Thuringiensis Insecticidal Crystal Proteins Research Articles

Genetic transformation of common beans (Phaseolus vulgaris L.) through Agrobacterium tumefaciens carrying Cry1Ab gene.

Seed beetles are one of the most important causes of yield loss in bean production. It is essential to develop resistant varieties in the fight against these pests. Agrobacterium-based gene transformation is the most widely used breeding method worldwide to develop insect-resistant varieties. Embryonic axes and plumule explants were obtained from Agrobacterium tumefciens treated mature zygotic embryos of low and high raw protein-based common bean cultivars Akman 98 and Karacaşehir 90. Agrobacterium tumefaciens contained a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt Cry1Ab) controlled by the 35S promoter and NOS terminator sequences. The transformation event was genotype and explant dependent. The plumule explants could not withstand kanamycin-based selection pressure and died. It was possible to get two transgenic plants using embryonic axis explants of low protein cultivar Akman 98. These results were validated using GUS analysis, PCR, RT-PCR, bioassay analysis, and ELISA test from the samples taken from T0 and T1 generations. Bioassay tests showed that these plants were protected from the damage of legume seed insects (Bruchus spp.). The results are very encouraging and may help in producing better transgenic common bean germplasm leading to safe agriculture and reducing environmental pollutions.

The responses of soil nematode communities to Bt maize cultivation at four field sites across Europe

Transgenic maize expressing the Bacillus thuringiensis (Bt) insecticidal crystal (Cry1Ab) protein is poisonous to lepidopterans including the European Corn Borer (Ostrinia nubilalis). In many European countries, commercial cultivation of Bt maize is not allowed. One major reason is the potential variation of the environmental risk across different biogeographical regions. The aim of this study was to collect data about soil nematode communities as bioindicators of unintended effects across geographically diverse maize growing regions in Europe by sampling field sites in Denmark, Slovakia, and Sweden during 2013–2014, and in Spain during 2013. DKC3872YG (Bt maize line MON810) and its near-isogenic line DKC3871 were grown at the sites in Slovakia, Denmark, and Sweden and hybrids DKC6451YG (Bt maize line MON810) and its near-isogenic line DKC6450 were cultivated at the site in Spain. Dominating nematode genera in the maize fields regardless of the field site or maize variants were bacterial feeders Rhabditis, Acrobeloides; root-fungal feeders Filenchus; fungal feeders Aphelenchoides, Aphelenchus; and omnivores Eudorylaimus. A significant effect of the field site location on the total nematode abundance, nematode abundance in trophic groups, diversity of nematode genera, and ecological and functional nematode indices was detected. Significant annual variation was found in the Plant parasite and Structure indices. There were significant differences in the abundances of omnivores and root-fungal feeders and in the Maturity, Channel, and Enrichment indices between Bt and non-Bt maize plots in Denmark in 2013, and in the abundance of fungal feeders in Sweden (2013). On the other hand, no difference was found between the Bt and non-Bt plots at the sites in Spain and Slovakia or at any of the sites in 2014. The effect of the field site location and season on the soil nematode community was more pronounced than that of the Bt genetic modification. We conclude that Bt maize had only a limited or no effect on soil nematode communities.

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T0 transformants were selected on the basis of PCR and protein expression profile. T1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T0, T1, and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

Cloning, Expression, Purification, and Insecticidal Activity of a Novel Cry1Na3 Toxin From Bacillus thuringiensis BRC-ZYR2.

