Involvement of Sep38ß in the Insecticidal Activity of Bacillus thuringiensis against Beet Armyworm, Spodoptera exigua (Lepidoptera).

The p38 mitogen-activated protein kinases (MAPKs) are associated with insect immunity, tissue repair, and the insecticidal activity of Bacillus thuringiensis (Bt). Here, a p38 MAPK family gene (Sep38ß) was identified from Spodoptera exigua. Among the developmental stages, the transcription level of Sep38ß was the highest in egg, followed by that in prepupa and pupa. Sep38ß expression peaked in Malpighian tubules and the hemolymph of fifth instar larvae. Knockdown of Sep38ß or injection of SB203580 (a p38 MAPK inhibitor) significantly downregulated the SeDUOX expression and reactive oxygen species (ROS) level in the midgut, accounting for deterioration of the midgut to scavenge pathogens and enhancement of Bt insecticidal activity. In conclusion, all the results demonstrate that Sep38ß regulates the immune-related ROS level in the insect midgut, which suppresses the insecticidal activity of Bt against S. exigua by 17-22%. Our study highlights that Sep38ß is essential for insect immunity and the insecticidal activity of Bt to S. exigua and is a potential target for pest control.

Processing Properties and Potency of Bacillus thuringiensis Cry Toxins in the Rice Leaffolder Cnaphalocrocis medinalis (Guenée).

Different Cry toxins derived from Bacillus thuringiensis (Bt) possess different insecticidal spectra, whereas insects show variations in their susceptibilities to different Cry toxins. Degradation of Cry toxins by insect midgut extracts was involved in the action of toxins. In this study, we explored the processing patterns of different Cry toxins in Cnaphalocrocis medinalis (Lepidoptera: Crambidae) midgut extracts and evaluated the impact of Cry toxins degradation on their potency against C. medinalis to better understand the function of midgut extracts in the action of different Cry toxins. The results indicated that Cry1Ac, Cry1Aa, and Cry1C toxins could be degraded by C. medinalis midgut extracts, and degradation of Cry toxins by midgut extracts differed among time or concentration effects. Bioassays demonstrated that the toxicity of Cry1Ac, Cry1Aa, and Cry1C toxins decreased after digestion by midgut extracts of C. medinalis. Our findings in this study suggested that midgut extracts play an important role in the action of Cry toxins against C. medinalis, and the degradation of Cry toxins by C. medinalis midgut extracts could reduce their toxicities to C. medinalis. They will provide insights into the action of Cry toxins and the application of Cry toxins in C. medinalis management in paddy fields.

Extracellular biomineralization of uranium and its toxicity alleviation to Bacillus thuringiensis X-27.

Uranium biomineralization can slow uranium migration in the environment and thus prevent it from further contaminating the surroundings. Investigations into the uranium species, pH, inorganic phosphate (Pi) concentration, and microbial viability during biomineralization by microorganisms are crucial for understanding the mineralization mechanism. In this study, Bacillus thuringiensis X-27 was isolated from soil contaminated with uranium and was used to investigate the formation process of uranium biominerals induced by X-27. The results showed that as biomineralization proceeded, amorphous uranium-containing deposits were generated and transformed into crystalline minerals outside cells, increasing the overall concentration of uramphite. This is a cumulative rather than abrupt process. Notably, B. thuringiensis X-27 precipitated uranium outside the cell surface within 0.5 h, while the release of Pi into the extracellular environment and the change of pH to alkalescence further promoted the formation of uramphite. In addition, cell viability determination showed that the U(VI) biomineralization induced by B. thuringiensis X-27 was instrumental in alleviating the toxicity of U(VI) to cells. This work offers insight into the mechanism of U(VI) phosphate biomineralization and is a reference for bioremediation-related studies.

A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness.

Maintaining fitness during pathogen infection is vital for host survival as an excessive response can be as detrimental as the infection itself. Fitness costs are frequently associated with insect hosts countering the toxic effect of the entomopathogenic bacterium Bacillus thuringiensis (Bt), which delay the evolution of resistance to this pathogen. The insect pest Plutella xylostella has evolved a mechanism to resist Bt toxins without incurring significant fitness costs. Here, we reveal that non-phosphorylated and phosphorylated forms of a MAPK-modulated transcription factor fushi tarazu factor 1 (FTZ-F1) can respectively orchestrate down-regulation of Bt Cry1Ac toxin receptors and up-regulation of non-receptor paralogs via two distinct binding sites, thereby presenting Bt toxin resistance without growth penalty. Our findings reveal how host organisms can co-opt a master molecular switch to overcome pathogen invasion with low cost, and contribute to understanding the underlying mechanism of growth-defense tradeoffs during host-pathogen interactions in P. xylostella.

