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The effects of insect-resistant genetically engineered rice producing Cry1C protein derived from Bacillus thuringiensis (Bt) on Chrysoperla sinica (Tjeder) (Neuroptera: Chrysopidae) were assessed in laboratory bioassays. Survival and development of C. sinica larvae were not adversely affected when the larvae were fed a diet containing purified Cry1C protein at 200 µg/g fresh weight, representing a worst-case exposure scenario; in contrast, C. sinica larvae were adversely affected when the diet contained avidin or potassium arsenate. Life table parameters of C. sinica adults did not differ when the adults were fed with Bt or non-Bt rice pollen together with a 2-M sucrose solution. Life table parameters of C. sinica adults also did not differ when the adults were fed an artificial diet with or without purified Cry1C protein at a nominal concentration that was approximately 20 times higher than that in rice pollen; in contrast, C. sinica adults were adversely affected when the diet contained potassium arsenate. In all bioassays with lacewings, the bioactivity and stability of the Cry1C protein in the diet and Cry1C protein uptake by the lacewings were confirmed by enzyme-linked immunosorbent assay (ELISA) and by bioassays with a Cry1C-sensitive lepidopteran. These results demonstrate that neither larvae nor adults of C. sinica are sensitive to Cry1C protein at concentrations higher than those encountered in the field, demonstrating that the growing of Bt rice producing Cry1C protein is unlikely to pose a risk to C. sinica.

yCollege of Animal Science and Technology, Yangzhou University, Yangzhou, People’s Republic of ChinazState Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing,People’s Republic of China(Submitted 21 October 2013; Returned for Revision 7 January 2014; Accepted 27 January 2014)Abstract: Becauseoftheprevalenceofalgaeinricepaddyfields,theywillbeexposedtoBacillusthurigiensis(Bt)proteinsreleasedfromBt protein-expressing genetically engineered rice. To assess the effects of leachates extracted from Cry1Ca-expressing transgenic rice(T1C-19) straw on the microalga Chlorella pyrenoidosa, the authors added purified Cry1Ca (10mg/L, 100mg/L, and 1000mg/L) and 5concentrations of diluted extracts (5%, 10%, 20%, 40%, and 80%) from T1C-19 and the nontransformed control strain Minghui 63(MH63) to the medium of C. pyrenoidosa. The authors found that the growth curves of C. pyrenoidosa treated with purified Cry1Caoverlapped with the medium control; that the order of C. pyrenoidosa growth rates for the T1C-19 leachate concentrations was5%>10%>20%>control>40%>80%, and for the MH63 concentrations the order was 5%>10%>control>20%>40%>80%,but there were no statistical differences between the 20% T1C-19 or 20% MH63 leachate treatment and the medium control on day 8;and that after 7 d of culture, Cry1Ca could be detected in C. pyrenoidosa treated with different concentrations of T1C-19 leachate. Theresults demonstrated that Cry1Ca protein released from T1C-19 rice can be absorbed into C. pyrenoidosa but that purified Cry1Ca andleachatesfromT1C-19ricehavenoobviousadverseeffectsonthegrowthofC.pyrenoidosa.EnvironToxicolChem2014;33:1156–1162.# 2014 SETACKeywords: Bt-rice straw Cry1Ca protein Chlorella pyrenoidosa Safety assessment Nontarget effectINTRODUCTION

A semi-liquid artificial diet was established and found to be a suitable food source for Chrysoperla sinica larvae, comparable to aphid prey. Using the artificial diet, we established and validated a dietary exposure assay by using the insecticidal potassium arsenate (PA) as positive control. Dose-dependent responses were documented for all observed life-table parameters of C. sinica larvae such as survival rate, pupation rate, larval weight, and larval development time. Thus, the dietary assay can detect the effects of insecticidal compounds on the survival and development of C. sinica larvae. Using the established dietary assay, we subsequently tested the toxicity of Cry1Ab, Cry1Ac, and Cry2Aa proteins (which are produced by transgenic maize, cotton or rice plants) to C. sinica larvae. Artificial diets containing Galanthus nivalis agglutinin (GNA) or PA were included as positive controls. Survival and development of C. sinica larvae were not affected when the artificial diet contained purified Cry1Ab, Cry1Ac, or Cry2Aa at 200 μg/g diet. In contrast, C. sinica larvae were adversely affected when the diet contained PA and GNA. The stability and bioactivity of the Cry proteins in the diet and Cry protein uptake by the lacewing larvae were confirmed by bioassay with a Cry-sensitive insect species and by ELISA. The current study describes a suitable experimental system for assessing the potential effects of gut-active insecticidal compounds on green lacewing larvae. The experiments with the Cry proteins demonstrate that C. sinica larvae are not sensitive to Cry1Ab, Cry1Ac, and Cry2Aa.

Transgenic rice expressing Bacillus thuringiensis Berliner (Bt) protein can effectively control target insects including stem borers and leaf folders. However, the potential effects of Bt rice on nontarget organisms including nontarget herbivores have not been fully evaluated. In the current study, ecological fitness parameters of the nontarget herbivore, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae), fed on T1C-19 (Cry1C) or T2A-1 (Cry2A) rice were compared with non-Bt rice (MH63) under laboratory conditions. A 2-yr field trial was also conducted to monitor the population dynamics of N. cincticeps in the Bt and control rice plots using the vacuum-suction machine and yellow sticky card traps. Laboratory results showed that there were no significant differences in some of biological parameters including egg developmental duration, adult fresh weight, adult longevity, and oviposition period when N. cincticeps fed on Bt or non-Bt rice was compared. However, the survival rate of N. cincticeps nymphs fed on T2A-1 Bt rice plants was significantly higher than that on the control. When N. cincticeps fed on T1C-19 Bt rice plants, its nymphal duration was significantly longer and fecundity significantly lower compared with those fed on both T2A-1 Bt and non-Bt rice plants; the preoviposition period of N. cincticeps fed on T1C-19 and T2A-1 Bt rice was also significantly shorter than those on non-Bt rice. Nonetheless, both seasonal density and population dynamics of N. cincticeps adults and nymphs were similar between Bt (T1C-19 and T2A-1) and non-Bt rice plots under field conditions. In conclusion, our results indicate that our two tested Bt rice lines would not lead to higher population of N. cincticeps. Long-term experiments to monitor the population dynamics of N. cincticeps at large scale need to be carried out to confirm the current results.

