AN INTEGRATIVE APPROACH TO FALL ARMYWORM POPULATION GENOMICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1024939
• Grant No.: 2021-67013-33566
• Cumulative Award Amt.: $499,999.00
• Proposal No.: 2020-05818
• Project Start Date: Jan 1, 2021
• Project End Date: Dec 31, 2024
• Grant Year: 2021
• Program Code: [A1112]- Pests and Beneficial Species in Agricultural Production Systems

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Entomology

Non Technical Summary
The fall armyworm,Spodoptera frugiperda,has recently become a global invasive agricultural pest. Despite increased attention abroad, recent evidence indicates that our understanding of this pest's genetics, population biology and movement in its native range, namely the US, is severely lacking.This project will use population genomics, stable isotopes and behavioral assays to characterize the relationships between fall armyworm population structure, mating behavior, and dispersal patterns across the US. Based on strong preliminary data, our objectives are to:(1)Understand fall armyworm genetic structure across the entire US using population genomics. Specifically, we will(a)assess spatial and temporal population structure ofS. frugiperdaacross the US, and(b)identify diagnostic SNP markers to rapidly differentiate populations and their hybrids;(2)Test for evidence of north-to-south return migration in the US using stable isotope signatures; and(3)Assess the effect of photoperiod on the propensity for interstrain hybridization.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1070	75%
211	3110	1080	25%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1070 - Ecology; 1080 - Genetics;

Keywords

spodoptera frugiperda

fall armyworm

hybridization

pest movement

population genomics

stable isotopes

Goals / Objectives
Themajorobjectives of this study are to:Understand the population genetic structure of the fall armyworm,Spodopterafrugiperda,across the entire United States using a population genomics approach. Specifically, we will(a)assess spatial and temporal population structure ofS. frugiperdaacross all three US flyways, and(b)identify diagnostic SNP markers that can be used to rapidly differentiate populations and their hybrids.Determine if a significant return migration occurs inS. frugiperdausing stable isotope analysis.Assess the effect of photoperiod on the propensity for interstrain hybridization.

