Identification and characteristic analysis of an extracellular signal-regulated kinase from Ostrinia furnacalis Guenée.

The extracellular signal-regulated kinase (ERK) pathway, a critical genetic determinant, controls diverse physiological functions, including innate immunity, development, and stress response. In the current study, a full-length cDNA (1592bp) encoding the ERK gene (OfERK) was cloned from Ostrinia furnacalis Guenée (GenBank accession number: MF797866). The open reading frame of the OfERK gene encoded 364 amino acids and shared 96.43%-98.08% amino acid identities with other insect mitogen-activated protein kinases. For spatiotemporal analysis of the expression pattern, OfERK exhibited a significant peak expression on the 3rd day of the pupa stage and showed the highest expression in hemocytes specifically. Indirect immunofluorescence assays and immuno-electron microscopy revealed a wide distribution of the OfERK protein in hemocytes and epidermis. Moreover, the results demonstrated that the Bt Cry1Ab-activated toxin significantly induces the expression of OfERK. Other genes related to immune response, development, and stress response exhibited dynamic changes in expression after Cry1Ab oral treatment. The expression of OfERK was downregulated through RNA interference, and the correlation of its expression with other related genes was verified using quantitative real-time polymerase chain reaction. Our study provides valuable insights into the regulatory mechanism of ERK in insects for future studies.

Influence of Cry1Ab protein on growth and development of a predatory spider, Pardosa pseudoannulata, from protective perspectives.

The expression of Cry proteins in genetically modified rice varieties safeguards the crop from lepidopteran pests. These proteins have the potential to be transferred through the food chain to arthropods like planthoppers and predatory spiders, triggering defensive responses in these unintended organisms. Hence, we hypothesized that Cry protein might influence the growth and development of spiders by altering protective enzyme activities. The results showed that Cry1Ab protein could accumulate in tissues and subcellular organelles of Pardosa pseudoannulata from Nilaparvata lugens. Cry1Ab protein exposure prolonged the developmental duration in the 5th and 7th instar spiderlings but induced no alterations of other growth indicators, such as body length, median ocular area, and survival rate. In addition, Cry1Ab protein exerted no adverse impacts on several detoxifying enzymes (i.e., superoxide dismutase, catalase, glutathione peroxidase, and acetylcholine esterase) in muscle, midgut, ganglia, and hemolymph at subcellular components (i.e., microsome and cytoplasm). To further explore the effects of Cry1Ab protein on the spiderlings, we performed an integrated transcriptome analysis on spiderlings exposed to Cry1Ab protein. The results showed that Cry1Ab protein might prolong the development duration of P. pseudoannulata via the altered cuticle metabolism (e.g., chitin metabolic process and structural constituent of cuticle). In addition, the gene expression profile associated with detoxifying enzymes and three stress-responsive pathways (JAK/STAT, JNK/SAPK, and Hippo pathways) also displayed no significant alterations under Cry1Ab exposure. Collectively, this integrated analysis generates multidimensional insights to assess the effects of Cry1Ab protein on non-target spiders and demonstrates that Cry1Ab protein exerts no toxicity in P. pseudoannulata.

[Developmental toxicity of Cry1Ab protein in the embryonic stem-cell model].

