Prey-mediated effects of Mpp51Aa2-producing cotton on longevity and reproduction of Orius majusculus.

Genetically engineered (GE) cotton event MON 88702, producing Mpp51Aa2 (previously mCry51Aa2) from Bacillus thuringiensis (Bt), controls sucking pests, such as Lygus spp. (Hemiptera: Miridae) and thrips (Thysanoptera). Ingesting high doses of the insecticidal protein resulted in adverse effects on life table parameters of beneficial, predatory Orius spp. (Hemiptera: Anthocoridae). This triggered laboratory studies with more realistic food treatments, including different combinations of prey types with and without Bt protein to further characterize risks to this important group of non-target organisms. In this work, exclusive feeding of frozen spider mites (Tetranychus urticae, Acari: Tetranychidae) from Bt cotton confirmed adverse effects on longevity and fecundity of O. majusculus adults. Alternate feeding of Bt protein-containing spider mites and Bt-free Ephestia kuehniella (Lepidoptera: Pyralidae) eggs mitigated effects on longevity, but not on fecundity. When living larvae of Spodoptera littoralis (Lepidoptera: Noctuidae) from Bt cotton were fed to the predators, however, no effects on longevity and reproduction of female O. majusculus were observed, despite the fact that Bt protein concentrations in larvae were almost as high as concentrations in spider mites. When a diverse mix of prey species with various Bt protein concentrations is consumed in the field, it is unlikely that exposure of Orius spp. to Mpp51Aa2 is high enough to exert adverse effects on predator populations. MON 88702 cotton may thus be a valuable tool for integrated management of sucking pests.

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.

Prey-mediated effects of mCry51Aa2-producing cotton on the predatory nontarget bug Orius majusculus (Reuter).

Genetically engineered (GE) cotton, MON 88702, is protected against certain sucking pests, such as plant bugs and thrips, by producing mCry51Aa2, a modified protein from Bacillus thuringiensis (Bt). Predatory pirate bugs (Orius spp.), natural enemies contributing to biological pest control, are also sensitive to the insecticidal protein when exposed continuously to high concentrations. We evaluated effects of MON 88702 on Orius majusculus when fed prey types with different mCry51Aa2 concentrations. When neonates were provided exclusively Tetranychus urticae spider mites reared on MON 88702 (high mCry51Aa2 content), adverse effects on predator survival and development were confirmed, compared with specimens fed prey from near-isogenic non-Bt cotton. When fed a mixture of T. urticae and Ephestia kuehniella eggs (mCry51Aa2-free), predator life table parameters were similar to the treatment where eggs were fed exclusively. When mCry51Aa2-containing spider mites were provided for a limited time at the beginning or the end of juvenile development, effects were less pronounced. While pirate bug nymphs showed similar consumption rates for prey from Bt and non-Bt cotton, choice experiments revealed a preference for E. kuehniella eggs over spider mites. Lepidopteran larvae (Spodoptera littoralis, high mCry51Aa2 content) or cotton aphids (Aphis gossypii, mCry51Aa2-free) reared on MON 88702 as alternative prey did not result in adverse effects on O. majusculus. Our study suggests limited risk of mCry51Aa2-producing cotton for O. majusculus, because its sensitivity for the Bt protein is relatively low and its natural food consists of diverse prey species with varying concentrations of Bt protein.

No adverse dietary effect of a cisgenic fire blight resistant apple line on the non-target arthropods Drosophila melanogaster and Folsomia candida.

Genetic modification of apple cultivars through cisgenesis can introduce traits, such as disease resistance from wild relatives, quickly and without crossing. This approach was used to generate the cisgenic apple line C44.4.146, a 'Gala Galaxy' carrying the fire blight resistance gene FB_MR5. In contrast to traditionally bred apple cultivars, genetically modified (GM) plants need to undergo a regulatory risk assessment considering unintended effects before approval for commercial release. To determine potential unintended effects of C44.4.146, we assessed major leaf components and effects on the fitness of the decomposers Drosophila melanogaster (fruit fly) and Folsomia candida (collembolan), which were fed a diet amended with powdered apple leaf material. Leaf material of 'Gala Galaxy', several natural 'Gala' mutants, and the unrelated apple cultivar 'Ladina' were used for comparison. The genetic modification did not alter major leaf components and did not adversely affect survival, growth, or fecundity of the two decomposers. Consistent with previous studies with other GM crops, the differences between conventionally bred cultivars were greater than between the GM line and its non-GM wild type. These data provide a baseline for future risk assessments.

