Source: IOWA STATE UNIVERSITY submitted to NRP
RISK OF RESISTANCE TO CRY34/35AB1 BY WESTERN CORN ROOTWORM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1017079
Grant No.
2018-33522-28732
Cumulative Award Amt.
$500,000.00
Proposal No.
2018-02699
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Aug 31, 2023
Grant Year
2018
Program Code
[HX]- Biotechnology Risk Assessment
Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
(N/A)
Non Technical Summary
The western corn rootworm, Diabrotica virgifera virgifera, is the most serious pest of corn in the US Corn Belt, resulting in over $1 billion in lost yield and management costs each year. One of the primary tools used to manage western corn rootworm is transgenic Bt corn, which is genetically engineered corn that produces insecticidal protein derived from the bacterium Bacillus thuringiensis (Bt). However, in recent years, western corn rootworm has evolved resistance to three of the four Bt toxins that target this pest (i.e., Cry3Bb1, mCry3A, and eCry3.1Ab) with only one Bt toxin, Cry34/35Ab1, still showing generally consistent efficacy against this pest. In recent years, some cases of western corn rootworm resistance to Cry34/35Ab1 corn have been observed along with high levels of feeding injury Cry34/35Ab1 corn by western corn rootworm. The widespread evolution of Cry34/35Ab1 resistance represents a substantial risk to the continued efficacy of Bt corn targeting western corn rootworm. The goals of this project are to characterize the extent of Cry34/35Ab1 resistance within the landscape by sampling populations of western corn rootworm and conducting bioassays in the laboratory to measure the level of Bt resistance. Follow-up field studies in areas where resistance has been documented will be used to better characterize the effects of resistance on injury to Bt corn, and how to best manage Bt-resistant populations of western corn rootworm. Laboratory strains of Cry34/35Ab1-resistant western corn rootworm will be developed from western corn rootworm collected from fields where Cry34/35Ab1 resistance has been identified. These strains will be used to measure the inheritance of the resistance trait (i.e., dominant versus recessive) and whether or not resistance to Cry34/35Ab1 has associated fitness costs (i.e., whether or not Bt resistant individuals are less fit in the absence of Bt). Both the inheritance of resistance and whether or not fitness costs are present will provide critical information about how quickly Cry34/35Ab1 resistance is expected to evolve in the landscape, and thus, having this information will enable biotech companies and regulators to improve their approach for managing resistance. Finally, this work will use genotype-by-sequencing approaches to identify the genes associated with resistance. This information will enable more efficient monitoring of pest populations for resistance, by testing for specific genetic variants associated with resistance, and will enable the development of future transgenic technologies that target the mechanisms of Cry34/35Ab1 resistance. This research is aligned with program area 5c: understanding how pests overcome plant pest resistance traits conferred by genetic engineering.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110113050%
2111510113050%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1510 - Corn; 3110 - Insects;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
The goal of this project is to improve strategies for managing resistance of western corn rootworm, Diabrotica virgifera virgifera, to transgenic corn that produces the insecticidal protein Cry34/35Ab1 derived from the bacterium Bacillus thuringiensis (Bt). The western corn rootworm is the most serious pest of corn in the US Corn Belt, and beginning in the 1980s has been responsible for over $1 billion in annual costs to US farmers in terms of both lost yield and management costs. In Iowa and elsewhere, resistance by western corn rootworm to Cry34/35Ab1 threatens the utility of Bt corn as a management option for this serious agricultural pest. This threat is of heighted concern in several regions of the US Corn Belt where western corn rootworm has already evolved resistance to three of the four Bt toxins that target this pest, specifically Cry3Bb1, mCry3A, and eCry3.1Ab. The research funded by the grant will combine field and laboratory research to characterize the extent of Cry34/35Ab1 resistance by field populations of western corn rootworm, the effects of this resistance in terms of injury to corn and yield loss in the field, the risks associated with additional cases of resistance developing, and the genetic mechanisms by which western corn rootworm has developed Cry34/35Ab1 resistance. These goals will be achieved by accomplishing the following objectives:Objective 1. Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3)The goal of this objective is to track the evolution of resistance to Cry34/35Ab1 within the agricultural landscape by visiting fields where resistance is suspected, and then conducting bioassays to confirm resistance. Follow-up field experiments will be used to better understand the implications of resistance for root injury to Bt corn and survival of western corn rootworm in the field.Objective 2. Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3)The goal of this objective is to generate laboratory strains of western corn rootworm with field-derived genes for resistance to Cry34/35Ab1. These strains will be used in experiments to determine the genetic inheritance of resistance (i.e., whether resistance is inherited as a recessive or dominant trait) and the extent to which fitness costs accompany resistance (i.e., whether Cry34/35Ab1-resistant individuals are less fit then susceptible individuals in the absence of Bt corn). The risk of resistance to Cry34/35Ab1 is affected by both the inheritance of resistance and the extent to which fitness costs are present. Understanding these factors will allow pest management and resistance management approaches to be adjusted based on the risk of additional cases of resistance developing in the landscape.Objective 3. Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3)In this objective, we will apply cutting-edge genetic analysis approaches to identify the genes and genome regions associated with resistance to Cry34/35Ab1 in western corn rootworm. Such information can be applied to develop more efficient resistance-monitoring methods based on molecular markers, and can lead to the development of novel insecticidal toxins that can be used to kill Bt-resistant pests.
