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Widespread use of genetically engineered maize targeting the corn rootworm complex (Diabrotica species) has raised concerns about insect resistance. Twelve years of university field trial and farm survey data from 10 US Corn Belt states indicate that maize hybrids expressing toxins derived from the bacterium Bacillus thuringiensis (Bt maize) exhibited declining protection from rootworm feeding with increased planting while pest pressures simultaneously decreased. The analysis revealed a tendency to overplant Bt maize, leading to substantial economic losses; this was particularly striking in eastern Corn Belt states. Our findings highlight the need to go beyond the “tragedy of the commons” perspective to protect sustainable use of Bt and other crop biotechnology resources. We propose moving toward a more diversified and transparent seed supply. [ABSTRACT FROM AUTHOR]

Biopesticides, biological agents for pest control in plants, are becoming increasingly prevalent in agricultural practices. However, no established methodology currently exists to assess their quality, and there are currently no publicly available authenticity and purity evaluations of commercial products. This lack of data may represent risks because of their widespread dispersal in the environment. We evaluated the potential of whole genome sequencing (WGS) and metagenomics approaches, including nanopore long-read sequencing, to verify both authenticity (i.e., the labeled strain) and biological purity (i.e., the absence of any undesired genetic material) of commercial Bacillus thuringiensis bioinsecticides. Four commercially available bioinsecticidal products containing Bacillus thuringiensis serovar kurstaki strain HD-1 were collected from the European market as a case study. Two sequencing approaches were employed: WGS of isolates and metagenomics sequencing of all genetic material in a product. To assess authenticity, isolate WGS data were compared against the publicly available reference genome of the expected strain. Antimicrobial resistance gene content, insecticidal gene content, and single nucleotide polymorphism differences were characterized to evaluate similarity to the reference genome. To assess purity, metagenomic sequencing data were analyzed using read classification and strain differentiation methods. Additionally, long- and short-read data were used to assess potential large-scale structural variations. Our results confirmed all investigated products to be authentic and pure. With the increasing usage of biopesticides, it is crucial to have adequate quality control methods. Our proposed approach could be adapted for other biopesticides, and similar products, providing a standardized and robust approach to contribute to biopesticide safety. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis exopolysaccharide BPS-2 inhibits malondialdehyde secretion, enhances antioxidant enzyme activities, and significantly improves the antioxidant status of inflammatory cells. In the present study, the apparent morphology and spatial conformation of BPS-2 were analyzed further, and several functional properties were investigated. The results demonstrated that BPS-2 was a polymeric straight-chain polysaccharide with good thermal stability, exhibiting non-Newtonian properties and good antioxidant and anticancer activities. Notably, this study systematically investigated the impact of BPS-2 on the intestinal microbiota composition in patients diagnosed with ulcerative colitis. Through in vitro fermentation of fecal bacteria collected from six volunteers, it was found that BPS-2 exerted a positive influence on the intestinal flora of ulcerative colitis patients, augmenting the secretion of short-chain fatty acids and facilitating an increase in the relative abundance of Bifidobacterium spp. These results suggest that BPS-2 has the potential to be a food additive for suppressing ulcerative colitis and for other medically related applications. [ABSTRACT FROM AUTHOR]

The insecticidal Cry proteins from Bacillus thuringiensis are used in biopesticides or transgenic crops for pest control. The Cry1I protein family has unique characteristics of being produced during the vegetative rather than sporulation phase, its protoxins forming dimers in solution, and exhibiting dual toxicity against lepidopteran and coleopteran pests. The Cry1Ia protoxin undergoes sequential proteolysis from the N- and C-terminal ends, producing intermediate forms with insecticidal activity, while in some cases, the fully processed toxin is inactive. We investigated the oligomerization and toxicity of Cry1Ia intermediate forms generated through trypsinization (T-Int) and larval gut fluid (GF-Int) treatments, as well as the fully trypsinized protein (toxin). Heterologously expressed intermediate forms assembled into oligomers and showed similar toxicity to Cry1Ia protoxin against Ostrinia nubilalis (European corn borer) larvae, while the toxin form was ~30 times less toxic. In contrast, bioassays with Leptinotarsa decemlineata (Colorado potato beetle) larvae did not show significant differences in toxicity among Cry1Ia protoxin, T-Int, GF-Int, and fully processed toxin. These results suggest that the Cry1I mode of action differs by insect order, with N-terminal cleavage affecting toxicity against lepidopteran but not coleopteran larvae. This knowledge is essential for designing pest control strategies using Cry1I insecticidal proteins. [ABSTRACT FROM AUTHOR]