Recent documents issued by the State Council of China indicate that China's central government is paving the way to commercial cultivation of genetically engineered (GE) staple crops, with priority on insect-resistant GE (IRGE) maize expressing Bacillus thuringiensis (Bt) proteins. Multiple Bt maize lines have been developed in China, and their efficacy and benefits to human health and the environment have been demonstrated. We here consider issues regarding approval of GE lines for international trade, measures to be taken to ensure harmonious co-production of GE and non-GE crops, and management of evolution of Bt resistance in maize pests that should be addressed before commercialization of Bt maize in China.

The Asian corn borer, Ostrinia fumacalis (Guenee), is a major pest of maize in China. Transgenic Bt maize could provide an effective way to control this pest. However, the potential of resistance evolution has been documented in the laboratory—a Cry1Ab-resistant strain of Asian corn borer (Asian corn borer-AbR) could survive well on two Bt maize events, that is, MON810 and Bt11. Pyramided transgenic crops with multiple dissimilar Bt insecticidal proteins offer a superior route for pests control compared with that with one Bt single gene. In this study, two novel transgenic Bt maize events (N30 and V3), which contained a fused gene cry1Ab/cry2Aj and cry1Ab/vip3DA, respectively, were evaluated for their efficacy on protection against Cry1Ab-susceptible Asian corn borer (Asian corn borer-BtS) as well as their potential for dealing with the Asian corn borer-AbR. In laboratory bioassay, no neonate larvae of Asian corn borer-BtS fed on the whorl leaves, tassels, husks, silks, and kernels of N30 and V3 could survive at 96 h after infestation. In contrast, only 2.0–8.7% larvae died as they fed on non-Bt maize tissues. As Asian corn borer-AbR neonate larvae fed on N30 and V3 silks and kernels, 95.0% even more larvae were killed at 96 h after infestation, and all died at 168 h after infestation, while 10.0–11.67% larvae survived on non-Bt control. In the field test under artificial infestation at middle whorl leaf and silking stages of maize, there were no significant leaf, silk, and stalk damage on Bt maize plants by comparison with severe leaf feeding, stalk born, and ear damage in control. In addition, the expression profiles of a fused protein of Cry1Ab/Cry2Aj or Cry1Ab/Vip3DA in different tissues of N30 and V3 were also confirmed. In conclusion, it is clear that both Bt maize events (N30 and V3) show great potential for protecting maize from attack by Asian corn borer that has resistance to Cry1Ab protein.

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.

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.

Background Rice (Oryza sativa L.), which is a staple food for more than half of the world’s population, is frequently attacked by herbivorous insects, including the rice stem borer, Chilo suppressalis. C. suppressalis substantially reduces rice yields in temperate regions of Asia, but little is known about how rice plants defend themselves against this herbivore at molecular and biochemical level. Results In the current study, we combined next-generation RNA sequencing and metabolomics techniques to investigate the changes in gene expression and in metabolic processes in rice plants that had been continuously fed by C. suppressalis larvae for different durations (0, 24, 48, 72, and 96 h). Furthermore, the data were validated using quantitative real-time PCR. There were 4,729 genes and 151 metabolites differently regulated when rice plants were damaged by C. suppressalis larvae. Further analyses showed that defense-related phytohormones, transcript factors, shikimate-mediated and terpenoid-related secondary metabolism were activated, whereas the growth-related counterparts were suppressed by C. suppressalis feeding. The activated defense was fueled by catabolism of energy storage compounds such as monosaccharides, which meanwhile resulted in the increased levels of metabolites that were involved in rice plant defense response. Comparable analyses showed a correspondence between transcript patterns and metabolite profiles. Conclusion The current findings greatly enhance our understanding of the mechanisms of induced defense response in rice plants against C. suppressalis infestation at molecular and biochemical levels, and will provide clues for development of insect-resistant rice varieties. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0946-6) contains supplementary material, which is available to authorized users.

Rice and maize are important cereal crops that serve as staple foods, feed, and industrial material in China. Multiple factors constrain the production of both crops, among which insect pests are an important one. Lepidopteran pests cause enormous yield losses for the crops annually. In order to control these pests, China plays an active role in development and application of genetic engineering (GE) to crops, and dozens of GE rice and GE maize lines expressing insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) have been developed. Many lines have entered environmental release, field testing, and preproduction testing, and laboratory and field experiments have shown that most of the Bt rice and Bt maize lines developed in China exhibited effective control of major target lepidopteran pests on rice (Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrocis medinalis) and maize (Ostrinia furnacalis), demonstrating bright prospects for application. However, none of these Bt lines has yet been commercially planted through this writing in 2016. Challenges and perspectives for development and application of Bt rice and maize in China are discussed. This article provides a general context for colleagues to learn about research and development of Bt crops in China, and may shed light on future work in this field. Published version

Because of its ecological and economic importance, the honey bee Apis mellifera is commonly used to assess the environmental risk of insect-resistant, genetically modified plants. In the present study, feeding-exposure experiments were used to determine whether pollen from transgenic rice harms A. mellifera worker bees. In 1 experiment, the survival and mean acinus diameter of hypopharyngeal glands of adult bees were similar when bees were fed on pollen from Bt rice lines or from a non-Bt rice line, but bee survival was significantly reduced when they received pollen that was mixed with potassium arsenate as a positive control. In a second experiment, bee survival and hypopharyngeal gland development were not reduced when adult bees were fed on non-Bt pollen and a sucrose solution supplemented with Cry2A at 400 µg/g, Cry1C at 50 µg/g, or bovine serum albumin (BSA) at 400 µg/g, but bee survival and hypopharyngeal gland development were reduced when the diet was supplemented with soybean trypsin inhibitor as a positive control. In both experiments, the uptake of Cry proteins by adult bees was confirmed. Overall, the results indicate that the planting of Bt rice lines expressing Cry2A or Cry1C protein poses a negligible risk to A. mellifera worker bees. Environ Toxicol Chem 2017;36:1243-1248. © 2016 SETAC.

