Since their introduction in 2013, genetically modified soybeans (Glycine max [L.] Merr.) expressing the Cry1Ac Bacillus thuringiensis (Bt) protein and glyphosate tolerance (i.e., transgenic events MON87701 and MON89788) have increased crop yields while controlling major pests and improving weed management practices (Monsanto 2019; Horikoshi 2021b). The Americas account for 87% of global soybean production exerting unparalleled influence over the industry (FAOSTAT 2022). Notably, Argentina ranks as the third-largest global soybean producer, where transgenic varieties cover 97% of the crop acreage, 28% being Bt soybeans (SISA 2022). This large-scale adoption causes intense selection pressure on target pest populations, especially in the northern of the country, and can lead to shifts in the associated pest community with the resurgence of primary pests as well as secondary pest outbreaks (Martins-Salles et al. 2017; Páez Jerez et al. 2023). Hence, research efforts to develop effective insect resistance management (IRM) programs remain a priority.
Lepidoptera, mostly noctuid species, are major insect pest complexes in South American soybeans (Scoble 1992; Bradshaw et al. 2016; Suckling et al. 2017). These insects have contrasting inherent susceptibility to Cry1Ac and can be consequently placed in two main categories as follows: a) target species, such as Anticarsia gemmatalis Hübner, Rachiplusia nu (Guenée), Chrysodeixis includens (Walker), Helicoverpa gelotopoeon Dyar, Crocidosema aporema (Walsingham), Spilosoma virginica Fabricius, Colias lesbia (Fabricius), Heliothis virescens (Boddie) and Loxostege bifidalis (Fabricius); and b) non-target species, such as those belonging to the Spodoptera genus. The Plusiinae subfamily, including the sunflower looper (R. nu) and soybean looper (C. includens), is particularly noteworthy for its widespread presence in soybean fields throughout the Southern Cone and certain regions of Brazil (Perini et al. 2021). Their larvae feed on several high-value crops, such as soybean, sunflower (Helianthus annus L.), alfalfa (Medicago sativa L.), cotton (Gossypium hirsutum L.), and various aromatic plants (Eichlin and Cunningham 1978; Lafontaine and Poole 1991). Larvae of these species cause defoliation in different soybean growth stages, particularly the early reproductive stages, when flowering and pod initiation have started (Murúa et al. 2018; Páez Jerez et al. 2023).
One practical means to safeguard global agricultural production is to minimize the losses by arthropod pests, often applying curative control measures. The development of pest resistance to control measures is a significant global challenge that raises several concerns, including increased risk of pest resurgence, management costs and other unintended effects on beneficial insects, such as natural enemies and pollinators, as well as shifts in pest species dominance, status, and pesticide susceptibility with overall ecosystem stress (Han et al. 2019; Desneux et al. 2022). Biorational selective insecticides are used increasingly in soybean fields, but these modern tools are also vulnerable to the risks of the evolution of resistance in the target organisms (Andow and Zwahlen 2006). Proposed resistance management strategies in transgenic Bt crops include maintaining refuge host plants and using high-dose Bt traits pyramided in Bt cultivars (Reisig and Kurtz 2018). The refuge consists of non-Bt host plants to reduce the high pressure on susceptible individuals, allowing susceptibility alleles to persist in the population. In South America, where Brazil and Argentina concentrate 57% of the global soybean production, an in-field refuge modality of 20% is recommended (Monsanto 2019) despite low adoption by growers maybe because the pest pressure in refuges is high (Páez Jerez et al. 2023) and growers are generally risk-averse. As a result, the crop management can be complex and fields often receive insecticide applications, rendering the refuge ineffective to delay the selection of resistant pest populations (Horikoshi et al. 2021a).
