Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium.

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Variable gut pH as a potential mechanism of tolerance to Bacillus thuringiensis subsp. israelensis toxins in the biting midge Culicoides sonorensis.

BACKGROUND: Toxins of Bacillus thuringiensis subsp. israelensis (Bti) are safer alternatives for controlling dipteran pests such as black flies and mosquitoes. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is an important pest of livestock in much of the United States and larval midges utilize semi-aquatic habitats which are permissive for Bti product application. Reports suggest that Bti products are ineffective at killing biting midges despite their taxonomic relation to black flies and mosquitoes. Here, we investigate the toxicity of a Bti-based commercial insecticide and its active ingredient in larval Culicoides sonorensis. A suspected mechanism of Bti tolerance is an acidic larval gut, and we used a pH indicator dye to examine larval Culicoides sonorensis gut pH after exposure to Bti. RESULTS: The lethal concentration to kill 90% (LC90) of larvae of the commercial product (386 mg/L) was determined to be almost 10 000 times more than that of some mosquito species, and no concentration of active ingredient tested achieved 50% larval mortality. The larval gut was found to be more acidic after exposure to Bti which inhibits Bti toxin activity. By comparison, 100% mortality was achieved in larval Aedes aegypti at the product's label rate for this species and mosquito larvae had alkaline guts regardless of treatment. Altering the larval rearing water to alkaline conditions enhanced Bti efficacy when using the active ingredient. CONCLUSION: We conclude that Bti is not practical for larval Culicoides sonorensis control at the same rates as mosquitos but show that alterations or additives to the environment could make the products more effective. © 2024 Society of Chemical Industry.

Involvement of miR-8510a-3p in response to Cry1Ac protoxin by regulating PxABCG3 in Plutella xylostella.

Overuse of insecticides has accelerated the evolution of insecticide resistance and created serious environmental concerns worldwide, thus incentivizing development of alternative methods. Bacillus thuringiensis (Bt) is an insecticidal bacterium that has been developed as a biopesticide to successfully control multiple species of pests. It operates by secreting several insect toxins such as Cry1Ac. However, metabolic resistance based on ATP-binding cassette (ABC) transporters may play a crucial role in the development of metabolic resistance to Bt. Here, we characterized an ABCG gene from the agricultural pest Plutella xylostella (PxABCG3) and found that it was highly expressed in a Cry1Ac-resistant strain, up-regulated after Cry1Ac protoxin treatment. Binding miR-8510a-3p to the coding sequence (CDS) of PxABCG3 was then confirmed by a luciferase reporter assay and RNA immunoprecipitation. miR-8510a-3p agomir delivery markedly reduced PxABCG3 expression in vivo and consequently decreased the tolerance of P. xylostella to Cry1Ac, while reduction of miR-8510a-3p significantly increased PxABCG3 expression, accompanied by an increased tolerance to Cry1Ac. Our results suggest that miR-8510a-3p could potentially be used as a novel molecular target against P. xylostella or other lepidopterans, providing novel insights into developing effective and environmentally friendly pesticides.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Bacillus thuringiensis Cry9Aa Insecticidal Protein Domain I Helices α3 and α4 Are Two Core Regions Involved in Oligomerization and Toxicity.

Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.

The chitin-binding domain of Bacillus thuringiensis ChiA74 inhibits gram-negative bacterial and fungal pathogens of humans and plants.

The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of ∼14 kDa and binds strongly to α-chitin, with Kd and Bmax of ∼4.7 ± 0.9 µM and 1.5 ± 0.1 µmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (∼0.31) than microcrystalline cellulose (∼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 µg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 µg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 µg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.

20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis ISPC-12: Purification, characterization, solution scattering and structural analysis.

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.

Channel Formation in Cry Toxins: An Alphafold-2 Perspective.

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Multiple cry Genes in Bacillus thuringiensis Strain BTG Suggest a Broad-Spectrum Insecticidal Activity.

The properties of Bacillus thuringiensis strains as a biopesticide with potent action against moths, beetles, and mosquitoes have been known for decades, with individual subspecies showing specific activity against a particular pest. The aim of the present work is to characterize strains that can be used for broad-spectrum pest control in agriculture. Twenty strains of B. thuringiensis were isolated from Bulgarian soil habitats. The strains were screened for genes encoding 12 different crystal (Cry) endotoxins by PCR with specific primer pairs. Seven of the isolates contained cry genes in their genomes. B. thuringiensis strains PL1, PL3, and PL20 contained at least three different cry genes, while B. thuringiensis serovar galleriae BTG contained at least four. Moreover, scanning electron microscopy (SEM) investigation revealed the production of bipyramidal (PL1, PL3, PL20), polygonal (PL1), cubic (BTG), and spherical crystals (BTG and PL20). Potentially containing the most cry genes, the BTG genome was sequenced and annotated. It comprises 6,275,416 base pairs, does not contain plasmids, has a GC content of 35.05%, and contained 7 genes encoding crystal toxins: cry1Ab35, cry1Db, cry1Fb, cry1Ib, cry2Ab, cry8Ea1, and cry9Ba. This unique combination would possibly enable the simultaneous pesticidal action against pest species from orders Lepidoptera, Coleoptera, Diptera, and Hemiptera, as well as class Gastropoda. Whole-genome sequencing provided accurate information about the presence, localization, and classification of Cry toxins in B. thuringiensis BTG, revealing the great potential of the strain for the development of new broad-spectrum bio-insecticides.

