Cry Endotoxin Research Articles

Field Evolved Resistance in Earias vittella (Lepidoptera: Noctuidae) From Punjab, Pakistan Against Commercial Formulations of Bacillus thuringiensis kurstaki.

Transgenic commercial cotton expressing Bacillus thuringiensis (Bt) Cry endotoxins or vegetative Vip toxins provide protection to cotton against bollworm attack. Continuous exposure of these targeted pests to cry toxins and to Bt commercial spray formulations has resulted in the development of resistance through natural selection. Spotted bollworm Earias vittella (Noctuidae: Lepidoptera) is considered to be one of the most destructive pests of cotton and okra crops in South Asia including Pakistan and has developed resistance to various synthetic insecticides. In the present study, the level of resistance in field populations of the spotted bollworm E. vittella against Bt Cry toxins has been evaluated for the first time. We collected twelve populations of E. vittella from three districts of Punjab, Pakistan for testing against four commercial Bt formulations containing different strains of B. thuringiensis subspecies kurstaki (Btk) with a range of Cry toxins. Low to high levels of resistance were found in the field populations compared with a laboratory-reared susceptible population of E. vittella (resistance ratios 6 to 111-fold). These results suggest that E. vittella has developed resistance against different Cry toxins after continuous exposure to Bt cotton in field. In order to prevent field control failures, regular insecticide resistance monitoring programs are required together with the use of integrated management approaches, including the use of Bt cotton varieties expressing two or more toxins to delay the development of resistance against Bt toxins in E. vittella.

The level of Cry1Ac endotoxin and its efficacy against H. armigera in Bt cotton at large scale in Pakistan

ABSTRACT A biophysical survey was conducted in 15 cotton-growing districts of Pakistan. Four hundred cotton growers were approached and inquired about the production technology of Bt cotton. Further, 25 strip tests using combo strips (Cry1Ac, Cry2Ab, Vip3Aa and Cp4, EPSPS gene) were performed at each farmer’s field. Out of 10,000 total-tested samples, farmers claimed 9682 samples as Bt and 318 samples as non-Bt. After performing a strip test, 1009 and 87 samples were found false negative and false positive, respectively. Only 53 samples were found positive for Cry2Ab, 214 for EPSPS and none for Vip3Aa gene. Quantification of Cry endotoxin and bioassay studies were performed by taking leaves from upper, middle, and lower canopies, and fruiting parts at approximately 80 days after sowing from 89 varieties. Expression was highly variable among different canopies and fruiting parts. Moreover, Cry endotoxin expression and insect mortality varied significantly among varieties from 0.26 µg g−1 to 3.54 µg g−1 with mortality ranging from 28 to 97%, respectively. Highest Cry1Ac expression (3.54 µg g−1) and insect mortality (97%) were observed for variety FH-142 from DG Khan. Cry endotoxin expression varied significantly across various plant parts, i.e., IUB-13 variety from upper canopy documented 0.34 µg g−1 expression with 37% insect mortality in Layyah to 3.42 µg g−1 expression and 96% insect mortality from DG Khan. Lethal dose, LD95 (2.20 µg g−1) of Cry1Ac endotoxin was optimized for effective control of H. armigera. Our results provided evidence of practical resistance in H. armigera and way forward.

Seven years of continuously planted Bt corn did not affect mineralizable and total soil C and total N in surface soil