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Debate over resistance management tactics for genetically engineered (GE) crops expressing insecticidal toxins is not new. For several decades, researchers, regulators, and agricultural industry scientists have developed strategies to limit the evolution of resistance in populations of lepidopteran and coleopteran pests. A key attribute of many of these events was insecticide resistance management (IRM) strategies designed around a presumed high-dose expression sufficient to kill 99.5% of exposed larvae for some of the main target pests in corn, Zea mays L. and cotton, Gossypium hirsutum L. In contrast, other target pests did not meet this high-dose criterion. Similarly, the recent release of ThryvOn cotton that expresses thysanopteran and hemipteran active Mpp51Aa2.834_16 toxin is not high dose, working on a combination of behavioral and sublethal effects to suppress populations. This unique mode of control has generated considerable uncertainty about what IRM strategies will be most effective to limit field-evolved resistance to this unique spectrum of pests. The goal of this manuscript is to present several knowledge gaps that exist in proposed Mpp51Aa2 IRM plans, focusing on its activity on thrips, Frankliniella spp. Addressing these gaps will be crucial to limit resistance and preserve the benefits that this technology may provide by alleviating reliance on conventional insecticides and seed treatments. Broadly, these considerations will be important for future GE events that are non-high dose but remain valuable components of a more holistic insect management programs that integrate multiple tactics to reduce conventional insecticide use for challenging pests. [ABSTRACT FROM AUTHOR]

The strategic use of refuge areas is a well-known method for delaying the development of pest resistance to Bacillus thuringiensis (Bt) crop. A lesser-known method to control against resistance development is sterile insect releases. In this article, an agent-based simulation model is used to test the effectiveness of combining the use of Bt sugarcane, refuge areas, and sterile insect releases as an integrated strategy against Eldana saccharinaWalker (Lepidoptera: Pyralidae) infestation and resistance development. Individual insects are modeled with their own genetic traits on a simulated sugarcane field that represents either Bt or refuge area.The model is applied to 2 hypothetical case studies. In the first experiment, resistance development and infestation dynamics in Bt sugarcane without refuge areas are considered using various sterile:wild sterile release ratios, and different release distributions. In the second experiment, the inclusion of a refuge area in Bt sugarcane is considered, using various sterile:wild releases ratios and different release distributions. A trade-off between sterile insect releases and the use of the refuge area was observed, and could, in some cases, work against each other. [ABSTRACT FROM AUTHOR]

Background: Aedes aegypti is the primary vector of dengue, chikungunya, and Zika viruses in Colombia. Various insecticides, including pyrethroid, organophosphate, and carbamate insecticides; growth regulators; and biological insecticides, such as Bacillus thuringiensis var. israelensis, have been used to control Ae. aegypti populations. However, organophosphates such as malathion, pirimiphos-methyl, and temephos have been used over the last decade owing to the high resistance to pyrethroids. Methods: This study assessed the susceptibility to organophosphates in 14 Ae. aegypti populations from the Córdoba department in northern Colombia. Moreover, possible resistance mechanisms were investigated by determining the activity levels of α-esterases, β-esterases, mixed function oxidases (MFOs), glutathione S-transferases (GSTs), and insensitive acetylcholinesterase (iAChE). Additionally, the Ace-1 gene was sequenced to identify mutations at the target site of action. Results: The populations were susceptible to temephos and malathion but resistant to fenitrothion, and in three of them, to pirimiphos-methyl. Alterations in the enzyme activity levels of α-esterases and β-esterases, GST, and iAChE were observed among the populations, with high enzyme activity levels of α and β esterases associated with resistance to fenitrothion. No mutations were identified in the Ace-1 gene. Conclusions: These findings are highly relevant for vector control programs in the region, as they allow for adjustments in resistance management strategies and improve the effectiveness of interventions against these arboviruses. [ABSTRACT FROM AUTHOR]

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS taxonomic units (TUs). The TUs in the QPS list were updated based on a verification, against their respective authoritative databases, of the correctness of the names and completeness of synonyms. Of 54 microorganisms notified to EFSA between April and September 2024 (33 as feed additives, 17 as food enzymes or additives, 4 as novel foods), 50 were not evaluated because: 12 were filamentous fungi, 1 was Enterococcus faecium and 8 were Escherichia coli (all excluded from the QPS evaluation), and 29 were TUs that already have a QPS status. One notification (Ensifer adhaerens) was already evaluated in a previous Panel Statement. Another notification (Enterococcus lactis) was already evaluated in the previous 3- year QPS cycle and was reassessed within this document. Two TUs were notified for the first time and were assessed for a possible QPS status: Serratia plymuthica and Lacticaseibacillus huelsenbergensis. Bacillus thuringiensis and Bacillus nakamurai have been assessed for a possible QPS status in response to internal requests. The following was concluded on the five assessed TUs. L. huelsenbergensis can be granted the QPS status based on its close relatedness to several other QPS Lacticaseibacillus species. E. lactis is not recommended for the QPS status due to insufficient information on safety. S. plymuthica and B. thuringiensis are not recommended for the QPS status due to safety concerns. B. nakamurai cannot be recommended for the QPS list due to a lack of body of knowledge for its use in the food and feed chain. [ABSTRACT FROM AUTHOR]

Genetically modified crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) are currently the most efficient and safest method of pest control worldwide. However, the prolonged planting period has led to a reduction in the efficacy of Bt crops due to the evolution of pest resistance in the field. This review paper examines the resistance status of lepidopteran pests to Bt crops under field conditions, elucidates the molecular mechanism underlying their resistance to Bt Cry toxins, and discusses resistance management strategies based on these mechanisms. Extensive research has demonstrated that mutations and alterations in expression patterns of midgut receptor genes are closely associated with Bt resistance. As our understanding of molecular mechanisms progresses, several innovative approaches such as DNA molecular detection techniques, engineering modified Cry toxins, and combining Bt toxin with RNAi technology have been developed for effective pest control measures. Future research will further unravel the intricate molecular mechanisms underlying this phenomenon to develop scientifically sound integrated pest management strategies. [ABSTRACT FROM AUTHOR]

【Objective】The objective of this study is to develop an expression vector that enables rapid and efficient expression of insecticidal genes in Bacillus thuringiensis (Bt), aiming to increase the efficiency of gene discovery and functional research in Bt.【Method】The pUC18 vector was employed for the construction of a rapid visual expression vector, namely p1Ac-GFP, under the guidance of the cry1Ac gene promoter p1Ac and fused with green fluorescent protein (GFP). Subsequently, an analysis was conducted on its biological activity, alkaline solubility, anti-trypsin stability, and culture conditions.【Result】There was no statistically significant disparity in the insecticidal activity between the Cry1Ac protein expressed through p1Ac-GFP guidance in Escherichia coli and the protein derived from Bt. Meanwhile, the Cry1Ac protein expressed by p1Ac-GFP was solubilized in a 50 mmol/L Na2CO3 solution, and the resulting soluble fraction was enzymatically cleaved by trypsin to yield a 60 kD biologically active core fragment. Furthermore, it was observed that the host strain of p1Ac-GFP emited green fluorescence, with fluorescence intensity of the bacterial solution exhibiting a good linear relationship with expressed Bt protein concentration. The optimization results obtained from culture conditions demonstrated that optimal culture time for Bt Cry1Ac protein expression using p1Ac-GFP was 37℃ over a period of 48 h at a culture broth to bottle volume ratio of 5/5.【Conclusion】The fluorescence intensity of the bacterial solution, when p1Ac-GFP is utilized to express Bt protein, can serve as a direct indicator of concentration for biological activity assays. This facilitates the rapid verification of the insecticidal activity function of Bt genes. [ABSTRACT FROM AUTHOR]

Simple Summary: The housefly (Musca domestica) is one of the most common species in urban areas and a mechanical vector of various pathogens affecting human and animal health worldwide. To control this vector, the bacterium Bacillus thuringiensis (Bt) offers a safe alternative to chemical insecticides that is highly specific, environmentally safe, and economically viable. In this study, we evaluated the effectiveness of 12 Bt strains isolated from substrates collected in Macapá, Brazil, on the postembryonic development of housefly. Six strains caused high mortality rates (70–100%) and carried specific cry and vip genes. Scanning electron microscopy revealed diverse crystal morphologies, suggesting bioinsecticidal potential against this and other pest species. These findings demonstrate the promising potential of Brazilian Bt strains for developing bioinsecticides to manage this muscoid dipteran. The high pathogenic activity of Bacillus thuringiensis (Bt) strains against various insect orders has positioned it as the most effective, environmentally safe, and sustainable approach to integrated insect management. We aimed to identify Bt strains capable of effectively controlling the housefly Musca domestica, a species of significant medical, veterinary, and public health concern. Twelve Bt strains from Macapá, Brazil, were tested against housefly larvae. PCR was employed to detect genes encoding Cry and Cyt proteins and Vips. Six strains exhibited 70 to 100% larval mortality, with five containing cry genes, none harboring cyt genes, and four carrying vip genes. Scanning electron microscopy revealed the production of crystal inclusions with distinct morphologies: spherical (TR4J, SOL5DM, SOL6RN), cuboidal and bipyramidal (TRO1TN and TRO2MQ), and spherical and bipyramidal (UNI2MA). The potential presence of genes from the cry1, cry2, vip1, and vip3 families suggests that these strains also exhibit bioinsecticidal activity against other muscoid flies and insect pests across various orders. This study underscores the bioactive potential of these Brazilian Bt strains for developing new bioinsecticides. [ABSTRACT FROM AUTHOR]

Nacobbus sp. is a phytoparasitic nematode that affects economically important crops. Due to the negative environmental and health impacts of chemical nematicides, sustainable alternatives, such as the use of biological control agents, are being developed. Rhizobacteria are microorganisms that can suppress nematode reproduction by producing different metabolites, induce systemic resistance in plants and stimulate plant growth. The aim of this study was to investigate whether the inoculation with commercial formulations of plant-growth promoting rhizobacteria (PGPR) can mitigate the deleterious effects of the nematode N. aberrans s.l. and promote the growth of pepper plants grown in greenhouses under biotic stress. Different bacteria were tested in vitro before being inoculated into pepper plants. Plants were inoculated with Bacillus subtilis, Bacillus thuringiensis and Azospirillim brasilense at transplant and then every 15 days, in the presence and absence of N. aberrans s.l.. The pathogen reproduction factor was 5.34; 2.09; 0.88 and 12.46 for A. brasilense B. subtilis, B. thuringiensis and control, respectively. In both nematode-parasitized and unparasitized plants, the rhizobacteria increased the soluble proteins (54%) and chlorophyll (18%) content and exerted a positive effect on net photosynthesis, transpiration, stomatal conductance and increased water use efficiency, height, stem diameter, fresh and dry weight. This beneficial effect was also verified by a lower accumulation of proline and sugars, metabolites used by plants as osmoregulators in stress situations. These results showed that B. thuringiensis and B. subtilis perform better in promoting pepper growth in the presence of the nematode and in the biocontrol of this pathogen. [ABSTRACT FROM AUTHOR]

Lysinibacillus sphaericus PKA17, Bacillus cereus PKA18 and Bacillus thuringiensis PKA19, isolated from the intestine of adult indigenous magur (Clarias magur), were evaluated as putative probiotics for the cultivation of C. magur fingerlings. A total of 180 magur fingerlings (average weight 4.01 ± 0.01 g) were randomly distributed into four dietary groups in triplicate. The fingerlings were cultivated for 120 days into continuous-flow experimental rectangular cemented tanks (195 × 105 × 90 cm3; 1600 l; 5 cm bottom mud). Probiotics were mixed in feed (PT1: 2 × 106L. sphaericus PKA17 cells; PT2: 2 × 106B. cereus PKA18 cells; PT3: 2 × 106B. thuringiensis PKA19 cfu/kg of basal feed where, PT = Probiotic treated) and fed to different dietary groups of fish. However, the control feed comprises of basal ingredients, and was not supplemented with any bacterial isolate. Fish carcass composition revealed significantly (p < 0.05) higher amounts of carbohydrate (2.8 ± 0.06 g kg−1), iron (2.21 ± 0.01 mg 100 g−1), fat-soluble vitamins and amino acid in fish fed PT1 than other groups. Fish fed PT1 feed also displayed a considerably higher protein efficiency ratio (2.05 ± 0.01) and lower feed conversion ratio (1.23 ± 0.01). Higher carcass protein content (164.9 ± 0.23 g kg−1) was observed in fish fed PT3 feed followed by PT1 and PT2. Fish fed probiotic-supplemented feed (PT1, PT2, and PT3) showed higher amounts of high-density lipoprotein and calcium than the control-fed fish. High survivability was observed in fish fed probiotic-feed compared to the control group. The results suggest that feed (PT1, PT2, PT3) incorporated with these probiotics might enhance the growth of C. magur in captivity, which may promote the cultivation of this endangered species. [ABSTRACT FROM AUTHOR]

Simple Summary: The soil bacterium Bacillus thuringiensis (Bt) produces protein crystals during sporulation. Cry toxins, a major component of these protein crystals, are insecticidal proteins with pore-forming activity. Cry toxins are environmentally friendly biopesticides used to create Bt-incorporating genetically modified crops (BT-GMCs) that confer resistance to insect pests. However, public acceptance of BT-GMCs is lacking. A major concern over BT-GMCs is the allergenicity of Cry toxins. Specifically, the pore-forming activity of Cry toxins may directly induce intestinal mast cells to release histamine. In this study, three lepidopteran-specific Cry toxins, Cry1Aa, Cy1Ab, and Cry1Ac, were treated with simulated digestive fluids under different conditions. Cry1A toxins exhibited differing sensitivity to simulated digestive fluids, and digests of Cry1A toxins contained the α4–α5 helical hairpin of domain I, which is thought to be the transmembrane domain. To determine whether Cry1A toxins directly induce histamine release, intact and digested Cry1A toxins were applied to RBL-2H3 cultured rat mast cells. However, no histamine secretion from these mast cells was detected, even when samples were analyzed using HPLC. The results of our study provide important data supporting the safety of Cry1A toxins and potentially BT-GMCs, and reducing anxiety over their safety should help increase public acceptance. Public acceptance of genetically modified crops engineered with Bacillus thuringiensis (Bt) insecticidal protein genes (BT-GMCs), which confer resistance to various lepidopteran insect pests, is generally lacking. As a major concern over BT-GMCs is the allergenicity of insecticidal proteins, alleviating safety concerns should help increase public acceptance. In this study, three lepidopteran-specific Bt toxins, Cry1Aa, Cy1Ab, and Cry1Ac, were treated with simulated digestive fluids under various conditions. Western blotting using antiserum raised against individual segments (α-helices of domain I and β-sheets of domains II and III) of Cry1Aa showed that digestion produces a variety of polypeptides. In particular, the transmembrane α4–α5 of domain I, which may retain the ability to form pores, was the most resistant to digestion. Intact Cry1A toxins and these digests were then applied to RBL-2H3 cultured rat mast cells to determine whether the toxins directly induce histamine release. However, fluorescence microscopy revealed no specific binding of Cry1A toxins to RBL-2H3 cultured rat mast cells. In addition, neither the OPA method nor HPLC analysis detected significant histamine release from mast cells treated with Cry1A toxins and these digests. Our results provide important data supporting the safety of Cry1A toxins and potentially BT-GMCs. [ABSTRACT FROM AUTHOR]

