Location: Southern Insect Management Research
2018 Annual Report
Objectives
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.
Approach
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 36-month reporting period of this project. Annual assessments of conventional and transgenic insecticide resistance in numerous populations of key heliothine pests of cotton have continued. Transgenic insecticide assays were conducted using commercially available Bacillus thuringiensis (Bt) toxins, purified Bt proteins, and transgenic plants expressing different combinations of Bt toxins. Additionally, collections of insects from both Bt and non-Bt crops were examined for their susceptibilities to conventional insecticides. Variability in responses were observed for the tested populations. This was largely dependent on collection date, location and host. Data from these assessments will allow for detection and response to an implied insect resistance event in the Midsouth cropping region.
Quantifying the relationship between the relative susceptibility of bollworm to Bt proteins as measured in laboratory bioassays to the actual survival and damage on Bt plants is necessary to understand the economic impact of insects with varying degrees of susceptibility on cotton. We conducted large field cage experiments to link insect survival and damage on dual- and multi-gene cotton plants with laboratory estimates of two bollworm colonies with different levels of susceptibility to Bt toxins. This study provided evidence that decreased measurements of laboratory susceptibility in bollworms to Bt toxins resulted in higher levels of insect survival and plant damage on dual- and multi-gene Bt cottons relative to a susceptible colony. This information will allow for better predictions of bollworm control in Bt cottons based on laboratory measurements of susceptibility to Bt toxins.
Wild populations of Helicoverpa species from Florida, Iowa, New York, Pennsylvania, Texas, and Virginia were collected for the third consecutive season. Genomic 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.
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.
Accomplishments
1. Completed a study on optimal use of synthetic genome editing reagents in the bollworm, Helicoverpa zea. This is the first study to quantitate the efficiency of fully synthetic CRISPR reagents in an insect species. The results by ARS scientist at Stoneville, Mississippi, indicate that genome editing rates exceeding 90% can be routinely achieved in bollworm using optimal reagent concentrations.
2. A functional analysis of ABCC2 transporter, one of the receptors involved in Bacillus thuringiensis Cry1Ac toxin mode of action in bollworm, was completed using optimized genome editing reagents. Bioassays indicated that complete knock out of the ABCC2 transporter failed to yield high levels of resistance in bollworm. This demonstrated that, although the ABCC2 transporter is a receptor for Cry1Ac, mutations in this receptor alone will not lead to high level of resistance to Cry1Ac in bollworm. A functional analysis of ABCC2 transporter, one of the receptors involved in Bacillus thuringiensis Cry1Ac toxin mode of action in bollworm, was completed using optimized genome editing reagents. Bioassays indicated that complete knock out of the ABCC2 transporter failed to yield high levels of resistance in bollworm. This research by ARS scientist at Stoneville, Mississippi, demonstrated that, although the ABCC2 transporter is a receptor for Cry1Ac, mutations in this receptor alone will not lead to high level of resistance to Cry1Ac in bollworm.
3. A leaf tissue-based assay for heliothine pests of cotton was developed. This assay method by an ARS scientist at Stoneville, Mississippi, will serve as a linkage between meridic diet-based measurements of heliothine susceptibility to transgenic insecticides and survival on dual- and multi-gene Bt cottons.
4. An antibody specific for invasive old world bollworm (OWB), Helicoverpa armigera, was developed and validated. This antibody developed by an ARS scientist at Stoneville, Mississippi, is capable to distinguishing OWB from Helicoverpa zea and other morphologically similar noctuid moths.
Review Publications
Castagnola, A., Jackson, J., Perera, O.P., Oppert, C., Eda, S., Jurat-Fuentes, J. 2017. Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication. Insect Biochemistry and Molecular Biology. 5:e3886. https://doi.org/10.7287/peerj.preprints.2878v1.
Little, N., Mullen, R.M., Allen, K.C., Tyler, H.L. 2017. Leaf tissue assay for lepidopteran pests of Bt cotton. Southwestern Entomologist. 42(4):953-958. https://doi.org/10.3958/059.042.0414.
Gundersen, D.E., Adrianos, S.L., Allen, M.L., Becnel, J.J., Chen, Y., Choi, M.Y., Estep, A., Evans, J.D., Garczynski, S.F., Geib, S.M., Ghosh, S.B., Handler, A.M., Hasegawa, D.K., Heerman, M.C., Hull, J.J., Hunter, W.B., Kaur, N., Li, J., Li, W., Ling, K., Nayduch, D., Oppert, B.S., Perera, O.P., Perkin, L.C., Sanscrainte, N.D., Sim, S.B., Sparks, M., Temeyer, K.B., Vander Meer, R.K., Wintermantel, W.M., James, R.R., Hackett, K.J., Coates, B.S. 2017. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Applications of RNA interference and CRISPR gene editing technologies in pest control. Trends in Entomology. 13:109-137.
Pearce, S., Clark, D., East, P., Elfekih, S., Gordon, K., Jermiin, L., Mcgaughran, A., Oakeshott, J., Papanikolaou, A., Perera, O.P. 2017. Genomic basis for the pest status of two Helicoverpa species. BMC Biology. 15:63. https://doi.org/10.1186/s12915-017-0402-6.
Perera, O.P., Little, N., Pierce III, C.A. 2018. CRISPR/Cas9 mediated high efficiency knockout of the eye color gene vermillion in Helicoverpa zea (Boddie). PLoS One. 13(5):e0197567. https://doi.org/10.1371/journal.pone.0197567.