OBJECTIVE: To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell. METHODS: Cry1Ab protein was tested in seven dose groups (31.25, 62.50, 125.00, 250.00, 320.00, 1 000.00, and 2 000.00 µg/L) on mouse embryonic stem cells D3 (ES-D3) and 3T3 mouse fibroblast cells, with 5-fluorouracil (5-FU) used as the positive control and phosphate buffer saline (PBS) as the solvent control. Cell viability was detected by CCK-8 assay to calculate the 50% inhibitory concentration (IC50) of the test substance for different cells. Additionally, Cry1Ab protein was tested in five dose groups (125.00, 250.00, 320.00, 1 000.00, and 2 000.00 µg/L) on ES-D3 cells, with PBS as the solvent control and 5-FU used for model validation. After cell treatment, cardiac differentiation was induced using the embryonic bodies (EBs) culture method. The growth of EBs was observed under a microscope, and their diameters on the third and fifth days were measured. The proportion of EBs differentiating into beating cardiomyocytes was recorded, and the 50% inhibition concentration of differentiation (ID50) was calculated. Based on a developmental toxicity discrimination function, the developmental toxicity of the test substances was classified. Furthermore, at the end of the culture period, mRNA expression levels of cardiac differentiation-related markers (Oct3/4, GATA-4, Nkx2.5, and ß-MHC) were quantitatively detected using real-time quantitative polymerase chain reaction (qPCR) in the collected EBs samples. RESULTS: The IC50 of 5-FU was determined as 46.37 µg/L in 3T3 cells and 32.67 µg/L in ES-D3 cells, while the ID50 in ES-D3 cells was 21.28 µg/L. According to the discrimination function results, 5-FU was classified as a strong embryotoxic substance. There were no statistically significant differences in cell viability between different concentrations of Cry1Ab protein treatment groups and the control group in both 3T3 cells and ES-D3 cells (P>0.05). Moreover, there were no statistically significant differences in the diameter of EBs on the third and fifth days, as well as their morphology, between the Cry1Ab protein treatment groups and the control group (P>0.05). The cardiac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group (P>0.05). 5-FU significantly reduced the mRNA expression levels of ß-MHC, Nkx2.5, and GATA-4 (P < 0.05), showing a dose-dependent trend (P < 0.05), while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend (P < 0.05). However, there were no statistically significant differences in the mRNA expression levels of mature cardiac marker ß-MHC, early cardiac differentiation marker Nkx2.5 and GATA-4, and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group (P>0.05). CONCLUSION: No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 µg/L was observed in this experimental model.

How safe is our food we eat? An electrochemical lab-on-kitchen approach towards combinatorial testing for pesticides and GMOs; A case study with edamame.

In our daily life, as consumers we are constantly made aware of the impact of pesticides and other modifications to food products derived from genetically modified organisms (GMO's) that have an impact on human health. In our connected world, there is an immense interest for on-demand information about food quality prior to consumption. The gold standard method to detect pesticides or GMOs residues in food is complex and is not amenable to rapid consumer use. In this study, we demonstrate the feasibility of an electrochemical portable sensing approach for the simultaneous direct detection of spiked pesticides chlorpyrifos (Chlp) and GMOs protein Cry1Ab in real edamame soy matrix. The immunoassay based two-plex sensing platform was fabricated using respective antibody's Chlp on one side and Cry1Ab on other side. A simple lab-on-kitchen level preparation of matrix has been demonstrated and sensor response was tested using non-faradaic electrochemical impedance spectroscopy (EIS), which showed a linear response in Cry1Ab/Chlp concentrations from 0.3 ng/mL to 243 ng/mL with limit of detection 0.3 ng /mL for both the target antigens (Cry1Ab and Chlp) respectively. The spiked and recovery test results fall within ± 20% error in real sample matrix which demonstrates the performance of the our platform with maximum residue limit (MRL) for the given targets. Such electrochemical portable multi-analyte direct sensing tool with simple matrix processing protocol can be a future commercial field-testing tool for use at everyday consumer level.

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

Assessing the effects of Bt maize on the non-target pest Rhopalosiphum maidis by demographic and life-history measurement endpoints.

