Bacillus Thuringiensis Toxins Research Articles

CHEMICAL COMPOSITION AND INSECTICIDAL EFFICACY OF VITEX NEGUNDO L. ESSENTIAL OIL AGAINST FALL ARMYWORM, SPODOPTERA FRUGIPERDA (J. E. SMITH)

The fall armyworm, Spodoptera frugiperda, is a key pest ofmaize crops in tropical and subtropical regions and has developed resistanceagainst synthetic pesticides and Bacillus thuringiensis toxins. Thus,the experiments were designed to evaluate the chemical analysis andinsecticidal efficacy of Vitex negundo against S. frugiperda.Chemical analysis of V. negundo essential oil revealed a total of 36compounds, and the oil contained more monoterpenes (68.16%) than sesquiterpenes(22.21%) and diterpenes (13.56%). For the ovicidal and insecticidal activities,the essential oil was found to be most effective, with a median lethalconcentration (LC50) value of 51.31 µL/mL and 0.29 L/larvae, respectively.Larval antifeedant and growth inhibitory activity of essential oil werepositively correlated (R2 = 0.9645) and (R2 = 0.9727) with oil concentrations,respectively. Therefore, the V. negundo essential oil can be used forthe development of bioinsecticides to control S. frugiperda, a highlyinvasive and polyphagous pest, which will replace chemical pesticides.

Toxicity of Cry- and Vip3Aa-Class Proteins and Their Interactions against Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is one of the most important insect pests affecting corn crops worldwide. Although planting transgenic corn expressing Bacillus thuringiensis (Bt) toxins has been approved as being effective against FAW, its populations' resistance to Bt crops has emerged in different locations around the world. Therefore, it is important to understand the interaction between different Bt proteins, thereby delaying the development of resistance. In this study, we performed diet-overlay bioassays to evaluate the toxicity of Cry1Ab, Cry1Ac, Cry1B, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, Vip3Aa11, Vip3Aa19, and Vip3Aa20, as well as the interaction between Cry1Ab-, Cry1F-, Cry2Ab-, and Vip3Aa-class proteins against FAW. According to our results, the LC50 values of Bt proteins varied from 12.62 ng/cm2 to >9000 ng/cm2 (protein/diet), among which the Vip3Aa class had the best insecticidal effect. The combination of Cry1Ab and Vip3Aa11 exhibited additive effects at a 5:1 ratio. Cry1F and Vip3Aa11 combinations exhibited additive effects at 1:1, 1:2, and 5:1 ratios. The combination of Cry1F and Vip3Aa19 showed an antagonistic effect when the ratio was 1:1 and an additive effect when the ratio was 1:2, 2:1, 1:5, and 5:1. Additionally, the combinations of Cry1F and Vip3Aa20 showed antagonistic effects at 1:2 and 5:1 ratios and additive effects at 1:1 and 2:1 ratios. In addition to the above combinations, which had additive or antagonistic effects, other combinations exhibited synergistic effects, with variations in synergistic factors (SFs). These results can be applied to the establishment of new pyramided transgenic crops with suitable candidates, providing a basis for FAW control and resistance management strategies.

Negative association between host plant suitability and the fitness cost of resistance to Bacillus thuringiensis (Bacillales:Bacillaceae).

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.

Knockdown of MAPK p38-linked genes increases the susceptibility of Chilo suppressalis larvae to various transgenic Bt rice lines

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.

Laboratory evaluation of toxicity of selected insecticides against egg and larval stages of cotton pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae)

