Enhancing insecticidal efficacy of Bacillus thuringiensis Cry1Ab through pH-sensitive encapsulation.

Nanotechnology is a promising way to enhance the stability of Bacillus thuringiensis (Bt) insecticidal proteins under environmental conditions. In this work, two emulsions were prepared through the Pickering emulsion technique, stabilized by Cu2+-SQDs/S-CN nanocomposites and by GO nanosheets. In addition, a pH-sensitive polymer was incorporated into these emulsions, allowing the Bt protein, Cry1Ab, to be released in an alkaline pH environment, as it occurs in the lepidopteran pests' gut. The effectiveness of these two nanomaterials in protecting Cry1Ab from degradation, and therefore enhancing its pesticidal activity, was assessed by exposing samples of the purified unprotected protein and encapsulated protein to high-intensity UV light and 40°C temperature treatments. The UV treatment results were evaluated using SDS-PAGE analysis and pointed out that Cry1Ab could be structurally protected by the emulsions. The bioassays with first instar larvae of the lepidopteran pest Ostrinia nubilalis confirm the nanomaterial protection to UV and temperature treatments, i.e., decreasing about half the degradation rate and increasing up to 12-fold the residual activity after UV treatment. Our results indicate that encapsulation could be an effective strategy to improve the effectiveness of Cry1Ab under environmental conditions. KEY POINTS: • Pickering emulsions are effective for solubilized Cry1Ab encapsulation. • Structural and toxicity Cry1Ab properties are enhanced by pH-sensitive encapsulation. • Cu2+-SQDs/S-CN and GO nanomaterials improve the efficacy of Bt insecticides.

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats / Abundancia, distribución y expresión de genes de proteínas cristalinas nematicidas en cepas de Bacillus thuringiensis de diversos hábitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.(AU)

Mpp23Aa/Xpp37Aa Insecticidal Proteins from Bacillus thuringiensis (Bacillales: Bacillaceae) Are Highly Toxic to Anthonomus grandis (Coleoptera: Curculionidae) Larvae.

The beetle Anthonomus grandis Boheman, 1843, is the main cotton pest, causing enormous losses in cotton. The breeding of genetically modified plants with A. grandis resistance is seen as an important control strategy. However, the identification of molecules with high toxicity to this insect remains a challenge. The susceptibility of A. grandis larvae to proteins (Cry1Ba, Cry7Ab, and Mpp23Aa/Xpp37Aa) from Bacillus thuringiensis Berliner, 1915, with toxicity reported against Coleopteran, has been evaluated. The ingestion of different protein concentrations (which were incorporated into an artificial diet) by the larvae was tested in the laboratory, and mortality was evaluated after one week. All Cry proteins tested exhibited higher toxicity than that the untreated artificial diet. These Cry proteins showed similar results to the control Cry1Ac, with low toxicity to A. grandis, since it killed less than 50% of larvae, even at the highest concentration applied (100 µg·g-1). Mpp/Xpp proteins provided the highest toxicity with a 0.18 µg·g-1 value for the 50% lethal concentration. Importantly, this parameter is the lowest ever reported for this insect species tested with B. thuringiensis proteins. This result highlights the potential of Mpp23Aa/Xpp37Aa for the development of a biotechnological tool aiming at the field control of A. grandis.

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

El sistema inmune de los insectos: algo más complejo que simples barreras / The insect immune system: something more complex than simple barriers

En los últimos años se ha producido un aumento de toma de conciencia sobre la importancia económica y social de los insectos. Aunque algunos pueden ser perjudiciales ocasionando problemas desalud púbica o pérdidas económicas relacionadas con las plagas en cultivos, muchos son esencialesen procesos como la polinización, además de tener aplicaciones como la producción de biomasa o suuso como agentes de control biológico. Todos estos factores han contribuido a que se produzca unaumento del número de estudios relacionados con el sistema inmune de los insectos, lo cual ha ayudado a conocer el funcionamiento de estos animales. Se han observado similitudes con el sistema inmune de los mamíferos, además de descubrir la denominada sensibilización del sistema inmune (máscomúnmente conocida por su terminología inglesa “immune priming”), lo que ha provocado que serompa con la asunción de simplicidad que tradicionalmente se les ha asociado. Todo esto ha permitidola optimización de muchas aplicaciones importantes. (AU)

Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis.