Project Methods
Objective 1: Characterize the genetic structure ofS. frugiperdaacross the entire United States using a population genomics approach.Moth traps baited with pheromone lures will be set up in 2021 and 2020 at eleven locations across the three hypothesizedS. frugiperdaflywaysin the United States. Samples will be preserved in 95% ethanol at −20°C until DNA is extracted from the thorax of collected moths using the Qiagen Puregene DNA tissue extraction kit. The mtDNA host strain haplotype will be determined according to Nagoshi et al. (2006). DNA from 12 individuals of each host strain haplotype per site will be sent to the Texas A&M AgriLife Genomic and Bioinformatics Service for genomic sequencing on the Illumina NovaSeq.All sequences will be aligned to the publishedS. frugiperdachromosome map (Liu et al. 2019) and filtered using the programFastQScreen. ThedDocentpipeline and vcftools will be used to call and filter SNPs (Puritz et al., 2014).Expected/observed heterozygosity, Fstvalues, principal component analyses, and other summary statistics will be calculated in R to test specific hypotheses relating to spatiotemporal population structure (Jombart, 2008). The programFastSTRUCTUREwill be used to create population structure plots visualizing the data (Raj et al., 2014).If significant population structure is identified during the population genomic analysis as expected, we will also pinpoint diagnostic SNPs to differentiate host strains, hybrids, and/or geographic populations. We will develop diagnostic SNP TaqManTMassays to rapidly genotype individuals within their lifetime in order to establish true breeding C and R strains for the Objective 2 and 3 experiments.Objective 2:Determine if a significant return migration(north to south) occurs in fall armyworm moths using stable isotope analysis.Previously, tap water in southern Texas was determined to have nearly the highest concentrations of both δ2H and δ18O in the continental US while tap water in Colorado and the Rocky Mountains had the lowest concentrations of both isotopes (Bowen et al. 2007, Figure 5). Therefore, tap water from PD Sword's facility in College Station, TX and PD Gilligan's facility in Fort Collins, CO will be used to create four field relevant water standards that differ in their makeup of δ2H and δ18O.These standards include:1. Pure tap water from College Station, TX (high in both isotopes)2. Pure tap water from Fort Collins, CO (low in both isotopes),3. A mixture of 2 parts College Station tap water to 1 part For Collins tap water,4. A mixture of 1 part College Station tap water to 2 parts Fort Collins tap water.96 sorghum plants will be grown individually in the greenhouse and watered with one of the four water treatments above (24 plants per treatment) every 5 days.When the plants reach the 5-leaf stage, they will be infested with fall armyworm caterpillars and continue to be watered. Newly-eclosed adult moths from each plant will be sexed and stored in -80°C for isotope analysis. A minimum of 12 male moths from each treatment will be analyzed for hydrogen and oxygen isotopes.Plastic universal moth traps baited with pheromone lures (Scentry Biologicals, Billings, MT)will also be placed at four locations along both the central and eastern fall armyworm flyways. Moth samples will be collected during the first week of the month for September, October, and November of both 2020 and 2021. These samples will then be stored at -80°C until isotope analysis. All water and mothisotope samples will be submitted to theStable Isotopes for Biosphere Science Laboratory (SIBS) at Texas A&M University for analysis.The laboratory reared moths described above will be used to establish a correlation between δ2H and δ18O in the water used to irrigate a host plant and the δ2H and δ18O in field collected moths. Using the calculated correlation equation, the δ2H and δ18O in the wing tissues of field samples will be converted to the expected δ2H and δ18O values of the water used to irrigate the larval host plants. These data will then be used in program IsoMAP (Bowen et al. 2020) to depict the probable geographic origin of each moth collection.Objective 3: Assess the effects of photoperiod on the mating propensity of hybridization.Fall armyworm caterpillars will be collected from corn, sorghum, and Bermuda grass at the Texas A&M University Field Laboratory outside College Station, TX. Individuals will be genotyped for C- or R- strain determination prior to mating using the TaqManTMassays developed in Objective 1. All individuals homozygous for the same host strain will be mated to create pure C- and R- strain family lines.Newly emerged neonates (<8hrs old) will be placed in individual 2oz condiment cups containing artificial diet.Offspring from each mating pair will be evenly divided between three incubators set with different photoperiods approximating those experienced by insects in the summer in the south Texas overwintering site when hybridization was rare (L:D 12:12), along with a shorter (L:D 14:10) and longer (L:D 10:14) scotophase as would be experienced by insects further north in the summer or during the winter in the south, respectively. Upon reaching the pupal stage, pupae will be sexed and placed in a clean cup for eclosion. Within one day of eclosing, one virgin female and two virgin males will be placed together in a plexiglass mating cage inside one of the three incubators. There will be six different mating group combinations. These mating groups include control assays where a female has access to two males of the same host strain, no choice assays where the female has access to two males of the opposite host strain, and choice assays where the female has access to one male of each host strain. Each mating cage will be equipped with camera monitoring system to capture a photo of the insect behavior every minute throughout the scotophase when the moths are most active. To avoid interference by pheromones released from females in other cages, each cage will be supplied with charcoal filtered compressed air from outside the incubator.Every replicate mating combination will be screened for 4-days or until the female moth dies, whichever occurs first. The resulting camera data will be analyzed and the following behaviors will be recorded: female calling, male calling, copulation, and oviposition. Specifically, we will record the time of each mating, the time males and females initiate calling, total time spent copulating, time of oviposition, # eggs laid, and female mate choice. We will screen 12 mating pairs per incubator at a time, or 2 replicates per treatment. In total, we will screen 24 replicates over a six-month period. Behavioral data will be analyzed using the statistical methods described by Schöfl et al. (2011).After behavioral assays, all viable neonates from the choice mating groups will be genotyped using the TaqManTMassays created in Objective 1. This will serve as a validation of the behavioral mating data. If a female copulates with both males, the relative proportion of hybrid vs. true strain offspring will be determined.

