Cry Proteins Research Articles

Bacillus thuringiensis Cry14A family proteins as novel anthelmintics against gastrointestinal nematode parasites.

Bacillus thuringiensis crystal (Cry) proteins have been expressed in commercial transgenic crops for nearly 30 years, providing safe and effective control of insect pests and significantly reducing the application of hazardous chemical pesticides. B. thuringiensis crystal proteins have also been shown to target parasitic nematodes, including plant parasitic nematodes. Recently, transgenic soybean crops expressing Cry14Ab have been shown to provide control against the soybean cyst nematode Heterodera glycines, marking the first time a crystal protein is being commercialized in transgenic crops for control of a nematode pest. However, apart from H. glycines and the free-living nematode, Caenornabditis elegans, the breadth of nematode activity of Cry14Ab, e.g., against gastrointestinal parasitic nematodes (GINs), has not been reported. Here we study the efficacy of Cry14Ab against a wide range of gastrointestinal nematode parasites (GINs) in vitro and in vivo. We find that Cry14Ab is effective in vitro against the barber's pole worm Haemonchus contortus larvae, small strongyles cyathostomin larvae, the hookworm Ancylostoma ceylanicum adults, the roundworm Ascaris suum L4 larvae, and the whipworm Trichuris muris adults. In rodents infected with GIN parasites, Cry14Ab is effective as an in vivo anthelmintic against the hookworms A. ceylanicum and N. americanus, against the mouse parasite Heligmosomoides polygyrus bakeri, and against the roundworm A. suum. Cry14Ab also variably reduces the reproduction of the whipworm T. muris in vivo. Using optimized profile Markov Models, we looked for other putative anthelmintic Cry proteins and, within this list, identified a Bt crystal protein, GenBank accession no. MF893203, that we produced and demonstrated intoxicated GINs. This protein, with 90% amino acid identity to Cry14Ab, is active against C. elegans, A. ceylanicum adults, and A. suum L4 larvae in vitro. MF893203 was given the official designation of Cry14Ac. Cry14Ac is also an effective in vivo anthelmintic against A. ceylanicum hookworms in hamsters and intestinal A. suum in mice. Taken together, our results demonstrate that Cry14Ab and Cry14Ac have wide therapeutic utility against GINs.

Resistance to Cry14A family Bacillus thuringiensis crystal proteins in Caenornabditis elegans operates via the nhr-31 transcription factor and vacuolar-type ATPase pathway.

Bacillus thuringiensis (Bt) has been successfully used commercially for more than 60 years for biocontrol of insect pests. Since 1996, transgenic plants expressing Bt crystal (Cry) proteins have been used commercially to provide protection against insects that predate on corn and cotton. More recently, Bt Cry proteins that target nematodes have been discovered. One of these, Cry14Ab, has been expressed in transgenic soybean plants and found to provide significant protection against the soybean cyst nematode, Heterodera glycines. However, to date there has been no description of high-level resistance to any Cry14A family protein in nematodes. Here, we describe forward genetic screens to identify such mutants using the nematode Caenorhabditis elegans. Although non-conditional screens failed to identify highly resistant C. elegans, a conditional (temperature-sensitive) genetic screen identified one mutant, bre-6(ye123) (for Bt protein resistant), highly resistant to both Cry14Aa and Cry14Ab. The mutant comes at a high fitness cost, showing significant delays in growth and development and reduced fecundity. bre-6(ye123) hermaphrodites are only weakly resistant to copper intoxication, indicating that the mutant is not highly resistant to all insults. Backcrossing-whole genome sequencing was used to identify the gene mutated in ye123 as the nuclear hormone receptor nhr-31. RNAi, DNA rescue, and CRISPR analyses confirm that resistance to Cry14Aa intoxication in bre-6(ye123) is due to mutation of nhr-31 and was renamed nhr-31(ye123). As predicted for a mutation in this gene, nhr-31(ye123) animals showed significantly reduced expression of most of the subunits of the C. elegans vacuolar ATPase (vATPase). Mutants in the vATPase subunits unc-32 and vha-7 also show resistance to Cry14Aa and/or Cry14Ab. These data demonstrate that nhr-31 and the vATPase play a significant role in the intoxication of C. elegans by Cry14A family proteins, that reduction in vATPase levels result in high resistance to Cry14A family proteins, and that such resistance comes at a high fitness cost. Based on the relative difficulty of finding resistant mutants and the fitness cost associated with the vATPase pathway, our data suggest that transgenic Cry14Ab plants may hold up well to resistance by nematode parasites.

