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Hybridizing transgenic Bt with non- counters resistance in

Peng Wana,b, Dong Xub, Shengbo Congb, Yuying Jiangc, Yunxin Huangd, Jintao Wangb, Huaiheng Wub, Ling Wanga,b, Kongming Wua,1, Yves Carrièree, Andrea Mathiase, Xianchun Lie, and Bruce E. Tabashnike

aState Key Laboratory for of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China; bInstitute of Plant Protection and Soil Fertility, Hubei Academy of Agricultural Sciences, Wuhan 430064, People’s Republic of China; cNational Agro-Technical Extension and Service Center, Beijing 100026, People’s Republic of China; dSchool of Resource and Environmental Sciences, Hubei University, Wuhan 430062, People’s Republic of China; and eDepartment of Entomology, University of Arizona, Tucson, AZ 85721

Edited by Charles J. Arntzen, Arizona State University, Tempe, AZ, and approved April 5, 2017 (received for review January 10, 2017)

Extensive cultivation of crops genetically engineered to produce requirements (15, 17). However, mixtures increase oppor- insecticidal proteins from the bacterium (Bt) has tunities for larval movement between Bt and non-Bt plants or suppressed some major pests, reduced sprays, enhanced plant tissues, which could accelerate resistance evolution by pest control by natural enemies, and increased grower profits. How- raising the of resistance or by reducing the survival of ever, these benefits are being eroded by evolution of resistance in susceptible insects (13–15, 17). Despite potentially profound pests. We report a strategy for combating resistance by crossing implications of the seed mixture strategy for the sustainability of transgenic Bt plants with conventional non-Bt plants and then cross- Bt crops, large-scale field tests of its efficacy have been lacking.

ing the resulting first-generation (F1) progeny and sowing the Here, we test a version of the seed mixture strategy by com- second-generation (F2) . This strategy yields a random mixture paring predictions from computer modeling with field monitoring within fields of three-quarters of plants that produce Bt toxin and data for the pink bollworm (Pectinophora gossypiella) based on – one-quarter that does not. We hypothesized that the non-Bt plants evaluation of resistance in 96 sets of insects collected during 2005 in this mixture promote survival of susceptible insects, thereby delay- 2015 from 17 sites in six provinces in the Yangtze River Valley of China (total n = 66,648 larvae tested in bioassays). The millions of