BackgroundThe cryptic nature of pink bollworm Pectinophora gossypiella (Saunders) larvae enables its reduced vulnerability to insecticidal control. Further, the development of resistance against Bacillus thuringiensis (Bt) toxins posed a serious threat to transgenic cotton cultivation. This necessitated determining the critical timing of spray applications on the control effectiveness. This study assessed the influence of egg age (freshly laid vs. three-day-old) and the location of larvae (directly exposed to the insecticide residues on the boll rind vs. burrowed inside the bolls) on insecticide control efficacy.ResultsThe results revealed a significant decrease in the ovicidal activity of tested insecticides with an increase in the age of eggs from one day old to three days old (paired t-test, P < 0.05). The larvae directly exposed to the insecticide residues on the boll rind were more susceptible (> 80% mortality) than the larvae exposed after they had burrowed inside the bolls (< 49% mortality). The inhibitory effects of tested insecticides on developmental biology were more pronounced in the experiment on pre-larval release insecticide treatment compared with insecticide treatment given post-larval release and entry inside the bolls.ConclusionEgg age influences the insecticide susceptibility, as does the larval location, directly exposed vs burrowed inside the bolls. Older eggs and the larvae that had burrowed inside the green bolls of cotton were relatively less susceptible to the insecticide treatments. The toxic effects of insecticides on egg and larval stages were primarily ephemeral. These findings are significant for devising a comprehensive strategy for pink bollworm management on a sustainable basis.

Soil microbes from conservation agriculture systems reduce growth of Bt-resistant western corn rootworm larvae

AbstractSustainable agricultural management practices aimed at improving soil health can alter the soil microbiome, which can influence plant health and defenses against insects. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major belowground pest of corn. Pest management relies heavily on the planting of transgenic crops expressing Bacillus thuringiensis (Bt) toxins. In this study, we ask how corn-WCR interactions via the soil microbiome are affected by a conservation management system (extended corn-soybean-wheat rotation with cover crops under no-till) compared with a conventional management system (corn-soybean rotation under mulch tillage and no cover crops) when combined with transgenic Bt corn. To do this, we applied soil microbes from the conservation and conventional management systems to two corn lines, one producing Bt and one non-Bt. We then reared Bt-resistant and Bt-susceptible WCR on inoculated seedlings to examine plant and insect changes in fitness. We found that Bt was effective against susceptible larvae in both soil treatments. Bt-resistant larvae were ~ 20% smaller when reared in the presence of soil microbes from the conservation management system. Thus, control of Bt-resistant WCR may be improved in a conservation system without sacrificing Bt effectiveness in susceptible insects. Comparing the microbial communities using 16S rRNA sequencing revealed that management practices influenced the microbiomes associated with the soil and the plant rhizosphere, but not WCR. Our findings suggest value for growers in utilizing conservation management practices, such as no-till and cover crops, in agricultural systems through bottom-up changes to plant–insect interactions via the soil microbiome.

Development of a soybean leaf disc assay for determining oral insecticidal activity in the lepidopteran agricultural pest Helicoverpa armigera

Pest insects pose a heavy burden on global agricultural industries with small molecule insecticides being predominantly used for their control. Unwanted side effects and resistance development plagues most small molecule insecticides such as the neonicotinoids, which have been reported to be harmful to honeybees. Bioinsecticides like Bacillus thuringiensis (Bt) toxins can be used as environmentally-friendly alternatives. Arachnid venoms comprise another promising source of bioinsecticides, containing a multitude of selective and potent insecticidal toxins. Unfortunately, no standardised insect models are currently available to assess the suitability of insecticidal agents under laboratory conditions. Thus, we aimed to develop a laboratory model that closely mimics field conditions by employing a leaf disk assay (LDA) for oral application of insecticidal agents in a bioassay tray format. Neonate larvae of the cotton bollworm (Helicoverpa armigera) were fed with soybean (Glycine max) leaves that were treated with different insecticidal agents. We observed dose-dependent insecticidal effects for Bt toxin and the neonicotinoid insecticide imidacloprid, with imidacloprid exhibiting a faster response. Furthermore, we identified several insecticidal arachnid venoms that were active when co-applied with sub-lethal doses of Bt toxin. We propose the H. armigera LDA as a suitable tool for assessing the insecticidal effects of insecticidal agents against lepidopterans.

Effect of Bt toxin on net-spinning caddisfly Stenopsyche marmorata (Trichoptera, Stenopsychidae)

The Bacillus thuringiensis (Bt) toxin targets lepidopteran, dipteran and coleopteran pests. Despite their close taxonomic relationship to Lepidoptera, few studies have examined the hazard of Bt toxins on Trichoptera. We performed laboratory exposure tests to examine the effects of Bt var. aizawai on filter-feeding caddisflies, particularly Stenopsyche marmorata. In the continuous exposure test, the mortalities ranged from 0% at 1 mg/l to 100% at 20 mg/l 24 hours after exposure to Bta. The mortality at each concentration increased from 24 to 48 hours. In limited contact, mortality increased with contact time. Acclimatisation and water quality also affected larval mortality. Our results suggest that higher concentration and longer exposure to Bta affect or affect the increase of larval mortality.

Sequential and Simultaneous Interactions of Plant Allelochemical Flavone, Bt Toxin Vip3A, and Insecticide Emamectin Benzoate in Spodoptera frugiperda.

Target pests of genetically engineered crops producing both defensive allelochemicals and Bacillus thuringiensis (Bt) toxins often sequentially or simultaneously uptake allelochemicals, Bt toxins, and/or insecticides. How the three types of toxins interact to kill pests remains underexplored. Here we investigated the interactions of Bt toxin Vip3A, plant allelochemical flavone, and insecticide emamectin benzoate in Spodoptera frugiperda. Simultaneous administration of flavone LC25 + Vip3A LC25, emamectin benzoate LC25 + Vip3A LC25, and flavone LC15 + emamectin benzoate LC15 + Vip3A LC15 but not flavone LC25 + emamectin LC25 yielded a mortality significantly higher than their expected additive mortality (EAM). One-day pre-exposure to one toxin at LC5 followed by six-day exposure to the same toxin at LC5 plus another toxin at LC50 showed that the mortality of flavone LC5 + Vip3A LC50, emamectin benzoate LC5 + Vip3A LC50, and Vip3A LC5 + emamectin benzoate LC50 were significantly higher than their EAM, while that of flavone LC5 + emamectin benzoate LC50 was significantly lower than their EAM. No significant difference existed among the mortalities of Vip3A LC5 + flavone LC50, emamectin benzoate LC5 + flavone LC50, and their EAMs. The results suggest that the interactions of the three toxins are largely synergistic (inductive) or additive, depending on their combinations and doses.

Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

More than 10 years after commercialization, Vip3A-expressing MIR162 remains highly efficacious in controlling major Lepidopteran maize pests: laboratory resistance selection versus field reality

MIR162, a maize event that expresses Vip3Aa20 (Vip3A) approved for commercial cultivation around 2010, has been excellent for control of major Lepidopteran pests. However, development of fall armyworm (FAW) resistance to Vip3A is a serious concern. Resistant colonies selected in the laboratory can serve as valuable tools not only for better understanding of Vip3A's mode of action (MOA) and mechanism of resistance (MOR) but also for screening novel leads of new MOA that will help control FAW in case resistance to Vip3A in the field becomes a reality. We selected a Vip3A-resistant FAW strain, FAWVip3AR, by subjecting a FAW founder population containing field genetics to Vip3A exposure. FAWVip3AR had >9800-fold resistance to Vip3A by diet surface overlay bioassays and resistance was stable. Feeding bioassays using detached leaf tissues or whole plants indicated that FAWVip3AR larvae readily fed and completed the full life cycle on Vip3A-expressing MIR162 maize plants and leaf tissues that killed 100% of susceptible larvae. Yet, FAWVip3AR faced at least two challenges. First, FAWVip3AR suffered an apparent disadvantage (incomplete resistance) when feeding on MIR162 in comparison to FAWVip3AR feeding on Vip3A-free isoline AX5707 maize; and second, FAWVip3AR showed a fitness costs in comparison to a Vip3A-susceptible strain when both fed on AX5707. We also demonstrated that, >10 years after commercialization, MIR162 and Vip3A remain highly efficacious against field populations of three major Lepidopteran pests from different geographic locations and FAW strains resistant to other Bacillus thuringiensis (Bt) toxins that are currently on the market.

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila.

Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.

Genetic Engineering of Escherichia coli BL21 (DE3) with a codon-optimized insecticidal toxin complex gene tccZ.

