Zea Populations Research Articles

Inheritance of Cry2Ab2 resistance in two Helicoverpa zea (Lepidoptera: Noctuidae) populations resistant to single- and dual-Bacillus thuringiensis proteins

Cry2Ab2 is a Bacillus thuringiensis (Bt) protein that has been pyramided with Cry1A.105 in transgenic maize and Cry1Ac in cotton to control some major lepidopteran pests including the corn earworm/bollworm, Helicoverpa zea (Boddie). However, the widespread occurrence of resistance of this pest to the pyramided Cry1A/Cry2A crops in the southern region of the United State has become a threat to the sustainability of the technology. In this study, multiple genetic crosses and backcrosses were developed to characterize the inheritance of Cry2Ab2 resistance in two H. zea populations resistant to the single protein, Cry2Ab2 (RR2Ab) and the dual proteins in Bt maize, Cry1A.105/Cry2Ab2 (RRVT2P). Diet-overlay bioassays with F1 hybrids from reciprocal crosses between a susceptible and the resistant populations showed that the Cry2Ab2 resistance in both RR2Ab and RRVT2P was inherited autosomally and non-recessively. Segregation tests in F2 and backcrossed generations indicated that the resistance was likely controlled by more than one locus. The effective dominance levels of the resistance estimated at each of three discriminating concentrations were similar between RR2Ab and RRVT2P, ranging from incompletely dominant to incompletely recessive. The similar inheritance observed in RR2Ab and RRVT2P suggests that the Cry2Ab2 resistance is independent from the Cry1A.105 resistance. The non-recessive inheritance of the resistance could be an important factor causing the widespread resistance of this insect to the Cry1A/Cry2A crops in the southern region. Implications of the observed inheritance properties in Bt crop resistance management are discussed.

Longitudinal trials illustrate interactive effects between declining Bt efficacy against Helicoverpa zea (Lepidoptera: Noctuidae) and planting dates of corn.

Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) has evolved resistance to insecticidal toxins from Bacillus thuringiensis (Bt) Berliner (Bacillales: Bacillaceae) expressed in genetically engineered corn, Zea mays L. This study provides an overview of field trials from Georgia, North Carolina, and South Carolina evaluating Bt and non-Bt corn hybrids from 2009 to 2022 to show changes in susceptibility in H. zea to Bt corn. The reduction in kernel injury relative to a non-Bt hybrid averaged across planting dates generally declined over time for Cry1A.105 + Cry2Ab2 corn. In addition, there was a significant interaction with planting date used as a covariate. The reduction in kernel injury remained above 80% and did not vary with planting date from 2009 to 2014, whereas a significant decline with planting date was found in this reduction from 2015 to 2022. For Cry1Ab + Cry1F corn, the reduction in kernel injury relative to a non-Bt hybrid averaged across planting dates did not vary among years. The reduction in kernel injury significantly declined with planting date from 2012 to 2022. Kernel injury as a proxy for H. zea pressure was greater in late-planted trials in non-Bt corn hybrids. Our study showed that Bt hybrids expressing Cry1A.105 + Cry2Ab2 are now less effective in later planted trials in reducing H. zea injury; however, this was not the case during the earlier years of adoption of corn expressing these 2 toxins when resistance alleles were likely less frequent in H. zea populations. The implications for management of H. zea and for insect resistance management are discussed.

Rapid Adaptation and Interspecific Introgression in the North American Crop Pest Helicoverpa zea.

Insect crop pests threaten global food security. This threat is amplified through the spread of nonnative species and through adaptation of native pests to control measures. Adaptations such as pesticide resistance can result from selection on variation within a population, or through gene flow from another population. We investigate these processes in an economically important noctuid crop pest, Helicoverpa zea, which has evolved resistance to a wide range of pesticides. Its sister species Helicoverpa armigera, first detected as an invasive species in Brazil in 2013, introduced the pyrethroid-resistance gene CYP337B3 to South American H. zea via adaptive introgression. To understand whether this could contribute to pesticide resistance in North America, we sequenced 237 H. zea genomes across 10 sample sites. We report H. armigera introgression into the North American H. zea population. Two individuals sampled in Texas in 2019 carry H. armigera haplotypes in a 4 Mbp region containing CYP337B3. Next, we identify signatures of selection in the panmictic population of nonadmixed H. zea, identifying a selective sweep at a second cytochrome P450 gene: CYP333B3. We estimate that its derived allele conferred a ∼5% fitness advantage and show that this estimate explains independently observed rare nonsynonymous CYP333B3 mutations approaching fixation over a ∼20-year period. We also detect putative signatures of selection at a kinesin gene associated with Bt resistance. Overall, we document two mechanisms of rapid adaptation: the introduction of fitness-enhancing alleles through interspecific introgression, and selection on intraspecific variation.