ing evolution of resistance. To test this hypothesis, we compared SCIENCES small-scale farmers in this region first planted transgenic cotton predictions from computer modeling with data monitoring pink boll- AGRICULTURAL worm (Pectinophora gossypiella) resistance to Bt toxin pro- producing Bt toxin Cry1Ac in 2000, with the pink bollworm as one duced by transgenic cotton in an 11-y study at 17 field sites in six of the primary targets (18). The larvae of this devastating world- provinces of China. The frequency of resistant individuals in the field wide pest feed primarily on cotton seeds within bolls, and sus- increased before this strategy was widely deployed and then de- ceptible larvae are killed by the Cry1Ac in Bt cotton (6, 19). clined after its widespread adoption boosted the percentage of Previous work in the Yangtze River Valley documented a small but statistically significant increase in resistance of the pink non-Bt cotton plants in the region. The correspondence between – – the predicted and observed outcomes implies that this strategy coun- bollworm to Cry1Ac from 2005 2007 to 2008 2010 (18). The percentage of populations with one or more larvae surviving at tered evolution of resistance. Despite the increased percentage of non-Bt cotton, suppression of pink bollworm was sustained. Unlike other resistance management tactics that require regulatory inter- Significance vention, growers adopted this strategy voluntarily, apparently be- cause of advantages that may include better performance as well as Crops genetically engineered to produce insecticidal proteins lower costs for seeds and . from the bacterium Bacillus thuringiensis (Bt) kill some major pests and reduce use of insecticide sprays. However, evolution sustainability | evolution | resistance management | genetically modified | of pest resistance to Bt proteins decreases these benefits. We refuge report a strategy for combating resistance by crossing transgenic Bt plants with conventional non-Bt plants and then sowing the enetically engineered crops that produce insecticidal pro- second-generation seeds. This strategy yields a random mixture Gteins from the bacterium Bacillus thuringiensis (Bt) have within fields of three-quarters of plants that produce Bt protein been planted globally on a cumulative total of over 732 million and one-quarter that does not. An 11-y field study in China shows this strategy countered resistance to Bt cotton of pink ha since 1996 (1). The benefits of these Bt crops include pest ’ suppression, reduced insecticide use, enhanced biological control, bollworm, one of the world s most devastating pests. This out- and increased farmer profits (2–7). However, increasingly rapid come illustrates that non-Bt plants in a seed mixture can boost evolution of resistance to Bt crops by pests has eroded these benefits survival of susceptible insects and delay resistance. (8–11). The main strategy for delaying pest resistance to Bt crops “ ” Author contributions: K.-M.W. and B.E.T. designed research; P.W., D.X., S.B.C., Y.Y.J., J.T.W., aims to increase the survival of susceptible insects with refuges of H.H.W., and L.W. performed research; A.M. and B.E.T. conducted modeling; Y.H. and B.E.T. host plants that do not produce Bt toxins (9). Although refuges can analyzed data; and K.-M.W., Y.C., X.L., and B.E.T. wrote the paper. delay insect adaptation to Bt crops (2, 3, 9, 12), the optimal spatial Conflict of interest statement: B.E.T. is a coauthor of a patent on modified Bacillus scale for planting refuges remains unresolved (13–15). Also, because thuringiensis toxins, “Suppression of Resistance in Insects to Bacillus thuringiensis Cry Toxins, refuges are often perceived to cause short-term economic sacrifices Using Toxins that Do Not Require the Cadherin Receptor” (patent nos. CA2690188A1, CN101730712A, EP2184293A2, EP2184293A4, EP2184293B1, WO2008150150A2, and for growers, they are usually imposed by regulations. WO2008150150A3). CropScience, Dow AgroSciences, DuPont Pioneer, , Before 2010, regulations in the United States mandated ref- and did not provide funding to support this work, but may be affected finan- uges of non-Bt plants in blocks consisting of separate fields, rows, cially by publication of this paper and have funded other work by B.E.T. Y.C. has received or strips (14). In 2010, the regulations were modified to include funding from DuPont Pioneer, but DuPont Pioneer did not provide funding for this work. mixtures of Bt and non-Bt seeds generating a random array of Bt Syngenta, Dupont, Bayer Crop Science, FMC, and Gowan have funded other work by X.L. and non-Bt plants side by side within fields (14). By 2015, all This article is a PNAS Direct Submission. farmers surveyed in the US Corn Belt planted such seed mixtures Freely available online through the PNAS open access option. for some or all of their corn (16). Seed mixtures have several 1To whom correspondence should be addressed. Email: [email protected]. advantages relative to block refuges, particularly convenience for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. farmers and elimination of noncompliance with block refuge 1073/pnas.1700396114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1700396114 PNAS | May 23, 2017 | vol. 114 | no. 21 | 5413–5418 Downloaded by guest on September 27, 2021 the diagnostic concentration (9 μg of Cry1Ac per milliliter of 56% in 2008–2010 (n = 27 populations) to 0% in 2011–2015 (n = diet) increased from 0% in 2005–2007 to 56% in 2008–2010 45 populations) (Fisher’s exact test: P < 0.0001; Fig. 1A). Also, (total n = 51 populations; Fisher’s exact test: P < 0.0001; Fig. 1A the median percentage survival at the diagnostic concentration and SI Appendix, Fig. S1 and Table S1). In addition, the median declined from 1.6% in 2008–2010 to 0% in 2011–2015 (Mann– percentage survival at the diagnostic concentration increased Whitney U test: U = 945, P < 0.0001; Fig. 1B). The mean re- from 0% in 2005–2007 to 1.6% in 2008–2010 (Mann–Whitney U sistance ratio, which is a less sensitive indicator than survival at the test: U = 504, P < 0.001; Fig. 1B). The resistance ratio, which was diagnostic concentration, decreased slightly but not significantly, calculated as the concentration of Cry1Ac killing 50% of larvae from 4.1 in 2008–2010 to 3.7 in 2011–2015 (t test: t = 0.77, df = 70, = (LC50) for a field population divided by the LC50 for the sus- P 0.55; Fig. 1C). In control tests, survival at the diagnostic ceptible laboratory strain QJ-S tested in the same year, rose from concentration was 0% for the susceptible laboratory strain QJ-S a mean of 2.5 in 2005–2007 to 4.1 in 2008–2010 (t test: t = 2.2, every year from 2005 to 2015 (n = 72 larvae per year, total of df = 49, P = 0.03; Fig. 1C and SI Appendix, Table S2). 792 larvae), indicating consistency of the bioassays over time. Without major changes, rapid increases in resistance were To understand the unexpected decrease in survival at the di- anticipated after 2010 because refuges of non-Bt cotton varieties agnostic concentration of Cry1Ac, we investigated the possibility had decreased to only 6% of all cotton hectares planted (18) and that the percentage of non-Bt cotton plants increased in 2010 evolution of resistance is an exponential process expected to and subsequent years because growers purchased and planted accelerate after more than 1% of the population is resistant (20). seed from second-generation (F2) cotton hybrids. Because of However, the results reported here show that pink bollworm , F1 and F2 cotton hybrids typically have a higher yield resistance to Cry1Ac did not increase from 2008–2010 to 2011– than their parent varieties (21, 22). Whereas production of F1 2015 (Fig. 1). The percentage of populations with one or more hybrid seeds requires costly hand , self-pollination of larvae surviving at the diagnostic concentration declined from F1 hybrids produces F2 hybrid seeds (21, 22). Crossing Bt cotton with non-Bt cotton creates F1 hybrids that make Bt toxin and are hemizygous for this trait because they have one copy of the Bt and no corresponding (23, 24). Self-pollination by such F1 hybrids creates F2 hybrid seeds expected to consist of 25% Bt homozygotes and 50% hemizygotes that produce Bt toxin and 25% non-Bt homozygotes that do not (23). From 2010 to 2015, we determined the percentage of cotton seeds containing Cry1Ac using immunoassays with 100 seeds tested individually per assay in 140 assays of 16 varieties and 68 hybrids recommended for planting in the Yangtze River Valley (total of 14,000 seeds tested; Fig. 2 and SI Appendix, Fig. S2 and Tables S3–S6). We detected Cry1Ac in 99% (SE = 0.6%) of seeds from six Bt cotton varieties, which is close to the expected 100%. In 10 non-Bt cotton varieties, Cry1Ac occurred in 23% (SE = 2) of seeds, which indicates contamination of the non-Bt cotton seed supply. For the 68 hybrids, the percentage of seeds containing Cry1Ac clusters into two groups: 23 hybrids with a range of 89–100% (mean = 96%, SE = 0.5%) and 45 hybrids with a range of 39–84% (mean = 67%, SE = 1%; SI Appendix, Fig. S2). We refer hereafter to these groups as F1 and F2 hybrids, respectively, because they are likely to consist pre- dominantly of these hybrids. However, the observed percentage of seeds containing Cry1Ac is lower than the expected 100% for F1 hybrids and 75% for F2 hybrids, which implies that some of these F1 and F2 hybrids contain seeds from non-Bt cotton vari- eties, other hybrids, or both. Based on our classification of the hybrids, the price was 35% higher for seeds from F1 than F2 hybrids (SI Appendix, Table S7), which corresponds with the higher production cost of the F1 hybrids. Although our analysis focuses primarily on 2010–2015, we also surveyed 12 major seed companies to estimate the percentage of F2 hybrids planted from 2004 to 2009 (SI Appendix, Table S8). To calculate the total percentage of non-Bt cotton plants for each year, we multiplied the proportion of non-Bt cotton seeds for each type of cotton (SI Appendix, Tables S3–S6 and S8)by the percentage of cotton hectares planted with the correspond- ing type of cotton during that year (Fig. 2A and SI Appendix, Tables S8–S11), and then summed the resulting percentages. The overall percentage of non-Bt cotton plants declined from a maximum of 37% in 2004 to 12% in 2008 and 2009, then nearly doubled to 23% in 2010, and was 25–27% from 2011 to 2015 (Fig. 2B). The initial decrease in non-Bt cotton percentage re- flects reduced planting of non-Bt cotton varieties, whereas re- Fig. 1. Pink bollworm resistance to Bt toxin Cry1Ac in the Yangtze River Valley, 2005–2015. (A) Percentage of populations with one or more survivors placement of Bt cotton varieties and F1 hybrids with F2 hybrids caused the subsequent increase (Fig. 2). In each year from at the diagnostic concentration of Cry1Ac (SI Appendix, Table S1). (B) Per- – centage of survivors in each population (SI Appendix, Table S1). (C) Resistance 2010 to 2015, F2 hybrids accounted for 78 84% of the total non- = ratio, the LC50 divided by the LC50 for the susceptible QJ-S laboratory strain Bt cotton plants (mean 81%; Fig. 2B), which provided an tested in the same year as an internal control (SI Appendix, Table S2). Values exceptional opportunity to test hypotheses about how this seed in B and C are means with SE. Data for 2005–2010 are from ref. 18. mixture affects the evolution of resistance.

