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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...

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.

SUMMARY Advancements 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., Significance Statement We compared the biological variation at mRNA and metabolite levels among four genetically engineered rice lines and nine conventionally bred rice cultivars that represent a range of genetic and phenotypic diversity in rice. The results provide evidence that genetic engineering does not cause unintended effects that go beyond conventional cross‐breeding in rice.

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.

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.

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 μg/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.

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.

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

Normally, when different species of herbivorous arthropods feed on the same plant this leads to fitness reducing competition. We found this to be uniquely 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 plant quality, particularly for the stemborer because the planthopper fully suppresses caterpillar-induced production of proteinase inhibitors. It also drastically 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 uncovered cooperation between herbivores is telling of the exceptional adaptations resulting from the evolution of plant-insect interactions, and points out mechanistic vulnerabilities that can be targeted to control two 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.

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 rm, R0, 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.

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species.

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 Nilaparvata 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 pre-infestation 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.

Summary 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.

Harmonia axyridis (Pallas) is a common and abundant predator in China and may be exposed to Cry toxins that are produced in Bt crops either by feeding on plant parts or by feeding on target or nontarget herbivorous insects. A new Bt maize line, expressing the Cry1Ab/Cry2Aj fused protein, has been developed and should be rigorously assessed for the ecological risks on the natural enemy. Laboratory experiments were carried out to study the effects of this Bt maize on nontarget predator H. axyridis via bitrophic interaction of adult H. axyridis feeding on Bt maize pollen and tritrophic interaction of H. axyridis consuming the lepidopteran prey. Spodoptera exigua (Hübner) neonate larvae were used to transfer Bt protein because they could survive after ingesting transgenic cry1Ab/cry2Aj maize kernels in the previous study. ELISA bioassays confirmed that the Bt protein could be transferred, but diluted through Bt maize-prey-predator. Life history parameters such as survival, development, weight, fecundity, and egg hatching rate were not significantly different when H. axyridis consumed prey that had been reared on Bt maize compared with prey reared on a nontransformed parental control. Furthermore, feeding directly on Bt maize pollen also had no detrimental effects on fitness, survival, and weight of female and male adults. In conclusion, our results indicate that transgenic cry1Ab/cry2Aj maize poses no ecological risks on the nontarget predator H. axyridis.

Summary Bt rice can control yield losses caused by lepidopteran pests but may also harm nontarget species and reduce important ecosystem services. A comprehensive data set on herbivores, natural enemies, and their interactions in Chinese rice fields was compiled. This together with an analysis of the Cry protein content in arthropods collected from Bt rice in China indicated which nontarget species are most exposed to the insecticidal protein and should be the focus of regulatory risk assessment.

Summary Transgenic Bt rice expressing the insecticidal proteins derived from Bacillus thuringiensis Berliner (Bt) has been developed since 1989. Their ecological risks towards non‐target organisms have been investigated; however, these studies were conducted individually, yielding uncertainty regarding potential agroecological risks associated with large‐scale deployment of Bt rice lines. Here, we developed a meta‐analysis of the existing literature to synthesize current knowledge of the impacts of Bt rice on functional arthropod guilds, including herbivores, predators, parasitoids and detritivores in laboratory and field studies. Laboratory results indicate Bt rice did not influence survival rate and developmental duration of herbivores, although exposure to Bt rice led to reduced egg laying, which correctly predicted their reduced abundance in Bt rice agroecosystems. Similarly, consuming prey exposed to Bt protein did not influence survival, development or fecundity of predators, indicating constant abundances of predators in Bt rice fields. Compared to control agroecosystems, parasitoid populations decreased slightly in Bt rice cropping systems, while detritivores increased. We draw two inferences. One, laboratory studies of Bt rice showing effects on ecological functional groups are mainly either consistent with or more conservative than results of field studies, and two, Bt rice will pose negligible risks to the non‐target functional guilds in future large‐scale Bt rice agroecosystems in China.

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.

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.

Many lines of insect-resistant genetically modified (IRGM) corn and rice containing Bacillus thuringiensis (Bt) insecticidal genes have been developed and undergone different environmental biosafety assessments stages in China, showing robust application prospects. The potential of targeted pests to develop resistance to Bt crops is widespread, which threatens the sustainable utility of IRGM corn and rice. In this study, the potential risks of target pest complexes developing resistance to IRGM corn and rice are evaluated. Theoretical and empirical studies implementing precautionary insect resistance management (IRM) strategies to delay resistance evolution are summarized and challenges to IRM are discussed. Additionally, solutions facing these challenges are proposed. Finally, directions for future studies in developing IRGM corn and rice and IRM plans are discussed.

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.

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.

