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ARS Home » Southeast Area » Stoneville, Mississippi » Southern Insect Management Research » Research » Research Project #429232

Research Project: Innovative Strategies for Insect Resistance Management in Bt Cotton

Location: Southern Insect Management Research

2016 Annual Report

Objective 1: Determine impacts of Bt toxins on pest insect biology, assess population dynamics, pest behavior, and host-plant relationships that enhance resistance, and develop management strategies to mitigate evolution of insect resistance to host plant expressed insecticidal genes. Sub-objective 1A: Determine the impacts of transgenic crops producing two or more Bt toxins on population ecology and phenology of heliothines in cotton. Sub-objective 1.B: Evaluate optimal management strategies to delay resistance of heliothines to transgenic cotton. Objective 2: Determine genetic diversity of bollworm populations and impacts of changes in allele frequencies of loci known to be associated with resistance to Bt toxins and insecticides. Sub-objective 2.A: Determine genetic diversity of bollworm populations and allele frequencies of loci known to be associated with resistance to Bt toxins and insecticides. Sub-objective 2.B: Evaluate the allele frequency changes during selection with Bt toxins and insecticides. Objective 3: Determine impacts of insecticide resistance on management of lepidopteran pests and develop environmentally sound strategies to manage pest complexes in transgenic cropping systems. Sub-objective 3.A: Determine impacts of insecticide resistance on management of bollworm. Sub-objective 3.B: Evaluate IPM tactics for optimal management of pests in transgenic cotton.

The impacts of transgenic crops producing two or more Bacillus thuringiensis (Bt) toxins on population ecology and phenology of bollworm (BW) will be studied using replicated field experiments structured to examine multi-generational effects of selection by different sequences of transgenic crops (Bt-crops) and non-Bt crops. Experiments will be conducted using 1/16th acre field cages during the first three years of the project followed by five-acre field plots during the remainder of the project. Paired treatments will compare Bt-crop varieties with non-Bt counterparts (near isolines). Experimental crops inside cages will be infested with pupae reared from early season larval collections. Insect densities, species composition, survival on a given host, and crop damage data will be used to predict relationships between within-season selection of Bt-crop hosts and the effects of selection on population dynamics of BW. Sentinel plots of cotton and corn will be established on a spatial gradient representative of the range of latitudes within the Mississippi Delta and used to evaluate the effects of supplementary insecticide control of BW on primary Bt and non-Bt crop hosts. Different Bt crop varieties will be paired and planted with a non-Bt isoline. One replication of the Bt variety and its non-Bt isoline will be sprayed with chlorantraniliprole if and when recommended threshold for BW is reached. Other plots will receive no sprays for BW throughout the growing season. Non-target pests on the experimental plots will be controlled as needed with blanket applications of insecticides with no or low lepidopteran activity. Larval collections will be used to determine species composition infesting plots. Crop damage, species composition, and survival from each crop will be analyzed using each location as a replicate in a split plot design to determine the effects of supplementary control of BW in Bt and non-Bt crops on yield.Molecular markers will be used to evaluate genetic diversity of BW populations and impacts of changes in allele frequencies of loci associated with resistance to Bt toxins and insecticides. Allele frequencies in insects collected during the first three years of the project period will be compared with data from insects collected from 2002-2006. Identification of loci under selection will help us evaluate the impacts of field selection on BW over time. In addition, we will be able to estimate the mutation rates of the genes associated with Bt resistance and use those estimates in Bt resistance prediction models. A BW strain tolerant to Bt toxin Cry1Ac will be used to identify genomic regions responding to selection. Impacts of insecticide resistance on management of lepidopteran pests will be determined by mutating target receptor genes to generate insecticide resistance in BW lines with high tolerance to Bt toxins. Fitness costs of dual resistance will be evaluated using controlled experiments. Integrated pest management tactics utilizing various combinations of chemical and microbial agents will be evaluated to develop environmentally sound strategies to the management pest complexes in transgenic cropping systems.

Progress Report
Substantial progress was made in research planned for the first reporting period of this project. Annual assessments of insecticide resistance in multiple populations of key heliothine pests of cotton, corn, and soybean were conducted using commercially available Bacillus thuringiensis (Bt) toxins and transgenic plants with different combinations of Bt toxins. Multiple populations of bollworms and tobacco budworms were also screened against a commercially available formulation of Bt (Dipel) and plant tissue from commercially-grown Bt crops. This includes first generation (F1) screens for physiological changes in heliothines associated with exposure to conventional and transgenic insecticides and associated effects on survival in field environments. Cage studies, which contain various combinations of Bt and non-Bt cotton and corn, were established. Data are being collected. Research on five sentinel plot locations of cotton grown on a North to South gradient in the Mississippi Delta cropping region. Laboratory and field evaluations of a microbial pesticide for supplemental control of bollworms in undisturbed Bt cotton (ie. no synthetic insecticides applied for control of non-target pests) are ongoing. This includes laboratory studies with a nucleopolyhedrovirus of Helicoverpa armigera (HaNPV). Field evaluations are in progress. HaNPV is being evaluated against a pyrethroid, and an anthrinilic diamide in large sprayed and unsprayed Bt and non-Bt cottons growing in production fields. One year of field data is complete and year two of data collection is in progress. In laboratory assays, neonate and third instar Helicoverpa zea larvae were exposed to microbial and chemical insecticides on non-Bt and Bt cotton leaves. Mortality of larvae exposed to untreated Bt cotton was generally comparable to that of Bt cotton treated with microbial and chemical insecticides. Laboratory and field studies were initiated to measure concentration-response of field collected heliothine larvae to Dipel (Bacillus thuringiensis) incorporated into a meridic diet. Field collected larvae appear to respond similarly to laboratory-reared insects. Preliminary results suggest that resulting in insect mortality, may be independent of larval size at exposure to the diet. Pheromone trap collections of Helicoverpa species were initiated in Florida, Iowa, New York, Pennsylvania, Texas, and Virginia. DNA extractions are being carried out from moth collections for use in genotyping assays and for detecting invasive old world bollworm (OWB), Helicoverpa armigera using a high throughput detection system developed at the USDA-ARS Southern Insect Management Research Unit. In addition, a high-throughput sequence analysis-based genotyping system was developed for Helicoverpa species. This system has the potential to analyze more than 3,500 genetic loci from the genomes of Helicoverpa species. In addition to generating genotype data for population genetic analyses, this system could be used to detect invasive OWB and its hybrids. Cloned genomic DNA was used to characterize ryanodine receptor (RyR) gene, the target of diamide insecticides, of Helicoverpa zea. Annotated gene sequences were used for designing reagents for gene editing by technology based on clustered regularly interspersed short palindromic repeats (CRISPR). Population genetic analysis of Helicoverpa zea collected in two locations in Pennsylvania was completed. The results indicated asymmetrical immigration of H. zea due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of this pest to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species.

1. Population genetic analysis. A population genetics analysis of Helicoverpa zea revealed asymmetrical immigration from peripheral source populations that maintained high genetic diversity, but low genetic divergence in the species. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species such as invasive old world bollworm, Helicoverpa armigera.

2. Genotype System. A genotyping system using high-throughput sequence analysis that is suitable for conducting population genetic analysis and detection of invasive old world bollworm was developed for Helicoverpa species.

3. Microbial insecticides. Microbial insecticides were evaluated in laboratory and production-level environments to produce environmentally sound management of heliothines in cotton. These findings provide benchmark comparisons for insect resistance management with microbial and chemical insecticides in Bt and non-Bt cottons and strategic optimization of the need to spray transgenic Bt cotton in IRM programs.


Review Publications
Perera, O.P., Allen, K.C., Jain, D., Purcell, M., Little, N., Luttrell, R.G. 2015. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1. Journal of Insect Science. 15(1):155. doi:1093/jisesa/iev137.
Luttrell, R.G., Teague, T.G., Brewer, M.J. 2015. Cotton insect pest management. In: D. D. Fang and R. G. Percy (Eds.) Cotton. Agronomy Monograph 57, American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, WI. Book Chapter. Pp. 509-546.
Shirk, P.D., Perera, O.P., Shelby, K., Furlong, R.B., LoVullo, E.D., Popham, H.J. 2015. Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths. Gene. 574(1):121-139.
Perera, O.P., Walsh, T.K., Luttrell, R.G. 2016. Complete mitochondrial genome of Helicoverpa zea (Boddie) and expression profiles of mitochondrial-encoded genes in early and late embryos. Journal of Insect Science. 16(1):40. 1–10. doi: 10.1093/jisesa/iew023.
Seymour, M., Perera, O.P., Fescemyer, H.W., Jackson, R.E., Fleischer, S.J., Abel, C.A. 2016. Peripheral genetic structure of Helicoverpa zea indicates asymmetrical panmixia. Ecology and Evolution. 6(10):3198-3207. doi: 10.1002/ece3.2106.