Dawson Kerns

Helicoverpa zea Boddie (corn earworm or bollworm) is a primary target of transgenic crops expressing insecticidal Cry and Vip3Aa proteins from Bacillus thuringiensis (Bt). Widespread resistance to Cry toxins has increased reliance on Vip3Aa. We evaluated Vip3Aa susceptibility in 71 field populations of H. zea collected across the southeastern USA from 2018 through 2025 using diet-overlay bioassays of F₁ progeny. Vip3Aa1 was used in 2018 and Vip3Aa39 from 2019 through 2025. Median lethal concentrations (LC₅₀) were estimated and resistance ratios calculated relative to two susceptible laboratory colonies, a field-collected susceptible and the commercially available Benzon colony. Resistance ratios depended strongly on the susceptible reference used. Three populations evaluated in 2018 had resistance ratios ≥ 10 relative to the field-collected susceptible colony, whereas all Benzon-referenced resistance ratios were 50 values showed no temporal trend from 2019 through 2025, when all populations were evaluated with Vip3Aa39. These results provide no evidence of a sustained regional decline in Vip3Aa susceptibility in this region, but they highlight the importance of stable susceptible benchmarks, protein quality control, and complementary approaches for long-term resistance monitoring.

Transgenic crops producing insecticidal Bacillus thuringiensis (Bt) proteins have revolutionized the control of some pests. However, the evolution of resistance by pests has reduced the efficacy of Bt crops. Pest resistance to Bt crystalline (Cry) proteins has spurred a shift to crops that produce the Bt vegetative insecticidal protein Vip3Aa together with Cry proteins. With widespread field-evolved practical resistance of the major pest Helicoverpa zea (corn earworm or bollworm) to Cry proteins in the United States, Vip3Aa is the only Bt protein in transgenic corn and cotton that is highly effective against many populations. We generated five strains of H. zea with > 100-fold resistance to Vip3Aa in a susceptible genetic background using F ₂ screens of field populations from Louisiana, Mississippi, and Texas followed by crosses to a susceptible strain and additional laboratory selection with Vip3Aa. The results from diet bioassays show that in these Vip3Aa-resistant strains, cross-resistance to Cry1Ab, Cry1Ac, Cry1A.105, Cry1Fa, and Cry2Ab was not consistent and ranged 1100-fold: from 16-fold negative cross-resistance to 65-fold positive cross-resistance. By crossing a Vip3Aa-resistant strain with a Cry-resistant strain followed by selection with both types of Bt protein, we generated a strain highly resistant to Cry1Ac, Cry2Ab, and Vip3Aa. Cry1Ac and Cry2Ab interacted synergistically with Vip3Aa against this triple-resistant strain, such that combinations of the Cry proteins and Vip3Aa caused higher mortality than expected from their individual toxicity. The observed synergy may increase the durability of Bt crops producing combinations of these proteins against H. zea .

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has become a key plant-incorporated protectant (PIP) in transgenic crops targeting lepidopteran pests, particularly as resistance increasingly compromises the efficacy of Cry protein PIPs. More than a decade after its commercial deployment, Vip3Aa performance remains efficacious but increasingly vulnerable. Field screens have detected unexpectedly high baseline frequencies of Vip3Aa-resistant alleles and have produced highly resistant strains in several major pests, including Helicoverpa spp., Spodoptera spp., and Mythimna separata. Although structure–function experiments and studies on resistance to Vip3Aa have identified altered midgut processing and impaired receptor binding as candidate resistance mechanisms, the underlying genetic determinants remain poorly understood. Moreover, resistance to Vip3Aa appears to diverge from canonical Cry protein resistance pathways, underscoring the need for dedicated mechanistic studies. This review critically examines the available experimental evidence on Vip3Aa resistance mechanisms, highlighting major knowledge gaps and proposing research priorities to inform resistance monitoring and extend the durability of Vip3Aa-based pest control.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has become a key plant-incorporated protectant (PIP) in transgenic crops targeting lepidopteran pests, particularly as resistance increasingly compromises the efficacy of Cry protein PIPs. More than a decade after its commercial deployment, Vip3Aa performance remains efficacious but increasingly vulnerable. Field screens have detected unexpectedly high baseline frequencies of Vip3Aa resistance alleles and have produced highly resistant strains in several major pests, including Helicoverpa spp., Spodoptera spp., and Mythimna separata. Although structure-function experiments and studies on resistance to Vip3Aa have identified altered midgut processing and impaired receptor binding as candidate resistance mechanisms, the underlying genetic determinants remain poorly understood. Moreover, resistance to Vip3Aa appears to diverge from canonical Cry protein resistance pathways, underscoring the need for dedicated mechanistic studies. This review critically examines the available experimental evidence on Vip3Aa resistance mechanisms, highlighting major knowledge gaps and proposing research priorities to inform resistance monitoring and extend the durability of Vip3Aa-based pest control.

BACKGROUND: Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long‐term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field‐derived H. zea strains (M1‐RR, AC4‐RR, R2‐RR and R15‐RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70‐RR).RESULTS: Maternal effects and sex linkage were absent, and the effective dominance level (D_ML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 μg cm⁻², in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1‐RR and LT#70‐RR; another shared by R2‐RR and R15‐RR; and a distinct one for AC4‐RR.CONCLUSION: Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transgenic corn and cotton that express Cry and Vip insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt), have been valuable tools for the management of lepidopteran pests. In 2019, a Vip3Aa-resistant strain of Helicoverpa zea (CEW-Vip-RR) was isolated from F ₂ screens of field populations in Texas. Characterizing the resistance mechanism in this strain is important for predicting the sustained efficacy of current commercial Bt traits and guiding the development of future transgenic traits. Resistance to insecticidal proteins in Bt traits is commonly associated with reduced toxin binding, with the exception of Vip3Aa resistance being associated to altered proteolytic processing in the insect host gut. Therefore, Vip3Aa protoxin processing was tested by incubation with midgut fluids from CEW-Vip-RR relative to a susceptible strain (CEW-SS). Finding no significant processing differences, alterations in Vip3Aa binding were tested by comparing binding of radiolabeled and biotinylated Vip3Aa toxin to midgut brush border membrane vesicles (BBMV) from CEW-Vip-RR and CEW-SS larvae. Specific Vip3Aa binding to CEW-Vip-RR BBMV in these experiments was consistently reduced when compared with CEW-SS BBMV. These results support that an altered Vip3Aa-receptor is associated with resistance in CEW-Vip-RR. Understanding this resistance mechanism could have important implications for resistance management decisions, considering widespread Cry1 and Cry2 resistance in H. zea populations. IMPORTANCE: Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant-incorporated protectant in Bt crops that consistently provides excellent control of H. zea . The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.

Transgenic corn and cotton expressing Cry and Vip insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt), have been a valuable tool for the management of lepidopteran pests. In 2019, a Vip3Aa-resistant strain of Helicoverpa zea (CEW-Vip-RR) was isolated from F ₂ screens of field populations in Texas. Characterizing the resistance mechanism in this strain is important for predicting the sustained efficacy of current commercial Bt traits and guiding the development of future transgenic traits. Resistance to insecticidal proteins in Bt traits is commonly associated with reduced toxin binding, with the exception of Vip3Aa resistance being associated to altered proteolytic processing in the insect host gut. Therefore, Vip3Aa protoxin processing was tested by incubation with midgut fluids from CEW-Vip-RR relative to a susceptible strain (CEW-SS). Finding no significant processing differences, alterations in Vip3Aa binding were tested by comparing binding of radiolabeled and biotinylated Vip3Aa toxin to midgut brush border membrane vesicles (BBMV) from CEW-Vip-RR and CEW-SS larvae. Specific Vip3Aa binding to CEW-Vip-RR BBMV in these experiments was consistently reduced when compared with CEW-SS BBMV. These results support that an altered Vip3Aa- receptor is associated with resistance in CEW-Vip-RR. Understanding this resistance mechanism could have important implications for resistance management decisions considering widespread Cry1 and Cry2 resistance in H. zea populations. IMPORTANCE: Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton producing insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant incorporated protectant in Bt crops consistently providing excellent control of H. zea . The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.

BACKGROUND: Diet‐overlay bioassays suggest that Helicoverpa zea (Lepidoptera: Noctuidae) field populations have developed resistance to some of the Bt insecticidal proteins that are constituents of the pyramids expressed in the second and third generation Bt cotton technologies. Unfortunately, these bioassays are not always a reliable indicator for how a seemingly resistant population will perform in an actual cotton field, and thus, leaf tissue bioassays have been suggested as a method to better assess field performance. However, bollworm larvae typically prefer to feed on floral tissue rather than leaf tissue, and an alternative cotton structure type may be more ideal for use in plant tissue‐based bioassays. A series of diet‐overlay bioassays using Bt proteins and Bt cotton plant tissue were conducted with laboratory susceptible (Bz‐SS) and resistant (Cry‐RR, resistant to Cry1Ac and Cry2Ab) H. zea strains to determine if plant tissue overlays could detect resistance and which cotton plant structure type would be most ideal for use in bioassays.RESULTS: Results suggest that diet overlays using lyophilized plant tissue were able to detect resistance. Lyophilized tissue from white flowers was most ideal for use in bioassays, whereas tissue from non‐Bt bolls and leaves affected larval health and behavior, confounding assay results.CONCLUSION: Overlays using white flower tissue could potentially be used to supplement Bt protein overlays and provide an improved assessment of larval performance on Bt cotton technologies. © 2021 Society of Chemical Industry.