Immunolocalization and Ultrastructure Show Ingestion of Cry Protein Expressed in Glycine max by Heterodera glycines and Its Mode of Action.

Great interest exists in developing a transgenic trait that controls the economically important soybean (Glycine max) pest, soybean cyst nematode (SCN, Heterodera glycines), due to its adaptation to native resistance. Soybean plants expressing the Bacillus thuringiensis delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2s) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay. Here, we show that consistent with expectations for a Cry toxin, Cry14Ab has a mode of action unique from the native resistance sources Peking and PI 88788. Further, we demonstrate in planta the ingestion and localization of the Cry14Ab toxin in the midgut of nematodes feeding on roots expressing Cry14Ab using immunogold labeling and transmission electron microscopy. We observed immunolocalization of the toxin and resulting intestinal damage primarily in the microvillus-like structure (MvL)-containing region of the midgut intestine but not in nematodes feeding on roots lacking toxin. This demonstrated that Cry14Ab was taken up by the J2 SCN, presumably through the feeding tube within the plant root cell that serves as its feeding site. This suggests that relatively large proteins can be taken up through the feeding tube. Electron microscopy showed that Cry14Ab caused lysis of the midgut MvL membrane and eventual degradation of the MvL and the lysate, forming particulate aggregates. The accumulated electron-dense aggregate in the posterior midgut intestine was not observed in SCN in nonCry14Ab-expressing plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome mining of Bacillus thuringiensis strain SY49.1 reveals novel candidate pesticidal and bioactive compounds.

Bacillus thuringiensis SY49.1 (Bt SY49.1) strain has promising insecticidal and fungicidal activity against phytopathogens and pests. Therefore, we selected this strain for whole-genome sequencing (WGS), annotation and analysis, with the aim of identifying genes responsible for producing putative pesticidal toxins, antimicrobial metabolites and plant growth-promoting features. Our results showed that the SY49.1 genome is 6. 32 Mbp long with a GC content of 34.68%. Genome mining revealed the presence of multiple gene inventories for the biosynthesis of bioactive compounds such as insecticidal delta endotoxins, secondary metabolites, and several plant growth-promoting proteins. Multiple sequence alignment revealed residue variations in the toxic core of Cry1Ab when compared with known Cry1Ab sequences from Bt nomenclature databases. This suggests that the cry1Ab of SY49.1 is a new kind of its group. Among the predicted secondary metabolites, we found a kurstakin with a predicted peptide that differs from the known kurstakin peptide available in the NORINE database. In addition, lipopeptides extracted from SY49.1 suppressed the growth of Verticillium dahliae and Fusarium oxysporum. We anticipate that the complete genome of Bt SY49.1 may provide a model for properly understanding and studying antimicrobial compound mining, genetic diversity among the B. cereus group, and pathogenicity against insects. This is the first report on the WGS and mining of the Bt strain isolated from Turkey. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Plasmid vector(s) in Bacillus thuringiensis harbor genes for insect pest control and for neglected infectious diseases in humans

Plasmids (circular DNA molecules) represent an ingenious strategy for horizontal gene transfer in prokaryotes and eukaryotic cells. Plasmids harbored in bacteria are responsible for the spread of traits such as antibiotic resistance, virulence factors, and the machinery for the horizontal gene transfer e.g., type IV secretion systems. Remarkably, Bacillus thuringiensis (Bt) cryptic plasmids encode and carry genes that, under the host environment, replicate and concomitate with sporulation, producing parasporal crystalline proteins of two major types, crystalline (Cry) and cytolytic (Cyt), the former toxic against different orders of insects such as Lepidopterans, Coleopterans, and Dipterans (Cry proteins, MW 50–130 KDa); Cyt proteins, produced by B. thuringiensis subspecies israelensis (Bti)(MW 27-kDa) are toxic against Dipterans, i.e., mosquitoes and black flies. The X-Ray tridimensional structure for both types of toxins, formed by three domains, mostly of beta sheets antiparallel (Domain II and Domain III) linked through loops of different lengths. Domain I is a bundle of alpha helices. This structure is characterized by five conserved blocks, implying a conservation in the mode of action. Cyt proteins possess two alpha helices and some beta sheets with a structure similar to the antimicrobial peptides. Indeed, the mode of action proposed is mediated by the toxin-lipid interaction that hypothetically could result in transmembrane ionic channel formation. Several pieces of evidence support the action of both toxins in insects and mammals. The question is to what extent these Bt/Bti plasmid-encoded Cry or Cyt genes can be applied as bioinsecticides individually or in combination with Lysinibacillus sphaericus. The feasibility of being considered a promising and safe biological strategy for crop pests and vector-borne neglected infectious diseases is an issue pinpointed in the present review.

