Insecticidal Cry, or Bt, proteins are produced by the soil-endemic bacterium, Bacillus thuringiensis and some genetically modified crops. Their environmental fate depends on interactions with soil. Little is known about the toxicity of adsorbed proteins and the change in toxicity over time. We incubated Cry1Ac and Cry2A in contrasting soils subjected to different treatments to inhibit microbial activity. The toxin was chemically extracted and immunoassayed. Manduca sexta was the target insect for biotests. Extractable toxin decreased during incubation for up to four weeks. Toxicity of Cry1Ac was maintained in the adsorbed state, but lost after 2 weeks incubation at 25 °C. The decline in extractable protein and toxicity were much slower at 4 °C with no significant effect of soil sterilization. The major driving force for decline may be time-dependent fixation of adsorbed protein, leading to a decrease in the extraction yield in vitro, paralleled by decreasing solubilisation in the larval gut. (Résumé d'auteur)

The common soil arthropod Folsomia candida can survive well when fed only maize pollen and thus may be exposed to insecticidal proteins by ingesting insect-resistant genetically engineered maize pollen containing Bacillus thuringiensis (Bt) proteins when being released into the soil. Laboratory experiments were conducted to assess the potential effects of Cry1Ab/Cry2Aj-producing transgenic Bt maize (Shuangkang 12–5) pollen on F. candida fitness. Survival, development, and the reproduction were not significantly reduced when F. candida fed on Bt maize pollen rather than on non-Bt maize pollen, but these parameters were significantly reduced when F. candida fed on non-Bt maize pollen containing the protease inhibitor E-64 at 75 μg/g pollen. The intrinsic rate of increase (rm) was not significantly reduced when F. candida fed on Bt maize pollen but was significantly reduced when F. candida fed on non-Bt maize pollen containing E−64. The activities of antioxidant-related enzymes in F. candida were not significantly affected when F. candida fed on Bt maize pollen but were significantly increased when F. candida fed on non-Bt pollen containing E−64. The results demonstrate that consumption of Bt maize pollen containing Cry1Ab/Cry2Aj has no lethal or sublethal effects on F. candida.

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 μg L−1) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 μL L-1) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors.

Sustained cultivation of Bacillus thuringiensis (Bt) transgenic crops requires stable transgene expression under variable abiotic conditions. We studied the interactions of Bt toxin production and chronic ozone exposure in Bt cry1Ac-transgenic oilseed rape and found that the insect resistance trait is robust under ozone elevations. Bt Cry1Ac concentrations were higher in the leaves of Bt oilseed rape grown under elevated ozone compared to control treatment, measured either per leaf fresh weight or per total soluble protein of leaves. The mean relative growth rate of a Bt target herbivore, Plutella xylostella L. larvae was negative on Bt plants in all ozone treatments. On the non-transgenic plants, larval feeding damage was reduced under elevated ozone. Our results indicate the need for monitoring fluctuations in Bt toxin concentrations to reveal the potential of ozone exposure for altering dosing of Bt proteins to target and non-target herbivores in field environments experiencing increasing ozone pollution. Elevated atmospheric ozone can induce fluctuations in insecticidal protein concentrations in transgenic plants.

Insect gut microbiotas have a variety of physiological functions for host growth, development, and immunity. Bacillus thuringiensis (Bt) is known to kill insect pests by releasing insecticidal protoxins, which are activated in the insect midgut. However, the interplay among Bt infection, host immunity, and gut microbiota are still unclear. Here we show that Bt Cry1Ac protoxin interacts with the gut microbiota to accelerate the mortality of P. xylostella larvae. Cry1Ac protoxin was found to cause a dynamic change in the midgut and hemocoel microbiota of P. xylostella, with a significant increase in bacterial load and a significant reduction in bacterial diversity. In turn, loss of gut microbiota significantly decreased the Bt susceptibility of P. xylostella larvae. The introduction of three gut bacterial isolates Enterococcus mundtii (PxG1), Carnobacterium maltaromaticum (PxCG2), and Acinetobacter guillouiae (PxCG3) restored sensitivity to Bt Cry1Ac protoxin. We also found that Cry1Ac protoxin and native gut microbiota can trigger host midgut immune response, which involves the up-regulation of expression of Toll and IMD pathway genes and most antimicrobial peptide genes, respectively. Our findings further shed light on the interplay between insect gut microbiota and host immunity under the Bt toxin killing pressure, and this may provide insights for improving the management of Bt resistance and lead to new strategies for biological control of insect pests.

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.

