转基因作物风险评估的监测和田间试验设计

1、Risks转基因作物风险评估的监测和田间试验设计Rick Hellmich and Jarrad PrasifkaUSDAARS, Corn Insects & Plant Genetics Research Unit美国农业部-农业研究局，玉米昆虫和植物遗传研究所 Dept. Entomology, Iowa State University昆虫系,依阿华州立大学AgriculturalResearchServiceTopics 主题Challenges for field entomologists 田间昆虫学家的挑战Development of guidelines for te

2、sting non-target effects in the field 制定检测非靶作用的指导规则 Plot size小区尺寸 Sampling 采样 taxa abundance, statistical power 种属多寡，统计方式 trap efficiency 截淤效率 timing 时机Selecting taxa 选择种属 ecological functional groups 生态作用群Hypothesis driven studies 推动研究的假设Influences on Maize Agroecosystem 玉米农业生态系统的影响因素uPrevious crop

3、 and farming practices 以前的作物和耕作方式uFarming disturbances 耕作干扰 Tillage 耕耘 fertilizer/herbicides 肥料除草剂 planting 栽培 secondary tillage 二次耕作 harvesting 收割uNatural disturbances 自然干扰 local storm flooding 当地的洪水 temperature extremes 极端天气 plant diseases and pests 植物的病虫害Before We Walk into Field Plot我们走进田间试验地前Ho

4、w do disturbances of Bt protein in cropscompare with other disturbances?怎样比较作物的Bt蛋白干扰与其它的干扰因素？What is the signal tonoise ratio? 什么是信噪比？Undisturbed Pool 未受干扰的游泳池GM Crop Signal? 转基因作物信号？uSmall disturbance小干扰 uLarge disturbance大干扰Can this signal be detected?这样的信号可以检测到吗？Background Noise?背景噪音？uChildren j

5、umping 小孩跳水Background Noise?背景噪音？uLarger person jumping 大人跳水Background Noise? 背景噪音？uNear Chaos 附近混乱的杂音Ecological Risk Assessment 生态风险评估uTier 1 梯度 1 Simple, worst-case scenarios 简单的，最坏的情况 High estimated environmental concentration (EEC) 高估计环境浓度 （EEC）uTier 2 梯度 2 Use plant material (1X EEC) 使用植物材料 (1X

6、 EEC)uTier 3 梯度 3 Long-term laboratory, greenhouse or semi-field tests 长期实验室，温室或半田间试验uTier 4 梯度 4 Field tests 田间试验uField Monitoring 田间监测LABFIELD试验田间uTier 1 梯度 1 Simple, worst-case scenarios 简单的，最坏的情况 High estimated environmental concentration (EEC) 高估计环境浓度 （EEC）uTier 2 梯度 2 Use plant material (1X EE

7、C) 使用植物材料 (1X EEC)uTier 3 梯度 3 Long-term laboratory, greenhouse or semi-field tests 长期实验室，温室或半田间试验uTier 4 梯度 4 Field tests 田间试验uField Monitoring 田间监测Ecological Risk Assessment 生态风险评估LABFIELD试验田间Challenges for Field Trials 田间试验的难点uMethods criticized 被垢病的方法 effects of Bt crops likely subtle Bt作物的影响可能很

8、细微 Scientific Advisory Panel (USEPA 2001-2002)科学顾问团 （美国环保局 20012002）uKey issues 主要问题 choice of indicator taxa 标识种属的选择 plot size 小区尺寸 replication 重复 statistical analysis 统计分析Collecting Insects in the Field田间采集昆虫Sticky Cards 捕虫粘纸u thrips 牧草虫 u leafhoppers 叶蝉u corn leaf aphids 玉米缢管蚜虫u long-legged flies

9、 长腿蝇u minute pirate bug 小暗色花蝽 u fairyflies 缨小蜂Pitfall Traps 诱捕陷阱u centipedes 唇足类 u collembolans 弹尾目昆虫Sminthuridae 圆跳虫科 u spiders 蜘蛛wolf spiders 狼蛛u carabid larvae, adults 步行虫的幼虫和成虫Pterostichus, Harpalus spp.u rove beetle larvae, adults 隐翅虫的幼虫和成虫Litter Bags 废物袋 Visual Counts 目视计数u green lacewing eggs

10、 草蛉虫卵u green lacewing larvae 草蛉虫卵幼虫u lady beetle larvae 瓢虫幼虫u lady beetle adults 瓢虫成虫Harmonia axyridisColeomegilla maculataCoccinella septempunctataGalen Divelys Lab (Univ. Maryland) 实验室 （马里兰大学）3 year study: Bt, non-Bt, insecticide 3年研究： Bt, 非Bt, 杀虫剂12 orders 12个目112 families 112个科203 taxonomic grou

11、ps 203个分类群562,434 individuals counted 562，434 计数的个体Carabids identified to genus 步行虫识别到属Development of Guidelines指导的制定uCollaborative effort 协作者 USDAARS 美国农业部农业服务局 Corn Insects & Crop Genetics Research Unit 玉米昆虫和作物遗传研究所 University Maryland 马里兰大学 USDAAPHIS 美国农业部动植物检疫局 Industry 产业界uResearch issues 研

