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The expression of Bacillus thuringiensis (Bt) toxins in transgenic cotton confers resistance to insect pests. However, it has been demonstrated that its effectiveness varies among cotton cultivars and different tissues. In this study, we evaluated the expression of Bt protein in 28 cotton cultivars and selected 7 cultivars that differed in Bt protein expression for transcriptome analysis. Based on their Bt protein expression levels, the selected cultivars were categorized into three groups: H (high Bt protein expression), M (moderate expression), and L (low expression). In total, 342, 318, and 965 differentially expressed genes were detected in the H vs. L, M vs. L, and H vs. M comparison groups, respectively. And three modules significantly associated with Bt protein expression were identified by weighted gene co-expression network analysis. Three hub genes were selected to verify their relationships with Bt protein expression using virus-induced gene silencing (VIGS). Silencing GhM_D11G1176, encoding an MYC transcription factor, was confirmed to significantly decrease the expression of Bt protein. The present findings contribute to an improved understanding of the mechanisms that influence Bt protein expression in transgenic cotton. [ABSTRACT FROM AUTHOR]

The expression of Bacillus thuringiensis (Bt) toxins in transgenic cotton confers resistance to insect pests. However, it has been demonstrated that its effectiveness varies among cotton cultivars and different tissues. In this study, we evaluated the expression of Bt protein in 28 cotton cultivars and selected 7 cultivars that differed in Bt protein expression for transcriptome analysis. Based on their Bt protein expression levels, the selected cultivars were categorized into three groups: H (high Bt protein expression), M (moderate expression), and L (low expression). In total, 342, 318, and 965 differentially expressed genes were detected in the H vs. L, M vs. L, and H vs. M comparison groups, respectively. And three modules significantly associated with Bt protein expression were identified by weighted gene co-expression network analysis. Three hub genes were selected to verify their relationships with Bt protein expression using virus-induced gene silencing (VIGS). Silencing GhM_D11G1176, encoding an MYC transcription factor, was confirmed to significantly decrease the expression of Bt protein. The present findings contribute to an improved understanding of the mechanisms that influence Bt protein expression in transgenic cotton.

Simple Summary: Corn and cotton that produce insecticidal toxins derived from Bacillus thuringiensis (Bt) are widely adopted in the United States to control corn earworm/cotton bollworm, Helicoverpa zea (Boddie), which has resulted in major benefits to growers and the general public. However, resistance evolution in H. zea populations has become a major threat to the sustainability of these crops. Bt sweet corn producing the same toxins as Bt field corn is more attractive to H. zea than field corn and, thus, can function as a sentinel plant to detect early stages of resistance. As part of an existing sentinel monitoring network, this study evaluated changes in H. zea resistance during 2020–2022 by estimating the phenotypic and resistance allele frequencies for toxins in sentinel Bt corn. Transgenic corn and cotton that produce Cry and Vip3Aa toxins derived from Bacillus thuringiensis (Bt) are widely planted in the United States to control lepidopteran pests. The sustainability of these Bt crops is threatened because the corn earworm/bollworm, Helicoverpa zea (Boddie), is evolving a resistance to these toxins. Using Bt sweet corn as a sentinel plant to monitor the evolution of resistance, collaborators established 146 trials in twenty-five states and five Canadian provinces during 2020–2022. The study evaluated overall changes in the phenotypic frequency of resistance (the ratio of larval densities in Bt ears relative to densities in non-Bt ears) in H. zea populations and the range of resistance allele frequencies for Cry1Ab and Vip3Aa. The results revealed a widespread resistance to Cry1Ab, Cry2Ab2, and Cry1A.105 Cry toxins, with higher numbers of larvae surviving in Bt ears than in non-Bt ears at many trial locations. Depending on assumptions about the inheritance of resistance, allele frequencies for Cry1Ab ranged from 0.465 (dominant resistance) to 0.995 (recessive resistance). Although Vip3Aa provided high control efficacy against H. zea, the results show a notable increase in ear damage and a number of surviving older larvae, particularly at southern locations. Assuming recessive resistance, the estimated resistance allele frequencies for Vip3Aa ranged from 0.115 in the Gulf states to 0.032 at more northern locations. These findings indicate that better resistance management practices are urgently needed to sustain efficacy the of corn and cotton that produce Vip3Aa. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) Cyt1Aa toxin shows toxicity to mosquitoes, to certain coleopteran pests and also to red blood cells (RBC). However, its mode of action in the different target cells is not well defined. This protein is a single α-β domain pore-forming toxin, where a β sheet is wrapped by two α-helices layers. The Cyt1Aa α-helix hairpin in the N-terminal has been proposed to be involved in initial membrane binding and oligomerization, while the β sheet inserts into the membrane to form a pore that lyze the cells. To determine the role of the N-terminal α-helix hairpin region of Cyt1Aa in its mode of action, we characterized different single point mutations located in helices α-1 and α-2. Eight cysteine substitutions in different residues were produced in Bt, and we found that three of them: Cyt1AaA65C, Cyt1AaL85C and Cyt1AaN89C, lost insecticidal toxicity against Aedes aegypti larvae but retained similar or increased hemolytic activity towards rabbit RBC. Analysis of toxin binding and oligomerization using Ae. aegypti midgut brush border membrane vesicles showed that the three Cyt1Aa mutants non-toxic to Ae. aegypti were affected in oligomerization. However, these mutants were still hemolytic. Our data shows that oligomerization of Cyt1Aa toxin is essential for its toxicity to Ae. aegypti but not for its toxicity against RBC indicating that the mode of action of Cyt1Aa is different in these distinct target membranes.