5414 | www.pnas.org/cgi/doi/10.1073/pnas.1700396114 Wan et al. Downloaded by guest on September 27, 2021 confirmed previous reports that okra (Abelmoschus esculentus) and tomato (Lycopersicon esculentum) support pink bollworm larval development (19, 27), but no survival occurred on eggplant (Solanum melongena), cucumber (Cucumis sativus), apple (Malus domestica), or kidney bean (Phaseolus vulgaris)(SI Appendix, Table S13). In field experiments, however, pink bollworm larvae and adults were common in cotton, yet none were found in okra or tomato (SI Appendix, Tables S14 and S15). Thus, it is unlikely that non-Bt host plants other than cotton contributed substantially to the observed delay in evolution of pink bollworm resistance to Bt cotton. A potential drawback of the increased percentage of non-Bt cotton plants from 2011 to 2015 is higher population density of pink bollworm. However, the mean number of pink bollworm eggs per 100 cotton plants was not higher in 2011–2015 (5.4) than in 2006–2010 (11) (Fig. 4A and SI Appendix, Table S16). Furthermore, no significant association occurred between pink bollworm eggs per cotton plant and year from 2011 to 2015 (linear regression: r2 = 0.05, df = 3, P = 0.72; Fig. 4A), indicating no steady rise in population density during this period. Relative to the 5 y before Bt cotton was planted (1995–1999), when the mean number of pink bollworm eggs per 100 cotton plants was 94 (SE = 9.4), the abundance in 2011–2015 declined by 96%. In parallel, the mean number of insecticide sprays applied yearly against pink bollworm per cotton field decreased from 2.0 in 1995–1999 to 0.64 in 2011–2015, a 69% reduction (Fig. 4B and SI Appendix, Table S17). Moreover, sprays were 27% fewer in 2011–2015 relative to 2006–2010 (mean = 0.88) (Mann– = =