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. KEY POINTS: • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites.

Effect of Cry Toxins on Xylotrechus arvicola (Coleoptera: Cerambycidae) Larvae.

The beetle Xylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly; the latter through the proliferation of wood fungi, which can invade the inside of the plant, decreasing the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A, and Cry23/37) was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development. Cry7Ab and Cry1Ba were the most effective in controlling X. arvicola larvae due to the significant reduction in larvae survival (32.9 and 25.9 days, respectively), and by causing serious alterations in the larvae during the remaining months of their development. The developmental stage of the prepupal and pupal stages was not affected by the previous ingestion of Cry proteins. The Cry proteins tested could be applied to control X. arvicola larvae since they were able to kill them and cause serious alterations in the larvae during the remaining months of development that followed. The data presented suggest that these Cry proteins can be used as bioinsecticides against the larvae of this insect, applying them only at the moment when the larvae hatch from the egg outside the grapevine wood (this would only be useful and justified if the economic threshold is exceeded) in order to avoid the rapid evolution of resistance against these toxins since not all of the larvae were killed and thus increase vine wood protection.

Genomics and Proteomics Analyses Revealed Novel Candidate Pesticidal Proteins in a Lepidopteran-Toxic Bacillus thuringiensis Strain.

Discovery and identification of novel insecticidal proteins in Bacillus thuringiensis (Bt) strains are of crucial importance for efficient biological control of pests and better management of insect resistance. In this study, the Bt strain KhF, toxic for Plodia interpunctella and Grapholita molesta larvae, underwent genomics and proteomics analyses to achieve a better understanding of the bases of its pathogenicity. The whole-genome sequencing results revealed that the KhF strain contained nine coding sequences with homologies to Bt insecticidal genes. The lepidopteran toxic mixture of spores and crystals of this Bt strain was subjected to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to assess the protein composition. The results of the proteomic analyses, combined with the toxin gene sequences, revealed that two of the main components of the crystals were two new candidate pesticidal proteins, named KhFA and KhFB. These proteins showed a similarity lower than 36% to the other known Bt toxins. The phylogenetic analysis showed that the KhFA and KhFB grouped with the newly denominated Xpp and Mpp (former ETX/Mtx) pesticidal protein groups, respectively. Altogether, this study has led to the discovery of two novel candidate pesticidal toxins in the lepidopteran toxic KhF strain.

The Independent Biological Activity of Bacillus thuringiensis Cry23Aa Protein Against Cylas puncticollis.

The Cry23Aa/Cry37Aa proteins from Bacillus thuringiensis (Bt) have been described toxic to Cylas puncticollis larvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity toward C. puncticollis larvae has been assessed. The results showed that both proteins were toxic for C. puncticollis larvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity against C. puncticollis larvae. Also, the binding behavior of Cry23Aa protein to midgut receptors of C. puncticollis larvae has been determined. According to our results, Cry23Aa binds to C. puncticollis brush border membrane vesicles (BBMV) specifically and independently of Cry37Aa. Due to the lack of common binding sites, Cry23Aa can be pyramided with Cry3Aa protein for better management of C. puncticollis.

Insecticidal Activity of Bacillus thuringiensis Proteins Against Coleopteran Pests.

Bacillus thuringiensis is the most successful microbial insecticide agent and its proteins have been studied for many years due to its toxicity against insects mainly belonging to the orders Lepidoptera, Diptera and Coleoptera, which are pests of agro-forestry and medical-veterinary interest. However, studies on the interactions between this bacterium and the insect species classified in the order Coleoptera are more limited when compared to other insect orders. To date, 45 Cry proteins, 2 Cyt proteins, 11 Vip proteins, and 2 Sip proteins have been reported with activity against coleopteran species. A number of these proteins have been successfully used in some insecticidal formulations and in the construction of transgenic crops to provide protection against main beetle pests. In this review, we provide an update on the activity of Bt toxins against coleopteran insects, as well as specific information about the structure and mode of action of coleopteran Bt proteins.

Study of the Bacillus thuringiensis Cry1Ia Protein Oligomerization Promoted by Midgut Brush Border Membrane Vesicles of Lepidopteran and Coleopteran Insects, or Cultured Insect Cells.

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

Toxicity of five Cry proteins against the insect pest Acanthoscelides obtectus (Coleoptera: Chrisomelidae: Bruchinae).

