes às espécies alvo da tecnologia Bt foram 10 vezes menores na soja Bt do que em soja não-Bt . Utetheisa ornatrix e Elaphria deltoides foram registradas na soja pela primeira vez, observando-se larvas de ambas espécies na soja não-Bt e as de U. ornatrix também na soja Bt. Somente as larvas de A. gemmatalis se correlacionaram (p 0,05) negativamente com a precipitação. Este estudo forneceu informações em campo sobre a abundância e riqueza de espécies na soja não- Bt, associada aos efeitos da soja Bt. A importância do monitoramento das lagartas é substancial, a fim de tomar a melhor decisão de controle, considerando-se os diferentes níveis de infestação entre cultivares como critério.