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:The target audience for this project included researchers, extension personnel, the agricultural industry, land managers and the general public interested in the management and biology of the fall armyworm (Spodoptera frugiperda) as a global migratory agricultural pest. Project findings were disseminated internationally in publicly accessible peer-reviewed journal articles along with presentations at a wide variety of international, national, state and local research conferences. Audience members ranged from established university, government and industryprofessionals to undergraduate university students. Changes/Problems:Objective 1. This objective was fully accomplished with project results either successfully published or in preparation for publication in peer-reviewed journal articles. Objective 2. Although samples were collected as planned,we experienced an extensive delay in receiving the isotope data for our wing samples as outlined in Objective 2 of this grant. The SIBS facility at Texas A&M where we submitted our wing samples had a change in personnel and then ran into some technical challenges with their elemental analyzer equipment (TCEA).Ultimately, the SIBS facility honored our original agreement and sent the samples to an alternative provider for analysis. Immediately prior to the submission of this final report, we began receiving the long-awaited data. We anticipate the analysis of these results to be completed and published in 2025 as originally planned. Objective 3. The biggest challenges that we encountered during this reporting period was in the completion of the behavioral mating assays as outlined in Objective 3. In addition to the delay in receiving the necessary environmental chambers to conduct the experiments, we had also originally proposed to use camcorders to film moth mating behavioral parameters to determine the timing of female calling, male calling, copulation, and oviposition. We ran into technical challenges filming and scoring these behaviors within a recording. Therefore, we have focused our efforts on determining the number, viability, and genotype of the offspring to determine the rates of hybridization across our treatments. We alsoneeded torestart the lengthy experimentafter discovering some cross-contamination of strains in the breeding populations. Fortunately, this was discovered prior to any genotyping, thus ensuring the integrity of the results once obtained. The completed work including final data collection, analysis and write-up will be included as a dissertation chapter in PhD student, Andie Miller's, dissertation which she is scheduled to defend by the end of 2025. The results will also be prepared for dissemination in a peer-reviewed publication to be submitted in the same timeframe according to our original plan. What opportunities for training and professional development has the project provided?This project directly and indirectly supported training and development activities for a Postdoctoral Research Associate (Dr. Ashley Tessnow), a PhD student (Andie Miller), and four undergraduate researchers (Miriam Torres, Jonathan Riggan, Megan Kopecki, and Randall Hobday). Undergraduate researchers initially conducted their own undergraduate research projects for course credit (ENTO491) investigating fall armyworm pheromone trapping effects and allochronic mating behavior under the mentorship of PhD student Andie Miller and PI Sword. Following those successful experiences, three of the students wanted to continue gaining experience and went on to become paid undergraduate research assistants. In addition to these long-term undergraduate students, we had the opportunity to put together a short workshop for four Florida A&M students through the NEXGEN Undergraduate Research Experience. During this 6-hour workshop, we talked with students about fall armyworm monitoring and management and brought the students to the field to show them a few methods for monitoring pest populations. We then returned to the molecular lab, talked about the importance of pest identification, and had each student learn to extract DNA and genotype a moth sample. The students described this as a rewarding experience that gave them a better understanding of the importance of agricultural research. The project also supported the professional development of trainees through successful publication of seven peer-reviewed publications and book chapters based on contributions from their work - with several more anticipated. The research reported in these publications reflects multidisciplinary trainee experiences provided through the integration of molecular biology, genetics, ethology, and field ecology as well as bioinformatics and other statistical analyses involved in data analysis. In addition to reporting project results in the scientific literature, results arising from trainee activities have been presented in a total of 24 conference presentations at local, national and international conferences to date, providing valuable experience in scientific communication. This project fostered direct professional interactions between postdoctoral research associate Ashley Tessnow and project personnel Rob Meagher and Rod Nagoshi at the USDA-ARS-CMAVE laboratory in Gainesville, Florida. This interaction led to Dr. Tessnow being hosted at their laboratory as a Visiting Scholar during the final year of the project, after which she successfully obtained a USDA-Oak Ridge Institute for Science Education (ORISE) postdoctoral fellowship to continue her work. PhD student Andy Miller gained valuable field experience in a collaborative context by conducting a large-scale field experiment that involved field work in Florida with Rob Meagher. Miller is set to graduate with a PhD in 2025 and is currently seeking employment at either the postdoctoral or assistant professor level. One of our undergraduate researchers, Megan Kopecki, has been accepted into the Master's degree program at Texas State University to pursue a degree in Integrated Agricultural Sciences. Two others continue to be employed as research assistants in the lab. How have the results been disseminated to communities of interest?Project findings have been disseminated globally in publicly accessible peer-reviewed journal articles along with presentations at a wide variety of international, national, state and local research conferences. Audience members ranged from established university, government and industryprofessionals to undergraduate university students. We have also made full genome sequencing data for 412 fall armyworm moths available on NCBI. This data can be downloaded and used by researchers globally who may be interested in further exploring or comparing specific aspects of the fall armyworm genome. We also conducted outreach activities to engage with the general public by providing hands on exposure to live insects and some of the automated tracking technologies that we utilized in our research along with an infographic poster summarizing our research findings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. The first objective of this grant was to characterize