Cry1Ab Toxin Research Articles

Toxicity of Bacillus thuringiensis-Derived Pesticidal Proteins Cry1Ab and Cry1Ba against Asian Citrus Psyllid, Diaphorina citri (Hemiptera).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera), is an important pest of citriculture. The ACP vectors a bacterium that causes huanglongbing (HLB), a devastating and incurable disease of citrus. The bacterium Bacillus thuringiensis (Bt) produces multiple toxins with activity against a diverse range of insects. In efforts to provide additional control methods for the ACP vector of HLB, we identified pesticidal proteins derived from Bt for toxicity against ACP. The trypsin proteolytic profiles of strain-derived toxins were characterized. Strain IBL-00200, one of six strains with toxins shown to have basal activity against ACP was selected for liquid chromatography-mass spectrometry (LC-MS/MS) identification of the individual Cry toxins expressed. Toxicity assays with individual toxins derived from IBL-00200 were then performed. The activated form of the Cry toxins Cry1Ab and Cry1Ba were toxic to ACP with LC50 values of approximately 120 µg/mL. Disruption of the midgut epithelium was associated with the toxicity of both the IBL-00200-derived toxin mixture, and with Cry1Ba. With further optimization of the efficacy of Cry1Ab and Cry1Ba, these toxins may have practical utility against ACP. Bt toxins with activity against ACP may provide an additional tool for management of ACP and the associated HLB disease, thereby providing a more sustainable and environmentally benign approach than repeated application of broad-spectrum insecticides.

Effect of Transgenic Maize on Abundance of the Corn Flea Beetle,Chaetocnema pulicariaMelsheimer, as a Non-Target Pest

Effect of transgenic maize Agrisure® VipteraTM 3111 event with Cry1Ab and Vip3Aa20 toxins of Bacillus thuringiensis Berliner that provide resistance to Lepidoptera and mCry3A to Coleoptera was tested on abundance of the corn flea beetle, Chaetocnema pulicaria Melsheimer, at Sinaloa, Mexico and compared to isoline maize with and without insecticide applied. Ten randomly selected plants were carefully inspected every 2 weeks, and abundance, frequency, and fluctuation of the pest were recorded. Abundance of C. pulicaria during crop development was a total of 2,239 insects on the three hybrids at both locations and did not differ among the hybrids. One-hundred percent frequency was found throughout the growing season on the three hybrids. The study showed that transgenic maize expressing Bt toxins did not negatively affect the abundance of C. pulicaria.

Impact of Bt cotton expressing single (Cry1Ac) and dual toxins (Cry1Ac and Cry2Ab) on the fitness of the predator Chrysoperla zastrowi sillemi (Esben-Petersen): prey-mediated tri-trophic analysis

Transgenic Bt cotton with genes from soil inhabiting spore forming bacterium Bacillus thuringiensis Berliner produces δ-endotoxin for the control of lepidopteran insects. The prey-mediated effects of Cry protein on the third trophic level is the most realistic exposure pathway that needs to be addressed as an important component of environment risk assessment. The green lacewing, Chrysoperla zastrowi sillemi (Esben-Petersen) (Neuroptera: Chrysopidae) is the most important generalist predator in the cotton ecosystem in India. The tri-trophic interactions involving Bt cotton expressing single (Cry1Ac) and dual toxins (Cry1Ac and Cry2Ab) fed herbivores, i.e. mealybug, Phenacoccus solenopsis Tinsley, whitefly Bemisia tabaci (Gennadius) and leafhopper, Amrasca biguttula biguttulla (Ishida) on the fitness of C. zastrowi sillemi, were studied. The development, survival and body weight of C. zastrowi sillemi had no deleterious effect as there were insignificant differences in any of the studied fitness parameters regardless of having consumed prey fed on Bollgard, Bollgard II and non-Bt cotton plants. The feeding potential of C. zastrowi sillemi on mealybug was also not different on Bt or non-Bt cotton plants. ELISA studies confirmed the presence of Cry proteins in Bt cotton leaves; however, no Cry1Ac or Cry2Ab protein was detected in prey herbivores (P. solenopsis, B. tabaci and A. biguttula biguttula) or in the predator C. zastrowi sillemi. It could be concluded that transgenic cotton that expresses single (Cry1Ac) or dual (Cry1Ac and Cry2Ab) toxins had no apparent effect on the fitness of the predator through its preys P. solenopsis, B. tabaci and A. biguttula biguttulla.