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

Aedes cadherin (AaeCad, AAEL024535) has been characterized as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins. However, its role in development is still unknown. In this study, we modified the cadherin gene using ZFN and TALEN. Even though we obtained heterozygous deletions, no homozygous mutants were viable. Because ZFN and TALEN have lower off-targets than CRISPR/Cas9, we conclude the cadherin gene is essential for Aedes development. In contrast, in lepidopteran insects loss of a homologous cadherin does not appear to be lethal, since homozygous mutants are viable. To analyze the role of AaeCad in vivo, we tagged this protein with EGFP using CRISPR-Cas9-mediated homologous recombination and obtained a homozygous AaeCad-EGFP line. Addition of Aedes Rad51 mRNA enhanced the rate of recombination. We then examined AaeCad protein expression in most tissues and protein dynamics during mosquito development. We observe that AaeCad is expressed in larval and adult midgut-specific manner and its expression pattern changed during the mosquito development. Confocal images showed AaeCad has high expression in larval caecae and posterior midgut, and also in adult midgut. Expression of AaeCad is observed primarily in the apical membranes of epithelial cells, and not in cell-cell junctions. The expression pattern observed suggests AaeCad does not appear to play a role in these junctions. However, we cannot exclude its role beyond cell-cell adhesion in the midgut. We also observed that Cry11A bound to the apical side of larval gastric caecae and posterior midgut cells exactly where AaeCad-EGFP was expressed. Their co-localization suggests that AaeCad is indeed a receptor for the Cry11A toxin. Using this mosquito line we also observed that low doses of Cry11A toxin caused the cells to slough off membranes, which likely represents a defense mechanism, to limit cell damage from Cry11A toxin pores formed in the cell membrane.

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

Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry11Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry11Aa, Cry11Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry11Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show thatCry11Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry11Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry11Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry11Aa and Cry11Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Leopoldo Palma reports financial support was provided by University of Valencia. Leopoldo Palma reports a relationship with University of Valencia that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Sudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of Bacillus thuringiensis (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020-2021, two Bt transgenic cotton varieties, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20 °C) with different durations (12 h, 24 h and 48 h) were applied during the peak boll-setting period., Results: As the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the threshold temperature which caused a significant reduction in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17 °C to 19 °C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both free amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content., Conclusion: In summary, when the daily average temperature was ≤ 19 °C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production., (© 2024. The Author(s).)