Whitney U test: U 24, P 0.02). SCIENCES

Whereas previous work has emphasized problems caused by AGRICULTURAL interbreeding between transgenic and nontransgenic plants (13– Fig. 2. Cotton planted in the Yangtze River Valley of China from 2004 to 2015. 15), we discovered that increased planting of F2 hybrids from (A) Percentage of cotton hectares planted to F1 hybrid cotton, F2 hybrid cotton, crosses between Bt and non-Bt cotton was associated with non-Bt cotton varieties, and Bt cotton varieties (SI Appendix, Tables S8–S11). (B) delayed evolution of pink bollworm resistance to Bt cotton and Percentage of cotton hectares consisting of plants not producing Cry1Ac: total continued suppression of this voracious pest. Despite extensive and contributions by F2 hybrids and non-Bt cotton varieties. The contribution of global monitoring of resistance to Bt crops during the past two F1 hybrids plus Bt cotton to the percentage of cotton hectares consisting of ≤ = decades and some examples of fluctuations in resistance allele plants not producing Cry1Ac was 2.5% each year (mean 1.4%). frequency (2, 9, 28), previous reports have not documented any cases where resistance was markedly delayed or reversed after We used computer modeling to evaluate two hypotheses: (a) the threshold of 1% resistant individuals was exceeded in a re- all non-Bt cotton plants act as refuges, including the non-Bt gion for several years, as reported here. The seed mixture generated with F2 hybrids analyzed here may cotton plants in fields of F2 hybrid cotton, or (b) only non-Bt cotton plants in fields of non-Bt cotton varieties act as refuges. In be especially effective against pink bollworm because of the substantial fitness cost associated with its resistance to Cry1Ac both scenarios, we simulated the period from the end of the and its recessive inheritance of resistance (25), even when season in 2008 to 2015 using a previously described one-locus, feeding on bolls from F hybrids, Cry1Ac-producing bolls from two-allele population genetic model, with fitness values for each 1 F hybrids (SI Appendix, Table S18), or a mixture of Bt and non- genotype on Bt and non-Bt cotton plants based on empirical data 2 Bt seeds (24). Selfing of F plants in subsequent generations is (25, 26) (SI Appendix, Table S12). Under hypothesis (a), which 2 yielded an annual mean refuge of 23% non-Bt cotton plants, re- sistance evolution was delayed, which corresponds with the ob- served outcome in the field (Fig. 3). In striking contrast, hypothesis (b) yielded an annual mean refuge of only 4.1%, and the predicted percentage of resistant individuals surged to 100% by 2013 (Fig. 3). These comparisons between the observed and predicted outcomes support hypothesis (a) but not hypothesis (b). Sensitivity analyses varying assumptions about refuges and insect fitness show that the predicted delay in resistance evolu- tion associated with the increased percentage of non-Bt cotton plants provided by F2 hybrids is robust (SI Appendix, Figs. S3 and S4). The correspondence between the outcome in the field and modeling results under hypothesis (a) demonstrates that the increase in the overall percentage of non-Bt cotton plants, in- cluding F2 hybrids, is sufficient to explain the observed lack of increase in resistance from 2011 to 2015 (Fig. 3). Nevertheless, Fig. 3. Predicted versus observed evolution of pink bollworm resistance to Bt this result does not exclude the alternative hypothesis that an toxin Cry1Ac in the Yangtze River Valley of China from 2008 to 2015. The increase in the relative abundance of non-Bt host plants other predictions are from computer simulations of hypotheses proposing that (a) all than cotton contributed to this delay in resistance. To address non-Bt cotton plants act as refuges, including the non-Bt cotton plants in fields

this alternative hypothesis, we tested the potential of six other of F2 hybrid cotton or (b) only non-Bt cotton plants in fields of non-Bt cotton common crops in the Yangtze River Valley to serve as pink varieties act as refuges. The observed values are bioassay data for survival at bollworm host plants. The results of laboratory experiments the diagnostic concentration of Cry1Ac (Fig. 1B and SI Appendix,TableS1).

Wan et al. PNAS | May 23, 2017 | vol. 114 | no. 21 | 5415 Downloaded by guest on September 27, 2021 artificial diet in the laboratory, and their F1 progeny were tested on a diet containing concentrations of Cry1Ac ranging from 0 to 9 μg of Cry1Ac per milliliter of diet, which is a diagnostic concentration that kills all or nearly all susceptible larvae but few or no resistant larvae. Neonates were tested in- dividually, with 24 neonates tested per concentration per replicate and three replicates for each concentration. After 21 d in darkness at 29 ± 1°C, live fourth instars and pupae were scored as survivors. Each year, as an in- ternal control, we used the same methods to test the susceptible strain QJ-S, which was started with insects collected from Qianjiang, Hubei province, in 2004 and reared in the laboratory without exposure to toxin. Results from 2005 to 2010 were reported previously (18) and are included here for comparison with the new data for 2011–2015.