Simple Insecticide Bean-Dip Bioassay Shows Pyrethroid Susceptibility of Helicoverpa zea (Lepidoptera: Noctuidae) Populations in Virginia Varies across Locations and Years

Abstract Corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is a major insect pest of many agricultural crops in Virginia. Pyrethroids have been the major insecticide class used to control the pest for decades; however, H. zea resistance to pyrethroids has been widely documented in the southeastern and midwestern United States. To assess the pyrethroid susceptibility of H. zea in Virginia, we collected larval populations from the same five locations in 2021 and 2022 and used a quick end user–friendly bioassay to assess survival of larvae after feeding on insecticide-dipped edamame [Glycine max (L.) Merrill] pods. Pyrethroid efficacy varied between years and locations, with some 2021 H. zea populations showing reduced pyrethroid susceptibility, but not in 2022. This variability is most likely influenced by factors such as overwintering survivability in Virginia. Thus, the need for continued annual monitoring is important to track efficacy trends and grower recommendations. The simple bean-dip bioassay could be used as an efficient monitoring tool in the future, with pyrethroid susceptibility determined in 48 h from field collection.

An Extended Investigation of Unexpected Helicoverpa zea (Boddie) Survival and Ear Injury on a Transgenic Maize Hybrid Expressing Cry1A/Cry2A/Vip3A Toxins.

The wide occurrence of resistance to Cry1A and Cry2A insecticidal toxins from Bacillus thuringiensis (Bt) in the corn earworm/bollworm Helicoverpa zea (Boddie) leaves the Vip3A toxin produced during the vegetative stage of Bt as the only fully active toxin expressed in transgenic crops to control H. zea in the U.S.A. During 2021, the first unexpected survival of H. zea and injury (UXI) on a maize hybrid expressing Cry1A.105, Cry2Ab2, and Vip3Aa in Louisiana, U.S.A. were observed in two sentinel plots used for resistance monitoring. A follow-up intensive investigation was conducted with two H. zea populations established from larvae collected from the two UXI plots. The main goal of this study was to reveal if the unexpected damage was due to resistance development in the insect to the Bt toxins expressed in the maize hybrid. Diet-overlay bioassays showed that the two populations were highly resistant to Cry1A.105, moderately resistant to Cry2Ab2, but still highly susceptible to Vip3Aa when compared to a reference susceptible strain. In 10 d assays with detached ears, the larvae of the two UXI populations exhibited survival on ears expressing only Cry toxins but presented near 100% mortality on maize hybrids containing both cry and vip3A transgenes. Multiple field trials over three years demonstrated that natural H. zea populations in Louisiana were highly resistant to maize expressing only Cry toxins but remained susceptible to all tested hybrids containing cry and vip3A genes. Altogether, the results of this study suggest that the observed UXIs in Louisiana were associated with a resistance to Cry toxins but were not due to a resistance to Vip3A. The possible causes of the UXIs are discussed. The results generated and procedures adopted in this study help in determining thresholds for defining UXIs, assessing resistance risks, and documenting field resistance.

Extended Sentinel Monitoring of Helicoverpa zea Resistance to Cry and Vip3Aa Toxins in Bt Sweet Corn: Assessing Changes in Phenotypic and Allele Frequencies of Resistance.