A toxin complex consists of a high-molecular-weight group of toxins that exhibits insecticidal activity against insect pests. These toxins are a promising alternative to Bacillus thuringiensis (Bt) toxins that have been extensively utilized in insect pest control. Herein, a codon-optimized insecticidal gene (tccZ) (381 bp) identified in Pantoea ananatis strain MHSD5 (a bacterial endophyte previously isolated from Pellaea calomelanos) was ligated into the pET SUMO expression vector and expressed in Escherichia coli BL21 (DE3). We report the success of cloning the tccZ gene into the pET SUMO vector and ultimately the transformation into E. coli BL21 (DE3) competent cells. However, despite conducting a time course of expression as well as isopropyl β-d-1-thiogalactopyranoside (IPTG) dosage optimization to determine optimal conditions for expression, TccZ protein expression could not be detected on Stain-Free and Coomassie-stained SDS-PAGE gels.

EFFECT OF SAW GINNING ON THE FIBRE QUALITY OF Bt AND NON-Bt COTTON

Bt cotton refers to a plant which has Bacillus thuringiensis (Bt) toxins in many of its cells. This naturally occurring soil bacterium will be used to reduce insect damage from bollworm, pink bollworm, and budworm. Therefore, farmers who are cultivating the Bt cotton variety will not be subjected to spray pesticides to control these worms. Whereas in this study, the non-Bt cotton (DP-90) refers to the commercially known variety which have no Bacillus thuringiensis (Bt) toxins in its cells. During the cultivation of non-Bt cotton varieties, the control of bollworms is done through the application of pesticides, which is a costly exercise in terms of cost of pesticides, spray equipment and labour. Along with the cost of cultivation, the best index to cotton quality is the performance of the fibres during spinning at the textile mill. In the present study the effect of saw ginning to the quality of both varieties was studied. The result could help the spinners to predict the preperformance of both varieties when subjected to the mechanical action of modern high rotating spinning machines parts. Ginning results a significant effect (at 0.05 level of significance) in all other measured fibre quality properties (upper half mean length, length uniformity index, short fibre content by number and by weight, level of neps, single fibre tenacity and elongation) of both Bt and non-Bt (DP-90) cotton varieties. The impact of ginning on the studied fibre quality properties was relatively severer on Bt cotton varieties than non-Bt (DP-90) varieties.

Resistance Bioassays and Allele Characterization Inform Analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) Introduction Pathways in Asia and Australia.

The fall armyworm (FAW) Spodoptera frugiperda (Smith; Lepidoptera: Noctuidae) is present in over 70 countries in Africa, Asia, and Oceania. Its rapid dispersal since 2016 when it was first reported in western Africa, and associated devastation to agricultural productivity, highlight the challenges posed by this pest. Currently, its management largely relies on insecticide sprays and transgenic Bacillus thuringiensis toxins, therefore understanding their responses to these agents and characteristics of any resistance genes enables adaptive strategies. In Australia, S. frugiperda was reported at the end of January 2020 in northern Queensland and by March 2020, also in northern Western Australia. As an urgent first response we undertook bioassays on two Australian populations, one each from these initial points of establishment. To assist with preliminary sensitivity assessment, two endemic noctuid pest species, Helicoverpa armigera (Hübner; Lepidoptera, Noctuidae) and Spodoptera litura (Fabricius; Lepidoptera, Noctuidae), were concurrently screened to obtain larval LC50 estimates against various insecticides. We characterized known resistance alleles from the VGSC, ACE-1, RyR, and ABCC2 genes to compare with published allele frequencies and bioassay responses from native and invasive S. frugiperda populations. An approximately 10× LC50 difference for indoxacarb was detected between Australian populations, which was approximately 28× higher than that reported from an Indian population. Characterization of ACE-1 and VGSC alleles provided further evidence of multiple introductions in Asia, and multiple pathways involving genetically distinct individuals in Australia. The preliminary bioassay results and resistance allele patterns from invasive S. frugiperda populations suggest multiple introductions have contributed to the pest's spread and challenge the axiom of its rapid 'west-to-east' spread.

