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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

2017 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 second reporting period of this project. Annual assessments of insecticide resistance in multiple populations of key heliothine pests of cotton 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. Cage studies, which contain various combinations of Bt and non-Bt cotton and corn, were completed. Laboratory and field experiments were used to determine the effectiveness of microbial insecticides relative to synthetic chemicals for environmentally sound management of bollworm in Bt cotton. Neonate and third instar larval survivals were evaluated on Bollgard II and non-Bt cotton leaf tissue treated with the following microbial and chemical insecticides: 1) a commercial formulation of Bacillus thuringiensis, 2) a Heliothis nuclear polyhedrosis virus (NPV), lambda-cyhalothrin, and chlorantraniliprole. Production level field evaluations were also conducted for two years in Bollgard II and non-Bt cottons. During both years of the field study, all chemical and microbial treatments were successful in suppressing bollworm larval densities in non-Bt cotton below economic threshold levels. However, net returns above bollworm control in non-Bt cotton were diminished as larval densities increased. There was no economic benefit to supplemental control of bollworms in Bt cotton at the larval densities observed during the study. These data 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 non-Bt and Bt cotton in IRM programs. Pheromone trap collections of Helicoverpa species from Florida, Iowa, New York, Pennsylvania, Texas, and Virginia continued for a second year in a row. DNA extractions were 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. This detection system was adopted by USDA APHIS for screening OWB in pheromone trap collections across the United States. A high-throughput sequence analysis-based genotyping system developed for Helicoverpa species was used to determine genome level changes in OWB over 30-year period. Genome data from preserved insect specimens from 1983 to 1993 were compared with samples collected in 2014. Cloned genomic DNA was used to characterize two receptors for Bt toxin in bollworm. Genomic scaffolds containing ATP binding cassette (ABC) transporter genes ABCA2 and ABCC2, which are receptors for Cry1Ac and Cry2Ab, respectively, were annotated and submitted to GenBank. Annotated gene sequences were used for designing reagents for gene editing by technology based on clustered regularly interspersed short palindromic repeats (CRISPR) to evaluate function of different protein domains in the receptors. CRISPR genome editing in bollworm was optimized using guide RNA (gRNA) designed to the phenotypic marker tryptophan oxygenase (TO) gene. TO gene mutants of bollworm produced yellowish-pink eyes in bollworm in contrast to vermillion eye color in other moths and flies.

1. Completed and published a comparative analysis of transposable element (TE) sequences identified in a bacterial artificial chromosome (BAC) library of bollworm, Helicoverpa zea. ARS researchers in Stoneville, Mississippi found that bollworm genome contained three major groups of TEs that are commonly found among lepidopteran species and approximately 14% of the transposable elements were non-randomly distributed across the genome sequences suggesting that some TE integrations may be clustered within the genome. This information will be important for describing transposons in the eventual bollworm whole genome sequence, isolating genome regions involved in insecticide resistance traits, and will help increase scientific understanding of genetic variation within this species as well as evolutionary relationships between related Helicoverpa species.

2. A high-throughput method for detecting invasive old world bollworm (OWB). A high-throughput method for detecting invasive old world bollworm (OWB) was developed and published in collaboration with USDA APHIS. Technology previously developed by ARS researchers in Stoneville, Mississippi, for rapid identification of OWB was further enhanced to detect one OWB in a pool of 1,000 moths using a droplet digital PCR (ddPCR) based method. This method is being used by USDA APHIS for analyzing bulk trap collections from across the USA to detect OWB invasions.

3. Optimized microinjection parameters for genome editing in bollworm using an eye color gene. Adult moths emerging from embryos injected with reagents targeting tryptophan oxygenase (TO) gene were phenotypically and genetically evaluated by ARS researchers in Stoneville, Mississippi, for mutations that change wild type eye color in bollworm. Of the 68 injected embryos surviving to adulthood, 57 (83.8%) had yellowish-pink eye color, indicating that targeted genome level mutations can be achieved at high frequency in bollworm. These results will help in selecting precise microinjection parameters for future genome editing experiments in bollworm.

Review Publications
Coates, B.S., Abel, C.A., Perera, O.P. 2016. Estimation of long terminal repeat element content in the Helicoverpa zea genome from high-throughput sequencing of bacterial artificial chromosome pools. Genome. 60(4):310-324. doi:10.1139/gen-2016-0067.
Little, N., Luttrell, R.G., Allen, K.C., Perera, O.P., Parys, K.A. 2017. Effectiveness of microbial and chemical insecticides for supplemental control of bollworm on Bt and non-Bt cottons. Journal of Economic Entomology. 110(3):1039-1051. doi:10.1093/jee/tow323.
Little, N., Catchot, A.L., Allen, K.C., Gore, J., Musser, F.R., Cook, D.R., Luttrell, R.G. 2017. Supplemental control with diamides for Heliothines1 in Bt Cotton. Southwestern Entomologist. 42(1):15-26. doi:10.3958/059.042.0102.
Zink, F.A., Tembrock, L.R., Timm, A.E., Farris, R.E., Perera, O.P., Gilligan, T.M. 2017. A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples. PLoS One. doi:10.1371/journal.pone.0178704.