Transgenic corn and cotton that produce Cry and Vip3Aa toxins derived from Bacillus thuringiensis (Bt) are widely planted in the United States to control lepidopteran pests. The sustainability of these Bt crops is threatened because the corn earworm/bollworm, Helicoverpa zea (Boddie), is evolving a resistance to these toxins. Using Bt sweet corn as a sentinel plant to monitor the evolution of resistance, collaborators established 146 trials in twenty-five states and five Canadian provinces during 2020-2022. The study evaluated overall changes in the phenotypic frequency of resistance (the ratio of larval densities in Bt ears relative to densities in non-Bt ears) in H. zea populations and the range of resistance allele frequencies for Cry1Ab and Vip3Aa. The results revealed a widespread resistance to Cry1Ab, Cry2Ab2, and Cry1A.105 Cry toxins, with higher numbers of larvae surviving in Bt ears than in non-Bt ears at many trial locations. Depending on assumptions about the inheritance of resistance, allele frequencies for Cry1Ab ranged from 0.465 (dominant resistance) to 0.995 (recessive resistance). Although Vip3Aa provided high control efficacy against H. zea, the results show a notable increase in ear damage and a number of surviving older larvae, particularly at southern locations. Assuming recessive resistance, the estimated resistance allele frequencies for Vip3Aa ranged from 0.115 in the Gulf states to 0.032 at more northern locations. These findings indicate that better resistance management practices are urgently needed to sustain efficacy the of corn and cotton that produce Vip3Aa.

Helicoverpa genus on the edge of the continental U.S.: Flight phenology, analysis of hybrid presence, and insecticide performance in high-input field crops in Puerto Rico.

The genus Helicoverpa includes several agricultural pests globally. Helicoverpa armigera was reported in several countries in South America in 2013, and in Puerto Rico, in 2014. This territory is considered an agricultural hub, with a high-input system of seed production in the southern region of the island, and also at the edge of the continental U.S. Possible natural dispersion of populations of H. armigera from the Caribbean or other Central American regions poses a continuing risk to the U.S. This study was performed during the post-detection scenario of H. armigera in Puerto Rico, from 2018 to 2021. A year-round pheromone trapping program of adult males indicated an increase in the population from October to March and differences in the occurrence of Helicoverpa spp. between the municipalities Juan Diaz and Salinas. The proportion of H. armigera/H. zea and detection of congeneric hybrids between these species were assessed based on genital morphology and DNA analysis. Interestingly, neither H. armigera nor expected hybrids were detected in the present study. The susceptibility of H. zea populations to the insecticides Spinetoram, Emamectin benzoate, Chlorantraniliprole, and Esfenvalerate was assessed, and an overall significant effect of insecticide susceptibility was detected. Chlorantraniliprole and Emamectin benzoate had the highest efficacy. These results contribute to the Integrated Pest Management and Insect resistance management programs to Helicoverpa spp. in Puerto Rico. In addition, provide validated information to be considered in mitigation plans, in the scenario of an invasion of H. armigera in the continental U.S.

Selection, effective dominance, and completeness of Cry1A.105/Cry2Ab2 dual-protein resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)

In the U.S., Helicoverpa zea (Boddie) is a major pest targeted by both transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins. Resistance of insect to Bt maize and cotton containing cry1A and cry2A genes has widely occurred in the U.S. In this study, two trials were performed to investigate larval survival and development of a Cry1A.105/Cry2Ab2 dual-protein resistant (VT2P-RR), a susceptible, and an F1 heterozygous (VT2P-RS) populations of H. zea on ears of nine Bt and three non-Bt maize hybrids. The Bt maize hybrids evaluated represent five common pyramided traits expressing two or three of the Cry1A.105, Cry1Ab, Cry1F, Cry2Ab2, and Vip3Aa20 proteins. In the laboratory, neonates of the three H. zea populations were inoculated on silks of ears collected from maize at R1–R2 plant stages; and larval survivorship was checked 10 d after neonate release. All three insect populations survived normally on non-Bt maize ears. Varied numbers of VT2P-RR and VT2P-RS survived on ears of Cry1A.105/Cry2Ab2 maize, while all larvae of the three populations died or could not develop on ears of Vip3Aa20-expressing maize. The results demonstrated that the dual-protein resistant H. zea was not cross-resistant to Vip3Aa20-expressing maize, and thus traits with vip3Aa20 gene should be effective to manage Cry1A.105/Cry2Ab2-resistant H. zea. The resistance in VT2P-RR was determined to be incomplete on Cry1A.105/Cry2Ab2 maize. The effective dominance levels varied greatly, from recessive to incompletely dominant, depending on maize hybrids and trials, suggesting that proper selection of maize hybrids could be important for mitigating the Cry1A.105/Cry2Ab2 resistance. The data generated should aid in modeling multiple-protein Bt resistance in H. zea.

Pest population dynamics are related to a continental overwintering gradient

Overwintering success is an important determinant of arthropod populations that must be considered as climate change continues to influence the spatiotemporal population dynamics of agricultural pests. Using a long-term monitoring database and biologically relevant overwintering zones, we modeled the annual and seasonal population dynamics of a common pest, Helicoverpa zea (Boddie), based on three overwintering suitability zones throughout North America using four decades of soil temperatures: the southern range (able to persist through winter), transitional zone (uncertain overwintering survivorship), and northern limits (unable to survive winter). Our model indicates H. zea population dynamics are hierarchically structured with continental-level effects that are partitioned into three geographic zones. Seasonal populations were initially detected in the southern range, where they experienced multiple large population peaks. All three zones experienced a final peak between late July (southern range) and mid-August to mid-September (transitional zone and northern limits). The southern range expanded by 3% since 1981 and is projected to increase by twofold by 2099 but the areas of other zones are expected to decrease in the future. These changes suggest larger populations may persist at higher latitudes in the future due to reduced low-temperature lethal events during winter. Because H. zea is a highly migratory pest, predicting when populations accumulate in one region can inform synchronous or lagged population development in other regions. We show the value of combining long-term datasets, remotely sensed data, and laboratory findings to inform forecasting of insect pests.

A Chromosome-Scale Genome Assembly of a Helicoverpa zea Strain Resistant to Bacillus thuringiensis Cry1Ac Insecticidal Protein.

Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

Estimation of resistance allele frequencies to Cry1A.105 and Cry2Ab2 in the corn earworm (Lepidoptera: Noctuidae) with F2 isolines generated from a mass-mating method

The corn earworm/bollworm, Helicoverpa zea (Boddie), in the United States is targeted by transgenic corn and cotton expressing Bacillus thuringiensis (Bt) proteins. Previous studies have shown that resistance to Cry1A/Cry2A Bt corn has occurred in the field. In this study, we established 103 F2 isolines by mass-mating of H. zea populations collected during 2018 and 2019 in four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. Dissection of mated females showed that at least 92.7% of the isolines were originated from single matings, suggesting that the applied mass-mating method is a suitable approach to establish H. zea F2 isolines. F2 screen showed that resistance allele frequencies in these isolines were high to the two Bt proteins expressed in MON 89034 corn hybrids, averaging 0.405 for Cry1A.105 and 0.330 for Cry2Ab2. The observed high resistance allele frequencies confirm that resistance to Cry1A/Cry2A in the insect is common in the southeastern region. In addition, the F2 screen showed that resistance allele frequencies (0.490 for Cry1A.105 and 0.541 for Cry2Ab2) in the populations collected in 2019 were significantly greater than the frequencies (0.312 for Cry1A.105 and 0.081 for Cry2Ab2) in the populations collected in 2018, indicating that selection for the Cry resistance was likely still ongoing in the field. Information generated from this study is useful to assess resistance risk, refine resistance modeling, and improve IRM programs to mitigate Cry resistance in H. zea for the sustainable use of Bt crop technology in the United States.

Resistance Allele Frequency to Cry1Ab and Vip3Aa20 in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Louisiana and Three Other Southeastern U.S. States.

The corn earworm/bollworm, Helicoverpa zea (Boddie), is a pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1Ab and Vip3Aa20 are two common Bt toxins that are expressed in transgenic maize. The objective of this study was to determine the resistance allele frequency (RAF) to Cry1Ab and Vip3Aa20 in H. zea populations that were collected during 2018 and 2019 from four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. By using a group-mating approach, 104 F2 iso-lines of H. zea were established from field collections with most iso-lines (85) from Louisiana. These F2 iso-lines were screened for resistance alleles to Cry1Ab and Vip3Aa20, respectively. There was no correlation in larval survivorship between Cry1Ab and Vip3Aa20 when the iso-lines were exposed to these two toxins. RAF to Cry1Ab maize was high (0.256) and the RAFs were similar between Louisiana and the other three states and between the two sampling years. In contrast, no functional major resistance allele (RA) that allowed resistant insects to survive on Vip3Aa20 maize was detected and the expected RAF of major RAs with 95% probability was estimated to 0 to 0.0073. However, functional minor RAs to Vip3Aa20 maize were not uncommon; the estimated RAF for minor alleles was 0.028. The results provide further evidence that field resistance to Cry1Ab maize in H. zea has widely occurred, while major RAs to Vip3Aa20 maize are uncommon in the southeastern U.S. region. Information that was generated from this study should be useful in resistance monitoring and refinement of resistance management strategies to preserve Vip3A susceptibility in H. zea.

Soybeans as a non-Bt refuge for Helicoverpa zea in maize-cotton agroecosystems

Geospatial models are crucial for identifying likely ‘hot-spots’ of Bt resistance evolution in Helicoverpa zea (Lepidoptera: Noctuidae), thereby improving regional insecticide resistance management (IRM) strategies and planted refuge compliance. To characterize H. zea distributions in relation to land use, we used historical trapping data collected from 2008 to 2019 in North Carolina to model the spatial and temporal abundance of H. zea populations across Bt-dominated landscapes. Helicoverpa zea abundance was standardized across site-year observations, and candidate landscape composition and configuration predictors of H. zea abundance were obtained. Spatiotemporal Bayesian hierarchical models were developed to make posterior predictions of H. zea abundance from environmental covariates, and results were used to generate interpolation prediction maps to visualize H. zea abundance across the sampled region. Our results suggest inverse distance weighted (IDW) soybeans is the most important predictor of H. zea abundance through time in row crop agroecosystems in North Carolina. Soybeans in North Carolina and southeastern U.S. likely serves as a critical non-Bt refuge for delaying H. zea resistance to Bt toxins in landscapes dominated by Bt maize and cotton. Moreover, soybean abundance can be used to predict the spatial abundance of H. zea in this region. Results can be applied to understand population dynamics of H. zea in landscapes dominated by genetically engineered (GE) crops expressing Bt toxins and will enable the development of sound insect resistance management strategies of H. zea populations to GE toxins targeting noctuid pests of maize and cotton. This work will also drive future geospatial studies investigating environmental predictors of resistance evolution in arthropod pests to GE technologies in crop production systems.

Economic Injury Levels for Bt-resistant Helicoverpa zea (Lepidoptera: Noctuidae) in Cotton.

Thresholds for Helicoverpa zea (Boddie) in cotton Gossypium hirsutum L. have been understudied since the widespread adoption of Bt cotton in the United States. Our study was possible due to the widespread presence of H. zea populations with Cry-toxin resistance. We initiated progressive spray timing experiments using three Bt cotton brands (Deltapine, Stoneville, and Phytogen) widely planted across the U.S. Cotton Belt expressing pyramided toxins in the Cry1A, Cry2, and Vip3Aa19 families. We timed foliar insecticide treatments based on week of bloom to manipulate H. zea populations in tandem with crop development during 2017 and 2018. We hypothesized that non-Bt cotton, cotton expressing Cry toxins alone, and cotton expressing Cry and Vip3Aa19 toxins would respond differently to H. zea feeding. We calculated economic injury levels to support the development of economic thresholds from significant responses. Pressure from H. zea was high during both years. Squares and bolls damaged by H. zea had the strongest negative yield associations, followed by larval number on squares. There were fewer yield associations with larval number on bolls and with number of H. zea eggs on the plant. Larval population levels were very low on varieties expressing Vip3Aa19. Yield response varied across experiments and varieties, suggesting that it is difficult to pinpoint precise economic injury levels. Nonetheless, our results generally suggest that current economic thresholds for H. zea in cotton are too high. Economic injury levels from comparisons between non-Bt varieties and those expressing only Cry toxins could inform future thresholds once H. zea evolves resistance to Vip3Aa19.

Extended investigation of field-evolved resistance of the corn earworm Helicoverpa zea (Lepidoptera: Noctuidae) to Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins in the southeastern United States

Previous studies have reported that the corn earworm/bollworm, Helicoverpa zea (Boddie), has developed field resistance to pyramided Bacillus thuringiensis (Bt) Cry1A/Cry2A maize and cotton in certain areas of the southeastern United States. The objective of the current study was to determine the current status and distribution of the resistance to Cry1A.105 and Cry2Ab2 in H. zea. In the study, 31 H. zea populations were collected from major maize planting areas across seven southeastern states of the United States during 2018 and 2019 and assayed against the two Bt proteins. Diet over-lay bioassays showed that most of the populations collected during the two years were significantly resistant to the Cry1A.105 protein. Most of the populations collected during 2019 were also resistant to Cry2Ab2, while significant variances were observed in the susceptibility of the populations collected during 2018 to Cry2Ab2. The results showed that Cry1A.105 and Cry2Ab2 resistance in H. zea is widely distributed in the regions sampled. The resistance to Cry1A.105 appeared to have plateaued, while selection for Cry2Ab2 resistance is likely still occurring. Thus, effective measures for mitigating the Cry1A/Cry2A resistance need to be developed and implemented to ensure the sustainable use of Bt crop biotechnology.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Brazil: the Big Outbreak Monitored by Light Traps.

The first detection of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Brazil pointed to a gloomy future for South American agriculture in the 2012/2013 harvest, and insecticide sales increased exponentially during 2013 and 2014. However, after this outbreak, the population of this insect returned to manageable levels. This study documents data on H. armigera moth collection using light traps during the outbreak in two locations: (a) western region of the state of Bahia, including population levels of H. armigera and main noctuid pests during the big outbreak (February and March 2013); (b) Federal District, including monthly data for five growing seasons (July 2012-June 2017). During the outbreak, an average of 527.3 moths were collected per trap (cotton crop, March) in the western region of Bahia each night, while the maximum average in the Federal District was 75.4 moths (January). The number of moths decreased in the following growing seasons, and in October 2016, the maximum number was 2.8 moths per trap. Aspects related to the variations of H. armigera and Helicoverpa zea (Boddie)populations, including the study of phenology and relationship with climatic factors and Oceanic Niño Index, were analyzed and discussed. All aspects are approached to contribute to the management of lepidopteran pests, especially H. armigera, in the productive systems of the Brazilian Cerrado.

Populations of Helicoverpa zea (Boddie) in the Southeastern United States are Commonly Resistant to Cry1Ab, but Still Susceptible to Vip3Aa20 Expressed in MIR 162 Corn.

sThe corn earworm, Helicoverpa zea (Boddie), is a major pest targeted by pyramided Bacillus thuringiensis (Bt) corn and cotton in the U.S. Cry1Ab is one of the first insecticidal toxins used in Bt crops, while Vip3A is a relatively new toxin that has recently been incorporated into Cry corn with event MIR 162 and Cry cotton varieties to generate pyramided Bt traits targeting lepidopteran pests including H. zea. The objectives of this study were to determine the current status and distribution of the Cry1Ab resistance, and evaluate the susceptibility to Vip3Aa20 expressed in MIR 162 corn in H. zea in the southeastern U.S. During 2018 and 2019, 32 H. zea populations were collected from non-Bt corn (19 populations), Cry corn (12), and Cry/Vip3A cotton (1) across major corn areas in seven southeastern states of the U.S. Susceptibility of these populations to Cry1Ab and Vip3Aa20 was determined using diet-overlay bioassays. Compared to a known susceptible insect strain, 80% of the field populations were 13- to >150-fold resistant to Cry1Ab, while their response to Vip3Aa20 ranged from >11-fold more susceptible to 9-fold more tolerant. Mean susceptibility to each Bt toxin was not significantly different between the two groups of the populations collected from non-Bt and Bt crops, as well as between the two groups of the populations collected during 2018 and 2019. The results show that resistance to Cry1Ab in H. zea is widely distributed across the region. However, the Cry1Ab-resistant populations are not cross-resistant to Vip3Aa20, and H. zea in the region is still susceptible to the Vip3Aa20 toxin. Vip3Aa20 concentrations between 5 and 10 µg/cm2 may be used as diagnostic concentrations for susceptibility monitoring in future. Additional studies are necessary to elucidate the impact of the selection with Bt corn on resistance evolution in H. zea to Vip3A cotton in the U.S.

Seed blends of pyramided Cry/Vip maize reduce Helicoverpa zea populations from refuge ears

The Vip3A, a vegetative Bacillus thuringiensis (Bt) gene, has been introduced into many Cry maize and cotton varieties that can manage the recently occurred Cry1/Cry2 resistance in Noctuidae pests including Helicoverpa zea (Boddie) in the USA. A seed blend refuge has been used for providing susceptible insect populations for Bt maize resistance management. Four field trials were deployed in this study to investigate the effects of Bt protein contamination due to gene flow in seed blend refuges of Viptera maize containing Cry1Ab and Vip3Aa20 genes with 0–30% refuges on the survival, development, and reproduction of the parental and F1 generations of H. zea. Viptera maize is highly effective against H. zea and likely expresses a ‘high dose’ for the insect. Compared to the survival on the structured refuge, seed blends reduced approximately 70% of the pupal and adult productions from refuge ears, and the reductions were not related to the refuge percentage. Pupae from the seed blend refuge weighed 22.1% less than those from the structured refuge, but the body mass reduction did not significantly affect the reproduction per female. All field populations were highly resistant to Cry1Ab, but susceptible to Vip3Aa20. Fitness of the F1 generations from the structured and seed blend refuges in diet rearing was generally similar. Data generated from the study can be used in simulation modeling to evaluate the feasibility of seed blends for resistance management.

Sweet Corn Sentinel Monitoring for Lepidopteran Field-Evolved Resistance to Bt Toxins.

As part of an insect resistance management plan to preserve Bt transgenic technology, annual monitoring of target pests is mandated to detect susceptibility changes to Bt toxins. Currently Helicoverpa zea (Boddie) monitoring involves investigating unexpected injury in Bt crop fields and collecting larvae from non-Bt host plants for laboratory diet bioassays to determine mortality responses to diagnostic concentrations of Bt toxins. To date, this monitoring approach has not detected any significant change from the known range of baseline susceptibility to Bt toxins, yet practical field-evolved resistance in H. zea populations and numerous occurrences of unexpected injury occur in Bt crops. In this study, we implemented a network of 73 sentinel sweet corn trials, spanning 16 U.S. states and 4 Canadian provinces, for monitoring changes in H. zea susceptibility to Cry and Vip3A toxins by measuring differences in ear damage and larval infestations between isogenic pairs of non-Bt and Bt hybrids over three years. This approach can monitor susceptibility changes and regional differences in other ear-feeding lepidopteran pests. Temporal changes in the field efficacy of each toxin were evidenced by comparing our current results with earlier published studies, including baseline data for each Bt trait when first commercialized. Changes in amount of ear damage showed significant increases in H. zea resistance to Cry toxins and possibly lower susceptibility to Vip3a. Our findings demonstrate that the sentinel plot approach as an in-field screen can effectively monitor phenotypic resistance and document field-evolved resistance in target pest populations, improving resistance monitoring for Bt crops.

Temporal Variation in Genetic Composition of Migratory Helicoverpa Zea in Peripheral Populations.

Migrant populations of Helicoverpa zea (Boddie) captured during 2002, 2005, 2016, and 2018 from Landisville and Rock Springs in Pennsylvania, USA were genotyped using 85 single nucleotide polymorphism (SNP) markers. Samples (n = 702) genotyped were divided into 16 putative populations based on collection time and site. Fixation indices (F-statistics), analysis of molecular variance, and discriminant analysis of principal components were used to examine within and among population genetic variation. The observed and expected heterozygosity in putative populations ranged from 0.317–0.418 and 0.320–0.359, respectively. Broad range of FST (0.0–0.2742) and FIS (0.0–0.2330) values indicated different genotype frequencies between and within the populations, respectively. High genetic diversity within and low genetic differentiation between populations was found in 2002 and 2005. Interestingly, high genetic differentiation between populations from two collection sites observed in 2018 populations was not evident in within-site comparisons of putative populations collected on different dates during the season. The shift of H. zea population genetic makeup in 2018 may be influenced by multiple biotic and abiotic factors including tropical storms. Continued assessment of these peripheral populations of H. zea will be needed to assess the impacts of genetic changes on pest control and resistance management tactics.