12、究问题 plot size effects 小区尺寸效应 sampling protocols (e.g. timing, replication) 采样方式 （如，时机和重复） improved identification 改良的标识 alternative methods of analysis 替代分析方法Evaluation of plot sizes试验小区尺寸评价uAccount for mobility of key taxa 阐释主要种属的流动性uSmall plots reduce differences 小小区减少差异 lowers chance to detect ef

13、fects (positive or negative) 降低检测到效应的几率（阳性或阴性）uSmallest realistic size sought 寻找最小的可行尺寸 suitable for pre- or post- registration 适于注册前和注册后Evaluating Plot Size试验小区尺寸评价uNon-transgenic corn hybrid 非转基因玉米杂交uVaried plot size from 0.01 to 0.53 ha (9, 18, 36 m) 各种试验小区尺寸，从0.01到0.53（9，18，36m)uPlots disturbed

14、(insecticides) 2X 干扰的小区（杀虫剂） 2X Sprayed喷杀虫剂9 mSprayed喷杀虫剂 18 mSprayed喷杀虫剂 36 mControl对照Sprayed喷杀虫剂Ladybird larvae 瓢虫幼虫瓢虫幼虫Aphids 蚜虫蚜虫Leafhoppers叶禅叶禅C. maculataSprayed喷杀虫剂Control对照MediumSized Plots 中等尺寸中等尺寸小区( (Sprayed)Sprayed)对照 中等（喷杀虫剂）采样时间典型系数小区LeafhoppersLadybird larvaeLacewing larvaeThripsLacewi

15、ng eggs采样时间 大 中 小典型系数015304550658095Days after plantingCoccinellid (l) / 10 plants (SE)9 m18 m36 mSMLSLSL小区种植后天数龟纹瓢虫（I）10株 小 大小 大小中 大小区05101550658095Days after plantingOrius / plot (SE)9 m18 m36 mSMSMLSL花蝽地块种植后天数小 大小中 大小 中小区uInfluence of plot size 小区尺寸的影响Small plots conceal effects小小区掩盖效应“Best” size

16、 is case dependent“最好”尺寸是依情况而定的Small Plot Penalty小小区的差异Prasifka et al. 2005Environmental Entomology环境昆虫学Control对照Chem杀虫剂Transgenic转基因Insects / Plot昆虫地块Sampling Issues: 采样问题： Species Abundance and Replication种属多寡和重复051015202530354045507/57/97/308/28/138/209/49/1710/110/180200400600800100012001400Impo

17、rtance of Insect Abundance昆虫富集度的重要性Date sampled采样时间Collembola per plot弹尾目昆虫地块Coefficient of variation 变异系数uReplication needed to detect potential differences (%) 检测到潜在差异（）需要的重复uTotal cost estimates for non-target field testing are important 非靶作用田间试验总费用的估计是很重要的Replicates required必须的重复Statistical powe

18、r统计幂00.81246810121410%20%30%40%Refining Sampling Methods采样方法的优化uEpigeal (ground-dwelling) taxa 穴居类Often small, nocturnal 经常很小，夜间出没uTrapping method comparison 捕捉方法比较Pitfall traps 诱捕陷阱Litter bags (above / below) 废物袋 （上下）Comparing Trapping Methods捕捉方法比较Carabid Beetle步行虫Location variation 位置变异R

19、elative ground beetle (Coleoptera: Carabidae) species abundance, collected in pitfall traps at the Sorenson and Deep Loess Research Station (DLRS) locations. 步行虫(Coleoptera: Carabidae)相对种属的富集度，采集于Sorenson and Deep Loess科研基地(DLRS) SpeciesSorenson2001Sorenson2002DLRS Plot 1DLRS Plot 2DLRS Plot 3DLRS P

20、lot 41Chlaenius erythropus 1%-10%6%1%1%2Elaphrops granarius34%7%-1%3Harpalus pensylvanicus11%25%49%43%80%89%4Poecilus lucublandus28%27%-1%1%1%5Pterostichus coracinus1%1%2%36%1%1%Lopez et al. 2005Carabid Trap Fluctuations步行虫捕获的变动Poecilus lucublandusElaphrops granariusMicrolestes linearis28-Jun 2-Jul

21、23-Jul 30-Jul 2-Aug 13-Aug 27-Aug 25-Sep 9-Oct 23-OctHarpalus pensylvanicus28-Jun 2-Jul 23-Jul 30-Jul 2-Aug 13-Aug 27-Aug 25-Sep 9-Oct 23-OctBt Non-BtInsecticideAverage # Beetles per trapAverage # Beetles per trapLopez et al. 2005Sample Timing 采样时机Chrysoperla rufilabris (Green lacewing)00.050.10.150

22、.50.40.456/21/20016/28/20017/5/20017/12/20017/19/20017/26/20018/2/20018/9/20018/16/20018/23/20018/30/20019/6/20019/13/20019/20/20019/27/200110/4/200110/11/2001Dates of Visual ExaminationsAverage Number per ObservationTransgenic Mon810IsolineIsoline/InsecticideInsecticide杀虫剂平均数观察观察时间转基因 Mo

