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- Materials and methods
- Data archiving statement
- Literature cited
- Supporting Information
Evolution of resistance by insect pests threatens the long-term benefits of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Previous work has detected increases in the frequency of resistance to Bt toxin Cry1Ac in populations of cotton bollworm, Helicoverpa armigera, from northern China where Bt cotton producing Cry1Ac has been grown extensively for more than a decade. Confirming that trend, we report evidence from 2011 showing that the percentage of individuals resistant to a diagnostic concentration of Cry1Ac was significantly higher in two populations from different provinces of northern China (1.4% and 2.3%) compared with previously tested susceptible field populations (0%). We isolated two resistant strains: one from each of the two field-selected populations. Relative to a susceptible strain, the two strains had 460- and 1200-fold resistance to Cry1Ac, respectively. Both strains had dominant resistance to a diagnostic concentration of Cry1Ac in diet and to Bt cotton leaves containing Cry1Ac. Both strains had low, but significant cross-resistance to Cry2Ab (4.2- and 5.9-fold), which is used widely as the second toxin in two-toxin Bt cotton. Compared with resistance in other strains of H. armigera, the resistance in the two strains characterized here may be especially difficult to suppress.
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- Materials and methods
- Data archiving statement
- Literature cited
- Supporting Information
The insecticidal proteins of Bacillus thuringiensis (Bt) kill some major insect pests, but are harmless to vertebrates and most other organisms (Mendelsohn et al. 2003; Sanahuja et al. 2011; Pardo-López et al. 2013). Corn and cotton plants genetically engineered to produce Bt toxins have provided many benefits including pest suppression, reduced use of insecticide sprays, conservation of natural enemies, increased yield, and higher farmer profits (Wu et al. 2008; Carpenter 2010; Hutchison et al. 2010; National Research Council 2010; Tabashnik et al. 2010; Edgerton 2012; Kathage and Qaim 2012; Lu et al. 2012). Since 1996, farmers worldwide have planted transgenic crops producing Bt toxins on a cumulative total of more than 480 million ha, including 70 million hectares in 2012 (James 2012). The most widely used Bt proteins are crystalline (Cry) toxins, particularly three toxins that kill lepidopteran larvae: Cry1Ab in Bt corn, Cry1Ac in Bt cotton, and Cry2Ab in second-generation Bt corn and Bt cotton (Tabashnik et al. 2009b).
The primary threat to the continued efficacy of Bt toxins is evolution of resistance by pests (Tabashnik 1994; Gould 1998; Ferré and Van Rie 2002; Tabashnik et al. 2009b, 2013). Field-evolved (or field-selected) resistance is defined as a genetically based decrease in susceptibility of a population to a toxin caused by exposure of the population to the toxin in the field (Tabashnik et al. 2009b). Field-evolved resistance associated with reduced efficacy of Bt toxins has been reported in some populations of seven pest species: two targeted by Bt sprays (Tabashnik et al. 1990; Janmaat and Myers 2003) and five targeted by Bt crops (Luttrell et al. 2004; Van Rensburg 2007; Tabashnik et al. 2008, 2013; Storer et al. 2010; Dhurua and Gujar 2011; Gassmann et al. 2011). Other cases of significant decreases in susceptibility to the Bt toxins in transgenic crops including ‘incipient resistance’ and ‘early warning’ of resistance have been detected in at least four additional pest species (Downes et al. 2010; Alcantara et al. 2011; Zhang et al. 2011; Huang et al. 2012; Wan et al. 2012).
In particular, increases in the frequency of resistance to Cry1Ac have been reported in populations of the major cotton pest, cotton bollworm (Helicoverpa armigera), from northern China, where Bt cotton that produces Cry1Ac has been grown intensively for more than a decade (Liu et al. 2010; Zhang et al. 2011, 2012a). Decreased susceptibility to Cry1Ac in populations of H. armigera from China has been documented with monitoring data from a number of studies based on comparisons over time within populations exposed intensively to Bt cotton and between populations that differ in their history of exposure to Bt cotton (Wu et al. 1999; Li et al. 2007, 2010; Yang et al. 2007; An et al. 2010; Liu et al. 2010; Zhang et al. 2011, 2012a). Nonetheless, the maximum percentage of resistant individuals reported in a population is 2.6% (compared with 0% for susceptible populations), and Bt cotton producing Cry1Ac has continued to provide substantial control of this pest in China (Zhang et al. 2011). Thus, the small but statistically significant increases in the frequency of resistance noted above provide an early warning of resistance that could become a more serious problem (Zhang et al. 2011).
