The conventional approach to preventing yield loss from insects is the use of insecticides. Funding from the Iowa Soybean Association has revealed the repeated use of pyrethroids (e.g., ?-cyhalothrin and bifenthrin) has resulted in resistance. Insecticide-resistant soybean aphids are found in southern Minnesota and northwestern Iowa (Hanson et al. 2017, Menger et al. 2020, Valmorbida et al. 2020) and complicates profitable soybean production. Since pyrethroids are less expensive than other insecticides, switching to a new insecticide will cost farmers more to protect yield (Dean et al. 2020). If farmers do not switch and continue to use pyrethroids, there is the potential to lose 25-40% yield if an outbreak of insecticide-resistant aphids occurs.

Insecticide use is likely to increase, as the frequency of outbreaks increases in the Midwest. Starting in 2018, several counties in western Iowa experienced yield losses due to a new soybean pest, the soybean gall midge (Gagné et al. 2019). Larval feeding inside stems can lead to plant death. As of 2021, 31 Iowa counties have been confirmed, adding to the 109 counties in Iowa, Minnesota, Missouri, Nebraska, and South Dakota), with a range of infestation severity. Yield losses observed in commercial fields during 2019 and 2020 ranged from 1-50%, where dead plants were found mostly at field edges. Unlike the soybean aphid, little is known about the biology and how best to managing the gall midge. The recent loss of chlorpyrifos has increased farmers dependence on a few groups of insecticides. With fewer options, there is an increasing need to prevent the occurrence and spread of resistance. This proposal aims to develop new management practices to minimize gall midge damage and to address resistance not only in the soybean aphid but potentially all insects that attack crops.

OBJECTIVE 1: Soybean pesticide field efficacy evaluations
Co-PI Hodgson runs the nation’s largest soybean aphid efficacy evaluation program. Hodgson explores insecticides insect-resistant plants. In the summers of 2023-2025, experimental plots at high-risk locations in Iowa will be used to evaluate seed treatments and foliar insecticides. Foliar applications will be based on our established treatment threshold for soybean aphid or our ‘best guess’ for midges. Yield will be collected and compared to pest abundance for each treatment.
Obj. 1a. Soybean aphid efficacy. At one location, 20-30 treatments will be replicated four times in a randomized complete block design using a 30" row spacing. Treatments will include a range of insecticidal groups and application sites. Aphid populations will be monitored weekly in addition to secondary pest activity (e.g., beetles, mites, stink bugs, caterpillars, etc.). Aphids will be collected to determine the frequency of pyrethroid-resistant mutations before and after insecticides are applied.
Obj. 1b. Soybean gall midge efficacy. At one location, 20 treatments will be replicated four times in a randomized complete block design using a 30" row spacing. Midge larval will be monitored weekly in addition to secondary pest activity (e.g., soybean aphid, beetles, mites, stink bugs, etc.). Foliar applications will be based on our observation of overwintering adult emergence. At this point, that is our “best guess” for targeting adult movement into soybean.

