Supplementary Information

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1 Supplementary Information Large-scale test of the natural refuge strategy for delaying insect resistance to transgenic Bt crops Lin Jin 1, Haonan Zhang 1, Yanhui Lu 2, Yihua Yang 1, Kongming Wu 2, Bruce E. Tabashnik 3, Yidong Wu 1 1 College of Plant Protection, Nanjing Agricultural University, Nanjing, China. 2 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. 3 Department of Entomology, University of Arizona, Tucson, Arizona, USA. Address correspondence to Y.W. (wyd@njau.edu.cn). This Supplementary Information contains: Supplementary Figures 1-4 Supplementary Tables 1-6 1

2 Supplementary Figure 1. Computer simulations of the effect of initial percentage of resistant individuals on evolution of resistance to Bt cotton by H. armigera in northern China. For these simulations, conditions were similar to those in Fig. 2: we modeled two alleles at each of three independently segregating loci controlling survival on Bt cotton (Online Methods). The resistance allele was recessive (h = 0) at one locus, additive (h = 0.5) at the second locus, and dominant (h = 1) at the third locus. The mean refuge percentage was 56%. We varied the initial percentage of resistant individuals from 0.36 to 1.5%, which represents the 95% confidence interval for the mean value of 0.93% (data from ref. 33). 2

3 Supplementary Figure 2. Percentage of individuals resistant to Cry1Ac with alleles conferring different levels of dominance. We estimated the percentage of individuals resistant to Cry1Ac based on larval survival at a diagnostic concentration of the F 1 progeny obtained from mass matings of field-derived adults. We crossed the survivors of these bioassays individually with adults from a susceptible strain (SCD) and inferred the genotype of the survivors based on the survival of the resulting single-pair families (see experimental design in Fig. 5 and data in Supplementary Tables 4 and 6). 3

4 Supplementary Figure 3. Performance on non-bt cotton plants of H. armigera from a susceptible strain (SCD), a resistant strain derived from a field-selected population in northern China (AY2), and their F 1 progeny (AY2 X SCD). Bars represent means; vertical lines show 95% confidence intervals. (a) Survival (%) from neonate to adult emergence (n = 120 for each bar). (b) Percentage of adults that were female (n = 40 for SCD, n =47 for F 1, and n = 33 for AY2). (c) Fertile eggs laid per female (n = 20 for SCD, n = 20 for F 1, and n = 15 for AY2). Performance did not differ significantly between the resistant strain and the susceptible strain for any of the three traits measured: (a) Fisher s exact test, P = 0.40, (b) Fisher s exact test, P = 0.81, and (c) Mann-Whitney U-test, P =

5 Supplementary Figure 4. Computer simulations of the effect of a dominant 50% fitness cost on evolution of resistance to Bt cotton by H. armigera in northern China with no natural refuge. The refuge percentage was 2% based on 2% of all cotton ha planted with non-bt cotton. The conditions for these simulations were identical to those for Fig. 1a, except that fitness on non- Bt cotton was 0.5 for rs and rr, which yields a dominant fitness cost of 50% (compared with no fitness cost on non-bt cotton in Fig. 1a). As in Fig. 1a, with no natural refuge, the projected percentage of resistant individuals exceeded 50% by 2011, 95% by 2012, and 98% by 2013 with all three levels of dominance. 5

6 Supplementary Table 1. Parameter values used in computer simulations. Parameter Value(s) Source Fitness on Bt cotton Fig. 6 ss rr rs: recessive (h = 0) rs: additive (h = 0.5) rs: dominant (h = 1) Survival at the diagnostic concentration of Cry1Ac a Zhang et al. 34 ss rr rs: recessive (h = 0) rs: additive (h = 0.5) rs: dominant (h = 1) Fitness on non-bt host plants ss rs, rr (no fitness cost, Figs. 1-3) rs, rr (50% dominant cost, Suppl. Fig. 4) by definition Supplementary Fig. 3 hypothetical Initial percentage of resistant individuals b 0.36, 0.93, 1.5% Zhang et al. 33 Initial resistance allele frequency with 0.93% resistant individuals b One resistance allele (r) in the population (Fig. 1 and Supplementary Fig. 3) h = h = h = Three resistance alleles (r x, r y, and r z ) (Figs. 2 and 3) Supplementary Table 6 r x (h x = 0) r y (h y = 0.5) r z (h z = 1) Generations per year on cotton 3 Mean effective refuge (%) c 2, 40, 50, 56, 70, 90 a 1 microgram Cry1Ac per cm 2 diet surface. b Values for 2010, the initial year simulated. The default value was 0.93%, see Online Methods for details including initial resistance allele frequencies with 0.36 and 1.5% resistant individuals. c Mean effective refuge percentage for the three generations during which H. armigera fed on cotton each year. 6

7 Supplementary Table 2. Area planted with host plants of H. armigera in northern China and their potential contribution as refuges. Host plant Area planted (mean per year) a Generation 2 b Generations 3-4 b million ha % of total host plant area Refuge contribution relative to non-bt cotton (SE) c Effective refuge area (million ha) d Effective refuge % e Refuge contribution relative to non-bt cotton c Effective refuge area (million ha) d Bt cotton Effective refuge % e Non-Bt cotton f Corn g (0.04) g Peanut (0.4) (0.4) Legumes (0.4) (0.4) Other h Total i i a 2010 to 2012 data for the six provinces where we monitored resistance (Anhui, Hebei, Henan, Hubei, Jiangsu, and Shandong) from the Chinese Ministry of Agriculture. b In northern China, H. armigera feeds on wheat during its first generation, and cotton and other host plants during its second to fourth generations 26. c Mean H. armigera moths (and standard error) produced per ha relative to non-bt cotton based on data from from field cage experiments for corn, peanut and legumes (see Online Methods). The value for legumes is based on the field cage data for soy. In calculating the effective refuge %, we did not include contributions from Bt cotton and other host plants (melons, sesame, sorghum and vegetables as listed by the Chinese Ministry of Agriculture). 7