Bacillus thuringiensis produces a variety of insecticidal crystal proteins (ICPs). Genome sequencing is a promising strategy for detecting and identifying B. thuringiensis ICPs, which are of great interest to the biocontrol field. In this study, a novel ICP gene was cloned from B. thuringiensis BRC-ZYR2 based on genomic data from 454 GS-FLX Titanium sequencing and an analysis of the results using the B. thuringiensis Toxin_Scanner ( http://bcam.hzaubmb.net/BtToxin_scanner/index.php ). cry1Na3 designated by the B. thuringiensis Toxin Nomenclature Committee, encoded a 601-amino acid, 68.0-kDa protein that exhibited 95% identity with Cry1Na1 and 99% identity with Cry1Na2. Cry1Na3 contained three conserved domains commonly found in three-domain ICPs. Cry1Na3 was toxic to Plutella xylostella (L.) and Ostrinia furnacalis (Guenée), with LC 50 values of 3.69 μg/ml and 31.30 μg/ml, respectively. However, Laodelphax striatellus (Fallén) nymphs were unaffected when fed purified Cry1Na3 (250 μg/ml) in their diet. Spodoptera exigua (Hübner) and Colaphellus bowringi (Baly) larvae survived even when the concentration of Cry1Na3 protein reached 500 μg/ml. Cry1Na3 is a promising agent for the control of lepidopteran insect pests.

Crystal structure of Cry51Aa1: A potential novel insecticidal aerolysin-type β-pore-forming toxin from Bacillus thuringiensis

The structures of several Bacillus thuringiensis (Bt) insecticidal crystal proteins have been determined by crystallographic methods and a close relationship has been explicated between specific toxicities and conserved three-dimensional architectures. In this study, as a representative of the coleopteran- and hemipteran-specific Cry51A group, the complete structure of Cry51Aa1 protoxin has been determined by X-ray crystallography at 1.65 Å resolution. This is the first report of a coleopteran-active Bt insecticidal toxin with high structural similarity to the aerolysin-type β-pore forming toxins (β-PFTs). Moreover, study of featured residues and structural elements reveal their possible roles in receptor binding and pore formation events. This study provides new insights into the action of aerolysin-type β-PFTs from a structural perspective, and could be useful for the control of coleopteran and hemipteran insect pests in agricultures.

Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exigua

Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to “pyramid” two or more Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops.

English

Stem borers (Chilo partellus) are important field insect pests of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) in Africa. They account for more than 30% yield losses depending on the composition of the pest community. C. partellus larvicide like Bacillus thuringiensis have been widely and effectively used in C. partelluscontrol programs, but the industrial production of theses bacilli is expensive. Here we have attempted to develop three cost-effective media, based on legumes, potato, and whey. Growth and production of the insecticidal proteins from these bacteria were satisfactory; protein concentration yields of 27.60 mg/ml, spore counts of 5.60 × 108 CFU/ ml and first-instar Chilo partellus larvicidal activity (LC50) of 78 µg/l were obtained with a 72 h culture of this bacterium. Therefore, this investigation suggests that legume, potato and whey-based culture media are more economical and effective for the industrial production of B. thuringiensis insecticidal crystal proteins. Key words: Bacillus thuringiensis, larvicidal, Chilo partellus, insecticidal crystal proteins, LC50, optical density,sulphate polyacrylamide gel electrophoresis (SDS-PAGE), spore counts.

The specific responses of Acari community to Bt cotton cultivation in agricultural soils in northern China

Transgenic cotton expressing the Bacillus thuringiensis (Bt) insecticidal crystal (Cry1A) protein effectively controls the cotton bollworm and thus has been planted extensively in China. However, the large-scale release of Bt cotton may have undesirable effects on soil fauna due to Bt protein accumulation and the pleiotropic effects of genetic manipulation. A survey of soil mites was carried out monthly for two consecutive years (2009–2010) in Bt and non-Bt cotton fields. The soil mites were extracted using modified Tullgren funnels and were identified to the genus level, when possible. The results suggested that the effects of Bt cotton on soil mite community size were time dependent and taxonomic group specific. The cumulated abundance over a year was always higher in non-Bt fields for Oribatida; this effect was statistically significant in 2010 for Prostigmata and Astigmata. The changes in the community variables tested were similar between Bt and non-Bt cotton fields in 2009, whereas the taxonomic group richness, Shannon–Weaver index and evenness index were significantly different between Bt and non-Bt cotton fields in 2010. Additionally, sharp inter-annual fluctuations in the community composition of the soil mites were found, accompanied with the replacement of some taxonomic groups. Finally, the dominances of some taxonomic groups were significantly different between Bt and non-Bt cotton fields. Bt cotton cultivation fostered Laelapidae populations while inhibited Tectocepheus abundance in 2009. However, Bt cotton cultivation negatively impacted the abundances of Scheloribates and Nothrus in 2010. In conclusion, Bt cotton cultivation exerted specific impacts on soil mites.