The main strategy for delaying evolution of pest resistance to Bt crops relies on refuges of host plants that do not produce Bt toxins, which promotes survival of pests susceptible to Bt toxins (Gould 1998; Tabashnik et al. 2004). Ideally, most of the rare resistant pests surviving on Bt crops will mate with the relatively abundant susceptible pests from nearby refuges. If inheritance of resistance is recessive, the progeny from such matings will die on Bt crops, substantially delaying the evolution of resistance. Conversely, if inheritance of resistance is dominant, the progeny from matings between resistant and susceptible adults will survive on Bt crops, and refuges will be less effective for delaying resistance.
The refuge strategy has been used with first generation Bt plants that produce a single Bt toxin and with more recently introduced Bt crop ‘pyramids’ that produce two or more Bt toxins that kill a given pest (Zhao et al. 2005; Brévault et al. 2013). Pyramids are expected to be most effective for delaying evolution of resistance if adaptation to one toxin in the pyramid does not cause cross-resistance to the other toxin(s) in the pyramid (Zhao et al. 2005; Tabashnik et al. 2009a; Brévault et al. 2013). Whereas Bt cotton producing only Cry1Ac is grown in China, second-generation Bt cotton plants producing toxins Cry1Ac and Cry2Ab are grown in Australia, India, and the United States (Tabashnik et al. 2013).
Because the refuge and pyramid strategies for delaying resistance require understanding of the dominance of resistance and cross-resistance, we evaluated these traits in two resistant strains of H. armigera isolated from populations in two provinces of northern China where Bt cotton has been grown extensively for more than a decade. We quantified dominance of resistance with the parameter h, which varies from 0 for completely recessive resistance to 1 for completely dominant resistance (Liu and Tabashnik 1997). Whereas h for resistance to a diagnostic concentration of Cry1Ac ranged from 0 to 0.66 (mean = 0.19) in 14 previously studied strains of H. armigera (Kaur and Dilawari 2011; Zhang et al. 2012a,b), it was 1 for both strains analyzed here, indicating completely dominant resistance. Also in contrast to previous results for H. armigera (Akhurst et al. 2003; Xu et al. 2005; Luo et al. 2007; Liang et al. 2008; Yang et al. 2009; Zhang et al. 2012b), both resistant strains had minor but significant cross-resistance to Cry2Ab.
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The results from this study are consistent with previous studies reporting evidence of field-evolved resistance of H. armigera to Cry1Ac in northern China (Li et al. 2007; Liu et al. 2010; Zhang et al. 2011, 2012a). Survival at the diagnostic concentration of Cry1Ac was significantly higher in 2011 (this study) for Anyang (2.3%) and Qiuxian (1.4%) from northern China compared with two previously tested populations from northwestern China that had limited exposure to Bt cotton (0%, 0 of 1296, Zhang et al. 2011) (Fisher's exact test, P < 0.0001 for Anyang and Qiuxian tested separately). For Anyang, survival at the diagnostic concentration in 2011 was significantly higher than in 2005 (1.2%, 123 of 9984, Yang et al. 2007) (chi-squared = 10.7, df = 1, P = 0.001), but did not differ significantly from 2010 (2.6%, 33 of 1248, Zhang et al. 2011) (Fisher's exact test, P = 0.62). For Qiuxian, survival at the diagnostic concentration increased significantly from 2010 (0.2%, 2 of 888, Zhang et al. 2011) to 2011 (Fisher's exact test, P = 0.003).
The two resistant strains of H. armigera from northern China analyzed here, AY2 from Anyang and QX7 from Qiuxian, had dominant resistance to a diagnostic concentration of Cry1Ac in diet (h = 1.0 for both strains) and dominant resistance to leaves of Bt cotton containing Cry1Ac (h = 0.94 and 0.97, respectively). Assuming that at least one dominant resistance allele occurred in each set of individuals sampled from the field during 2011 to start each strain, we estimate the minimum frequency of individuals carrying a dominant resistance allele was 0.0045 for Anyang (1 of 222 field-collected moths) and 0.0056 for Qiuxian (1 of 178 field-collected moths). Because the frequency of individuals with resistance to Cry1Ac conferred by all alleles in 2011 was 0.023 for Anyang and 0.014 for Qiuxian (Results), we estimate the minimum percentage of resistant individuals carrying the dominant resistance alleles detected here as 20% for Anyang (0.0045/0.023) and 40% for Qiuxian (0.0056/0.014).
AY2 and QX7 had resistance ratios for Cry1Ac of 1200 and 460, as well as cross-resistance to Cry1Aa (>260- and 100-fold, respectively), Cry1Ab (69- and 34-fold, respectively), and Cry2Ab (5.9- and 4.2-fold, respectively) (Tables 1 and 2). For two other strains of H. armigera with high levels of resistance to Cry1Ac based on cadherin mutations (540-fold for SCD-r1 and 140-fold for XJ-r15), the magnitude of cross-resistance was lower to each toxin (Yang et al. 2009; Zhang et al. 2012b). However, as seen with AY2 and QX7, cross-resistance of SCD-r1 and XJ-r15 was highest to Cry1Aa (41- and 27-fold, respectively), intermediate to Cry1Ab (31- and 6.3-fold), and lowest to Cry2Ab (1.2- and 1.4-fold) (Yang et al. 2009; Zhang et al. 2012b).
Among 26 Cry1Ac-resistant strains of H. armigera from Australia, China, India, and Pakistan (Table 3), AY2 and QX7 have the resistance traits that appear to be most difficult to suppress. The resistance ratios for these two strains are among the highest (median for 22 other strains = 120-fold, range = 13–5400). The dominance (h) of resistance to Cry1Ac for AY2 and QX7 either at a diagnostic concentration in diet or in Bt cotton leaves containing Cry1Ac is higher than any reported previously (range of h for other strains = 0–0.66). Based on LC50 values, h was 0.85 for AY2 and 0.83 for QX7, which is similar to the maximum reported for 13 other strains (median = 0.39, range = 0–0.85). In contrast to the significant cross-resistance to Cry2Ab in AY2 and QX7 (Table 2), cross-resistance to Cry2Ab or Cry2Aa was not significant for any of the seven previously analyzed Cry1Ac-resistant strains of H. armigera considered individually (Table 3). However, the cross-resistance ratio for Cry2Ab or Cry2Aa was greater than one in eight of nine Cry1Ac-resistant strains of H. armigera (Table 2, median = 1.4, range = 1.0–5.9). Overall, for these nine strains, selection with Cry1Ac significantly decreased susceptibility to the Cry2A toxins (signed-rank test, one-tailed P < 0.005). Moreover, including the data for strains AY2 and QX7 reported here and the 21 selection experiments with H. armigera and seven other species of lepidopteran pests reviewed by Brévault et al. (2013), the cross-resistance ratio between Cry1A and Cry2A toxins was greater than one in 20 of 23 cases (median = 1.6, range = 0.32–420), with significant cross-resistance detected when all of the data are considered collectively (signed-rank test, one-tailed P = 0.0003).
Table 3. Resistance to Cry1Ac and cross-resistance to Cry2Ab in Cry1Ac-selected strains of H. armigera.
|Country (regiona)||Location||Yearb||Strain||RRc||Dominance (h) d||Cry2Ab CRRh||References|
|Cry1Ac (DCe)||Cry1Ac (LC50f)||Bt cottong|
|China (N)||Anyang||2009||AY9||88||0.00|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY16|| ||0.00|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY27|| ||0.00|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY148|| ||0.00|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY440||47||0.04|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY335||89||0.13|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY-r15||82||0.33||0.63|| || ||Zhang et al. (2012b)|
|China (N)||Anyang||2009||AY423||660||0.64|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2009||AY441||95||0.66|| || || ||Zhang et al. (2012a)|
|China (N)||Anyang||2011||AY2||1200||1.0||0.85||0.94||5.9||This paper|
|China (N)||Gaoyangi||2001||SCD-r1||440|| ||0.00|| ||1.2j||Yang et al. (2009)|
|China (N)||Gaoyangi||2001||SCD-r1||540||0.00||0.04|| || ||Zhang et al. (2012b)|
|China (N)||Gaoyang||2001||GYBT||560|| ||0.24|| ||1.4i||Xu et al. (2005)|
|China (N)||Langfang||2000||LFR10||250k|| || || ||1.0k||Luo et al. (2007)|
|China (N)||Qiuxian||2011||QX7||460||1.0||0.83||0.97||4.2||This paper|
|China (N)||Xiajin||2009||XJ-r15||140||0.65||0.68|| ||1.4||Zhang et al. (2012b)|
|China (N)||Xinxiang||1996||BtR||3000k|| ||0.28|| ||1.1k||Luo et al. (2007), e Liang et al. (2008)|
|China (NW)||Shache||2010||SC23||39||0.00|| || || ||Zhang et al. (2012a)|
|China (NW)||Shawan||2010||SW34||31||0.26|| || || ||Zhang et al. (2012a)|
|Australia||Mixedl|| ||BX||260|| ||0.39||0.00, 0.63m||1.4||Akhurst et al. (2003), Bird and Akhurst (2004, 2005)|
|India||Akola|| ||Cry1Ac- resistant||72|| || || ||1.1||Rajagopal et al. (2009)|
|India||Gujarat||2002||Res-Bt||93|| ||0.42||0.43|| ||Kranthi et al. (2006)|
|India||Gujarat||2006||BH-R||230|| ||0.85n|| || ||Nair et al. (2010)|
|India||Nagpur||2002||Res-Ac||210|| ||0.56|| || ||Kranthi et al. (2006)|
|India||Punjab||2005||BM-R||72||0.00o||0.31|| || ||Kaur and Dilawari (2011)|
|India||Tamil Nadu|| ||BCR||13|| ||0.37|| || ||Shanmugam et al. (2007)|
|Pakistan||Punjab||2010||Cry1Ac- SEL||5400p|| ||0.59|| || ||Alvi et al. (2012)|
Under selection for resistance, allele frequency is expected to increase faster for dominant resistance alleles than for recessive resistance alleles (Carrière and Tabashnik 2001). However, if dominant fitness costs are associated with the dominant resistance alleles found here, such costs could substantially slow the increase in these dominant alleles (Carrière and Tabashnik 2001). Dominant fitness costs would be especially effective for delaying resistance in this case because a high proportion of the host plants of H. armigera in northern China are crops other than cotton that do not produce Bt toxins and thus may act as refuges (Wu and Guo 2005). For example, from 2000 to 2006, Bt cotton accounted for a mean of only 7.5% of the total area planted to host plants of H. armigera each year in northern China (Wu et al. 2008).
Although complete assessment of costs in AY2 and QX7 will require additional work, some evidence suggests that a dominant fitness cost occurs in these strains. Both AY2 and QX7 had completely dominant resistance to Bt toxin Cry1Ac (h = 1.0) based on evaluations made after 10 successive generations of laboratory selection with Cry1Ac. However, for both strains, survival was only 33% for the single-pair F2 progeny produced from the initial matings between one resistant F1 male survivor from the diagnostic concentration test and one female from the susceptible strain SCD (Fig. 1). With the results from both strains pooled (n = 96), the 33% observed survival is lower than the 50% survival expected with completely dominant resistance (Fisher's exact test, P = 0.028), assuming that the resistant male was a heterozygote (Rs), so that each cross (Rs × ss) is expected to yield 50% Rs that survive and 50% ss that die. The lower than expected survival could have been caused by a dominant fitness cost that reduced the proportion of Rs individuals from the mating between the Rs male and the ss female that became second instars and were tested in the bioassays.
Mortality of both Rs and ss individuals caused by factors other than Cry1Ac also could have contributed to the lower than expected survival. Another possibility is that dominance increased during the subsequent 10 generations of selection, which could have been mediated by modifiers at one or more loci other than the primary resistance locus or by replacement of the initial resistance allele by a more dominant resistance allele at the same locus. However, genetic variation was limited within strains because each strain was started with a single resistant male and a single female from a susceptible laboratory strain. In a previous study with the laboratory-selected BtR strain of H. armigera, Liang et al. (2008) reported a slight decrease in dominance as resistance increased during 87 generations of selection.