Utility of Cry1Ja for Transgenic Insect Control.

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

Bioefficacy and molecular characterization of Bacillus thuringiensis strain NBAIR BtGa against greater wax moth, Galleria mellonella L.

Galleria mellonella, the greater wax moth has always been an important pest against honeybees and has remained a nightmare for beekeeping farmers. Management of G. mellonella in live honeybee colonies is very difficult because most current management practices can destroy whole honeybee colonies. In the present study, experiments were conducted to isolate and characterize Bacillus thuringiensis from infected greater wax moth cadavers and to evaluate their biocontrol ability against G. mellonella. The bioefficacy of these isolates has been evaluated against greater wax moth along with the standard strain HD-1. Among all the strains tested, NBAIR BtGa demonstrated higher efficacy compared to other strains, with an LC50 value of 125.17µg/ml, whereas HD-1 exhibited a significantly higher LC50 value of 946.61µg/ml. Considering the economic importance of NBAIR BtGa we performed whole genome sequencing of this strain resulting in the identification of a genome size of 5.96Mb consisting of 6888 protein-coding genes. Gene ontology analysis categorized these genes into three groups based on their roles, i.e., biological functions (2169 genes), cellular components (1900 genes), and molecular functions (2774 genes). Through insecticidal toxicity-related genes (ITRG) profiling of our strain across the genome by Bt toxin scanner and cry processor resulted in the identification of several Cry proteins namely Cry1Ab11, Cry1Ia44, Cry1Aa2, Cry2Af1, Cry1Da2, Cry1Eb1, Cry1Ab5, Cry1Cb2, Cry1Ac2. Besides Cry proteins, other ITRG genes, viz. Vip3Bb2, Zwittermicin A resistance proteins, Chitinase C, Mpp46Ab1, immune inhibitor A, Bmp1, Vpb4Ca1, and Spp1Aa1 were also reported, which show toxicity against lepidopteran pests. The studies were also conducted to test the biosafety of Bt toxins against honeybee larvae and adults, which showed strain NBAIR BtGa was more than 99% safer for honeybee larvae as well as adults. Thus, the data generated ascertains its effectiveness as a biocontrol agent and it can be used further for the development of bio formulation for the management of G. mellonella in honeybee colonies.

Synergism of Cry1 Toxins by a Fusion Protein Derived from a Cadherin Fragment and an Antibody Peptide.

Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

An overview of the production and use of Bacillus thuringiensis toxin.

The widespread utilization of traditional chemical pesticides has given rise to numerous negative impacts, leading to a surge in interest in exploring environmentally friendly alternatives. Bacillus thuringiensis (Bt), a bacterium renowned for its insecticidal properties, produces Cry proteins during its lifecycle. These proteins have distinct advantages over traditional chemical pesticides, including higher environmental safety, broader insecticidal spectra, and lower pesticide residues. Consequently, the discovery and application of Bt hold immense significance in plant disease and pest management, as well as in plant protection. Currently, Bt preparations occupy a prominent position as the world's largest and most widely used biopesticides. This article comprehensively reviews the fundamental aspects, insecticidal mechanisms, practical applications, and fermentation technologies related to Bt.