Corn, Zea mays L. (Poales: Poaceae), growers in the US Cotton Belt are required to plant 20% of total corn acres to non-Bt hybrids for resistance management (non-Bt refuge). Most growers do not meet this requirement, in part, because they perceive non-Bt hybrids to yield less than Bt hybrids. We planted multiple non-Bt and Bt hybrids from a single company in small-plot replicated trials at a single location from 2019 to 2023, as well as in small-plot replicated trials at multiple locations during 2022 and 2023. In the single location, we measured kernel injury from corn earworm, Helicoverpa zea Boddie (Lepidoptera: Noctuidae), and we recorded yield at all locations. In the single location trial, yields only separated among hybrids in 3 out of 5 years. In the multiple location trial, yields were variable between both years. We found that Bt hybrids tended to yield higher than non-Bt hybrids overall, but this was influenced by the inclusion of non-Bt hybrids that had a lower overall genetic yield potential in the environments we tested them in. In both tests, when hybrids were analyzed during each year, both Bt and non-Bt hybrids were among the statistically highest yielders. Our study demonstrates the importance of comparing multiple Bt and non-Bt hybrids to draw yield comparisons. This highlights the need for corn seed company breeders to put effort into improving yield for non-Bt hybrids. Hopefully this effort will translate into increased planting of non-Bt refuge corn for growers in the US Cotton Belt., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America.)

Exopolysaccharides (EPSs) are carbohydrate polymers that are synthesized and secreted into the extracellular during the growth of microorganisms. Bacillus thuringiensis (Bt) is a type of entomopathogenic bacterium, that produces various insecticidal proteins and EPSs. In our previous study, the EPSs produced by Bt strains were first found to enhance the toxicity of insecticidal crystal proteins against Plutella xylostella . However, the response of the intestinal bacterial communities of P. xylostella under the action of EPSs is still unelucidated. In this study, 16S rRNA amplicon sequencing was used to characterize the intestinal bacterial communities in P. xylostella treated with EPSs alone, Cry1Ac protoxin alone, and both the Cry1Ac protoxin and EPSs. Compared with the control group, alpha diversity indices, the Chao1 and ACE indices were significantly altered after treatment with EPSs alone, and no significant difference was observed between the groups treated with Cry1Ac protoxin alone and Cry1Ac protoxin + EPSs. However, compared with the gut bacterial community feeding on Cry1Ac protoxin alone, the relative abundance of 31 genera was significantly changed in the group treated with Cry1Ac protoxin and EPSs. The intestinal bacteria, through the oral of Cry1Ac protoxin and EPSs, significantly enhanced the toxicity of the Cry1Ac protoxin towards the axenic P. xylostella . In addition, the relative abundance of the 16S rRNA gene in the chloroplasts of Brassica campestris decreased after adding EPSs. Taken together, these results show the vital contribution of the gut microbiota to the Bt strain-killing activity, providing new insights into the mechanism of the synergistic insecticidal activity of Bt proteins and EPSs.

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% (LC 90 ) 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., (© 2024 Society of Chemical Industry.)