The study evaluated the effect of Bacillus thuringiensis (Bt) and Trichoderma asperellum (Ta) as biostimulants in hydroponically grown tendril pea (Pisum sativum) microgreens. A completely randomized experimental design was conducted under controlled conditions; the study included the root pea-spraying treatment with Bt, Ta, and their combination, alongside a non-inoculated control. The results showed that treatments with Ta significantly enhanced growth parameters, such as stem length and diameter, stipular leaf area, and fresh weight of the microgreens. Specifically, fresh biomass increased by 140% and dry biomass by 80% with Ta compared to the control, while combined treatment with Bt and Ta reduced nitrates by 39.6%. Bt and Bt + Ta increased chlorophyll b and carotenoids contents, suggesting improved photosynthetic activity. No significant differences in visual appearance were observed. In conclusion, the application of Ta and Bt can enhance tendril pea microgreens' yield and certain biochemical (chlorophyll b and carotenoids) qualities without affecting their visual quality, supporting the application of these microorganisms as biostimulants. These findings underscore the potential to scale these treatments in commercial hydroponic systems, offering a sustainable approach to optimizing the production of this emerging crop type. [ABSTRACT FROM AUTHOR]

NupR is a nucleoside permease regulator belonging to the GntR family, mainly regulating nucleoside transport in Bacillus thuringiensis. A conserved binding site for NupR was found in the promoter region of plcR. This study aimed to investigate the regulation of the virulence regulator PlcR by NupR and its impact on Bt virulence. We demonstrated that NupR can directly repress the expression of plcR. The expression of plcR can be induced by glucose and nucleosides. Glucose impacts the expression of plcR mainly through Spo0A, while the induction effect of nucleosides may be due to the production of ribose through nucleoside catabolism. In addition, NupR regulates the expression of the PlcR regulon, including hemolysin, phospholipase C, papR, and oligopeptide permease, which could result in the culture supernatant of BMB171 being less virulent to sf9 cells compared to the nupR knockout strain. The results combine the nutritional status of cells with virulence to form a regulatory loop, providing new ideas and research foundations for the study of bacterial virulence. [ABSTRACT FROM AUTHOR]

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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. [ABSTRACT FROM AUTHOR]

Abstract Strong and shifting selective pressures of the Anthropocene are rapidly shaping phenomes and genomes of organisms worldwide. Crops expressing pesticidal proteins from Bacillus thuringiensis (Bt) represent one major selective force on insect genomes. Here we characterize a rapid response to selection by Bt crops in a major crop pest, Helicoverpa zea. We reveal the polygenic architecture of Bt resistance evolution in H. zea and identify multiple genomic regions underlying this trait. In the genomic region of largest effect, we identified a gene amplification event, where resistant individuals showed variation in copy number for multiple genes. Signals of this amplification increased over time, consistent with the history of field-evolved Bt resistance evolution. Modern wild populations from disparate geographical regions are positive for this variant at high, but not fixed, allele frequencies. We also detected selection against single copy variants at this locus in wild H. zea collected from Bt expressing plants, further supporting its role in resistance. Multiple genes were annotated in this genomic region, and all appeared to be significantly upregulated in Bt resistant H. zea. We functionally characterized genes within the copy number variant (CNV), providing insight into their potential roles in resistance evolution. Our findings reveal the nature of rapid genome evolution in a major crop pest following anthropogenic selection and highlight the role that CNVs can have in rapid evolutionary responses.

Abstract The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, has evolved resistance to nearly every management tactic utilized in the field. This study investigated the resistance mechanisms in a WCR strain resistant to the Bacillus thuringiensis (Bt) protein eCry3.1Ab using dsRNA to knockdown WCR midgut genes previously documented to be associated with the resistance. ATP-binding cassette transporter (ABCC4), aminopeptidase-N, cadherin, and cathepsin-B were previously found to be differentially expressed in eCry3.1Ab-resistant WCR larvae when compared to susceptible larvae after feeding on maize expressing eCry3.1Ab and its near-isoline. Here we compared the susceptibility of resistant and susceptible WCR larvae to eCry3.1Ab protein in presence or absence of dsRNA targeting the above genes using 10-day diet overlay toxicity assays. Combining ABCC4 dsRNA with eCry3.1Ab protein increased susceptibility to Bt protein in WCR-resistant larvae, but the other three genes had no such effect. Among 65 ABC transport genes identified, several were expressed differently in resistant or susceptible WCR larvae, fed on eCry3.1Ab-expressing maize versus its isoline, that may be involved in Bt resistance. Our findings provide strong evidence that ABCC4 is indirectly involved in WCR resistance to eCry3.1Ab protein by enhancing the effects of Bt-induced toxicity.