Project Methods
Objective 1. Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3)Fields will be visited in response to reports of high levels of feeding injury by corn rootworm to corn containing Cry34/35Ab1. The goals of the field visits are to 1) sample roots and measure feeding injury by corn rootworm, and 2) sample western corn rootworm adults. Western corn rootworm adults from each field will be housed separately in a small mesh cage to collect eggs. Field populations will be tested for resistance using a plant-based bioassay. Field populations will be tested alongside Bt-susceptible control populations that were collected from throughout the US, and were brought into laboratory culture before Bt corn was commercialized for management of western corn rootworm. Field populations and control populations will be tested against several Bt and non-Bt corn hybrids. We will analyze data on survival in plant-based bioassays with a mixed-model analysis of variance. A significant interaction between hybrid and population type would indicate that survival differed for field populations versus control populations on the various hybrids, and pairwise comparisons will be applied to understand the nature of these differences. Significantly higher survival for field population compared to control populations on a Bt hybrid would indicate resistance to a particular Bt hybrid (for example Cry34/35Ab1 corn or Cry3Bb1 corn). In addition to analyzing survival in plant-based bioassays, we also will analyze data on developmental rate, as measured by the proportion of larvae that reach the third and final instar, to further understand the extent to which populations are resistant to various Bt hybrids. The statistical model used will be the same as that used to measure survival, and pairwise comparisons will be applied to understand the factors contributing to a significant interaction between hybrid and population type. Faster development among field populations compared to control populations on Bt hybrids will provide additional evidence of resistance.During years two and three, we will return to fields that experienced greater than one node of root injury to corn containing the Cry34/35Ab1 protein and we will conduct follow-up field studies. Three fields with a high level of root injury to Cry34/35Ab1 corn during the previous year will be compared to two control field that receive an artificial infestation of a diapausing laboratory strain of western corn rootworm that has never been exposed to Bt corn. At each location both Bt and non-Bt hybrids will be evaluated. Illinois style emergence cages will be placed over live corn plants to measure the number of adults emerging per plant following established methods. Additionally, we will sample roots in each experimental plot and score root injury based on the 0 to 3 node injury scale. We will analyze data on survival to adulthood for western corn rootworm and root injury with mixed-model analysis of variance. A significant hybrid by population interaction would mean there was a difference between population types for how the various corn hybrids affected the response variable (i.e., root injury or rootworm survival). Pairwise comparisons will be conducted to understand the nature of this interaction. Of particular interest will be the extent to which corn with Cry34/35Ab1, either alone or as a pyramid, suffers higher levels of root injury and has a greater number of adult rootworm emerging per plant for field populations compared to control populations.Objective 2. Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3)When fields are visited in response to high levels of feeding injury to Cry34/35Ab1 corn, as part of Objective 1, an additional sample of adult western corn rootworm will be made, and males from these collections will be crossed with virgin non-diapausing females from a Bt-susceptible laboratory strain. The progeny of this initial cross will display the non-diapausing phenotype but will possess field-derived genes for resistance to Cry34/35Ab1. Insects then will be selected on root mats of Cry34/35Ab1 for multiple generations. To measure inheritance of resistance, resistant and susceptible strains will be crossed to generate heterozygous progeny. These heterozygous crosses, along with the parental resistant and susceptible lines, will be reared on Cry34/35Ab1 and non-Bt corn to measure survival to adulthood. Fitness costs will be tested by rearing resistant and susceptible larvae on non-Bt corn. Adult insects will be placed in small cages and housed in an incubator to measure survival and fecundity. Data on survival to adulthood from the experiments measuring inheritance of resistance will be analyzed with mixed-model analysis of variance. A significant strain by hybrid interaction would indicate that the strains differed in their survival on the hybrids. If the heterozygous crosses have greater survival than the susceptible strain on Cry34/35Ab1 corn, this would indicate that resistance to Cry34/35Ab1 corn is non-recessive. We will calculate inheritance to quantify the inheritance of resistance in the selected strains. Data on the various life-history variables collected in the fitness cost experiment (e.g., survival to adulthood, adult longevity and fecundity) will be compared between a Cry34/35Ab1-resisant strain and a susceptible strain with an analysis of variance. If significant effects are present, it will indicate that the means differ between a resistant strain and a susceptible strain, and to the extent that a resistant strain has lower fitness (e.g., lower survival to adulthood or lower fecundity) it will mean that fitness costs accompany resistance to Cry34/35Ab1.Objective 3. Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3)To identify the genetic basis of Cry34/35Ab1 resistance in western corn rootworm, pedigree and genome re-sequencing approaches will be combined. Laboratory strains of Cry34/35Ab1-resistant western corn rootworm will be generated as part of Objective 2. After the initial two generations of selection on Cry34/35Ab1 a sub-strain will be taken from two different stains in Objective 2, selected again on Cry34/35Ab1, and then used for this objective. Sub-strains will be reared on non-Bt corn or Cry34/35Ab1 corn with live adults collected and DNA extracted. Genetic libraries will be constructed based on genotype-by-sequence analysis. In short, this approach will identify the underlying genetic loci by 1) mapping in silico pooled short read sequences separately for survivors on Cry34/35Ab1 corn and non-Bt corn in a bulk analysis of segregating nucleotide polymorphisms; and 2) identifying markers from parental and F2 individual for linkage mapping. Subsequently, a Pool-seq approach will be applied. Genomic DNA from each replicate by treatment will be submitted to the Iowa State University's DNA Facility for ~400-bp insert paired-end library construction. Raw data will be trimmed of low quality sequence, and mapped to the draft genome sequence. Subsequent alignments and SNP predictions will be conducted. Genome scaffold sequences with significantly reduced nucleotide and genetic diversity indices, estimated between pools of Cry34/35Ab1 survivors compared to insects from non-Bt corn, will be deemed putatively linked to gene(s) involved in resistance. The scaffold positions of short reads from GBS libraries in proximity to quantitative trait loci for Cry34/35Ab1 resistance will be used as secondary confirmation of a genome region involved in Cry34/35Ab1 resistance. Genes from the annotated draft genome located within scaffolds linked to resistance will be interrogated for amino acid changes fixed differently between Cry34/35Ab1 survivors compared to insects from non-Bt corn to determine putative Cry34/35Ab1 resistance traits in western corn rootworm.