Database of non-target invertebrates recorded in field experiments of genetically engineered Bt maize and corresponding non-Bt maize.

OBJECTIVES: To assess potential non-target effects of genetically engineered/modified (GM) maize that produces insecticidal proteins from Bacillus thuringiensis (Bt), numerous field experiments have been conducted worldwide. Field data are often variable and influenced by uncontrolled factors and meta-analyses can recognize general effects with increased statistical power compared to individual studies. This database represents a comprehensive collection of experimental field data on non-target invertebrates in Bt and non-Bt maize. It was created for a systematic review with the question if growing Bt maize changes abundance or ecological function of non-target animals compared to growing of non-GM maize. Systematic literature searches identified relevant data. Authors were contacted for additional information or raw data if needed and a critical appraisal scheme was developed and applied to each data record. DATA DESCRIPTION: The database contains 7279 records of non-target invertebrate abundance, activity density, or predation or parasitism extracted from 120 articles. Records for individual species and life stages, but also aggregated data are available. Each record represents a comparison of invertebrates in Bt and non-Bt maize and includes means, standard deviations and sample sizes. Additional variables characterize publication details, experimental setup, cultivars, Bt proteins, geographic location, field management, insecticide treatments, sampling details, and taxonomy.

No Adverse Effects of Stacked Bacillus thuringiensis Maize on the Midge Chironomus riparius.

Material from genetically engineered maize producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) may enter aquatic ecosystems and expose nontarget organisms. We investigated the effects on life table parameters of the midge Chironomus riparius (Diptera: Chironomidae) of SmartStax maize leaves, which contain six different Cry proteins targeting Lepidoptera and Coleoptera pests, in two plant backgrounds. For midge development and emergence, 95% confidence intervals for the means of six conventional maize lines (Rheintaler, Tasty Sweet, ES-Eurojet, Planoxx, EXP 258, and EXP 262), were used to capture the natural range of variation. For reproduction, lowest and highest means were used. The natural range of variation allows one to judge whether observed effects between Bt maize and the closest non-Bt comparator are likely to be of biological relevance. No adverse effects on C. riparius were observed with any Bt maize line compared with the respective non-Bt counterpart. Development time was shorter when females were fed Bt maize than when they were fed non-Bt maize, but this effect was not considered adverse. Development time, emergence ratio, sex ratio, and larvae/egg rope measured for Bt maize were within the natural range of variation. Fecundity for the Bt lines was equal to or higher than that for the conventional lines. Future risk assessment studies may consider plant background effects and the natural range of variation to judge the relevance of observed differences between particular genetically engineered and non-genetically engineered plants. Environ Toxicol Chem 2022;41:1078-1088. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Addressing the challenges of non-target feeding studies with genetically engineered plant material - stacked Bt maize and Daphnia magna.

Previous studies reported adverse effects of genetically engineered maize that produces insecticidal Cry proteins from Bacillus thuringiensis (Bt) on the water flea Daphnia magna. In the current study, effects of flour, leaves, or pollen from stacked Bt maize that contains six Bt proteins (SmartStax) in two plant backgrounds on life table parameters of D. magna were investigated. Adverse effects were observed for Bt maize flour, originating from different production fields and years, but not for leaves or pollen, produced from plants grown concurrently in a glasshouse. Because leaves contained eight to ten times more Cry protein than flour, the effects of the flour were probably not caused by the Cry proteins, but by compositional differences between the plant backgrounds. Furthermore, considering the natural range of variation in the response of D. magna to conventional maize lines, the observed effects of Bt maize flour were unlikely to be of biological relevance. Our study demonstrates how Cry protein effects can be separated from plant background effects in non-target studies using Bt plant material as the test substance and how detected effects can be judged for their biological relevance.

Performance of Daphnia magna on flour, leaves, and pollen from different maize lines: Implications for risk assessment of genetically engineered crops.