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) is a prevalent pollen consumer in corn fields and is therefore exposed to insecticidal proteins contained in the pollen of insect-resistant transgenic corn cultivars expressing Cry proteins derived from Bacillus thuringiensis (Bt). In the present study, the potential effect of Cry1Ab/2Aj- or Cry1Ac-containing transgenic Bt corn pollen on the fitness of P. japonica larvae was evaluated. The results show that the larval developmental time was significantly shorter when P. japonica larvae were fed pollen from Bt corn cultivars rather than control pollen but that pupation rate, eclosion rate, and adult fresh weight were not significantly affected. In the feeding experiments, the stability of the Cry proteins in the food sources was confirmed. When Bt corn pollen passed through the gut of P. japonica, 23% of Cry1Ab/2Aj was digested. The results demonstrate that consumption of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac has no detrimental effect on P. japonica larvae; the shortened developmental time of larvae that consumed these proteins was likely attributable to unknown differences in the nutritional composition between the Bt-transgenic and control corn pollen.

Transformation of rice with genes encoding insecticidal Cry proteins from Bacillus thuringiensis (Bt) should confer high resistance to target lepidopteran pests, such as Chilo suppressalis, and low toxicity to non-target organisms, such as silkworm Bombyx mori. Five purified Cry proteins that have been used for plant transformation were tested using dietary exposure assays. The susceptibility of C. suppressalis larvae to the five insecticidal proteins in the decreasing order was: Cry1Ca>Cry1Ab>Cry1Ac>Cry2Aa>Cry1Fa. However, the toxicities of the Cry proteins to B. mori were in the order: Cry1Fa>Cry1Ca>Cry2Aa>Cry1Ab>Cry1Ac. The Cry1Ca, Cry1Ab and Cry1Ac proteins exhibited relatively high toxicity to C. suppressalis larvae, with EC50 values of 16.4, 45.8 and 89.6ng/g, respectively. The toxicities of the three Cry proteins to B. mori larvae were 8, 14, and 22times lower, with EC50 values of 138.3, 628.4 and 1939.2ng/g, respectively. The Cry1Fa and Cry2Aa proteins showed high toxicity to B. mori larvae, with EC50 values of 135.7 and 373.9ng/g, respectively, but low toxicity to C. suppressalis larvae, with EC50 values of 6092.1 and 1208.5ng/g, respectively. We thus conclude that Cry1Ab, Cry1Ac and Cry1Ca are appropriate for transforming rice to control lepidopteran rice pests. In contrast, Cry1Fa and Cry2Aa are not appropriate due to their high toxicity to silkworm larvae and low activity against the target pest.

In this study, we analyze the genetic diversity and phylogeny of photoperiod related genes Hd1 and Ehd1 in Oryza rufipogon populations from Hainan Island and Guangdong province separated by the Qiongzhou Strait. The levels of variations in Hd1 and Ehd1 of O. rufipogon from Hainan Island were higher than those from Guangdong province, although their polymorphism distribution trends were similar. The haplotype phylogenetic analysis showed that the genetic diversity of O. rufipogon from Hainan Island was richer than that from Guangdong province. The phylogenic networks of individuals based on either Hd1 or Ehd1 or the combination of these genes indicated that the differentiation of O. rufipogon did not occur due to geographic isolation. Our findings suggest that different ecological niches seem to have significant impacts on the genetic differentiation of O. rufipogon populations.

Adults of Chrysoperla sinica Tjeder are prevalent pollen feeders in rice fields in China. They are therefore exposed to insecticidal proteins when foraging insect-resistant genetically engineered rice pollen containing Bacillus thuringiensis (Bt) proteins. Laboratory bioassays were conducted to evaluate the potential impacts of Cry2Aa-expressing transgenic rice (T2A-1) pollen on fitness of adult C. sinica. Survival, pre-oviposition period, fecundity and adult dry weight did not differ between lacewings that had consumed Bt or non-Bt rice pollen. To ensure lacewing adults were not sensitive to Cry2Aa protein independent from the plant background and to add certainty to the toxicity assessment, C. sinica adults were fed with an artificial diet containing Cry2Aa protein at an approximately 10 times higher concentration than in rice pollen. Artificial diet containing potassium arsenate (PA) was included as a positive control. No difference was detected for any of the life-table parameters tested between Cry2Aa-containing and pure diet. In contrast, the pre-oviposition, fecundity and dry weight of C. sinica were significantly adversely affected by feeding on PA-containing diet. In both bioassays, the uptake of Cry protein by adult C. sinica and the bioactivity of Cry protein in the food sources were confirmed by ELISA measurements and sensitive insect bioassays, respectively. These results indicate that adults of C. sinica are not affected by Cry2Aa-expressing rice pollen and are not sensitive to Cry2Aa protein at concentrations exceeding the levels in pollen, thus demonstrating that growing Bt rice expressing Cry2Aa protein will pose a negligible risk to adult C. sinica.

The Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is an important rice, Oryza sativa L., pest in China and difficult to control with conventional pest management. To develop and optimize integrated pest management strategies, efficient and economic artificial diet and rearing protocols are desirable. A new artificial diet based on soybean, Glycine max (L.) Merr., powder and fresh water bamboo, Zizania caduciflora (Turcz.) Hand.-Mazz, was formulated and rearing technique was developed. Fitness parameters including larval development, immature survival, pupal weight, pupation, adult emergence, egg hatchability, and oviposition were measured to evaluate the performance of C. suppressalis fed on the diet over 15 successive generations. C. suppressalis reared on the artificial diet showed better performance with shorter developmental stage, similar larval survival rate and fecundity, and heavier pupae compared with that fed on rice plants and fresh water bamboo. A positive correlation was observed between number of eggs laid per female and number of generations reared on the diet. Larval development time tended to be shortened with successive rearing on the artificial diet. These results indicated that C. suppressalis adapted well to the artificial diet and successive rearing conditions. The diet could serve as a viable alternative to natural host plants for consecutive rearing of the insect. In addition, the diet is inexpensive (US$1.5/1,000 g) and easy to make. The better preserve ability of the diet required only one diet replacement during the rearing process. The successful development of the diet and rearing technique provides a very useful tool for refining stem borer pest management techniques.

