Transgenic Bacillus Thuringiensis Research Articles

Expression of Cry1Ab/2Aj Protein in Genetically Engineered Maize Plants and Its Transfer in the Arthropod Food Web

While transgenic Bacillus thuringiensis (Bt) maize provides pest resistance and a reduced application of chemical pesticides, a comprehensive environmental risk assessment is mandatory before its field release. This research determined the concentrations of Bt protein in plant tissue and in arthropods under field conditions in Gongzhuling City, northeastern China, to provide guidance for the selection of indicator species for non-target risk assessment studies. Bt maize expressing Cry1Ab/2Aj and non-transformed near-isoline were grown under identical environmental and agricultural conditions. Cry1Ab/2Aj was detected in plant tissues and arthropods collected from Bt maize plots during pre-flowering, flowering, and post-flowering. The expression of Cry1Ab/2Aj varied across growth stages and maize tissues, as well as in the collected arthropods at the three growth stages. Therefore, representative species should be chosen to cover the whole growing season and to represent different habitats and ecological functions. Dalbulus maidis (Hemiptera: Cicadellidae), Rhopalosiphum padi (Hemiptera: Aphididae), Heteronychus arator (Coleoptera: Scarabaeidae), and Somaticus angulatus (Coleoptera: Tenebrionidae) are suitable non-target herbivores. Propylea japonica (Coleoptera: Coccinellidae), Paederus fuscipes (Coleoptera: Staphylinidae), Chrysoperla nipponensis (Neuroptera: Chrysopidae), and spiders are suggested predators. Apis cerana and Apis mellifera ligustica (both Hymenoptera: Apidae) represent pollinators and Folsomia candida (Collembola: Isotomidae) decomposers.

Sensitivities of Fall Armyworm (Spodoptera frugiperda) Populations in Different Regions of China to Four Bt Proteins

Fall armyworm (FAW), Spodoptera frugiperda, invaded the south of China in December 2018 and has since posed a huge threat to crop production in China. However, transgenic Bacillus thuringiensis (Bt) corn can efficiently control the damage caused by FAWs. In fact, the Chinese government has issued biosafety certificates for several Bt corn hybrids expressing any one of four Bt proteins, Vip3A, Cry1F, Cry1Ab, and Cry2Ab, or combinations thereof, to control FAWs. These Bt corn events are soon to be commercialized in China. Therefore, it is necessary to monitor and evaluate whether the FAW has developed resistance to any of the Bt corn hybrids planted in fields in China. To address this issue, we collected 11 geographical populations of FAWs and determined the sensitivity of each to the aforementioned four purified Bt proteins as assessed by diet surface overlay bioassays. The ranges for the 50% lethal concentration (LC50) of the four Bt proteins to all FAW populations were as follows: 11.42–88.33 ng/cm2 (for Vip3A), 111.21–517.33 (Cry1F), 135.76–1108.47 (Cry1Ab), and 994.42–5492.50 (Cry2Ab). The corresponding ranges for the 50% growth inhibition concentrations (GIC50) were 1.43–14.86, 2.35–138.97, 1.58–464.86, and 25.01–1266.07 ng/cm2. The lethal effects and growth inhibition effects of the four Bt proteins on FAW were in the same order of Vip3A > Cry1F > Cry1Ab > Cry2Ab. A comparison with published LC50 values of Bt proteins towards sensitive FAW populations revealed that all 11 FAW populations in this study were sensitive to Vip3A, Cry1F, and Cry1Ab. This study provides foundational data for monitoring and controlling the resistance of Bt corn to FAW in China.

Genome-scale analysis of ABC transporter genes and characterization of the ABCC type transporter genes in the oriental armyworm, Mythimna separata (Walker)

The oriental armyworm Mythimna separata is a polyphagous, migratory corn pest in China and other Asian countries. Transgenic Bacillus thuringiensis (Bt) corn may effectively control this insect pest. Several reports have suggested that ATP-binding cassette (ABC) transporter proteins may act as receptors that bind Bt toxins. However, our knowledge about ABC transporter proteins in M. separata is limited. We identified 43 ABC transporter genes in the M. separata genome by bioinformatics prediction. Evolutionary tree analysis grouped these 43 genes into 8 subfamilies, ABCA to ABCH. Among the 13 ABCC subfamily genes, the transcript levels of MsABCC2 and MsABCC3 were upregulated. In addition, RT-qPCR analyses of these two potentials showed that both were predominantly expressed in the midgut tissue. Knock-down of MsABCC2, but not MsABCC3, decreased Cry1Ac susceptibility as indicated by increased larval weight and reduced larval mortality. This suggested that MsABCC2 might play a more important role in Cry1Ac toxicity and that it is a putative Cry1Ac receptor in M. separata. Together, these findings provide unique and valuable information for future elucidating of the role of ABC transporter genes in M. separata, which is highly valuable and important for the long-term application of Bt insecticidal protein.