Unveiling gene expression regulation of the Bacillus thuringiensis Cry3Aa toxin receptor ADAM10 by the potato dietary miR171c in Colorado potato beetle.

BACKGROUND: The Colorado potato beetle (CPB) is a worldwide devastating pest of potato plants and other Solanaceae characterized by its remarkable ability to evolve resistance to insecticides. Bacillus thuringiensis (Bt) Cry3Aa toxin represents an environmentally safe alternative for CPB control but larvae susceptibility to this toxin has been reported to vary depending on the host plant on which larvae feed. To gain more insight into how nutrition mediates Bt tolerance through effects on gene expression, here we explored the post-transcriptional regulation by microRNAs (miRNAs) of the CPB-ADAM10 gene encoding the Cry3Aa toxin functional receptor ADAM10. RESULTS: The lower CPB-ADAM10 gene expression in CPB larvae fed on potato plants cv. Vivaldi than those fed on potato cv. Monalisa or tomato plants was inversely related to Cry3Aa toxicity. By high-throughput sequencing we identified seven CPB miRNAs and one potato miRNA predicted to base pair with the CPB-ADAM10 messenger RNA. No differential expression of the endogenous lde-miR1175-5p was found in larvae feeding on any of the two potato plant varieties. However, statistically significant increased amounts of potato stu-miR171c-5p were detected in CPB larvae fed on potato cv. Vivaldi compared to larvae fed on potato cv. Monalisa. CONCLUSION: Our results support a role for dietary miRNAs in Bt toxicity by regulating the CPB-ADAM10 gene encoding the Cry3Aa toxin receptor ADAM10 in CPB larvae and opening up the possibility of exploiting plant natural variation in miRNAs to provide more sustainable potato crop protection against CPB. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Selective Adsorption and Efficient Degradation of Petroleum Hydrocarbons by a Hydrophobic/Lipophilic Biomass Porous Foam Loaded with Microbials.

Highly efficient elimination of petroleum pollution is of great importance for addressing environmental issues and social sustainability. In this study, we demonstrate a novel strategy for efficient elimination of petroleum pollution by selective adsorption of it by an ultralight hydrophobic/lipophilic microorganism-loaded biomass porous foam (BTS-MSFT4@MTMS) followed by a green degradation of adsorbates under mild conditions. The porous structure of biomass porous foam (MSFT) could provide plenty of room for immobilization of Bacillus thuringiensis (BTS), while a simple surface modification of the MSFT load with a BTS strain (BTS-MSFT4) by methyltrimethoxysilane (MTMS) could change its wettability from hydrophilic to lipophilic, which makes selective adsorption of hydophobic petroleum pollution from water for biodegradation possible. As expected, using a petroleum n-hexadecane solution with a concentration of 3% as a model oily wastewater, the as-prepared BTS-MSFT4@MTMS possesses both a superior selective adsorption of ca. 99% and high degradation activity with a high degradation rate of up to 86.65% within 8 days under the conditions of 37 °C, 120 r min-1, and pH = 7, while the degradation rates for the BTS-MSFT4 and the free BTS strain were measured to be only 81.62 and 65.65%, respectively, under the same conditions. In addition, the results obtained from the study on environment tolerance show that the BTS-MSFT4@MTMS exhibits a strong tolerance under different conditions with various pHs, temperatures, and initial concentrations. Compared with the existing methods for removal of petroleum pollution by direct adsorption of petroleum pollution via superoleophilic porous materials or applying free microorganisms for biodegradation only, which suffers the drawbacks of low selectivity or poor efficiency, our method has great advantages of cost-effectiveness, scalable fabrication, and high efficiency without secondary pollution. Moreover, such a two-in-one strategy by integration of both selective adsorption and biodegradation into biodegradable BTS-MSFT4@MTMS may particularly have great potential for practical application in environmental remediation.

Performance of Daphnia magna on flour, leaves, and pollen from different maize lines: Implications for risk assessment of genetically engineered crops.