Abstract Background The pink bollworm, Pectinophora gossypiella (Saunders), is a devastating global pest of cotton that has caused substantial economic damage to Bt Bollgard-II® cotton plants in recent years due to the evolution of Bt resistance. The associated fitness cost is assumed to be one of the factors delaying the development of resistance against Bt transgenic crops. Hence, the present study was undertaken to assess the biological performance of pink bollworms by comparing the life history and demographic parameters of a resistant (Field-R) and susceptible (Lab-S) population. Results Prolonged larval duration (23.40 days in Field-R vs 18.80 days in Lab-S population), total life cycle (male = 50.00 vs 42.80 days; female = 53.60 vs 46.20 days), reduced fecundity (100.60 vs 154.20 eggs/female) and fertility (88.00 vs 138.00 fertile eggs/female) was observed. The demographic parameters indicated a significant reduction in the net reproductive rate (184.27 vs 276.72), innate capacity for increase in number (0.11 vs 0.15), finite rate of increase in number (1.12 vs 1.16 female progenies produced/female/day), weekly multiplication rate (2.16 vs 2.86), potential fecundity (545.06 vs 634.11 eggs), number of hypothetical F2 females (33 955.65 vs 76 572.41), but longer mean length of generation (47.54 vs 37.74 days) and population doubling time (6.30 vs 4.62 days) in Field-R compared with Lab-S population. A stage-specific life table demonstrated the differences in survival rates between susceptible and resistant populations at various life stages, with the resistant population having higher generation mortality (0.22 vs 0.19). Conclusions The study confirms the involvement of fitness costs associated with Bt resistance in P. gossypiella. Despite reduced reproductive fitness, the resistant population tried prolonging the larval stage as a compensatory mechanism to repair the damaged host tissues due to Bt intoxication and for accumulation of enough nutrient reserves for normal pupation and adult emergence. Presence of a high proportion of double Bt-resistant larvae in the field coupled with continued noncompliance with refug planting certainly favours the flaring up of this monophagous pest despite the observed fitness costs. The resistance cannot be effectively reversed unless suitable alternative management strategies are deployed.

High-yield strains of cold-active lipase are screened to provide production information for the industrial development of lipase. A cold-active lipase-producing strain was isolated from Xing'an larch forest soil in Mohe County using the Rhodamine B plate method. Its morphological, physiological, and biochemical characteristics were identified, and combined with the 16S rDNA gene sequence, the strain was determined to be Bacillus thuringiensis. The lipase gene Lip240 was cloned using the Bacillus thuringiensis genome as a template, and heterologous expression and enzymatic properties of Lip240 were analyzed. The results showed that the recombination lipase Lip240 had an optimal reaction temperature of 30 ℃, and could maintain more than 60% activity when treated at 4~30 ℃ for 6 hours, thus a cold-active lipase. Besides, its optimal pH was 8.0. Additionally, Ca2+, Mn2+, Fe2+, Fe3+, and Cr3+ had a certain enhancing effect on the activity of Lip240. Notably, Lip240 had a good organic solvent tolerance, while SDS could significantly (P

Abstract Key message Aerial application of Bacillus thuringiensis Berliner var. kurstaki (Btk) every second year to stands of white spruce (Picea glauca (Moench)Voss.), black spruce (Picea mariana Mill.) and balsam fir (Abies balsamea (L.) Mill.) is the most cost-effective spraying scenario for reducing the impact of spruce budworm (Choristoneura fumiferana Clemens) on wood production, providing a similar level of forest protection, but at lower cost, to the standard scenario currently used in which 50% of current year’s foliage is protected every year. Context Insect outbreaks can have significant effects on forest productivity and various formulations of Bacillus thuringiensis Berliner var. kurstaki (Btk) are used to reduce their damage. In the Province of Québec, Canada, control programs aim to protect at least 50% of current-year foliage to limit tree mortality, but little information exists on the long-term cost-effectiveness of such programs. Aims Our goal was to evaluate the benefit/cost ratio and the efficacy of different Btk protection scenarios in reducing coniferous tree mortality and growth losses over a 11-year period. We hypothesized that less-intensive protection approaches (Btk applications every 2 or 3 years) may provide similar levels of protection but with higher cost-effectiveness ratios than the standard program currently used in Quebec. Methods In 2007, we established nineteen 100-ha experimental units in Quebec’s Côte-Nord region to determine the efficacy and cost-effectiveness of different Btk spraying scenarios for reducing tree mortality and volume losses in coniferous stands dominated by mature balsam fir trees (Abies balsamea (L.) Mill., with white spruce (Picea glauca (Moench) Voss.) and black spruce (P. mariana Mill.) as companion species. Tree mortality was monitored annually in three circular plots of 400 m2 within each experimental unit. Growth losses were evaluated using stem analyses. Results Mortality was much higher in balsam fir than in black spruce and white spruce (respectively 74.4%, 13.8% and 5.9% in untreated stands) in all protection scenarios. The application of Btk every 2 years reduced balsam fir mortality to a level similar to the standard scenario (10.3% vs 7.15%, respectively) at a much lower cost. Growth losses have also been reduced but not to the same extent as in the standard scenario. Conclusion Spraying Btk every 2 years provides effective protection to balsam fir and is the most cost-effective scenario. A less intensive use of Btk would mitigate impact on non-target lepidoptera and allow protecting other areas, such as habitat of the woodland caribou, a threatened species which avoids disturbed areas.