Genetically engineered corn (Zea mays L.) containing a gene from the soil bacterium Bt (Bacillus thuringiensis) constitutes a large proportion of all corn planted in the United States. In a number of studies, Bt plant residues have been reported to have higher lignin content and to decompose slower than those of non-Bt plants, possibly due to the presence of the Cry endotoxin. We hypothesize that after multiple years of continuous cultivation of Bt corn, the combined results of alleged differences in Bt corn residues and of Cry endotoxin presence will be reflected in soil characteristics, specifically in an increase in soil total carbon (C) and nitrogen (N) levels. We collected soil samples at 0–7.5 cm depth in 2006 from continuous Bt and non-Bt corn treatments in an randomized complete block design experiment with four replications. The experiment was established in 1999 at the Kellogg Biological Station long-term ecological research (LTER) site in southwest Michigan. We found that, after 7 years of Bt cropping, neither total soil C and N nor soil C mineralized during a 35-day incubation were significantly different between Bt and non-Bt corn treatments (P > 0.05). Total soil C was equal to 7.3 g kg−1 and 7.4 g kg−1, in Bt and non-Bt corn, respectively, with a standard error of the means (SEM) = 0.2, and total N was 0.67 g kg−1 (SEM 0.02) in both treatments. Post-hoc power analysis indicated that, given the number of samples collected in this study and the observed level of variability, the minimal differences between the Bt and non-Bt treatments that could be detected as statistically significant at α = 0.05 with a power of 0.80 were equal to 1.0 g kg−1, 0.14 g kg−1 and 0.125 g kg−1 for total C, total N, and soil C mineralized during a 35-day incubation, respectively. The results indicate that continuous Bt corn production during a 7-year period did not lead to sizeable changes in total soil C and N.

Assessment of Chrysoperla plorabunda Longevity, Fecundity, and Egg Viability When Adults Are Fed Transgenic Bt Corn Pollen

Abstract The widespread planting of transgenic corn containing Bacillus thuringiensis (Bt) Cry endotoxin in its tissues for insect pest control raises the potential for influence on many non-target species including pollen-feeding species of Chrysopidae. This study was conducted to assess fitness parameters associated with longevity, fecundity, and egg viability of adult Chrysoperla plorabunda (Fitch) (Neuroptera: Chrysopidae) when fed Bt corn pollen. Bt products tested with their respective non-Bt near-isolines were Event 176 (Cry1Ab), MON810 (Cry1Ab), and TC1507 (Cry1F). Females fed pollen from Event 176 lived significantly longer than those fed pollen from its non-Bt near-isoline. Males fed pollen from TC1507 showed a trend for living longer than males fed its non-Bt near-isoline pollen, but there was no difference for females regarding this event. The mean number of eggs produced per female per day was significantly less for those fed MON810 pollen compared with females fed pollen from the non-Bt near...

Ingestion and excretion of two transgenic Bt corn varieties by slugs

The release of transgenic Bacillus thuringiensis (Bt) corn expressing various Cry endotoxins has raised concern that these endotoxins are disseminated in the food web and may adversely affect non-target beneficial organisms, such as predators and organisms of the decomposer food web. We therefore investigated in a laboratory study, whether the Cry1Ab and Cry3Bb1 protein from Bt corn could potentially be transferred to such organisms by measuring the Cry protein content in the two common agricultural slug pests Arion lusitanicus and Deroceras reticulatum and their feces. We measured Cry1Ab and Cry3Bb1 protein concentration in leaves, intestines, and feces of corn leaf-fed slugs using ELISA and determined how much of the ingested protein is excreted by the slugs. Cry3Bb1 concentration in leaves of DKC5143Bt corn was significantly higher than Cry1Ab concentration in leaves of N4640Bt corn. While slugs were feeding on corn leaves, the Cry3Bb1 and Cry1Ab proteins were found in intestines and feces of both slug species. Bt protein concentrations in intestines of Cry3Bb1 corn-fed slugs were in both slug species higher than in Cry1Ab corn fed slugs, whereas no differences between Cry3Bb1 and Cry1Ab protein in feces were found. After slugs had ceased feeding on Bt corn, Cry1Ab was detectable in fresh slug feces for a significantly longer time and often in higher amounts than the Cry3Bb1. Our results indicate that both Cry proteins are likely to be transferred to higher trophic levels and to the decomposer food web. Since different Bt proteins seem to vary in their degradation, they have different transfer probabilities. This should be considered in risk assessments for non-target arthropods.

