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BACKGROUND: RNA interference (RNAi) eliminates or decreases gene expression by disrupting the target mRNA or by interfering with translation. Recently, RNAi technique was applied to generate new crop traits which provide protection against pests. To establish the environmental risk assessment protocol of RNAi LMO in lab scale, we developed dsRNA expression system using E. coli and tested acute oral toxicity assay to honey. METHOD AND RESULTS: The dsRNA expression vector, L4440, was chosen and cloned 240 bp of Snf7 and GFP gene fragment. To develop the maximum dsRNA induction condition in E. coli, we tested induction time, temperature and IPTG concentration in media. To estimate the risk assessment of dsRNA to honey bee, it has been selected and cultured with dsRNA supplement for 48 hours according to OECD guideline. As a result, the optimum condition of dsRNA induction was 37 , 4 hours and 0.4 ℃ mM IPTG concentration and the difference between Snf7 and GFP dsRNA molecules from E. coli was not significant in survival and behavior to honey bee. Furthermore, blast search results indicated that effective match of predicted dsRNA fragments were not existed in honey bee genome. CONCLUSION:In this study, we developed and tested the acute oral toxicity of dsRNA using E. coli expression system to honey bee.

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The RNA interference (RNAi) has been considered as an important genetic tool and applied to develop a new living modified (LM) crop trait which is an improvement of nutrient quality or pest management. The RNAi of DvSnf7 has been used for resistance to LM maize and the Western Corn Rootworm which is a major agricultural pest for the US Corn Belt. Most of the environmental risk assessments (ERA) of double strand RNA (dsRNA) have been performed using in vitro transcript products, and not in vivo expressed product. A large amount of dsRNA was required for the acute toxicity assay of water fleas. Therefore development of massive dsRNA purification techniques is critical. Daphnia, a freshwater microcrustacean, is a model organism for studying cellular and molecular mechanism involved in life history traits and ecotoxicology. In this study, we established the massive dsRNA purification method using Escherichia coli and implemented acute toxicity assays to Daphnia magna. As a result, the present RNase A and DNase I, dsRNA was efficiently purified without any special techniques or equipment. Even though purified dsRNA existed during the acute toxicity test, lethality or abnormal behavior were not observed in D. magna. These results indicated that GFP and DvSnf7 dsRNA were not significantly affected to D. magna due to their lack of sequence matching in its genome. The purification method of dsRNA and the acute toxicity assay of water fleas using purified dsRNA would be suitable for the toxicological studies of LMOs to aquatic non‐target organisms.

BtPlus is a group of biopesticides that are made of Bacillus thuringiensis and immunosuppressant. A new BtPlus that exhibits high insecticidal activity against mosquito larvae has been investigated in control efficacy in field conditions and its environmental safety against aquatic system. This study assessed the control efficacy of BtPlus against mosquito larvae with two different application methods. In aerial spraying application (100 mL per 3.3 m 2 ), BtPlus was effective at 50% or above formulation concentrations to control mosquito larvae. For a direct application to aqueous mosquito habitat, a semi-field mimicking paddy rice field was constructed. In this condition, BtPlus showed 80% and 100% control efficacies at 0.1% and 0.2% concentrations, respectively. BtPlus also showed 40% mortality against adults at 0.1% concentration in 10% sugar bait. However, its control efficacies against adults were much less than against larvae. Safety assessment of BtPlus against ecosystem was evaluated using young carp (Cyprinus carpio), a water flea (Daphnia magna), and a honey bee (Apis mellifera). BtPlus did not give any adverse effects on these nontarget organisms. Based on these results, BtPlus can be applied to control mosquitoes by direct aqueous application to paddy rice field.