Percentage of Cotton Seeds Containing Cry1Ac. We used immunoassays to determine the percentage of cotton seeds producing Cry1Ac for cotton hybrids, Bt cotton varieties, and non-Bt cotton varieties (SI Appendix, Tables S3–S6). In each year from 2010 to 2015, we purchased 1,200–1,375 g of seeds of each of 20–30 cotton hybrids and varieties that were recommended that year by the local government. Each year, we randomly selected 100 seeds from each hybrid or variety and divided them into four groups with 25 seeds per replicate. We tested each seed individually for Cry1Ac in a total of 140 assays (total n = 14,000 seeds tested individually) of six varieties of Bt cotton, 10 varieties of non-Bt cotton, and 68 hybrids. We used Cry1Ab/Cry1Ac immunoassay test strips (Aochuangjinbiao Bio- tech) according to the manufacturer’s instructions. Each replicate of 25 seeds had a separate positive and negative control. Each seed was stored in a 2-mL CryoPure tube (Sarstedt) with a 6-mm-diameter oil-free steel ball and 400 μL of leaching solution (Aochuangjinbiao Biotech). After centrifugation at 5,000 rpm for 18 seconds in a bead mill homogenizer (Precellys 24; Bertin Technologies), we added 600 more μL of leaching solution before analyzing each sample with a test strip. After 5 min, the appearance of a quality control line indicated the strip worked properly and the appearance of a second line indicated detection of Cry1Ac. For each of the 68 hybrids, if the mean percentage of seeds producing Cry1Ac was >87.5% (mean of the expected 100% for F seeds and 75% for F Fig. 4. Pink bollworm population density and insecticide sprays in the 1 2 seeds), the hybrid was classified as F . If the mean percentage of seeds Yangtze River Valley, 1995–2015. (A) Number of F pink bollworm eggs per 1 1 producing Cry1Ac was <87.5%, the hybrid was classified as F . Based on this 100 cotton plants was determined annually for 150 plants per county in each 2 criterion, which aligns with a gap in the observed distribution (SI Appendix, of 12–24 counties (mean = 20 counties, total sample = 63,300 plants; SI Fig. S2), we classified 23 hybrids as F (n = 38 assays, mean = 96.3%, SE = 0.5%, Appendix, Table S16). (B) Sprays targeting pink bollworm annually per cot- 1 range = 89–100%; SI Appendix,TableS5)and45hybridsasF (n = 82 assays, ton field based on ∼10 fields per county for 14–16 counties per year (SI 2 mean = 67.1%, SE = 1.4%, range = 39–84%; SI Appendix,TableS6). Appendix, Table S17). Values in both panels are means with SE.

Percentage of Cotton Planted to F1 and F2 Hybrid Cotton, Bt Cotton, and Non-Bt expected to maintain roughly 25% Bt homozygotes, 50% hemi- Cotton. For each year from 2004 to 2015, we calculated the percentage of zygotes, and 25% non-Bt homozygotes. The F cotton hectares planted to hybrid cotton, Bt cotton, and non-Bt cotton using 2 data from China’s Ministry of Agriculture for the six provinces in the Yangtze mixture planted in China differs from the standard seed mixtures River Valley we studied (Anhui, Hubei, Hunan, Jiangsu, Jiangxi, and Sichuan). used now for managing resistance to Bt corn in the United Because the Ministry’s data do not distinguish between F1 and F2 hybrids, we States, which do not entail hybridization and have either 90% or used two methods to calculate the proportion of hybrid cotton planted with 95% homozygous Bt seeds combined in seed bags with 10% or each of these two hybrids. For 2004–2009, we used responses regarding

5% homozygous non-Bt seeds, respectively (15). planting of F1 and F2 hybrids from a telephone survey of 12 seed companies The major advantage of a seed mixture generated with F2 (SI Appendix, Table S8). For 2010–2015, we used the data from the per- hybrids is a built-in refuge of roughly one-quarter non-Bt plants, centage of seeds containing Cry1Ac for the 68 hybrids (SI Appendix, Tables which could be especially important in developing countries S5 and S6) in conjunction with the data from Ministry of Agriculture on the where planting of separate refuges is low or nil. Whereas com- hectares planted to each of the 68 hybrids (SI Appendix, Tables S9 and S10). pliance with refuge requirements is considered a key factor that For each year, we summed the hectares planted to the 23 F1 hybrids and, delayed evolution of pink bollworm resistance to Bt cotton in the separately, to the 45 F2 hybrids. We calculated the proportion of hybrid United States, the scarcity of non-Bt cotton refuges probably cotton planted to F1 (or F2) hybrids in each year as the hectares planted to hastened this pest’s resistance to Bt cotton in (8, 9). In the the F1 (or F2) hybrids divided by the total hectares planted to the F1 plus F2 hybrids. For 2010–2015, the mean percentage of total hectares planted to Yangtze River Valley, millions of farmers have planted F2 hybrid = cotton not as a resistance management strategy, but apparently the 68 tested hybrids was 58.4% (SE 6.3). For 1 y, 2010, we have the data on the percentage of hybrids consisting of F2 hybrids for both methods: 52% for immediate benefits that may include higher yield relative to from the survey versus 77% based on analysis of Cry1Ac in seeds and hect- parental varieties, reduced expenses for insecticides relative to ares planted to each hybrid. This contrast suggests that the survey may non-Bt cotton, and reduced cost of seeds relative to F hybrids. 1 underestimate the planting of F2 hybrids. The efficacy of this strategy for managing resistance in other regions and against other pests remains to be determined. Percentage of Cotton Planted to Non-Bt Cotton. To calculate the percentage of cotton hectares planted to non-Bt cotton plants in each year, we multiplied Materials and Methods the proportion of non-Bt cotton seeds (1 minus the proportion containing Monitoring Pink Bollworm Resistance to Cry1Ac. We conducted field sampling Cry1Ac based on the assays described above) for each of the four types of