Background: Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests for nearly 30 years. Dose of a Bt crop is key to assessing the risk of resistance evolution because it affects the heritability of resistance traits. Western corn rootworm (Diabrotica virgifera virgifera, LeConte), a major pest of maize, has evolved resistance to all commercially available Bt traits targeting it, and threatens resistance to future transgenic traits. Past research shows the dose of Bt maize targeting western corn rootworm can be confounded by larval density-dependent mortality. We conducted a 2-year field study at two locations to quantify larval density-dependent mortality in Bt and non-Bt maize. We used these results to calculate dose for our method and compared it to three previously published methods. Additionally, adult emergence and root injury were analyzed for predicting initial egg density., Results: Increased pest density caused greater proportions of larvae to die in Bt maize than in non-Bt maize. All methods for calculating dose produced values less than high-dose, and stochastic variation had the greatest impact on dose at high and low pest densities. Our method for calculating dose did not produce values positively correlated with pest density while the three other methods did., Conclusion: To achieve the most accurate calculation of dose for transgenic maize targeting western corn rootworm, density-dependent mortality should be taken into account for both transgenic and non-transgenic maize and assessed at moderate pest densities. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Genetically modified (GM) crops, expressing Bacillus thuringiensis (Bt) insecticidal toxins, have substantially transformed agriculture. Despite rapid adoption, their environmental and economic benefits face scrutiny due to unsustainable agricultural practices and the emergence of resistant pests like Spodoptera frugiperda, known as the fall armyworm (FAW). FAW's adaptation to Bt technology in corn and cotton compromises the long-term efficacy of Bt crops. To advance the understanding of the genetic foundations of resistance mechanisms, we conducted an exploratory comparative transcriptomic analysis of two divergent FAW populations. One population exhibited practical resistance to the Bt insecticidal proteins Cry1A.105 and Cry2Ab2, expressed in the genetically engineered MON-89Ø34 - 3 maize, while the other population remained susceptible to these proteins. Differential expression analysis supported that Cry1A.105 and Cry2Ab2 significantly affect the FAW physiology. A total of 247 and 254 differentially expressed genes were identified in the Cry-resistant and susceptible populations, respectively. By integrating our findings with established literature and databases, we underscored 53 gene targets potentially involved in FAW's resistance to Cry1A.105 and Cry2Ab2. In particular, we considered and discussed the potential roles of the differentially expressed genes encoding ABC transporters, G protein-coupled receptors, the P450 enzymatic system, and other Bt-related detoxification genes. Based on these findings, we emphasize the importance of exploratory transcriptomic analyses to uncover potential gene targets involved with Bt insecticidal proteins resistance, and to support the advantages of GM crops in the face of emerging challenges., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bacillus thuringiensis ( Bt ) toxins are potential alternatives to synthetic insecticides for the control of lepidopteran pests. However, the evolution of resistance in some insect pest populations is a threat and can reduce the effectiveness of Bt toxins. In this review, we summarize the results of 161 studies from 20 countries reporting field and laboratory-evolved resistance, cross-resistance, and inheritance, mechanisms, and fitness costs of resistance to different Bt toxins. The studies refer mainly to insects from the United States of America (70), followed by China (31), Brazil (19), India (12), Malaysia (9), Spain (3), and Australia (3). The majority of the studies revealed that most of the pest populations showed susceptibility and a lack of cross-resistance to Bt toxins. Factors that delay resistance include recessive inheritance of resistance, the low initial frequency of resistant alleles, increased fitness costs, abundant refuges of non- Bt, and pyramided Bt crops . The results of field and laboratory resistance, cross-resistance, and inheritance, mechanisms, and fitness cost of resistance are advantageous for predicting the threat of future resistance and making effective strategies to sustain the effectiveness of Bt crops.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis., (© 2024. The Author(s).)

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., (© 2024 Wiley Periodicals LLC.)