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Screening of indigenous Bacillus thuringiensis for dipteran active cry gene profiles and potential toxicity against melon fruit fly, Zeugodacus cucurbitae (Coquillett)

BackgroundMelon fruit fly, Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae), is a devastating polyphagous pest attacking large number of fruits and vegetables causing huge economic yield losses across the world. Management of this notorious pest is very challenging as the larvae feed inside the fruit. Hence, the present research study aimed to screen the indigenous Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strains causing toxicity to larvae and to identify the insecticidal toxicity-related genes present in respective strains. In the present study, 50 indigenous B. thuringiensis (Bt) strains along with one reference strain were screened against second-instar larvae Z. cucurbitae. All the strains were analyzed for presence of 21 dipteran active cry genes.ResultsMortality in Z. cucurbitae larvae due to Bt strains ranged from 16 to 92%. PCR results revealed that each strain tested positive for a minimum of three cry genes to maximum of nine cry genes. Among the cry genes, cry1A, cry2A, cry1C, cry19, cry11 and cry70 were detected in high frequency of 100, 88, 84, 74, 58 and 54%, respectively. Bioassay studies revealed that ten out of fifty strains displayed more than 50% mortality. Hence, these ten strains, along with the reference strain, were further tested for mortality for the calculation of the median lethal concentration (LC50). The LC50 values ranged between 38.48 and 105.18 μg/ml. The lowest LC50 found for the strain NBAIR Bt107 was 38.48 μg/ml and was on par with the reference strain (Bti 4Q1) (31.3 μg/ml).ConclusionIndigenous Bt strains displayed a toxicity against the larvae of Z. cucurbitae. The probable dipteran active cry genes responsible for toxicity were interpreted. Thus, the Cry toxins from Bt can play a very important role in the management of Z. cucurbitae.

Continued decline in sublethal effects of Bt toxins on Helicoverpa zea (Lepidoptera: Noctuidae) in field corn.

The majority of field corn, Zea mays L., in the southeastern United States has been genetically engineered to express insecticidal toxins produced by the soil bacterium, Bacillus thuringiensis (Bt). Field corn is the most important mid-season host for corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), which has developed resistance to all Cry toxins in Bt corn. From 2020 to 2023, corn earworm pupae were collected from early- and late-planted pyramided hybrids expressing Bt toxins and non-Bt near-isolines in North and South Carolina (16 trials). A total of 5,856 pupae were collected across all trials, with 55 and 88% more pupae collected in later-planted trials relative to early plantings in North and South Carolina, respectively. Only 20 pupae were collected from hybrids expressing Cry1F + Cry1Ab + Vip3A20 across all trials. Averaged across trials, Cry1A.105 + Cry2Ab2 hybrids reduced pupal weight by 6 and 9% in North and South Carolina, respectively, relative to the non-Bt near-isoline. Cry1F + Cry1Ab hybrids reduced pupal weight on average by 3 and 8% in North and South Carolina, respectively, relative to the non-Bt near-isoline. The impact of the Bt toxins on pupal weight varied among trials. When combined with data from 2014 to 2019 from previous studies, a significant decline in the percent reduction in pupal weight over time was found in both states and hybrid families. This study demonstrates a continued decline in the sublethal impacts of Bt toxins on corn earworm, emphasizing the importance of insect resistance management practices.

Positive selection analysis of Cyt proteins from Bacillus thuringiensis: A conservative trend driven by negative (purifying) selection

Bacillus thuringiensis is a Gram-positive entomopathogenic bacterium that produces different pesticidal proteins: vegetative insecticidal proteins (Vpb1/Vpa2, Vip3, and Vpb4) during vegetative growth, which are secreted to the culture medium, and δ–endotoxins (Cry and Cyt) during sporulation, which accumulate into parasporal crystals. Cyt proteins are the smaller subset of δ–endotoxins targeting Diptera species. While Cry and Vip3 proteins undergo positive selection, our analysis suggests that Cyt proteins evolve following a conservative trend driven negative (purifying) selection.

A Shared Receptor Suggests a Common Ancestry between an Insecticidal Bacillus thuringiensis Cry Protein and an Anti-Cancer Parasporin.

Cry toxins, produced by the bacterium Bacillus thuringiensis, are of significant agronomic value worldwide due to their potent and highly specific activity against various insect orders. However, some of these pore-forming toxins display specific activity against a range of human cancer cells whilst possessing no known insecticidal activity; Cry41Aa is one such toxin. Cry41Aa has similarities to its insecticidal counterparts in both its 3-domain toxic core structure and pore-forming abilities, but how it has evolved to target human cells is a mystery. This work shows that some insecticidal Cry toxins can enhance the toxicity of Cry41Aa against hepatocellular carcinoma cells, despite possessing no intrinsic toxicity themselves. This interesting crossover is not limited to human cancer cells, as Cry41Aa was found to inhibit some Aedes-active Cry toxins in mosquito larval assays. Here, we present findings that suggest that Cry41Aa shares a receptor with several insecticidal toxins, indicating a stronger evolutionary relationship than their divergent activities might suggest.

