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.

Chilo suppressalis is a major target pest of transgenic rice expressing the Bacillus thuringiensis (Bt) Cry1C toxin in China. The evolution of resistance of this pest is a major threat to Bt rice. Since Bt functions by binding to receptors in the midgut (MG) of target insects, identification of Bt functional receptors in C. suppressalis is crucial for evaluating potential resistance mechanisms and developing effective management strategies. ATP-binding cassette (ABC) transporters have been vastly reported to interact with Cry1A toxins, as receptors and their mutations cause insect Bt resistance. However, the role of ABC transporters in Cry1C resistance to C. suppressalis remains unknown. Here, we measured CsABCC2 expression in C. suppressalis Cry1C-resistant (Cry1C-R) and Cry1C-susceptible strains (selected in the laboratory) via quantitative real-time PCR (qRT-PCR); the transcript level of CsABCC2 in the Cry1C-R strain was significantly lower than that in the Cry1C-susceptible strain. Furthermore, silencing CsABCC2 in C. suppressalis via RNA interference (RNAi) significantly decreased Cry1C susceptibility. Overall, CsABCC2 participates in Cry1C mode of action, and reduced expression of CsABCC2 is functionally associated with Cry1C resistance in C. suppressalis.

RNAi-based insect-resistant genetically engineered (IRGE) crops represent a promising approach for pest management by suppressing gene expressions or translation. A developed microRNA-mediated IRGE rice line expressing endogenous Chilo suppressalis Csu-novel-260 shows significant resistance to target pests. The nontarget insect Apis mellifera is an important pollinator used as a surrogate species for the ecological risk assessment of IRGE plants. To simulate a worst-case scenario, the full-length C. suppressalis and A. mellifera disembodied (dib) cDNAs were cloned. The dib 3'- untranslated regions shared 58.06% nucleotide sequence similarity between C. suppressalis and A. mellifera. No potential Csu-novel-260 binding site in Amdib was detected through the bioinformatics analysis. A dietary RNAi toxicity assay of the impacts of ingested Csu-novel-260 on A. mellifera adults showed that the survival rates of RNAi-treated A. mellifera did not significantly differ from those in the blank control (CK) and negative control (NC) treatments. The Csu-novel-260 uptake by A. mellifera peaked at 8 days postfeeding and then gradually decreased. The Amdib expression was not affected by the RNAi assay days or treatments. These results suggest that A. mellifera adults are not susceptible to high doses of Csu-novel-260 in the dietary RNAi assay and that the impact of miRNA-mediated IRGE plants on A. mellifera is negligible.

Use of genetically engineered plants that express insecticidal Cry proteins derived fromBacillus thuringiensis(Bt) have been proven efficacious for managing lepidopteran pests. However, in some cases herbivores that are not targeted by theBttrait have increased in importance. It has been suggested that reduced caterpillar damage toBtcrops 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 ofMythimna separataon aphid populations inBtcorn. We compared the performance ofRhopalosiphum maidisfeeding on non-Btcorn plants that had been infested byM. separatalarvae or were uninfested. The results showed that caterpillar-infested corn plants significantly reduced the fitness ofR. maidisleading 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-resistantBtcorn than on non-Btcorn when plants were infested withM. separata, since the caterpillars caused very little damage to theBtplants. The current findings indicate that reducedM. separatainfestation could benefit aphid development inBtcorn.Btcorn 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.

BACKGROUND:Chilo suppressalis and Cnaphalocrocis medinalis are destructive rice pests co-occurring in major rice-growing areas in China. RNA interference (RNAi)-based insect-resistant genetically engineered (IRGE) crops provide a promising approach for pest management by suppressing gene expression or translation. A microRNA (miRNA)-mediated IRGE rice line expressing endogenous Chilo suppressalis miRNA Csu-novel-260, showing significant resistance against Chilo suppressalis, provides an attractive control strategy for Chilo suppressalis by suppressing the expression of the disembodied (dib) gene expression. However, whether this transgenic line also shows the resistance against Cnaphalocrocis medinalis remains unknown. RESULTS:A spatiotemporal expression analysis of Csu-novel-260 in the transgenic rice line was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine the paddy field pest exposure dose. In diet feeding assays, a chemically synthesized Csu-novel-260 agomir at 200 fmol g-1 significantly inhibited Chilo suppressalis pupation. However, larval development, survival and pupal weight were not significantly affected. Additionally, the transgenic line significantly affected Cnaphalocrocis medinalis pupation but not larval survival. The qRT-PCR showed that Csdib and Cmdib expression levels were significantly suppressed when the two pests fed on the transgenic line. Additionally, the transgenic line significantly decreased Cry1C-resistant and Cry1C-susceptible Chilo suppressalis larval survival in detached rice tissue feeding assays, indicating that Cry1C-resistant Chilo suppressalis was not cross-resistant to Csu-novel-260 expressed in miRNA-mediated IRGE rice. CONCLUSION:Our study demonstrated that miRNA-mediated IRGE rice significantly inhibited Chilo suppressalis and Cnaphalocrocis medinalis pupation. The results provide a new viewpoint for the application of RNAi-based plants and the inspiration for environmental risk assessment.