Non-target effects of genetically engineered (GE) plants on aquatic Daphnia magna have been studied by feeding the species with different maize materials containing insecticidal Cry proteins from Bacillus thuringiensis (Bt). The results of those studies were often difficult to interpret, because only one GE plant was compared to one related non-GE control. In such a setting, effects of the Cry proteins cannot be distinguished from plant background effects, in particular when the test species is nutritionally stressed. In the present study, we tested the suitability of three different maize materials, i.e., flour, leaves and pollen, from five diverse non-GE maize lines (including EXP 258, a breeding line that is closely related to a SmartStax Bt maize) as exclusive food sources for D. magna. The parameters recorded included survival, sublethal endpoints such as body size, number of moltings to first offspring, time to first offspring, number of individuals in first clutch, total number of clutches, total number of offspring, average number of offspring per clutch, and population measures such as net reproductive rate R0, generation time T and intrinsic rate of increase rm. The results showed that D. magna can survive, grow and reproduce when fed only maize materials, although the performance was poorer than when fed algae, which indicates nutritional stress. Large differences in life table and population parameters of D. magna were observed among the different maize lines. Our results suggest that confounding effects caused by nutritional stress and plant background might explain some of the conflicting results previously published on the effects of Bt crops on D. magna. Using 95% confidence intervals for the means of the five maize lines for all measured parameters of D. magna performance in our study, we captured the natural range of variation. This information is useful for the interpretation of observed differences in D. magna performance between a GE plant and its non-GE comparator as it helps judging whether observed effects are of biological relevance. If differences between a GE and comparator line are observed and their biological relevance needs to be assessed in future risk assessments of GE maize, 1) the data on natural variation of the different parameters generated by previous studies can be informative (e.g. data from our study for maize fed D. magna); 2) for additional experiments the inclusion of multiple unrelated non-GE comparators should be considered; In addition, it should be taken into account that nutritional stress can affect the outcome of the study.

ß-carotene and Bacillus thuringiensis insecticidal protein differentially modulate feeding behaviour, mortality and physiology of European corn borer (Ostrinia nubilalis).

Maize with enhanced ß-carotene production was engineered to counteract pervasive vitamin A deficiency in developing countries. Second-generation biofortified crops are being developed with additional traits that confer pest resistance. These include crops that can produce Bacillus thuringiensis Berliner (Bt) insecticidal proteins. Currently, it is unknown whether ß-carotene can confer fitness benefits through to insect pests, specifically through altering Ostrinia nubilalis foraging behaviour or development in the presence of Bt insecticidal toxin. Therefore the effects of dietary ß-carotene plus Bt insecticidal protein on feeding behaviour, mortality, and physiology in early and late instars of O. nubilalis larvae were investigated. The results of two-choice experiments showed that irrespective of ß-carotene presence, at day five 68%-90% of neonates and 69%-77% of fifth-instar larvae avoided diets with Cry1A protein. Over 65% of neonate larvae preferred to feed on diets with ß-carotene alone compared to 39% of fifth-instar larvae. Higher mortality (65%-97%) in neonates fed diets supplemented with ß-carotene alone and in combination with Bt protein was found, whereas <36% mortality was observed when fed diets without supplemented ß-carotene or Bt protein. Diets with both ß-carotene and Bt protein extended 25 days the larval developmental duration from neonate to fifth instar (compared to Bt diets) but did not impair larval or pupal weight. Juvenile hormone and 20-hydroxyecdysone regulate insect development and their levels were at least 3-fold higher in larvae fed diets with ß-carotene for 3 days. Overall, these results suggest that the effects of ß-carotene and Bt protein on O. nubilalis is dependent on larval developmental stage. This study is one of the first that provides insight on how the interaction of novel traits may modulate crop susceptibility to insect pests. This understanding will in turn inform the development of crop protection strategies with greater efficacy.

Metal-immobilizing and urease-producing bacteria increase the biomass and reduce metal accumulation in potato tubers under field conditions.

In this study, the effect of two metal-immobilizing bacterial strains, Serratia liquefaciens CL-1 and Bacillus thuringiensis X30, on the availability of Cd and Pb and the metal accumulation in potato tubers, as well as the underlying mechanisms in metal-contaminated soils were characterized. Moreover, the impacts of the strains on metal immobilization, pH, and NH4+ concentration in metal-contaminated soil solutions were evaluated. Strains CL-1 and X30 increased tuber dry weight by 46% and 40%, reduced tuber Cd and Pb contents by 68-83% and 42-47%, and decreased the Cd and Pb translocation factors by 61-70% and 30-34%, respectively, compared to the controls. Strains CL-1 and X30 decreased the available Cd and Pb contents by 52-67% and 30-44% and increased the NH4+ content by 55% and 31%, pH, urease activity by 70% and 41%, and relative abundance of ureC gene copies by 37% and 20% in the rhizosphere soils, respectively, compared with the controls. Reduced Cd and Pb concentrations and increased pH and NH4+ concentration were found in the bacteria-inoculated soil solution compared to the controls. These results suggested that the strains reduced tuber metal uptake through decreasing the metal availability and increasing the pH, ureC gene relative abundance and urease activity as well as decreasing the metal translocation from the leaves to tubers. These results may provide an effective metal-immobilizing bacteria (especially strain CL-1)-enhanced approach to reduce metal uptake of potato tubers in metal-polluted soils.