Silence of Aminopeptidase N 2gene reveals the trade-offs for acquiring Cry1Ac resistance in Plutella xylostella.

Bacillus thuringiensis (Bt) is widely used as a biopesticide worldwide. To date, at least eight pest species have been found to be resistant to Bt in the field. As the first pest that was reported having resistance to Bt in the field, considerable research has been done on the mechanisms of Bt resistance in Plutella xylostella. However, whether the acquisition of Bt resistance by P. xylostella comes at a fitness cost is also a valuable question. In this study, Aminopeptidase-N 2 (APN2), a Cry toxin receptor gene of P. xylostella, was knocked down by RNA interference, resulting in improved resistance to Cry1Ac. It was also found that larval mortality of APN2 knockdown P. xylostella was significantly higher than that of the control, while the pupation rate, pupal weight, eclosion rate, fecundity (egg/female), hatchability, and female adult longevity were significantly lower in APN2 knockdown P. xylostella than in the control. These results illustrate that if Cry1Ac resistance was obtained only through the reduction of APN2 expression, P. xylostella would need to incur some fitness costs for it.

Exploring the Therapeutic Potential of Benzoxazolone Derivatives on the Circadian Clock: An In Silico and Hypothetical Approach

Objective: Pain conditions exhibit variations linked to circadian rhythms. Circadian rhythms, regulated by clock proteins, impact melatonin levels and immune functions. Melatonin, structurally similar to indomethacin, a non-steroidal anti-inflammatory drug, serves as an alternative in pain-related conditions. Literature suggests a correlation between melatonin-dependent regulation of circadian rhythm and reductions in pain complaints. 2(3<i>H</i>)-Benzoxazolone, known for its anti-inflammatory and analgesic properties, is a promising scaffold for drug design. In this study, pharmacophore analysis focused on benzoxazolone derivatives, evaluating their impact on clock proteins, and providing insights into potential chronotherapeutic implications. Methods: Molecular docking and dynamics simulations were conducted on CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 clock proteins using benzoxazolone and its 5-substituted derivatives. Molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) calculations were employed to analyze binding free energies. Benzoxazolone derivatives, especially 5-nitro-2-benzoxazolone and 5-fluoro-2-benzoxazolone, exhibited elevated binding affinities on clock proteins compared to melatonin and indomethacin, the reference molecules. The interaction profiles and stability of complexes were maintained throughout the simulations. Results: The results suggest a regulatory impact on CRY proteins, emphasizing their role in circadian rhythm and pain modulation. In addition, the benzoxazolone ring and its derivatives, structurally resembling the core structure of melatonin and indomethacin, demonstrate promising affinities on clock proteins. Conclusion: These findings provide preliminary data and hypothetically propose benzoxazolone derivatives as potential candidates for dual functionality in analgesic activity and circadian rhythm regulation, warranting further in vitro and clinical investigations.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

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.

Cry Toxins from <i>Bt</i> Bollgard II® Cotton Seeds: An Affordable Source for Insect Bioassays

The relative expression of Bacillus thuringiensis (Bt) Cry1Ac and Cry2Ab toxins from seeds of Bt Bollgard II® cotton was quantified using Enzyme Linked Immunosorbent Assays (ELISA) method. The ELISA technique involves binding a sample or calibrator to a solid-phase antibody, forming a "sandwich complex" with an enzyme-labeled antibody. The resulting enzyme substrate reaction produces a product proportional to the antigen concentration. The study reveals that each gram of Bt Bollgard II® seed powder contains 3.25 μg Cry1Ac and 182.32 μg Cry2Ab, with Cry2Ab expression 56 times higher. Variability in Cry protein expression is influenced by factors like temperature, genetic background, and plant maturity. The research emphasizes the importance of Bt plants, particularly Bt Bollgard II® seed powder as a valuable source for studying insect responses to combined Cry1Ac + Cry2Ab expression in bioassay studies.

Biocidal Activity of the Bacillus thuringiensis 3D Cry Toxins, Molecular Crosstalk at the Insect Midgut with Implication in Insect Resistance Development

Biocidal Activity of the Bacillus thuringiensis 3D Cry Toxins, Molecular Crosstalk at the Insect Midgut with Implication in Insect Resistance Development