The C-terminal protoxin region of Bacillus thuringiensis Cry1Ab toxin has a functional role in binding to GPI-anchored receptors in the insect midgut

Bacillus thuringiensis Cry toxins are used worldwide for controlling insects. Cry1Ab is produced as a 130-kDa protoxin that is activated by proteolytic removal of an inert 500 amino-acid-long C-terminal region, enabling the activated toxin to bind to insect midgut receptor proteins, leading to its membrane insertion and pore formation. It has been proposed that the C-terminal region is only involved in toxin crystallization, but its role in receptor binding is undefined. Here we show that the C-terminal region of Cry1Ab protoxin provides additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) insect receptors. ELISA, ligand blot, surface plasmon resonance, and pulldown assays revealed that the Cry1Ab C-terminal region binds to both ALP and APN but not to cadherin. Thus, the C-terminal region provided a higher binding affinity of the protoxin to the gut membrane that correlated with higher toxicity of protoxin than activated toxin. Moreover, Cry1Ab domain II loop 2 or 3 mutations reduced binding of the protoxin to cadherin but not to ALP or APN, supporting the idea that protoxins have additional binding sites. These results imply that two different regions mediate the binding of Cry1Ab protoxin to membrane receptors, one located in domain II-III of the toxin and another in its C-terminal region, suggesting an active role of the C-terminal protoxin fragment in the mode of action of Cry toxins. These results suggest that future manipulations of the C-terminal protoxin region could alter the specificity and increase the toxicity of B. thuringiensis proteins.

Densidades y Daños de Insectos Plaga en Algodonero Convencional y Bt en la Comarca Lagunera, México

The genetically modified cotton expressing Cry toxins from the bacteria Bacillus thuringiensis (transgenic or Bt cotton) was introduced to Mexico in 1996, achieving a high degree of adoption by cotton producers, due to its high effectiveness against lepidopteran pests, which resulted in a reduction of the use of insecticides and a significant increase in crop yields. This study was conducted to determine and compare the populations and levels of damage of the main insect pests in commercial conventional (non-Bt cotton) and transgenic cotton (Bt cotton) fields in the Comarca Lagunera, Mexico during 2016, after 20 years of planting Bt cotton. In four fields of each type of cotton samplings were carried out from June 30 to August 27 to evaluate densities and damages of bollworm, Helicoverpa zea (Boddie); beet armyworm, Spodoptera exigua (Hubner); pink bollworm, Pectinophora gossypiella (Saunders); boll weevil, Anthonomus grandis (Boheman); conchuela, Chlorochroa ligata (Say); and sweetpotato whitefly, Bemisia tabaci (Gennadius). Bollworm and beet armyworm populations were significantly lower in Bt cotton than in conventional cotton, with reductions from 70-100% and 90-98%. Damage to squares and bolls by these two pests were lower in Bt cotton than in conventional cotton, with reductions from 82 to 84%. The pink bollworm and tobacco budworm were not detected in any type of cotton. Damage to cotton bolls by boll weevils and conchuelas were low and did not differ between types of cotton. Sweetpotato whitefly populations did not differ between types of cotton and increased at the end of the crop season. These results show that Bt cotton (Bollgard II) expressing Cry1Ac and Cry2Ab toxins continues to be effective against bollworm and beet armyworm; while the pink bollworm and tobacco budworm are practically eradicated from this region. The Bt cotton showed no effectiveness against sweetpotato whitefly, boll weevil, and conchuela, as was to be expected because they are non-target pests.

