The first report of field-evolved resistance to a Bt crop was published in 2006 and it involved populations of Helicoverpa zea collected in the US in 2003-2004 in Bollgard® cotton, which showed greatly reduced susceptibility to the toxin Cry1Ac produced by the Bt crop (Ali et al., 2006). From that moment and up to the date, over 15 cases of practical resistance, which according to Tabashnik et al.
(2014) is “field-evolved resistance that reduces pesticide efficacy and has practical consequences for pest control”, have been reported worldwide (Tabashnik and Carrière, 2017). Additionally, there are other cases in which resistance alleles have been detected in the field, but control failure has not occurred (Huang et al., 2012; Zhang et al., 2014; Jin et al., 2015). Reported cases of resistance mainly involve Bt maize and single-toxin events. Resistance has been reported in populations of seven pest species, including six Lepidoptera and one Coleoptera species. It is noteworthy to mention that four out of the six lepidopteran species in which resistance has been detected are noctuids. The first report of field-evolved resistance to Bt maize expressing Cry1Ab protein
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corresponded to populations of Busseola fusca from South Africa (Van Rensburg, 2007). Field populations of Spodoptera frugiperda resistant to Bt maize that expresses the toxin Cry1F were detected in Puerto Rico, Brazil and continental United States (US) just a few seasons after Bt maize hybrids were first deployed (Storer et al., 2010; Farias et al., 2014a; Huang et al., 2014). More recently, field-evolved resistance to Bt sweet maize that expresses different Cry toxins was reported in Helicoverpa zea populations from the US (Dively et al., 2016).
Spodoptera frugiperda is the only species in which resistance has been detected in different countries (insular and continental US, Brazil and Argentina), whereas resistance to different Bt toxins has been also reported in H. zea, Diabrotica virgifera virgifera and Pectinophora gossyipiella (Table 1.2).
Resistance evolution has often been associated with poor compliance of the requirements of the HDR strategy (Table 1.2). In some countries farmers have failed to plant a proportion high enough of refuges or they have failed to plant them as instructed, either by placing them too far from the Bt crop, by not making them as desirable as their transgenic counterpart (for instance by not irrigating them) or by spraying them excessively with insecticides to prevent economic damage, thus killing susceptible individuals (Van Rensburg, 2007; Dhurua and Gujar, 2011; Farias et al., 2014a; Dively et al., 2016; Omoto et al., 2016; Grimi et al., 2018). In most cases of field-evolved resistance, the Bt varieties deployed did not represent a high-dose against the target pest, which sometimes was coupled with non-recessive inheritance of resistance (Gassmann et al., 2011). On the other hand, the climate of some areas along with the cropping regimes adopted could have played a role in some cases (Van Rensburg, 2007; Dhurua and Gujar, 2011;
Kranthi, 2015). Thus, in tropical regions where some pests can be found throughout the year with overlapping generations and multiple Bt crops are grown every season, the selective pressure exerted on pests is huge and can lead to early resistance development (Storer et al., 2010; Farias et al., 2014a; Kranthi, 2015;
Omoto et al., 2016). Additionally, cross-resistance has been suggested to contribute to resistance evolution in some cases in which a pest became resistant to several Bt toxins in a narrow time period (Dively et al., 2016; Omoto et al., 2016; Zukoff et al., 2016; Grimi et al., 2018).
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Table 1.2. Reported cases of field-evolved practical resistance. Each case involves a pest species, a Bt toxin and a specific location. (Modified from Tabashnik and Carrière, 2017)
(Lepidoptera: Noctuidae) Maize Cry1Ab South
Africa 8 1, 2, 3, 8 Van Rensburg, 2007; Campagne et al., 2013 Diatraea saccharalis
(Lepidoptera: Crambidae) Maize Cry1F Argentina 4 3 Grimi et al., 2018
Cry1A.105 Argentina 4 3, 5 Grimi et al., 2018
(Lepidoptera: Gelechiidae) Cotton Cry1Ac India 6 1, 3 Dhurua and Gujar, 2011; Mohan et al., 2016;
Nair et al., 2016
Cry2Ab India 8 3, 4, 7, 8 Fabrick et al., 2015; Kranthi, 2015
Striacosta albicosta
(Lepidoptera: Noctuidae) Maize Cry1Fa US 10 1 Eichenseer et al., 2008; DiFonzo et al., 2016;
Ostrem et al., 2016
a Number of years elapsed between the year when the Bt variety expressing the toxin was marketed and the moment when resistance was first detected.
b Factors classified as: 1= Not high dose; 2= Non-recessive inheritance; 3= Low refuge compliance; 4= Tropical conditions allowing multiple crops per season and overlapping generations of the target pest; 5= Cross-resistance between toxins (suspected or known); 6= Delayed development in resistant individuals leading to assortative mating; 7=
Deployment of pyramids containing a toxin previously used in the area; 8= Agricultural practices favoring resistance development.
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1.7. Objectives
The general aim of this thesis is to optimize the ongoing IRM strategies of Bt maize concerning three noctuid pests of this crop: the primary pest Sesamia nonagrioides, the secondary pest Mythimna unipuncta, and a potentially invasive pest in the EU, Spodoptera frugiperda.
More specifically, we have addressed the following objectives:
To assess population variation in the susceptibility to the Cry1Ab toxin of S.
nonagrioides in the Ebro Valley and of M. unipuncta in areas with high-adoption (Ebro Valley) or no-adoption (Galicia) of Bt maize.
To evaluate the performance of S. nonagrioides on cultivated and wild host plants and its potential implications for Bt maize resistance management.
To assess the frequency of resistance alleles to Bt maize in Spanish populations of S. nonagrioides using an F2 screen.