OBJECTIVE 2: Using RNAi to restore pyrethroid susceptibility among resistant soybean aphids
Our prior ISA-funded research found four mutations in the voltage-gated sodium channel (vgsc) gene in aphids collected in Minnesota and Iowa that are resistant to pyrethroids. These mutations change the vgsc protein targeted by pyrethroids, reducing the insecticides ability to kill aphids. These resulted in genetic markers that detect aphids with the vgsc mutations. These markers found that 82.7% of field-collected aphids in Iowa were resistant. We have designed RNAi (dsvgsc-h1) to selectively kill aphids by silencing the vgsc gene, killing 33% of the pyrethroid resistant aphids. The dsvgsc-h1 applied along with a pyrethroid increased aphid mortality 20-fold compared to pyrethroid alone, killing heterozygous insecticide-resistant aphids. We will explore if adding RNAi to a commonly used, inexpensive insecticide kills both susceptible and resistant aphids.
Obj. 2a. Determine the optimal dsRNA sequence and concentration for aphid mortality (Year 1 & 2)
We will compare aphid mortality after topical application of a mixture of four dsRNAs, each specific to one of the four vgsc mutations and to dsRNA that attacks all mutations. The effect of dsRNA treatments will be tested alone and in combination with a pyrethroid against resistant and susceptible aphid genotypes kept at ISU. After selecting the optimal dsRNA sequence to maximize aphid mortality, we will determine the optimal concentration of both dsRNA and pyrethroid on aphid-infested soybean plants. We will measure the number of dead aphids on plants infested with 100 aphids of a single genotype across a range of concentrations. We will test the lowest concentration that provides consistent mortality in a greenhouse-based experiment with treatments applied using a backpack sprayer.
Obj. 2b. Assess effects of optimized insecticidal dsRNA on non-target species (Year 3)
Although RNAi has been approved for the SmartStax® Pro corn in the US, it has not yet been approved for use as a foliar spray. An environmental risk assessment is a key component for regulated use of an insecticide by the EPA. Since our dsRNAs only kill insecticide resistant soybean aphids, we do not anticipate an impact on non-target species. We further anticipate survival will be greatest when the genetic diversity is greatest between the target (soybean aphid) and non-target organisms. To convince agribusiness that this approach is viable, we will conduct a test of the dsRNA against a commonly found aphid, corn leaf aphid (, and an aphid predator, multi-colored Asian lady beetle (.

OBJECTIVE 3: Extension communication strategies
Co-PI Erin Hodgson and collaborator Ashley Dean will conduct extension programs summarizing efficacy data drawing directly from Obj. 1 and 2. Field day demonstrations and winter programs will be coordinated with ISU Extension and Outreach, Iowa Soybean Association, industry partners, and other organizations. Potential ISU Extension and Outreach programs include: Integrated Crop Management Conference, Crop Advantage Series, and field days with ISU. In addition, research findings will be published in a Yellow Book (www.ent.iastate.edu/soybeanresearch/content/extension), and deliver real-time updates on soybean pests in the summer, they will continue posting informal articles to ICM Blog and contributing articles to ICM News.

Expected Stakeholder Deliverables:
• Raise awareness of pyrethroid resistance among farmers and ag industry [short term knowledge];
• Implement scouting and adopt economic thresholds for soybean gall midge [long term skill];
• Reduce insecticide use, including seed and foliar treatments [long term skill];
• Understand the implications of aphid genetic resistance to insecticides [long term knowledge];
• Improve profit margins by reducing input costs [long term knowledge].

Specific Extension Outputs and Metrics for Soybean (Oct 1, 2022 to March 31 2023):
Newsletter Articles, Blog Posts, etc.: 2
Lectures and Field Days: 7
Face-to-face contacts: 458
Erin Hodgson (@erinwhodgson) Twitter Followers: 3,158 [167,000 impressions]
Ashley Dean (@ashleyn_dean) Twitter Followers: 312 [19,760 impressions]

Update:
OBJECTIVE 1: Soybean pesticide field efficacy evaluations
During the initial period of this grant (Oct 1, 2022 to March 31 2023), we accomplished the following per each of the three objectives.
1. The plots established during the 2022 growing season, which is not immediately covered by this grant, were harvested and evaluated with funding from this grant. We are preparing a similar evaluation for 2023.
The insecticide evaluations conducted during 2022 were conducted in plots at one ISU Research Farm in northwestern Iowa in 2022. In total, we evaluated 26 replicated treatments with six insecticidal group/subgroups. The plots were initially colonized by soybean aphid in late June, and populations remained very low throughout the summer. There were a few other soybean pests present (e.g., spider mites, stink bugs, grasshoppers) but economic populations were not evident. Natural enemies, such as beetles, flies, lacewings, and wasps, were also present throughout the reproductive stages. The threshold was never reached, so plots were sprayed on 17 August when plants were at the R5 (beginning seed set) growth stage. Soybean aphid populations in the untreated control peaked on 16 August with 17.4 aphids per plant. Since soybean aphid populations were well below the economic threshold, no yield losses were expected or observed. Average treatment yields ranged from 45.4 to 57.1 bushels per acre.

We are accumulating request from various agribusinesses to evaluation products during the 2023 growing season.

OBJECTIVE 2: Using RNAi to restore pyrethroid susceptibility among resistant soybean aphids
This research topic attracted the interest of a visiting professor from Brazil, Dr. Jonas Arnemann, of the Federal University of Santa Maria who assisted us in conducting some of the early tests of our RNAi probes. The preliminary evidence was strong enough to further attract the attention of his graduate student, Bruna Wojahn. She continued tests of several RNAi probes on multiple aphid genotypes that represent aphids both resistant and susceptible to insecticides. Bruna returned to Brazil in November of 2022, and is returning to August in 2023 to complete the objectives of this first year as well as those scheduled for the second year of this project (in the proposal to be submitted on May 8th, 2023).

To determine the optimal dsRNA sequence and concentration for killing insecticide-resistant soybean aphids we used five colonies of aphids kept in the Soybean Entomology laboratory in the ATRB at ISU. These five colonies have distinct genetic backgrounds (Table 1): four are resistant to pyrethroids and have different mutations in the voltage-gated sodium channel (VGSC) associated with resistance to pyrethroids, The fifth colony is susceptible to pyrethroids and does not have any of these mutations (i.e. wild type).

Working with Dr. Coates, dsRNA was synthetized from each colony. A section of dsRNA was developed from each colony that included the regions where point mutations associated with insecticide resistance are found. Once we created these dsRNA, our objective was to determine if these five sets dsRNA would selectively kill the resistant aphids. When dsRNA is applied to these insects with regions in their genome that correspond to them, it will interfere with the function of the gene. Therefor we refer to these sections of dsRNA as an RNAi treatment. A subset of our colonies are heterozygotes, meaning they have a mutated version and the wild type version of the VGSC gene. Therefore we can test if RNAi shuts down the mutated version could return the resistant aphid to being susceptible to the insecticide. To accomplish all of this, we have to test if RNAi applied alone or in combination with a pyrethroid can increase aphid mortality.
To measure the impact of the potential RNAi treatments, we conducted assays using a small section of aphid infested soybean leaf. Each leaflet had 10 aphids from a unique colony. The treatments included lambda-cyhalothrin alone, a unique isolated dsRNA from each colony alone, or the dsRNA with lambda-cyhalothrin. Each unique section of dsRNA was tested in individual experiments using plates with soybean leave disks, where the insecticide treatment was applied by dipping the leaf disks in lambda-cyhalothrin solution and the probe was applied by topical delivery method, where 1 ul was displayed on the dorsal side of the soybean aphid abdomen. A total of 30 separate experiments were conducted to evaluate the impact of the five dsRNA strands on the five colonies. Aphid mortality was assessed 24 hours after the treatments were applied. For purposes of this report, we have summarized a subset of the experiments, combining the results of the various dsRNA treatments.

Table 1. Aphid colonies kept at ISU and used in these experiments.
Colony Mutation Name
Nashua, IA L1014F (heterozygous) MtA Rs
Kanawha, IA L1014F (homozygous) MtA RR
MN L1014F + M918I (heterozygous) MtAB Rs
Darwin, MN L925M + M918L (heterozygous) MtCB Rs
Boone, IA Wild type Wild type

Overall, we were able to increase mortality of insecticide resistant aphids by adding dsRNA to the insecticide (Figure 1 see attached). The summary in figure 1 shows the average mortality for each probe applied to the four colonies of resistant aphids and the wild type. These data suggest that combining dsRNA and insecticides can manage aphids that are both susceptible and resistant to an insecticide, at the same time in one application.