Non-target effects of genetically engineered (GE) plants on aquatic Daphnia magna have been studied by feeding the species with different maize materials containing insecticidal Cry proteins from Bacillus thuringiensis (Bt). The results of those studies were often difficult to interpret, because only one GE plant was compared to one related non-GE control. In such a setting, effects of the Cry proteins cannot be distinguished from plant background effects, in particular when the test species is nutritionally stressed. In the present study, we tested the suitability of three different maize materials, i.e., flour, leaves and pollen, from five diverse non-GE maize lines (including EXP 258, a breeding line that is closely related to a SmartStax Bt maize) as exclusive food sources for D. magna. The parameters recorded included survival, sublethal endpoints such as body size, number of moltings to first offspring, time to first offspring, number of individuals in first clutch, total number of clutches, total number of offspring, average number of offspring per clutch, and population measures such as net reproductive rate R0, generation time T and intrinsic rate of increase rm. The results showed that D. magna can survive, grow and reproduce when fed only maize materials, although the performance was poorer than when fed algae, which indicates nutritional stress. Large differences in life table and population parameters of D. magna were observed among the different maize lines. Our results suggest that confounding effects caused by nutritional stress and plant background might explain some of the conflicting results previously published on the effects of Bt crops on D. magna. Using 95% confidence intervals for the means of the five maize lines for all measured parameters of D. magna performance in our study, we captured the natural range of variation. This information is useful for the interpretation of observed differences in D. magna performance between a GE plant and its non-GE comparator as it helps judging whether observed effects are of biological relevance. If differences between a GE and comparator line are observed and their biological relevance needs to be assessed in future risk assessments of GE maize, 1) the data on natural variation of the different parameters generated by previous studies can be informative (e.g. data from our study for maize fed D. magna); 2) for additional experiments the inclusion of multiple unrelated non-GE comparators should be considered; In addition, it should be taken into account that nutritional stress can affect the outcome of the study.

Fate of multiple Bt proteins from stacked Bt maize in the predatory lady beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae).

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) can be transferred from genetically engineered crops to herbivores to natural enemies. For the lady beetle Harmonia axyridis, we investigated potential uptake of Cry proteins from the gut to the body and intergenerational transfer. Third and fourth instar H. axyridis fed with pollen or spider mites from SmartStax maize contained substantial amounts of Cry1A.105, Cry1F, Cry2Ab2, Cry3Bb1, and Cry34Ab1. Cry protein concentrations in lady beetle larvae were typically one order of magnitude lower than in the food. When H. axyridis larvae were fed Bt maize pollen, median amounts of Cry protein in the non-feeding pupae were below the limit of detection except for small amounts of Cry34Ab1. No Cry protein was detected in pupae when spider mites were used as food. Cry protein concentrations decreased quickly after H. axyridis larvae were transferred from pollen or spider mites to Bt-free food. Aphids contained very low or no detectable Cry protein, and no Cry protein was found in H. axyridis larvae fed with aphids, and in pupae. When H. axyridis adults were fed with Bt maize pollen (mixed with Ephestia kuehniella eggs), the median concentrations of Cry proteins in lady beetle eggs were below the limit of detection except for Cry34Ab1 in eggs laid later in adult life. No Bt protein was detected in eggs laid by H. axyridis females fed with aphids from Bt maize. Our results confirm previous observations that Cry proteins are degraded and excreted quickly in the arthropod food web without evidence for bioaccumulation. Despite the fact that small amounts of Cry proteins were detected in some samples of the non-feeding pupal stage of H. axyridis as well as in eggs, we conclude that this route of exposure is unlikely to be significant for predators or parasitoids in a Bt maize field.

Stacked Bt Proteins Pose No New Risks to Nontarget Arthropods.

Concerns have been raised that multiple insecticidal proteins produced by genetically engineered (GE) crops may interact unexpectedly and pose new threats to biodiversity and nontarget organisms. We reviewed the literature to assess whether this concern is justified and whether the current regulatory framework needs to be adapted to address this concern.

Transgenic Winter Wheat Expressing the Sucrose Transporter HvSUT1 from Barley does not Affect Aphid Performance.