Cyclosporin A as a Potential Insecticide to Control the Asian Corn Borer Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae).

Simple SummaryWhile chemical insecticides are conventionally effective for pest control, they also cause severe problems, such as insect resistance and environmental pollution. Alternative insecticides with distinct mechanisms are needed. Cyclosporin A (CsA), a polypeptide produced by fungi, is widely used as an immunosuppressant in organ transplantation. This study used Ostrinia furnacalis, a major pest of maize in Asia, as a model to investigate the insecticidal activity of CsA. CsA had marked insecticide activity against O. furnacalis larvae. Furthermore, sublethal doses of CsA caused negative effects on larval, pupal and adult development. CsA treatments suppressed reproduction, significantly decreasing the mating rate, ovary size, and numbers and hatchability of eggs. CsA showed excellent combined toxicities when mixed with five common insecticides, respectively. Importantly, CsA acts by suppressing calcineurin, which represents a novel insecticidal target. Thus, CsA has potential as a new insecticide for sustainable pest management.The long-term use of chemical insecticides has caused serious problems of insect resistance and environmental pollution; new insecticides are needed to solve this problem. Cyclosporin A (CsA) is a polypeptide produced by many fungi, which is used to prevent or treat immune rejection during organ transplantation. However, little is known about the utility of CsA as an insecticide. Therefore, this study evaluated the insecticidal activity of CsA using Ostrinia furnacalis as a model. The results demonstrated that CsA was toxic to O. furnacalis with LC50 values of 113.02 μg/g and 198.70 μg/g for newly hatched neonates and newly molted third-instar larvae, respectively. Furthermore, CsA treatment had sublethal effects on the development of O. furnacalis, and significantly reduced the fecundity of adults; this suggests that CsA has great potential to suppress O. furnacalis populations. Further analysis revealed that CsA suppressed calcineurin activity in larvae. CsA had independent or synergistic toxic effects on O. furnacalis when combined with β-cypermethrin, indoxacarb, emamectin benzoate, azadirachtin, and the Bacillus thuringiensis toxin Cry1Ac, which suggests that CsA can help prevent or manage resistance. Our study provides detailed information on the potential of CsA as an insecticide for controlling lepidopterans.

Resistance of Cabbage Loopers to Bacillus thuringiensis (Bt) Toxin Cry1F and to Dual-Bt Toxin WideStrike Cotton Plants.

The Cry proteins from Bacillus thuringiensis (Bt) are major insecticidal toxins in formulated Bt sprays and are expressed in genetically engineered Bt crops for insect pest control. However, the widespread application of Bt toxins in the field imposes strong selection pressure on target insects, leading to the evolution of insect resistance to the Bt toxins. Identification and understanding of mechanisms of insect resistance to Bt toxins are an important approach for dissecting the modes of action of Bt toxins and providing knowledge necessary for the development of resistance management technologies. In this study, cabbage looper (Trichoplusia ni) strains resistant to the transgenic dual-Bt toxin WideStrike cotton plants, which express Bt toxins Cry1Ac and Cry1F, were selected from T. ni strains resistant to the Bt formulation Bt-DiPel. The WideStrike-resistant T. ni larvae were confirmed to be resistant to both Bt toxins Cry1Ac and Cry1F. From the WideStrike-resistant T. ni, the Cry1F resistance trait was further isolated to establish a T. ni strain resistant to Cry1F only. The levels of Cry1F resistance in the WideStrike-resistant and the Cry1F-resistant strains were determined, and the inheritance of the Cry1F-resistant trait in the two strains was characterized. Genetic association analysis of the Cry1F resistance trait indicated that the Cry1F resistance in T. ni isolated in this study is not shared with the Cry1Ac resistance mechanism nor is it associated with a mutation in the ABCC2 gene, as has so far been reported in Cry1F-resistant insects. IMPORTANCE Insecticidal toxins from Bacillus thuringiensis (Bt) are highly effective for insect control in agriculture. However, the widespread application of Bt toxins exerts strong selection for Bt resistance in insect populations. The continuing success of Bt biotechnology for pest control requires the identification of resistance and understanding of the mechanisms of resistance to Bt toxins. Cry1F is an important Bt toxin used in transgenic cotton, maize, and soybean varieties adopted widely for insect control. To understand the mode of action of Cry1F and mechanisms of Cry1F resistance in insects, it is important to identify Cry1F-specific resistance and the resistance mechanisms. In this study, Trichoplusia ni strains resistant to commercial "WideStrike" cotton plants that express Bt toxins Cry1Ac and Cry1F were selected, and a Cry1F-specific resistant strain was isolated. The isolation of the novel Cry1F-specific resistance in the T. ni provided an invaluable biological system to discover a Cry1F-specific novel resistance mechanism.

