Location: Corn Insects and Crop Genetics Research
2017 Annual Report
Objectives
Objective 1: Improve knowledge of the ecology, genetics, and behavior of key corn pests, especially corn rootworm and lepidopteran species, such as European corn borer, corn earworm and western bean cutworm, in relation to pest abundance and insect resistance to transgenic corn.
Sub-objective 1.A. Correlate genetic markers with phenotypic traits of interest in European corn borer, western corn rootworm and western bean cutworm.
Sub-objective 1.B. Determine how larval movement and adult dispersal influence insect resistance to transgenic corn.
Objective 2: For corn agro-ecosystems, determine potential impacts of changing farming practices on the demographics and ecology of pest and non-pest arthropods, such as the monarch butterfly.
Sub-objective 2.A. Assess the potential value of neonicotinoid insecticide seed treatments to growers of major row crops under different agronomic conditions.
Sub-objective 2.B. Develop strategies for improving monarch butterfly habitat in modern farm landscapes.
Objective 3: Characterize genetic and biochemical responses associated with corn defenses to rootworm and lepidopteran caterpillar pest injury to enhance conventional and transgenic crop protection strategies.
Sub-objective 3.A. Develop genetic markers and genomic tools for western corn rootworm, European corn borer, western bean cutworm, and other pests of corn.
Sub-objective 3.B. Characterize genetic regulation of surface lipids on corn silks and assess protective capacity of these lipids on corn earworm feeding.
Approach
Field-resistance in western corn rootworm (WCR) to Cry3Bb1 Bt toxins and in European corn borer (ECB) to Cry1Fa and Cry1Ab toxins will be mapped by using genotyping-by-sequencing (GBS) protocols and single nucleotide polymorphism (SNP) markers. Pedigrees will be constructed from Bt resistant and susceptible individuals of both species. GBS will be performed by constructing genomic DNA libraries from non-size-selected fragments, to which adapters with a unique barcode are ligated. A population mapping approach using a high density of SNP markers will be taken to identify loci that differ significantly between E- and Z-race ECB. The premise of population mapping is similar to quantitative trait loci (QTL) mapping, but SNPs with skewed frequencies between races are assumed to result from either selection for race-specific adaptations or genetic drift facilitated by restricted gene flow. GBS-derived SNP markers will be used to estimate gene flow among WCR populations. The strategy will be to estimate Wright's neighborhood area for WCR, the radius of which constitutes a measure of the typical distance genes move per generation. Dispersal and survival rates of fall armyworm (FAW) will be measured in field plot trials. FAW egg masses will be placed in the whorl of vegetative-stage corn plants surrounded by uninfested plants. Living and dead larvae on plants will be counted periodically using destructive sampling. Planting arrangements will include various combinations of Bt and non-Bt isoline plants. Flight behavior and capacity of WCR will be characterized and compared across three Cry3Bb1-resistant and two susceptible strains using flight mills to determine the degree to which resistance affects dispersal. In collaboration with other ARS laboratories and as a NP304 effort, a comprehensive review of the literature will be conducted to provide information on the usefulness of seed treatment with neonicotinoid insecticide in controlling target pests and protecting crops from yield loss. A series of studies will be conducted related to milkweed species selection (larval performance and oviposition preference), and milkweed plant establishment (determining patch sizes and maximizing sustainability of milkweeds). Initially, studies will focus on four Asclepias species found in Iowa: common milkweed, A. syriaca, swamp milkweed, A. incarnata, butterfly weed, A. tuberosa, and whorled milkweed, A. verticillata. Also, studies will be conducted to determine how to maintain these plants in the landscape while avoiding loss from plant competition. This will require identifying the best companion plants for the targeted milkweed species. Two sources of corn germplasm will be used to screen for silk activity against corn earworm (CEW). These corn lines and CEW resistant checks will be grown in the field. Emerged silks will be harvested, immediately frozen in liquid nitrogen, lyophilized, and ground to a powder using a knife mill. One cohort of powdered silks will be used for surface lipid metabolic analysis, while another will be used for CEW bioassays, allowing the metabolomic analysis and CEW feeding studies to be conducted in parallel.