Bt corn hybrids expressing mCry3A and eCry3.1Ab Proteins protect corn roots against western corn rootworm injury.

BACKGROUND: The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is one of the most serious pests of corn (Zea mays L.) In 2017 and 2018, studies were conducted in fields with and without known unexpected root injury to Cry3Bb1, to determine root protection by Bt corn hybrids expressing both mCry3A and eCry3.1Ab insecticidal crystal proteins, and hybrids expressing either mCry3A or eCry3.1Ab only against the WCR root injury. Node injury was evaluated using the Iowa State University 0-3 node-injury scale (NIS), and the consistency of root protection was also determined. RESULTS: In 2017, with medium to high larval feeding pressure, the Bt corn hybrids expressing both mCry3A and eCry3.1Ab in the breeding stack, molecular stack, and Bt corn hybrid expressing eCry3.1Ab only, sustained low node injury compared with Bt corn hybrid expressing mCry3A only, and the non-Bt corn. In 2018, with low larval feeding pressure in most of the locations, node injury was not different for the Bt and Non-Bt corn hybrids. Across all locations in both years, the Bt corn hybrids expressing both mCry3A and eCry3.1Ab provided better and consistent node injury protection. CONCLUSION: Bt corn hybrids expressing both mCry3A and eCry3.1Ab proteins provided better root protection and consistency than the Bt corn hybrid expressing mCry3A only, and non-Bt. Therefore, stacking of Bt traits will be the best option for managing insect resistance. © 2023 Society of Chemical Industry.

Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography.

Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To improve our understanding of the nature of their oligomers in the toxic actions and synergistic effects, we performed the atomic force microscopy to probe the surfaces of their natively grown crystals, and used the L-weight filter to enhance the structural features. By L-weight filtering, molecular sizes of the Cry11Aa and Cyt1Aa monomers obtained are in excellent agreement with the three-dimensional structures determined by x-ray crystallography. Moreover, our results show that the layered feature of a structural element distinguishes the topographic characteristics of Cry11Aa and Cyt1Aa crystals, suggesting that the Cry11Aa toxin has a better chance than Cyt1Aa for multimerization and therefore cooperativeness of the toxic actions.

Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with ß-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

Optimized high-yield preparation of alkaline-solubilizable crystalline inclusion of the Bacillus thuringiensis Cry4Aa δ-endotoxin expressed in Escherichia coli.

The native Cry4Aa δ-endotoxin produced exclusively in Bacillus thuringiensis during sporulation as a ∼130-kDa inactive protoxin is confined within the parasporal crystalline inclusion that dissolves at alkaline pH in the midgut lumen of mosquito larvae. Here, the recombinant Cry4Aa toxin over-expressed in Escherichia coli at 30 °C as an alkaline-solubilizable inclusion was found inevitably lost during isolation from the cell lysate (pH ∼6.5) of which host cells were pre-suspended in distilled water (pH ∼5.5). When 100 mM KH2PO4 (pH 5.0) was used as host cell-suspending buffer, the cell lysate's pH became more acidic (pH 5.5), allowing the expressed protoxin to be entirely retained in the form of crystalline inclusion rather than a soluble form, and thus high-yield recovery of the partially purified inclusion was obtained. Upon dialysis of the alkaline-solubilized protoxin against the KH2PO4 buffer, the protoxin precipitate was efficiently recovered and still exhibited high toxicity to Aedes aegypti mosquito larvae. Additionally, the precipitated protoxin was completely resolubilized in 50 mM Na2CO3 buffer (pH 9.0) and proteolytically processed by trypsin to produce the 65-kDa activated toxin comprising ∼47- and ∼20-kDa fragments. In silico structural analysis suggested that His154, His388, His536 and His572 were involved in a dissolution of the Cry4Aa inclusion at pH 6.5, conceivably through interchain salt bridge breakage. Altogether, such an optimized protocol described herein was effective for the preparation of alkaline-solubilizable inclusions of the recombinant Cry4Aa toxin in large amounts (>25 mg per liter culture) that would pave the way for further structure-function relationship studies of different Cry toxins.