and diet bioassays to monitor pink bollworm resistance to Bt toxin Cry1Ac cotton (F1 and F2 hybrid cotton, Bt cotton, and non-Bt cotton) by the per- as described previously (18). Briefly, for 2011–2015, we sampled 60–700 centage of cotton hectares planted of the corresponding type of cotton in (mean = 329) pink bollworm larvae per site from seven to 10 sites per year in that year (Fig. 2A), and then summed the four resulting percentages. For

the Yangtze River Valley (SI Appendix, Fig. S1; totals: 45 sets of larvae and each of the 23 F1 hybrids and 45 F2 hybrids, we calculated the mean pro- 14,799 larvae sampled). Larvae from each site were mass-reared on an portion of non-Bt seeds for 2010–2015. For each year from 2010 to 2015, we

5416 | www.pnas.org/cgi/doi/10.1073/pnas.1700396114 Wan et al. Downloaded by guest on September 27, 2021 calculated the hectares of non-Bt cotton plants for each F1 hybrid by mul- refuges. Population mean fitness was calculated with the standard tiplying the mean proportion of non-Bt cotton seeds for that hybrid by the equation (37): hectares planted to that hybrid in that year. We calculated the percentage = 2 + + 2 of non-Bt cotton plants for all F1 hybrids for each year from 2010 to 2015 as WM p Wrr 2pqWrs q Wss. [5] 100-fold the total hectares of non-Bt cotton plants for all F1 hybrids divided We iterated Eq. 1 with a computer program to project changes in the r allele by the total hectares of all F1 hybrids. We used the same approach to cal- frequency over successive generations. culate the percentage of non-Bt cotton for all F2 hybrids for each year from 2010 to 2015. For 2004–2009, when seeds were not tested for Cry1Ac, we For each year, we simulated three generations, corresponding to the three used the mean proportion of non-Bt cotton seeds based on all assays from annual generations of pink bollworm in the Yangtze River Valley (38). We modeled evolution of resistance from the end of the third generation of 2010 to 2015 for F1 hybrids and separately for F2 hybrids. Relative to the 2008 (end of the season) to the end of the third generation of 2015 (end of extensive data on Cry1Ac in seeds for F1 hybrids (38 assays) and F2 hybrids (82 assays), which enabled calculation of the proportion of non-Bt cotton the season). We started with 2008 because 2008 is the first year that resistant individuals were detected in the Yangtze River Valley (18), which allowed us seeds separately for each year from 2010 to 2015, we had fewer assays for to use the monitoring data to set the percentage of resistant individuals (rr) varieties of Bt cotton (nine assays) and non-Bt cotton (11 assays). Accord- at the end of 2008 as the observed value of 0.88% (18). ingly, we used the mean proportion of non-Bt cotton seeds from 2010 to 2015 to estimate the percentage of non-Bt cotton plants for all years for Bt To make robust comparisons between predicted and observed outcomes, we cotton varieties (mean = 0.013, SE = 0.006, n = 9 assays of six varieties; SI used the means for parameters in the model (SI Appendix, Table S12) and for the Appendix, Table S3) and non-Bt cotton varieties (mean = 0.77, SE = 0.02, n = observed percentage of resistant individuals in field populations based on 11 assays of 10 varieties; SI Appendix, Table S4). survival at the diagnostic concentration of Cry1Ac (SI Appendix,TableS1). We also conducted sensitivity analyses with the model to assess the potential effects on resistance evolution of variation in the refuge percentage and variation in Computer Simulations. To assess the potential effects of F hybrid cotton on 2 the fitness of rr on Bt cotton and non-Bt cotton, which yields variation in in- the evolution of pink bollworm resistance to Bt cotton in the Yangtze River complete resistance and fitness cost (39), respectively. In sensitivity analyses, we Valley of China, we used a previously described deterministic population modeled four assumptions about which cotton plants act as refuges: (i)hy- genetic model of resistance to a Bt crop (26, 29) with some minor modifi- pothesis (a): all non-Bt cotton plants, including plants in fields of F hybrids, F cations. We chose this simple, previously described population genetic 2 1 hybrids, non-Bt cotton, and Bt cotton; (ii) hypothesis (b): only non-Bt cotton model for several reasons: to make clear the link between the model and the plants in fields of non-Bt cotton varieties; (iii) hypothesis (c): only non-Bt cotton hypothesized increase in refuge percentage associated with planting F2 plants in fields of F2 hybrid cotton; and (iv) hypothesis (d): non-Bt cotton plants hybrids, to enable incorporation of realistic biological parameters for pink in fields of F1 hybrids, non-Bt cotton varieties, and Bt cotton varieties (but not F2 bollworm in the Yangtze River Valley, to examine projected outcomes of