Normally, when different species of herbivorous arthropods feed on the same plant this leads to fitness-reducing competition. We found this to be different for two of Asia's most destructive rice pests, the brown planthopper and the rice striped stem borer. Both insects directly and indirectly benefit from jointly attacking the same host plant. Double infestation improved host plant quality, particularly for the stemborer because the planthopper fully suppresses caterpillar-induced production of proteinase inhibitors. It also reduced the risk of egg parasitism, due to diminished parasitoid attraction. Females of both pests have adapted their oviposition behaviour accordingly. Their strong preference for plants infested by the other species even overrides their avoidance of plants already attacked by conspecifics. This cooperation between herbivores is telling of adaptations resulting from the evolution of plant-insect interactions, and points out mechanistic vulnerabilities that can be targeted to control these major pests.

The potential risk of Bt (Bacillus thuringiensis) crops on non-target organisms (NTOs) has drawn a lot of public concerns. Despite a series of risk assessments of Bt crops on NTOs has been conducted, a quantitative approach which could support a precise judgment of their safety is required. In the present work, hazard quotient (HQ) was applied in the safety evaluation of three Bt rice events (Cry1Ab, Cry1C and Cry2Aa rice) on NTOs. Eight NTOs in different functional guilds associated with Bt rice were selected to conduct the tests. The results showed that the HQs of three Bt rice events for eight NTOs were all below the trigger value 1, while the HQ of Cry1Ab rice for one target pest Chilo suppressalis was three times higher than 1. Our results assured the reliability of the HQ and indicated that the three Bt rice events would pose no risks to the eight NTOs. Further testing of three Bt proteins on biological parameters of one NTO Nasonia virtipennis under no observed adverse effect concentration (NOAEC) confirmed the robustness of HQ assessment. We recommend that the HQ could be applied in tier-1 risk assessments of Bt crops on NTOs as a reference data standard, which would provide more clear and credible safety information of transgenic crops for the public and policy makers.

通过添加玉米穗和水稻茎粉并改变其他饲料组分的比例,我们在前期研发的半合成人工饲料的基础上优化并发展了一种新的大螟人工饲料.大螟能在该饲料上成功完成世代发育,与取食天然寄主茭白相比,取食该人工饲料的大螟呈现较快的发育速率、高的幼虫存活率和成虫产卵量.可见,该饲料的饲养效果显著优于茭白,可适用于大螟的大规模饲养.

Genetically modified crops expressing insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been grown commercially since 1996 and have successfully controlled target pests, reduced insecticide usage and increased yields. A major concern is that Bt crops may trigger outbreaks of non-target herbivores against which they are not effective, by reducing bio-control services provided by natural enemies, as seen with broad-spectrum insecticides.

The potential risks of Bt rice on non-target arthropods (NTAs) should be evaluated and defined before commercial production. Recently, effects of Bt rice on NTAs under abiotic and biotic stress conditions attracted much attention. Here we reported the effects of Bt rice T1C-19 (Cry1C rice) on the non-target herbivore, Nilaparvata lugens (rice brown planthopper, BPH) with or without RDV ( rice dwarf virus ) infection conditions. BPH showed no feeding and oviposition preference between Bt rice T1C-19 and its non- Bt parental rice Minghui 63 (MH63), as well as between RDV-infected and RDV-free rice plants. Meanwhile, rice type, RDV infection status, and their interaction had little impacts on the survival, development and fecundity of BPH. By comparison with non- Bt control, Bt rice T1C-19 with or without RDV infection had no significant effects on the life-table parameters of BPH including r m , R 0 , T , DT and λ . Thus, it could be concluded that Bt rice T1C-19 doesn’t affect the ecological fitness of BPH either under RDV stress or not.

Plants typically release large quantities of volatiles in response to herbivory by insects. This benefits the plants by, for instance, attracting the natural enemies of the herbivores. We show that the brown planthopper (BPH) has cleverly turned this around by exploiting herbivore-induced plant volatiles (HIPVs) that provide safe havens for its offspring. BPH females preferentially oviposit on rice plants already infested by the rice striped stem borer (SSB), which are avoided by the egg parasitoid Anagrus nilaparvatae, the most important natural enemy of BPH. Using synthetic versions of volatiles identified from plants infested by BPH and/or SSB, we demonstrate the role of HIPVs in these interactions. Moreover, greenhouse and field cage experiments confirm the adaptiveness of the BPH oviposition strategy, resulting in 80% lower parasitism rates of its eggs. Besides revealing a novel exploitation of HIPVs, these findings may lead to novel control strategies against an exceedingly important rice pest.