Characterization of transgenic rice expressing fusion protein Cry1Ab/Vip3A for insect resistance

Management of resistance development of insect pests is of great importance for continued utilization of Bt crop. The high-dose/refuge and pyramid (gene stacking) strategy are commonly employed to delay the evolution of insect resistance. Due to the anticipated difficulty for deployment of mandatory refuge for transgenic crops in China, where the size of farmer is quite small, stacking of genes with different modes of action is a more feasible strategy. Here we report the development of transgenic rice expressing a fusion protein of Cry1Ab and Vip3A toxin. Analysis of trypsin proteolysis suggested that the fusion protein is equivalent to the combination of Cry1Ab and Vip3A protein. The transgenic plants expressing the fusion protein were found to be highly resistant to two major rice pests, Asiatic rice borer Chilo suppressalis (Lepidoptera: Crambidae) and rice leaf folder Cnaphalocrocis medinalis (Lepidoptera: Crambidae), while their agronomic performances showed no significant difference compared to the non-transgenic recipient rice. Therefore, the transgenic rice may be utilized for rice pest control in China.

Probing active microbes involved in Bt-containing rice straw decomposition.

Transgenic Bacillus thuringiensis (Bt) rice extends significant protection against insect pests and meets the increasing demands for food and energy. Many studies have been conducted investigating the impacts of Bt rice to the agricultural ecosystem, but much less attention has been given to efforts attempting to determine how the presence of Bt rice influences and shapes the microbial community, especially the active microbes. Stable isotope probing and high-throughput sequencing were employed to explore the active microbes involved in Bt-containing straw decomposition. Compared to its near isoline, the Bt straw contained higher contents of total N, total P, total K, lignin, cellulose, and Cry1Ab toxin protein. These chemical differences did not affect the decomposition rate but significantly changed the active microbial decomposer communities. During the decomposition of Bt-containing straw, fungi were more affected than bacteria. Agromyces, Terrabacter, Microbacterium, Glycomyces, and Kribbella were the most representative unique (existed only in the Bt treatments and appeared at the early stage) bacterial genera, and Trichoderma was the most representative unique fungal genus in the Bt straw decomposition. By using similarity index calculation and function prediction, the significant differences between Bt straw and non-Bt straw treatments were found to be transient for both microbial taxa and functional traits. These results suggested that Bt rice has a significant but transient impact on soil microbes in terms of microbial straw decomposition.

ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.

Development of an immunochromatographic assay for the specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin

Cry1Ab has been widely used in genetically modified (GM) crops and its amino acid sequence had high identity to Cry1Ac toxin. Existing nanogold immunochromatographic strips cannot distinguish Cry1Ab from Cry1Ac toxin. In this study, a rapid (5–6 min), qualitative nanogold immunochromatographic strip was successfully developed for the specific detection of Cry1Ab toxin. The assay was based on double antibody sandwich format with the visual detection limit (vLOD) of 0.1 μg mL−1. The results of immunochromatographic assay were all positive validated against the DAS-ELISA (recoveries between 109.6 and 111.8%). In addition, 10%, 5% and 0% error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1, 0.2, 0.5 and 1 μg mL−1, respectively, demonstrating the reproducibility of the test strip. Furthermore, the test strip could be stored for 3 months under dry conditions without significant loss of sensitivity. Furthermore, the practical sample analysis results showed that the test strip was able to detect the presence of Cry1Ab in GM materials containing as low as 0.5% MON 810 Bt maize which indicated the practical value of the test strip. To our knowledge, this is the first report on the detection of Cry1Ab by immunochromatographic assay without interference from Cry1Ac toxin.

Spatial distribution and losses by grain destroying insects in transgenic corn expressing the toxin Cry1Ab.

Insect pests are one of the factors that most impact plant yield. The magnitude of the losses and the spatiotemporal pest distribution in crops is a result of their interactions with the environment. Therefore, the understanding of the causes of production losses and the pest spatial patterns is important for the development of suitable sampling plans and pest management programs. Thus, this study aimed to quantify grain losses caused by insects and to determine the spatial distribution pattern of arthropod pest species in Bt and non-Bt corn. The prevailing insect pests in the corn ears were the earworm and fall armyworm caterpillars (Helicoverpa spp. and Spodoptera frugiperda), the cornsilk fly (Euxesta spp.), the maize weevil (Sitophilus zeamais), and the square-necked grain beetle (Cathartus quadricollis). The non-Bt corn was more attacked by the caterpillars and the weevil, while Bt corn was more affected by the cornsilk fly Euxesta spp. Spatial dependence was significant for the damage caused by the caterpillars, the grain beetle and the maize weevil in both the Bt and non-Bt corn genotypes. The range of the damage caused by the insects was between 9.0–9.7 m for the caterpillars, 6.9–12.20 m for the cornsilk fly, 10.7–80.4 m for the square-necked grain beetle, and 51.9–170.7 m for the maize weevil. The pattern of the spatial distribution of pest damage in both corn genotypes (i.e., Bt and non-Bt corn) was similar with a prevalence of moderate to strong spatial dependence and aggregate damage distribution. The plants near to the sampling points exhibited injury and infestation levels similar to those of the sampled plants.