The effectiveness of the refuge strategy depends on various aspects of pest biology, as highlighted by previous studies (Carrière et al. 2015, 2016; Tabashnik and Carrière 2017; Dively et al. 2019). For instance, if adults of the target pest species prefer to oviposit on Bt plants rather than on non-Bt ones, this preference may increase the rate of Bt resistance evolution by increasing the proportion of the population under selection pressure (Téllez-Rodríguez et al. 2014). While some studies with lepidopteran species targeted by Bt crops have shown that moths do not discriminate between Bt and non-Bt plants (Kumar 2004; Van den Berg and Van Wyk 2007; Hardke et al. 2012; Sun et al. 2013; Gonçalves et al. 2020), another study (Téllez-Rodríguez et al. 2014) suggests that Spodoptera frugiperda (J.E. Smith) has a strong oviposition preference for Bt over non-Bt maize. Although the oviposition pattern in polyphagous insect herbivores is typically non-selective (Ramaswamy 1988), this behavior in soybean pests could compromise the efficacy of the refuge strategy and lead to faster evolution of resistance to Bt soybean.
In 2020, one case of field-evolved resistance to MON 87701 × MON 89788 soybean was reported for R. nu and C. aporema populations in Brazil (Horikoshi et al. 2021b). Field resistance to Bt occurs when a target pest develops a genetically mediated ability to feed and complete its development on one or more commercial lines of a Bt crop under field conditions (Moar et al. 2008). Insecticide resistance can surge in a population not only through exposure to lethal concentrations that eliminate susceptible individuals, but also through sublethal exposure levels that enhance the survival and reproduction of the resistant population (Guedes et al. 2017). If larvae that are not susceptible to Bt toxin move from Bt plants to non-Bt plants or larger larvae that are less susceptible move from non-Bt plants to Bt plants, it could increase the selection for resistance leading to the survival of heterozygous individuals and a higher rate of resistance evolution (Erasmus et al. 2016). Ingestion of sublethal doses of Bt has various effects on lepidopteran larvae. For instance, it can cause rapid paralysis of the gut and cessation of feeding, leading to decreased larval feeding rates, reduced larval weights, and delays in larval development (Hesketh and Hails 2015). Non-Bt plants could also produce reduced numbers of susceptible moths if susceptible larvae moved from non-Bt plants to Bt plants, decreasing refuge efficiency. This reduction should impact primarily Bt plants with single proteins and highly mobile lepidopteran larvae (Carroll et al. 2012). While resistance cases have not yet been reported in Argentinian populations of R. nu, concerns have been raised due to the occurrence of large numbers of individuals in Bt-fields reported by growers (Szwarc et al. 2022). For C. includens, field populations have shown high susceptibility to Cry1Ac toxin (Yano et al. 2016; Horikoshi et al. 2021a), and resistance has not been recorded. However, larvae of the soybean looper are abundant in non-Bt soybean fields and refuges, making this species subject to high selection pressure from Cry1Ac soybeans and at risk of developing field resistance to Cry1Ac if adequate resistance management practices are not implemented.
Evolutionary and ecological factors often co-determine the dynamics of ecological communities (Fussmann et al. 2007). For instance, the number of generations per crop season and the fitness costs of insecticide resistance can affect the frequency of resistance alleles in pest populations (Higginson et al. 2005; Gassmann et al. 2009). These parameters are crucial for predicting the lifetime of a Bt trait and the need for locally-adapted control measures to manage new pest outbreaks. Furthermore, the development of pest resistance may increase the use of broad-spectrum synthetic insecticides, leading to higher economic and environmental costs of pest control. The fall armyworm, for example, evolved practical resistance to single-toxin Bt maize in only three years after its introduction, resulting in the withdrawal of the Bt technology from the market in Puerto Rico (Huang 2021). For major soybean-producing countries, such as Argentina, Brazil, Paraguay, and Uruguay, surges in pest resistance to Bt soybean could have substantial economic and ecological consequences.
In this study, we aimed to test the hypothesis that some populations of R. nu in Argentina have developed reduced susceptibility to Cry1Ac Bt protein due to intense selection pressure from 10 years of Bt soybean usage. Initially, we collected larvae from Bt and non-Bt soybean fields with greater-than-expected plant damage by the plusiine R. nu and C. includens. Subsequently, we conducted concentration-response bioassays to determine the susceptibility of the larvae to purified Cry1Ac protein. We also assessed the sublethal effect of consuming Cry1Ac over some time on survival, larval stage duration, weight, and pupae formation. Finally, we investigated the oviposition preference of the sunflower looper moths on non-Bt and Bt soybean plants or their foliage. We discuss the implications of our findings for insect resistance management in Bt soybeans.