SUMMARYAdvancements in ‐omics techniques provide powerful tools to assess the potential effects in composition of a plant at the RNA, protein and metabolite levels. These technologies can thus be deployed to assess whether genetic engineering (GE) causes changes in plants that go beyond the changes introduced by conventional plant breeding. Here, we compare the extent of transcriptome and metabolome modification occurring in leaves of four GE rice lines expressing Bacillus thuringiensis genes developed by GE and seven rice lines developed by conventional cross‐breeding. The results showed that both types of crop breeding methods can bring changes at transcriptomic and metabolic levels, but the differences were comparable between the two methods, and were less than those between conventional non‐GE lines were. Metabolome profiling analysis found several new metabolites in GE rice lines when compared with the closest non‐GE parental lines, but these compounds were also found in several of the conventionally bred rice lines. Functional analyses suggest that the differentially expressed genes and metabolites caused by both GE and conventional cross‐breeding do not involve detrimental metabolic pathways. The study successfully employed RNA‐sequencing and high‐performance liquid chromatography mass spectrometry technology to assess the unintended changes in new rice varieties, and the results suggest that GE does not cause unintended effects that go beyond conventional cross‐breeding in rice.

Interspecific interactions are complex in agro-ecosystems and could be affected by agricultural technologies including transgenic crop planting. Few studies focused on the effects of Bt crops on the interspecific interactions of non-target organisms. Here we assessed the effects of transgenic cry2A rice (Cry2A rice) on the interspecific interaction between two rice planthoppers, namely, Nilaparvata lugens (the brown planthopper, BPH) and Sogatella furcifera (the white-backed planthopper, WBPH). Cry2A rice showed no significant effects on most biological parameters of these two rice planthoppers, except for wet weight of BPH female adults and development duration of WBPH female nymphs. In contrast, interspecific interactions between BPH and WBPH showed significant impacts on their biological parameters, no matter on Cry2A rice or non-transgenic control. In two-factor analysis combing rice line and interspecific interaction together, the interaction between these two factors did not affect most biological parameters of neither planthopper species, except for development duration of BPH female nymphs and WBPH nymphs (both male and female). Additionally, the egg distributions of BPH and WBPH had no significant differences between Cry2A and non-Cry2A treatments. Results of field experiments showed that Cry2A rice did not affect their population densities at most sampling dates in a five-year survey, and the interaction between BPH and WBPH showed no significant differences in both Cry2A and non-Cry2A rice paddies. In conclusion, our tested Cry2A rice would not affect the interspecific interactions between BPH and WBPH based both laboratory and field results.

Transgenic rice lines expressing Bacillus thuringiensis (Bt) toxins have been successfully developed for the control of Chilo suppressalis . However, the evolution of insect resistance is a major threat to Bt rice durability. Bt toxins function by binding specific receptors in the midgut of target insects; specifically, cadherin proteins have been identified as Cry toxin receptors in diverse lepidopteran species. Here, we report the functional roles of cadherin CsCad in the midgut of C . suppressalis in Cry1Ab and Cry1C toxicity. We expressed a recombinant truncated CsCad peptide (CsCad-CR11-MPED) in Escherichia coli that included the eleventh cadherin repeat and MPED region. Based on ligand blotting and ELISA binding assays, the CsCad-CR11-MPED peptide specifically bound Cry1Ab with high affinity but weakly bound Cry1C. The CsCad-CR11-MPED peptide significantly enhanced the susceptibility of C . suppressalis larvae to Cry1Ab but not Cry1C. Furthermore, the knockdown of endogenous CsCad with Stealth siRNA reduced C . suppressalis larval susceptibility to Cry1Ab but not Cry1C, suggesting that CsCad plays differential functional roles in Cry1Ab and Cry1C intoxication in C . suppressalis . This information directly enhances our understanding of the potential resistance mechanisms of C . suppressalis against Bt toxins and may assist in the development of effective strategies for delaying insect resistance.

Adult Chrysoperla sinica Tjeder is a common pollen feeder in maize fields. They are thus directly exposed to insecticidal proteins by consumption of genetically engineered maize pollen containing Bacillus thuringiensis（Bt） proteins. Here we assessed the potential effects of Cry1 Ab/2 Aj-or Cry1 Ac-containing Bt maize pollen on the fitness of adult C. sinica via a dietary-exposure assay under laboratory conditions. Survival, pre-oviposition, fecundity and adult dry weight did not differ between adult C. sinica consuming Bt or the corresponding non-Bt maize pollen. The stability of the Cry protein in the food sources and uptake of the Cry protein by adult C. sinica during the feeding experiment were confirmed by ELISA. These results demonstrate that adult C. sinica are not affected by the consumption of Cry1 Ab/2 Aj-or Cry1 Ac-containing maize pollen, suggesting that production of Bt maize expressing cry1 Ab/2 Aj or cry1 Ac genes will pose a negligible risk to adult C. sinica.