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC 50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T 0 and T 1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC 50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T 0 and T 1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Bacillus thuringiensis (Bt) and its crystal toxin or δ-endotoxins (Cry) offer great potential for the efficient control of crop pests. A vast number of pests can potentially infect the same host plant, either simultaneously or sequentially. However, no effective Bt-Cry protein has been reported to control both aphids and plant parasitic nematodes due to its highly specific activity., Results: Our study indicated that the Cry5Ba2 protein was toxic to the green peach aphid Myzus persicae, which had a median lethal concentration (LC 50 ) of 9.7 ng μL -1 and fiducial limits of 3.1-34.6 ng μL -1 . Immunohistochemical localization of Cry5Ba2 revealed that it could bind to the apical tip of microvilli in midgut regions. Moreover, transgenic tobacco plants expressing Cry5Ba2 exhibited significant resistance to Myzus persicae, as evidenced by reduced insect survival and impaired fecundity, and also intoxicated the Meloidogyne incognita as indicated by a decrease in galls and progeny reproduction., Conclusion: In sum, we identified a new aphicidal Bt toxin resource that could simultaneously control both aboveground and belowground pests, thus extending the application range of Bt-based strategy for crop protection. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Transgenic corn and cotton that produce Cry and Vip3Aa toxins derived from Bacillus thuringiensis (Bt) are widely planted in the United States to control lepidopteran pests. The sustainability of these Bt crops is threatened because the corn earworm/bollworm, Helicoverpa zea (Boddie), is evolving a resistance to these toxins. Using Bt sweet corn as a sentinel plant to monitor the evolution of resistance, collaborators established 146 trials in twenty-five states and five Canadian provinces during 2020–2022. The study evaluated overall changes in the phenotypic frequency of resistance (the ratio of larval densities in Bt ears relative to densities in non-Bt ears) in H. zea populations and the range of resistance allele frequencies for Cry1Ab and Vip3Aa. The results revealed a widespread resistance to Cry1Ab, Cry2Ab2, and Cry1A.105 Cry toxins, with higher numbers of larvae surviving in Bt ears than in non-Bt ears at many trial locations. Depending on assumptions about the inheritance of resistance, allele frequencies for Cry1Ab ranged from 0.465 (dominant resistance) to 0.995 (recessive resistance). Although Vip3Aa provided high control efficacy against H. zea, the results show a notable increase in ear damage and a number of surviving older larvae, particularly at southern locations. Assuming recessive resistance, the estimated resistance allele frequencies for Vip3Aa ranged from 0.115 in the Gulf states to 0.032 at more northern locations. These findings indicate that better resistance management practices are urgently needed to sustain efficacy the of corn and cotton that produce Vip3Aa.

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 (IC 50 ) values of 0.12-9.86 μg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC 50 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

BackgroundAlthough much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion.ResultsIn this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses.ConclusionsThis study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae.

Abstract Biopesticides based on the entomopathogenic bacterium, Bacillus thuringiensis toxins, have shown high ability to control several pests belonging to order Lepidoptera. Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera:Noctuidae), is a major pest that attacks various crops in Egypt, and Cry1C toxin-based formulations are heavily used to its control. A laboratory investigation was conducted to study the resistance development in S. littoralis against Cry1C toxin. Thus, a field strain of S. littoralis was selected and maintained under laboratory conditions for 12 generations. The selection pressure resulted in 32.12-fold of resistance ratio after 12 generations. The hydrolysis of Cry1C toxin by the midgut extracts of the susceptible line, and the resistant line was compared. Results revealed that Cry1C toxin was hydrolyzed more rapidly in the resistant line than the susceptible one. The Cry1C toxin was processed till 1 hour after incubation in the susceptible line; but in case of resistant line, the Cry1C toxin was degraded in 1 hour after incubation.