Helix \u03b1-3 inter-molecular salt bridges and conformational changes are essential for toxicity of Bacillus thuringiensis 3D-Cry toxin family

Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.

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

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

The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins

The cadherin-like protein in lepidopteran insects, known as a receptor for Bacillus thuringiensis Cry1A toxins, is a single-pass membrane protein that can be divided into extracellular and intracellular regions. The extracellular region is important for toxin binding and oligomerization, whereas the role of the intracellular region during Cry1A intoxication is unclear. In the present study, we generated a deletion mutant of Bombyx mori cadherin-like protein (BtR175) that lacked the intracellular region to investigate its role in mediating Cry1A toxicity. Like wild-type BtR175, the mutant protein conferred susceptibility to Cry1Aa and Cry1Ab toxins in Sf9 cells, suggesting that the intracellular region is not required to mediate intoxication. The deletion mutant maintained another role of cadherin-like proteins; that it, synergistic activity with B. mori ABC transporter C2 (ABCC2) when mediating Cry1Aa and Cry1Ab toxicity. In addition, we evaluated the effects of reagents that have been reported to inhibit Cry1A toxicity (e.g., protein kinase A inhibitors, EDTA, and sucrose) on Cry1A toxicity in BtR175-expressing cells. Our results suggest that Cry1Aa-induced cell death in BtR175-expressing cells was not caused by signal transduction but by osmotic lysis. Overall, our data indicate that BtR175 mediates the toxicity of Cry1Aa and Cry1Ab toxins entirely via its extracellular region. They also indicate that the synergism between cadherin-like protein and ABCC2 occurs outside of cells or in the cell membrane.

Phage-Mediated Competitive Chemiluminescent Immunoassay for Detecting Cry1Ab Toxin by Using an Anti-Idiotypic Camel Nanobody.

Cry toxins have been widely used in genetically modified organisms for pest control, raising public concern regarding their effects on the natural environment and food safety. In this work, a phage-mediated competitive chemiluminescent immunoassay (c-CLIA) was developed for determination of Cry1Ab toxin using anti-idiotypic camel nanobodies. By extracting RNA from camels' peripheral blood lymphocytes, a naive phage-displayed nanobody library was established. Using anti-Cry1Ab toxin monoclonal antibodies (mAbs) against the library for anti-idiotypic antibody screening, four anti-idiotypic nanobodies were selected and confirmed to be specific for anti-Cry1Ab mAb binding. Thereafter, a c-CLIA was developed for detection of Cry1Ab toxin based on anti-idiotypic camel nanobodies and employed for sample testing. The results revealed a half-inhibition concentration of developed assay to be 42.68 ± 2.54 ng/mL, in the linear range of 10.49-307.1 ng/mL. The established method is highly specific for Cry1Ab recognition, with negligible cross-reactivity for other Cry toxins. For spiked cereal samples, the recoveries of Cry1Ab toxin ranged from 77.4% to 127%, with coefficient of variation of less than 9%. This study demonstrated that the competitive format based on phage-displayed anti-idiotypic nanobodies can provide an alternative strategy for Cry toxin detection.

Yield Losses in Transgenic Cry1Ab and Non-Bt Corn as Assessed Using a Crop-Life-Table Approach.

