Binding properties of Bacillus thuringiensis Cry4A toxin to the apical microvilli of larval midgut of Culex pipiens.

Cry4A is a dipteran-specific delta-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.     

Binding properties of Bacillus thuringiensis Cry1C delta-endotoxin to the midgut epithelial membranes of Culex pipiens

The Cry1C delta-endotoxin from Bacillus thuringiensis is toxic to both lepidopteran and dipteran insect larvae. To analyze the dipteran-specific insecticidal mechanisms, we investigated the properties of Cry1C binding to the epithelial cell membrane of the larval midgut from the mosquito Culex pipiens in comparison with dipteran-specific Cry4A. Immunohistochemical staining of the larval midgut sections from Culex pipiens showed that Cry1C and Cry4A bound to the microvilli of the epithelial cells. The Cry1C binding to brush border membrane vesicles from the mosquito larvae was specific and irreversible, and did not compete with Cry4A. By ligand blotting analyses, we detected several Cry1C-binding proteins, the Cry1C binding to which did compete with excess unlabeled Cry4A. These results suggested that Cry1C and Cry4A recognized the same binding site(s) on the epithelial cell surface but that their interaction with the target membrane differed.     

Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity

A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. 125I-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. AgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Interaction of Bacillus thuringiensis svar. israelensis Cry toxins with binding sites from Aedes aegypti (Diptera: Culicidae) larvae midgut

This work shows in vitro processing of Bacillus thuringiensis svar. isralensis Cry toxins and the capacity of the active fragments to bind the midgut microvilli of Aedes aegypti larvae. Processing of Cry11Aa, Cry4Aa and Cry4Ba yielded double fragments of 38-30, 45-20 and 45-18 kDa, respectively. Competition assays showed that all active (125)I-Cry toxins are able to specifically bind to brush border membrane fractions and they might share a common class of binding sites. The values of IC(50) suggested that toxins do not display high affinity for the receptors from brush border membrane fractions, while dissociation assays showed that binding was irreversible, indicating the insertion of toxins in the cell membrane.     

Cytopathological effect of Bacillus thuringiensis israelensis endotoxins on the intestines of Aedes aegypti mosquito larvae

The dynamics of pathological changes in the intestine of Aedes aegypti larvae under the influence of toxins Cry11A and Cry4B produced by Bacillus thuringiensis israelensis was studied by means of electron microscope. Most significant ultrastructure changes in the intestine of the second instar larvae were observed in the midgut. The cytoplasm of cells disintegrated, and elongated lacunae appeared. The number of microvilli decreased, or they disappeared in the result of destruction. The peritrophic membrane displaced to the lumen of midgut. Any changes in epithelial cells and cuticle in time of foregut and hindgut were not observed in a comparison to control. The toxin Cry4B caused the most effective destruction of the midgut epithelium.     

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.     

Activation process of dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp. israelensis.

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST-20-kDa fusion protein (GST-20) nor the GST-45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST-60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.     

A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.     

Lack of detrimental effects of Bacillus thuringiensis Cry toxins on the insect predator Chrysoperla carnea: a toxicological, histopathological, and biochemical analysis

The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.     

In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae)

Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.     

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.     

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the Cry1A class of Bt toxins have been identified: an aminopeptidase N (APN-1) and a 270 kDa anionic glycoconjugate (BTR-270). Studies have shown that APN-1 has a relatively weak affinity and a very narrow specificity to Cry1Ac, the only Cry1A toxin that it binds. In contrast, BTR-270 binds all toxins that are active against L. dispar larvae, and the affinities for these toxins to BTR-270 correlate positively with their respective toxicities. In this study, an immunohistochemical approach was coupled with fluorescence microscopy to localize APN-1 and BTR-270 in paraffin embedded midgut sections of L. dispar larvae. The distribution of cadherin and alkaline phosphatase in the gut tissue was also examined. A strong reaction indicative of polyanionic material was detected with alcian blue staining over the entire epithelial brush border, suggesting the presence of acidic glycoconjugates in the microvillar matrix. The Cry1A toxin-binding sites were confined to the apical surface of the gut epithelial cells with intense labeling of the apical tips of the microvilli. APN-1, BTR-270, and alkaline phosphatase were found to be present exclusively along the brush border microvilli along the entire gut epithelium. In contrast, cadherin, detected only in older gypsy moth larvae, was present both in the apical brush border and in the basement membrane anchoring the midgut epithelial cells. The topographical relationship between the Bt Cry toxin-binding molecules BTR-270 and APN-1 and the Cry1A toxin-binding sites that were confined to the apical brush border of the midgut cells is consistent with findings implicating their involvement in the mechanism of the action of Bt Cry toxins.     

Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

Background

Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.

Methodology/Principal Findings

Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that ¹²⁵I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances ¹²⁵I-Cry35Ab1 specific binding, and that ¹²⁵I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with ¹²⁵I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with ¹²⁵I-Cry3Aa, or ¹²⁵I-Cry8Ba.

Conclusions/Significance

Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.     

取食转Bt基因抗虫玉米后亚洲玉米螟幼虫中肠的组织病理变化

利用透射显微镜（TEM）观察亚洲玉米螟Ostrinia furnacalis (Guenée)幼虫取食了表达Cry1Ab杀虫蛋白的转Bt基因玉米心叶组织后中肠的组织病理变化, 以探讨转Bt基因玉米对亚洲玉米螟的致病机理, 为其合理、安全和持续利用提供理论依据。结果表明：亚洲玉米螟取食Bt玉米后中肠细胞及其细胞器发生了明显的病变。取食Bt玉米12 h后中肠细胞开始病变, 首先微绒毛脱落、内质网开始肿胀, 24 h后内质网肿胀、增多, 杯状细胞杯腔增大, 48 h后微绒毛大量脱落, 细胞开始空泡化, 随着取食时间的增加, 细胞空泡化程度加剧, 在感染前期细胞间的病变程度差异较大。微绒毛脱落、内质网肿胀断裂是在多数取食Bt玉米的亚洲玉米螟中肠细胞发生的普遍病变。由此表明, 人工修饰的Cry1Ab基因导入到玉米染色体组中所表达的杀虫蛋白可使玉米螟幼虫中肠细胞发生病变, 最终导致其死亡。     

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 ¹⁴²³GVLSLNFQ¹⁴³⁰ 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.     

Lack of Detrimental Effects of Bacillus thuringiensis Cry Toxins on the Insect Predator Chrysoperla carnea: a Toxicological, Histopathological, and Biochemical Analysis

The effect of Cry proteins of Bacillus thuringiensis on the green lacewing (Chrysoperla carnea) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed Helicoverpa armigera larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated H. armigera larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from Spodoptera exigua but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.     

A binding site for Bacillus thuringiensis Cry1Ab toxin is lost during larval development in two forest pests

The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/microl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with (125)I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.     

Proteolysis, histopathological effects, and immunohistopathological localization of delta-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of lepidopteran olive tree pathogenic insect Prays oleae

Considering the fact that Prays oleae is one of the most pathogenic insects to the olive tree in the Mediterranean particularly in Tunisia, the mode of action of Cry insecticidal toxins of Bacillus thuringiensis kurstaki in Prays oleae midgut was investigated. The proteolysis of Bacillus thuringiensis δ-endotoxins in the midgut was a key step in determining their potency against Prays oleae. The latter's proteases activated the δ-endotoxins early, yielding stable toxins. The in vitro and in vivo binding of these toxins to Prays oleae larvae midgut was studied immunohistochemically, evidencing a midgut columnar cell vacuolization, microvilli damage, and then a pass of epithelium cell content into the larvae midgut. Moreover, Bacillus thuringiensis toxins were shown to bind to the apical microvilli of the midgut epithelial cells. The in vitro study of the interaction of Prays oleae midgut proteins with biotinylated Bacillus thuringiensis toxins allowed the prediction of four suitable receptor proteins in Prays oleae.