Enhancement of Purified Human Colon Cancer-Specific Parasporal Toxin from Bacillus thuringiensis-LDC-501.

Parasporal inclusion protein of Bacillus thuringiensis-LDC-501 (Bt-LDC-501) exhibits selective cytocidal action towards human colon cancer cells. The yield of this parasporal protein was minimum in the normal culture. In order to increase the yield of protein from Bt-LDC-501 various agro-based cost-efficient nutrient sources such as corn steep liquor (CSL), sesame oil cake extract (SOC), groundnut oil cake extract (GOC), neem oil cake extract (NOC), rice bran extract (RB), wheat bran extract (WB), red gram hull extract (RGH), green gram hull extract (GGH), black gram hull extract (BGH), Mysore gram hull extract (MGH), and maize flour waste extract (MFW) were screened. Statistical experimental designs such as Plackett-Burman design (PBD) and response surface methodology (RSM) were the tools employed for the optimization of medium. Groundnut cake extract (GOC) served as a potential carbon and nitrogen source, as it induced twofold higher production of parasporal protein. Among the optimized seven media components KH2PO4, K2HPO4, GOC, NaCl, MgSO4, MnSO4, and FeSO4, the concentrations of GOC, NaCl, and MgSO4 have significant effect on parasporin production as well as cytotoxicity against colon cancer cell line, HCT-116. Bt-LDC-501 was found to produce 0.88 mg/ml of parasporal protein in optimized medium. In the un-optimized medium, the yield was 0.23 mg/ml only. This indicated that there was 382% of increase in the production of Parasporal protein. Parasporin protein with the molecular weight of 27 kDa has been purified with the purification fold of 27.1. It showed a LC50 value of 0.91 and 1.21 µg/ml against colorectal cancer cell lines such as HCT-116 and HCT-15, respectively. Purified parasporin exhibited stable cytocidal activity between pH 4.0 and 9.0 at room temperature. The present study revealed that the quantity and quality of media composition were necessary for eliciting cytocidal activity against human colon cancer and the importance of alternate cost-effective production of clinically significant parasporin. Moreover, this is the first report regarding optimization of media components for parasporal protein production from Bt.

Meeting technical challenges for protein characterization and surrogate equivalence studies that resulted from insecticidal protein co-expression in maize event MZIR098.

Safety assessment of genetically modified plants includes protein characterization to confirm the intended trait protein expression. In addition, to conduct safety tests, the large amount of purified protein needed is usually met through the use of a surrogate, microbially produced protein source. Characterization of the eCry3.1Ab and mCry3A proteins as derived from Event MZIR098 maize was challenging because of the difficulty in purifying/isolating these proteins that are of similar molecular weight and have considerable shared sequence and immunogenicity. This also applies to establishing the biochemical equivalence to the microbially produced surrogate proteins, as highly-purified plant protein is required. While use of crude plant extracts facilitated functional equivalence testing with the surrogate proteins, a separate technical challenge had to be met. The eCry3.1Ab and mCry3A proteins display differentiated modes of action toward CRW pests, however, with the same overall target pest spectrum, no differential test organism existed to allow equivalence testing for one insecticidal protein in the presence of the other. To establish that the microbially produced proteins are suitable surrogates for the plant-produced proteins, the challenges in the protein purification and bioactivity testing had to be addressed. This article describes technical solutions to assess and characterize the insecticidal proteins in this new event and thereby confirm equivalence/suitability of the microbially produced protein surrogates.

Strategical isolation of efficient chicken feather-degrading bacterial strains from tea plantation soil sample.