23、n 810正常正常杀虫剂(草蛉虫)Sample Timing 采样时机Insecticide（小暗色花蝽 ）平均数观察观察时间杀虫剂转基因 Mon 810正常正常杀虫剂Standardization difficult标准化的困难uPlot size and protocols likely will be dependent on transgenic product (stressor) 小区尺寸和程序很可能决定于转基因产品（刺激因素） taxa emphasis 分类的重要性uCase by case 案例分析 above ground 在地面上 below ground 在地面下Sel

24、ecting Representative Taxa选择有代表性的分类uWhich species will be exposed? 哪些种属暴露了？ Common sense selections 常识性选择 eg., carabid beetles for rootworm active maize 如，根线虫活跃的玉米则选择步行虫uFunctional groups 功能性群uUncomplicated identification 简单识别uTaxa associated with target insects (?) 与靶昆虫有关的分类 e.g., sap beetles that

25、are attracted to insect damage 如，吸引到昆虫损害的地方的露尾甲虫 specialist parasitoids of target species 靶昆虫的拟寄生虫Bt exposure for Carabidae during anthesis?步行虫在花期暴露在Bt下？2 mmElaphropus xanthopus (Dejean)Tree of Life Project, David R. Maddison = Collembola 弹尾目弹尾目Cry34/35 Bt = Pollen 花粉花粉?Test for Bt proteins with ELI

26、SAELISA方法检测Bt蛋白 Herbivores 食草类Saprophytes 腐生类Parasitoids 拟寄生类Predators 捕食类Pollinators 传粉类Seed dispersers 种子散播类Herbivores 食草类Saprophytes 腐生类Predators 捕食类Parasitoids 拟寄生类Pollinators 传粉类Seed dispersers 种子散播类0.000.200.400.600.801.003691215-50%-30%-20%0.000.200.400.600.801.0036912150.000.200.400.600.801.

27、003691215CicadellidaeDiabrotica spp.ThysanopteraNumber of replicatesMedian statistical power (1 )Relationship between power and replication for non-target HERBIVORES非非靶作用食草类幂和重复的关系Plotted curves estimate the median power to detect a -50, -30 or -20% change in abundance for indicated non-target taxa.

28、用曲线估计的中值幂来检测特定非靶作用种属50，30或20富集度的差异中值统计幂（1）重复次数ChloropidaeEntomobryidaeNitidulidaeNumber of replicatesMedian statistical power (1 )0.000.200.400.600.801.003691215-50%-30%-20%0.000.200.400.600.801.0036912150.000.200.400.600.801.003691215Relationship between power and replication for non-target SAPROPH

29、YTES 非非靶作用腐生类幂和重复的关系Plotted curves estimate the median power to detect a -50, -30 or -20% change in abundance for indicated non-target taxa.用曲线估计的中值幂来检测特定非靶作用种属50，30或20富集度的差异中值统计幂（1）重复次数LycosidaeOrius spp.CoccinellidaeNumber of replicatesMedian statistical power (1 )0.000.200.400.600.801.003691215-5

30、0%-30%-20%0.000.200.400.600.801.0036912150.000.200.400.600.801.003691215Relationship between power and replication for non-target PREDATORS 非非靶作用捕食类幂和重复的关系Plotted curves estimate the median power to detect a -50, -30 or -20% change in abundance for indicated non-target taxa。用曲线估计的中值幂来检测特定非靶作用种属50，30

31、或20富集度的差异中值统计幂（1）重复次数ScelionidaeTrichogrammatidaeMymaridaeNumber of replicatesMedian statistical power (1 )0.000.200.400.600.801.003691215-50%-30%-20%0.000.200.400.600.801.0036912150.000.200.400.600.801.003691215Relationship between power and replication for non-target PARASITOIDS 非非靶作用拟寄生类幂和重复的关系Pl

32、otted curves estimate the median power to detect a -50, -30 or -20% change in abundance for indicated non-target taxa.用曲线估计的中值幂来检测特定非靶作用种属50，30或20富集度的差异中值统计幂（1）重复次数Processing samples and identifying taxa are challenging 样本处理和分类是很有挑战性的Brian Wiegmann, NC StateDavid Yeates, CSRIO AustraliaRudolf Meier,

33、 U. SingaporeMarkus Friedrich, Wayne StateGreg Courtney, Iowa S组装生命之树双翅目拥有150，000品种的双目翅是昆虫中在这个星球上最多样化的生物之一0.00.81.027-Jun9-Jul23-Jul6-Aug13-Aug4-Sep18-SepSample ending onMacrocentrus +/- SEBtIsoChem采样结束时间 Bt 普通 杀虫剂长距茧蜂 SE02468101225-Jun9-Jul16-Jul30-Jul 13-Aug4-Sep12-Sep 30-SepSample ending onSap beetles +/- SEBtIsoChem露尾甲虫SE Bt 普通 杀虫剂采样结束时间Different Approaches to Field Test