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Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenee) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

This study reported the functionality integration of zeolitic imidazolate framework-8 (ZIF-8) with horseradish peroxidase (HRP) and streptavidin (SA) for the synthesis of a HRP&SA/ZIF-8 nanocomposite through one-pot coprecipitation. The synthesized HRP&SA/ZIF-8 nanocomposite was then employed as the ideal signal tag for application in the enzyme-linked immunosorbent assay (ELISA) and exhibited excellent sensitivity, selectivity and accuracy in the detection of insecticidal crystalline (Cry) protein Cry1Ab as a transgenic biomarker with a detection limit of 4.8 pg/mL. This proposed method provides a new way for the detection of transgenic biomarkers in food and may inspire further integration of a variety of biomolecules into ZIF-8 for applications ranging from biosensing, biomedicine, and catalysis to energy.

To learn the spatial and temporal distribution of Bt proteins in Bt maize leaves, Bt protein levels of Bt maize leaves and their corresponding non-transgenic lines were detected and analysed by the ELISA method. The results showed that Bt proteins were expressed in the leaves of Bt maize varieties during all periods of their growth, while no such Bt proteins were detected in the leaves of non-transgenic maize varieties. The range of leaf Bt proteins was 1.08∼5.81 µg.g−1 FW, which could effectively protect Bt maizes from the corn borer pest during their growth periods. Furthermore, the younger leaves of Bt maizes had significantly lower levels of Bt proteins than their older leaves. In most periods, Bt protein content of Bt maize leaves under drought stress was lower than that under normal growth conditions, but Bt protein content under nutrition stress was higher than that under normal growth conditions. The results provided a full pattern of the spatial and temporal distribution of Bt proteins in Bt maize leaves.

Bt (Bacillus thuringiensis) corn is one of the top three large-scale commercialized anti-insect transgenic crops around the world. In the present study, we tested the Bt protein content, defense chemicals contents, and defense enzyme activities in both the leaves and roots of Bt corn varieties 5422Bt1 and 5422CBCL, as well as their conventional corn 5422 seedlings, with two fully expanded leaves which had been treated with 2.5 mM exogenous salicylic acid (SA) to the aboveground part for 24 h. The result showed that the SA treatment to the aboveground part could significantly increase the polyphenol oxidase activity of conventional corn 5422, the Bt protein content, and peroxidase activities of Bt corn 5422Bt1, as well as the polyphenol oxidase and peroxidase activity of Bt corn 5422CBCL in the leaves. In the roots, the polyphenol oxidase and peroxidase activity of conventional corn 5422, the polyphenol oxidase and superoxide dismutase activities of Bt corn 5422Bt1, the DIMBOA (2,4-dihydroxy-7-methoxy-2H, 1, 4-benzoxazin-3 (4H)-one) content, and four defense enzymes activities of Bt corn 5422CBCL were systematically increased. These findings suggest that the direct effect of SA application to aboveground part on the leaf defense responses in Bt corn 5422CBCL is stronger than that in non-Bt corn. Meanwhile, the systemic effect of SA on the root defense responses in Bt corn 5422CBCL is stronger than that in conventional corn 5422 and Bt corn 5422Bt1. It can be concluded that the Bt gene introduction and endogenous chemical defense responses of corns act synergistically during the SA-induced defense processes to the aboveground part. Different transformation events affected the root defense response when the SA treatment was applied to the aboveground part.

Reproductive organs of Bacillus thuringiensis transgenic cotton, which contribute to cotton final yield, have low insect resistant efficacy, so it is important to improve their insect resistance. This study was conducted to find out the impact of different urea spray doses on the expression of Cry1A protein in boll shell of Bt cotton (Sikang 1 and Sikang 3), and nitrogen metabolism in this process was also studied to uncover the physiological mechanism. The experiment with six urea doses was organized during peak boll stage in 2017 and 2018. The results showed that urea spray could significantly increase boll shell insecticidal protein contents in both cultivars, with the highest Bt protein content observed at 28–32 kg ha−1 urea dose. In addition, urea spray increased the contents of soluble protein and free amino acid and the activities of GS, GOGAT, GOT, and GPT, but decreased the activities of peptidase and protease in boll shell. Correlation analysis showed that the amount of boll shell Bt protein was positively correlated with levels of soluble protein and amino acid, and activities of GS, GOGAT, GOT, and GPT, but negatively correlated with peptidase and protease activities. Thus, this study demonstrated that higher protein synthesis ability and lower proteolysis ability were related to increased Bt protein content in urea-sprayed boll shell.

Bollgard® III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex® cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity.

Background: To know the effects of transgenic crops on soil microorganisms, animals and other expected hazards due to the introduction of GM crops into the environment is critical both scientifically and environmentally. The work was conducted to study the effect of insecticidal Bt protein on Rats and Earthworms. Methods: For this purpose, animals like rat and soil organisms like Earthworm were selected. Rats were selected on the basis of its 95% homology on genomic, cellular and enzymatic level with human while earthworm were preferred on the basis of their direct contact with soil to evaluate the impact of Bt (Cry1AC) crop field soil on earthworm, secreted by root exudates of Bt cotton. Several physical, molecular, biochemical and histological analyses were performed on both Rats/Earthworms fed on standard diet (control group) as well containing Bt protein (experimental group). Results: Molecular analyses such as immune Dot blot, SDS-PAGE, ELISA and PCR, confirmed the absence of Cry1Ac protein in blood and urine samples of rats, which were fed with Bt protein in their diet. Furthermore, histological studies showed that there was no difference in cellular architecture in liver, heart, kidney and intestine of Bt and non-Bt diet fed rats. To see the effect of Bt on earthworm two different groups were studied, one with transgenic plant field soil supplemented with grinded leaves of cotton and second group with non-Bt field soil. Conclusions: No lethal effects of transgenic Bt protein on the survival of earthworm and rats were observed. Bradford assay, Dipstick assay ELISA demonstrated the absence of Cry1Ac protein in the mid-gut epithelial tissue of earthworm. The results of present study will be helpful in successful deployment and commercial release of genetically modified crop in Pakistan.

Higher boll worm survival rates were detected after high temperature presented during square period in Bt cotton. The objective of this study was to investigate the effects of high temperature level on the Bt efficacy of two different types of Bt cotton cultivars at squaring stage. During the 2011 to 2013 cotton growth seasons, high temperature treatments ranged from 34 to 44°C in climate chambers, and field experiments under high temperature weather with various temperature levels were conducted to investigate the effects of the high temperature level on square Bt protein concentration and nitrogen metabolism. The climate chamber experiments showed that the square insecticidal protein contents reduced after 24 h elevated temperature treatments for both cultivars, whereas significant declines of the square insecticidal protein contents were detected at temperature >38°C, and only slightly numerical reductions were observed when temperature below 38°C. Similar high temperature responses were also observed at the two field experimental sites in 2013. Correspondingly, high temperature below 38°C seems have little effect on the square amino acid concentrations, soluble protein contents, glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) activities as well as protease and peptidase activities; however, when the temperature was above 38°C, reduced soluble protein contents, enhanced amino acid concentrations, decreased GPT and GOT activities, bolstered protease and peptidase activities in square were detected. In general, the higher the temperature is (>38°C), the larger the changes for the above compound contents and key enzymes activities of the square protein cycle. The findings indicated that the unstable insect resistance of the square was related to high temperature level during square stage.

Background Pardosa pseudoannulata is a prevailing spider species, and has been regarded as an important bio-control agent of insect pests in farmland of China. However, the available genomic and transcriptomic databases of P. pseudoannulata and their venom are limited, which severely hampers functional genomic analysis of P. pseudoannulata. Recently high-throughput sequencing technology has been proved to be an efficient tool for profiling the transcriptome of relevant non-target organisms exposed to Bacillus thuringiensis (Bt) protein through food webs. Results In this study, the transcriptome of the venom apparatus was analyzed. A total of 113,358 non-redundant unigenes were yielded, among which 34,041 unigenes with complete or various length encoding regions were assigned biological function annotations and annotated with gene ontology and karyotic orthologous group terms. In addition, 3726 unigenes involved in response to stimulus and 720 unigenes associated with immune-response pathways were identified. Furthermore, we investigated transcriptomic changes in the venom apparatus using tag-based DGE technique. A total of 1724 differentially expressed genes (DEGs) were detected, while 75 and 372 DEGs were functionally annotated with KEGG pathways and GO terms, respectively. qPCR analyses were performed to verify the DEGs directly or indirectly related to immune and stress responses, including genes encoding heat shock protein, toll-like receptor, GST and NADH dehydrogenase. Conclusion This is the first study conducted to specifically investigate the venom apparatus of P. pseudoannulata in response to Bt protein exposure through tritrophic chain. A substantial fraction of transcript sequences was generated by high-throughput sequencing of the venom apparatus of P. pseudoannulata. Then a comparative transcriptome analysis showing a large number of candidate genes involved in immune response were identified by the tag-based DGE technology. This transcriptome dataset will provide a comprehensive sequence resource for furture molecular genetic research of the venom apparatus of P. pseudoannulata. Electronic supplementary material The online version of this article (10.1186/s12896-017-0392-z) contains supplementary material, which is available to authorized users.

In recent years, different Bacillus thuringiensis (Bt) toxin-encoding genes have been combined or 'stacked' in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).