Figure S1: Key volatile compounds emitted by non-Bt rice plants (from 20:00 pm to 4:00 am) that were damaged by Chilo suppressalis larvae for 72 h (A) or remained undamaged (B). IS = internal standard. The peak numbers of all compounds are consistent with those listed in Table 1.; Figure S2: Number of eggs laid by female Chilo suppressalis on undamaged or caterpillar-damaged Bt or non-Bt rice plants (greenhouse cage experiment). Caterpillar symbols indicate damage by two 3rd instar of C. suppressalis. Each choice test was performed with 15 to 20 replicates, each consisting of a group of 10 pairs of C. suppressalis adults. The number of egg masses represents the total egg masses laid on two rice plants. Asterisks indicate significant differences: *P < 0.05; ** P < 0.01; n.s. indicates no significance (P > 0.05) (paired-sample t-test).; Figure S3: Performance of Chilo suppressalis larvae feeding on rice plants that were either undamaged or damaged by 3rd instars of conspecifics. Two-day old larvae of C. suppressalis were individually placed on caterpillar-damaged or undamaged non-Bt rice plants for 7 days. Forty insects were tested in each treatment. Asterisks indicate significant differences between the undamaged and caterpillar-damaged treatments (P

The ecological risks to nontarget organisms should be rigorously assessed before Bt crops are released. Here, the impacts of a new Cry1Ab/Vip3H rice line on arthropod communities in rice agroecosystems were evaluated across 3 yr. Arthropods collected via vacuum were sorted into five guilds. The abundance and proportion of each guild as well as community-level parameters were determined in Cry1Ab/Vip3H and control rice fields. Changes in arthropod species assemblage over sampling dates were investigated by principal response curves (PRCs). Cry1Ab/Vip3H rice did not exert significant impacts on the seasonal density and proportion of each guild, except parasitoids. Detritivore seasonal density, but not its relative abundance, was significantly affected by Cry1Ab/Vip3H rice. Four community indices (species richness S , Shannon–Wiener index H ′, Simpson index D , and evenness index J′ ) were similar between rice types. PRCs revealed a slight community difference between rice types in the past two tested years, with rice types accounting for 1.0–3.5% of the variance among arthropod communities. However, sampling dates explain 32.1–67.6% for these community differences. Of the 46 taxa with higher species weights, 26.1% of the taxa were significantly different, including seven taxa with higher abundance and five with lower density in Cry1Ab/Vip3H rice fields. These differences may be attributed to change in abundance of prey or hosts but not to direct effects of Bt proteins. We infer that this new Cry1Ab/Vip3H rice line poses no unintended ecological risks to the arthropod community.

Assessing the potential effects of insect-resistant genetically engineered (GE) plants on collembolans is important because these common soil arthropods may be exposed to insecticidal proteins produced in GE plants by ingestion of plant residues, crop pollen, or root exudates. Laboratory studies were conducted to evaluate the potential effects of two Bacillus thuringiensis (Bt)-rice lines expressing Cry1C and Cry2A in pollen and leaves and of their non-Bt conventional isolines on the fitness of the collembolan Folsomia candida and on the activities of its antioxidant-related enzymes, superoxide dismutase and peroxidase, and of its detoxification-related enzymes, glutathione reductase and glutathione S-transferase. Survival, development, reproduction, and the intrinsic rate of increase (rm) were not significantly reduced when F. candida fed on the Bt rice pollen or leaf powder than on the non-Bt rice materials; these parameters, however, were significantly reduced when F. candida fed on non-Bt rice pollen or non-Bt leaf-based diets containing the protease inhibitor E-64 at 75 μg/g. The activities of the antioxidant-related and detoxification-related enzymes in F. candida were not significantly affected when F. candida fed on the Bt rice materials, but were significantly increased when F. candida fed on the non-Bt rice materials containing E-64. The results demonstrate that Cry1C and Cry2A are not toxic to F. candida, and also indicate the absence of unintended effects on the collembolan caused by any change in plant tissue nutritional composition due to foreign gene transformation.

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K2Cr2O7), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C.

BACKGROUND Chilo suppressalis and Sesamia inferens are important lepidopteran rice pests that occur concurrently in rice-growing areas of China. The development of transgenic rice expressing Cry1A insecticidal proteins has provided a useful strategy for controlling these pests. RESULTS This study evaluated the baseline susceptibilities of C. suppressalis and S. inferens to Cry1A, as well as their responses to selection with Cry1A. Wide geographic variation in susceptibility was observed across all field populations. Within a given population, the LC50 of both Cry1Ab and Cry1Ac against S. inferens was drastically higher than that of C. suppressalis. Large LC50 differences (74.6-fold) were detected between the two species for Cry1Ab in the Poyang population, while small differences (3.6-fold) were detected for Cry1Ac in the Changsha population. The Cry1Ac LC50 of C. suppressalis and S. inferens increased 8.4- and 4.4-fold after 21 and eight selection generations respectively. Additionally, the estimated realised heritabilities (h2) of Cry1Ac tolerance were 0.11 in C. suppressalis and 0.292 in S. inferens. CONCLUSIONS S. inferens exhibited a significantly lower susceptibility and more rapidly evolved resistance to Cry1A compared with C. suppressalis. Therefore, S. inferens is more likely to evolve increased resistance, which threatens the sustainability of rice expressing Cry1A protein. © 2014 Society of Chemical Industry