Release of genetically modified (GM) plants has sparked off intensive debates worldwide partly because of concerns about potential adverse unintended effects of GM plants to the agro system and the safety of foods. In this study, with the aim of revealing the molecular basis for unintended effects of a single site insertion GM Kemingdao (KMD) rice transformed with a synthetic cry1Ab gene, and bridging unintended effects of KMD rice through clues of differentially expressed genes, comparative transcriptome analyses were performed for GM KMD rice and its parent rice of Xiushui11 (XS11). The results showed that 680 differentially expressed transcripts were identified from 30-day old seedlings of GM KMD rice. The absolute majority of these changed expression transcripts dispersed and located over all rice chromosomes, and existed physical distance on chromosome from the insertion site, while only two transcripts were found to be differentially expressed within the 21 genes located within 100 kb up and down-stream of the insertion site. Pathway and biology function analyses further revealed that differentially expressed transcripts of KMD rice were involved in certain biological processes, and mainly implicated in two types of pathways. One type was pathways implicated in plant stress/defense responses, which were considerably in coordination with the reported unintended effects of KMD rice, which were more susceptible to rice diseases compared to its parent rice XS11; the other type was pathways associated with amino acids metabolism. With this clue, new unintended effects for changes in amino acids synthesis of KMD rice leaves were successfully revealed. Such that an actual case was firstly provided for identification of unintended effects in GM plants by comparative transciptome analysis.

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.

In this study, the non-target effects of Bt rice "KMD2" expressing a Cry1Ab protein on the performance of the brown planthopper (BPH), Nilaparvata lugens, over multiple generations were evaluated under laboratory and field conditions. In the laboratory, BPH was reared to observe the impact of the Bt rice as compared to its parental non-Bt cultivar Xiushui 11, while the population dynamics and oviposition performance of BPH were investigated in the field. The survival of BPH nymphs fed Bt and non-Bt rice did not differ significantly. The nymph developmental duration of BPH was significantly delayed by the Bt rice by comparison with the non-Bt rice for the 1st and 2nd but not the 4th generation. Most importantly, the fecundity of BPH on the Bt rice was significantly decreased in every generation when compared with the non-Bt rice. In the field investigations, the population density of BPH nymphs was significantly lower in the Bt rice field. However, the temporal pattern of population dynamics of BPH adults was similar between the Bt and non-Bt rice, presumably due to migratory interference of the adults. In the Bt rice field, the percentage of tillers with eggs and the number of eggs per tiller were also significantly lower from tillering to mature stage. Additionally, Cry1Ab protein could not be detected in guts from single BPH adults. In general, our results suggest that the Bt rice "KMD2" could not stimulate an outbreak of BPH.

Transgenic phytase maize (Zea maysL.) line BVLA430101 was the first transgenic maize obtained the security certification in 2009 in China. However, the construct of the phytase gene expression cassette and the specific detection method have not been reported yet. In this study, the phytase gene expression cassette was identified, which include maize legumin promoter, signal peptide, phytase gene, and maize legumin terminator. The construct-specific qualitative and quantitative PCR methods of BVLA430101 maize were established based on the transition of signal peptide and phytase gene using a maize taxon-specific gene zSSIIb as the endogenous gene. The detection limit for the conventional qualitative PCR was 200 haploid genome copies of BVLA430101. The absolute limit of quantification of the real-time PCR was about 20 haploid genome copies. In addition, two known BVLA430101 contents (5 and 1%) of mixed genomic DNA (V/V) were quantified using the developed real-time PCR detection system, which indicated that the developed quantitative method can be employed reliably for transgenic phytase maize BVLA430101 measurement.

To assess the potential dietary effects of insecticidal substances on the predacious ladybird beetle, Coleomegilla maculata De Geer, a Tier-1 laboratory testing system was developed. Artificial diets using shrimp eggs were developed, and a tier-1 bioassay examining C. maculata development and survival was designed based on those diets. To further measure the artificial diet in a Tier-1 testing system, larvae of C. maculata were fed the diet treated with different concentrations of an inorganic stomach poison, potassium arsenate (PA), or a cysteine protease inhibitor, E-64. The results demonstrated that the testing system was capable of detecting the dietary effects of both substances on the survival and development of C. maculata. With increasing concentrations of PA in the diet, fewer larvae developed to adults, with only 22.7% larvae surviving to the adult stage in the treatment with the highest content of PA (32 μg/g of diet). Likewise, dose-dependent responses also were found for other life-table parameters of C. maculata. Similar to the assays with PA, the survival rates of C. maculata consistently decreased with increasing E-64 content in the diet. Survival analysis showed that the insects fed E-64 at 50, 150, and 450 μg/g in the diet had significantly lower survival rates compared with those on the untreated artificial diet. The study presented here describes a robust testing system that will be useful for assessing the potential hazard (or toxicity) effects after dietary exposure of insecticidal compounds produced by GE plants or conventional insecticides on the ladybird predator, C. maculata.