The most commercialized Bt maize plants in Europe were transformed with genes which express a truncated form of the insecticidal delta-endotoxin (Cry1Ab) from the soil bacterium Bacillus thuringiensis (Bt) specifically against Lepidoptera. Studies on the effect of transgenic maize on non-target arthropods have mainly converged on beneficial insects. However, considering the worldwide extensive cultivation of Bt maize, an increased availability of information on their possible impact on non-target pests is also required. In this study, the impact of Bt-maize on the non-target corn leaf aphid, Rhopalosiphum maidis, was examined by comparing biological traits and demographic parameters of two generations of aphids reared on transgenic maize with those on untransformed near-isogenic plants. Furthermore, free and bound phenolics content on transgenic and near-isogenic plants were measured. Here we show an increased performance of the second generation of R. maidis on Bt-maize that could be attributable to indirect effects, such as the reduction of defense against pests due to unintended changes in plant characteristics caused by the insertion of the transgene. Indeed, the comparison of Bt-maize with its corresponding near-isogenic line strongly suggests that the transformation could have induced adverse effects on the biosynthesis and accumulation of free phenolic compounds. In conclusion, even though there is adequate evidence that aphids performed better on Bt-maize than on non-Bt plants, aphid economic damage has not been reported in commercial Bt corn fields in comparison to non-Bt corn fields. Nevertheless, Bt-maize plants can be more easily exploited by R. maidis, possibly due to a lower level of secondary metabolites present in their leaves. The recognition of this mechanism increases our knowledge concerning how insect-resistant genetically modified plants impact on non-target arthropods communities, including tritrophic web interactions, and can help support a sustainable use of genetically modified crops.

Toxic effects of the combined cadmium and Cry1Ab protein exposure on the protective and transcriptomic responses of Pirata subpiraticus.

Cadmium (Cd) pollution poses a serious threat to agricultural production and paddy field fauna. Crystalline proteins (e.g., Cry1Ab and Cry1Ac) are secreted by Bacillus thuringiensis, which can manage pests via a complicated toxic mechanism and have been widely used for pest control due to the commercialization of transgenic crops (e.g., cotton and rice) that expresses Bt insecticidal proteins. Nonetheless, studies on the effects of combined stress of Cd and Cry1Ab protein on field indicator species are limited. In the present study, we showed that spiders, Pirata subpiraticus, fed with Cd-containing flies+Cry1Ab had dramatically higher Cd accumulation than that in the spiders fed with Cd-containing flies (p < 0.05). In addition, the enrichment of Cd led to the activation of the protective mechanism by elevating the concentrations of glutathione peroxidase, glutathione S-transferase, and metallothionein in the spiders (p < 0.05). An in-depth transcriptome analysis revealed that the activities of ion metal binding proteins, transporters, and channels might play essential roles in the Cd accumulation process. More importantly, the higher Cd concentration in the combined Cd+Cry1Ab exposure prolonged developmental duration of P. subpiraticus, due to the down-regulated cuticle proteins (CPs) encoding genes involved in the molting process, which was regulated by a series of putative transcriptional factors such as ZBTB and zf-C2H2. Collectively, this integrated analysis illustrates that the combined Cd+Cry1Ab exposure increases the adverse effects of Cd stress on the growth, antioxidase, and CPs encoding genes of P. subpiraticus, thus providing a research basis and prospect for the rationality of transgenic Cry1Ab crops in the cultivation of heavy metal contaminated soil.

[Protein efficiency ratio of genetically modified corn with Cry1Ab-ma gene].

OBJECTIVE: To evaluate the protein efficiency ratio(PER) of genetically modified corn with Cry1Ab-Ma gene and parental corn. METHODS: Sixty SD rats(60-80 g) were randomly divided into genetically modified corn group, parental corn group and casein control group, with 20 rats in each group and half male and half female. Casein was added to 10% of the diet in casein control group. When the protein content of the diets in the genetically modified corn group and parental corn group was still less than 10% according to the principle of maximum incorporation, the defective part was supplemented with casein. Rats were free to drink and eat for 28 days. Food intake and body weight of each group were recorded every week. Blood was collected at the end of the experiment to determine hematology and blood biochemical indexes. The main organs were weighed and organ/body weigh indexes were calculated. PER and corrected PER were calculated. RESULTS: The body weight of all the animals in each group showed an increasing trend, and the weight growth was normal. Although there were statistical differences in the individual indexes of end-stage hematology and blood biochemical indexes, there was no biological significance. There were no significant change in the organ/body weigh indexes. PER of genetically modified corn, parental corn and casein were 2.01±0.22, 1.77±0.30 and 3.64±0.48, respectively. The corrected PER of genetically modified corn and parent corn were 1.38 and 1.22, respectively. CONCLUSION: The PER of this batch of genetically modified corn with Cry1Ab-ma gene was better than that of parental corn, but worse than that of casein.