To illustrate the impact of genetically modified (GM) Bt maize on the natural enemy communities in the Huang-Huai-Hai summer maize-growing region in China, the abundance of 7 common predator taxa (Geocoris pallidipennis Costa, Harmonia axyridis (Pallas), lacewings, Orius sauteri (Poppius), Propylea japonica (Thunberg), spiders, and Staphylinidae) was quantitatively evaluated by comparing Bt-Cry1Ab DBN9936 and Bt-Cry1Ab/Cry2Aj Ruifeng 125 events to their near non-Bt isolines during the growing season from 2016 to 2019. A total of 10,302, 19,793, 13,536, and 5,672 individuals were observed during 4 years, and the abundance of each taxa on Bt maize varied between sample dates among those arthropod taxa. Shannon-Wiener diversity index of predator communities from 7 taxa showed very similar temporal dynamics and principal response curve analyses to examine community-level effects showed no significant differences in predator abundance in Bt maize compared with non-Bt maize. We conclude that the 2 Bt maize hybrids did not adversely affect the predator community in the Huang-Huai-Hai summer maize-growing region of China., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the Americas, transgenic crops producing insecticidal proteins from Bacillus thuringiensis Berliner (Bt, Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.) rapidly evolved in some FAW populations, pyramided Bt maize hybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Transgenic crops producing Bacillus thuringiensis (Bt) toxins are commonly used for controlling insect pests. Nearby refuges of non-Bt host plants play a central role in delaying the evolution of resistance to Bt toxins by pests. Pervasive fitness costs associated with resistance, which entail lower fitness of resistant than susceptible individuals in refuges, can increase the ability of refuges to delay resistance. Moreover, these costs are affected by environmental factors such as host plant suitability, implying that manipulating refuge plant suitability could improve the success of the refuge strategy. Based on results from a previous study of Trichoplusia ni resistant to Bt sprays, it was proposed that low-suitability host plants could magnify costs. To test this hypothesis, we investigated the association between host plant suitability and fitness costs for 80 observations from 30 cases reported in 18 studies of 8 pest species from 5 countries. Consistent with the hypothesis, the association between plant suitability and fitness cost was negative. With plant suitability scaled to range from 0 (low) to 1 (high), the expected cost was 20.7% with a suitability of 1 and the fitness cost increased 2.5% for each 0.1 decrease in suitability. The most common type of resistance to Bt toxins involves mutations affecting a few types of midgut proteins to which Bt toxins bind to kill insects. A better understanding of how such mutations interact with host plant suitability to generate fitness costs could be useful for enhancing the refuge strategy and sustaining the efficacy of Bt crops., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1B M and Cry1C M ; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing Cry M genes, and to remove or alter sequences that might adversely affect their activity in plants., Results: To assess the insecticidal efficacy of the Cry1B M /Cry1C M genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1B M /Cry1C M expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1B M /Cry1C M expression. Protein accumulation for Cry1C M ranged from 5.18 to 176.88 µg Cry1C M /g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1B M /Cry1C M genes, with a similar range of Cry1C M transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1B M /Cry1C M genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves., Conclusions: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1 M genes in GM crop plants in the future., (© 2024. The Author(s).)

Competing Interests: Declaration of competing interest No financial support was provided for the preparation of this article.