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.

Development and Evaluation of the Cotton Leaf Curl Kokhran Virus-Burewala Bidirectional Promoter for Enhanced Cry1Ac Endotoxin Expression in Bt Transgenic Cotton

Fluctuation in Cry1Ac endotoxin levels expressed in transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.) can result in a variation in efficacy throughout the growing season. Here, a green tissue-specific strong promoter of the cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) C1 gene is reported that can direct consistently high levels of Cry1Ac endotoxin expression in transformed cotton plants. The objective of this study was to investigate the capacities of the CLCuKoV-BuC1 promoter to drive transcription of Cry1Ac and stably express endotoxin in mature leaves and bolls of transgenic cotton plants, compared to the traditional CaMV35S promoter. The Cry1Ac gene expression cassettes were constructed under the control of a bidirectional promoter and transformed into cotton ‘MNK-786′. The expression of Cry1Ac constructs was evaluated in transient and stable expression systems using Nicotiana tabacum ‘Rustica’ and cotton plants, respectively. Accumulation of the Cry1Ac expressed in two resultant transgenic cotton plants harboring the constructs driven by the CLCuKoV-BuC1 and CaMV35S promoter, respectively, was analyzed using a commercially available enzyme-linked immunosorbent assay. In leaves and bolls of two cotton plants shown to express CLCuKoV-BuC1-Cry1Ac (CLCuV-Ac), the Cry1Ac protein accumulated at 400 and 300 ng g−1 per fresh tissue weight, respectively, whereas no toxin was detectable in the roots. In contrast, CaMV35S-Cry1Ac transgenic cotton plants accumulated three times less Cry1Ac protein than those transformed with CLCuV-Ac. Results indicate that the greatest amount of Cry1Ac endotoxin accumulated in transgenic cotton when expression was driven by the CLCuKoV-BuC1 compared to the CaMV35S promoter. Thus, the CLCuKoV-BuC1 promoter offered more robust transgene expression in cotton plants than the traditional CaMV35S promoter. The newly validated CLCuV-Ac promoter of begomoviral origin offers an exciting alternative as a robust promoter for genetic engineering of cotton and other plants.

Effect of Transgenic Cotton Expressing Bt Cry1Ac or Cry1Ab/Ac Toxins on Lacewing Larvae Mediated by Herbivorous Insect Pests.

A simple food chain (plant, insect pests, and predatory arthropods) in an agro-ecosystem was set up here as a model system to elucidate the potential effect of transgenic Bacillus thuringiensis (Bt) cotton on non-target organisms. The system included transgenic/non-transgenic cotton, neonate larvae of three herbivorous insects (Spodoptera exigua, Helicoverpa armigera, and S. litura), and predatory lacewing larvae (Chrysopa spp.), which represent the first, second, and third trophic levels, respectively. The results showed that transgenic treatments and different densities of prey had significant effects on both body-weight gain of neonate herbivorous larvae and the number of prey captured by lacewing larvae, respectively. It was found that Bt toxin could persist at the third trophic level in lacewing larvae. The diet mixture bioassay showed that body-weight gain of lacewing larvae was significantly affected by various treatments, especially at lower concentrations of plant-expressed Bt toxin in the diet mixture, which caused significant decreases in body-weight gain. In contrast, synthetic Bt toxin at higher concentrations in the diet did not show this effect. Thus, we inferred that Bt toxin indirectly affected the growth of the lacewings and the lacewings may not be susceptible to Bt toxin or are able to metabolize it.

Impact of Transgenic Cry1Ab/2Aj Maize on Abundance of Non-Target Arthropods in the Field.