Fusing the vegetative insecticidal protein Vip3Aa7 and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvae

Bacillus thuringiensis insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) have been widely used as a kind of safe bio-insecticides. A problem that has been of concern worldwide is how to improve their insecticidal activities. In this study, to determine the synergism between VIPs and ICPs effect on insecticidal activity, a construct that produces a chimeric protein of the Vip3Aa7 and the N terminus ofCry9Ca, named V3AC9C, was expressed in Escherichia coli BL21 cells. In additional experiments, the V3AC9C chimeric protein, the single Vip3Aa7, and the single N terminus of Cry9Ca were treated with trypsin. SDS-PAGE showed that the V3AC9C could be processed into two single toxins. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the chimeric protein was markedly better than either of the single toxins. Interestingly, the toxicity of the chimeric protein was 3.2-fold higher than a mixture of the Vip3Aa7 and Cry9Ca toxins (mass ratio of 1:1). The synergism factor (SF) of chimeric protein containing Vip3Aa7 and Cry9Ca was calculated to be 4.79. The SF in mixture of toxins is only 1.46. Hence, the effect was more than the sum of the Vip3Aa7 and Cry9C activities. Analysis of the protein's solubility showed that the Vip3Aa7 helped the N terminus of Cry9Ca to dissolve in an alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly resulted from this increase in solubility. These results lay a foundation for the development of a new generation of bio-insecticides and multi-gene transgenic plants.

Genetically modified Bt maize lines containing cry3Bb1, cry1A105 or cry1Ab2 do not affect the structure and functioning of root-associated endophyte communities

Transgenic maize expressing Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins may contribute to a decreased need for chemical pesticides, providing benefits for human health and the environment. However, there is public concern regarding the environmental safety of transgenic crops, which relates also to potential adverse effects on plant-microbe interactions by affecting the plant-associated, endophytic microflora. Thus, we compared the endophytic bacterial communities in plants of the three transgenic Bt maize lines MON89034 (cry3Bb1), MON88017 (cry1A.105 and cry2Ab2) and the stacked event MON88017xMON89034 (cry1A.105 and cry2Ab2, cry3Bb1), with those of the respective near-isogenic line, and with those in plants of three additional, conventional maize lines. The maize plants were grown in a containment system on two different soils which were commonly used for maize cultivation in Lower Austria.We obtained 700 bacterial endophytes and characterized them regarding their phylogenetic diversity and specific plant growth promoting functions, including the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and indole-3-acetic acid (IAA). Both the soil environment and the plant cultivar had an effect on the phylogenetic diversity of the endophytic communities, but there were no specific effects of the transgenic varieties. Diversity measures of endophytic isolates were not different in Bt- versus non-Bt maize varieties. A variable number of isolates showed ACC deaminase activity irrespective of the soil and plant cultivar. Generally, the number of high IAA producers appeared to be affected by the soil environment but not by the plant cultivar. Cultivation-independent profiling via terminal restriction fragment polymorphism (T-RFLP) analysis of the 16S rRNA gene revealed that maize root endophytes were differentially composed dependent on soil type and plant cultivar, but they were not discriminated with respect to Bt- versus non-Bt varieties. In conclusion, maize endophyte communities studied were highly diverse and showed a high potential for plant growth promotion, emphasizing their importance in modern agricultural systems irrespective of the maize cultivar, including Bt-modifications.

Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons

A reference plasmid, pMD-CCS, was constructed to detect multiple target genes in the insect resistant cotton (Gossypium hirsutum L.) varieties in China. This plasmid contained cowpea trypsin inhibitor (CpTI), Bacillus thuringiensis insecticidal crystal protein (cry1A), and cotton endogenous gene Stearoyl-acyl carrier protein desaturase (Sad1) sequences targeting the key exogenous gene types. The real-time quantitative PCR methods for CpTI and cry1A were established using pMD-CCS as the reference material (RM). The dosagess of CpTI and cry1A from 9 insect resistant cotton varieties were determined. The average CpTI dosages were 0.018–0.020 copies per genome and the average cry1A dosages were 1.377–2.136 copies per genome in the 3 insect resistant cotton varieties including Kemian 3. The average cry1A dosages were 0.887–2.564 copies per genome in 6 varieties including Ezamian 1-F1. The standard deviations of the quantitative measurement ranged from 0.001 to 0.049. These results demonstrated that pMD-CCS could be used as the RM for the quantitative measurement of CpTI and cry1A genes in insect resistant cotton varieties.

Advances in effects of insecticidal crystal proteins released from transgenic Bt crops on soil ecology

With the large scale cultivation of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystal proteins in the world, the problem of environmental safety caused by these Bt crops has received extensive attention. These insecticidal crystal proteins can be released into the soil continuously in the growing period of Bt plants. If their accumulation of the insecticidal crystal proteins exceeds consumption by insect larvae and degradation by the environmental factors, these insecticidal crystal proteins could constitute a hazard to non-target insects and soil microbiota. There are three main ways to release insecticidal crystal proteins into soil for Bt plants: root exudates, pollen falling, and crop reside returning. The Bt insecticidal crystal proteins released into soil can be adsorbed rapidly by active soil particles and the absorption equilibrium attained within 1-3 h. The adsorption protects Bt insecticidal crystal proteins against soil microbial degradation or enzyme degradation, which leads to remarkable prolong of the persistence of insecticidal activity. The change of soil microorganism species is an important index for evaluating the effect of Bt plants on soil ecology. The research showed that these insecticidal crystal proteins released by the Bt plant root exudates or Bt organism had no toxicity to the soil earthworms, nematodes, protozoa, bacteria and fungi; however, it could reduce the mycelium length of the arbuscular mycorrhizal fungi (AMF) and restrain AMF to form invasion unit. The influencing degree of Bt protein on soil enzyme activity varied with the releasing modes or growth period of Bt crops. Bt Cry1Ab protein can be taken up from soil by parts of following crops; however, different results were obtained with different commercial kits. To better understand the soil ecological evaluation about the insecticidal crystal proteins released from transgenic Bt crops, this review provides a comprehensive overview about the release, adsorption and residue of Bt insecticidal crystal proteins in soil, as well as their effects on soil protozoa, soil microorganism, soil enzyme activity and following crops.

New Vector pBUC3 can Express <i>Bt</i> <i>Cry1Ac31</i> during the Vegetative Phase in <i>Bacillus thuringiensis</i>

The feature of Bacillus thuringiensis ( B. t .) is the ability to generate parasporal crystal protein during the spore-forming process. In order to realize the expression of the Bt Cry genes independent on spore formation, we attempted to design new vector that could express Bacillus thuringiensis insecticidal crystal protein in the vegetable growth phase in this research. First, we designed a pair of specific primers based on cry 3Aa promoter gene sequence to amplify cry3Aa promoter from B.t. subsp. Tenebrions ; then the DNA fragment with blunt-end was ligated to the cross-species vector pBU4 to build new pBUC3 vector which would be non-dependent sporulation vector expressing in the vegetative growth phase of Bacillus thuringiensis . In order to validate the vector whether express B.t cry gene in the vegetative phase, The B.t cry1Ac31 gene was ligated to the pBUC3 vector to be transformed into B.t non crystal mutant IPS strain by electronic transformation approach. Through the examinations by the PCR, microscopic observation and SDS-PAGE, the results showed that B.t cry1Ac31 was expressed in cell growth vegetable phase. Our study suggested that the feature of cry 3Aa promoter being non-dependent on spore formation might be useful expression element that could achieve the cry gene of Bacillus thuringiensis expressing in vegetative phase.