Winter wheat expressing the sucrose transporter HvSUT1 from barley (HOSUT) has an increased yield potential. Genetic engineering should improve cultivars without increasing susceptibility to biotic stresses or causing negative impacts on ecosystem services. We studied the effects of HOSUT wheat on cereal aphids that feed on the sugar-rich phloem sap. Three HOSUT winter wheat lines, their conventional parental cultivar Certo, and three conventional cultivars were used. Clip cage experiments in the greenhouse showed no differences in life-table parameters of Rhopalosiphum padi and Sitobion avenae (Hemiptera: Aphididae) on transgenic lines compared to Certo, except higher fecundity of S. avenae on one HOSUT line. Population development of both aphid species over three weeks on caged flowering tillers did not reveal differences between the HOSUT lines and Certo. When aphids were monitored in a Swiss field study over two years, no differences between HOSUT lines and Certo were observed. We conclude that HOSUT wheat did not have consistent effects on aphids compared to the parental cultivar and measured parameters were generally in the range observed for the conventional winter wheat cultivars. Thus, HOSUT wheat is unlikely to suffer from increased aphid damage.

Constitutive and induced insect resistance in RNAi-mediated ultra-low gossypol cottonseed cotton.

BACKGROUND: Besides fibers, cotton plants also produce a large amount of seeds with a high oil and protein content. The use of these seeds is restricted by their high contents of the terpenoid gossypol, which is harmful to humans and livestock. Using a genetic engineering approach, "Ultra-low gossypol cottonseed" (ULGCS) plants were produced by knocking down an enzyme that catalyzes the formation of a precursor of gossypol. This was accomplished via RNAi-mediated silencing of the target gene using a seed-specific α-globulin promotor. Since gossypol is also a crucial defense mechanism against leaf-feeding herbivores, ULGCS plants might possess lower herbivore resistance than non-engineered plants. Therefore, we tested the constitutive and inducible direct insect resistance of two ULGCS cotton lines against the African cotton leafworm, Spodoptera littoralis. RESULT: The herbivore was equally affected by both ULGCS lines and the control (Coker 312) line when feeding on fully expanded true leaves from undamaged plants and plants induced by jasmonic acid. When plants were induced by caterpillar-damage, however, S. littoralis larvae performed better on the ULGCS plants. Terpenoid analyses revealed that the ULGCS lines were equally inducible as the control plants. Levels of terpenoids were always lower in one of the two lines. In the case of cotyledons, caterpillars performed better on ULGCS cotton than on conventional cotton. This was likely caused by reduced levels of gossypol in ULGCS cotyledons. CONCLUSION: Despite those effects, the insect resistance of ULGSC cotton can be considered as largely intact and the plants may, therefore, be an interesting alternative to conventional cotton varieties.

Genetically Engineered Crops: Importance of Diversified Integrated Pest Management for Agricultural Sustainability.

As the global population continues to expand, utilizing an integrated approach to pest management will be critically important for food security, agricultural sustainability, and environmental protection. Genetically engineered (GE) crops that provide protection against insects and diseases, or tolerance to herbicides are important tools that complement a diversified integrated pest management (IPM) plan. However, despite the advantages that GE crops may bring for simplifying the approach and improving efficiency of pest and weed control, there are also challenges for successful implementation and sustainable use. This paper considers how several GE traits, including those that confer protection against insects by expression of proteins from Bacillus thuringiensis (Bt), traits that confer tolerance to herbicides, and RNAi-based traits that confer resistance to viral pathogens, can be key elements of a diversified IPM plan for several different crops in both developed and developing countries. Additionally, we highlight the importance of community engagement and extension, strong partnership between industry, regulators and farmers, and education and training programs, for achieving long-term success. By leveraging the experiences gained with these GE crops, understanding the limitations of the technology, and considering the successes and failures of GE traits in IPM plans for different crops and regions, we can improve the sustainability and versatility of IPM plans that incorporate these and future technologies.

Reduced caterpillar damage can benefit plant bugs in Bt cotton.