8 d Area planted (mean per year) for a crop multiplied by the refuge contribution relative to non-bt cotton for that crop. For example, million ha of peanut planted X 0.78 adults produced per ha relative to non-bt cotton = 2.07 million effective refuge ha. e Effective refuge area divided by (area planted to Bt cotton plus total effective refuge area). For example, in generation 2, the total effective refuge area is 2.83 million ha, which is divided by (2.685 million ha Bt cotton million ha total effective refuge area) to yield a total effective refuge % of 51.3%. f The percentage of cotton planted to non-bt cotton was 1.8% (0.18/[ ]), which we rounded to 2% for simulations. g Because of the timing of planting, corn can contribute as a refuge during the third and fourth generations, but not the second generation of H. armigera 26. Corn is planted at two different times (referred to as early and late) in northern China 26. For simplicity, we assumed that half of the corn was planted early and contributed as a refuge during generation 3, and the other half was planted late and contributed as a refuge during generation 4. Therefore, to estimate the refuge contribution of corn relative to non-bt cotton for generations 3 and 4, we divided the production of adults per ha of corn relative to non-bt cotton (0.20) by two, which yields h Sesame, sorghum, vegetables and melons (as listed by the Chinese Ministry of Agriculture). i In simulations based on the data in this table, we set the refuge percentage at 51% for generation 2 and 59% for generations 3 and 4. For brevity, we report the mean effective refuge % for these conditions as 56%, which is the mean effective refuge percentage for generations 2 to 4 ([51% + 59% + 59%]/3). 8

9 Supplementary Table 3. Host plants of H. armigera in six provinces of northern China. Values are means for 2010 to 2012 based on data from the Chinese Ministry of Agriculture. Province Total host plants Non-cotton Corn Peanut Legumes Other d (million ha) a host plants b (%) c (%) c (%) c (%) c (%) c Anhui Hebei Henan Hubei Jiangsu Shandong a Cotton, corn, peanut, legumes, melons, sesame, sorghum and vegetables. b Corn, peanut, legumes, melons, sesame, sorghum and vegetables. c Host plants other than cotton (ha) divided by total host plant (ha) times 100%. d Melons, sesame, sorghum and vegetables. 9

10 Supplementary Table 4. Survival at a diagnostic concentration of Cry1Ac for F 1 progeny of H. armigera from northern China. Province Site Survival (%) a Number of F 1 larvae tested b Number collected c 2010 d Anhui Anqing (Aq) Hebei Anci (Ac) Hebei Cangxian (Cg) Hebei Gaoyang (Gy) Hebei Nanpi (Np) Hebei Qiuxian (Qx) Hebei Quzhou (Qz) Henan Anyang (Ay) Henan Kaifeng (Kf) Henan Nanyang (Ny) Hubei Jingzhou (Jz) Hubei Qianjiang (Qj) Jiangsu Yancheng (Yc) Shandong Caoxian (Co) Shandong Huimin (Hm) Shandong Juye (Jy) Shandong Xiajin (Xj) Mean or total e a Survival of F 1 larvae at diagnostic concentration (1 μg Cry1Ac per cm 2 diet). b Number of F 1 larvae tested at the diagnostic concentration. c Number of eggs (from Anqing, Jingzhou, Qianjiang, and Yancheng) or moths (all other sites) collected from the field. d 2010 data reported previously by Zhang et al. 33 e Mean for survival and total for number of larvae tested or collected. 10

11 Supplementary Table 5. Survival at a diagnostic concentration of Cry1Ac for F 1 progeny of H. armigera from two field sites in northwestern China and from a susceptible laboratory strain (SCD). Province Site Survival (%) a Number of F 1 larvae tested b Number collected c 2010 d Northwestern China Xinjiang Shache (Sc) Xinjiang Shawan (Sw) Susceptible laboratory strain SCD e Mean or total f a Survival of F 1 larvae at diagnostic concentration (1 μg Cry1Ac per cm 2 diet). b Number of F 1 larvae tested at the diagnostic concentration. c Number of moths collected from the field. d 2010 data reported previously by Zhang et al. 33 e Susceptible strain from Côte D Ivoire (see Methods). f Mean for survival and total for number of larvae tested or collected. 11

12 Supplementary Table 6. Survival of progeny from single-pair families that we generated by crossing a susceptible moth (SCD) with a field-derived resistant moth (survivor of diagnostic concentration of Cry1Ac from among F 1 progeny of field-collected insects, see Fig. 5). Year Survival in single-pair families a No. of larvae tested No. of families <15% d 15 to 30 to 50% Total Mean b Range c <30% <50% , , , , All years , a Survival at the diagnostic concentration of Cry1Ac. b Mean number of larvae tested per family. c Range in number of larvae tested per family. d Survival <15% indicates recessive inheritance of resistance; the proportion of families with non-recessive resistance is one minus the proportion of families with recessive resistance: in 2010, in 2011, in 2012, and in