Progress 09/01/18 to 08/31/23

Outputs
Target Audience:The target audience included agricultural companies, biotechnology companies, extension specialists, farmers, members of the scientific community, regional agronomists, regulators, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of this project, this grant provided training for multiple Ph.D. students who planned and conducted experiments, analyzed data, and presented research results at scientific conferences and in peer-reviewed publications. Additional training opportunities were provided for several undergraduate students who assisted with experiments. Graduate students gained experience in experimental design and data analysis, application of bioassays to measure the resistance of insects to transgenic crops, insect rearing, and molecular analysis. Undergraduate students gained experience with conducting laboratory research and rearing insect strains. Additional training opportunities for graduate students included attending regional and national meetings of the Entomological Society of America to present research results from this work and working with the Project Director to write and publish peer-reviewed journal articles from the results of this research. How have the results been disseminated to communities of interest?Research results were disseminated through research presentations at scientific conferences, including the annual and north central branch meetings of the Entomological Society of America, and through presentations at various US universities. Slides summarizing this research were made available to the department's extension entomologist, Dr. Erin Hodgson, and to Iowa State University field agronomists for use in meetings with clientele. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Western corn rootworm is among the most serious pests of corn in the United States Corn Belt. Each year, US farmers suffer economic losses in excess of two billion dollars as a result of yield losses and management costs associated with corn rootworm. The challenges associated with the management of this pest have increased over time due to the evolution of resistance, particularly resistance to genetically engineered corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Currently, there are four Bt traits available to manage corn rootworm: eCry3.1Ab, mCry3A, Cry3Bb1, and Cry34/35Ab1. However, resistance to three of these four traits is widespread in the Midwest, with Cry34/35Ab1 constituting the only Bt trait that has substantial efficacy against western corn rootworm in the US Corn Belt. Research conducted under this grant has included completing bioassays for western corn rootworm sampled from fields where resistance was suspected due to high levels of rootworm feeding injury to corn with the Cry34/35Ab1 trait. Data from these bioassays will help farmers and crop advisors to more effectively manage western corn rootworm by increasing the knowledge of where Cry34/35Ab1 resistance is present. Managing these more resistant populations with a greater diversity of tactics, such as crop rotation and relying less on the Cry34/35Ab1 trait, should increase the longevity of Cry34/35Ab1 as one management tool that can be used against this serious agricultural pest. Additionally, we conducted research to measure the inheritance and fitness costs of Cry34/35Ab1 resistance. These data will enable estimates of how quickly Cry34/35Ab1 resistance may evolve in additional western corn rootworm populations. With this information, biotech companies and federal regulators can improve their approaches for managing resistance to Bt corn by western corn rootworm. Finally, we developed family pedigrees and recombinant inbred lines from Cry34/35Ab1-resistant strains and are currently completing molecular analyses on the genetic basis of Cry34/35Ab1 resistance, which will enable the development of genetic markers for resistance. Molecular markers will greatly simplify screening western corn rootworm populations for Bt resistance and enable results to be provided to farmers, regulators, and biotech companies in a matter of weeks, as opposed to more than a year with current bioassay approaches. Objective 1... Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3). Over the course of this project, western corn rootworms were sampled from fields that experienced high levels of feeding injury to corn that produced Cry34/35Ab1, either alone or in a pyramid with Cry3. In total, a four-year data set has been generated that contains multiple years of bioassay results and feeding injury to Bt corn in the field. These results will provide valuable information on the prevalence of western corn rootworm resistance to Cry34/35Ab1 and other Bt traits within the agricultural landscape. Farmers, agronomists, and other members of the agricultural community will be able to use this information to modify agricultural practices, specifically reducing the extent to which corn rootworm is managed with the Cry34/35Ab1 trait for multiple growing seasons, which should, in turn, reduce cases of pest resistance and increase the longevity of Cry34/35Ab1 as a tool to manage western corn rootworm. Additionally, federal regulators and biotechnology companies can use these data to better understand the extent of pest resistance in the field and to refine approaches to manage resistance. Objective 2... Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3). For this objective, we generated two laboratory strains of western corn rootworm that possessed field-evolved resistance to Cry34/35Ab1, Waterloo, and Dubuque. These strains came from fields that experienced high levels of feeding injury to corn with Cry34/35Ab1 and were subsequently selected for resistance to Cry34/35Ab1 corn in the laboratory. We have completed our proposed experiments to characterize inheritance and fitness costs associated with Cry34/35Ab1 resistance in western corn rootworm. These results were presented at scientific conferences this year and are currently being prepared for publication. Additionally, we analyzed and published the results of an experiment measuring inheritance and fitness costs for a western corn rootworm strain with laboratory-selected resistance to Cry34/35Ab1. Data from these experiments are important because they can be used to predict how quickly resistance will evolve and spread within the agricultural landscape. Biotechnology companies and federal regulators can use this information to improve resistance management strategies for Bt corn that targets western corn rootworm. Objective 3... Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3). For this objective, we successfully generated multiple family lines (i.e., pedigrees) from both Waterloo and Dubuque, which were subsequently reared on non-Bt corn and Cry34/35Ab1 corn to generate selected and unselected individuals for use in genetic analyses of resistance. Additionally, work was completed propagating and sampling recombinant inbred lines that will be used for complimentary genetic analyses with the pedigrees. Data analysis is still ongoing, but based on preliminary results, these experiments will enable us to identify regions of the western corn rootworm genome and specific genes associated with Cry34/35Ab1 resistance. This will have several benefits to scientists, biotechnology companies, federal regulators, and farmers. These benefits include more rapid identification of resistance traits in western corn rootworm populations, the development of novel transgenic traits that target Bt-resistant individuals, and a more accurate assessment of resistance levels in the field.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Smith, E. M., Shrestha, R. B. and Gassmann, A. J. 2023. Inheritance and fitness costs of laboratory-selected resistance to Gpp34/Tpp35Ab1 corn in western corn rootworm. Journal of Economic Entomology 116:565-573
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Smith, E. M., Shrestha, R., and Gassmann, A. J. 2022. Evaluating inheritance and fitness costs for western corn rootworm with laboratory-selected resistance to Gpp34/Tpp35Ab1 corn. Entomological Society of America. Vancouver, British Columbia, Canada (talk)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Smith, E. M., Coates, B. S., and Gassmann, A. J. 2023. Inheritance and fitness costs of field-evolved resistance to Gpp34/Tpp35Ab1 maize by western corn rootworm (Coleoptera: Chrysomelidae). Entomological Society of America. National Harbor, Maryland (talk)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Smith, E. M., Coates, B. S., and Gassmann, A. J. 2023. Inheritance and fitness costs of field-evolved resistance to Gpp34/Tpp35Ab1 maize by western corn rootworm (Coleoptera: Chrysomelidae). European Congress of Entomology. Heraklion, Crete, Greece (talk)
  • Type: Other Status: Published Year Published: 2023 Citation: Gassmann, A. J. 2023. Challenges and opportunities for management of western corn rootworm with transgenic corn. Iowa State University and Bayer Technology Sharing Meeting. Iowa State University. Ames, IA (Outreach Presentation)


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience included agricultural companies, biotechnology companies, extension specialists, farmers, members of the scientific community, regional agronomists, regulators, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the past year, research conducted under this grant provided training for one Ph.D. student who planned and conducted experiments, and one undergraduate student who assisted with experiments. The Ph.D. student attended both the national and north central branch meetings of the Entomological Society of America and presented research results from this work. How have the results been disseminated to communities of interest?Research results were disseminated through research presentations at the annual and north central branch meetings of the Entomological Society of America and at the International Congress of Entomology. Slides summarizing this research were made available to the department's extension entomologist, Dr. Erin Hodgson, and to Iowa State University field agronomists for use in meetings with clientele. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, laboratory experiments will be conducted to test for the presence of fitness costs of resistance to Cry34/35Ab1 and the inheritance of Cry34/35Ab1 resistance in the Waterloo strain. Molecular analyses will be run to determine genes and molecular markers associated with resistance for family pedigrees that were generated during the past year.

Impacts
What was accomplished under these goals? Western corn rootworm is among the most serious pests of corn in the United States Corn Belt. Each year US farmers suffer economic losses in excess of one billion dollars as a result of yield losses and management costs associated with corn rootworm. The challenges associated with management of this pest have increased over time due to the evolution of resistance, particularly resistance to genetically engineered corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Currently, there are four Bt traits available to manage corn rootworm: eCry3.1Ab, mCry3A, Cry3Bb1 and Cry34/35Ab1. However, resistance to three of these four traits is widespread in the Midwest, with Cry34/35Ab1 constituting the only Bt trait that has efficacy against western corn rootworm in the US Corn Belt. Over the past year, research conducted under this grant has included conducting bioassays for western corn rootworm sampled from fields where resistance was suspected due to high levels of rootworm feeding injury to corn with the Cry34/35Ab1 trait. Data from these bioassays will help farmers and crop advisors to more effectively manage western corn rootworm by increasing the knowledge of where Cry34/35Ab1 resistance is present. Managing these more resistant populations with a greater diversity of tactics, such as crop rotation and relying less on the Cry34/35Ab1 trait, should increase the longevity of Cry34/35Ab1 as one management tool that can be used against this serious agricultural pest insect. Additionally, during the past year, we conducted research to test the inheritance and fitness costs of Cry34/35Ab1 resistance. These data will enable estimates of how quickly Cry34/35Ab1 resistance may evolve in additional western corn rootworm populations. With this information, biotech companies and federal regulators can improve their approaches for managing resistance to Bt corn by western corn rootworm. Finally, we developed family pedigrees and recombinant inbred lines from Cry34/35Ab1-resistant strains, which will enable us to conduct molecular analyses on the genetic basis of Cry34/35Ab1 resistance and develop genetic markers for resistance. Molecular markers will greatly simplify screening western corn rootworm populations for Bt resistance, and enable results to be provided to farmers, regulators and biotech companies in a matter of weeks, as opposed to more than a year with current bioassay approaches. Objective 1... Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3). During the fall of 2021, we completed scoring bioassays for populations sampled during 2020. This included quantifying larval survival on Bt corn for western corn rootworm populations that were sampled from fields that experienced high levels of feeding injury to corn that produced Cry34/35Ab1, either alone or in a pyramid with Cry3. As part of this grant, a three-year data set has been generated that contains multiple years of bioassay results for western corn rootworm populations sampled from fields with high levels of feeding injury to Bt corn. These results will provide valuable information on the prevalence of western corn rootworm resistance to Cry34/35Ab1 and other Bt traits within the agricultural landscape. Farmers, agronomists and other members of the agricultural community will be able to use this information to modify agricultural practices, specifically reducing the extent to which corn rootworm is managed with the Cry34/35Ab1 trait for multiple growing seasons, which should in turn reduce cases of pest resistance and increase the longevity of Cry34/35Ab1 as a tool to manage western corn rootworm. Objective 2... Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3). During the first year of this study, two laboratory strains of western corn rootworm were generated that possessed field-evolved resistance to Cry34/35Ab1, Waterloo and Dubuque. We continued to culture these strains in the laboratory, rearing larvae on Cry34/35Ab1 corn to maintain the field evolved resistance traits in these populations. These strains were used extensively to advance research under this objective and under objective 3. For this objective, we conducted an experiment to measure fitness costs of resistance to Cry34/35Ab1 corn in the Dubuque strain. Previously, we measured the inheritance of Cry34/35Ab1 resistance in this strain. Additionally, data analysis was conducted on the results of an experiment measuring inheritance in fitness costs for a western corn rootworm strain with laboratory-selected resistance to Cry34/35Ab1. Data from these experiments are important because they can be used to predict how quickly resistance will evolve and spread within the agricultural landscape. Biotech companies and regulators can use this information to refine how Bt resistance by western corn rootworm is managed. Objective 3... Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3). During the previous reporting period, multiple family lines (i.e., pedigrees) were generated from both Waterloo and Dubuque. During this reporting period, these pedigrees were reared on non-Bt corn and Cry34/35Ab1 corn to generate selected and unselected individuals that were used in genetic analyses of resistance. Additionally, work was completed propagating and sampling recombinant inbred lines that will be used for complimentary genetic analyses with the pedigrees. These genetic analyses will be conducted during the upcoming year and will enable the identification of genes and molecular markers that are associated with Cry34/35Ab1 resistance. This will have several benefits to scientists, biotech companies, federal regulators and farmers. These benefits include more rapid identification of resistance traits in western corn rootworm populations, development of novel transgenic traits that target Bt resistance individuals, and a more accurate assessment of resistance levels in the field.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Gassmann, A. J. 2022. Pest adaptation in a transgenic landscape: western corn rootworm and Bt maize. International Congress of Entomology, Helsinki, Finland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Gassmann, A. J., Shrestha, R. B. and St. Clair, C. R. 2021. Western corn rootworm resistance to Bt corn within the midwestern agricultural landscape. Entomological Society of America. Denver, Colorado, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Smith, E., Shrestha, R., and Gassmann, A. J. 2022. Evaluating fitness costs in western corn rootworm with laboratory-selected resistance to Gpp34Ab1/Tpp35Ab1 corn. North Central Branch Meeting, Entomological Society of America. Minneapolis, Minnesota
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Smith, E., Coates, B., and Gassmann, A. J. 2021. Characterizing the inheritance of resistance to Bacillus thuringiensis Gpp34Ab1/Tpp35Ab1 insecticidal protein in western corn rootworm. Entomological Society of America. Denver, Colorado


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience included agricultural companies, biotechnology companies, extension specialists, farmers, members of the scientific community, regional agronomists, regulators, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the past year, research conducted under this grant provided training for three Ph.D. students who planned and conducted experiments, and one undergraduate student who assisted with experiments. Due to COVID-19, the students involved in this research did not attend any scientific meetings during this reporting period, but will attend multiple scientific conferences during the coming year. How have the results been disseminated to communities of interest?Research results were disseminated through a symposium presentation at the annual meeting of the Entomological Society of America, and two research presentations at US universities (all were given virtually due to COVID-19). Slides summarizing this research were made available to the department's extension entomologist, Dr. Erin Hodgson, and to Iowa State University field agronomists for use in meetings with clientele. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, laboratory experiments will be conducted to test for the presence of fitness costs of resistance to Cry34/35Ab1 and the inheritance of Cry34/35Ab1 resistance. Molecular analyses will be run to determine molecular markers associated with resistance for family pedigrees that were generated during the past year.