The ladybird Propylea japonica, adults of the green lacewing Chrysoperla nipponensis and the honey bee Apis mellifera are common pollen feeders in many crop systems. They could therefore be directly exposed to Cry proteins in Bacillus thuringiensis (Bt)-transgenic crop fields by ingestion of pollen. They, or closely related species, are therefore often selected as surrogate test species in non-target risk assessment of Bt plants. In the current study, we evaluated the potential effects of the ingestion of Bt maize pollen containing the Cry1Ab/Cry1Ac fusion protein on various life-table parameters of the three pollen-feeding non-target species in laboratory feeding assays. The results showed that pupation rate and male adult fresh weight of P. japonica were significantly increased when fed pollen from Bt maize compared to control maize pollen, but other test life-table parameters were not affected. For the other two species, none of the tested life-table parameters (survival, pre-oviposition period, fecundity and adult fresh weight for C. nipponensis; survival and mean acinus diameter of hypopharyngeal glands for A. mellifera) differed between non-Bt and Bt maize pollen treatments. ELISA measurements confirmed the stability and uptake of the Cry protein by all three species during the feeding bioassays. In addition, a sensitive insect bioassay confirmed the bioactivity of the Cry1Ab/Cry1Ac protein in the Bt maize pollen used. Overall, the results suggested that the three pollen feeders are not sensitive to the Cry1Ab/Cry1Ac protein, and planting of the Bt maize variety will pose a negligible risk to P. japonica, adult C. nipponensis and adult A. mellifera.

The proliferation and differentiation of stem cell populations allow the midgut to grow/regenerate in lepidopteran insect. Basic epithelial regenerative functions can be assessed in vitro by purifying these stem and mature cell populations. Therefore, we isolated and purified stem and mature cells from the midgut of C. suppressalis larvae by density gradient centrifugation and observed the morphologies of these cells. A flow cytometry method was used to monitor C. suppressalis stem cell proliferation and differentiation under different cell culture conditions. We observed high proportions of the stem and differentiating cells in third- and fourth-instar larvae, respectively, indicating that, in larvae, stem cells rapidly proliferate early in development and are strongly differentiated at late stages. Incubation in medium supplemented with fat body extract and ecdysone resulted in a significantly increased proportion of stem cells, not of the differentiating cells, indicating that co-culture with fat body extract and ecdysone stimulates the proliferation of C. suppressalis stem cells. Viability bioassays showed that Cry1Ab displayed significant cytotoxic effects on the midgut cell culture of C. suppressalis. The proportion of differentiating cells was significantly increased after a 48-h exposure to sublethal doses of Cry1Ab toxin, and peaked at the Cry1Ab concentration of 0.3 mu g/ml, demonstrating that epithelial cells with strong regenerative capacity via the differentiation of stem cells. These results improve our understanding of C. suppressalis stem cell biology and illustrate the potential role of the enhanced midgut regeneration induced by stem cell proliferation or differentiation as a reparation mechanism to Bt toxin.

Cost-effective Bt resistance monitoring programmes exist to manage insect resistance to Bt crops. F2 screening is widely used in detecting rare resistant alleles. One way to establish numerous isofemale lines for F2 bioassays is acquiring many field-trapped copulated females. However, it is difficult to obtain sufficient Chilo suppressalis isofemale lines because females have low mating rates and fecundity. We developed a new method to establish C. suppressalis isofemale lines with field-collected egg masses. No significant difference in detection ability was observed in the F2 screen between the old and new methods. Moreover, the new method is economical, convenient and efficient.

With 20% of the world's population but just 7% of the arable land, China has invested heavily in crop biotechnology to increase agricultural productivity. We examine research on insect-resistant genetically engineered (IRGE) crops in China, including strategies to promote their sustainable use. IRGE cotton, rice, and corn lines have been developed and proven efficacious for controlling lepidopteran crop pests. Ecological impact studies have demonstrated conservation of natural enemies of crop pests and halo suppression of crop-pest populations on a local scale. Economic, social, and human health effects are largely positive and, in the case of Bt cotton, have proven sustainable over 20 years of commercial production. Wider adoption of IRGE crops in China is constrained by relatively limited innovation capacity, public misperception, and regulatory inaction, suggesting the need for further financial investment in innovation and greater scientific engagement with the public. The Chinese experience with Bt cotton might inform adoption of other Bt crops in China and other developing countries.