Enhancing Cry1Ac toxicity by expression of the Helicoverpa armigera cadherin fragment in Bacillus thuringiensis

Insect cadherin proteins localized in the midgut epithelium were identified as receptors for Bacillus thuringiensis insecticidal crystal proteins (Cry toxins). These cadherins facilitated toxin monomer oligomerization and mediated oligomer binding to secondary receptors. It has been reported that Manduca sexta, Helicoverpa armigera, Anopheles gambiae and Diabrotica virgifera cadherin toxin binding regions function as synergists for Cry1A, Cry4Ba and Cry3A toxicity against target insects. In the present study, the toxin binding region fragment of the H. armigera cadherin (hacad1) gene was cloned and fused with the promoter of the cry3Aa gene. The fusion gene pro3Aa-hacad1 and the cry1Ac gene were inserted into shuttle vector pHT304 and introduced into B. thuringiensis acrystalliferous strain BMB171 for coexpression (resulting in recombinant strain BMB1073). SDS-PAGE and mass spectrum analysis showed that BMB1073 could express HaCad1 and Cry1Ac proteins together. Bioassay results demonstrated that insecticidal activities against H. armigera and Spodoptera exigua could be increased 5.1-fold and 6.5-fold, respectively, by BMB1073 compared with the strain which can only express the Cry1Ac protein. Our discovery showed that coexpression of HaCad1 and Cry1Ac toxin in B. thuringiensis enhanced the insecticidal activity of Cry1Ac toward Lepidoptera insects. This finding also revealed a novel strategy for engineering strains and transgenic plants with higher insecticidal activity.

Characterisation and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain JF19-2

The Bacillus thuringiensis isolate JF19-2, a native organism found in soil samples from the Sichuan Basin in western China, contains a bipyramidal crystal harbouring one insecticidal crystal protein (about 70 kDa). Interestingly, a novel cry2A-type gene, encoding a 635 amino acid protein with 92% homology to cry2Ab1, was found and cloned from this strain. According to the nomenclature principles adopted for B. thuringiensis insecticidal crystal proteins, this gene represents a novel haplotype cry gene and was designated as cry2Ag1 by the Bacillus thuringiensis Pesticide Crystal Protein Nomenclature Committee. Cry2Ag1 was expressed in Escherichia coli BL21(DE3)pLysS cells, and the results of insecticidal activity assays showed that Cry2Ag1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. These results strongly suggest that the cloning of the cry2Ag1 gene is not only of interest to researchers studying insecticidal crystal genes, but may ultimately serve as a solution to the increasing resistance of pests to currently used insecticides.

Helicoverpa armigera cadherin fragment enhances Cry1Ac insecticidal activity by facilitating toxin-oligomer formation

The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity. It has been reported that Manduca sexta cadherin fragment CR12-MPED and Anopheles gambiae cadherin fragment CR11-MPED enhance the insecticidal activity of Cry1Ab and Cry4Ba to certain lepidopteran and dipteran larvae species, respectively. This study reports that a Helicoverpa armigera cadherin fragment (HaCad1) containing its toxin binding region, expressed in Escherichia coli, enhanced Cry1Ac activity against H. armigera larvae. A binding assay showed that HaCad1 was able to bind to Cry1Ac in vitro and that this event did not block toxin binding to the brush border membrane microvilli prepared from H. armigera. When the residues (1423)GVLSLNFQ(1430) were deleted from the fragment, the subsequent mutation peptide lost its ability to bind Cry1Ac and the toxicity enhancement was also significantly reduced. Oligomerization tests showed that HaCad1 facilitates the formation of a 250-kDa oligomer of Cry1Ac-activated toxin in the midgut fluid environment. Oligomer formation was dependent upon the toxin binding to HaCad1, which was also necessary for the HaCad1-mediated enhancement effect. Our discovery reveals a novel strategy to enhance insecticidal activity or to overcome the resistance of insects to B. thuringiensis toxin-based biopesticides and transgenic crops.