Impacts
What was accomplished under these goals? IMPACT: Western corn rootworm arethe most serious pests of corn in the United States Corn Belt. Each year US farmers suffer economic losses in excess of one billion dollars as a result of yield losses and management costs associated with corn rootworm. The challenges associated with management of this pest have increased over time due to the evolution of pesticide resistance, particularly resistance to genetically engineered corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Currently, there are four Bt traits available to manage corn rootworm: eCry3.1Ab, mCry3A, Cry3Bb1 and Cry34/35Ab1. However, resistance to three of these four traits is widespread within the Midwest, with Cry34/35Ab1 constituting the only Bt trait that remains generally effective against western corn rootworm within the US Corn Belt. Over the past year, research conducted under this grant has included visits to fields where resistance is suspected due to high levels of rootworm feeding injury to corn with the Cry34/35Ab1 trait. In addition to quantifying feeding injury to Cry34/35Ab1, we collected samples of adult western corn rootworm, the progeny of which were used in bioassays to test for resistance to various Bt traits including Cry34/35Ab1. Data from these field visits and bioassays will help farmers and crop advisors to more effectively manage western corn rootworm by increasing the knowledge of where Cry34/35Ab1 resistance is present. Managing these more resistant populations with a greater diversity of tactics, such as crop rotation and relying less on the Cry34/35Ab1 trait, should increase the longevity of Cry34/35Ab1 as one management tool that can be used against this serious agricultural pest insect. During the past year, we also developed single family pedigrees from Cry34/35Ab1-resistant strains that were initiated in year one of this project. These family pedigrees will enable us to conduct molecular analyses on the genetic basis of Cry34/35Ab1 resistance during the coming year, and develop genetic markers for resistance. Molecular markers will greatly simplify screening western corn rootworm populations for Bt resistance, and enable results to be provided to farmers, regulators and biotech companies in a matter of weeks, as opposed to more than a year with current bioassay approaches. Objective 1... Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3). During 2020, fields in Iowa were visited in response to high levels of feeding injury from western corn rootworm to corn that produced Bt toxin Cry34/35Ab1. Roots were sampled to quantify rootworm feeding injury and adults were sampled for subsequent bioassays to quantify resistance to Bt corn within these populations. Additionally, during the spring and summer of 2021, populations sampled during 2020 were tested for resistance to Cry34/35Ab1 and other Bt traits in transgenic corn targeting corn rootworm. As part of this grant, a three-year data set has been generated that contains multiple years of bioassay results for western corn rootworm populations sampled from fields with high levels of feeding injury. These results will provide valuable information on the prevalence of western corn rootworm resistance to Cry34/35Ab1 and other Bt traits within the agricultural landscape. Farmers, agronomists and other members of the agricultural community will be able to use this information to modify agricultural practices, specifically reducing the extent to which corn rootworm is managed with the Cry34/35Ab1 trait for multiple growing seasons, which should in turn reduce cases of pest resistance and increase the longevity of Cry34/35Ab1 as a tool to manage western corn rootworm. Objective 2... Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3). During the first year of this study, two laboratory strains of western corn rootworm were generated that possessed field-evolved resistance to Cry34/35Ab1, Waterloo and Dubuque. These strains were used extensively during the past year to advance research under objective 3, and will be used during the coming year to complete research under this objective. To date, we have successfully measured the inheritance of Cry34/35Ab1 resistance in one strain, Dubuque. Results from this experiment found that resistance was non-recessive, and this is important because population-genetics theory predicts that resistance traits that are inherited in a non-recessive manner evolve more quickly than those that are inherited in a recessive manner. As such, there appears to be a heighted risk of additional populations of western corn rootworm evolving resistance to Cry34/35Ab1. Biotech companies and regulators can use this information to refine how Bt resistance by western corn rootworm is managed. Objective 3... Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3). During the past year, substantial progress was made under this objective. Multiple family lines (i.e., pedigrees) were generated from both Waterloo and Dubuque. These pedigrees were propagated for multiple generations, with individuals from each pedigree then reared on either non-Bt corn or Cry34/35Ab1 corn. Genetic analysis of these individuals during the coming year will enable the identification of genes and molecular markers associated with Cry34/35Ab1 resistance. This will have several benefits to scientists, biotech companies, federal regulators and farmers. These benefits include more rapid identification of resistance traits in western corn rootworm populations, development of novel transgenic traits that target Bt resistance individuals, and a more accurate assessment of resistance levels in the field.

Publications

  • Type: Other Status: Published Year Published: 2021 Citation: Texas A&M University. Department of Entomology. 2021. Resistance to Bt corn by western corn rootworm: effects of pest biology, the pest-crop interaction, and the agricultural landscape on resistance. (presentation given remotely due to COVID-19)
  • Type: Other Status: Published Year Published: 2021 Citation: Michigan State University. Department of Entomology. 2021. Resistance to Bt corn by western corn rootworm: effects of pest biology, the pest-crop interaction, and the agricultural landscape on resistance. (presentation given remotely due to COVID-19)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Gassmann, A. J., St. Clair, C. R., Shrestha, R. B. and Kropf, A. L. 2020. Western corn rootworm and Bt maize: effects of the pest-crop interaction and pest biology on resistance. Entomological Society of America. Virtual conference due to COVID-19.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The target audience included agricultural companies, biotechnology companies, extension specialists, farmers, members of the scientific community, regional agronomists, regulators, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Research conducted under this grant has provided training for two Ph.D. students who planned and conducted experiments, and two undergraduate students who assisted with experiments. Additionally, one Ph.D. student attended the national meeting of the Entomological Society of America and gave a poster presentation on their Ph.D. research, which they will carry out as part of this grant. How have the results been disseminated to communities of interest?Research results were disseminated through the publication of a peer-reviewed journal article, a symposium presentation at the annual meeting of the Entomological Society of America, and an invited presentation at a meeting focused on pest resistance, which was held at Rothamsted Research in Harpenden, England. Slides summarizing this research were made available to the department's extension entomologist, Dr. Erin Hodgson, and to Iowa State University field agronomists for use in meetings with clientele. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, bioassay will be conducted to test for resistance to Cry34/35Ab1 and other Bt traits in populations of western corn rootworm that were sampled throughout Iowa during 2020. These populations were collected from fields where a high level of rootworm feeding injury was observed to corn that produces Cry34/35Ab1, and resistance to Cry34/35Ab1 is suspected. Laboratory experiments will be conducted to test for the presence of fitness costs of resistance to Cry34/35Ab1 and the inheritance of Cry34/35Ab1 resistance traits. Family pedigrees will be initiated to enable experiments aimed at determining molecular markers associated with resistance.