A 2-yr field trial was conducted to assess the impacts of two new transgenic Bt rice lines, T1C-19 expressing Cry1C protein and T2A-1 expressing Cry2A protein, on the arthropod community sampled via vacuum. All the arthropods were classified into five guilds, including herbivores, parasitoids, predators, detritivores, and others. The seasonal density and dominance distribution of each guild and community-level indices (species richness, Shannon-Wiener diversity index, Simpson diversity index, and evenness index) were compared among rice types. Principal response curves were used to investigate the differences of entire arthropod community of Bt rice plots relative to non-Bt rice plots. The results showed no significant difference was detected in the community-level indices and dominance distribution of guilds between Bt and non-Bt rice plots. The seasonal density of herbivores, detritivores, and others as well as density of the arthropod overall community were also not significantly affected by rice types in either year, although the density of predators and parasitoids in Bt rice plots was significantly lower than those in non-Bt rice plots. The lower abundances of Braconidae, Eulophidae, Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), and Theridiidae in Bt rice plots are likely attributed to the lower abundances of prey species or hosts. Principal response curves revealed that arthropod community in Bt was similar with that in non-Bt rice plots. In conclusion, our findings indicate that these two tested Bt rice lines had no marked negative effects on the arthropod community in the paddy fields.

As genetic engineering in plants is increasingly used to control agricultural pests, it is important to determine whether such transgenic plants adversely affect non-target organisms within and around cultivated fields. The cry1Ab/1Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into rice line Minghui 63 (MH63). We evaluated the effect of transgenic cry1Ab/1Ac rice (Huahui 1, HH1) on paddy frogs by comparing HH1 and MH63 rice paddies with and without pesticide treatment. The density of tadpoles in rice fields was surveyed at regular intervals, and Cry1Ab/1Ac protein levels were determined in tissues of tadpoles and froglets collected from the paddy fields. In addition, Rana nigromaculata froglets were raised in purse nets placed within these experimental plots. The survival, body weight, feeding habits, and histological characteristics of the digestive tract of these froglets were analyzed. We found that the tadpole density was significantly decreased immediately after pesticide application, and the weight of R. nigromaculata froglets of pesticide groups was significantly reduced compared with no pesticide treatment, but we found no differences between Bt and non-Bt rice groups. Moreover, no Cry1Ab/1Ac protein was detected in tissue samples collected from 192 tadpoles and froglets representing all four experimental groups. In addition, R. nigromaculata froglets raised in purse seines fed primarily on stem borer and non-target insects, and showed no obvious abnormality in the microstructure of their digestive tracts. Based on these results, we conclude that cultivation of transgenic cry1Ab/1Ac rice does not adversely affect paddy frogs.

The effect of purified Cry1Ca insecticidal protein on the growth of Chlorella pyrenoidosa was studied in a three-generation toxicity test. The C. pyrenoidosa medium with a density of 5.4×105 cells/mL was subcultured for three generations with added Cry1Ca at 0, 10, 100, and 1000 µg/L, and cell numbers were determined daily. To explore the distribution of Cry1Ca in C. pyrenoidosa and the culture medium, Cry1Ca was added at 1000 µg/L to algae with a high density of 4.8×106 cells/mL, and Cry1Ca content was determined daily in C. pyrenoidosa and the culture medium by enzyme-linked immunosorbent assays. Our results showed that the growth curves of C. pyrenoidosa exposed to 10, 100, and 1000 µg/L of Cry1Ca almost overlapped with that of the blank control, and there were no statistically significant differences among the four treatments from day 0 to day 7, regardless of generation. Moreover, the Cry1Ca content in the culture medium and in C. pyrenoidosa sharply decreased under exposure of 1000 µg/L Cry1Ca with high initial C. pyrenoidosa cell density. The above results demonstrate that Cry1Ca in water can be rapidly adsorbed and degraded by C. pyrenoidosa, but it has no suppressive or stimulative effect on algae growth.

Because of the rapid development of transgenic maize, the potential effect of transgene flow on seed purity has become a major concern in public and scientific communities. Setting a proper isolation distance in field experiments and seed production is a possible solution to meet seed-quality standards and ensure adventitious contamination of products is below a specific threshold. By using a Gaussian plume model as basis and data recorded by meteorological stations as input, we have established a simple regionally applicable maize gene-flow model for prediction of the maximum threshold distances (MTD) at which gene-flow frequency is equal to or lower than a threshold value of 1 or 0.1 % (MTD1%, MTD0.1%). After optimization of the model variables, simulated outcrossing rate was a good fit to data obtained from field experiments (y = 1.156x, R 2 = 0.8913, n = 30, P