hybrids) (Fig. 3 and SI Appendix,Figs.S3andS4). Based on experimental results SCIENCES different assumptions about refuges and fitness values using the same basic (25), the standard fitness of rr was 0.16 on Bt cotton and 0.46 on non-Bt cotton. AGRICULTURAL model, and to make the modeling results readily verifiable by readers. In sensitivity analyses, we also multiplied the fitness of rr by 1.25 on Bt cotton Furthermore, as reported previously (26), the results from this model match (0.16 × 1.25 = 0.20) and on non-Bt cotton (0.46 × 1.25 = 0.58), which tends to precisely with the results of Gould (30) and are similar to results from much make resistance evolve faster by reducing the negative effect of incomplete more complex models, such as the results of Gustafson et al. (31). The resistance on Bt cotton and by reducing the fitness cost on non-Bt cotton. In a standard values for parameters and the alternative values used in sensitivity complementary sensitivity analysis, we divided the fitness of rr by 1.25 on Bt analyses are listed in SI Appendix, Table S12. cotton (0.16/1.25 = 0.13) and on non-Bt cotton (0.46/1.25 = 0.37), which tends We assumed that resistance to Cry1Ac is controlled by two at a single to slow evolution of resistance by increasing the negative effect of incomplete autosomal locus: a recessive allele r conferring resistance and an allele s con- resistance on Bt cotton and by increasing the fitness cost on non-Bt cotton. ferring susceptibility. This assumption is based on previous analyses of pink bollworm resistance to Cry1Ac in many laboratory-selected strains from Ari- Host Plant Experiments: Larval Feeding in the Laboratory. We conducted larval zona and in a strain derived from three field-selected populations in India (32– feeding experiments to test a non-Bt variety of cotton (Simian3) and six other 35). Data reported here for a laboratory-selected resistant strain (AQ47) of crops common in the Yangtze River Valley as potential pink bollworm host plants: pink bollworm derived from the Yangtze River Valley also demonstrate au- okra, tomato, eggplant, cucumber, kidney bean, and apple. The following pro- tosomal recessive inheritance of resistance to Cry1Ac in bolls of Bt cotton, F1 cedures were followed for all crops except apple. The crops were planted at the hybrid cotton, and F2 hybrid cotton (SI Appendix, Table S18). In addition, Wuhan experimental farm of the Hubei Academy of Agricultural Sciences in mid- preliminary data indicate that in several strains of pink bollworm established April 2014. No insecticides were used throughout the growing season. In mid- recently from populations in the Yangtze River Valley, resistance to Cry1Ac August, for each crop, we collected >50 fruits (i.e., bolls, pods, other fruits), is conferred by mutations in the same cadherin gene that is associated with one to three fruits collected per plant. We bought apples from a super- with recessive resistance to Cry1Ac in pink bollworm from Arizona and India market and soaked them in water for 2 h to reduce potential residues. (32–36). We used the data from China reported here and the extensive, pre- For the fruits used in experiments, the approximate diameter was 2 cm for viously published empirical data on pink bollworm tested in greenhouse or cotton, and it was 7 cm for tomato and apple; the approximate length was 12 cm field experiments to estimate the relative fitness of each genotype on Bt for okra, 10 cm for eggplant, 25 cm for cucumber, and 15 cm for kidney beans. cotton and non-Bt cotton (25) (SI Appendix, Tables S12 and S18). To reduce the All fruits were rinsed with water in the laboratory, air-dried,andplacedinplastic influence of differences between strains unrelated to resistance, we estimated boxes (43 × 32 × 22 cm). The number of fruits per box was 34 for cotton and okra; fitness on non-Bt cotton using data only from experiments with resistant and 54 for tomato, eggplant, cucumber, and apple; and 108 for kidney bean. For susceptible strains that shared a common genetic background (i.e., the re- each of the seven crops, we tested three replicates with one replicate per box. sistant strain was derived from the susceptible strain via laboratory selection). We used a soft fine brush to put neonates (less than 1 h old) of the susceptible We assumed random mating among all adults and projected the change in QJ-S strain of pink bollworm on the fruits. The number of neonates per fruit was r allele frequency from one generation to the next as follows: five for cotton, okra, and kidney bean and 10 for the others. After inoculation, the boxes were closed and held at 29 ± 1°C,60± 10% relative humidity (RH), Δp = pqðp½Wrr − Wrs + q½Wrs − WssÞ=WM, [1] and 13 h light/11 h dark. One day later, we counted the number of entrance holes under a stereoscopic microscope. Five days after inoculation, we put soft where p and q are the frequencies of the r and s alleles, respectively; WM is paper into each box to facilitate pupation of larvae emerging from fruit. Larvae the population mean fitness; and Wrr,Wrs, and Wss are the fitnesses of rr, rs, emerged and pupated over a period of more than 2 wk. We stopped tracking and ss, respectively. Fitness for each genotype was calculated as follows: pupation after no larvae had emerged for 3 consecutive days from any fruit.

Wrr = BtWrr · PBt + RefWrr · PRef, [2] Host Plant Experiments: Larval Infestation in the Field. We measured in- festation of non-Bt cotton (Simian3), okra, and tomato by pink bollworm Wrs = BtWrs · PBt + RefWrs · PRef, [3] larvae in field plots at the Wuhan experimental farm of the Hubei Academy of Agricultural Sciences in 2014 and 2015. We planted the crops in a total of six Wss = BtWss · PBt + RefWss · PRef, [4] fields, consisting of the following three pairs of crops in adjacent fields:

where PBt is the proportion of Bt cotton plants and PRef is the proportion of cotton and okra, cotton and tomato, and okra and tomato. Within each pair, refuge (non-Bt cotton plants), and fitnesses for rr, rs,andss,respectively,are each crop was planted on 1.8 × 36 m and separated from the other crop in

BtWrr,BtWrs, and BtWss on Bt plants and RefWrr,RefWrs, and RefWss in the pair by 0.5 m. The distance between paired fields was 2 m. Crops were

Wan et al. PNAS | May 23, 2017 | vol. 114 | no. 21 | 5417 Downloaded by guest on September 27, 2021 planted in mid-April, and fruits were sampled six times: early, middle, and Insecticide Sprays Targeting Pink Bollworm. We obtained the data on insecticide late August and September. Each sample consisted of 100 randomly col- sprays targeting pink bollworm from the National Agricultural Technology Ex- lected fruits of each crop. The diameter was ∼2 cm for cotton bolls and 10 cm tension and Service Center, which has a network of plant protection stations that for tomatoes, and okra length was ∼12 cm. All samples were stored in plastic report these data. The number of sprays targeting pink bollworm in each of 14– boxes (43 × 32 × 22 cm) covered with 100-mesh gauze, and held in the 16 counties per year is a mean based on approximately 10 cotton fields per county. laboratory at 29 ± 1 °C, 60 ± 10% RH, and 13 h light/11 h dark. After 10 d, we checked the fruits for emergence holes, and then opened the fruits to check for Dominance of Resistance to Cotton Bolls Producing Cry1Ac in a Pink Bollworm live larvae. We calculated the total number of pink bollworm larvae per fruit as Strain from the Yangtze River Valley. We determined the dominance of pink the number of emergence holes plus the number of live larvae per fruit. bollworm resistance to Cry1Ac-producing bolls by testing four types of larvae derived from the Yangtze River Valley: resistant (AQ47 strain), susceptible × Host Plant Experiments: Adult Males Caught in Pheromone Traps. We used traps (QJ-S strain), F1 progeny from resistant females susceptible males, and F1 × baited with pink bollworm female sex pheromone (Pherobio Technology Co.) to progeny from susceptible females resistant males (additional details are catch male pink bollworm moths at field sites in four counties (provinces): Anqing provided in SI Appendix). We used survival on bolls containing Cry1Ac of the (Anhui), Anxiang (Hunan), Qianjiang (Hubei), and Wuxue (Hubei). Cotton, okra, resistant strain, the susceptible strain, and their F1 progeny to calculate and tomato were planted in each county, with roughly 50 km separating fields of dominance (h) of resistance (which ranges from 0 for completely recessive to the three different crops within a county (four counties × three field sites per 1 for completely dominant) as described previously (41). county = total of 12 field sites). At each of the 12 sites, three fields of the same crop were planted with one trap in each field (total of three traps per site × Data Analysis. We analyzed diet bioassay data with probit regression (42) to 12 sites = 36 traps). Males caught in each trap were counted daily from June determine LC50 values and their 95% fiducial limits, as well as slopes of the through September. For data analysis, the moths caught were totaled for each concentration-mortality lines. We calculated the resistance ratio as the LC50 for of the 12 traps per crop for each of the 4 mo of the field study. a strain divided by the LC50 for the susceptible QJ-S strain tested in the same year. We used Fisher’s exact test to determine if the proportion of populations with one or more survivors at the diagnostic concentration differed between Pink Bollworm Population Density. To evaluate pink bollworm population 2008–2010 and 2011–2015. We used the Mann–Whitney U test to determine if density, we used the number of first-generation pink bollworm eggs per percentage survival at the diagnostic concentration differed between 2008– 100 cotton plants. A total of 150 cotton plants per county were sampled every 4 d 2010 and 2011–2015. in 12–24 counties of six provinces of the Yangtze River Valley (Anhui, Hubei, Hunan, Jiangsu, Jiangxi, and Sichuan) during June and July each year from ACKNOWLEDGMENTS. We thank Yidong Wu for his thoughtful comments. 1995 to 2015. For each sampled plant, a thorough visual whole-plant survey was This work was supported by National Natural Science Foundation of China conducted to count the pink bollworm eggs, and each egg found was removed. Grants 31210103921 and 31321004, China’s Key Project for Breeding Genet- Pink bollworm females do not distinguish between Bt and non-Bt cotton for ically Modified Organisms Grant 2016ZX08012-004, and US Department of oviposition (40), and the type of cotton plant (Bt or non-Bt) was not recorded. Agriculture Risk Assessment Grant 2014-33522-22214.

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