Impacts
What was accomplished under these goals? Overall impact statement: Western corn rootworm is the most serious pests of corn in the United States Corn Belt. Each year US farmers suffer economic losses in excess of one billion dollars as a result of yield losses and management costs associated with corn rootworm. The challenges associated with management of this pest have increased over time due to the evolution of resistance, particular resistance to genetically engineered corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Currently, there are four Bt traits available to manage corn rootworm: eCry3.1Ab, mCry3A, Cry3Bb1 and Cry34/35Ab1. However, resistance to three of these four traits is widespread within the Midwest, with Cry34/35Ab1 constituting the only Bt trait that remains generally effective against western corn rootworm within the US Corn Belt. Over the past year, research conducted under this grant has included visits to fields where resistance is suspected due to high levels of rootworm feeding injury to corn with the Cry34/35Ab1 trait. In addition to quantifying feeding injury to Cry34/35Ab1, we collected samples of adult western corn rootworm which will enable us to run bioassays to test for resistance. Furthermore, bioassays were conducted for populations sampled in 2019. Data from these field visits and bioassays will help farmers and crop advisors to more effectively manage western corn rootworm by increasing the knowledge of where Cry34/35Ab1 resistance is present. Managing these more resistant populations with a greater diversity of tactics, such as crop rotation, and relying less on the Cry34/35Ab1 trait should increase the longevity of Cry34/35Ab1 as one management tool that can be used against this serious agricultural pest insect. During the past year, we also undertook research to test the inheritance and fitness costs of Cry34/35Ab1 resistance. These data will enable estimates of how quickly Cry34/35Ab1 resistance may evolve in additional western corn rootworm populations. With this information, biotech companies and federal regulators can improve their approaches for managing resistance to Bt corn by western corn rootworm. Objective 1... Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3). Fields throughout Iowa were visited in response to high levels of feeding injury from western corn rootworm to corn that produced Bt toxin Cry34/35Ab1. Roots were sampled to quantify rootworm feeding injury and adults were sampled for subsequent bioassays to quantify resistance to Bt corn within these populations. Additionally, multiple populations sampled during 2019 were tested for resistance to Cry34/35Ab1 and other Bt traits in transgenic corn targeting corn rootworm. Results from these bioassays will provide information on the occurrence of western corn rootworm resistance to Cry34/35Ab1 and other Bt traits within the agricultural landscape. Farmers, agronomists and other members of the agricultural community can use this information to modify agricultural practices, specifically reducing the extent to which corn rootworm is managed with the Cry34/35Ab1 for multiple growing season, which should in turn reduce cases of pest resistance and increase the longevity of Cry34/35Ab1 as a tool to manage western corn rootworm. Objective 2... Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3). During the first year of this study, two laboratory strains of western corn rootworm with field-evolved resistance to Cry34/35Ab1, Waterloo and Dubuque, were generated. During the past year, for one of these strains, we conducted experiments to characterized the genetic inheritance of Cry34/35Ab1 resistance and the extent to which this resistance has any accompanying fitness costs. Both of these parameters are key factors that affect how quickly a pest population develops resistance to Bt corn in the field. Information gained through this research objective will enable biotech companies and federal regulators to improve strategies for managing resistance to Bt corn by western corn rootworm. Objective 3... Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3). Research under this objective will be initiated during the third year of the project. The insect strains that will be used for this research were successfully generated in the laboratory during year 1. These strains, Waterloo and Dubuque, were produced by collecting Cry34/35Ab1 resistant insects from the field and crossing them into a non-diapausing background, thereby enabling more rapid study in a laboratory setting. During year three, these strains will be used in molecular analyses to determine the genetic basis of Cry34/35Ab1 resistance, which will have several benefits to scientists, biotech companies, federal regulators and farmers. These benefits include more rapid identification of resistance traits in populations, development of novel transgenic traits that target Bt resistance individuals, and a more accurate assessment of resistance levels in the field.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Gassmann, A. J. 2019. Field-evolved resistance to Bt maize by western corn rootworm: patterns of resistance, potential causes and management approaches. Resistance '19. Rothamsted Research, Harpenden, England.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Smith, E., Coates, B., and Gassmann, A. J. 2019. Evaluating resistance to Cry34/35Ab1 by western corn rootworm. Entomological Society of America. St. Louis, Missouri
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Gassmann, A. J., Shrestha, R. B., Kropf, A. L., St. Clair, C. R. and Brenizer, B. D. 2020. Field-evolved resistance by western corn rootworm to Cry34/35Ab1 and other Bacillus thuringiensis traits in transgenic maize. Pest Management Science 76:268-276
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Gassmann, A. J. 2019. Bt resistance by western corn rootworm and the potential for IPM to delay resistance. Entomological Society of America. St. Louis, Missouri, USA.


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:The target audience included agricultural companies, biotechnology companies, extension specialists, farmers, members of the scientific community, regional agronomists, regulators, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Research conducted under this grant has provided training for two Ph.D. students who planned and conducted experiments, and four undergraduate students who assisted with experiments.Additionally, one Ph.D. student attended the national meeting of the Entomological Society of America and gave a poster presentation on their Ph.D. research, which they will carry out as part of this grant. How have the results been disseminated to communities of interest?Research results were disseminated through the publication of a peer-reviewed journal article, and presentations at the annual meeting of the Entomological Society of America, including a keynote talk as part of a symposium organized by the insecticide resistance action committee (IRAC). Slides summarizing this research were made available to the department's extension entomologist, Dr. Erin Hodgson, and to Iowa State University field agronomists for use in meetings with clientele. What do you plan to do during the next reporting period to accomplish the goals?During the coming year, bioassay will be conducted to test for resistance to Cry34/35Ab1 and other Bt traits in populations that have been sampled throughout Iowa, during 2019, from fields where a high level of rootworm feeding injury was observed to corn that produces Cry34/35Ab1. Laboratory experiments will be initiated to test for the presence of fitness costs of resistance to Cry34/35Ab1 and the inheritance of Cry34/35Ab1 resistance traits. Family pedigrees will be initiated for future experiments to determine molecular markers that are associated with resistance.