To investigate the genetic basis of resistance to Bt maize in two populations of S. frugiperda resistant to Cry1F maize.
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2.1. Introduction
Spain is the only European country where transgenic maize expressing the protein Cry1Ab (Bt maize) has been grown continuously since it was first commercialized in 1998, accounting for 91% of the total Bt maize area in the EU in 2017 (ISAAA, 2017). From 1998 to 2013 adoption of Bt maize increased steadily in Spain, and since that year it has stabilized at around 25-30% of all maize grown in the country (estimated from the data available at http://www.mapama.gob.es/en/), with regional differences in adoption (Fig. 1.12B). Continued exposure to the high levels of toxin expressed by the plants exerts a strong selective pressure on pests and could lead to resistance development (Tabashnik, 1994; Gould, 1998), which would threaten the efficacy of Bt maize in suppressing pest damage. The high and continued use of Bt maize in the Ebro Valley and the presence of 2-3 generations per year of the target pests Sesamia nonagrioides and Ostrinia nubilalis (González-Nuñez et al., 2000) render this area as the only hotspot in Europe where resistance has a higher probability of evolving (EFSA Panel on GMO, 2012).
In the European Union (EU), the approval of a genetically modified plant for commercial cultivation is subject to regulation at EU level. Thus, according to the Directive 2001/18/EC and the Regulation (EC) 1829/03, post-market environmental monitoring (PMEM) is a mandatory requirement for commercial release of GM crops (EC, 2001; 2003). With regards to Bt maize, one of the objectives of the PMEM is to assess the potential resistance development of target pests’ populations to the protein Cry1Ab expressed in the GM crop, as a consequence of the high selection pressure these populations are subjected to in the field (MARM, 2010). With this purpose, an insect resistance monitoring program was implemented across the EU in order to detect, in a timely manner, shifts in susceptibility of the target pests to the Bt protein that could be indicative of resistance development. This program establishes that monitoring efforts should focus in areas where the ecology of the pest and the high adoption rates of Bt maize make resistance more likely to develop (EFSA, 2015).
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A post-market resistance monitoring program for Bt maize was initiated in Spain in 1998 (MARM, 2010). This program allowed for the establishment of the baselines of susceptibility to Cry1Ab toxin of Spanish populations of S.
nonagrioides and O. nubilalis from the most representative regions where Bt maize is grown and several generations of these pests are present per year: Northeast Spain (Ebro Valley), Central Spain and Southwest Spain (González-Núñez et al., 2000).
Subsequently, between 1999 and 2015, resistance monitoring included biannual samplings in the aforementioned Bt maize growing regions. In each of the three zones, susceptibility of field populations to the protein Cry1Ab was estimated by dose-response bioassays, and the values obtained were compared with baseline susceptibility values determined earlier in the same areas and with the susceptibility of a control laboratory strain, in order to evaluate whether significant changes in this parameter were taking place (EFSA, 2015; Farinós et al., 2018). The results indicated that susceptibility of both S. nonagrioides and O. nubilalis to the protein Cry1Ab has not decreased over time either in the Ebro Valley or in any other areas of Spain between 1999 and 2015 (Farinós et al., 2004; 2011; 2018; Castañera et al., 2016; Thieme et al., 2017). More recently, the regulatory agency of the EU has stated the need to improve resistance monitoring strategies in an attempt to lower detection limits of resistance alleles from frequencies of 5% to 3% (EFSA Panel on GMO, 2016). Gaining knowledge on the smaller-scale variation of susceptibility to Cry1Ab protein in the Ebro Valley, the area in the EU where resistance is more prone to develop, would help optimize sampling strategies.
The secondary noctuid pest Mythimna unipuncta has shown a much lower susceptibility to the toxin Cry1Ab expressed in MON 810 Bt maize cultivars. This species has been reported to attack maize fields in the Ebro Valley sporadically causing important yield losses (López et al., 2000; Eizaguirre et al., 2010).
Moreover, it was observed that a small percentage of larvae of this pest can survive and complete their development MON 810 maize (Eizaguirre et al., 2010;
González-Cabrera et al. 2013). These findings indicate that the concentration of Cry1Ab expressed by transgenic maize hybrids represents a “low dose” scenario for M. unipuncta, favouring the potential development of resistance in field populations (Pérez-Hedo et al., 2012; González-Cabrera et al., 2013). Additionally,
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they hinted at the risk of resistance evolution in field populations of M. unipuncta and the importance of taking this species into consideration in Bt maize resistance monitoring programs (González-Cabrera et al., 2013; García et al., 2015). To our knowledge, no studies have evaluated the susceptibility to Cry1Ab protein of M.
unipuncta populations from areas where Bt maize has never been grown commercially. Obtaining information on the variation in susceptibility to Cry1Ab protein between areas of high adoption and areas with no history of Bt maize cultivation would help determine whether the high selective pressure that populations from the Ebro Valley are subjected to could have led to lower susceptibility to Cry1Ab protein in comparison with populations from areas where Bt maize has never been sown.
This chapter has two main objectives. The first one is to study the variation in the susceptibility to Cry1Ab protein between (interpopulation variation) and within (intrapopulation variation) populations of the target pest S. nonagrioides from the hotspot area of the Ebro Valley, in order to optimize sampling strategies and to improve the resistance monitoring plan by determining whether it should cover large areas or focus in smaller zones where susceptibility is lower. The second objective is to discern whether the susceptibility to Cry1Ab protein varies between field populations of M. unipuncta subjected to high or no selective pressure of Bt maize, so as to learn whether this pest could become a threat to the sustainability of the Bt crop.