Transgenic rice Kefeng 6 is a transformation event containing two insect-resistant genes, cry1Ac and SCK (modified CpTI gene) in China. In order to monitor the probable release of Kefeng 6 in the future and execute the labeling requirements, it is necessary to develop a rapid and reliable detection method. In this study, both the 5′ and 3′-junction sequences spanning the plant DNA and the integrated gene construct of the rice event Kefeng 6 were isolated by genome walking and long-distance PCR (LD-PCR), successively. Multiple copies of truncated SCK gene and cry1Ac gene were found to integrate into the host rice genome. The event-specific real-time detection method for Kefeng 6 event based on its 5′-junction sequence was established using one plasmid molecule pMD-KF6 containing both 5′-junction sequence and rice endogenous gene gos9 sequence as the reference material (RM) with an absolute limit of quantification (LOQa) around 10 template copies. Thereafter, three different transgenic amounts of w/w mixed samples (5, 1, and 0.5%, respectively) were quantified to assess the performance characteristics of the established real-time PCR method. The accuracy expressed as bias deviated from the 4.00–26.00%, the precision expressed as standard deviation (SD) and relative standard deviation (RSD) deviated from 0.03–0.19 and 3.42–4.76%, respectively. Based on the earlier results, we concluded that the qualitative and quantitative PCR assays were reliable and accurate for Kefeng 6 measurement, and the reference plasmid pMD-KF6 could be a good substitute for the reference material for Kefeng 6 quantification.

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC50 of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.

Six transgenic rice, Oryza sativa L., lines (G6H1, G6H2, G6H3, G6H4, G6H5, and G6H6) expressing a fused Cry1Ab/Vip3H protein, were evaluated for resistance against the Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), and the stem borer Sesamia inferens (Walker) (Lepidoptera: Noctuidae) in the laboratory and field. The bioassay results indicated that the mortality of Asiatic rice borer and S. inferens neonate larvae on six transgenic lines from seedling to filling stage was up to 100% at 168 h after infestation. The cumulative feeding area by Asiatic rice borer neonate larvae on all transgenic lines was significantly reduced compared with the untransformed parental 'Xiushui 110' rice. A 2-yr field evaluation showed that damage during the vegetative stage (deadheart) or during the reproductive stage (whitehead) caused by Asiatic rice borer and S. inferens for transgenic lines was much lower than the control. For three lines (G6H1, G6H2, and G6H6), no damage was found during the entire growing period. Estimation of fused Cry1Ab/Vip3H protein concentrations using PathoScreen kit for Bt-Cry1Ab/1Ac protein indicated that the expression levels of Cry1Ab protein both in main stems (within the average range of 0.006-0.073% of total soluble protein) and their flag leaves (within the average range of 0.001-0.038% of total soluble protein) were significantly different among six transgenic lines at different developmental stages. Both laboratory and field researches suggested that the transgenic rice lines have considerable potential for protecting rice from attack by both stem borers.

The area planted with insect-resistant genetically engineered crops expressing Bacillus thuringiensis ( Bt ) genes has greatly increased in many areas of the world. Given the nearby presence of non- Bt crops (including those planted as refuges) and non-crop habitats, pests targeted by the Bt trait have a choice between Bt and non- Bt crops or weeds, and their host preference may greatly affect insect management and management of pest resistance to Bt proteins. In this study, we examined the oviposition preference of the target pest of Bt rice, Chilo suppressalis , for Bt versus non- Bt rice plants as influenced by previous damage caused by C. suppressalis larvae. The results showed that C. suppressalis females had no oviposition preference for undamaged Bt or non- Bt plants but were repelled by conspecific-damaged plants whether Bt or non- Bt . Consequently, C. suppressalis egg masses were more numerous on Bt plants than on neighbouring non- Bt plants both in greenhouse and in field experiments due to the significantly greater caterpillar damage on non- Bt plants. We also found evidence of poorer performance of C. suppressalis larvae on conspecific-damaged rice plants when compared with undamaged plants. GC-MS analyses showed that larval damage induced the release of volatiles that repelled mated C. suppressalis females in wind tunnel experiments . These findings suggest that Bt rice could act as a dead-end trap crop for C. suppressalis and thereby protect adjacent non- Bt rice plants. The results also indicate that the oviposition behaviour of target pest females should be considered in the development of Bt resistance management strategies.

Genetically modified insect-resistant rice lines containing the cry1Ac gene from Bacillus thuringiensis (Bt) or the CpTI (cowpea trypsin inhibitor) gene developed for the management of lepidopterous pests are highly resistant to the major target pests, Chilo suppressalis (Walker), Cnaphalocrocis medinalis (Guenee), and Scirpophaga incertulas (Walker), in the main rice-growing areas of China. However, the effects of these transgenic lines on Sesamia inferens (Walker), an important lepidopterous rice pest, are currently unknown. Because different insect species have varying susceptibility to Bt insecticidal proteins that may affect population dynamics, research into the effects of these transgenic rice lines on the population dynamics of S. inferens was conducted in Fuzhou, southern China, in 2005 and 2006. The results of laboratory, field cage, and field plot experiments show that S. inferens has comparatively high susceptibility to the transgenic line during the early growing season, with significant differences observed in larval density and infestation levels between transgenic and control lines. Because of a decrease in Cry1Ac levels in the plant as it ages, the transgenic line provided only a low potential for population suppression late in the growing season. There is a correlation between the changing expression of Cry1Ac and the impact of transgenic rice on the population dynamics of S. inferens during the season. These results indicate that S. inferens may become a major pest in fields of prospective commercially released transgenic rice, and more attention should be paid to developing an effective alternative management strategy.

Geographic variability in susceptibility of field-collected Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae) to Bacillus thuringiensis (Berliner) was studied to establish a geographic baseline for comparison of future population responses to the increased use of B. thuringiensis-based insect control products. Colonies of C. medinalis were established from 10 populations collected from 10 different provinces in the major rice (Oryza savita L.)-growing regions of China. Populations were evaluated for their susceptibility to Cry1Ac and Cry1Ab endotoxin proteins with the leaf-dip bioassay. The LC50 values to Cry1Ac and Cry1Ab were estimated for the 10 populations. Bioassay results indicated that the ranges of LC50 in the second instars of C. medinalis to Cry1Ac and Cry1Ab were from 3.77 to 208.22 mg ([AI])/liter and 0.22-7.05 mg ([AI])/liter, respectively. The relative ratios in susceptibility between the most susceptible and the most tolerant populations were beyond 50-fold for Cry1Ac and 30-fold for Cry1Ab. Moreover, there was a significant positive correlation between susceptibilities to the two toxins tested, suggesting that insect populations that are relatively tolerant to one protein are also relatively tolerant to the other.