Bacillus thuringiensis Cry1Ab Domain III ß-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith).

The fall armyworm, Spodoptera frugiperda, is an invasive maize pest that has spread from the Americas into Africa and Asia and causes severe crop damage worldwide. Most populations of S. frugiperda show low susceptibility to Bacillus thuringiensis (Bt) Cry1Ab or Cry1Ac toxins, which have been proved to be effective against several other lepidopteran pests. In addition, S. frugiperda has evolved resistance to transgenic maize expressing Cry1Fa toxin. The specificity and toxicity of Cry toxins are determined by their binding to different larval midgut proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), and cadherin (CAD), among other proteins, by means of exposed domain II loop regions and also by the domain III ß-sheets ß-16 and ß-22. Here, we analyzed different Cry1Ab mutants with mutations in the domain III ß-22 region. Alanine-scanning mutagenesis of this region revealed that all mutants showed increased toxicity against a nonsusceptible Cry1Ab S. frugiperda population. Further analysis of the mutant toxin Cry1AbS587A (bearing a mutation of S to A at position 587) revealed that, compared to Cry1Ab, it showed significantly increased toxicity to three other S. frugiperda populations from Mexico but retained similar toxicity to Manduca sexta larvae. Cry1AbS587A bound to brush border membrane vesicles (BBMV), and its higher toxicity correlated with higher binding affinities to APN, ALP, and CAD recombinant proteins. Furthermore, silencing the expression of APN1 and CAD receptors in S. frugiperda larvae by RNA interference (RNAi) showed that Cry1AbS587A toxicity relied on CAD expression, in contrast to Cry1Ab. These data support the idea that the increased toxicity of Cry1AbS587A to S. frugiperda is in part due to an improved binding interaction with the CAD receptor.IMPORTANCESpodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III ß-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.

Fluorescent analyses of Bacillus thuringiensis Cry1Fa and Cry1Ab toxin binding sites on brush border membrane vesicles of Ostrinia nubilalis (Hübner), Diatraea grandiosella (Dyar), and Helicoverpa zea (Boddie) larvae.

Bacillus thuringiensis (Bt) Cry1Fa and Cry1Ab proteins are important Cry toxins due to their high, selective toxicity against a number of lepidopteran species, including important pests of corn and cotton. Competition binding assays are a classical tool for investigating Cry toxin interactions with target pest insects. We developed a fluorescence-based binding assay and assessed Cry1Fa and Cry1Ab toxin binding to brush border membrane preparations from lepidopteran corn pests including Ostrinia nubilalis (European corn borer, ECB), Diatraea grandiosella (south western corn borer, SWCB), and Helicoverpa zea (corn earworm, CEW). Homologous and heterologous competition binding assays with fluorophore-(Alexa488)-labeled Cry1Fa toxin showed that Cry1Fa shares binding site(s) with Cry1Ab toxin in ECB, and SWCB for which Cry1Ab has higher affinity than Cry1Fa. Apart from the shared binding sites, Cry1Ab and Cry1Fa bind an additional site(s) in ECB and SWCB. In CEW, Cry1Fa and Cry1Ab each, has a high affinity binding site(s), which binds the heterologous toxin with low affinity. The Cry1Ab-Cry1Fa toxin binding models for ECB, SWCB and CEW based on our results are considered in the context of what is known about acquired cross-resistance against Cry1Ab and Cry1Fa toxins.

[Immune function effect of F3 rats fed with genetically modified maize harboring Cry1Ab and epsps genes].

OBJECTIVE: To evaluate the effects of genetically modified maize with Cry1Ab and epsps genes on immune function in F3 rats. METHODS: A total of 180 weaning SD rats for F0 generation were randomly divided into three groups, which were treated with AIN-93 G feed control diet, parental maize diet and genetically modified maize diet respectively. After three generations of breeding, antibody producing cells determination, concanavalin A(ConA)-induced lymphocyte transformation test, natural killer(NK)cells activities assay, whole blood lymphocyte subtype detection, delayed type hypersensitivity test and immunity organ index were performed. RESULTS: There were no significant differences between parental maize diet and genetically modified maize diet in terms of the number of antibody-producing cells, ConA-induced spleen lymphocyte proliferation, NK cell activity, whole blood lymphocyte subsets, delayed type hypersensitivity and thymus index(P>0. 05). CONCLUSION: Under the conditions of this experiment, no significant effects were found on immune function of F3 SD rats through the three generation development study of genetically modified maize with CrylAb and epsps genes.