Chicken feather waste is generally insufficiently utilized despite its high content of protein, constituting an environmental issue. Biodegradation of the waste with enabling microbes provides an advantageous option among the available solutions. In this study, an efficient whole feather-degrading strain was strategically isolated from a soil sample taken from a local tea plantation that has little or nothing to do with feathers. The strain was identified as Bacillus thuringiensis (designated as FDB-10) according to the cloned complete 16S rRNA sequence. The FDB-10 could efficiently degrade briefly heat-treated whole feather (102 °C, 5 min; up to 90% of a maximum concentration of 30 g/L) in a salt medium supplemented with 0.1 g/L yeast extract within 24 h (37 °C, 150 rpm). Addition of carbon sources (glycerol, glucose, starch, Tween 20, Tween 80, 1.25 g/L as glycerol) to the fermentation medium could improve the degradation. However, significant inhibition could be observed when the added carbon source reached the amount usually adopted in the investigation of carbon source preference (1%). Nitrogen source (NH4Cl, (NH4)2SO4, peptone) adversely influenced the performance of the strain. When the molar concentrations of NH4+ were equal for the two salt, the inhibitory effect on degradation of whole feathers was similar. Entirely different from other reported feather-degrading strains showing a preference to melanin-free feather substrates, the strain isolated in this study could degrade melanin-containing feather equally efficiently, and higher protease activity could be detected in the digest mix. As a plus, the strain could degrade feathers in rice wash produced in daily cooking, indicating its potential use in the simultaneous treatment of rice cooker wastewater produced by a rice processing plant. All these results imply that the FDB-10 is a strain with great potential in the biodegradation of feather waste.

Pest management through Bacillus thuringiensis (Bt) in a tea-silkworm ecosystem: status and potential prospects.

Bacillus thuringiensis (Bt) is a soil bacterium that forms spores containing crystals comprising one or more Cry or Cyt proteins having potential and specific insecticidal activity. Different strains of Bt produce different types of toxins, affecting a narrow taxonomic group of insects. Therefore, it is used in non-chemical pest management, including inherent pest resistance through GM crops. The specificity of action of Bt toxins reduces the concern of adverse effects on non-target species, a concern which remains with chemical insecticides as well. To make use of Bt more sustainable, new strains expressing novel toxins are actively being sought globally. Since Bt is successfully used against many pests including the lepidopteran pests in different crop groups, the insecticidal activity against Samia cynthia (Drury) (Eri silkworm) and Antheraea assamensis Helfer (Muga silkworm) becomes a concern in the state of Assam in India which is a predominantly tea- and silk-producing zone. Though Bt can be used as an effective non-chemical approach for pest management for tea pests in the same geographical region, yet, it may potentially affect the silk industry which depends on silkworm. There is a need to identify the potentially lethal impact (through evaluating their mortality potential) of local Bt strains on key silkworm species in North Eastern India. This will allow the use of existing Bt for which the silkworms have natural resistance. Through this review, the authors aim to highlight recent progress in the use of Bt and its insecticidal toxins in tea pest control and the potential sensitivity for tea- and silk-producing zone of Assam in India.

Heme sensing in Bacillus thuringiensis: a supplementary HssRS-regulated heme resistance system.

Several Gram-positive pathogens scavenge host-derived heme to satisfy their nutritional iron requirement. However, heme is a toxic molecule capable of damaging the bacterial cell. Gram-positive pathogens within the phylum Firmicutes overcome heme toxicity by sensing heme through HssRS, a two-component system that regulates the heme detoxification transporter HrtAB. Here we show that heme sensing by HssRS and heme detoxification by HrtAB occur in the insect pathogen Bacillus thuringiensis We find that in B. thuringiensis, HssRS directly regulates an operon, hrmXY, encoding hypothetical membrane proteins that are not found in other Firmicutes with characterized HssRS and HrtAB systems. This novel HssRS-regulated operon or its orthologs BMB171_c3178 and BMB171_c3330 are required for maximal heme resistance. Furthermore, the activity of HrmXY is not dependent on expression of HrtAB. These results suggest that B. thuringiensis senses heme through HssRS and induces expression of separate membrane-localized systems capable of overcoming different aspects of heme toxicity.

Toxin stability improvement and toxicity increase against dipteran and lepidopteran larvae of Bacillus thuringiensis crystal protein Cry2Aa.

