Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with ¹²⁵ I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC ₃ (5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis , we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.

Transgenic maize producing the Cry1Ab toxin of Bacillus thuringiensis (Bt maize) was approved for cultivation in the European Union (EU) in 1998 to control the corn borers Sesamia nonagrioides (Lefèbvre) and Ostrinia nubilalis (Hübner). In the EU since then, Cry1Ab is the only Bt toxin produced by Bt maize and Spain is the only country where Bt maize has been planted every year. In 2021, about 100,000 hectares of Bt maize producing Cry1Ab were cultivated in the EU, with Spain accounting for 96% and Portugal 4% of this area. In both countries, Bt maize represented less than 25% of all maize planted in 2021, with a maximum regional adoption of 64% Bt maize in northeastern Spain. Insect resistance management based on the high-dose/refuge strategy has been implemented in the EU since 1998. This has been accompanied by monitoring to enable early detection of resistance. The monitoring data from laboratory bioassays show no decrease in susceptibility to Cry1Ab had occurred in either pest as of 2021. Also, control failures have not been reported, confirming that Bt maize producing Cry1Ab remains effective against both pests. Conditions in the EU preventing approval of new genetically modified crops, including maize producing two or more Bt toxins targeting corn borers, may limit the future effectiveness of resistance management strategies.