Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Farmers, agribusiness, scientists studying insect pests of soybeans, insecticide resistance, sustainable agriculture. Changes/Problems:We have been delayed in conducting the amplicon sequencing of our aphid samples. This delay is because several of the samples had very low concentrations of DNA. Multiple bouts of PCR were needed to amplify the DNA to levels suitable for amplification. We could use a no-cost extension as amplicon sequencing will likely extended into the summer of 2024. What opportunities for training and professional development has the project provided?Funding from this project was used to partially fund an MS student in Entomology, Matthew Pfab, and PhD student, Bruna Wojahn. This training included field work, PCR and bio-assays to test insecticide resistance phenotype. Funding was also used to hire undergraduates to maintain soybean aphid colonies used to confirm that the molecular markers confirm presence of mutations associated with insecticide resistance. How have the results been disseminated to communities of interest?We have shared our results at several meetings noted above. This include presentations by our co-PIs (Dr. Brad Coates) and students (Matthew Pfab). What do you plan to do during the next reporting period to accomplish the goals?We need to complete the amplicon DNA sequencing. This is time-consuming and dependent upon the availability of sequencing equipment through the ISU DNA sequencing center. We also need to compile our data and submit manuscripts for peer-reviewed publications.
Impacts
What was accomplished under these goals?
Objective. 1: Discover genetic markers for lambda-cyhalothrin resistance within soybean aphid populations. (Years 1-2) We have accomplished this objective and published these results (Valmorbida et al. 2022a, 2022b) Objective. 2: Use molecular marker(s) to describe changes in the frequency of pyrethroid resistance among field collected soybean aphids. (Years 2-3) We have finished the field work portion of this objective, collecting soybean aphids from commercial fields in the northern third of Iowa. The aphids collected from these fields have been processed for amplicon DNA sequencing. This processing has resulted in over 4000 individual aphids that will be sequenced. From these individuals we will determine for each individual the type and number of mutations found within them that are associated with insecticide resistance. These specimens have been sent to a sequencing center and we are awaiting the samples to be processed to begin analyzing these data to determine the frequency of aphid-resistance before and after insecticides were applied in a commercial, conventional cropping system. We have also completed the field work associated with the insecticide evaluations conducted by co-PI Dr. Erin Hodgson. This experiment was conducted during the summer and included 4 replications of the following insecticides applied to small plots of soybeans (10ft by 40 ft): lambda-cyhalothrin, thiamethoxam (as a seed treatment), thiamethoxam (seed treatment) and lambda-cyhalothrin, thiamethoxam (foliar-applied and lambda-cyhalthorin, Pyrifluquinanzon (1.2 fl oz), Pyrifloquinanzon (1.6 fl oz), as well as an untreated control. Aphids were counted and collected from these plots before and after insecticide applications. These aphids were processed for amplicon sequencing. Preliminary analysis of the field data revealed evidence of lambda-cyhalothrin resistant aphids, as those plots had aphid populations that increased after the insecticides were applied. Objective 3: Use findings to create applied extension outputs. (Years 2-3) Our co-PI, Erin Hodgson, is waiting for the completion of the amplicon sequencing. Once completed, she will work with our team to prepare extension outputs that reveal the frequency of insecticide-resistant aphids in both her insecticide efficacy trial and the commercial fields. We will determine if the alternative modes of action (thiamethoxam, pyrifluquinazon) provide protection from these mutants. Finally, we will report on how economical it is for farmers to switch alternative active ingredients or alternative methods of management.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Valmorbida, I., J.D. Hohenstein, B.S. Coates, J.G. Bevilaqua, J. Menger, E.W Hodgson, R.L. Koch and M.E. ONeal. 2022a. Association of voltage-gated sodium channel mutations with field-evolved pyrethroid resistant phenotypes in soybean aphid and genetic markers for their detection. Sci Rep 12, 12020. https://doi.org/10.1038/s41598-022-16366-1
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Valmorbida, I., B.S. Coates, E.W. Hodgson, M.Ryan, and M.E. ONeal. 2022b. Evidence of enhanced reproductive performance and lack of fitness costs among soybean aphid, Aphis glycines, with varying levels of pyerthroid resistance. Pest Management Science. https://doi.org/10.1002/ps.6820.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Soybean farmers, agribusiness that supports soybean production, especially companies that manufacture and sell insecticides. Changes/Problems:There are two methods used to evaluate resistance to pyrethroids in soybean aphid. Resistance phenotype in soybean aphid populations can be evaluated using an established leaf-dip bioassay procedure. Resistance associated genotypes can be assayed with molecular techniques. L1014F, M918I, L925M, and M918L are point mutations in the soybean aphid voltage-gated sodium channel (vgsc) gene. These four mutations have been identified and evaluated with PCR assays. I am currently running four different PCR assays to identify each mutation, according to established protocol. PCR assays have some advantages over leaf-dip bioassays. Leaf-dip bioassays require live samples to reproduce and create populations large enough to test. This can take time to set up in comparison to molecular assays, which can be performed immediately after field collection using DNA extracted from dead aphids. As a result, molecular assays can be performed on a larger group of aphids in a shorter period. Leaf-dip bioassays ignore the variability of conditions found in field populations due to the use of uniform individuals (Robertson 2017). Due to this, genotyping a variety of field-collected samples better reflects population target site resistance. Despite these advantages, PCR based molecular assays have some weaknesses when being used for population studies. Assays become less efficient when mutation number increases. Multiple PCR reactions are used to identify each mutation and each mutation requires its own gel to evaluate results. With the four mutations found in the soybean aphid, the time needed increases 4-fold. This is less efficient when dealing with population studies. Another issue is that PCR assays can only report mutations targeted by individually designed primers. It is possible that the current four mutation study is ignoring vgsc mutations found in other Aphididae. These mutations could appear with less frequency than the four identified, but it is important to know if they exist for a clear image of target site resistance in soybean aphid. A third issue is that PCR assays do not report the aphid's haplotype. Haplotype analysis has been used to explore genetic diversity in population studies of cyclical and obligate parthenogenic species, other members of Aphididae, and soybean aphid. Haplotype analysis would evaluate the conditionality of mutation combinations being associated with resistance based on the DNA strand both mutations are on. The study is about the change in allele frequency after pyrethroid application. Haplotype information will additionally provide more context for how alleles are changing due to the soybean aphid's life cycle being cyclically parthenogenetic. Lastly, PCR assays are expensive at the scale and mutation count that is currently being worked at. In 2007, LCR, the assay I am using for the M918I and L925M genotypes cost $2.10 per aphid. Not accounting for inflation, the price increases to $10,000 for 5000 aphids and $20,000 for two mutations. Amplicon sequencing is a recent, high-throughput procedure that will reduce time and money spent in genotyping soybean aphid populations, while simultaneously providing more detailed data to answer the research question. Previously expensive, high-throughput procedures like amplicon sequencing are now commonplace and have been used to sequence genes of animals, plants, and viruses. Three reactions would be performed for each aphid to sequence the two currently known vgsc mutation sites and genotype all four mutations currently being explored. This is about the same time needed to genotype one mutation in the previous procedure. It is still possible for contamination to occur in PCR,but it can be evaluated on a gel before sequencing. This is the only gel that will be ran per sample batch, which is ¼ the gels ran in the previous procedure. The Illumina Sequencing unit used will read each aphid's vgsc fragment thousands of times, per the guidelines from the Iowa State DNA Facility. This form of self-checking ensures information on its vgsc genotype will be accurate. Since the output is the base sequence of the vgsc mutation sites, it is also possible to evaluate 5000 soybean aphids for other mutations associated with pyrethroid resistance in Aphididae and other taxonomic groups. During the summer of 2023, we will combine our samples from this most recent survey with those from 2022, and use amplicon sequencing to detect mutations in each aphid. What opportunities for training and professional development has the project provided?We have used these activities to train an MS student in entomology, as well as two undergraduates hired to assist the student. This training includes learning how to find the soybean aphid in soybean fields, process it for molecular analysis and use that molecular analysis with the genetic markers. In addition to these field and laboratory skills, the students had to learn about various aspects of farming so that they could gain access to cropfields where soybean aphids might be present. How have the results been disseminated to communities of interest?We have shared our results with colleagues at the 2022 North Central Branch meeting of the Entomological Society of America (ESA) and the 2022 National meeting of the ESA. We have also shared our laboratory work with the Iowa State University Research Foundation (ISURF). We explored the potential for patenting and/or licensing this technology and the RNA primers generated from our experiments. This resulted in an invention disclosure that was shared with the USDA Agricultural Research Services administrators at the Corn Insects and Crop Genetics Research Unit. The USDA declined to pursue this path, and ISURF referred us to the Office of Intellectual Property and Technology Transfer (OIPTT at Iowa State University. We prepared a brief summary of our results which OIPTT has shared anonymously with agribusiness in an attempt to generate interest in licensing this technology. We have yet to hear back from our contact at OIPTT regarding interest in licensing this technology. What do you plan to do during the next reporting period to accomplish the goals?We will repeat our field survey, working with a growing number of farmers interested in assisting us in developing our marker-based approach to detecting insecticide resistant aphids. We are also continuing with our laboratory experiments in an attempt to determine how robust the markers are for detecting multiple mutations within a single insect. This is an attempt to address issues of sample contamination and excessive costs in processing a sample (see Changes/Problems section).
Impacts
What was accomplished under these goals?
Impact statement: We completed several activities related to our three objectives that allowed us to incorporate commercial farmers and a commodity organization (Iowa Soybean Association) in the process of collecting data. These are related to our three objectives. Objective 1: Discover genetic markers for lambda-cyhalothrin resistance within soybean aphid populations. (Years 1-2). We completed a significant portion of this objective by publishing the mutations we have found to be associated with pyrethriod resistance in the soybean aphid (Valmorbida et al. 2022a and 2022b). These publically accessible articles describe four mutations (M918I, M918L, L925M and L1014F) in the voltage gated sodium channel of the soybean. In Valmorbida 2022a, we describe how these mutations can be identified with genetic markers. We have also conducted several experiments to confirm the contribution of the various mutations to the resistance. This work has opened up a heretofore unexplored potential pest management method. We are exploring if the RNA-primers used to confirm that the mutation are responsible for resistance could be used to silence the mutations. If silenced, we could restore susceptibility to the insecticide. If possible, we could include these primers in an application of an insecticide so that both the resistant and susceptible aphids are killed. We have shared this innovation with USDA and Iowa State University, to explore the potential for commercialization. We are currently exploring how to more quickly process individual aphids through an amplicon process (described briefly in this report). This method has been developed an used with other insects. We are exploring if it can be used to more cost-effectively detect these four mutations in the several thousand aphids we have collected per objective 2. Objective 2: Use molecular marker(s) to describe changes in the frequency of pyrethroid resistance among field collected soybean aphids. We worked with several farmers, identified with help from the Iowa Soybean Association, to track aphids in soybean fields of Iowa (described previously). We are currently attempting to use the amplicon method to identify the various mutations in each aphid. Objective 3: Use findings to create applied extension outputs We have employed some of our findings within a variety of extension outputs and presentations by students working on this project. This includes two oral presentations given by a MS student (Matthew Pfab). With the future completion of the activities to detect mutations from the field, we will produce additional extension outputs, informing farmers of the risk of insecticide-resistance in their soybean fields.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Valmorbida, I., J.D. Hohenstein, B.S. Coates, J.G. Bevilaqua, J. Menger, E.W Hodgson, R.L. Koch and M.E. ONeal. 2022a. Association of voltage-gated sodium channel mutations with field-evolved pyrethroid resistant phenotypes in soybean aphid and genetic markers for their detection. Sci Rep 12, 12020. https://doi.org/10.1038/s41598-022-16366-1
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Pfab, M.R., Valmorbida, I, ONeal, M.E., Coates, B.S., and Hodgson, E.W. (November 2022) Measuring the frequency of mutations associated with pyrethroid resistance in Aphis glycines. 10-minute presentation. Entomological Society of America National Meeting, Vancouver, CA.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Valmorbida, I., B.S. Coates, E.W. Hodgson, M.Ryan, and M.E. ONeal. 2022b. Evidence of enhanced reproductive performance and lack of fitness costs among soybean aphid, Aphis glycines, with varying levels of pyerthroid resistance. Pest Management Science. https://doi.org/10.1002/ps.6820.
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Research colleagues, agricultural professionals (e.g. crop consultants, agricultural Industry representatives), and governmental agencies (e.g. EPA) responsible for the development and implementation of IRM plans for plant protectants, especially insecticides. Changes/Problems:Project was impacted by limitations to laboratory and field research imposed by COVID-19 protocols with specific limitations affecting each unit are described below. For Co-PI Coates, the USDA-ARS Corn Insects & Crop Genetics Research Unit (CICGRU), Ames, IA COVID-19 pandemic response severely impacted laboratory and field research operations. ARS has enacted a COVID-19 response whereby only Area Office approved "mission critical" field work is allowed to continue, where risk of exposure to personnel to complete work on this grant has not been allowed. Specifically, work in Objective 1 to generate and analyze soybean aphid genome and RNA-seq data hindered due to limited time allowances for laboratory work and on-site access to computational resources. What opportunities for training and professional development has the project provided?This project was conducted with assistance from a graduate student, Ivair Valmorbida, and two undergraduate students, Matthew Pfab and Paige Hollenberg. How have the results been disseminated to communities of interest?We have shared these results with the Iowa Resistance Management Program (IPRMP) at thier annual meeting on 21 May, 2021. THe IPRMP is a unique program that brings multiple stakeholders within the state of Iowa to address the problems assosciated with resistance in crop pests. Details can be found at https://www.ipm.iastate.edu/about-the-iprmp. We also are scheduled to share our preliminary results with other entomologists and agribusiness at the national meeting of the Entomological Society of America in November of 2021. What do you plan to do during the next reporting period to accomplish the goals?Objective 1)Discover genetic markers for lambda-cyhalothrin resistance within soybean aphid populations. (Years 1-2). 1a: Determine the role of detoxification gene expression in lambda-cyhalothrin resistant soybean aphids carrying the kdr mutation. Short read genome re-sequencing data from soybean aphid lines carrying different mutations in the voltage-gated sodium channel (VGSC) gene will be aligned to a reference whole genome assembly. From this alignment all nucleotide variants will be predicted including those in genome regions encoding the VGSC and gene annotated with putative detoxification functions. Additionally, RNA-seq data will be assembled by genome reference guided method and depth (read count) of RNA-seq data aligned to each predicted gene will be used as a proxy for gene expression. Interspecific comparison of differences in gene expression between soybean aphid lines with different VGSC mutations and varying levels of pyrethroid resistance will be made, and up- and down-regulated detoxification genes will be predicted. Realtime PCR will be used to validate any predicted differential expression. Genome re-sequencing data from the same soybean aphid lines will be used to predict mutations in the VGSC, as well as in differentially-expressed detoxification genes. Mutations resulting in putative amino acid changes will also be predicted in the coding sequence of detoxification genes. 1b: Develop genetic markers for mutations associated with changes in detoxification genes associated with lambda-cyhalothrin resistant soybean aphids. Genetic markers will be developed to detect predicted mutations in soybean aphid detoxification genes that are differentially-expressed or show a significant number of amino acid changes. The markers will be validated by PCR-RFLP, ligase chain reaction, or direct Sanger sequencing assays prior to development of detection methods on a high-throughput platform for downstream population screening in Objective 2. Objective 2) Use molecular marker(s) to describe changes in the frequency of pyrethroid resistance among field collected soybean aphids. (Years 2-3). Soybean aphids will be collected pre- and post-application of field-applied rates of a pyrethroid insecticide from locations in the upper-Midwest. Individuals from all collections will be exposed to a diagnostic pyrethroid concentration to estimate proportion of phenotypic resistant aphids. The genetic markers developed in Objective 1 for detection of the VGSC mutations M918I, M918L, L925M, and L1014F will be used to genotype all survivor and moribund aphids across collection sites, and association of genotypes with resistance (survivorship following pyrethroid exposure in laboratory bioassay and/or field applied rates of a pyrethroid) will be made.
Impacts
What was accomplished under these goals?
The project year, we continued to monitor pyrethroid-resistant soybean aphids. Soybean aphids samplings were (and still are being) conducted in Iowa to determine the geographic distribution and the frequency of resistant aphids. Aphids from each location will be used for DNA extraction. Our different molecular markers will be used to determine if the aphids have one or more of the mutations in the voltage-gated sodium channel associated with pyrethroid resistance. In some locations, soybean aphids will be collected before and after a foliar insecticide spray. By using our molecular markers, we will be able to determine changes in the frequency of resistant allele pre and post-insecticide application. See below for additional outcomes. Objective 1)Discover genetic markers for lambda-cyhalothrin resistance within soybean aphid populations. (Years 1-2). 1a: Determine the role of detoxification gene expression in lambda-cyhalothrin resistant soybean aphids carrying the kdr mutation. - 25% complete, Synergists, chemicals the inhibit the function of a subset of detoxification enzymes, were used to determine their addition to exposure bioassays in the laboratory did not impact the level of resistance. This indicated for at least one soybean aphid line that detoxification pathways may not be involved in the pyrethroid resistance mechanism. RNA-seq data is currently being collected, and will be analyzed in year 2 in order to determine association of any differences in expression with pyrethroid resistance. Outcomes: Results indicate that a subset of detoxification enzymes with their function inhibited by synergists may not be involved in the pyrethroid resistance mechanism in field-collected soybean aphid lines. 1b: Develop genetic markers for mutations associated with changes associated with lambda-cyhalothrin resistant soybean aphids. - 50% complete. In addition to the heterozygous knockdown resistance (kdr) L1014F mutation in the soybean aphid voltage gated sodium channel (VGSC) at identified in preliminary data for our proposal, Sanger sequencing of the VGSC domain II segments 4 through 6 (DIIS4-S6) identified a homozygous kdr genotype in a line collected from Kanawha, Iowa. These data also identified an additional mutation predicted to results in an amino acid change at position 918 from a methionine to isoleucine (M918I) in the MN1 isofemale line, SBA-MN1-2017_Iso. This mutation was heterozygous among clones from the SBA-MN1-2017_Iso line. Sequencing data from an isofemale line initiated from a 2019 field collection from Darwin, Minnesota showed two additional amino acid changing mutations M918L and L925M, where the line was heterozygous for both mutations. Analogous Sanger sequencing of the VGSC DIIIS6, and DIVS4-S6 reported to have mutations conferring pyrethroid resistance in other insects found no additional amino acid changing mutations, thereby leading us to conclude these regions are not involvement in A. glycines resistance. All Sanger sequencing data has been submitted to the public database at GenBank (https://www.ncbi.nlm.nih.gov/). Phenotypes of the Darwin population showed an elevated level of resistance to the pyrethroid insecticide bifenthrin compared to pyrethroid lambda-cyhalothrin that was not predicted in any other soybean aphid line. In order to facilitate the screening of large numbers of individuals from field populations, we developed genetic markers analogous to the BstEII PCR-RFLP marker that detects the L1014F mutation shown in the proposal preliminary data. Specifically, we developed and validated an SspI PCR-RFLP assay to detect the M918L mutation, as well as two ligase chain reaction (LCR)-based assays that independently detect the mutations M918I and L925M. Collections were not made during 2020 due to labor and travel restrictions during COVID-19 pandemic, but initial screening of populations in Iowa during summer 2021 indicate that a majority of genotypes among survivors of field applied rates of lambda-cyhalothrin are heterozygous for the kdr mutation (full results pending). To date, RNA has been successfully extracted from all soybean aphid lines and are submitted for sequencing. Genomic DNA was extracted and short read full genome sequence data collected with greater than or equal to 29.1X estimated genome coverages. Outcomes: Four validated genetic markers and associations made with mutations they detect with pyrethroid resistance provide a valuable tool for predicting resistance status and demographic changes in soybean aphid populations. Furthermore, the markers may prove useful for evaluating the efficacy of insect resistance management and integrated pest management strategies.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Valmorbida, I., Hohenstein, J.D., Coates, B.S., Bevilaqua, J.G., Menger, J., Hodgson, E.W., Koch, R.L. and ONeal, M.E. Association of voltage-gated sodium channel mutations with field-evolved pyrethroid resistant phenotypes in soybean aphid and genetic markers for their detection. Scientific Reports, In review. 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Valmorbida, I., Hohenstein, J., Coates, B.S., Hodgson, E., and ONeal, M.E. Research Update on Insecticide Resistant Soybean Aphids. IPRMP Annual Meeting. 21 May 2021.
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