Current Patents Related to Bacillus thuringiensis Insecticidal Crystal Proteins

This work categorizes a number of patents related to Bacillus thuringiensis insecticidal crystal proteins. The patents are classified into groups according to the type of toxins appearing in the claims. The purpose of the summary is to promote the application of B. thuringiensis insecticidal crystal proteins and the development of patentable technologies.

A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase

Bacillus thuringiensis insecticidal crystal proteins bind to cell-surface receptors which represent a family of aminopeptidases [APN (aminopeptidase N)] present on the brush border membrane of insect midgut cells of susceptible insects leading to pore formation and death of the insect. We report here for the first time the presence of a novel APN in the fat body of the moth Achaea janata. Northern blotting detected at least one APN-specific transcript in the fat body, whereas two transcripts of different sizes were detected in the midgut. We have cloned two full-length APN cDNAs of 3015 bp and 2850 bp from fat body and midgut respectively, which encode proteins of 1004 and 950 amino acids. These two APNs share only 33% amino acid sequence identity, but both display the typical APN features, such as the N-terminal signal peptide, several putative glycosylation sites, C-terminal glycosylphosphatidylinositol anchor signal, the APN-specific zinc-binding/gluzincin motif HEXXHX(18)E and gluzincin motif GAMENWG. The fat body APN manifested a variation in its expression with respect to tissue and developmental stage. In spite of the abundance of the APN transcript in the fat body, fairly low APN activity was detected in this tissue. The fat-body- and midgut-specific APNs showed differential interaction with various Cry1A toxins. Besides, the level of toxicity of different Cry subtypes varied enormously with mode/site of delivery, such as intrahaemocoelic injections and feeding bioassays. These data indicate that the fat body might be a potential alternative Cry toxin target site in the moth.

Inheritance and cross-resistance of Bacillus thuringiensis insecticidal crystal protein Cry1Ac resistance in cotton bollworm Helicoverpa armigera Hubner population from Tamil Nadu, India

Cotton bollworm, Helicoverpa armigera Hubner selected for five generations with Bacillus thuringiensis insecticidal crystal protein Cry1Ac in the laboratory developed 12.98-fold resistance. Resistance and susceptible populations were mass crossed to study the dominance of resistance. The Cry1Ac—selected (BCR) population showed 5.8-fold resistance to Cry1Aa and 5.04-fold resistance to Cry1Ab. The degree of dominance (D) was 0.34 and 0.40 for the R × S and S × R hybrids, respectively, which indicates incomplete recessive character of Cry1Ac resistance in the population. The estimated realized heritability (h2) and response quotient (Q) of resistance for Cry1Ac were 0.52 and 0.15, respectively. This indicated the lower phenotypic variation in the selected population. The resistance risk assessment based on h2 indicated that the resistance would increased tenfold after <9 generations for Cry1Ac in the resistant population. The results show the ability of H. armigera to develop resistance against Cry1Ac and cross-resistance to Cry1Aa and Cry1Ab.

Changes in Permeability of Brush Border Membrane Vesicles from Spodoptera littoralis Midgut Induced by Insecticidal Crystal Proteins from Bacillus thuringiensis

Bacillus thuringiensis insecticidal crystal proteins (ICPs) are thought to induce pore formation in midgut cell membranes of susceptible insects. Cry1Ca, which is significantly active in Spodoptera littoralis, made brush border membrane vesicles permeable to KCl (osmotic swelling was monitored by the light scattering technique); the marginally active ICPs Cry1Aa, Cry1Ab, and Cry1Ac did not.