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.

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 Cry1Ab-expressing Bt rice straw return on juvenile and adult Eisenia fetida.

A 90 day experiment was conducted in the laboratory to investigate the potential effects of transgenic Cry1Ab-expressing rice (Bacillus thuringiensis (Bt) rice: T775 and its F1 hybrid) straw return on earthworm Eisenia fetida, compared to non-Bt rice (TYHZ) straw. Juvenile E. fetida could survive, grow up, mature and reproduce offspring well in a Bt rice treated test during the whole experiment. The significantly higher relative growth rate (RGR) was found in earthworms from Bt rice treatment than from non-Bt rice treatment on the 7th day. The period of sexual maturity for earthworms from Bt rice treatments was shortened significantly, compared to non-Bt rice treatments. Adult E. fetida survived with weight loss under Bt rice treatments. On the 7th and 15th day, earthworm RGR decreased and glutathione peroxidase (GSH-PX) activity increased under Bt rice straw treatments. Significantly fewer offspring were produced by earthworms from Bt rice than non-Bt rice treatments on the 60th and 75th day. Enzyme-linked immunosorbent assay (ELISA) determined a sharp decrease of Cry1Ab in straw mixed soil along with the experimental time, regardless of juvenile or adult earthworm treatments. Cry1Ab concentration in the earthworms from the juvenile group was significantly higher than those from the adult group. Bt rice straw return had significant effects on soil nutrients, especially on the content of total and available phosphorus. In view of two bioassays, Bt rice (T775 and its F1 hybrid) straw return presented different effects on E. fetida from the juvenile (no deleterious effect) and adult (a little negative effect) groups, that were not directly related to Cry1Ab presence and nutrient differences among the three rice variety treatments.

microRNA profiling between Bacillus thuringiensis Cry1Ab-susceptible and -resistant European corn borer, Ostrinia nubilalis (Hübner).

Transgenic maize hybrids that express insecticidal Bacillus thuringiensis (Bt) crystalline (Cry) protein toxins effectively protect against the European corn borer, Ostrinia nubilalis, a devastating maize pest. Field monitoring and laboratory selections have detected varying levels of O. nubilalis resistance to Cry1Ab toxin. MicroRNAs (miRNAs) are short noncoding RNAs that are involved in post-transcriptional gene regulation. Their potential roles in the evolution of Bt resistance, however, remain largely unknown. Sequencing of small RNA libraries from the midgut of Cry1Ab-susceptible and resistant O. nubilalis larvae resulted in the discovery of 277 miRNAs, including 248 conserved and 29 novel. Comparative analyses of miRNA expression profiles between the laboratory strains predicted 26 and nine significantly up- and down-regulated transcripts, respectively, in the midgut of Cry1Ab resistant larvae. Amongst 15 differentially regulated miRNAs examined by quantitative real-time PCR, nine (60%) were validated as cosegregating with Cry1Ab resistance in a backcross progeny. Differentially expressed miRNAs were predicted to affect transcripts involved in cell membrane components with functions in metabolism and binding, and the putative Bt-resistance genes aminopeptidase N and cadherin. These results lay the foundation for future investigation of the potential role of miRNAs in the evolution of Bt resistance.

Comparative compositional analysis of transgenic potato resistant to potato tuber moth (PTM) and its non-transformed counterpart.

In this study, the compositions of transgenic potatoes (TPs) resistant to potato tuber moth (Phthorimaea operculella) were compared with those of its non-transgenic (NTP) counterparts. The light inducible promoter, phosphoenolpyruvate carboxylase led to the expression of Cry1Ab only in the leaves and light-treated tubers of the TPs. No significant differences were found in the moisture, ash, dry weight, total soluble protein, carbohydrate, starch, fiber, ascorbate, cations, anions, fatty acids, and glycoalkaloids contents of TP and NTP. Moreover, light treatment significantly affected the contents of ascorbate, acetate and nitrite anions, palmitic, stearic and linolenic fatty acids, α-haconine and α-solanine glycoalkaloids in TP and NTP tubers. While, significant differences were observed in the amino acid contents in light-treated tubers of TPs than the NTP ones. Although, light treatment in potato tubers resulted in marked metabolic changes, all the variations observed in the metabolites compositions were found to be within the desired reference ranges for potato plants. In conclusion, the results indicated that the TPs were substantially and nutritionally equivalent to the NTP counterparts.

A Novel Fluorescent Nanoparticle for Sensitive Detection of Cry1Ab Protein In Vitro and In Vivo.

Here, we report the synthesis and characterization of CoFe2O4 doping Ag2S dendrimer-modified nanoparticles (CoFe2O4-Ag2S DMNs) in Cry1Ab protein detection and imaging. The near-infrared Ag2S quantum dots were first prepared by using the thermal decomposition method, followed by modification of the water-soluble quantum dots using the method of solvent evaporation and ligand exchange, and finally the fluorescent magnetic bifunctional nanoparticles were obtained by binding with CoFe2O4. As-prepared CoFe2O4-Ag2S DMNs were characterized by fluorescence (FL) spectroscopy and transmission electron microscopy (TEM). Results showed that Ag2S DMNs could sensitively detect Cry1Ab both in vitro and in vivo. In vitro, the enhanced FL intensity as a function of the concentration is notably consistent with the Langmuir binding isotherm equation in the range of 0-200 ng/mL of Cry1Ab proteins. The detection limit of this method was found to be 0.2 ng/mL. Meanwhile, the fluorescence wavelength was extended to the second near-infrared range (NIR-II, 1.0~1.4 µm), which enables in vivo imaging. This study highlights the importance of NIR QDs doping magnetic materials as a new method to trace Bacillus thuringiensis (Bt) in insects and their potential applications in in vivo NIR tissue imaging.

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three-dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organisms.

Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc.

Transcriptomic response of wolf spider, Pardosa pseudoannulata, to transgenic rice expressing Bacillus thuringiensis Cry1Ab protein.

BACKGROUND: Bacillum thuringiensis (Bt) toxin produced in Cry1-expressing genetically modified rice (Bt rice) is highly effective to control lepidopteran pests, which reduces the needs for synthetic insecticides. Non-target organisms can be exposed to Bt toxins through direct feeding or trophic interactions in the field. The wolf spider Pardosa pseudoannulata, one of the dominant predators in South China, plays a crucial role in the rice agroecosystem. In this study, we investigated transcriptome responses of the 5th instar spiders fed on preys maintained on Bt- and non-Bt rice. RESULTS: Comparative transcriptome analysis resulted in 136 differentially expressed genes (DEGs) between spiderlings preying upon N. lugens fed on Bt- and non-Bt rice (Bt- and non-Bt spiderlings). Functional analysis indicated a potential impact of Bt toxin on the formation of new cuticles during molting. GO and KEGG enrichment analyses suggested that GO terms associated with chitin or cuticle, including "chitin binding", "chitin metabolic process", "chitin synthase activity", "cuticle chitin biosynthetic process", "cuticle hydrocarbon biosynthetic process", and "structural constituent of cuticle", and an array of amino acid metabolic pathways, including "alanine, asparatate and glutamate metabolism", "glycine, serine and theronine metabolism", "cysteine and methionine metabolism", "tyrosine metabolism", "phenylalanine metabolism and phenylalanine", and "tyrosine and tryptophan biosynthesis" were significantly influenced in response to Cry1Ab. CONCLUSIONS: The Cry1Ab may have a negative impact on the formation of new cuticles during molting, which is contributed to the delayed development of spiderlings. To validate these transcriptomic responses, further examination at the translational level will be warranted.