The relative toxicity of Bt rice pollen to domestic silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), was assessed by a leaf-dip bioassay under laboratory conditions. Silkworm first instars were sensitive to pollen from Bt rice lines, B1 and KMD1, but were not sensitive to pollen from Bt rice line TT9–3. First instars were 1.34–2.12 times more sensitive to B1 pollen than older instars. Bioassays of subacute toxicity under a worst-case scenario suggested that continuous exposure to a sublethal dose of B1 pollen or equivalent doses of non-Bt rice pollen affected silkworm survival and development. Young larvae were more affected by continuous exposure to Bt pollen than older larvae but less affected by non-Bt pollen. Ultrastructural observations showed that Cry proteins associated with Bt pollen were released into the larval lumen and resulted in pathological midgut changes and negative impacts on silkworm survival and development. However, considering that the sublethal dose of Bt pollen (L...

Detached leaf bioassays, open field tests and cage tests were conducted to evaluate the control efficacy of two transgenic rice lines, expressing Cry1Ac and CpTI, against Cnaphalocrocis medinalis (Guenee) during 2005-2006 in Fuzhou, China. Bioassay results showed that cumulative feeding areas of C. medinalis on transgenic lines were significantly lower than that on control rice lines at different developmental stages. The corrected mortalities at 96 h after infestation on transgenic lines during six rice growth stages were >90% and 100% during experiments conducted in 2005 and 2006, respectively. In the open field test, there was no significant difference in egg density between transgenic and control lines during early days of infestation, but significant differences were detected in late season, due to serious damage on control lines. Larval densities on control lines were significantly higher than the low larval populations observed on transgenic lines during both seasons. The percentages of plants with folded leaves and percentages of folded leaves on transgenic lines were significantly lower than that on control lines with and without insecticide applications, during the entire season. In cage tests the cumulative numbers of C. medinalis adults derived from transgenic lines were significantly lower than that from control lines with and without insecticide treatments. The high level of efficacy of the two transgenic rice lines against C. medinalis may provide an important basis for reduced insecticide applications, an expansion of alternative pest-control strategies and insect resistance management of Bt rice in the future.

Transgenic rice containing insecticidal protein(s) from Bt (Bacillus thuringiensis) effectively control lepidopteran pests under laboratory and field conditions. However, Bt rice has not been widely commercialized. A major concern regarding the deployment of Bt rice is its potential impact on non-target arthropods, including herbivorous insect pests and biological control agents (predators and parasitoids) in the rice ecosystem. A 2-year field study was conducted at 3 sites in Zhejiang Province, China to assess the impacts of a homozygous transgenic japonica rice line KMD1 (Bt rice) containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner. The impacts on three planthoppers (Homoptera: Delphacidae), Sogatella furcifera (Horvath), Nilaparvata lugens (Stal) and Laodelphax striatellus (Fallen), and the natural enemy, Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), were evaluated using two sampling methods: vacuum-suction and Malaise trap. Population densities of planthoppers and C. lividipennis were not significantly affected by rice type (Bt/non-Bt) over the season at any of the 3 sites in each year, regardless of sampling methods. Both in Bt rice and non-Bt rice plots, S. furcifera was the predominant species of planthoppers as determined by either of the two sampling methods, and comprised >50% of the planthoppers at each site. No consistent effects of Bt rice and Bt rice × sampling date interaction on population dynamics of the predominant planthopper species, S. furcifera, and the predator, C. lividipennis, were observed throughout the sampling period. Overall, this 2-year field study indicates that, in comparison with non-Bt rice, Bt rice did not lead to higher planthopper populations and did not negatively affect the predator C. lividipennis. These results suggest that use of Bt rice may provide control of the key lepidopteran pests while promoting the key biological control agent of planthopppers, C. lividipennis.

In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 μg L-1 or 10 μg L-1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development.

Collembolans are common soil arthropods that may be exposed to insecticidal proteins produced in genetically engineered (GE) plants by ingestion of crop residues or root exudates. In the present study, a dietary exposure assay was validated and used to assess the lethal and sublethal effects of two Bacillus thuringiensis (Bt) insecticidal proteins, Cry1C and Cry2A, on Folsomia candida. Using the insecticidal compounds potassium arsenate (PA), protease inhibitor (E-64) and Galanthus nivalis agglutinin (GNA) mixed into Baker’s yeast, we show that the assay used can detect adverse effects on F. candida. Survival and development were significantly reduced when F. candida was fed a diet containing PA, E-64 and GNA at 9, 75 and 100 μg/g diet, respectively, but not when fed a diet containing 300 μg/g Cry1C or 600 μg/g Cry2A. The activities of test antioxidant-, detoxification- and digestion-related enzymes in F. candida were unaltered by a diet containing 300 μg/g Cry1C or 600 μg/g Cry2A, but were significantly increased by a diet containing 75 μg/g E-64. The results confirm that Cry1C and Cry2A are not toxic to F. candida at concentrations that are much higher than those encountered under field conditions.

We determined whether plant volatiles help explain why Cotesia chilonis (a parasitoid of the target pest Chilo suppressalis) is less abundant in Bt than in non-Bt rice fields. Olfactometer studies revealed that C. chilonis females responded similarly to undamaged Bt and non-Bt rice plants. Parasitoids preferred rice plants damaged by 3rd-instar larvae of C. suppressalis, but did not differentiate between caterpillar-infested Bt and non-Bt plants. According to GC-MS analyses of rice plant volatiles, undamaged Bt and non-Bt rice plants emitted the same number of volatile compounds and there were no significant differences in the quantity of each volatile compound between the treatments. When plants were infested with and damaged by C. suppressalis larvae, both Bt and non-Bt rice plants emitted higher numbers and larger amounts of volatile compounds than undamaged plants, but there were no significant differences between Bt and non-Bt plants. These results demonstrate that the volatile-mediated interactions of rice plants with the parasitoid C. chilonis were not disrupted by the genetic engineering of the plants. We infer that parasitoid numbers are lower in Bt than in non-Bt fields because damage and volatile induction by C. suppressalis larvae are greatly reduced in Bt fields.