In this study, we constructed crop life tables for Bacillus thuringiensis Berliner (Bt) Cry1Ab and non-Bt corn hybrids, in which yield-loss factors and abundance of predaceous arthropods were recorded during 2 yr at two locations. Corn kernel/grain was the yield component that had the heaviest losses and that determined the overall yield loss in the corn hybrids across years and locations. Yield losses in both corn hybrids were primarily caused by kernel-destroying insects. Helicoverpa zea (Boddie) and Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) were the key loss factors at one location, while at the other, the key loss factor was the silk fly larvae, Euxesta spp. (Diptera: Ulidiidae). Although the realized yield of corn grains was not different (P > 0.05) between Cry1Ab and non-Bt corn hybrids, the Bt corn hybrid reduced (P < 0.05) the damage by H. zea and S. frugiperda in three of the four field trials, particularly at the location where Lepidoptera were the key loss factors. As expected, no reduction in the abundance of predaceous arthropods was observed in Cry1Ab corn fields. Various species of natural enemies were recorded, particularly the earwig Doru luteipes (Scudder) (Dermaptera: Forficulidae), which was the most abundant and frequent predaceous insect. These results indicate that integration of pest management practices should be pursued to effectively minimize losses by kernel-destroying insects during corn reproductive stages when growing non-Bt or certain low-dose Bt corn cultivars for fall armyworm and corn earworm, such as those producing Cry1Ab or other Cry toxins.

Interaction of Fluorescently Labeled Cadherin G Protein-coupled Receptor with the Cry1Ab Toxin of Bacillus thuringiensis.

The Cry1Ab toxin produced by Bacillus thuringiensis binds to a conserved structural motif in the 12th ectodomain module (EC12) of BT-R1, a cadherin G protein-coupled receptor (GPCR) contained in the membrane of midgut epithelial cells of the tobacco hornworm Manduca sexta. Toxin binding transmits a signal into the cells and turns on a multi-step signal transduction pathway, culminating in cell death. Using chromatographically purified Cry1Ab and EC12 proteins, we demonstrated the direct formation of a stable complex between these two proteins in solution and visualized it on a native polyacrylamide gel. Moreover, we generated a fluorescent EC12 probe by converting the 36th residue to cysteine to enable maleimide-mediated conjugation of Alexa-488 fluorescent dye to EC12 by site-directed mutagenesis. In addition, we changed the 44th residue of EC12 to tryptophan, which greatly improved accuracy of protein quantification and traceability. Using the fluorescently labeled EC12 probe for direct and competitive binding assays, we were able to determine binding specificity in solution. These accomplishments will facilitate identification and characterization of the interface sequences for both the Cry1Ab toxin and BT-R1.

"The Defined Toxin-binding Region of the Cadherin G-protein Coupled Receptor, BT-R1, for the Active Cry1Ab Toxin of Bacillus thuringiensis".

The bacterium Bacillus thuringiensis (Bt) produces protoxin proteins in parasporal crystals. Proteolysis of the protoxin generates an active toxin which is a potent microbial insecticide. Additionally, Bt toxin genes have been introduced into genetically modified crops to produce insecticidal toxins which protect crops from insect invasion. The insecticidal activity of Cry toxins is mediated by specific interaction between toxins and their respective cellular receptors. One such toxin (Cry1Ab) exerts toxicity by first targeting the 12th ectodomain region (EC12) of the moth cadherin receptor BT-R1. Binding promotes a highly regulated signaling cascade event that concludes in oncotic-like cell death. We previously determined that conserved sequence motifs near the N- and C-termini of EC12 are critical for toxin binding in insect cells. Here, we have established that Cry1Ab specifically binds to EC12 as a soluble heterodimeric complex with extremely high affinity (Kd = 19.5 ± 1.6 nM). Binding assays using Cry1Ab toxin and a fluorescently labeled EC12 revealed that the heterodimeric complex is highly specific in that no such formation occurs between EC12 and other Cry toxins active against beetle and mosquito. Disruption of one or both terminal sequence motifs in EC12 eliminates complex formation. Until now, comprehensive biophysical characterization of Cry1Ab recognition and binding by the BT-R1 receptor was unresolved. The findings presented here provide insight on the molecular determinants in the Cry family of toxins and should facilitate the assessment and advancement of their use as pesticidal agents.

