The research team, led by Yucheng Wang and Fei Yang of the University of Minnesota together with David L. Kerns of Texas A&M University, Graham P. Head of Bayer Crop Science, Dawson Kerns of Louisiana State University, and Bruce E. Tabashnik of the University of Arizona, set out to answer two questions with direct consequences for resistance management. First, does selection for resistance to Vip3Aa cause cross-resistance to Cry proteins, which would undermine the pyramided crops that produce both toxin classes? Second, do Cry proteins and Vip3Aa interact synergistically against insects that are already resistant, a property that could extend the useful life of multi-toxin Bt crops?

To address these questions, the researchers generated five strains of H. zea carrying more than 100-fold resistance to Vip3Aa in a susceptible genetic background. The strains originated from F2 screens of field populations collected in Louisiana, Mississippi, and Texas between 2019 and 2020, a technique that exposes hidden recessive resistance alleles carried by mating pairs of field-collected insects. Each strain was then crossed repeatedly with a laboratory-susceptible strain, BZ-SS, obtained from a commercial supplier, and reselected with Vip3Aa in the laboratory. After five to six rounds of backcrossing and selection, the resulting strains, designated LT70-Vip, AC4-Vip, M1-Vip, R2-Vip, and R15-Vip, showed such profound resistance that even the highest concentration of Vip3Aa tested, 100 micrograms per square centimeter of diet, killed no more than 15 percent of larvae, implying resistance ratios exceeding 500-fold.

The team then tested these Vip3Aa-resistant strains against five Cry proteins used in commercial Bt crops: Cry1Ab, Cry1Ac, Cry1A.105, Cry1Fa, and Cry2Ab. The verdict was strikingly inconsistent. Across 23 bioassay comparisons, seven LC50 values were significantly higher than those of the susceptible strain, seven were significantly lower, and nine did not differ significantly, a seven-to-seven split that matches exactly what would be expected if no consistent cross-resistance existed. The overall mean cross-resistance ratio was 1.2, statistically indistinguishable from the value of 1.0 expected if selection with Vip3Aa had no effect on susceptibility to Cry proteins. Only Cry1Ac showed a statistically significant mean positive cross-resistance, and only one strain, AC4-Vip, displayed significant overall positive cross-resistance across the Cry proteins tested.

What astonished the researchers was not the absence of a trend but the sheer magnitude of the variation around it. The 23 cross-resistance ratios spanned an extraordinary 1100-fold range, from 0.06, indicating 17-fold negative cross-resistance to Cry1Fa in strain LT70-Vip, to 65-fold positive cross-resistance to Cry1A.105 in strain AC4-Vip. That range is roughly 49 to 70 times wider than those reported in previous reviews of cross-resistance between Vip3 and Cry proteins in other lepidopteran species. The authors suggest that the genetic diversity of the field-derived resistance alleles, which complementation tests place at three distinct loci among the five strains, likely contributes to this variability, as does the unknown mechanism of resistance in most of the strains. Only LT70-Vip has a biochemically characterized mechanism, reduced binding of Vip3Aa to midgut brush border membrane vesicles, and as expected under the receptor model of Bt toxicity, that strain showed no significant cross-resistance to any Cry protein.

The second half of the study tackled synergy, an interaction in which two toxins combined kill more insects than would be predicted from the independent action of each toxin alone. Previous surveys suggested synergy between Cry and Vip proteins is relatively rare, appearing in only about 19 percent of bioassay evaluations reviewed in 2020, though more recent studies on fall armyworm and peach fruit moth reported far higher frequencies. Critically, nearly all prior synergy work used susceptible insects. To test synergy against resistant insects, the team created a triple-resistant strain, TRE-RR, by crossing the Vip3Aa-resistant LT70-Vip strain with a Cry-resistant strain derived from Texas field corn, then selecting successive generations with all three toxins. The resulting strain exhibited resistance ratios of 265 for Cry1Ac, 495 for Cry2Ab, and greater than 96 for Vip3Aa.

Against this formidable laboratory adversary, synergy emerged clearly. In diet bioassays conducted at two concentrations, combinations of Vip3Aa with Cry1Ac produced significantly lower observed survival than expected under independent action at both concentrations tested, and Cry2Ab combined with Vip3Aa was synergistic at the higher concentration. Most remarkably, the three-toxin combination of Cry1Ac, Cry2Ab, and Vip3Aa was synergistic at both concentrations, meaning that even larvae equipped with resistance to every toxin individually died more often when the toxins were delivered together than their separate toxicities would predict. The finding is consistent with the authors’ earlier analysis of field data from Bt corn showing synergy between Cry1Ab and Vip3Aa against susceptible H. zea, but it is, to the authors’ knowledge, the first demonstration of Cry-Vip synergy against a strain resistant to both toxin classes.

The practical implications are substantial. If synergy between Cry proteins and Vip3Aa operates in the field against resistant H. zea, as it appears to against susceptible populations, it would kill some insects that survive either toxin alone, reducing the intensity of selection for Vip3Aa resistance and potentially slowing its evolution. That would be welcome news for an industry and a grower community watching early warning signs of Vip3Aa resistance accumulate in the United States, where practical resistance to Vip3Aa has already been documented in Brazil. The synergy finding also carries a cautionary note for resistance monitoring. Current surveillance relies partly on sentinel plots of corn producing Cry1Ab plus Vip3Aa, from which a phenotypic frequency of resistance is calculated by comparing larval abundance in Bt ears with that in non-Bt ears. If synergy kills some larvae that are resistant to either toxin alone, this approach could underestimate the true frequency of Vip3Aa resistance, a discrepancy the authors recommend resolving by comparing estimates from F2 screens with those from sentinel plots.

The study also refines the scientific understanding of how Bt resistance evolves and spreads. Because Vip3Aa and Cry proteins bind to different midgut receptors, strong cross-resistance between them was not expected, and the new results largely vindicate that expectation while revealing how messy the underlying biology can be. Mechanisms other than altered receptor binding, such as changes in toxin processing within the midgut, can confer broad cross-resistance, and the authors note that determining the resistance mechanisms in the remaining strains could explain the puzzling differences between R2-Vip and R15-Vip, which share a resistance locus yet differed 13-fold in their mean cross-resistance to Cry proteins. For now, the message for resistance management is cautiously optimistic: pyramiding Vip3Aa with Cry proteins remains a sound strategy, the lack of consistent cross-resistance means Cry resistance alleles should not compromise Vip3Aa efficacy, and synergy may quietly buy precious time for one of agriculture’s most valuable pest control technologies as its chief adversary continues to adapt.

Subject of Research: Cross-resistance and synergy between the Bt toxins Vip3Aa and Cry proteins in Vip3Aa-resistant strains of the corn earworm Helicoverpa zea

Article Title: Evaluating cross-resistance and synergy between Vip3Aa and Cry proteins from Bt in six strains of Helicoverpa zea derived via F2 screens

Article References: Evaluating cross-resistance and synergy between Vip3Aa and Cry proteins from Bt in six strains of Helicoverpa zea derived via F2 screens. (n.d.). https://doi.org/10.1007/s44297-026-00073-8

Image Credits: AI Generated

DOI: 10.1007/s44297-026-00073-8

Keywords: Helicoverpa zea, corn earworm, Bt crops, Vip3Aa, Cry proteins, cross-resistance, synergy, insect resistance, transgenic crops, resistance management, Bacillus thuringiensis, pest control