Anatomical damage caused by Bacillus thuringiensis variety israelensis in yellow fever mosquito Aedes aegypti (L.) larvae revealed by micro-computed tomography.

With micro-computed tomography techniques, using the single-distance phase-retrieval algorithm phase contrast, we reconstructed enhanced rendered images of soft tissues of Aedes aeqypti fourth instar larvae after Bti treatment. In contrast to previous publications based on conventional microscopy, either optical or electron microscopy, which were limited to partial studies, mostly in the form of histological sections, here we show for the first time the effects of Bti on the complete internal anatomy of an insect. Using 3D rendered images it was possible to study the effect of the bacterium in tissues and organs, not only in sections but also as a whole. We compared the anatomy of healthy larvae with the changes undergone in larvae after being exposed to Bti (for 30 min, 1 h and 6 h) and observed the progressive damage that Bti produce. Damage to the midgut epithelia was confirmed, with progressive swelling of the enterocytes, thickening epithelia, increase of the vacuolar spaces and finally cell lysis, producing openings in the midgut walls. Simultaneously, the larvae altered their motility, making it difficult for them to rise to the surface and position the respiratory siphon properly to break surface tension and breathe. Internally, osmotic shock phenomena were observed, resulting in a deformation of the cross-section shape, producing the appearance of a wide internal space between the cuticle and the internal structures and a progressive collapse of the tracheal trunks. Taken together, these results indicate the death of the larvae, not by starvation as a consequence of the destruction of the epithelia of the digestive tract as previously stated, but due to a synergic catastrophic multifactor process in addition to asphyxia due to a lack of adequate gas exchange.

Purification, characterization and proteolytic processing of mosquito larvicidal protein Cry11Aa from Bacillus thuringiensis subsp. israelensis ISPC-12.

Cry11Aa is the most potent mosquito larvicidal protein of Bacillus thuringiensis subsp. israelensis (Bti). Development of resistance against insecticidal proteins including Cry11Aa is known but no field resistance was observed with Bti. The phenomenon of increasing resistance in insect pests necessitates the development of new strategies and techniques to enhance efficacy of insecticidal proteins. Recombinant technology offers better control over the molecule and allows modification of protein to achieve maximal effect against target pests. In this study, we standardised protocol for recombinant purification of Cry11Aa. Recombinant Cry11Aa found active against larvae of Aedes and Culex mosquito species and LC50 were estimated. Detailed biophysical characterization provides crucial insights into stability and in-vitro behaviour of the recombinant Cry11Aa. Moreover, trypsin hydrolysis doesn't improve overall toxicity of recombinant Cry11Aa. Proteolytic processing suggests domain I and II are more prone to proteolysis in comparison to domain III. Significance of structural features for proteolysis of Cry11Aa was observed after performing molecular dynamics simulations. Findings reported here are contributing significantly in method for purification, understanding in-vitro behaviour and proteolytic processing of Cry11Aa which could facilitate in efficient utilisation of Bti for insect pests and vectors control.

Potential control of the infective stage of Taenia pisiformis using Bacillus thuringiensis GP526 strain.

The GP526 strain of Bacillus thuringiensis has been referred as an in vitro helminthicide on various stages of Dipylidium caninum and Centrocestus formosanus. Our study addresses the in vitro ovicidal activity of GP526 strain spore-crystal complex on Taenia pisiformis eggs, evaluating induced damage microscopically. The eggs exposed to the total extract containing spores and crystals show damage after 24 hours, with loss of integrity on the eggshell, and an ovicidal activity of 33% at 1mg/ml. The destruction of the embryophore was observed after 120 h with a 72% of ovicidal activity at 1 mg/ml. The LC50 was 609.6 µg/ml, dose that causes a 50% of lethality on the hexacanth embryo, altering the oncosphere membrane. The spore-crystal proteins were extracted, and the protein profile was obtained by electrophoresis, finding a major band of 100 kDa suggestive of an S-layer protein, since an S-layer was immunodetected in both, spores and extracted proteins. The protein fraction containing the S-layer protein presents adhesion to the T. pisiformis eggs, and 0.4 mg/ml of the protein induces a lethality of 21.08% at 24 h. The characterization of molecular mechanisms of ovicidal activity will be an important contribution, so the characterization of the proteins that make up the extract of the GP526 strain, would be useful to support the biological potential for control of this cestodiasis and other parasitosis. B. thuringiensis is shown as a potent helminthicide on eggs, with useful potential for biological control of this cestodiasis.

In vivo and in silico comparison analyses of Cry toxin activities toward the sugarcane giant borer.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin-receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.