Formulation for optimizing Bacillus thuringiensis production

Bacillus thuringiensis Berliner, a gram positive aerobic bacterium, produces parasporal crystal (Cry) toxins that are highly specific and effective against insect species. During the course of isolation of native strains, B. thuringiensis AUG-5 was found the most effective with a wide range of activity against lepidopterans. Hence, different media were evaluated for its growth and development. Increase in concentration of the Luria Bertani [(LB), composed of casein, yeast extract and sodium chloride in 2:1:2 w/w)] medium in the fermentation broth from 1 to 2% increased colony forming unit (CFU), spore and also Cry1Ac and Cry2Ab toxin content. However, further increase of LB concentration to 3% adversely affected bacterial growth and development. Addition of 1% Wesson salt in 1% LB broth significantly increased spore, CFU counts, and also that of Cry1Ac but not of Cry2Ab. Spore and CFU counts in media were positively correlated and cell mass negatively correlated with Cry1Ac and Cry2Ab contents. Of all media substituting LB with agro products, medium consisting of 2% wheat flour, 2% soybean meal and 1% Wesson salt could be considered as an alternative to LB medium to achieve economy of largescale production costs. Spore-crystal complexes of Medium II and III were most toxic to the neonates of cotton bollworm, Helicoverpa armiger a and tobacco caterpillar, Spodoptera litura at 10 I¼g/g, and differed significantly from those of Medium LB-2X and LB-3X and Cry2Ab2. Cry1Ac was most toxic to H. armigera at 1 I¼g/g and less toxic to S. litura than Cry2Ab.

Insight into the Mode of Action of Celangulin V on the Transmembrane Potential of Midgut Cells in Lepidopteran Larvae.

Celangulin V (CV) is the main insecticidal constituent of Celastrus angulatus. The V-ATPase H subunit of the midgut cells of lepidopteran larvae is the putative target protein of CV. Here, we compared the effects of CV on the midgut membrane potentials of Mythimna separata and Agrotis ipsilon larvae with those of the Cry1Ab toxin from Bacillus thuringiensis and with those of inactive CV-MIA, a synthetic derivative of CV. We investigated the changes in the apical membrane potentials (Vam) and basolateral membrane potentials (Vbm) of the midguts of sixth-instar larvae force-fed with the test toxins. We also measured the Vam and Vbm of larval midguts that were directly incubated with the test toxins. Similar to the effect of Cry1Ab, the Vam of CV-treated midguts rapidly decayed over time in a dose-dependent manner. By contrast, CV-MIA did not influence Vam. Meanwhile, the Vam of A. ipsilon larval midguts directly incubated with CV decayed less than that of M. separata larval midguts, whereas that of larvae force-fed with CV did not significantly change. Similar to Cry1Ab, CV did not affect the Vbm of isolated midguts. CV significantly inhibited V-ATPase activity in a dose-dependent manner. Therefore, CV initially inhibits V-ATPase in the apical membrane and affects intracellular pH, homeostasis, and nutrient transport mechanisms in lepidopteran midgut cells.

Identification and Characterization of Hyphantria cunea Aminopeptidase N as a Binding Protein of Bacillus thuringiensis Cry1Ab35 Toxin.

The fall webworm, Hyphantria cunea (Drury) is a major invasive pest in China. Aminopeptidase N (APN) isoforms in lepidopteran larvae midguts are known for their involvement in the mode of action of insecticidal crystal (Cry) proteins from Bacillus thuringiensis. In the present work, we identified a putative Cry1Ab toxin-binding protein, an APN isoform designated HcAPN3, in the midgut of H. cunea by ligand blot and mass spectrometry. HcAPN3 was highly expressed throughout all larval developmental stages and was abundant in the midgut and hindgut tissues. HcAPN3 was down-regulated at 6 h, then was up-regulated significantly at 12 h and 24 h after Cry1Ab toxin treatment. We expressed HcAPN3 in insect cells and detected its interaction with Cry1Ab toxin by ligand blot assays. Furthermore, RNA interference (RNAi) against HcAPN3 using oral delivery and injection of double-stranded RNA (dsRNA) resulted in a 61–66% decrease in transcript level. Down-regulating of the expression of HcAPN3 was closely associated with reduced susceptibility of H. cunea to Cry1Ab. In addition, the HcAPN3E fragment peptide expressed in Escherichia coli enhanced Cry1Ab toxicity against H. cunea larvae. This work represents the first evidence to suggest that an APN in H. cunea is a putative binding protein involved in Cry1Ab susceptibility.