Bt maize and mycotoxins: the current state of research

The commercialisation of Bt (Bacillus thuringensis) maize expressing the Cry endotoxin specific for some types of Lepidopteran and Coleopteran pests has lead researchers to study the reduction in mycotoxin concentrations in Bt hybrids compared to the correspondent isogenic plants. Indeed, insect damage is one of the main inoculation pathways of mycotoxinogenic moulds. The present study aims to evaluate, according to the scientific literature published to date, the real efficiency of Bt maize hybrids in reducing the mycotoxin problem. The results obtained from the analysis of the literature do not show significant variations in the content of aflatoxins, zearalenone and trichothecenes, between Bt hybrids and corresponding isogenic control plants. The only mycotoxins where Bt hybrids have any effect are the fumonisin group, but even in this case studies in commercially planted fields have shown that their effect is mitigated by many biotic and abiotic factors.

Structure of the Functional Form of the Mosquito Larvicidal Cry4Aa Toxin from Bacillus thuringiensis at a 2.8-Angstrom Resolution

The Cry4Aa delta-endotoxin from Bacillus thuringiensis is toxic to larvae of Culex, Anopheles, and Aedes mosquitoes, which are vectors of important human tropical diseases. With the objective of designing modified toxins with improved potency that could be used as biopesticides, we determined the structure of this toxin in its functional form at a resolution of 2.8 angstroms. Like other Cry delta-endotoxins, the activated Cry4Aa toxin consists of three globular domains, a seven-alpha-helix bundle responsible for pore formation (domain I) and the following two other domains having structural similarities with carbohydrate binding proteins: a beta-prism (domain II) and a plant lectin-like beta-sandwich (domain III). We also studied the effect on toxicity of amino acid substitutions and deletions in three loops located at the surface of the putative receptor binding domain II of Cry4Aa. Our results indicate that one loop is an important determinant of toxicity, presumably through attachment of Cry4Aa to the surface of mosquito cells. The availability of the Cry4Aa structure should guide further investigations aimed at the molecular basis of the target specificity and membrane insertion of Cry endotoxins.

Soil microbial communities associated with Bt and non-Bt corn in three soils.

The effects of expression of Cry endotoxin by Bt corn (transgenic corn engineered to express Bacillus thuringiensis toxin) on soil microbial community structure were assessed in a growth chamber experiment. Two lines of transgenic corn expressing different Cry endotoxins were compared with their respective non-transgenic isolines in three soil types with differing textures. Phospholipid fatty acid (PLFA) profiles from bulk soil and community-level physiological profiles (CLPP) from the rhizosphere community were used to assess community structure. Differences in PLFA profiles due to soil type were significant, accounting for 73% of the total variability in the dataset. Differences in bacterial and fungal CLPP profiles due to soil type were statistically significant, but probably not biologically important, accounting for 6.3 and 3.8% of the total variability, respectively. Neither expression of Cry endotoxin nor corn line had a significant effect on microbial profiles, except in the high-clay soil where both factors significantly affected bacterial CLPP profiles (accounting for 6.6 and 6.1% of the variability in that soil, respectively). Expression of Cry endotoxin also significantly reduced the presence of eukaryotic PLFA biomarker in bulk soils, although it is unclear which groups of eukaryotes were affected. We conclude that the effects of transgenic Bt corn in this short-term experiment are small, and longer-term investigations are necessary.

Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence

Previously we demonstrated that the yield of Cry3A (70 kDa) can be increased as much as 10-fold when cry3A including its upstream STAB-SD mRNA stabilizing sequence is expressed in Bacillus thuringiensis under the control of cyt1A promoters. To determine whether the cyt1A promoters/STAB-SD combination ( cyt1AP/STAB) has broader applicability, we used it to synthesize two other Cry endotoxins in the 70-kDa mass range, Cry2A and Cry11A. Combination of cyt1AP/STAB with orfs 2 and 3 of the cry2A operon yielded about 4.4-fold the amount of Cry2A obtained with the wild-type cry2A operon. The yield of Cry11A obtained with a construct that contained the cyt1AP/STAB, cry11A and the 20-kDa protein gene was 1.3-fold the amount obtained with a construct similar to the wild-type operon. These results demonstrate that the cyt1AP/STAB combination can enhance synthesis of different Cry proteins significantly, but that the level of enhancement varies with the specific protein synthesized.