Bt cotton was genetically modified to produce insecticidal proteins targeting Lepidopteran pests and is therefore only minimally affected by caterpillar damage. This could lead to reduced levels of inherent, systemically inducible defensive compounds in Bt cotton which might benefit other important cotton herbivores such as plant bugs. We studied the effects of plant defense induction on the performance of the plant bug Lygus hesperus by caging nymphs on different food sources (bolls/squares) of Bt and non-Bt cotton which were either undamaged, damaged by Bt tolerant caterpillars, or treated with jasmonic acid (JA). Terpenoid induction patterns of JA-treated and L. hesperus-damaged plants were characterized for different plant structures and artificial diet assays using purified terpenoids (gossypol/heliocide H1/4) were conducted. Nymphs were negatively affected if kept on plants damaged by caterpillars or sprayed with JA. Performance of nymphs was increased if they fed on squares and by the Bt-trait which had a positive effect on boll quality as food. In general, JA-sprayed plants (but not L. hesperus infested plants) showed increased levels of terpenoids in the plant structures analyzed, which was especially pronounced in Bt cotton. Nymphs were not negatively affected by terpenoids in artificial diet assays indicating that other inducible cotton responses are responsible for the found negative effects on L. hesperus. Overall, genetically engineered plant defenses can benefit plant bugs by releasing them from plant-mediated indirect competition with lepidopterans which might contribute to increasing numbers of hemipterans in Bt cotton.

Differential Impact of Herbivores from Three Feeding Guilds on Systemic Secondary Metabolite Induction, Phytohormone Levels and Plant-Mediated Herbivore Interactions.

Phytochemical defense responses of plants are often herbivore-specific and can be affected by a herbivore's feeding mode. However, comprehensive studies documenting the impact of multiple herbivores from different feeding guilds on induced phytochemical responses in distal leaves and its consequences for plant-mediated herbivore interactions are limited and findings are inconsistent. We investigated how herbivory by leaf-chewing caterpillars, cell-content feeding spider mites and phloem-feeding aphids and whiteflies affect secondary metabolomes and phytohormone levels in youngest, non-damaged cotton leaves (distal leaves). Furthermore, bioassays with caterpillars were conducted to assess their performance on distal leaves of plants infested with different herbivores. Caterpillars, and to a lesser degree spider mites, led to a systemic induction of terpenoids with negative consequences for caterpillar performance in the bioassays. Both herbivores reduced levels of various nutrients and potentially antioxidative compounds. Caterpillar damage increased levels of jasmonoyl-L-isoleucine and abscisic acid (ABA), whereas spider mite infestation had no effect on phytohormone levels. Aphid and whitefly infestation did not systemically affect secondary metabolites. Aphids decreased salicylic acid levels while whitefly-infested plants contained increased ABA levels. Neither aphid nor whitefly infestation affected caterpillar performance. In general, feeding mode of a herbivore can affect systemically induced changes in phytochemistry and plant-mediated indirect interactions even though the two phloem-feeding herbivores triggered different phytohormonal responses. The observed reduction of nutrients and potentially antioxidative compounds upon caterpillar and spider mite herbivory underlines the importance of further elucidating the role of resource sequestration as a potential systemic defensive response following herbivory by chewers and cell-content feeding herbivores.

Bt rice could provide ecological resistance against nontarget planthoppers.

Genetically engineered (GE) rice lines expressing Lepidoptera-active insecticidal cry genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China. Field surveys indicated that Bt rice harbours fewer rice planthoppers than non-Bt rice although planthoppers are not sensitive to the produced Bt Cry proteins. The mechanisms underlying this phenomenon remain unknown. Here, we show that the low numbers of planthoppers on Bt rice are associated with reduced caterpillar damage. In laboratory and field-cage experiments, the rice planthopper Nilapavata lugens had no feeding preference for undamaged Bt or non-Bt plants but exhibited a strong preference for caterpillar-damaged plants whether Bt or non-Bt. Under open-field conditions, rice planthoppers were more abundant on caterpillar-damaged non-Bt rice than on neighbouring healthy Bt rice. GC-MS analyses showed that caterpillar damage induced the release of rice plant volatiles known to be attractive to planthoppers, and metabolome analyses revealed increased amino acid contents and reduced sterol contents known to benefit planthopper development. That Lepidoptera-resistant Bt rice is less attractive to this important nontarget pest in the field is therefore a first example of ecological resistance of Bt plants to nontarget pests. Our findings suggest that non-Bt rice refuges established for delaying the development of Bt resistance may also act as a trap crop for N. lugens and possibly other planthoppers.