Susceptibilities of the Invasive Fall Armyworm (Spodopterafrugiperda) to the Insecticidal Proteins of Bt maize in China

To control the fall armyworm (FAW), Spodoptera frugiperda (Smith), a serious threat to maize production in China, the Chinese government has issued biosafety certificates for transgenic insect-resistant maize expressing Bt (Bacillus thuringiensis) toxins including Bt-Cry1Ab maize (crop event DBN9936), Bt-Vip3Aa maize (event DBN9501), Bt-(Cry1Ab+Vip3Aa) maize with superimposed traits (event DBN9936 × DBN9501) and Bt-(Cry1Ab+Vip3Aa) maize with superimposed traits (event Bt11 × MIR162), but the susceptibility baselines of geographically distinct FAW populations to these events, which form the basis for managing resistance development in the pest to these events, are not clear. We used the diet-incorporated bioassays method to detect the susceptibilities of the seven FAW populations collected from Yunnan, Henan and Hubei provinces in China in 2021 to the insecticidal proteins of the four Bt maize events. The result showed that the susceptibilities of different geographical populations to Bt insecticidal proteins were significantly different. In the seven populations, the range in median lethal concentrations (LC50) of Cry1Ab expressed in DBN9936 was 0.87–2.63 μg/g, 0.14–0.30 μg/g for Vip3Aa expressed in DBN9501, 0.78–1.86 μg/g for Cry1Ab+Vip3Aa expressed in DBN9936 × DBN9501, and 0.36–1.42 μg/g for CryAb+Vip3Aa expressed in Bt11 × MIR162. The growth inhibition responses also showed that the susceptibilities varied with the different median growth inhibitory concentration (GIC50) ranges (0.38–1.22, 0.08–0.28, 0.28–0.87, and 0.24–0.78 μg/g, respectively). The variations in the ranges of the susceptibility baselines of the geographical populations of fall armyworm in China to the insecticidal proteins expressed in the four events provide a scientific basis for monitoring FAW population resistance to Bt maize and managing the populations using different Bt maize events.

Cry3Aa Toxin Is Not Suitable to Control Lepidopteran Pest Spodoptera littoralis (Boisd.).

The toxicity of the Bacillus thuringiensis (Bt) toxin Cry3Aa—originally used against the main potato pest, the Colorado potato beetle, Leptinotarsa decemlineata—was verified on this species and then evaluated against the Egyptian armyworm, Spodoptera littoralis, which is a pest of several economically important plants. Larvae of S. littoralis were fed a semi-artificial diet supplemented either with a recombinant or with a natural Bt toxin Cry3Aa and with the genetically engineered (GE) potato of variety Superior NewLeaf (SNL) expressing Cry3Aa. Cry3Aa concentration in the diet and the content in the leaves were verified via ELISA (enzyme-linked immunosorbent assay) and HPLC (high-performance liquid chromatography) during and at the end of the experiments. The biological effectiveness of the coleopteran-specific Cry3Aa with previous reports of activity against S. littoralis was tested on five different populations of S. littoralis larvae by monitoring 13 parameters involving development from penultimate instar, weight, the efficiency of food conversion to biomass, ability to reproduce, and mortality. Although some occasional differences occurred between the Cry3Aa treatments and control, any key deleterious effects on S. littoralis in this study were not confirmed. We concluded that the Cry3Aa toxin appears to be non-toxic to S. littoralis, and its practical application against this pest is unsuitable.

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.