Transgenic Bacillus thuringiensis (Bt) maize has broad prospects for application in China. Before commercialization, it is necessary to assess possible ecological impacts, including impacts on non-target arthropods (NTAs) in the field. In the present study, transgenic Bt maize expressing cry1Ab/2Aj and its corresponding non-transformed near isoline were planted under the same environmental and agricultural conditions, and arthropods in the field were collected during the three main growth stages of maize. In a one year trial, the results showed the composition of NTA communities in the transgenic and control maize fields were similar. There were no significant differences for community-level parameters of species richness (S), Shannon–Wiener diversity index (H′), evenness index (J) and Simpson’s dominant concentration (C) between the two types of maize fields. Likewise, a Bray–Curtis dissimilarity and distance analysis showed that Cry1Ab/2Aj toxin exposure did not increase community dissimilarities between Bt and non-Bt maize plots and that the structure of the NTAs community was similar on the two maize varieties. Furthermore, planting of the transgenic cry1Ab/2Aj maize did not affect the density or composition of non-target decomposers, herbivores, predators, parasitoids and pollinator guilds. In summary, our results showed that planting of Bt maize producing Cry1Ab/Cry2Aj proteins do not adversely affect population dynamics and diversity of NTAs.

Transcriptomic and Metabolomic Responses in Cotton Plant to Apolygus lucorum Infestation.

With the wide-scale adoption of transgenic Bacillus thuringiensis (Bt) cotton, Apolygus lucorum (Meyer-Dür) has become the most serious pest and has caused extensive yield loss in cotton production. However, little is known about the defense responses of cotton at the seedling stage to A. lucorum feeding. In this study, to elucidate the cotton defense mechanism, cotton leaves were damaged by A. lucorum for 0, 4, 12 and 24 h. The transcriptomic results showed that A. lucorum feeding elicits a rapid and strong defense response in gene expression during the whole infestation process in cotton plants. Further analysis revealed that at each assessment time, more differentially expressed genes were up-regulated than down-regulated. The integrated analysis of transcriptomic and metabolic data showed that most of the genes involved in jasmonic acid (JA) biosynthesis were initially up-regulated, and this trend continued during an infestation. Meanwhile, the content levels of JA and its intermediate products were also significantly increased throughout the whole infestation process. The similar trend was displayed in condensed tannins biosynthesis. This research proved that, after plants are damaged by A. lucorum, the JA pathway mediates the defense mechanisms in cotton plants by promoting the accumulation of condensed tannins as a defense mechanism against A. lucorum. These results will help us to discover unknown defensive genes and improve the integrated pest management of A. lucorum.

Expression and Purification cry1Aa Gene

The cry gene encodes a crystal protein of Bacillus thuringiensis known as Cry toxin, which is toxic to major Lepidopteran insect pest. The effectiveness of Cry toxin against the rice yellow stem borer (YSB, Scirpophaga incertulas Wlk) has been reported. Transgenic Bacillus thuringiensis (Bt) rice has been developed by introducing the fusion gene cry1B::cry1Aa gene driven by the CaMV35S promoter to Javanica rice cv. Rojolele to improve its resistance against rice YSB. Resistance to YSB at a greenhouse and multi-site scales have been conducted and reported. Food and feed safety studies for the release and commercialization of genetically modified crops require a sufficient amount of purified protein sample. Therefore, a strategy to produce Cry1Aa protein in a short time with high productivity needs to be developed. In this study, the cry1Aa gene (1857 bp) was cloned into pRHA-SDM expression vector under the control of the pRHA promoter fused to a 6xHis tag on the C-terminal to produce pRHA-SDM-cry1Aa. The expression host used was Escherichia coli strain NiCo21, and protein purification was performed using IMAC Co2+ on the TALON® matrix. The results showed that the recombinant Cry1Aa protein, approximately 69 kDa, was detected using Western blot performed using anti-rabbit Cry1Aa polyclonal antibody and His detector nickel-NTA conjugated with HRP reporter enzyme. Expression and purification protocols that have been developed can be used to produce Cry1Aa protein which can be utilized for further protein studies.