Common varieties of genetically modified (GM) cotton increasingly display insect-resistant properties via expression of bacterial-derived toxins from Bacillus thuringiensis (Bt). This necessitates a deeper understanding of the possible effects of these crops on non-target insects. The mirid bug Apolygus lucorum is a major pest in cotton production in China, however, the effect of GM cotton on this non-target species is currently virtually unknown. This insect is exposed to these transgenic plants by consuming genetically modified (GM) leaves. In this study, laboratory experiments were conducted to assess the toxicity of CCRI41 and CCRI45, (genetically modified cotton varieties which express the toxins Cry1Ac and CpTI (Cowpea Trypsin Inhibitor)) on nymphs and adults of A. lucorum. There was no detectable increase in mortality after A. lucorum fed on GM cotton leaves for 20 days. While we detected trace amounts of Cry1Ac proteins in both A. lucorum nymphs and adults (<10 ng/g fresh weight), the expression of genes related to detoxification did not detectably differ from those feeding on non-GM cotton. Our binding assays did not show Cry1Ac binding to receptors on the midgut brush border membrane from either A. lucorum nymphs or adults. Our findings collectively indicate that feeding on leaves of the GM cotton varieties CCRI41 and CCRI45 have few toxic effects on A. lucorum.

Propylea japonica is a very important predator in agricultural ecosystems, which could be exposed to Bt protein. In this study, the bacterial community of P. japonica fed with normal food and food containing Cry2Ab protein was characterized for the first time using qPCR and high-throughput sequencing approaches. Results showed no effect of Cry2Ab on P. japonica development and reproduction. The most abundant bacterial phylum was Firmicutes, and the most abundant genus was Staphylococcus. The total bacteria copy number was not significantly different across four larval stages. Bacteria species composition was gathered more closely in feed on sucrose solution (sucrose-fed) than in larvae only fed on pea aphid (aphid-fed), the diversity indices of some operational taxonomic unit (OTU) were significantly different between sucrose-fed and aphid-fed samples. Different instar larval stages of P. japonica fed with sucrose solution containing Cry2Ab Bt protein and found no effect on microbial community composition and total bacteria copy numbers. However, effects on relative abundance of microbes, copy numbers of Corynebacterium 1 and Glutamicibacter arilaitensis were observed significantly lower in Bt-fed first and fourth larval stages. Low and high concentrations of Cry2Ab protein altered the microbial abundance relative to sucrose-fed P. japonica and copy numbers of G. arilaitensis and Staphylococcus xylosus were significantly lower in Bt-fed samples than control sucrose-fed. Our results are the first report showing that feeding on Cry2Ab protein does not alter microbial species composition in P. japonica, but effects gene copy number of some dominant bacteria. Further investigations are needed to assess the effect of copy number change on P. japonica.

The widespread cultivation of transgenic Bt cotton makes assessing the potential effects of this recombinant crop on non-target organisms a priority. However, the effect of Bt cotton on many insects is currently virtually unknown. The plant bug Adelphocoris suturalis is now a major pest of cotton in southern China and the beetle Haptoncus luteolus is one of the most ancient cotton pollinators. We conducted laboratory experiments to evaluate the toxicity of the Bt cotton varieties ZMSJ, which expresses the toxins Cry1Ac and Cry2Ab, and ZMKCKC, which expresses Cry1Ac and EPSPS, on adult A. suturalis and H. luteolus. No significant increase in the mortality of either species was detected after feeding on Bt cotton leaves or pollen for 7 days. Trace amounts of Cry1Ac and Cry2Ab proteins could be detected in both species but in vitro binding experiments found no evidence of Cry1Ac and Cry2Ab binding proteins. These results demonstrate that feeding on the leaves or pollen of these two Bt cotton varieties has no toxic effects on adult A. suturalis or H. luteolus.

With the commercialization of transgenic cotton that expresses Bt (Bacillus thuringiensis) insecticidal proteins, mirid bugs have become key pests in cotton and maize fields in China. Genetically engineered (GE) crops for controlling mirids are unavailable owing to a lack of suitable insecticidal genes. In this study, we developed and validated a dietary exposure assay for screening insecticidal compounds and for assessing the potential effects of insecticidal proteins produced by GE plants on Apolygus lucorum, one of the main mirid pests of Bt cotton and Bt maize. Diets containing potassium arsenate (PA) or the cysteine protease inhibitor E-64 were used as positive controls for validating the efficacy of the dietary exposure assay. The results showed that with increasing concentrations of PA or E-64, A. lucorum larval development time was prolonged and adult weight and fecundity were decreased, suggesting that the dietary exposure assay was useful for detecting the toxicity of insecticidal compounds to A. lucorum. This assay was then used to assess the toxicity of Cry1Ab, Cry1Ac, Cry1F, Cry2Aa, and Cry2Ab proteins, which have been transformed into several crops, against A. lucorum. The results showed that A. lucorum did not show a negative effect by feeding on an artificial diet containing any of the purified Cry proteins. No significant changes in the activities of digestive, detoxifying, or antioxidant enzymes were detected in A. lucorum that fed on a diet containing Cry proteins, but A. lucorum fitness was reduced when the insect fed on a diet containing E-64 or PA. These results demonstrate that A. lucorum is not sensitive to the tested Cry proteins and that the dietary exposure assay is useful for evaluating the toxicity of insecticidal compounds to this species.