BACKGROUND: Bacillus thuringiensis δ-endotoxins are the most widely used biopesticides for controlling economically important crop pests and disease vectors. Improving their efficacy is of great benefit. Here, an improvement in Cry2Aa δ-endotoxin toxicity was attempted via a cry gene over expression system using P20 from B. thuringiensis israelensis. RESULTS: The coexpression of Cry2Aa with P20 resulted in a seven fold increase in its production yield in B. thuringiensis. Generated crystals proved to be significantly more toxic (505.207 µg g-1 , 1.99 mg L-1 and 1.49 mg L-1 ) than the P20-lacking control (720.78 µg g-1 , 705.69 mg L-1 and 508.51 mg L-1 ) against Ephestia kuehniella, Aedes aegypti and Culex pipiens larvae respectively. In vitro, processing experiments revealed a P20-mediated protection of Cry2Aa against degradation under larval gut conditions. Thus, P20 could promote the maintenance of a tightly packaged conformation of Cry2Aa toxins in the larval midgut upon correct activation and binding to its membrane receptors. CONCLUSION: Based on their resistance against excessive proteolysis, Cry2Aa δ-endotoxins, produced in the presence of P20, could be considered as a successful control agent for E. kuehniella and an effective alternative for mosquito control, implying its possible exploitation in pest management programmes. © 2016 Society of Chemical Industry.

The mechanism of uranium transformation from U(VI) into nano-uramphite by two indigenous Bacillus thuringiensis strains.

The mechanism of uranium transformation from U(VI) into nano-uramphite by two indigenous Bacillus thuringiensis strains was investigated in the present work. Our data showed that the bacteria isolated from uranium mine possessed highly accumulation ability to U(VI), and the maximum accumulation capacity was around 400 mg U/g biomass (dry weight). X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) analyzes indicated that the U(VI) was adsorbed on the bacterial surface firstly through coordinating with phosphate, CH2 and amide groups, and then needle-like amorphous uranium compounds were formed. With the extension of time, the extracellular crystalline substances were disappeared, but some particles were appeared in the intracellular region, and these particles were characterized as tetragonal-uramphite. Moreover, the disrupted experiment indicated that the cell-free extracts had better uranium-immobilization ability than cell debris. Our findings provided the understanding of the uranium transformation process from amorphous uranium to crystalline uramphite, which would be useful in the regulation of uranium immobilization process.

Consumption of Bt rice pollen containing Cry1C or Cry2A does not pose a risk to Propylea japonica (Thunberg) (Coleoptera: Coccinellidae).

As a pollen feeder, Propylea japonica would be directly exposed to Cry proteins in Bacillus thuringiensis (Bt)-transgenic rice fields. The effect of Cry1C- or Cry2A-containing transgenic rice pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, larval developmental time of P. japonica was significantly longer when fed pollen from Bt rice lines rather than control pollen but other life table parameters were not significantly affected. In the second experiment, P. japonica was not affected when fed a rapeseed pollen-based diet containing purified Cry1C or Cry2A at concentrations that were >10-times higher than in pollen, but P. japonica was affected when the diet contained E-64 as a positive control. In both experiments, the stability and bioactivity of the Cry proteins in the food sources and the uptake of the proteins by P. japonica were confirmed. The results show that P. japonica is not sensitive to Cry1C or Cry2A proteins; the effect observed in the first experiment was likely attributable to unknown differences in the nutritional composition of Bt rice pollen. Overall, the data indicate that the growing of Cry1C- or Cry2A-transgenic rice should pose a negligible risk to P. japonica.

Response surface methodology: optimisation of antifungal bioemulsifier from novel Bacillus thuringiensis.

An antifungal bioemulsifier compound was produced from a novel strain of Bacillus thuringiensis pak2310. To accentuate the production and as the first step to improve the yield, a central composite design (CCD) was used to study the effect of various factors like minimal salts (1X and 3X), glycerol concentration (2% and 4%), beef extract concentration (1% and 3%), and sunflower oil concentration (2% and 4%) on the production of bioemulsifier molecule and to optimize the conditions to increase the production. The E 24 emulsification index was used as the response variable as the increase in surfactant production was seen to be proportional to increased emulsification. A quadratic equation was employed to express the response variable in terms of the independent variables. Statistical tools like student's t-test, F-test, and ANOVA were employed to identify the important factors and to test the adequacy of the model. Under optimum conditions (1X concentration of minimal salts (MS), 2.6% glycerol (v/v), 1% beef extract (w/v), and 2% sunflower oil (v/v)) a 65% increase in yield was produced.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

Testing pollen of single and stacked insect-resistant Bt-maize on in vitro reared honey bee larvae.

The ecologically and economic important honey bee (Apis mellifera) is a key non-target arthropod species in environmental risk assessment (ERA) of genetically modified (GM) crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops.