Location: Corn Insects and Crop Genetics Research
2019 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
Monarch butterfly (Danaus plexippus) populations have declined precipitously over the last two decades, largely attributed to declines of milkweed populations in agricultural landscapes across the monarch’s breeding range. A study was initiated using a combination of free flight and laboratory flight mill experiments to elucidate the monarch adult’s perceptual range for locating food and oviposition resources, particularly prairie plants for nectaring and milkweed plants for egg laying. This information will help determine the most efficient ways to spatially distribute habitat in the agricultural landscape where land that can be kept out of crop production is limited. The proximity of such habitat to crops presents some potential risks, including exposure to agrochemicals. Another study was initiated to determine whether female monarchs avoid laying eggs on milkweed that has absorbed a systemic insecticide, imitating possible runoff from neonicotinoid seed treatments. Preliminary results indicate that the presence of imidacloprid did not influence oviposition in female monarchs. This suggests that females are either incapable of detecting imidacloprid or oviposition is not based on its presence. Follow-up studies will examine if foliar application of insecticides influence female oviposition behavior.
Research is continuing towards developing strategies for increasing habitat for monarch butterflies to counter loss of milkweeds in herbicide-tolerant corn and soybeans. The third season is underway for 45 monarch/pollinator habitats that were planted November 2016, mostly on growers’ non-crop lands. Optimal habitat establishment occurred when weed populations were reduced before a fall planting. Weed seed banks will be problematic for some areas. Planting into bluegrass that had been killed with glyphosate or soybean stubble produced habitats with low weed pressure. A manuscript was published related to monarch caterpillar growth, development and oviposition on nine milkweed species native to Iowa. All the milkweed species were useful to monarch butterflies, but common milkweed, Asclepias syriaca, and swamp milkweed, A. incarnata, stood out as the most valuable. Another study investigated effects of different densities (one, five and ten plants per 25 m2) of common and swamp milkweed on monarch oviposition. Female monarchs oviposited in all three patch densities when given a choice of three patches. Optimal patch size, however, will depend on the goals of the land manager and their interest in a higher number of eggs per stem or a higher number of eggs per patch. A second year of a study was continued to determine the best way to establish common milkweed into brome grass areas.
Corn is a preferred host of fall armyworm and the pest has become resistant to several classes of insecticide and Bt corn grown in certain areas. There is a need to discover new sources of plant resistance to this pest to expand control options available to corn producers. In 2018, a study was conducted to test corn germplasm collected from Saint Croix, U.S. Virgin Islands, for resistance to leaf feeding by fall armyworm. Plants were grown in the field and artificially infested at a high level. Damage by fall armyworm feeding on leaves at 7 and 14 days differed significantly for the 13 corn genotypes tested. Fall armyworm feeding scores at 14 days for Saint Croix Group 1, Saint Croix Group 3, Saint Croix 2, and Saint Croix 7 were moderately resistant and not significantly different from one another. Individual plants in these populations were variable for resistance to leaf feeding, and scored between resistant and susceptible. Current efforts are focused on selecting within these populations for greater resistance to fall armyworm feeding on leaves.
Efforts continue to understand the genetic and biochemical mechanisms underpinning the corn earworm silk feeding resistance in corn Piura 208. A composite population from (GT119 x Piura 208) F1:2 families was used to generate >500 advanced intercross doubled haploid lines. Most of these doubled haploid lines were successfully seed increased in 2018.
The evolution of insecticide resistance in pest arthropod populations threatens the sustainability of corn production, and research to uncover the molecular mechanisms was conducted. A genome assembly for the western corn rootworm was completed after inclusion of read data from proximity-ligated library sequencing, and structural gene annotation was completed via the National Center for Biotechnology Information pipeline. Manual community annotation was organized by ARS and is ongoing using tools at the i5K-Workspace@NAL. Genome re-sequencing data was collected from pools of western corn rootworm differing in Bt Cry3Bb1 or organophosphate resistance, and data processing and alignment to the genome assembly are being conducted to predict genome regions under selection and associated with resistance. Candidate Bt resistance genes were annotated from a western corn rootworm transcriptome constructed from adult, egg and larval stages, and function of these genes was determined via targeted knockdown by RNA interference (RNAi). The whole genome sequence assembly of the western bean cutworm was improved by the addition of long-read sequence data. RNA sequencing data is being collected for gene annotation and applications to determine the genetic basis of western bean cutworm field-evolved Cry1F resistance. Soybean aphid biotype-specific mutations were identified within genome re-sequencing data mapped to the reference whole genome assembly. A subset of soybean aphid variant nucleotide sites were validated and shown to be capable of differentiating biotypes. Also, direct DNA sequencing and a derived genetic marker for a specific mutation in the voltage gated sodium channel gene of soybean aphid was associated with field-evolved pyrethroid resistance. Genetic markers for soybean aphid biotype and resistance are being evaluated and may be useful for future evaluation of insect resistance management strategies. Within a Drosophila insecticide resistance model, the differential expression of microRNAs was correlated with a subset of differential expression of stress response genes, and differentially-expressed transcripts showed biased enrichment for those associated with mitochondrial function. Inducible transgenic RNAi-based gene knockdown experiments are being conducted to decipher the role of endogenous genes in mediating insecticide resistance. Ecological interaction can determine gene flow in pest insect populations and impact the spread of resistance alleles. Genome re-sequencing data from the European corn borer detected divergent regions on the Z-chromosome between strains that differ in diapause duration and identified specific mutations in two genes that function epistatically to influence the induction and duration corn borer overwintering. The development of single locus markers for European corn borer population screening are being developed and will be important tools for differentiating subpopulations and providing data to model the efficacy of insect resistance management models.
Laboratory bioassays documented that corn earworm collected from problem fields showing damage to Bt corn showed elevated resistance to Cry1Ac and tolerance to Cry1F toxins. This was the first case of such resistance in the Corn Belt. Laboratory experiments are ongoing to determine the inheritance of this corn earworm resistance trait. Populations of western bean cutworm were screened for survival on Vip3A corn tissues. Enzyme-linked immunosorbent assay (ELISA) results determined the variation in Cry1F toxin levels in corn tissues at plant development stages when western bean cutworm feed, and are important for estimating larval exposures during selection within the field. Experiments to determine the rate of cross pollination of non-Bt refuge corn plants by transgenic Bt pollen were initiated, with future results having implications in the creation of low-dose Bt exposures that may exacerbate the rate of resistance development in pests that feed on corn ears.
Accomplishments
1. Larval density affects dispersal of western corn rootworm. Resistance to transgenic Bt corn has developed in many populations of western corn rootworm threatening major economic losses. Knowledge gaps in dispersal behavior of western corn rootworm hinder our ability to predict resistance spread and to develop effective mitigation. Laboratory flight mills were used by ARS researchers from Ames, Iowa to characterize female western corn rootworm flight performance as a function of larval rearing density. Females from larvae reared under crowded conditions engaged in greater flight activity than those reared at low density. The results suggest that females emerging from high density populations in cornfields, including from Bt-resistant populations, are more likely to disperse, and disperse farther, than those from low density populations. Taking the effects of different dispersal rates into account in models used by regulatory agencies, industry, and public-sector scientists will be important for improving resistance management strategies for this pest.
2. Characterization of a trait that confers corn silk resistance to corn earworm. Corn earworm is a pest of corn causing yield and quality losses through herbivory and disease vectoring. The discovery and characterization of new plant resistance sources is vital to protect corn hybrids from future economic losses to this pest. To aid this effort, ARS researchers in Ames, Iowa developed a quantitative bioassay and tested it on four corn populations with contrasting alleles from corn Piura 208 (corn earworm resistant) with those from inbred line GT119 (corn earworm susceptible). The bioassay effectively determined that corn genotype accounted for 84% and 68% of the variance in corn earworm larval weights, and up to 60% of the variance in corn earworm pupal development, demonstrating both the success of the quantitative bioassay and the strength of the Piura 208 resistance mechanism. The bioassay also revealed backcrossing the corn lines resulted in incremental diminishing effects on corn earworm development, suggesting complex (i.e. multi-genic) inheritance of a threshold-dependent mechanism. This study demonstrated the utility of the new bioassay for corn pest researchers while providing useful information about the Piura 208 derived resistance factor to enable corn breeders the ability to incorporate the factor into elite germplasm to protect the crop from future damage by this pest.
Review Publications
Steele, L.D., Coates, B.S., Seong, K., Valero, C.M., Mittapalli, O., Clark, J.M., Pittendrigh, B.R. 2018. Variation in mitochondria-derived transcript levels associated with DDT resistance in the 91-R strain of Drosophila melanogaster (Diptera: Drosophilidae). Insect Science. 18(6). https://doi.org/10.1093/jisesa/iey101.
Roberson, H.M., Walden, K.O., Ruzzante, L., Reijnders, M., Waterhouse, R.M., Coates, B.S., Legeai, F., Gress, J.C., Biyiklioglu, S., Weaver, D.K., Wanner, K.W., Budak, H. 2018. Genome sequence of the wheat stem sawfly, Cephus cinctus, representing an early-branching lineage of the Hymenoptera, illuminates evolution of hymenopteran chemoreceptors. Genome Biology and Evolution. 10(11):2997-3011. https://doi.org/10.1093/gbe/evy232.
Abel, C.A., Van Roekel, J.W., Wilson, R.L. 2019. Cuphea lanceolata and Cuphea ignea seed increase using three pollinators in insect-proof cages in the field. Southwestern Entomologist. 44(1):95-98. https://doi.org/10.3958/059.044.0110.
Abel, C.A., Coates, B.S., Scott, M.P. 2019. Evaluation of maize germplasm from Saint Croix for resistance to leaf feeding by fall armyworm. Southwestern Entomologist. 44(1):99-103. https://doi.org/10.3958/059.044.0111.
Seong, K., Coates, B.S., Pittendrigh, B.R. 2019. Impacts of sub-lethal DDT exposures on microRNA and putative target transcript expression in DDT resistant and susceptible Drosophila melanogaster strains. Frontiers in Genetics. https://doi.org/10.3389/fgene.2019.00045.
Coates, B.S., Dopman, E.B., Wanner, K.W., Sappington, T.W. 2018. Genomic mechanisms of sympatric ecological and sexual divergence in a model agricultural pest, the European corn borer. Current Opinion in Insect Science. 26:50-56. https://doi.org/10.1016/j.cois.2018.01.005.
Pocius, V.M., Pleasants, J.M., Debinski, D.M., Bidne, K.G., Hellmich II, R.L., Bradbury, S.P., Blodgett, S.L. 2018. Monarch butterflies show differential utilization of nine Midwestern milkweed species. Frontiers in Ecology and Evolution. 6:169. https://doi.org/10.3389/fevo.2018.00169.