Recovery Characteristics of Cry1Ac Endotoxin Expression and Related Physiological Mechanisms in Bt Transgenic Cotton Squares after High-Temperature Stress Termination

High-temperature stress reduces the endotoxin expression of the insecticidal gene (Cry1Ac) in transgenic Bacillus thuringiensis (Bt) cotton and affects nitrogen metabolism. However, its effects on Cry1Ac endotoxin expression after high-temperature stress termination remain unclear. In order to investigate the effect of high-temperature stress on the expression of insecticidal proteins in Bt cotton squares, the conventional Sikang-1 cultivar and hybrid Sikang-3 cultivar were used as the experimental materials. The potted cotton plants in the squaring stage were moved to an artificial climatic chamber at 38 °C for 72 h and 96 h in 2017 and 2018, respectively, and plants were moved to the climate chamber where the control cotton plants were located (at 27 °C). Then, cotton squares were collected to measure the Bt protein concentration and nitrogen metabolism physiology at 0, 12, 24, 48, 72 and 96 h after high-temperature stress termination, respectively. The Cry1Ac endotoxin expression of the squares could be recovered to the corresponding control level and a longer recovery time was required as the high-temperature stress period increased. Therefore, the recovery degree of Cry1Ac endotoxin expression of cotton squares can be predicted according to the duration of high-temperature stress, which may provide a reference for the rational control of Helicoverpa armigera and related pests in cotton production.

Effects of transgenic Bacillus Thuringiensis maize (2A-7) on the growth and development in rats

This study aimed to determine the effects of genetically modified insect-resistant maize (2A-7) on the growth and development in developing rats. Rats were fed a diet formulated with 2A–7 maize and were compared with rats fed a diet formulated with non-transgenic maize (CK group) and rats fed AIN-93G diet (BC group). 2A-7 maize was formulated into diets at ratios of 82.4% (H group) and 20.6% (L group); non-transgenic maize was formulated into diets at a ratio of 82.4%. From the first day of pregnancy, adult rats were divided into four groups and fed with the above four diets, respectively. Weaning on postnatal day 21, the diets of offspring were consistent with their parents. The results showed that body weight, hematology, serum biochemistry, organ weight, organ coefficients and allergenicity of offspring fed with 2A–7 maize were comparable with those in the CK and BC groups. In physiological and behavioral development experiments, there was no statistically significant difference among groups. Although mCry1Ab proteins were detected in organs and serum, no histopathological changes were observed among groups. In conclusion, A-7 maize cause no treatment-related adverse effects on offspring, indicating that 2A-7 maize is safe for developing rats.

Trait-based comparison of transgenic Bt rice and its non-Bt counterpart in response to soil copper pollution.

Transgenic Bacillus thuringiensis (Bt) rice can provide economic and environmental benefits under the current increasing demand for food and socioeconomic pressures for sustainability. However, information about the ecological adaptation of Bt rice under nontarget environmental stress is still lacking. This study compared the adaptability of one Bt rice and its nontransgenic counterpart to soil copper (Cu) pollution in terms of agronomic and physiological traits. With Cu addition, grain yield and biomass of both cultivars were significantly decreased. Within the same Cu treatment, Bt rice exhibited higher biomass and close plant height, chlorophyll content, grain yield, and grain quality compared with non-Bt rice, except for the grain yield with a 35 mg kg-1 Cu addition with respect to which Bt rice was significantly lower by 22%. The Cu content in Bt rice was generally lower, whereas the antioxidant enzyme activity and lipid peroxidation were stronger than the non-Bt. These results demonstrated that Bt rice exhibited close adaptability but higher Cu tolerance compared with the non-Bt under Cu stress.

Differential impact of Bt-transgenic rice plantings on bacterial community in three niches over consecutive years

Transgenic-Bacillus thuringiensis (Bt) crops express insecticidal proteins, which can accumulate in plants and soil where they may influence microbial populations. The impact of Bt crops on bacterial communities has only been assessed under short-term, and results have been contradictory. Here, we analyzed the bacterial communities in three niches, rhizosphere soil (RS), root endosphere (RE) and leaf endosphere (LE), of three Bt rice and their non-Bt parental lines for three consecutive years by high-throughput sequencing. In principal coordinate analysis (PCoA) and PERMANOVA (Adonis) analysis, operational taxonomic units (OTUs) were clustered primarily by niche type and differed significantly in the RE and LE but not in the RS between each of three Bt lines compared with the non-Bt rice line, and not in each respective niche among the three Bt rice lines. The bacterial communities in the RS of different rice lines over the 3 years were clustered mainly by year rather than by lines. The differential bacterial taxa among the lines did not overlap between years, presumably because Cry proteins are rapidly degraded in the soil. A network analysis of RS bacterial communities showed that the network complexity and density for the three Bt rice lines did not decrease compared with those for the non-Bt line. In conclusion, our results demonstrated that bacterial communities differed significantly in RE and LE between Bt and non-Bt rice lines, but the differences were mild and transient, and had no adverse impact on RS over the 3 years. This study provides favorable evidence in support of the commercialization of Bt rice.

