Tier-1 assays for assessing the toxicity of insecticidal proteins produced by genetically engineered plants to non-target arthropods

In assessing an insect-resistant genetically engineered （IRGE） crop before its commercialization, researchers normally use so-called ＂Tier-1 assays＂ as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins （IPs） produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays： （i） methods for delivery of the IP to the test organisms; （ii） the need for and selection of compounds used as positive controls; and （iii） methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis （Lepidoptera： Crambidae）

Two transgenic rice lines （T2A-1 and T1C-19b） expressing cry2A and crylC genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis （Walker） （Lepidoptera： Crambidae）. The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a crylAb/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A- 1 〉 Huahui I 〉 T1C-19b among rice lines, and leaf 〉 stern 〉 root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar： higher level at early stages than at later stages with an exception that high Cry 1C level in T 1 C- 19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the j ointing stage with an exception for T1 C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest crylC is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.

Herbivore-induced rice resistance against rice blast mediated by salicylic acid

In agro-ecosystems,plants are important mediators of interactions between their associated herbivorous insects and microbes,and any change in plants induced by one species may lead to cascading effects on interactions with other species.Often,such effects are regulated by phytohormones such as jasmonic acid(JA)and salicylic acid(SA).Here,we investigated the tripartite interactions among rice plants,three insect herbivores(Chilo suppressalis,Cnaphalocrocis medinalis or Nilapai-vata lugens),and the causal agent of rice blast disease,the fungus Magnaporthe oryzae.We found that pre-infestation of rice by C.suppressalis or N.lugens but not by C.medinalis conferred resistance to M.oryzae.For C.suppressalis and N.lugens,insect infestation without fungal inoculation induced the accumulation of both JA and SA in rice leaves.In contrast,infestation by C.medinalis increased JA levels but reduced SA levels.The exogenous application of SA but not of JA conferred resistance against M.oryzae.These results suggest that preinfestation by C suppressalis or N.lugens conferred resistance against M.oryzae by increasing SA accumulation.These findings enhance our understanding of the interactions among rice plant,insects and pathogens,and provide valuable information for developing an ecologically sound strategy for controlling rice blast.

Use of a dietary exposure system for screening of insecticidal compounds for their toxicity to the planthopper Laodelphax striatellus

We developed a dietary exposure assay for screening insecticidal compounds for their toxicity and for assessing the side effects of insecticidal proteins produced by genetically engineered （GE） plants on the planthopper Laodelphax striatellus Fallen. The fitness bioassay confirmed that the diet fulfills the requirements to be used in the dietary exposure system. To validate the efficacy of the dietary exposure system, nymphs of L. striatellus were fed diets treated with different concentrations of an inorganic stomach poison, potassium arsenate （PA）, or a cysteine protease inhibitor, E-64. The results showed that with increasing concentrations of E-64, the larval development time was prolonged, the adult weight was reduced and the survival rate of L. striatellus was decreased. Similarly the survival rates of L. striatellus consistently decreased with increasing PA content in the diet. The data indicate that the dietary exposure assay is able to detect the effects of insecticidal compounds on L. striatellus. Subsequently, this assay was successfully used for assessing the potential toxicity of Cry2Aa. The results showed that L. striatellus larvae were not negatively affected when fed the artificial diet containing purified Cry2Aa at 300 μg/g diet. In the assay, the stability and bioactivity of crystal （Cry） proteins in the food sources were confirmed by enzyme-linked immunosorbent assay and sensitive-insect bioassays. These results show that L. striatellus is not sensitive to Cry2Aa. We conclude that the dietary exposure system is valid and useful for assessing the toxicity of insecticidal compounds produced by GE plants on planthoppers.

Reduced Mythimna separata infestation on Bt corn could benefit aphids

Use of genetically engineered plants that express insecticidal Cry proteins derived from Bacillus thuringiensis(Bt)have been proven efficacious for managing lepidopteran pests.However,in some cases herbivores that are not targeted by the Bt trait have increased in importance.It has been suggested that reduced caterpillar damage to Bt crops could lead to decreased levels of induced plant defensive compounds which might benefit other non-target herbivores.Here we investigated the potential effect of reduced damage by larvae of Mythimna separata on aphid populations in Bt corn.We compared the performance of Rhopalosiphum maidis feeding on non-Bt corn plants that had been infested by M.separata larvae or were uninfested.The results showed that caterpillar-infested corn plants significantly reduced the fitness of R.maidis leading to a prolonged nymphal development time,reduced adult longevity and fecundity compared to uninfested plants.Consequently,the population growth rate of corn aphids feeding on caterpillar-infested corn plants was significantly lower than on uninfested plants.As expected,the aphids performed significantly better on Lepidoptera-resistant Bt corn than on non-Bt corn when plants were infested with M.separata,since the caterpillars caused very little damage to the Bt plants.The current findings indicate that reduced M.separata infestation could benefit aphid development in Bt corn.Bt corn has the potential to be commercialized in China in the near future and aphids and other non-target pests should be monitored in the farming fields.