Whereas survival of the susceptible strain SCD was 9% in bioassays with leaves from China's popular GK19 variety of Bt cotton (Fig. 2 and Table S4), field data from 2001 and 2002 show that survival of larvae from susceptible populations of H. armigera on GK19 cotton was 8.2–18% (Wan et al. 2005). Thus, survival of the SCD strain in bioassays with GK19 cotton leaves was within the range of survival of susceptible field populations on GK19 plants in the field, which suggests that results from this bioassay are relevant to the field. Our leaf bioassays lasted only 5 days, which could boost survival relative to survival for longer periods required for complete larval development in the field. On the other hand, H. armigera larvae in the field eat a variety of plant parts, some of which have a much lower concentration of Cry1Ac than leaves, which could raise survival in the field relative to the leaf bioassays. In addition, in the field, the concentration of Cry1Ac declines in Bt cotton plants as they age (Wan et al. 2005), which increases survival of H. armigera larvae (Bird and Akhurst 2004, 2005). In greenhouse experiments with 15-week-old Bt cotton plants producing Cry1Ac, survival from neonate to adult was 62% for an H. armigera strain with a Cry1Ac resistance ratio of 97–440, 0% for a susceptible strain, and 39% for the F1 progeny of the resistant and susceptible strain, which had a Cry1Ac resistance ratio of 2–4 (Bird and Akhurst 2004, 2005). Similar to previous results with the Cry1Ac-selected Res-Bt strain of H. armigera from India (Kranthi et al. 2006) (Table 3), the results here with AY2 and QX7 show that dominance of resistance to Cry1Ac was similar whether measured in diet bioassays with Cry1Ac or in bioassays using Bt cotton leaves producing Cry1Ac (Figs 2 and 4, Table 3).
The high levels of resistance to Cry1Ac (1200- and 460-fold) and lower but significant cross-resistance to Cry2Ab (5.9- and 4.2-fold) of AY2 and QX7 raise concern about their potential resistance to two-toxin Bt cotton producing Cry1Ac and Cry2Ab. In bioassays with Bt cotton leaves containing Cry1Ac and Cry2Ab, survival was 13 times higher for the Cry1Ac-selected Res-Bt strain of H. armigera (32%) relative to a susceptible strain (2.4%) (Rajagopal et al. 2009), even though Res-Bt had only 72-fold resistance to Cry1Ac and no cross-resistance to Cry2Ab (Rajagopal et al. 2009). Similar results with the closely related pest species Helicoverpa zea show that survival from neonate to adult on Bt cotton producing Cry1Ac and Cry2Ab was 11 times higher for the Cry1Ac-selected GA-R strain (6.7%) relative to its unselected parent strain (0.6%), even though resistance of GA-R relative to GA was only 10-fold to Cry1Ac and twofold to Cry2Ab (Brévault et al. 2013).
While Cry1Ac is the only Bt toxin produced by transgenic cotton grown in China, two-toxin Bt cotton producing Cry1Ac and Cry2Ab has become the sole type of Bt cotton grown in Australia and the predominant type of Bt cotton grown in India and the United States (Tabashnik et al. 2013). An immediate switch in China to two-toxin Bt cotton producing Cry1Ac and Cry2Ab would probably slow the evolution of resistance to Bt cotton in H. armigera and in another major lepidopteran pest, Pectinophora gossypiella (Tabashnik et al. 2012). However, considering the increasing frequency of resistance of H. armigera in China to Cry1Ac and the concerns about an associated potential increase in survival on Bt cotton producing Cry1Ac and Cry2Ab described above, a shift to Bt cotton producing a toxin other than Cry1Ac or Cry2Ab could be particularly useful in China (Zhang et al. 2011).
Bt toxin Vip3Aa, which has no structural homology to Cry toxins (Estruch et al. 1996), is promising for controlling H. armigera populations (An et al. 2010; Mahon et al. 2012). Commercial varieties of three-toxin Bt cotton producing Vip3A, Cry1Ac, and Cry2Ab are under development, with availability in Australia and the United States expected in 2016 (Mahon et al. 2012). Susceptibility was not correlated between Cry1Ac and Vip3Aa within the Anci and Xiajin populations of H. armigera from northern China, and susceptibility was negatively associated between Cry1Ac and Vip3Aa across these two populations (An et al. 2010). In two Australian strains of H. armigera highly resistant to Vip3Aa, the mean LC50 of Cry1Ac was similar to that of a susceptible strain, while the mean LC50 of Cry2Ab was about fivefold lower than for a susceptible strain (Mahon et al. 2012). The frequency of recessive alleles conferring resistance to Vip3A was estimated as 0.008 in Australian populations of H. armigera, providing an indication of the potential for evolution of resistance to this toxin (Mahon et al. 2012). In addition to increasing the number and diversity of toxins in Bt cotton, integration of Bt cotton with other control tactics could help to delay the evolution of resistance and provide a more sustainable pest management system (Tabashnik et al. 2010).