Background The perennial O. rufipogon (common wild rice), which is considered to be the ancestor of Asian cultivated rice species, contains many useful genetic resources, including drought resistance genes. However, few studies have identified the drought resistance and tissue-specific genes in common wild rice. Results In this study, transcriptome sequencing libraries were constructed, including drought-treated roots (DR) and control leaves (CL) and roots (CR). Using Illumina sequencing technology, we generated 16.75 million bases of high-quality sequence data for common wild rice and conducted de novo assembly and annotation of genes without prior genome information. These reads were assembled into 119,332 unigenes with an average length of 715 bp. A total of 88,813 distinct sequences (74.42% of unigenes) significantly matched known genes in the NCBI NT database. Differentially expressed gene (DEG) analysis showed that 3617 genes were up-regulated and 4171 genes were down-regulated in the CR library compared with the CL library. Among the DEGs, 535 genes were expressed in roots but not in shoots. A similar comparison between the DR and CR libraries showed that 1393 genes were up-regulated and 315 genes were down-regulated in the DR library compared with the CR library. Finally, 37 genes that were specifically expressed in roots were screened after comparing the DEGs identified in the above-described analyses. Conclusion This study provides a transcriptome sequence resource for common wild rice plants and establishes a digital gene expression profile of wild rice plants under drought conditions using the assembled transcriptome data as a reference. Several tissue-specific and drought-stress-related candidate genes were identified, representing a fully characterized transcriptome and providing a valuable resource for genetic and genomic studies in plants.

Multiple lines of transgenic rice expressing insecticidal genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China, posing the prospect of increases in production with decreased application of pesticides. We explore the issues facing adoption of Bt rice for commercial production in China. A body of safety assessment work on Bt rice has shown that Bt rice poses a negligible risk to the environment and that Bt rice products are as safe as non-Bt control rice products as food. China has a relatively well-developed regulatory system for risk assessment and management of genetically modified (GM) plants; however, decision-making regarding approval of commercial production has become politicized, and two Bt rice lines that otherwise were ready have not been allowed to enter the Chinese agricultural system. We predict that Chinese farmers would value the prospect of increased yield with decreased use of pesticide and would readily adopt production of Bt rice. That Bt rice lines may not be commercialized in the near future we attribute to social pressures, largely due to the low level of understanding and acceptance of GM crops by Chinese consumers. Hence, enhancing communication of GM crop science-related issues to the public is an important, unmet need. While the dynamics of each issue are particular to China, they typify those in many countries where adoption of GM crops has been not been rapid; hence, the assessment of these dynamics might inform resolution of these issues in other countries.

As a pollen feeder, Propylea japonica would be directly exposed to Cry proteins in Bacillus thuringiensis (Bt)-transgenic rice fields. The effect of Cry1C- or Cry2A-containing transgenic rice pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, larval developmental time of P. japonica was significantly longer when fed pollen from Bt rice lines rather than control pollen but other life table parameters were not significantly affected. In the second experiment, P. japonica was not affected when fed a rapeseed pollen-based diet containing purified Cry1C or Cry2A at concentrations that were >10-times higher than in pollen, but P. japonica was affected when the diet contained E-64 as a positive control. In both experiments, the stability and bioactivity of the Cry proteins in the food sources and the uptake of the proteins by P. japonica were confirmed. The results show that P. japonica is not sensitive to Cry1C or Cry2A proteins; the effect observed in the first experiment was likely attributable to unknown differences in the nutritional composition of Bt rice pollen. Overall, the data indicate that the growing of Cry1C- or Cry2A-transgenic rice should pose a negligible risk to P. japonica.

Atrazine is the most commonly detected pesticide contaminant in ground and surface water. Previous studies have shown that atrazine is an endocrine disruptor owing to its adverse effects on the male reproductive system in several vertebrates, but very few molecular mechanisms for these effects have been revealed. In the present study, Xenopus laevis were exposed to 100 ppb of atrazine for 120 d, and then the isobaric tags for relative and absolute quantitation (iTRAQ) technique was used to detect global changes in protein profiles of the testes and ovaries. The results showed that 100 ppb of atrazine exposure adversely affected the growth of X. laevis and did not induce hermaphroditism but delayed or prevented the development of male seminiferous tubules. Proteomic analysis showed that atrazine altered expression of 143 and 121 proteins in the testes and ovaries, respectively, and most of them are involved in cellular and metabolic processes and biological regulation based on their biological processes. In addition, apoptosis, tight junctions, and metabolic pathways were significantly altered in the atrazine-treated gonads. Based on the above results, it is postulated that the reproductive toxicity of atrazine may be the result of disruption of tight junctions and metabolic signaling pathways and/or induction of apoptosis in germ cells. Environ Toxicol Chem 2015;34:1770–1777. © 2015 SETAC

The field efficacy of two transgenic rice lines, containing the Cry1Ac gene from Bacillus thuringiensis (Bt) and the CpTI (cowpea trypsin inhibitor) gene, on the striped stem borer, Chilo suppressalis (Walker), and population variation in susceptibility of C. suppressalis to the Bt insecticidal protein Cry1Ac were evaluated in Fuzhou, Fujian Province of China. The results of an open field test showed that the two transgenic lines exhibited high efficacy against natural infestations of C. suppressalis. In contrast, the untransformed parental control line showed serious damage symptoms not only in untreated plots, but also in plots treated with insecticides. The cumulative number of C. suppressalis adults derived from transgenic rice lines was significantly lower than that from control lines with and without chemical control treatments. Our results show that transgenic rice lines expressing Cry1Ac and CpTI have great potential for protecting rice from C. suppressalis damage with reduced use of inse...