Background: Insect-resistance genetically modified (GM) plants derived from Bacillus thuringiensis (Bt) have been cultivated to control pests, but continuous cultivation of Bt-transgenic plants at large-scale regions leads to the resistance evolution of target insects to transgenic plants. RNA interference (RNAi) technology is considered an effective strategy in delaying the resistance evolution of target insects., Results: We here developed a single transgenic oilseed rape (Brassica napus) line with hairpin RNA of the chitin-synthase 1 gene (CHS1) of Plutella xylostella (hpPxCHS1) and a pyramid transgenic B. napus line harboring hpPxCHS1 and Bt gene (Cry1Ac). Escherichia coli HT115 delivered hpPxCHS1 showed negative effects on the growth of P. xylostella. The single transgenic and pyramid transgenic B. napus significantly reduced the larval weight and length of P. xylostella and increased its lethality rate, with down-regulation expression of the PxCHS1 gene in insects., Conclusion: Compared to Bt-transgenic B. napus, pyramid-transgenic B. napus shorted the mortality time of P. xylostella, indicating that RNAi technology synergistic with Bt protein improves the effectiveness of controlling target insects. Our results proved that RNAi can delay the resistance evolution of target insects to Bt-transgenic plants. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets., Results: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure., Conclusion: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Insecticidal Bacillus thuringiensis Berliner (Bt) toxins produced by transgenic cotton (Gossypium hirsutum L.) plants have become an essential component of cotton pest management. Bt toxins are the primary management tool in transgenic cotton for lepidopteran pests, the most important of which is the bollworm (Helicoverpa zea Boddie) (Lepidoptera: Noctuidae) in the United States (U.S.). However, bollworm larvae that survive after consuming Bt toxins may experience sublethal effects, which could alter interactions with other organisms, such as natural enemies. Experiments were conducted to evaluate how sublethal effects of a commercial Bt product (Dipel) incorporated into artificial diet and from Bt cotton flowers impact predation from the convergent lady beetle (Hippodamia convergens Guérin-Méneville) (Coleoptera: Coccinellidae), common in cotton fields of the mid-southern U.S. Sublethal effects were detected through reduced weight and slower development in bollworm larvae which fed on Dipel incorporated into artificial diet, Bollgard II, and Bollgard 3 cotton flowers. Sublethal effects from proteins incorporated into artificial diet were found to significantly alter predation from third instar lady beetle larvae. Predation of bollworm larvae also increased significantly after feeding for three days on a diet incorporated with Bt proteins. These results suggest that the changes in larval weight and development induced by Bt can be used to help predict consumption of bollworm larvae by the convergent lady beetle. These findings are essential to understanding the potential level of biological control in Bt cotton where lepidopteran larvae experience sublethal effects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Bacillus thuringiensis (Bt) toxins have provided exceptional control of agricultural insect pests, however, over reliance on the proteins would potentially contribute to the development of field tolerance. Developing new sustainable insect pest control methods that target the mechanisms underlying Bt tolerance can potentially support the Bt control paradigm while also providing insights into basic insect physiology. The MAPK p38 pathway is strongly associated with Bt tolerance in Chilo suppressalis, a major pest of rice. To gain insights into how this pathway impacts tolerance, high-throughput screening of C. suppressalis larval midguts initially identified eight novel target genes. Increased larval sensitivity to the transgenic cry1Ca rice strain T1C-19 was observed following RNA interference-mediated knockdown of four of the genes, Cscnc, Csgcp, Cszfp26 and CsZMYM1. Similar enhanced sensitivity to the TT51 (expressing Cry1Ab/1Ac) and T2A-1 (expressing Cry2Aa) transgenic rice lines occurred when Cszfp26 and CsZMYM1 were knocked down. All four target genes are downstream of the MAPK p38 pathway but do not participate in negative feedback loop of the pathway. These results implicate Cscnc, Csgcp, Cszfp and CsZMYM1 in the C. suppressalis transgenic cry1Ca rice tolerance mechanism regulated by MAPK p38. These findings further enhance our understanding of the MAPK p38-dependent molecular mechanisms underlying Bt tolerance in C. suppressalis and open new avenues of tolerance management to develop., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins., Competing Interests: Declaration of competing interest Leilei Liu reports financial support was provided by the National Natural Science Foundation of China. Leilei Liu reports financial support was provided by the Natural Science Foundation of Hubei Province, China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Bacillus cereus sensu stricto ( s.s .) species comprises strains of biovar Thuringiensis ( Bt ) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins, also known as Bt toxins, or (ii) based on the presence of cry , cyt , and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt identification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90).IMPORTANCEStrains of Bacillus cereus sensu stricto ( s.s .) biovar Thuringiensis ( Bt ) are used as organic biopesticides . Bt is differentiated from the foodborne pathogen Bacillus cereus s.s . by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of whole-genome sequencing (WGS)-based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, identification of pesticidal sequences performed best overall at WGS-based Bt identification., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cadherin plays an important role in the toxicity of Bacillus thuringiensis Cry proteins. We previously cloned a full-length cadherin from Aedes aegypti larvae and reported this protein binds Cry11Aa toxin from B. thuringiensis subsp. israelensis with high affinity, ≈16.7nM. Based on these results, we investigated if Aedes cadherin is involved in the in vivo toxicity of Cry11Aa toxin to Ae. aegypti. We established a mosquito cell line stably expressing the full-length Aedes cadherin and transgenic mosquitoes with silenced Aedes cadherin expression. Cells expressing the Aedes cadherin showed increased sensitivity to Cry11Aa toxin. Cry11Aa toxin at 400nM killed approximately 37% of the cells in 3h. Otherwise, transgenic mosquitoes with silenced Aedes cadherin expression showed increased tolerance to Cry11Aa toxin. Furthermore, cells expressing Aedes cadherin triggered Cry11Aa oligomerization. These results show the Aedes cadherin plays a pivotal role in Cry11Aa toxicity to Ae. aegypti larvae by mediating Cry11Aa oligomerization. However, since high toxicity was not obtained in cadherin-expressing cells, an additional receptor may be needed for manifestation of full toxicity. Moreover, cells expressing Aedes cadherin were sensitive to Cry4Aa and Cry11Ba, but not Cry4Ba. However transgenic mosquitoes with silenced Aedes cadherin expression showed no tolerance to Cry4Aa, Cry4Ba, and Cry11Ba toxins. These results suggest that while Aedes cadherin may mediate Cry4Aa and Cry11Ba toxicity, this cadherin but is not the main receptor of Cry4Aa, Cry4Ba and Cry11Ba toxin in Ae. aegypti.