Progress Report
Resistance of western corn rootworm to transgenic Bacillus thuringiensis (Bt)-corn has been increasing in the Corn Belt, emphasizing the urgent need for better understanding of this pest's dispersal behavior, which strongly influences rate of resistance development and the rate at which resistance spreads after it develops. Genotyping-by-sequencing has been completed for adults collected in fields along two 120-mile transects in the species' native home range of eastern Colorado and western Kansas for both 2014 and 2016. Analyses of the 2014 samples indicate ~87.5% of the rootworm population dispersed an average of only 0.28km over their lifetime; however, the remaining 12.5% of the population migrated out of the natal field traveling up to 200km or further. Comparison of DNA profiles from the same locations between 2014 and 2016 is underway and will provide an independent temporal estimate of dispersal distances. Three novel viruses have been discovered that infect western corn rootworm and may provide an opportunity for developing management options. Comparison of viruses in collections of rootworms from several locations in 2015 including Iowa, eastern Colorado, and Pennsylvania, with earlier collections in 2013 and 2014, indicate large variability in virus prevalence from year to year within a location. Serious questions have arisen about the safety of neonicotinoid seed treatments of corn, soybean, cotton, and wheat to pollinators, especially honey bees, yet their use on seed is very widespread. Farmers tend to rely on them as "insurance" treatments against sporadic pests but the actual benefit derived is unclear. A major collaborative effort led by an ARS scientist in Ames, Iowa has completed reviews of the vast literature on the prevalence and risk posed by these pests, which have revealed the factors, sometimes region-dependent, that put certain fields at increased risk of infestation. The importance of considering individually-subeconomic pressure from multiple pests is now clear, but this aspect of pest management represents a major knowledge gap and formidable research challenge. To help address this knowledge gap, published and unpublished data are being collected and a meta-analysis will be performed to determine the value of neonicotinoid seed treatments to the farmer based on pest pressure and region of the country.
There was a western bean cutworm outbreak in Bt Cry1F hybrid cornfields in the Eastern Corn Belt during 2016. High levels of Cry1F toxin resistance were documented among western bean cutworm collected from Nebraska and Indiana, and a stakeholder response is being coordinated through the NC246 technical working group. Full-genome sequence data were collected from these insects and computational methods applied to filter and assemble reads from infecting microsporidia. Efforts are ongoing to assemble and annotate the western bean cutworm genome as well as to estimate gene expression differences in the midgut of resistant larvae. Laboratory screening of fall armyworm larvae documented high variation in levels of Cry1F toxin resistance and efforts are ongoing to describe the genetic mechanisms involved. A draft of the western corn rootworm genome was nearly finalized by joining fragmented gene coding regions into scaffolds and additional gene expression data from adult, egg and larval stages were generated to assist in annotation. Western corn rootworm genome scaffolds were applied for mapping short read genome sequence data from Cry3Bb resistant and susceptible larvae, which identified putative genome regions involved in evolution of the trait. Size estimates and reduced representation sequencing of the corn earworm genome described repetitive genome regions and are resources for a genome sequencing project. Research on European corn borer was aimed to estimate barriers to gene flow in the context of evolution and subsequent spread of Bt resistance. This past year: 1) an inversion was discovered on the Z-chromosome, which varies in frequency between populations of different voltinism (generations per year) and male pheromone reception phenotypes, and which contains gene(s) predicted to cause these differences; 2) influence of the highly divergent pheromone gland fatty acyl reductase (pgfar) gene was estimated with respect to the ecologically distinct E- and Z-strains: results indicated that gene flow remains high between strains, which homogenizes genetic variation in regions of the genome not located next to the pgfar gene; and 3) the degree of hybridization was estimated between European corn borer and closely related Asian corn borer in a region of China where the species co-exist.
A corn earworm bioassay was developed and tested to elucidate the genetic and biochemical basis of a new source of maize resistance to silk feeding derived from Peru landrace, Piura 208 (PI 503849). Four maize populations were developed from Piura 208 x GT119, a susceptible maize inbred, to test the bioassay. In replicated analyses of two populations of F1:2 families, corn genotype accounted for 84% and 68% of the variance in corn earworm larval weights, respectively, demonstrating both the success of the quantitative bioassay and the strength of the Piura 208 silk-feeding resistance mechanism. Corresponding analysis of two populations of BC1:2 families revealed no statistically significant effects of corn genotype, suggesting multigenic inheritance of resistance and/or a failure to capture key resistant alleles in backcrossing, two possibilities whose contributions could not be determined in this study. Technical factors in bioassay performance were assessed, primarily by analyzing 1600 corn earworm larvae raised on control diet (meridic with no corn silks added). Minor, but statistically significant contributions to corn earworm weight gain variance were revealed for factors in the preparation, incubation and evaluation phases of the bioassay, demonstrating the importance of randomization, stratification, replication, and variable-tracking across the many steps of the quantitative bioassay. Together, these findings indicate that the novel corn earworm resistance originating in Piura 208 is experimentally tractable using the bioassay as described and alleles already captured in the F1:2 families. Plans for using the bioassay for silk-resistance trait characterization and introgression into elite maize breeding lines are being developed.
Research is continuing towards developing strategies for increasing habitat for monarch butterflies to counter loss of milkweeds in corn and soybeans. Collaborations with grower groups has been facilitated by partnering with the Iowa Monarch Conservation Consortium. Forty-five monarch/pollinator habitats were planted November 2016 that were mostly associated with growers’ non-crop lands. Experiments were completed to investigate monarch caterpillar growth and development on nine milkweed species native to Iowa. A study to determine oviposition preferences of monarch females for the nine milkweed species was completed and another study is continuing for a third season to investigate effects of common and swamp milkweed densities on monarch oviposition.
The widespread use of Cry proteins in transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. One area of particular interest is the possible detection of Cry protein fragments by antibodies used in enzyme-linked immunosorbent assay (ELISA) kits. A study was conducted to develop a model system approach to generate environmentally relevant fragments. Seven different types of model systems were screened for their ability to generate fragments of the Cry1Ab protein of which five reliably generated Cry1Ab fragments. ELISA and bioassays with European corn borer were used to analyze these five model systems. The fragments from four were not detectable by ELISA and did not retain bioactivity. In another model system, the fragments generated were detectable by ELISA and retained bioactivity. The reason for this is not yet known. Despite this result, ELISAs appear to provide an accurate estimation of the amount of Cry proteins in the environment, as detectable fragments retained bioactivity, and non-detectable fragments did not retain bioactivity.
A review paper was published about the use of surrogate species to understand the potentially harmful effects of toxic pesticides including possible effects of proteins contained in genetically engineered insect resistant (GEIR) crops. Species commonly used in GEIR crop testing include beneficial organisms such as honeybees, arthropod predators, and parasitoids. The choice of appropriate surrogates is influenced by scientific factors, such as the knowledge of the mode of action and the spectrum of activity, as well as societal factors, such as protection goals that assign value to certain ecosystem services such as pollination or biological control. The primary reasons for using surrogates include the inability to test all possible organisms, the restrictions for using certain organisms in testing (e.g., rare, threatened, or endangered species), and the ability to achieve greater sensitivity and statistical power by using laboratory testing of certain species. The acceptance of surrogate species data can allow results from one region to be applied or “transported” for use in another region. On the basis of over a decade of using surrogate species to evaluate potential effects of GEIR crops it appears that the current surrogates have worked well to predict effects of GEIR crops that have been developed and it is expected that they should work well to predict effects of future GEIR crops based on similar technologies.
Accomplishments
1. Prevalence and risk factors associated with sporadic insect pests of seedling corn targeted by neonicotinoid seed treatments. Most corn planted in the U.S. is protected from several sporadic early-season pests by neonicotinoid seed treatments, usually without knowledge of the threat posed in a given field. Farmers often take a preventative management approach because it is impractical to directly assess populations of many of these unpredictable pests, but there is growing concern that indiscriminant use of these seed treatments are having negative environmental effects. ARS scientists in Ames, Iowa; Brookings, South Dakota; and Stoneville, Mississippi; undertook an extensive literature review of these sporadic pests to clarify the prevalence of economic infestations in different regions of the U.S., and the agronomic, biotic, and abiotic factors that affect the likelihood of attack. The summaries of prevalence and risk factors presented in a series of papers will help farmers and consultants better assess the value of preventative protection of seedling corn under the conditions at hand, and provide regulators and others with a better understanding of the complex issues involved in decision making at the farm-level.
2. RNAi does not persist in aquatic environments, suggesting this tactic poses a minimal risk to aquatic organisms. RNA interference (RNAi) represents the next wave of pest management technologies but the control tactic’s impact on non-target organisms needs to be fully understood. RNAi is initiated by double-stranded RNAs (dsRNA) that are ingested by the target pest and cleaved into small-interfering RNAs that silence a molecular target in the pest. The environmental fate of a non-bioactive dsRNA, representative of future insecticidal dsRNAs, was determined in three aquatic microcosms. The dsRNA was rapidly degraded in each microcosm, and was undetectable after 96 hours. Additionally, separation of the dsRNA into layers was not a major factor in the dissipation of dsRNA from water. Therefore, it can be expected that dsRNA will not persist in aquatic environments, and its use in agricultural settings should have little environmental impact on aquatic organisms.
Review Publications
Petzold-Maxwell, J.L., Siegfried, B.D., Hellmich II, R.L., Abel, C.A., Coates, B.S., Spencer, T.A., Horikoshi, R.J., Gassmann, A.J. 2017. Fitness costs associated with Cry1F resistance in the European corn borer. Journal of Applied Entomology. 141(1-2):67-79. doi:10.1111/jen.12356.
Li, X., Zhang, F., Coates, B.S., Zhang, Y., Zhou, X., Cheng, D. 2016. Comparative profiling of microRNAs in the winged and wingless English grain aphid, Sitobion avenae (F.) (Homoptera: Aphididae). Scientific Reports. 6:35668. doi: 10.1038/srep35668.
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.
Wenger, J.A., Cassone, B.J., Legeai, F., Johnston, S., Bansal, R., Yates, A.D., Coates, B.S., Michel, A. 2017. The whole genome sequence assembly of the soybean aphid, Aphis glycines. Insect Biochemistry and Molecular Biology. doi:10.1016/j.ibmb.2017.01.005.
Cheng, Y., Luo, L., Sappington, T.W., Jiang, X., Zhang, L., Frolov, A.N. 2016. Onset of oviposition triggers abrupt reduction in migratory flight behavior and flight muscle in the female beet webworm, Loxostege sticticalis. PLoS One. 11(11): e0166859. doi:10.1371/journal.pone.0166859.
Kozak, G.M., Wadsworth, C.B., Kahne, S.C., Bogdanowicz, S.M., Harrison, R.G., Coates, B.S., Dopman, E. 2017. A combination of sexual and ecological divergence contributes to the spread of a chromosomal rearrangement during initial stages of speciation. Molecular Ecology. 26(9):2331-2347. doi:10.1111/mec.14036.
Liu, S., Chen, Y., Sappington, T.W., Bonning, B.C. 2017. Genome sequence of the first coleopteran iflavirus isolated from western corn rootworm, Diabrotica virgifera virgifera LeConte. Genome Announcements. 5(6):e01530-16. doi: 10.1128/genomeA.01530-16.
Liu, S., Chen, Y., Sappington, T.W., Bonning, B.C. 2017. Genome sequence of Diabrotica virgifera virgifera virus 2, a novel small RNA virus of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Genome Announcements. 5(20):e00365-17. doi: 10.1128/genomeA.00365-17.
Liu, S., Chen, Y., Sappington, T.W., Bonning, B.C. 2017. Genome sequence of a novel positive sense, single-stranded RNA virus isolated from western corn rootworm, Diabrotica virgifera virgifera LeConte. Genome Announcements. 5(20):e00366-17. doi: 10.1128/genomeA.00366-17.
Qin, J., Jiang, X., Zhang, L., Sappington, T.W., Cheng, Y., Luo, L. 2017. Population projection and development of the loreyi leafworm, Mythimna loreyi, as affected by temperature: application of an age-stage, two-sex life table. Journal of Economic Entomology. 110(4):1583-1591. doi:10.1093/jee/tox138.
Zhang, T., Yaqi, S., Wanner, K., Coates, B.S., He, K., Wang, Z. 2017. Binding affinity of five PBPs to Ostrinia sex pheromones. BioMed Central (BMC) Molecular Biology. 18(1):4. doi: 10.1186/s12867-017-0079-y.
Sun, W., Huynh, B.L., Ojo, J.A., Coates, B.S., Kusi, F., Roberts, P.A., Pittendrigh, B.R. 2017. Comparison of complete mitochondrial DNA sequences between old and new world strains of the cowpea aphid, Aphis craccivora (Hemiptera: Aphididae). AGRI GENE. 4:23-29. doi:10.1016/j.aggene.2017.03.003.
Xie, W., Chen, C., Yang, Z., Guo, L., Wang, D., Chen, M., Huang, J., Wen, Y., Zeng, Y., Liu, Y., Yang, X., Xia, J., Tian, L., Cui, H., Wu, Q., Wang, S., Xu, B., Li, X., Tan, X., Ghanim, M., Qui, B., Pan, H., Chu, D., Delatte, H., Maruthi, M.N., Ge, F., Zhou, X., Wang, X., Wan, F., Du, Y., Luo, C., Yan, F., Preisser, E.L., Jiao, Z., Coates, B.S., Zhao, J., Gao, Q., Xia, J., Yin, Y., Liu, Y., Brown, J.K., Zhou, X., Zhang, Y. 2017. Genome sequencing of the sweet potato whitefly Bemisia tabaci MED/Q. Gigascience. 5(6):1-7. doi: 10.1093/gigascience/gix018.
Su, H., Tian, J., Naranjo, S.E., Romeis, J., Hellmich II, R.L., Shelton, A.M. 2014. Bacillus thuringiensis plants expressing Cry1Ac, Cry2Ab and Cry1F are not toxic to the assassin bug, Zelus renardii. Journal of Applied Entomology. 139:23-30.
Guo, Y., Tian, J., Shi, W., Dong, X., Romeis, J., Naranjo, S.E., Hellmich Ii, R.L., Shelton, A. 2015. The interaction of two-spotted spider mites, Tetranychus urticae Koch, with Cry protein production and predation by Amblyseius andersoni (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize. Transgenic Research. 25(1):33-44.
Miller, N.J., Sappington, T.W. 2017. Role of dispersal in resistance evolution and spread. Current Opinion in Insect Science. 21:68-74. doi: 10.1016/j.cois.2017.04.005.
Albright, V.C., Hellmich II, R.L., Coats, J.R. 2016. A review of Cry protein detection with enzyme-linked immunosorbent assays. Journal of Agricultural and Food Chemistry. 64(11)2175-2189. doi:10.1021/acs.jafc.5b03766.
