Research Project: Ecologically-based Management of Arthropods in the Maize Agroecosystem

Location: Corn Insects and Crop Genetics Research

2024 Annual Report

Objectives
Objective 1: Improve knowledge of the genetics, genomics, ecology, and behavior of key maize insect pests as they affect abundance and pest resistance to insecticidal agents, including those expressed in transgenic maize. Sub-objective 1.A. Determine genomic architecture of phenotypic traits within and among reproductive and ecological variants of European corn borer. Sub-objective 1.B. Determine how insect movement and genetics impact potential for development of resistance to GE insecticidal toxins. Sub-objective 1.C. Develop genomic and computational resources for study of key maize pests. Objective 2: Identify and functionally dissect contributions of maize alleles that confer host plant resistance to lepidopteran pests. Sub-objective 2.A. Identify contributing alleles and specialized metabolites conferring resistance to silk-feeding by corn earworm. Sub-objective 2.B. Characterize resistance and develop doubled haploid inbred lines of maize with leaf activity against fall armyworm. Objective 3: Determine potential impacts of farming practices in maize agro-ecosystems on the ecology and management of both arthropod pests and non-pests such as monarch butterflies and bees. Sub-objective 3.A. Develop a risk-based decision support tool for managing sporadic insect pests of seedling maize. Sub-objective 3.B. Develop strategies for improving monarch butterfly and bee habitat in agricultural landscapes.

Approach
European corn borer, corn rootworm, and western bean cutworm are the most serious pests of maize in the Corn Belt, while corn earworm and fall armyworm are major pests in the southern half of the United States. Genetically-engineered (GE) maize is an important management tool for these insect pests, but they have evolved resistance to GE crops in many areas, seriously threatening their continued effectiveness. This project will take an integrated approach toward developing management strategies and tools to use against these insect pests with emphases on insect resistance management to GE maize, insect ecology, insect genetics and genomics, and native host plant resistance. We will address critical knowledge gaps including maize insect pest population genetic dynamics and genomic function, dispersal behavior, efficacy of insect resistance monitoring, and insect resistance management strategies, including ways to incorporate a diversity of pest suppression tactics. Native host plant resistance to control insect pests can serve the latter function, and provide a cost-efficient sustainable management tool for growers who choose not to use GE maize. This project will study native resistance in maize to insect pests, particularly corn earworm and fall armyworm, so that low-input control options can be developed. Given concerns on the indirect effects of farming practices on non-target arthropods, including bees and monarch butterflies, project scientists will work with stakeholders to develop strategies for increasing habitat for monarch butterflies to counter loss of milkweeds in maize and soybean fields. In addition, researchers will provide growers a decision support tool to allow realistic assessment of when the use of seeds coated with neonicotinoid insecticide is justified in their particular fields to control sporadic seedling pests and when it is not, reducing overall insecticide input. Collectively, this research will result in maize pest management systems that are stable and reliable, cost effective for producers, and safe for growers, consumers and the environment.