No Interactions of Stacked Bt Maize with the Non-target Aphid Rhopalosiphum padi and the Spider Mite Tetranychus urticae.

In the agroecosystem, genetically engineered plants producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) interact with non-target herbivores and other elements of the food web. Stacked Bt crops expose herbivores to multiple Cry proteins simultaneously. In this study, the direct interactions between SmartStax® Bt maize producing six different Cry proteins and two herbivores with different feeding modes were investigated. Feeding on leaves of Bt maize had no effects on development time, fecundity, or longevity of the aphid Rhopalosiphum padi (Hemiptera: Aphididae), and no effects on the egg hatching time, development time, sex ratio, fecundity, and survival of the spider mite Tetranychus urticae (Acari: Tetranychidae). The results thus confirm the lack of effects on those species reported previously for some of the individual Cry proteins. In the Bt maize leaves, herbivore infestation did not result in a consistent change of Cry protein concentrations. However, occasional statistical differences between infested and non-infested leaves were observed for some Cry proteins and experimental repetitions. Overall, the study provides evidence that the Cry proteins in stacked Bt maize do not interact with two common non-target herbivores.

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators.

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%-50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%-30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.

Effects of purified or plant-produced Cry proteins on Drosophila melanogaster (Diptera: Drosophilidae) larvae.

Although genetically engineered crops producing insecticidal Cry proteins from Bacillus thuringiensis (Bt) are grown worldwide, few studies cover effects of Bt crops or Cry proteins on dipteran species in an agricultural context. We tested the toxicity of six purified Cry proteins and of Bt cotton and Bt maize tissue on Drosophila melanogaster (Diptera: Drosophilidae) as a surrogate for decomposing Diptera. ELISA confirmed the presence of Cry proteins in plant material, artificial diet, and fly larvae, and concentrations were estimated. Median concentrations in emerging adult flies were below the limit of detection. Bioactivity of purified Cry proteins in the diet was confirmed by sensitive species assays using Heliothis virescens (Lepidoptera: Noctuidae). Purified Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1F, or Cry2Aa, or leaf material from stacked Bt cotton (Bollgard II producing Cry1Ac and Cry2Ab) or Bt maize (SmartStax producing Cry1A.105, Cry1Fa2, Cry2Ab2, Cry3Bb1, Cry34Ab1 and Cry35Ab1) had no consistent effects on D. melanogaster survival, developmental time, adult body mass or morphometrics. However, D. melanogaster showed longer developmental time and smaller wing size when fed with cotton leaves from plants infested with H. virescens caterpillars compared to flies fed with leaves from uninfested plants, while no such effects were obvious for maize.

Stacked Bt maize and arthropod predators: exposure to insecticidal Cry proteins and potential hazards.

Genetically engineered (GE) crops with stacked insecticidal traits expose arthropods to multiple Cry proteins from Bacillus thuringiensis (Bt). One concern is that the different Cry proteins may interact and lead to unexpected adverse effects on non-target species. Bi- and tri-trophic experiments with SmartStax maize, herbivorous spider mites (Tetranychus urticae), aphids (Rhopalosiphum padi), predatory spiders (Phylloneta impressa), ladybeetles (Harmonia axyridis) and lacewings (Chrysoperla carnea) were conducted. Cry1A.105, Cry1F, Cry3Bb1 and Cry34Ab1 moved in a similar pattern through the arthropod food chain. By contrast, Cry2Ab2 had highest concentrations in maize leaves, but lowest in pollen, and lowest acquisition rates by herbivores and predators. While spider mites contained Cry protein concentrations exceeding the values in leaves (except Cry2Ab2), aphids contained only traces of some Cry protein. Predators contained lower concentrations than their food. Among the different predators, ladybeetle larvae showed higher concentrations than lacewing larvae and juvenile spiders. Acute effects of SmartStax maize on predator survival, development and weight were not observed. The study thus provides evidence that the different Cry proteins do not interact in a way that poses a risk to the investigated non-target species under controlled laboratory conditions.