Cry1Ab Toxin Research Articles

Inheritance and Fitness Cost of Laboratory-Selected Resistance to Cry1Ab in Hyphantria cunea Drury (Lepidoptera: Arctiidae)

Transgenic poplars have been used to control quarantine pests worldwide, such as the fall webworm (Hyphantria cunea, FW). However, the studies on the resistance mechanism of FW to Cry toxins are limited. This study obtained an FW strain with 45-fold resistance to Cry1Ab toxin by continuous screening in the laboratory. The resistance to Cry1Ab was autosomally completely recessive, and it had approximately 40-fold cross-resistance to Cry1Ac but no cross-resistance to Cry2Ab toxin. Analysis of indoor population life tables for the resistant and susceptible strains found no significant difference in the net reproduction rate (R0) between the two strains. The resistant strain had an insignificantly longer larval development duration and a comparable pupation rate, pupal weight, emergence rate, eggs laidper female, and egg hatching rate compared to the susceptible strain. This indicated that there was no fitness cost for Cry1Ab resistance. Our study helps in evaluating the risk of resistance to Cry1Ab toxin and the mechanisms of resistance to Cry1Ab toxin in FW.

Transcriptional regulation of Cry2Ab toxin receptor ABCA2 gene in insects involves GATAe and splicing of ant 5' UTR iron

Transcriptional regulation of Cry2Ab toxin receptor ABCA2 gene in insects involves GATAe and splicing of ant 5' UTR iron

Control Efficacy of the Bt Maize Event DBN3601T Expressing Cry1Ab and Vip3Aa Proteins against Beet Armyworm, Spodoptera exigua (Hübner), in China.

The beet armyworm, Spodoptera exigua (Hübner), is a major pest of maize, cotton, soybean, and many other crops globally. Despite the widespread deployment of Bt transgenic maize for pest control worldwide, the efficacy of Bt lepidopteran-resistant transgenic maize in managing S. exigua remains rarely studied. In this study, we quantified the expression level of pyramided Cry1Ab and Vip3Aa toxins in Bt maize (event DBN3601T) and evaluated their control efficiency against S. exigua under both laboratory and field conditions. The enzyme-linked immunosorbent assay (ELISA) results showed that the expression levels of Cry1Ab and Vip3Aa proteins in DBN3601T maize tissues followed a decreasing order as follows: V5-leaf > V8-leaf > VT-tassel > R2-kernel > R1-silk. Diet-overlay assay results showed that the LC50 values of Cry1Ab and Vip3Aa proteins against S. exigua larvae were 11.66 ng/cm2 and 27.74 ng/cm2, respectively, with corresponding GIC50 values at 1.59 ng/cm2 and 7.93 ng/cm2. Bioassay using various tissues of the DBN3601T maize indicated that after 7 days of infestation, mortality rates of neonates and third-instar larvae ranged from 86% to 100% and 58% to 100%, respectively. Mortality was highest on V5 and V8 leaves, followed by R2-kernel, VT-tassel, and R1-silk. Field trials demonstrated that DBN3601T maize exhibited significantly lower larval density, damage rate, and leaf damage score compared to non-Bt maize. Field cage trial showed that the control efficacy of DBN3601T maize at the vegetative stage could reach 98%. These findings provide a theoretical basis for utilizing Bt transgenic maize to enhance the sustainable management of S. exigua in Asia.

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

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

Engineering of Bacillus thuringiensis Cry2Ab toxin for improved insecticidal activity

Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin’s future application.

V-ATPase E mediates Cry2Ab binding and toxicity in Helicoverpa armigera

Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.

Reduced expression of the P-glycoprotein gene HaABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin but not Cry2Ab toxin in Helicoverpa armigera

Rapid evolution of pest resistance to Bt insecticidal proteins presents a serious threat to the sustainable use of Bt crops. The cotton bollworm has been extensively exposed to Bt cotton worldwide and has evolved resistance in laboratory and field. Previous studies have highlighted the significant roles played by the ABC transporter proteins in Bt resistance. In this study, the ORF of HaABCB1 was cloned and analyzed. The expression of HaABCB1 was detected in all developmental stages and tissues, with the highest expression in third instar larvae stage and hindgut tissue. Compared with susceptible strain, a remarkable decrease of HaABCB1 expression in Cry1Ac resistant strain while no significant change in Cry2Ab resistant strain were found. The HaABCB1 expression reduced after susceptible larvae induced by Cry1Ac, but no obvious expression changes after Cry2Ab exposure. RNAi-mediated down-regulation of HaABCB1 could lead to a significant reduction in larval susceptibility to Cry1Ac, but not to Cry2Ab, in susceptible strain. Genetic linkage analysis confirmed that decreased expression of the HaABCB1 mediates resistance to Cry1Ac, but not Cry2Ab resistance. This knowledge contributes to better understanding of the complex molecular mechanisms underlying Bt resistance and provide theoretical foundation for the development of new strategies for pest resistance management.

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

Baseline susceptibility of cabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) to Bacillus thuringiensis (Berliner) Cry toxins in Meghalaya

BackgroundCabbage butterfly, Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae), is one of the most important pests of cabbage and other cruciferous crops and accounts for > 40% yield reduction in the crops. An investigation on the baseline susceptibility of P. brassicae to Bacillus thuringiensis (Bt) Cry toxins in Meghalaya was evaluated for future exploitation in Bt resistance monitoring. Two different Cry toxins, Bt Cry1C and Cry2Ab, were screened against 11 different field populations of P. brassicae from the state of Meghalaya. LC50 was evaluated based on the response of larval mortality of P. brassicae using the leaf-dip bioassay method.ResultsThe baseline-susceptibility tests conducted on P. brassicae in 11 different field populations from Meghalaya revealed that Smit population strains seemed to show less tolerance to both the Bt Cry toxins (Cry1C and Cry2Ab). Compared to the Cry1C toxin, Cry2Ab was found more potent against P. brassicae. The median lethal concentrations, LC50 72 h, varied from 0.535 to 1.725 µg/ml for Cry2Ab and 0.546–1.803 µg/ml for Cry1C toxin. The screening using leaf-dip bioassay resulted in a tolerance ratio of 3.3-fold and 3.2-fold for Cry1C and Cry2Ab, respectively. The most tolerant strains of P. brassicae from Umiam and Pepbah regions were observed to show discriminating concentrations of 19.30 µg/ml for Cry1C and 24.03 µg/ml for Cry2Ab (LC99, 72 h).ConclusionsThe Cry2Ab toxin was found to be more virulent than Cry1C toxin for P. brassicae. Certain candidate discriminating concentrations for Cry1C and Cry2Ab can be used as benchmarks for future resistance monitoring of P. brassicae to Bt Cry toxins.

Seven Years of Monitoring Susceptibility to Cry1Ab and Cry1F in Asian Corn Borer.

Resistance monitoring in the Asian corn borer, Ostrinia furnacalis, is necessary to accommodate the commercial introduction and stewardship of Bt maize in China. The susceptibility of 56 O. furnacalis field populations, collected between 2015 and 2021 from the corn belt regions of China, to Cry1Ab and Cry1F toxins was determined. Neonate larvae (within 12 h after hatching) were placed on the surface of semi-artificial agar-free diet incorporating a series of concentrations of purified toxins, and mortality was evaluated after 7d. The median lethal concentration (LC50) values of Cry1Ab and Cry1F were 0.05 to 0.37 µg/g (protein/diet) and 0.10 to 1.22 µg/g, respectively. Although interpopulation variation in susceptibility to the toxins was observed, the magnitude of the differences was 5.8-fold and 8.3-fold for Cry1Ab and Cry1F, respectively. These results suggested that the observed susceptibility differences reflect natural geographical variation in response and not variation caused by prior exposure to selection pressures. Therefore, the O. furnacalis populations were apparently still susceptible to Cry1Ab and Cry1F across their range within China. The monitoring data established here will serve as a comparative reference for early warning signs of field-evolved resistance after the cultivation of Bt maize in China.

Rotation of Multiple Single-Gene Transgenic Crops Did Not Slow the Evolution of Resistance to Cry1F or Cry1Ie in Ostrinia furnacalis.

A common strategy for delaying the evolution of resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis is to ensure that insect pests are exposed to multiple toxins with different mechanisms of action (MoAs). This can take the form of planting crops in a rotation pattern when different crops expressing single toxins are available on the market. The efficacy of a rotation strategy is reliant on mathematical models based on biological assumptions. Here, we designed laboratory evolution experiments to test whether Bt-based insecticidal proteins with different MoAs used in rotation could delay resistance from developing in Asian corn borer (ACB), Ostrinia furnacalis. We investigated the proteins Cry1Ab, Cry1F, and Cry1Ie, which are widely utilized for commercial insect control. We found that rotation of multiple toxins did not slow the evolution of resistance to Cry1F or Cry1Ie. Furthermore, the evolution of ACB to the Cry1Ab toxin develops faster when Cry1F or Cry1Ie is present, as compared to Cry1Ab exposure only. Our results suggest that toxins used in a rotation fashion do not work as an effective strategy in delaying ACB resistance evolution to Cry toxins over one-toxin exposure. Our result highlights the need to better understand the biological factors leading to insecticidal protein resistance and to develop IRM strategies against target insects.

Monitoring Insect Resistance to Bt Maize in the European Union: Update, Challenges, and Future Prospects.

Transgenic maize producing the Cry1Ab toxin of Bacillus thuringiensis (Bt maize) was approved for cultivation in the European Union (EU) in 1998 to control the corn borers Sesamia nonagrioides (Lefèbvre) and Ostrinia nubilalis (Hübner). In the EU since then, Cry1Ab is the only Bt toxin produced by Bt maize and Spain is the only country where Bt maize has been planted every year. In 2021, about 100,000 hectares of Bt maize producing Cry1Ab were cultivated in the EU, with Spain accounting for 96% and Portugal 4% of this area. In both countries, Bt maize represented less than 25% of all maize planted in 2021, with a maximum regional adoption of 64% Bt maize in northeastern Spain. Insect resistance management based on the high-dose/refuge strategy has been implemented in the EU since 1998. This has been accompanied by monitoring to enable early detection of resistance. The monitoring data from laboratory bioassays show no decrease in susceptibility to Cry1Ab had occurred in either pest as of 2021. Also, control failures have not been reported, confirming that Bt maize producing Cry1Ab remains effective against both pests. Conditions in the EU preventing approval of new genetically modified crops, including maize producing two or more Bt toxins targeting corn borers, may limit the future effectiveness of resistance management strategies.

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

Bacillus thuringiensis affects reproductive capacity of adult rat offspring

ABSTRACT We investigated the effects of B. thuringiensis-based biological insecticides, XenTari and Dipel, and deltamethrin on the reproductive development of pups of pregnant rats. Twenty 90-day-old pregnant rats were divided randomly onto four equal groups: control group (GC) administered only water; XenTari group (GX) administered 1 mg XenTari (containing Cry1Ac toxin of B. thuringiensis)/100 g body weight; Dipel group (GDi) administered 1 mg Dipel (containing Cry1Aa, Cry1Ab and Cry1Ac toxins of B. thuringiensis)/100 g body weight; and a deltamethrin group (GDe) administered 2 mg deltamethrin (0.08 ml Keshet 25EC)/kg body weight as a positive control. Insecticides were administered by gavage at doses of 1 mg/100 g/day (GX and GDi), and 2 mg/kg/day (GDe) during pregnancy and lactation. Treatment with both biologic and synthetic insecticides reduced the weight gain of the mothers. The biological insecticides reduced the number, weight and length, and increased malformation and mortality of the offspring. In female offspring for all three groups administered insecticides, opening of the vagina was delayed, metestrus was increased and estrogen and progesterone levels were reduced compared to proestrus, estrus and metestrus of the cycle. The ovaries of female offspring of all three groups administered insecticides contained numerous tertiary and atretic follicles, few corpora lutea, primary and secondary follicles, and reduced estrogen receptors compared to controls. In male offspring, all three groups exposed to insecticides exhibited reduced testosterone levels. Histopathological changes in the testes including vacuolation and desquamation of the seminiferous epithelium were observed only in the GX and GDi groups. The number of androgen receptors was reduced significantly in the testes and testicular morphometry revealed reduced tubule diameter, height of the seminiferous epithelium and total tubule length compared to the control. The biological insecticides, XenTari and Dipel, administered in sublethal doses to pregnant rats, caused reproductive changes in the offspring similar to those of the insecticide, deltamethrin.

Biochemical characterization of alkaline phosphatase in midgut of Cry2Ab dosed, pink bollworm, Pectinophora gossypiella (Saunders)

Aim: The holistic management of pink bollworm is possible only after uncovering the underlying mechanism/s of pink bollworm resistance. The present study was carried out for investigating the effect of Cry2Ab toxin on alkaline phosphatase activity and expression of its isoforms in midgut of Pectinophora gossypiella. Methodology: Resistant and susceptible pink bollworm larval populations were subjected to Cry2Ab bioassays using F1 larvae in a 21 days diet-incorporation method. The total midgut protein was estimated by Bradford method. Alkaline phosphatase activity was estimated in midgut homogenates, Non-denaturing SDS-PAGE and Native polyacrylamide gel electrophoresis was performed by the following standard procedures. Results: The study showed increased alkaline phosphatase activity in pink bollworm midgut is correlated with insect resistance in response to Cry2Ab. The non-denaturing SDS-PAGE analysis of pink bollworm midgut alkaline phosphatase isozymes had differential banding patterns in resistant and susceptible populations. Yavatmal (R1), Hingoli (R4) and Parbhani (R5) showed similar banding patterns ranging from 65-95 kDa; Guntur (R2) and Hingoli (R3) showed near similar banding patterns ranging from 70-95 kDa; whereas susceptible population showed banding pattern in the range of 50-70 kDa. Interpretation: The expression profiles of alkaline phosphatase isomers in resistant and susceptible populations can be utilized as a biomarker to aid for the screening of PBW infected Bt -cotton fields and development of pink bollworm management strategies. Key words: Alkaline Phosphatase, Cry2Ab, Insect resistance, Pectinophora gossypiella, SDS-PAGE

Insecticidal Effect of Cry Toxins Produced by Bacillus thuringiensis on Diceraeus melacanthus (Dallas, 1851) and Euschistus heros (Fabricius, 1798) (Hemiptera: Pentatomidae)

Some species of the Heteroptera (Hemiptera) suborder are of great agricultural importance, mainly as pests, and their control is often necessary. The use of Bacillus thuringiensis, an entomopathogenic bacterium normally extracted from the soil and used in biological control, is an alternative to the chemical control of these insects. Mortality tests must be carried out in order to select and determine a viable toxic strain, but currently there is no validated methodology for conducting those tests. In this context, this research aimed to develop and improve a selective bioassay methodology to assess the toxic effect of B. thuringiensis Cry toxins on Diceraeus melacanthus (green-belly stink bug) and Euschistus heros (neotropical brown stink bug) nymphs. A bioassay methodology consisting of tubes and artificial diet was proposed. Bioassays with D. melacanthus and E. heros nymphs were performed incorporating Cry toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1F, Cry1G, Cry1Ia, Cry2Ab, Cry2A, Cry2Ae, Cry4A, Cry4B, Cry10, and Cry11Aa) into their liquid diet. The artificial feeding system developed in order to carry out the stink bug mortality tests was conducted. Among the toxins tested, we can highlight 2 causing 80-85% nymphal mortality on D. melacanthus, and 4 toxins causing 90-100% nymphal mortality for E. heros after 7 days of incubation. Both species are susceptible to different Cry toxins, with emphasis on Cry2Ab and Cry4B for D. melacanthus and Cry1B, Cry1G, Cry1Ia and Cry2Ab for E. heros.

In-silico Determination of Insecticidal Potential in Lepidopteron Specific Crystal Toxins with Midgut Alkaline Phosphatase Using Molecular Docking

Alkaline phosphatase (ALP) enzyme plays an important role in binding of lepidopteran specific insecticidal crystal toxins Cry1Ac and Cry1Ab on GPI-anchored membrane receptor. In most of the cases crystal toxin interaction with ALP or Aminopeptidase N (APN) mediated by the terminal N-acetyl galactosamine (GalNAc) moiety. Among toxins Cry1Ac (PDB Id-4ARY) and Cry1Ab (PDB Id - n642) expresses less binding affinity about (-5.79 and -5.62) to GalNAc in ALP or APN region, respectively. The diptera specific crystal toxins Cry4Ba structure resembles to the lepidopteran specific toxins as it mimics like Cry1Aa for binding to ALP receptor by showing least binding affinity about (-6.99). Cry4Ba may be repurposed along with the novel crystal toxins for lepidopteron insects as toxicity over Cry1Ac or Cry1Ab Bt which will effective to bind with ALP or APN for toxin receptor interaction and help to minimize the rate of resistance. The Cry4Ba pyramided in combination with other Cry genes in different crops like cotton and maize to increase crop protection by delaying would be an efficient strategy the insect resistance.

Baseline Susceptibility and Resistance Allele Frequency in Ostrinia furnacalis in Relation to Cry1Ab Toxins in China.

Asian corn borer (ACB), Ostrinia furnacalis (Guenée) is a destructive pest of corn and major target of transgenic corn expressing Bt (Bacillus thuringiensis) toxins in China. It is necessary to establish the baseline susceptibility of geographically distinct ACB populations to Cry1Ab protein and estimate the resistance alleles frequency. The median lethal concentration (LC50) and LC95 values of Bt toxin Cry1Ab for 25 geographically distinct populations collected in 2018–2019 ranged from 0.86 to 71.33, 18.58 to 5752.34 ng/cm2, respectively. The median effective concentration (EC50) and EC95 values ranged from 0.03 to 10.40 ng/cm2 and 3.75 to 172.86 ng/cm2, respectively. We used the F2 screening method for estimating the expected frequency of resistance alleles of the 13 ACB populations, to Bt corn (Bt11 × GA21) expressing the Cry1Ab toxin. The neonates could not survive on the leaves of transgenic maize Bt11 × GA21 with cry1Ab gene, the Cry1Ab resistance allele frequency was still low in each geographic population in the field, ranging from 0.0032–0.0048, indicating that the sensitivity of ACB to Cry1Ab was still at a high level, and there were no viable resistant individuals in the field at present. The susceptibility of 25 populations of ACB collected in China showed regional differences, although the Cry1Ab resistance allele frequency in these ACB populations is still at a low level. This provides essential knowledge for making the decision to commercialize Bt maize, and monitoring resistance development and evaluating resistance management strategies in the future in China.

Detection of a novel Cry2Ab toxin against Etiella zinckenella Treitschke from the Bacillus thuringiensis serovar canadensis SP142 strain

The soybean (Glycine max) is an important crop. The pod borer (Etiella zinckenella) is one of the most serious insects that attacks various Leguminosae. Common insecticidal controls are ineffective because of the insect’s growth properties. Use of resistant crop varieties offers stabilisation of the yield and has benefits over the use of insecticides. Bacillus thuringiensis is widely used as a bioinsecticide for pest control and a genetic material for pest-resistant transgenic plants. However, the resistance evolution of target insects is emerging as a major threat to the long-term efficacy of these applications. Studies on the detection of novel highly host-specific pesticidal proteins have been in urgent demand. A search for the source of Bt Cry toxins against E. zinckenella in the Vietnamese B. thuringiensis strain collection has been performed. The B. thuringiensis serovar canadensis SP142 is one of strains that resulted in more than 80% mortality to this pod borer. Its genome was estimated about 7.1 Mb and revealed a putative novel cry2Ab gene. The sequence analysis of cry2Ab gene revealed an open reading frame of 1 899 bp encoding a 633-amino acid protein with a calculated molecular mass of 70 kDa and 99.05% to 99.21% homology to known cry2Ab genes in the GenBank. There are eighteen different nucleotide sites which lead to five amino acid changes in Domain I and II. This gene was expressed in Escherichia coli BL21(DE3) and the purified Cry2Ab was toxic to E. zinckenella larvae with an LC<sub>50</sub> value of 1.74 µg/g diet. The novel Cry2Ab was designated as Cry2Ab39 by the Bacterial Pesticidal Protein Resource Center and its sequence was deposited in the GenBank (MN319700.1). This is a type of novel Cry2 toxin from B. thuringiensis against E. zinckenella, and it is important for breeding E. zinckenella-resistant soybeans.