Successful development and implementation of a practical proactive resistance management plan for Bt cotton in Australia.

This article describes the design and > 20 years of effective implementation of a proactive resistance-management plan for transgenic Bacillus thuringiensis (Bt) cotton that targets Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) in Australia, considering pest biology and ecology, insights from resistance-evolution modelling, and the importance of the human component to effective implementation. This is placed in the context of processes associated with adaptive resource management. Bt cotton has provided Australian cotton growers with technology to manage Helicoverpa species that previously challenged the industry's viability, while at the same time resulting in no detectable changes in the resistance allele frequency in field populations of either Helicoverpa species in eastern Australia. This is the most long-lived and successful global example of a proactive resistance management plan for an insect pest. Six key learnings important to the successful development and implementation of a proactive transgenic-crop resistance management plan are: the programme has to have a strong science base; there has to be broad stakeholder support at all levels; there has to be a strong implementation programme; the plan needs to be supported by auditing and enforced remediation of deviations from the mandated resistance management plan; A programme of rigorous and on-going resistance allele monitoring; an attitude of continuous improvement for all aspects of the resistance management plan. The lessons learnt from the deployment of Bt cotton in Australia are relevant globally and provide important guidelines for the deployment of transgenic crops for insect control wherever they are grown. © 2021 Society of Chemical Industry.

Degradation of transgenic Bacillus thuringiensis proteins in corn tissue in response to post-harvest management practices.

Knowledge of the persistence of Cry proteins in transgenic corn residue after harvest is necessary to assess the ecological risk to nontarget organisms. The amount of protein remaining in crop residue declines over time by a combination of microbial decomposition and leaching, both influenced by temperature, precipitation, and the amount of residue-soil contact. Here, we investigated how long biologically active Cry proteins persist in SmartStax corn residue expressing Cry1A.105, Cry1F, Cry2Ab2, Cry3Bb1, and Cry34/35Ab1, when subjected to four post-harvest practices (chisel plow tillage, flail mowing, cover crop planting, and undisturbed residue). Protein activity in residue samples collected up to 25weeks after harvest was measured by Ostrinia nubilalis feeding bioassays and cross validated with detection frequencies determined by ELISA. All corn residue remained above ground in the flail-mowed and undisturbed treatments, while the cover crop and chisel plow treatments left 88.3 and 39.6% of the residue remaining above ground, respectively. Cry proteins retained biological activity for as long as 24weeks after harvest when residue was left above ground with less soil contact, typical of no-till corn systems. ELISA detections were positively correlated with results of the feeding bioassays, which revealed the presence of active proteins beyond the point of ELISA detection.

Baseline Susceptibility and Laboratory Selection of Resistance to Bt Cry1Ab Protein of Chinese Populations of Yellow Peach Moth, Conogethes punctiferalis (Guenée).

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.

Does cotton bollworm show cross-resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab? A mini review

Since 1996, transgenic Bacillus thuringiensis (Bt) cotton has been commercially grown in numerous countries in an effort to stem the losses caused by key lepidopteran pests. However, the development of pest resistance to Bt toxins has jeopardized the continued utilization of Bt cotton. As a strategy designed to circumvent the development of resistance, Bt cotton varieties expressing two or more toxins targeting the same pest have been introduced. Nevertheless, from the perspective of long-term planting of Bt cotton, the potential risk of cross-resistance to these Bt toxins is a threat that cannot be ignored. In this paper, we review current research (including that based on the analysis of protein binding sites and resistance genes) on the resistance of cotton bollworm (Helicoverpa armigerd) to the Bt toxins Cry1Ac and Cry2Ab and the interrelationship between these toxins. On the basis of existing evidence, we assume that the actions of Cry1Ac and Cry2Ab against cotton bollworm are not completely independent, and then propose the “resistance-associated gene mutation potential hypothesis”. Although the mechanisms underlying the resistance of pests to Bt toxins are yet to be comprehensively elucidated, this hypothesis could undoubtedly have important implications for adopting “pyramid” strategy in the future. Further research is recommended to devise strategies to retard the development of H. armigera resistance to Bt cotton, either using different Bt toxins or their various combinations.