Abstract Cell motility increases the fitness of bacterial cells. Previous research focused on the transcriptional regulator CdsR, which represses cellular autolysis and promotes spore formation in Bacillus thuringiensis. However, the targets of CdsR are mostly unknown. Here, we reported a new function of CdsR in regulating cell motility. Mutation of cdsR results in increase of cell mobility, and a number of related genes were upregulated compared to wild type HD73. Thus, we investigated the transcription of the fla/che gene cluster, which involves in cell mobility and comprises eight operons/genes, including motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, and yjbJ-flgG. Additionally, the motAB2 operon was discovered, which consists of homologs genes motA2 and motB2 that are like motA1 and motB1. Through promoter-lacZ fusion assays and EMSA experiments, it was discovered that CdsR directly regulates the motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, yjbJ-flgG, and motAB2 operons by binding to their promoter regions. Importantly, it was confirmed that CdsR is a metalloregulator and the binding to promoter can be inhibited by Cu (II) ions. This research enhances our understanding of the regulation of cell mobility in B. thuringiensis.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, has evolved resistance to nearly every management tactic utilized in the field. This study investigated the resistance mechanisms in a WCR strain resistant to the Bacillus thuringiensis (Bt) protein eCry3.1Ab using dsRNA to knockdown WCR midgut genes previously documented to be associated with the resistance. ATP-binding cassette transporter (ABCC4), aminopeptidase-N, cadherin, and cathepsin-B were previously found to be differentially expressed in eCry3.1Ab-resistant WCR larvae when compared to susceptible larvae after feeding on maize expressing eCry3.1Ab and its near-isoline. Here we compared the susceptibility of resistant and susceptible WCR larvae to eCry3.1Ab protein in presence or absence of dsRNA targeting the above genes using 10-day diet overlay toxicity assays. Combining ABCC4 dsRNA with eCry3.1Ab protein increased susceptibility to Bt protein in WCR-resistant larvae, but the other three genes had no such effect. Among 65 ABC transport genes identified, several were expressed differently in resistant or susceptible WCR larvae, fed on eCry3.1Ab-expressing maize versus its isoline, that may be involved in Bt resistance. Our findings provide strong evidence that ABCC4 is indirectly involved in WCR resistance to eCry3.1Ab protein by enhancing the effects of Bt-induced toxicity. [ABSTRACT FROM AUTHOR]

Strong and shifting selective pressures of the Anthropocene are rapidly shaping phenomes and genomes of organisms worldwide. Crops expressing pesticidal proteins from Bacillus thuringiensis (Bt) represent one major selective force on insect genomes. Here we characterize a rapid response to selection by Bt crops in a major crop pest, Helicoverpa zea. We reveal the polygenic architecture of Bt resistance evolution in H. zea and identify multiple genomic regions underlying this trait. In the genomic region of largest effect, we identified a gene amplification event, where resistant individuals showed variation in copy number for multiple genes. Signals of this amplification increased over time, consistent with the history of field-evolved Bt resistance evolution. Modern wild populations from disparate geographical regions are positive for this variant at high, but not fixed, allele frequencies. We also detected selection against single copy variants at this locus in wild H. zea collected from Bt expressing plants, further supporting its role in resistance. Multiple genes were annotated in this genomic region, and all appeared to be significantly upregulated in Bt resistant H. zea. We functionally characterized genes within the copy number variant (CNV), providing insight into their potential roles in resistance evolution. Our findings reveal the nature of rapid genome evolution in a major crop pest following anthropogenic selection and highlight the role that CNVs can have in rapid evolutionary responses. [ABSTRACT FROM AUTHOR]

This paper presents a contrast of two different observation strategies viz a nonlinear observer and a classical extended Luenberger observer applied to a bioreactor system for Bacillus thuringiensis production. The performance of the two observers was evaluated under different conditions, both with and without state perturbations. Firstly, equal initial conditions were considered without the presence of white noise in the measurement of dissolved oxygen concentration in the culture medium. The performance was then analyzed by perturbing the maximum cell growth rate with equal and different initial conditions, and, finally, the performance of the observer with the presence of white noise was evaluated. The proposed observer performed better than the extended Luenberger observer against initial conditions different from the model. The results of this study are of great interest, as they provide insight into the estimation of the state of the dynamics for the B. thuringiensis bioreactor in a batch mode. In addition, these results provide valuable information for future research in the design of observers for B. thuringiensis bioprocessing. [ABSTRACT FROM AUTHOR]

Torenia fournieri L. is a popular ornamental plant in the genus Torenia, widely used in commercial landscaping, especially during the summer. Additionally, Torenia has served as a model ornamental plant in many studies exploring ornamental characteristics and pest control through genetic engineering. To date, no research has been reported on developing insect-resistant Torenia expressing genes from Bacillus thuringiensis (Bt). In this study, a recombinant vector carrying the Cry1Ab gene from Bt, pBI121-Cry1Ab, was constructed and transferred into T. fournieri via Agrobacterium tumefaciens-mediated transformation. A total of 13 shoots survived on the kanamycin selection medium, among which four putative transgenic lines, designated L1, L2, L7, and L11, were molecularly confirmed by PCR and Southern blot analysis, indicating successful integration of the Cry1Ab gene into the genomes of these lines. Quantitative real-time PCR and ELISA results further verified the successful expression of the Cry1Ab gene in the leaves of all four transgenic lines. Insect bioassay results demonstrated that all four transgenic lines showed strong resistance to the insect pest, Mythimna separata, with mortality rates ranging from 59.9% to 100.0%, in contrast to a larval mortality rate of 16.2% in the wild-type Torenia. Additionally, these transgenic lines significantly decreased in larval survival rates compared to those fed on wild-type plants. Furthermore, these transgenic lines activated superoxide dismutase (SOD) activity at 12 and 24 h, and catalase (CAT) activity at 72 h, while suppressing SOD activity at 72 h, and peroxidase (POD) activity over time. Our findings indicate that these transgenic lines exhibit high resistance to the insect pest and provide new insights into controlling insect pests in ornamental plants through genetic approaches. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) bacteria produce different pore forming toxins with insecticidal activity, including Cry and Vip3 proteins. While both Cry and Vip3 cause insect death by forming pores in susceptible lepidopteran larval midgut cells, their mechanisms of action differ. The Vip3Aa protoxin adopts a tetramer-structure, where each monomer has five distinct domains. Upon proteolytic activation, the Vip3 tetramer undergoes a large conformational change forming a syringe like structure that is ready for membrane insertion and pore formation. Here we show that Vip3Aa protoxin had low binding to Spodoptera frugiperda brush border membrane vesicles (BBMV) unlike the activated toxin that bound specifically in a concentration dependent way, suggesting that a structural change upon Vip3Aa proteolytic activation is required for efficient receptor binding. Consistently, the Vip3Aa protoxin showed no toxicity to Sf9 cells compared to the activated toxin. In contrast, Cry1Fa protoxin and its activated toxin, were both highly toxic to Sf9 cells. To identify the region of Vip3 involved in binding to BBMV proteins, different overlapping peptides from Vip3Aa covering domains III, IV and V were expressed, and binding analysis were performed against BBMV, showing that domain III is the primary binding domain. Additionally, domains III, IV and V amino acid residues that become exposed upon activation of Vip3Aa were identified. Mutagenesis of these exposed residues revealed three amino acids (K385, K526 and V529) located in two structural adjacent loops, domain III loop β5-β6 and loop α11-β16 that connects domains III and IV, that are crucial for binding to the midguts of S. frugiperda larvae and for toxicity. Our results demonstrate that proteolytic activation of Vip3Aa exposes a receptor binding region essential for its toxicity. Author summary: To exert toxicity, Vip3 tetrameric protoxins are activated by larval gut proteases facilitating the formation of lytic pores that destroy the midgut epithelium cells killing the susceptible larvae. Here, we demonstrate that the proteolytic activation of Vip3Aa protoxin is necessary for triggering receptor binding. Binding analysis of Vip3Aa protoxin and activated toxin to S. frugiperda midguts and toxicity assays against Sf9 cells, confirmed that proteolytic activation is necessary for an efficient binding to the insect midgut membranes and for toxicity. The analysis of residues from domains III, IV and V, that become exposed after Vip3Aa activation by proteolysis, allowed us to identify a structural binding epitope which plays an important role in receptor binding and toxicity. Our results indicate that the structural change that Vip3Aa undergoes after activation by larval midgut proteases, besides being important for membrane insertion and pore formation, also triggers receptor binding important for toxicity. [ABSTRACT FROM AUTHOR]