Over the years, genetically modified organism (GMO) development and regulation in China have regularly attracted international attention. In the early 1990s, China became the first country to commercialize a genetically modified (GM) crop with planting of a virus-resistant transgenic tobacco variety on a relatively large scale, while other parts of the world were debating about the risks of GMOs. A few years later, the GM tobacco was removed from cultivation because of strong pressure from outside of the country. This situation was not changed until late 1997, when an insect-resistant cotton variety was approved by the GMO Biosafety Committee in the Ministry of Agriculture for commercial cultivation in five provinces. This paper will give an overview of GMO development and biosafety research in China.

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) are prevalent predators and pollen feeders in East Asian maize fields. They are therefore indirectly (via prey) and directly (via pollen) exposed to Cry proteins within Bt-transgenic maize fields. The effects of Cry1Ie-producing transgenic maize pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, survival, larval developmental time, adult fresh weight, and fecundity did not differ between ladybirds consuming Bt or non-Bt maize pollen. In the second experiment, none of the tested lethal and sublethal parameters of P. japonica were negatively affected when fed a rapeseed pollen-based diet containing Cry1Ie protein at 200 μg/g dry weight of diet. In contrast, the larval developmental time, adult fresh weight, and fecundity of P. japonica were significantly adversely affected when fed diet containing the positive control compound E-64. In both experiments, the bioactivity of the Cry1Ie protein in the food sources was confirmed by bioassays with a Cry1Ie-sensitive lepidopteran species. These results indicated that P. japonica are not affected by the consumption of Cry1Ie-expressing maize pollen and are not sensitive to the Cry1Ie protein, suggesting that the growing of Bt maize expressing Cry1Ie protein will pose a negligible risk to P. japonica.

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.

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.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 (h(2) ) of Cry1Ac tolerance were 0.11 in C. suppressalis and 0.292 in S. inferens.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.

A marker-free Bt transgenic rice line, mfb-MH86, was recently developed in China, which contains a cry1Ab gene driven by a ubiquitin promoter. This Bt gene confers resistance to a range of lepidopteran species, including the striped stem borer, Chilo suppressalis (Walker). The expression of Cry1Ab protein in mfb-MH86 leaves, stems and leaf sheaths (hereinafter referred to as stems), and roots was evaluated throughout the rice-growing season using an enzyme-linked immunosorbent assay. In addition, mfb-MH86 resistance to C. suppressalis, a major pest of rice, was evaluated in a laboratory bioassay with field-collected rice stems. Cry1Ab protein levels of mfb-MH86 were highest in leaves (9.71-34.09 μg/g dry weight [DW]), intermediate in stems (7.66-18.51 μg/g DW), and lowest in roots (1.95-13.40 μg/g DW). In all tissues, Cry1Ab levels in mfb-MH86 were higher in seedling and tillering stages than in subsequent growth stages. In the laboratory bioassay, mortality of C. suppressalis after 6 d of feeding on mfb-MH86 stems was 100% throughout the rice-growing season; mortality of C. suppressalis when feeding on stems of the nontransformed isoline, MH86, ranged from 15.0 to 38.3%. The results indicate that Cry1Ab protein levels in mfb-MH86 stems are sufficient to protect plants against C. suppressalis throughout the rice-growing season. Although our results are promising, further comprehensive evaluations of mfb-MH86, including field surveys, will be needed before commercial use.

The effects of insect-resistant genetically engineered rice producing Cry1C protein derived from Bacillus thuringiensis (Bt) on Chrysoperla sinica (Tjeder) (Neuroptera: Chrysopidae) were assessed in laboratory bioassays. Survival and development of C. sinica larvae were not adversely affected when the larvae were fed a diet containing purified Cry1C protein at 200 µg/g fresh weight, representing a worst-case exposure scenario; in contrast, C. sinica larvae were adversely affected when the diet contained avidin or potassium arsenate. Life table parameters of C. sinica adults did not differ when the adults were fed with Bt or non-Bt rice pollen together with a 2-M sucrose solution. Life table parameters of C. sinica adults also did not differ when the adults were fed an artificial diet with or without purified Cry1C protein at a nominal concentration that was approximately 20 times higher than that in rice pollen; in contrast, C. sinica adults were adversely affected when the diet contained potassium arsenate. In all bioassays with lacewings, the bioactivity and stability of the Cry1C protein in the diet and Cry1C protein uptake by the lacewings were confirmed by enzyme-linked immunosorbent assay (ELISA) and by bioassays with a Cry1C-sensitive lepidopteran. These results demonstrate that neither larvae nor adults of C. sinica are sensitive to Cry1C protein at concentrations higher than those encountered in the field, demonstrating that the growing of Bt rice producing Cry1C protein is unlikely to pose a risk to C. sinica.

As a developing country with relatively limited arable land, China is making great efforts for development and use of genetically modified (GM) crops to boost agricultural productivity. Many GM crop varieties have been developed in China in recent years; in particular, China is playing a leading role in development of insect-resistant GM rice lines. To ensure the safe use of GM crops, biosafety risk assessments are required as an important part of the regulatory oversight of such products. With over 20 years of nationwide promotion of agricultural biotechnology, a relatively well-developed regulatory system for risk assessment and management of GM plants has been developed that establishes a firm basis for safe use of GM crops. So far, a total of seven GM crops involving ten events have been approved for commercial planting, and 5 GM crops with a total of 37 events have been approved for import as processing material in China. However, currently only insect-resistant Bt cotton and disease-resistant papaya have been commercially planted on a large scale. The planting of Bt cotton and disease-resistant papaya have provided efficient protection against cotton bollworms and Papaya ringspot virus (PRSV), respectively. As a consequence, chemical application to these crops has been significantly reduced, enhancing farm income while reducing human and non-target organism exposure to toxic chemicals. This article provides useful information for the colleagues, in particular for them whose mother tongue is not Chinese, to clearly understand the biosafety regulation and commercial use of genetically modified crops in China.

By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm(2) leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥ 150 grains/cm(2) leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori.