the corn earworm, Helicoverpa zea, is a major target pest of the insecticidal Vip3Aa protein used in pyramided transgenic Bt corn and cotton with Cry1 and Cry2 proteins in the U.S. The widespread resistance to Cry1 and Cry2 proteins in H. zea will challenge the long-term efficacy of Vip3Aa technology. Determining the frequency of resistant alleles to Vip3Aa in field populations of H. zea is critically important for resistance management. Here, we provided the first F 2 screen study to estimate the resistance allele frequency for Vip3Aa in H. zea populations in Texas, U.S. In 2019, 128 H. zea neonates per isofamily for a total of 114 F 2 families were screened with a diagnostic concentration of 3.0 μg/cm 2 of Vip3Aa39 protein in diet-overlay bioassays. The F 2 screen detected two families carrying a major Vip3Aa resistance allele. The estimated frequency of major resistance alleles against Vip3Aa39 in H. zea in texas from this study was 0.0065 with a 95% CI of 0.0014-0.0157. A Vip3Aa-resistant strain (RR) derived from the f 2 screen showed a high level of resistance to Vip3Aa39 protein, with a resistance ratio of >588.0fold relative to a susceptible population (SS) based on diet-overlay bioassays. We provide the first documentation of a major resistance allele conferring high levels of Vip3Aa resistance in a field-derived strain of H. zea in the U.S. Data generated from this study contribute to development of management strategies for the sustainable use of the Vip3Aa technology to control H. zea in the U.S. Genetically engineered crops producing insecticidal Cry and Vip proteins from the bacterium Bacillus thuringiensis (Bt) have been planted for control of insect pests for more than two decades 1. Field efficacy of these Bt crops has been outstanding in controlling most target species, resulting in substantial economic, environmental and social gains 2-7. However, with large scale adoption comes intense selection pressure for development of resistance and challenges for long-term sustainability 8,9. To date, field-evolved practical resistance to Bt crops has been globally reported in at least 21 cases 10-15. To delay insect resistance development, an insecticide resistance management (IRM) plan based on a "high-dose refuge" strategy has been implemented in the U.S 16. Monitoring for evolution of resistance in field populations of the target insect species is an essential component of this IRM plan to maintain sustainability of Bt crop technologies. The corn earworm/cotton bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is a major target pest of both Bt cotton and Bt corn in North America. Control of lepidopteran pests is achieved by the adoption of corn hybrids producing combinations of Cry1Ab, Cry1F, Cry1A.105, Cry2Ab2 and Vip3Aa20 insecticidal Bt proteins, and Bt cotton varieties producing combinations of Cry1Ac, Cry1F, Cry1Ab, Cry2Ab, Cry2Ae, and Vip3Aa19 17 .

Evolution of resistance by pests can reduce the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Because of the widespread resistance of Helicoverpa zea to crystalline (Cry) Bt toxins in the United States, the vegetative insecticidal protein Vip3Aa is the only Bt toxin produced by Bt corn and cotton that remains effective against some populations of this polyphagous lepidopteran pest. Here we evaluated H. zea resistance to Vip3Aa using diet bioassays to test 42,218 larvae from three lab strains and 71 strains derived from the field during 2016 to 2020 in Arkansas, Louisiana, Mississippi, Tennessee, and Texas. Relative to the least susceptible of the three lab strains tested (BZ), susceptibility to Vip3Aa of the field-derived strains decreased significantly from 2016 to 2020. Relative to another lab strain (TM), 7 of 16 strains derived from the field in 2019 were significantly resistant to Vip3Aa, with up to 13-fold resistance. Susceptibility to Vip3Aa was significantly lower for strains derived from Vip3Aa plants than non-Vip3Aa plants, providing direct evidence of resistance evolving in response to selection by Vip3Aa plants in the field. Together with previously reported data, the results here convey an early warning of field-evolved resistance to Vip3Aa in H. zea that supports calls for urgent action to preserve the efficacy of this toxin.

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

BACKGROUND: Crops genetically engineered to make insect-killing proteins from Bacillus thuringiensis (Bt) have revolutionized management of some pests. However, the benefits of such transgenic crops are reduced when pests evolve resistance to Bt toxins. We evaluated resistance to Bt toxins and Bt cotton plants using laboratory bioassays and complementary field trials focusing on Helicoverpa zea, one of the most economically important pests of cotton and other crops in the United States. RESULTS:The data from 235 laboratory bioassays demonstrate resistance to Cry1Ac, Cry1Fa, and Cry2Ab occurred in most of the 95 strains of H. zea derived from Arkansas, Louisiana, Mississippi, Tennessee, and Texas during 2016 to 2021. Complementary field data show efficacy decreased for Bt cotton producing Cry1Ac + Cry1Fa or Cry1Ac + Cry2Ab, but not Cry1Ac + Cry1Fa + Vip3Aa. Moreover, analysis of data paired by field site and year shows higher survival in bioassays was generally associated with lower efficacy of Bt cotton. CONCLUSIONS:The results confirm and extend previous evidence showing widespread practical resistance of H. zea in the United States to the Cry toxins produced by Bt cotton and corn, but not to Vip3Aa. Despite deployment in combination with Cry toxins in Bt crops, Vip3Aa effectively acts as a single toxin against H. zea larvae that are highly resistant to Cry toxins. Furthermore, Vip3Aa adoption is increasing and previous work provided an early warning of field-evolved resistance. Thus, rigorous resistance management measures are needed to preserve the efficacy of Vip3Aa against this highly adaptable pest.

The primary management tactic for lepidopteran pests of cotton in the United States of America (USA) is the use of transgenic cotton that produces Bacillus thuringiensis Berliner (Bt) toxins. The primary target pests of this technology are Helicoverpa zea (Boddie) and Heliothis virescens (F.) in the eastern and central Cotton Belt of the USA. Concerns over the evolution of resistance in H. zea to Bt toxins and scrutiny of the necessity of Bt crops has escalated. We reviewed published and unpublished data from field trials of Bt cotton in the eastern and central Cotton Belt of the USA through 2015 to evaluate the effectiveness of Bt cotton (Bollgard, Bollgard II, WideStrike, WideStrike 3, and TwinLink). Bt cotton reduced insecticide usage, reduced heliothine pest numbers and damage, and provided a yield benefit, but Bollgard II and WideStrike efficacy declined in the Midsouth over the period evaluated. In the Southeastern region, heliothine damage remained constant through 2015, but yield benefits declined from 2010 until 2015. Resistance of H. zea to several Bt toxins is the most plausible explanation for the observed changes in Bt cotton efficacy. The introduction of new Bt toxins such as found in Widestrike 3 and Twinlink may preserve the benefits of Bt crops. However, while both Widestrike 3 and Twinlink had less damage than Widestrike, damage levels of both were similar to Bollgard II.