Progress Report
In support of Objective 1, research to understand the genetic, genomic, population-level and ecological factors contributing to the development and spread of insecticide resistance in key maize pests was continued. Farmers grow maize hybrids genetically engineered (GE) to produce one or more Bacillus thuringiensis (Bt) insecticidal proteins to manage damage caused by corn earworm (CEW), European corn borer (ECB), western corn rootworm (WCR), and western bean cutworm (WBC). However, each of these pests have developed resistance to Bt proteins, resulting in economic losses and threatening the sustainability of GE maize. This has occurred despite use of resistance management tactics, including the use of non-Bt refuges which allow susceptible insects to emerge and mate with resistant insects to reduce the incidence of resistance in the next generation. Impacts of ecological variation in ECB on gene flow and potential for spread of Bt resistance. ECB has two races that differ in the chemical sex-attractant (pheromone) emitted by females and preferentially responded to by males, and ecotypes that differ in the larval development after diapause (a state of suspended development similar to hibernation) that determines number of annual reproductive generations (voltinism). The geographic range of pheromone races overlaps in the northeast United States. Mating between pheromone races is most reduced when populations also differ in voltinism. We showed that two genes involved in circadian rhythm determine ECB voltinism and the gene governing male pheromone response are linked on the sex chromosome. In some ECB strains, part of the sex chromosome is inverted. This inversion is most frequent in populations that differ in both pheromone race and voltinism, suggesting this inversion may limit gene exchange. The impact of this inversion on the spread of insecticide resistance genes between pheromone strains or voltinism types was unknown. Therefore, we developed three genome assemblies for ECB strains that differed in pheromones and/or voltinism, allowing the identification of genes located in the inversion. The research demonstrated that local gene expression was impacted by the inversion, indicating the inversion also impacts gene function. Furthermore, all genes located in the inversion do not recombine during sexual reproduction, meaning that they are inherited as a group in ECB populations. Although genes conferring resistance to Bt proteins are not located in the inversion, the inversion may impede the transfer of Bt resistance between populations that differ in pheromone race and voltinism. These ECB genomes assemblies were also used to investigating Bt resistance. Specifically, DNA sequence variation in inbred ECB lines was greatest between Cry1F resistant and susceptible larvae in a narrow region of the genome where a gene predicted to function in cell repair is located. This information is being used to develop genetic markers for detecting resistance in field populations. Impact of diapause on ECB migratory behavior. Each growing season, one generation of ECB adults arises from larvae that have remained dormant for several months in diapause to survive the winter, while the next generation of adults arise from larvae that have not gone through diapause. Diapause is a major physiological event in the life history of alternate generations, and it is unknown whether the resulting adults behave differently than those arising from direct development. We have placed several cohorts of ECB larvae, from an ARS laboratory colony, into diapause for emergence months later. Reproductive output (number of egg masses and duration of egg-laying period) of the resultant adult females will be compared to that of directly-developed adults from the same colony. This fundamental information is necessary to help characterize ECB movement ecology, because the portion of moths in a population that migrate do so during the pre-egg-laying period and mostly before mating. This pattern is characteristic of the “oogenesis-flight syndrome” common, but not universal, in migratory insects. We recently documented that ECB is a partial migratory species with >90% of females engaging in aseasonal, undirected migration away from their natal field before laying eggs, but most of the evidence is from adults that emerged from non-diapause larvae. If timing of egg-laying and/or eggs laid differ in the diapausing generation, it will be important information for parameterizing models of resistance development and mitigation of resistance to transgenic insecticidal corn. Insect resistance management (IRM) tactics impact growth and development of Bt susceptible CEW and WBC. A Bt IRM tactic used in the Corn Belt constitutes planting a blend of Bt corn seed with 5 or 10% non-Bt refuge corn, referred to as refuge in a bag (RIB). The non-Bt RIB plants ideally will produce a very large number of Bt susceptible insects that will mate with rare Bt resistant insects, effectively diluting out resistance in the next generation. However, cross-pollination of kernels on non-Bt RIB plants by pollen from adjacent Bt plants results in low-dose Bt expression in those kernels. The effect of low Bt doses on survival of susceptible CEW and WBC larvae remains unclear. Therefore, WBC and CEW larvae were fed artificial diets with differing amounts of kernel tissue from Bt, non-Bt, and non-Bt RIB plants. Larvae fed kernels from Bt and RIB refuge plants weighed significantly less than those fed non-Bt kernels, with no significant difference between larvae fed Bt or RIB refuge. These results indicate that Bt in cross-pollinated refuge kernels reduces the growth rate of susceptible insects, but whether this impacts their mating with resistant counterparts and effectiveness of resistance management strategies remains unknown. Identification of mutations associated with insecticide resistance. Research funded by a USDA-NIFA Crop Protection and Pest Management and conducted in collaboration with Iowa State University scientists used RNA interference (RNAi) to validate mutations in a gene expressed in the nervous system of insects are responsible for resistance to pyrethroid insecticides. Genetic markers that detect these mutations determined the distribution of resistance across Iowa, with information conveyed to extension agents. In collaboration with an ARS researcher in Stoneville, Mississippi, pyrethroid insecticide resistance in CEW from a Colorado field population in 2023 was linked to a gene acquired from a related species that is invasive in the U.S. This resistance gene was also detected in Iowa field populations from 2023 and at lower frequency in 2021. The WCR genome assembly generated in part by our team is being used for similar purposes. Specifically, we have developed genetic markers for two genes linked to resistance to GE maize expressing the Bt Cry3Bb1 protein and are currently validating the function of these two genes in Cry3Bb1 resistance using RNAi. Second, short high-throughput DNA sequence data from WCR resistant to Cry34/35Ab1 Bt maize were aligned to the WCR genome assembly and are currently being used to predict mutations associated with Bt resistance. Candidate genes putatively involved in Bt resistance were also identified in a chromosome-level genome assembly of the southern corn rootworm (SCR), completed in cooperation with the ARS Ag100Pest Initiative. Genes involved in mating and host plant preferences in SCR are being discovered in collaboration with ARS scientists in Columbia, Missouri. The corn planthopper (CPH) is an emerging pest in the eastern United States that causes reduced yields due to direct feeding and transmission of corn pathogenic viruses. Survival of this insect relies upon symbiotic bacteria in its cells to make certain essential vitamins and amino acids. To discover these critical genes, we cooperated with scientists at North Carolina State University to generate a chromosome-level genome assembly for CPH that included entire genomes for four different bacteria harbored by CPH. We compared genes in CPH and bacterial genomes to reveal those exclusively in the bacteria that produced essential dietary components. These genome assemblies are resources for identifying genes required for insect survival and developing novel insecticidal targets for pest control. Identifying and characterizing maize genes that confer host plant resistance to lepidopteran pests. In support of Objective 2 we continued examining the use of host plant resistance to control fall armyworm in maize. The first year of a two-year multi-location study evaluated fall armyworm leaf feeding resistance in inbred lines derived from maize population BS39 and GEMN-0095. Two derived inbred lines from BS39 performed as well as the resistant check, Mp708, at all locations tested. These inbred lines, along with moderately resistant genotypes 53SS4/GEMS-0026, B111/SCRO1, GEMN-0140/GEMN-0097, Ki3, NEI9008:NO826, and Suwan 1, were used as founders in a population development program that was initiated by making F1 crosses in 2023. After four cycles of intermating (planned for 2025 and 2026) and doubled haploid line production, this population will be used to identify sequence variants associated with leaf feeding resistance to fall armyworm. In related research, 362 maize inbred lines from two association panels (Nested Association Mapping and Ames Diversity Panel) were infested with CEW, ECB, and fall armyworm, and leaf feeding injury recorded. These inbred lines were also evaluated for the vegetative stage when the plants transitioned from immature to mature leaf production. This phenotypic information will be used to select maize genotypes for a large genome wide association study to identify genomic regions associated with resistance to leaf injury from lepidopterous insect pests.

Accomplishments
1. Insecticide resistance in corn earworm (CEW) acquired from an invasive species. The corn earworm (CEW) causes feeding damage to corn kernels, affecting yield and quality, especially in the sweet corn market. The old-world bollworm (OWBW) is native to Europe, Asia and Africa, and has invaded countries in South America and the Caribbean. Mating between OWBW and closely related CEW produces viable hybrids, allowing exchange of genes. A gene from OWBW that confers resistance to pyrethroid insecticides was previously detected in CEW populations in South America. ARS scientists in Ames, Iowa and Stoneville, Mississippi, detected the OWBW pyrethroid resistance gene at high frequency among pyrethroid resistant CEW from a field location in Colorado. This resistance gene was also detected at a high frequency among CEW from Iowa field populations collected in 2023, and at lower frequency among CEW in 2021. The degree to which this resistance gene has been integrated into the CEW population in the United States, as well as any impact on the efficacy of current management practices remains unknown, but is being addressed in research coordinated by ARS and APHIS along with university cooperators. This finding of emergence of pyrethroid resistance in CEW will be used to develop grower recommendations and evaluate IRM strategies.

2. Discovery and characterization of aseasonal, undirected migration in pest insects aids insect resistance management. Several insect pest species migrate in spring from southern locations where they can survive the winter, to northern locations where they exploit superabundant seasonal host crops. In contrast, many insect species survive hostile winter conditions in the north by entering diapause (a state of dormancy), and thus have no need to migrate between overwintering and breeding ranges. However, ARS researchers at Ames, Iowa have discovered a novel insect migration pattern called aseasonal, undirected migration. For some individuals of several pest species, migratory flight is nondirectional and occurs within the larger year-round distribution where others of its species already exist. Insect pests exhibiting this behavior include the European corn borer (ECB) and western corn rootworm (WCR), the two most destructive pests of corn in the U.S. In parts of North America, ECB and WCR have developed resistance to transgenic corn varieties widely used by farmers to control them. The spatial scale at which individuals move and intermate within the larger population contributes to the rate at which resistance allele spread, and thus has important implications for pest management practices, insect resistance management (IRM) strategies, and resistance mitigation programs. This information is being shared through presentations and consultations with regulatory, university, government, and industry scientists in the United States and Canada who will use the information to improve models and assessment of IRM strategies to combat resistance to control tactics.

Review Publications
Blanco, C., Hernandez, G., Conover, K., Portilla, M., Valentini, G., Alvarado, A., Fosado, A., Abel, C.A., Dively, G., Guzman, H., Israel, L., Occelli, L., Knolhoff, L., Corona, M., Vega, P., Macias, P., Ward, T., Urbano, N. 2024. Functional transgenes in Mexican maize: benefit and risks for insect pest management in Mexico and the United States. Annals of the Entomological Society of America. https://doi.org/10.1093/aesa/saae007.
Kar, S., Nagasubramanian, K., Elango, D., Carroll, M.E., Abel, C.A., Nair, A., Mueller, D.S., O'Neal, M.E., Singh, A.K., Sarkar, S., Ganapathysubramanian, B., Singh, A. 2023. Self-supervised learning improves classification of agriculturally important insect pests in plants. The Plant Phenome Journal. 6(1). https://doi.org/10.1002/ppj2.20079.
Dively, G.P., Kuhar, T.P., Taylor, S., Doughty, H.B., Holmstrom, K., Gilrein, D., Nault, B.A., Ingerson-Mahar, J., Huseth, A., Edward, T., Abel, C.A., Coates, B.S., et al. 2023. Extended sentinel monitoring of Helicoverpa zea resistance to Cry and Vip3Aa toxins in Bt sweet corn: Assessing changes in phenotypic and allele frequencies of resistance. Insects. 14(7):577. https://doi.org/10.3390/insects14070577.
Blanco, C., Conover, K., Dively, G., Hernandez, G., Portilla, M., Nava-Camberos, U., Abel, C.A., Williams, P., Hutchison, W. 2023. Severe Defoliation of Vegetative Maize Plants Does Not Reduce Grain Yield: Further Implications with Action Threshold. Southwestern Entomologist. https://doi.org/10.3958/059.048.0404.
Sappington, T.W., Spencer, J.L. 2023. Movement ecology of adult western corn rootworm; implications for management. Insects. https://doi.org/10.3390/insects14120922.
Yanarella, C.F., Fattel, L., Kristmundsdottir, A.Y., Lopez, M.D., Edwards, J.W., Campbell, D.A., Abel, C.A., Lawrence-Dill, C.J. 2024. Wisconsin diversity panel phenotypes: spoken descriptions of plants and supporting data. BMC Research Notes. 17. Article 33. https://doi.org/10.1186/s13104-024-06694-y.
Wang, Y., Yao, Y., Zhang, Y., Qian, X., Guo, D., Coates, B.S. 2024. A chromosome-level genome assembly of the soybean pod borer: Insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein. BMC Genomics. 25. https://doi.org/10.1186/s12864-024-10216-2.
Sappington, T.W. 2024. Critical facets of European corn borer adult movement ecology relevant to mitigating field resistance to Bt-corn. Insects. https://doi.org/10.3390/insects15030160.
Sappington, T.W. 2024. Aseasonal, undirected migration in insects: 'invisible' but common. iScience. 27. https://doi.org/10.1016/j.isci.2024.110040.
Wang, Y., Mikaelyan, A., Coates, B.S., Lorenzen, M.D. 2024. The genome of Arsenophonus sp. and its potential contribution in the corn planthopper, Peregrinus maidis. Insects. 15(2). https://doi.org/10.3390/insects15020113.