OBJECTIVE 3: Extension communication strategies

Co-PI Erin Hodgson and collaborator Ashley Dean are conducting extension programming summarizing efficacy data in soybean. Field day demonstrations and winter programs were coordinated with ISU Extension and Outreach, Iowa Soybean Association, industry partners, and other organizations. Examples include: ISU Extension and Outreach programs include: Integrated Crop Management Conference, Crop Advantage Series, and field days with ISU. In 2022, Erin and Ashley delivered real-time updates via text message on soybean pests during the summer, and they will continue posting updates to the Integrated Crop Management News and Blog.

Regional/invited extension presentations:
Hodgson, E. W. What happens when the easy button fails: insect management outlook. University of Missouri Crop Management Conference, Columbia, MO [175 people] 15 December 2022

Hodgson, E. W. Let’s get real about soybean insecticides: what’s in and works. Crop Pest Management Short Course, Minneapolis, MN [2 sessions; 225 people] 8 December 2021

Hodgson, E., and A. Dean. Why you “need” need assessment in your impactful extension program. ANR Extension Faculty Meeting, Ames, IA. [28 people]

Hodgson, E. Planter University: every seed counts. ANR Extension Professional Development, Lightning Talk Session. Ames, IA. [220 people] 11 October 2022.

Hodgson, E. W., and A. Dean. Throwing the kitchen sink at soybean gall midge. Iowa State University Extension and Outreach Crop Advantage Series Workshops.
- Storm Lake, IA. [11 people] 4 January 2023
- Webster City, IA. [2 sessions; 40 people] 11 January 2023
- Ankeny, IA. [2 sessions; 30 people] 17 January 2023
- Denison, IA. [15 people] 26 January 2023

Hodgson, E. W., and A. Dean. Direct destruction: how to manage late-season pests. Iowa State University Extension and Outreach Crop Advantage Series Workshops.
- Burlington, IA. [25 people] 5 January 2023
- Atlantic, IA. [66 people] 19 January 2023
- Davenport, IA. [25 people] 20 January 2023
- Le Mars, IA. [25 people] 20 January 2023

Hodgson, E. W., and A. Dean. Throwing the kitchen sink at soybean gall midge. Iowa State University Extension and Outreach Ag Chem Dealer Update.
- Nevada, IA. [20 people] 13 December 2022
- Crawfordsville, IA. [35 people] 14 December 2022

Hodgson, E. W., and A. Dean. Direct destruction: how to manage late-season pests. Iowa State University Extension and Outreach Integrated Crop Management Annual Conference, Ames, IA. [2 sessions; 175 people] 30 November 2022

Hodgson, E. W., and A. Dean. Throwing the kitchen sink at soybean gall midge. Iowa State University Extension and Outreach Integrated Crop Management Annual Conference, Ames, IA. [2 sessions; 190 people] 1 December 2022
Dean, A., and E. W. Hodgson. Throwing the kitchen sink at soybean gall midge. Iowa Advanced Agronomy Day, Growmark, Ames, IA [35 people] 21 November 2022



Webinars
Hodgson, E. W., B. Kolbe, and J. McMechan. Can pre-season tactics help with soybean gall midge management? Regional webinar for soybean gall midge, Virtual Delivery. [230 views live] 27 February 2023
Dean, A., and E. W. Hodgson. Tolerances revoked for chlorpyrifos: what are some other options? Loveland/Nutrien Regional Meeting Virtual Delivery [25 March 2022]
Hodgson, E. W., B. Kolbe, and J. McMechan. Using tillage to reduce overwintering soybean gall midge. Soybean gall midge discussion series, Virtual Delivery. [245 views live] 22 February 2022
Hodgson, E. W., and J. McMechan. Soybean gall midge ID, distribution, scouting tips, and injury score. Soybean gall midge discussion series, Virtual Delivery. [165views live] 15 February 2022

Peer-reviewed and peer-edited extension publications:
Coyle, D. R., and E. W. Hodgson. 2022. Guiding the next generation of extension professionals: there’s a method to the madness. American Entomologist. DOI: 10.1093/ae/tmac036.
Huseth, A. S., R. L. Koch, D. Reisig, J. A. Davis, S. V. Paula-Moraes, and E. W. Hodgson. 2021. Current distribution and population persistence of five caterpillars in U.S. soybean. Journal of Integrated Pest Management. DOI: 10.1093/jipm/pmab004.
Hodgson, E. W., R. L. Koch, J. A. Davis, D. Reisig, and S. V. Paula-Moraes. 2021. Identification and biology of common caterpillars in U.S. soybean. Journal of Integrated Pest Management. DOI: 10.1093/jipm/pmab006.
McMechan, A. J., E. W. Hodgson, A. Varenhorst, T. Hunt, R. Wright, and B. Potter. 2021. Soybean gall midge (Diptera: Cecidomyiidae), a new species causing injury to soybean in the United States. Journal of Integrated Pest Management. DOI: 10.1093.jipm/pmab001.
Hodgson, E. W., and A. Dean, V. Schmitt, and N. Seiter. 2020. Insect and mite management, pp. 109-116. In Hodgson, Licht, and Sisson [eds.], Field Crop Production Handbook, Iowa State University Extension and Outreach, Publication 3162.

View uploaded report

Update:
Summary of last year of ISA grant to O’Neal, Hodgson and Coates.

What it means for farmers:
Faculty at ISU are conducting extension and research to better equip Iowa farmers facing new and old insect pests. This includes a focus on the recently identified soybean gall midge and the soybean aphid that has developed resistance to insecticides.
The research team at ISU in collaboration USDA have developed a novel way to kill soybean aphids that are resistant to insecticides. This method includes coupling RNA probes developed from mutations found in insecticide-resistant aphids with the insecticide they are resistant to. This method killed 97% of the insecticide-resistant aphids exposed to both the probes and the insecticide.

Extension
As part of this grants third objective, Dr Erin Hodgson and her colleague Ashley Dean conducted a series of extension activities addressing insect pests of soybeans. Below is a list of these publications, meetings and events.
Hodgson, E, and A, Dean. “Speed Scout this year for soybean aphid” In ICM News 7 August 2023.
Dean, A, and E. Hodgson “Soybean gall midge adults now emerging” In ICM News 8 June 2023.
Dean, A, and E. Hodgson “Soybean aphid egg hatch is complete” In ICM News 4 May 2023
Hodgson, E. W. Updates on pest management strategies for field crop pests Certified
Entomologists of Mid-America Summer Business Meeting, Ames, IA. 12 August 2023
Hodgson, E. W., and A. Dean. Throwing the kitchen sink at soybean gall midge. Iowa State
University Extension and Outreach Crop Advantage Series Workshops
- Storm Lake, IA [11 people] 4 January 2023
- Webster City, IA [2 sessions; 40 people] 11 January 2023
- Ankeny, IA [2 sessions; 30 people] 17 January 2023
- Denison, IA [15 people] 26 January 2023
Hodgson, E. Soybean gall midge research updates. Regional field day, ENREEC, University of Nebraska-Lincoln, Mead, NE [4 sessions; 130 people] 25-26 July 2023
Hodgson, E. W., and A. Dean. Throwing the kitchen sink at soybean gall midge? Iowa State University Extension and Outreach CropsTV, January - March 2023
Hodgson, E. W., B. Kolbe, and J. McMechan. Can pre-season tactics help with soybean gall midge management? Regional webinar for soybean gall midge, Virtual Delivery [230 live views] 27 February 2023

Research
What was accomplished:
During 2023, we attempted to confirm if the multiple soybean aphid mutants we had identified in Iowa were contributing to insecticide resistance. Our previously funded projects had found soybean aphids in Iowa that were resistant to insecticides, with several revealed to be highly resistant to lambda-cyhalothrin (i.e. Warrior) and bifenthrin. Working with Dr Brad Coates of the USDA, we identified mutations in these aphids associated with changes in the voltage gated sodium channel targeted by insecticides.
These genotypes of these mutants were tested in Dr. O’Neal’s lab, revealing that a single aphid could have more than one mutation. Thanks to the collaboration with Dr. Coates, we were able to identify these mutations and generate RNA probes that could interfere with there expression. This process employs RNAi technology to silence the targeted genes. By silencing the mutations, the aphids would become susceptible to insecticide, confirming their contribution to insecticide resistance.
Our goal was to use apply probes that silence each individual mutation, to determine which is most responsible for insecticide resistance. To achieve this goal, we generated multiple colonies composed of aphids that were genetically identical (i.e. clones). Each represented a unique genotype of soybean aphids that varied in the form and number of mutations. As of the summer of 2023 we had six distinct genotypes, including both homozygous and heterozygous clones.
We were able to hire Bruna Wojahn, a PhD student at the Federal University of Santa Maria in Rio Grande do Sol, Brazil. Bruna has conducted an internship during the summer of 2022 and developed an assay that reveals the impact of RNA probes on soybean aphids. This assay involves directly applying the RNA dissolved in water to an aphid. Despite the very basic approach, Bruna demonstrated that directly applying RNA leads to gene silencing. Bruna had agreed to return to ISU, hired on an hourly basis to conduct additional experiments. These experiments would also be the basis of her PhD at Santa Maria.
In addition to answering some basic questions about which mutations in the soybean aphid is responsible insecticide resistance, we began exploring if silencing these mutations could be used to kill soybean aphids. If the RNAi converts the resistant aphids to susceptible again, then combining the RNA probe and insecticides, we could manage both susceptible and resistant aphids at the same time.
We planned to conduct experiments that could determine if combining RNA probes and insecticides could be a viable management option. This will involve a series of experiments that will demonstrate the potential for this approach using the colonies generated by O’Neal’s lab and the probes created by Dr. Coates using not only data generated in the most recent ISA grant, but also previous investments from the ISA in describing the soybean aphids genome.
Regardless of our results, we reached out to ISURF to determine if this approach could be patented. As of February 23, 2024, we have received a provisional patent, protecting this technology and allowing for future licensing for commercial development.
Set-backs:
We had two major setbacks in getting this work started. First, we experienced delays in getting Bruna the necessary visa to work in the US. She was eventually able to start in September 2023. We requested a no-cost extension into the fall, to allow Bruna the time to conduct our research.
A more substantial setback was the contamination of our soybean aphid colonies. Unfortunately, several of our insecticide resistant colonies were contaminated with susceptible aphids. The entire team had to first confirm the genetic identity of the remaining aphids, confirm their genotype with genetic markers generated by Dr. Coates, and then recreate the colonies from individual aphids. Because the soybean aphid reproduces asexually, we can identify the genotype of a mother and then use only her offspring if they are the appropriate genotype.
This re-start took all of October and November. Once completed, we had multiple colonies with different mutations that had been confirmed through genetic markers.
By the end of our no-cost extension, we were able to complete a large experiment that involved a clone of aphids that are the most resistant. Our initial experiment was designed to determine if combining an RNAi probe with an insecticide can kill aphids and then determine what concentration of probe is needed. The graph in the attachment summarizes our first set of results, completed during December 2023.

View uploaded report

What it means for farmers:
Faculty at ISU are conducting extension and research to better equip Iowa farmers facing new and old insect pests. This includes a focus on the recently identified soybean gall midge and the soybean aphid that has developed resistance to insecticides.

Expected Stakeholder Outcomes:
• increase understanding of soybean gall midge biology [long term knowledge];
• recognize soybean aphid, soybean gall midge, and other soybean pests [short term skill];
• improve general pest management approaches, including the effect of multiple pests and the potential for cumulative injury [short term skill];
• understand the implications for soybean aphid genetic resistance to insecticides [long term knowledge];
• improve profit margins by reducing input costs [long term knowledge].