Bacillus thuringiensis protein Vip3Aa does not harm the predator Propylea japonica: A toxicological, histopathological, biochemical and molecular analysis.

The ladybeetle Propylea japonica is a widely distributed natural enemy in many agricultural systems. P. japonica is often used as a test organism for safety assessments of transgenic Bacillus thuringiensis crops. Plant varieties expressing the Vip3Aa insecticidal protein are not currently commercially available in China. In this study, protease inhibitor E-64 was used as a positive control to examine the responses of P. japonica larvae to a high concentration of Vip3Aa proteins. Larvae that were fed E-64 had increased mortality and prolonged developmental period, but these parameters were unaffected when larvae were fed Vip3Aa. The epithelial cells of midguts were intact and closely connected with the basal membrane when larvae were fed Vip3Aa, but the epithelial cells degenerated in the E-64 treatment. The activities of antioxidative enzymes and expression levels of detoxification-related genes in P. japonica larvae were not altered after exposure to Vip3Aa; however, these biochemical and molecular parameters were significantly changed in the E-64 treatment. The results demonstrate that Vip3Aa protein is not harmful to the predator P. japonica.

Transgenic cotton and farmers' health in Pakistan.

Despite substantial research on the economic effects of transgenic insect-resistant Bacillus thuringiensis (Bt) cotton, there is still limited work on this technology’s impacts on human health. Due to the inbuilt insect resistance, Bt cotton requires fewer pesticide sprays than conventional cotton, which is not only advantageous from economic and environmental perspectives, but may also result in health benefits for farmers. Using socioeconomic and biophysical data from Pakistan, we provide the first evidence of a direct association between Bt gene expression in the plant and health benefits. A key feature of this study is that Bt cotton cultivation in Pakistan occurs in a poorly regulated market: farmers are often mistaken in their beliefs about whether they have planted Bt cotton or conventional cotton, which may affect their pesticide-use strategies and thus their pesticide exposure. We employ a cost-of-illness approach and variations in the measurement of Bt adoption to estimate the relationship between Bt cotton and farmers’ health. Bt adoption based on farmers’ beliefs does not reduce the pesticide-induced cost of illness. However, adoption based on measuring Bt gene expression is associated with significant health cost savings. Extrapolating the estimates for true Bt seeds to Pakistan’s entire Bt cotton area results in annual health cost savings of around US$ 7 million. These findings have important implications for the regulation of seed markets in Pakistan and beyond: improved regulations that ensure claimed crop traits are really expressed can increase the benefits for farmers and society at large.

Efficacy of Cry1Ac toxin from Bacillus thuringiensis against the beet armyworm, Spodoptera exigua (H\xfcbner) (Lepidoptera: Noctuidae)

Transgenic Bacillus thuringiensis (Bt) cotton is engineered to express delta endotoxin (Cry toxin) proteins in lepidopteran midgut when ingested by lepidopteran larvae such as the beet armyworm, Spodoptera exigua (Hübner). The efficacy of Bt technology depends on stage, age, and susceptibility of the tested population to Cry proteins. The baseline susceptibility bioassay test for first, second, and third larval instars of S. exigua, collected from Faisalabad, Multan, and Bahawalpur, compared with susceptible laboratory population was carried out in 2015 and 2016. The LC50 ranged from 0.45 to 2.52 μg ml−1, 1.08 to 5.74 μg ml−1, and 2.01 to 7.85 μg ml−1 for first, second, and third larval instars, respectively. The Bahawalpur population was highly resistant and showed 5.63, 5.30, and 3.89 variations than the susceptible population, followed by Multan 3.01, 3.71, 3.10, and Faisalabad 1.93, 2.41, 2.31 population for first, second, and third larval instars, respectively. The molt inhibitory concentration (MIC50) ranged from 0.04 to 0.56 μg ml−1, 0.08 to 0.99 μg ml−1, and 0.10 to 1.42 μg ml−1 for the three instars, respectively. The trend in lethal concentration and its respective resistance level was higher in 2016 than in 2015.