Impacts
What was accomplished under these goals? Overall impact statement: Western corn rootworm is the most serious pest of corn in the United States Corn Belt. Each year US farmers suffer economic losses in excess of one billion dollars, as a result of yield losses and management costs associated with corn rootworm. The challenges associated with management of this pest have increased over time due to the evolution of resistance, particular resistance to genetically engineered corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Currently, there are four Bt traits available to manage corn rootworm: eCry3.1Ab, mCry3A, Cry3Bb1 and Cry34/35Ab1. However, resistance to three of these four traits is widespread within the Midwest, with Cry34/35Ab1 constituting the only Bt trait that remains effective against western corn rootworm within the US Corn Belt.Research conducted under this grant has identified some western corn rootworm in Iowa with resistance to Cry34/35Ab1 and all other Bt traits, while other populations remain susceptible to Cry34/35Ab.This information will help farmers and crop advisors to more effectively manage western corn rootworm by increasing the diversity of tactics they apply to manage western corn rootworm, such as crop rotation, and relying less on the Cry34/35Ab1 trait. This, in turn, should increase the longevity of Cry34/35Ab1 as one management tool that can be used against this serious agricultural pest insect.Research that will be undertaken in the second year of this project will test the inheritance and fitness costs thus enabling estimates of how quickly Cry34/35Ab1 resistance may evolve in additional western corn rootworm populations.With this information, biotech companies and federal regulations can improve their approaches for managing resistance to Bt crops by western corn rootworm and other key insect pests in the United States. Objective 1... Measure resistance to Cry34/35Ab1 in field populations of western corn rootworm (Years 1-3). Multiple populations were tested for resistance to Cry34/35Ab1 and other Bt traits in transgenic corn targeting corn rootworm. Results of these assays revealed that some populations in Iowa were resistant to Cry34/35Ab1, and were resistant to all other Bt traits in transgenic corn that target western corn rootworm. Importantly, in some cases, these fields were associated with multiple years of corn cultivation and continuous use of the same Bt traits.Farms, agronomists and other members of the agricultural community can use this information to modify agricultural practices, specifically reducing the extent to which corn rootworm are managed with the Cry34/35Ab1 for multiple growing season, which should in turn reduce cases of pest resistance and increase the longevity of Cry34/35Ab1 as a tool to manage western corn rootworm. Objective 2... Characterize the inheritance and fitness costs of resistance to Cry34/35Ab1 (Years 1-3). Two laboratory strains, Waterloo and Dubuque, were initiated using western corn rootworm with field-evolved resistance to Cry34/35Ab1. These strains used adult western corn rootworm that were collected from fields where a high level of feeding injury to Cry34/35Ab1 corn was observed in that growing season.These field-collected adults were subsequently crossed with a non-diapausing laboratory strain of western corn rootworm.Unlike western corn rootworm from the field, which have only one generation per year, a non-diapausing strain has six or more generations per year, which increases the amount of research that can be conducted in a year. Thus, introgression of field-derived genes for resistance into a non-diapausing background will enhance our ability to study this emerging resistance in a laboratory setting.Currently, both strains are being selected on Cry34/35Ab1 corn in the laboratory to increase the level of resistance in the strains. During the coming months these strains will be used to quantify the genetic inheritance of Cry34/35Ab1 resistance and the extent to which this resistance has any accompanying fitness costs.Both of these parameters are key factors that affect how quickly a pest population develops resistance to Bt corn in the field Information gained through this research objective will enable biotech companies and federal regulators to improve strategies for managing resistance to Bt corn by western corn rootworm. Objective 3... Conduct genetic analyses to determine the genetic basis of resistance to Cry34/35Ab1 (Years 2-3). Research under this objective will be initiated during the second year of the project.The insect strains that will be used for this research have been successfully generated in the laboratory during year 1.These strains, Waterloo and Dubuque, were produced by collecting Cry34/35Ab1 resistant insects from the field and crossing them into a non-diapausing background, thereby enabling more rapid study in a laboratory setting.During years two and three, these strains will be used in molecular analyses to determine the genetic basis of Cry34/35Ab1 resistance, which will have several benefits to scientists, biotech companies, federal regulators and farmers. These benefits include more rapid identification of resistance traits in populations, development of novel transgenic traits that target Bt resistance individuals, and a more accurate assessment of resistance levels in the field.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Gassmann, A. J. 2019. Bt resistance by western corn rootworm and the potential for IPM to delay resistance. Entomological Society of America. St. Louis, Missouri, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Smith, E., Coates, B., and Gassmann, A. J. 2019. Evaluating resistance to Cry34/35Ab1 by western corn rootworm. Entomological Society of America. St. Louis, Missouri (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: ) Gassmann, A. J. and Coates, B. S. 2019. Risks of resistance to Cry34/35Ab1 by western corn rootworm. Annual Project Directors Meeting for Biotechnology Risk Assessment Grants Program. Riverdale, MD
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gassmann, A. J., Shrestha, R. B., Kropf, A. L., St. Clair, C. R. and Brenizer, B. D. 2019. Field-evolved resistance by western corn rootworm to Cry34/35Ab1 and other Bacillus thuringiensis traits in transgenic maize. Pest Management Science 76:268-276