The beetle Acanthoscelides obtectus (Say) causes severe post-harvest losses in the common bean (Phaseolus vulgaris). Under laboratory conditions, the susceptibility of A. obtectus to five coleopteran-specific Cry toxic proteins from Bacillus thuringiensis (Cry1Ba, Cry1Ia, Cry3Aa, Cry7Ab, and Cry23/37) was evaluated. After 30 days exposure, Cry proteins demonstrated high activity against A. obtectus adults (100% mortality). Proteins showed statistical differences in toxicity parameters compared to the control treatment, but the parameters were similar among them, and indicated that the final toxic effects can be observed after the 24th day. The toxic effects on A. obtectus larvae were evaluated indirectly by allowing adults to oviposit on treated beans and recording the emergence of F1 adults. All treatments resulted in a lower rate of successful emergence compared to the control treatment, ranging from 60% (Cry23/37) to 10% (Cry1Ia) reduction in eclosion. Finally, to evaluate the ability of Cry proteins to protect the beans against A. obtectus; the number of beans infested, the number of holes in each bean and bean weight loss were determined 45 days after the treatment. The parameters showed significant bean protection by all Cry proteins analyzed compared to control treatment. Cry23/37 showed the best results, however, results for the other proteins were similar. The proteins belong to different Cry protein families, which suggest that they could be used in combination to increase plant protection without compromising resistance management. Moreover, adult emergence and bean protection results indicate differences among the proteins, which may suggest different modes of action. Our results indicate that the studied Cry proteins can be applied for the control of A. obtectus larvae and adults.

Specific binding of Bacillus thuringiensis Cry1Ea toxin, and Cry1Ac and Cry1Fa competition analyses in Anticarsia gemmatalis and Chrysodeixis includens.

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper) are two important defoliation pests of soybeans. In the present study, we have investigated the susceptibility and brush border membrane-binding properties of both species to Bacillus thuringiensis Cry1Ea toxin. Bioassays performed in first-instar larvae demonstrated potent activity against both soybean pests in terms of mortality or practical mortality. Competition-binding studies carried out with 125Iodine-labelled Cry1Ea, demonstrated the presence of specific binding sites on the midgut brush border membrane vesicles (BBMV) of both insect species. Heterologous competition-binding experiments indicated that Cry1Ea does not share binding sites with Cry1Ac or Cry1Fa in either soybean pest. This study contributes to the knowledge of Cry1Ea toxicity and midgut binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ea with other Bt proteins aimed at controlling lepidopteran pests in soybeans.

Changes in gene expression and apoptotic response in Spodoptera exigua larvae exposed to sublethal concentrations of Vip3 insecticidal proteins.

The insecticidal Vip3 proteins from Bacillus thuringiensis (Bt), along with the classical Bt Cry proteins, are currently used in Bt-crops to control insect pests, since they do not share the same mode of action. Here we characterized the response of Spodoptera exigua larvae after Vip3 challenge. The expression profile of 47 genes was analyzed in larvae challenged with three concentrations of Vip3Ca. Results showed that the up-regulated genes were mainly involved in immune response, whereas the down-regulated genes were mainly involved in the digestion process. Other mechanisms of cellular response to the damage such as apoptosis were analyzed. For this analysis, sections from the midguts were examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The nuclei of the midgut epithelial cells were stained at the highest concentration of the Vip3Ca protein and at lower concentrations of Vip3Aa in agreement with the different potency of the two proteins. In addition, apoptosis was also examined by the analysis of the expression of five caspase genes. The present study shows that exposure of S. exigua larvae to sublethal concentrations of Vip3 proteins activates different insect response pathways which trigger the regulation of some genes, APN shedding, and apoptotic cell death.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis.

Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices.

Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM).

Unshared binding sites for Bacillus thuringiensis Cry3Aa and Cry3Ca proteins in the weevil Cylas puncticollis (Brentidae).

Bacillus thuringiensis Cry3Aa and Cry3Ca proteins have been reported to be toxic against the African sweetpotato pest Cylas puncticollis. In the present work, the binding sites of these proteins in C. puncticollis brush border vesicles suggest the occurrence of different binding sites, but only one of them is shared. Our results suggest that pest resistance mediated by alteration of the shared Cry-receptor binding site might not render both Cry proteins ineffective.