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.

Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.

Bacillus thuringiensis Cyt proteins are pore-forming toxins that have insecticidal activity mainly against dipteran insects. However, certain Cyt proteins have toxicity to some insect orders, but not toxicity of Cyt1Aa against lepidopteran larvae has been found. Insect specificity has been proposed to rely in specific binding to certain lipids on the brush border membrane of midgut cells since no protein receptors have been described so far. To determine the molecular basis of Cyt1Aa insect specificity we compared different steps of Cyt1Aa mode of action in a susceptible insect as the dipteran Aedes aegypti and also in the non-susceptible lepidopteran Manduca sexta. Our data shows that the lack toxicity of Cyt1Aa to M. sexta larvae does not rely on protoxin processing, membrane binding interaction, and oligomerization of Cyt1Aa since these steps were similar in the two insect species analyzed.

The Cry11Aa protein produced in Bacillus thuringiensis subsp. israelensis, a bacterial strain used worldwide for the control of Aedes aegypti larvae, binds midgut brush border membrane vesicles (BBMV) with an apparent K(d) of 29.8 nM. Previously an aminopeptidase N (APN), named AaeAPN2, was identified as a putative Cry11Aa toxin binding protein by pull-down assays using biotinylated Cry11Aa toxin (Chen et al., 2009. Insect Biochem. Mol. Biol. 39, 688-696). Here we show this protein localizes to the apical membrane of epithelial cells in proximal and distal regions of larval caeca. The AaeAPN2 protein binds Cry11Aa with high affinity, 8.6 nM. The full-length and fragments of AaeAPN2 were cloned and expressed in Escherichia coli. The toxin-binding region was identified and further competitive assays demonstrated that Cry11Aa binding to BBMV was efficiently competed by the full-length AaeAPN2 and the fragments of AaeAPN2b and AaeAPN2e. In bioassays against Ae. aegypti larvae, the presence of full-length and a partial fragment (AaeAPN2b) of AaeAPN2 enhanced Cry11Aa larval mortality. Taken together, we conclude that AaeAPN2 is a binding protein and plays a role in Cry11Aa toxicity.

Bacillus thuringiensis produces insecticidal Cry and Cyt proteins that are toxic to different insect orders. In addition, Cyt toxins also display haemolytic activity. Both toxins are pore-forming proteins that form oligomeric structures that insert into the target membrane to lyse cells. Cyt toxins play an important role in mosquitocidal activity since they synergize Cry toxins and are able to overcome resistance to Cry toxins. Cry and Cyt toxins interact by specific epitopes, and this interaction is important to induce the synergistic activity observed. It was proposed that Cyt toxins do not interact with protein receptors but directly interacting with the specific midgut cell lipids. Here, we analysed if oligomerization and membrane insertion of Cyt1Aa are necessary steps to synergize Cry11Aa toxicity. We characterized Cyt1Aa helix α-C mutants that were affected in oligomerization, in membrane insertion and also in haemolytic and insecticidal activities. However, these mutants were still able to synergize Cry11Aa toxicity indicating these steps are independent events of Cyt1Aa synergistic activity. Furthermore, the data indicate that formation of stable Cyt1Aa-oligomeric structure is a key step for membrane insertion, haemolysis and insecticidal activity.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the K D values between the toxins and BmABCC1 and BmABCC4 were 10 -5 to 10 -9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae., (© 2024. The Author(s).)

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.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p