the genetic population structure of fall armyworms sampled across the United States using a population genomics approach. To accomplish this goal, we setup an extensive fall armyworm trapping network that included 13 different locations spread across three proposed fall armyworm flyways in the US, collecting well over 2000 moth samples. Using a low coverage whole genome sequencing approach, we sequenced the DNA from 431 moth samples collected in 2021 and an additional 368 fall armyworm samples collected in 2022. Our analysis of the data collected in 2021 was completed and published as a first of its kind comprehensive population genomics assessment of the fall armyworm across its entire range in the continental US and Puerto Rico (Tessnow et al. 2025. BMC Genomics 26:116). We made several major findings. First, the fall armyworm as a species is divided into two genetically distinct strains in the United States, and the reproductive isolation between them is likely the result of incompatibilities on the Z-chromosome that limit hybridization. We also found different patterns of geographic sub-structuring between these strains, with the C-strain showing some reproductive isolation between populations originating in Florida, Texas and Puerto Rico, while the R-strain is completely admixed across these distant geographic locations. We also found, no evidence of the R-strain occurring in the western United States. Together, these findings indicate that the two fall armyworms strains likely exhibit differences in their migratory patterns and should be managed as two unique pests. When exploring the C-strain geographic populations in more detail, our data indicates that fall armyworms that overwinter in south Florida are under increased selection for detoxification and insecticide resistance than those populations that overwinter in south Texas. As such, it is increasingly important that we focus on tracking and managing the spread of insecticide resistance within this geographic population to better manage this pest. Analysis of the data obtained from sequencing the additional 368 fall armyworm samples collected in 2022 is ongoing and focused on: (1) addressing whether the patterns of population genetic structure observed in the 2021 dataset are consistent across years, and (2) whether we can identify the underlying genetic basis of a potential nuclear-mitochondrial genomic incompatibility that may finally provide an elusive causal mechanistic explanation for the existence and maintenance of the genetically distinct C- an R- fall armyworm strains. Objective 2. The second objective was to determine if a significant return migration (north to south) occurs in fall armyworm moths using stable isotope analysis. To do so, we conducted an experiment where we reared fall armyworm larvae on host plants irrigated with water sources that had a known isotopic ratio. We used the data from this experiment to develop a model that can be used to correlate the ratio of hydrogen isotopes found in wing chitin with the ratio of isotopes found in the water. We then collected moths from the field in both 2021 and 2022 to assess the isotopic profile found in the wing chitin of field collected individuals. The wing samples from both years were sent to the Stable Isotopes for Biosphere Science Laboratory (SIBS) facility at Texas A&M for elemental analysis with isotope ratio mass spectrometry (EA-IRMS). Unfortunately, the SIBS facility ran into technical challenges with their elemental analyzer equipment (TCEA) along with personnel turnover that delayed receipt of data. Ultimately, the SIBS facility honored our original agreement and sent the samples to an alternative provider at Utah State University for analysis. Immediately prior to the submission of this final report, we began receiving the long-awaited data. We anticipate the analysis of these results to be completed and published in 2025. Objective 3. Our final objective was to assess the effects of photoperiod on the hybridization rates between the two fall armyworm strains (C- and R-). To do so, we ordered new environmental cabinets with the specialized ability to set up three independently maintained photoperiod regimes within the same chamber. We also established homozygous C- and R-strain breeding lines in the laboratory to provide insects for the experiments. The proposed behavioral hybridization assays have taken much longer to complete than originally anticipated and needed to be reinitiated after discovering some cross-contamination of strains in the breeding populations. Fortunately, this was discovered prior to any genotyping, thus ensuring the integrity of the results once obtained. The completed work including final data collection, analysis and write-up will be included as a dissertation chapter in PhD student, Andie Miller's, dissertation which she is scheduled to defend by the end of 2025. The results will also be prepared for dissemination in a peer-reviewed publication to be submitted in the same timeframe. In addition to the primary objectives, we also used this project as an opportunity to further explore strain specific allochronic behaviors in both the field and lab. In the laboratory, we have conducted assessments of strain-specific allochronic variation across behaviors, traits and life stages including adult male and female activity patterns, timing of female pheromone expression, larval development time, egg lay, and timing of larval feeding, In the field, we used automated trapping devices to monitor the time when fall armyworm strains were active at night based on the timing of male captures in pheromone traps. Consistent with previous findings, we found that across geographic regions, C-strain males tend to be active in the first 5 hours after sunset, whereas R-strain males are active much later in the night (7+ hrs after sunset). However, there was substantial variation within each strain on any given night that may complicate the use of male pheromone capture times as a diagnostic trait for real-time strain specific monitoring and management applications. These results were successfully published as part of a special issue of the peer-reviewed journal,Frontiers in Plant Sciencesfocusing onMagnetobiology and Chronobiology: New Opportunities for Smart Phytoprotection(Miller et al. 2024. Frontiers in Plant Science. 15:1380624).

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Tessnow, A.E., Nagoshi, R., Meaghar, R., Gilligan, T.M., Sadd, B.M., Carri�re, Y., David, H.N., Fleischer, S.J., Richers, K., Palumbo, J.C., Porter, P. Rodrigues, J.C.V. & Sword, G.A. (2025) Genomic patterns of strain-specific genetic structure, linkage, and selection across fall armyworm populations. BMC Genomics 26:116. DOI:10.1186/s12864-025-11214-8
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Miller, A.C., Tessnow, A.E., Meagher, R.L., Nagoshi, R.N., Gilligan, T.M. & Sword, G.A. Assessing fall armyworm (Spodoptera frugiperda) allochronic behavior as a predictor of local strain composition in United States populations. (2024) Frontiers in Plant Science. 15:1380624. Special Issue Magnetobiology and Chronobiology: New Opportunities for Smart Plant Protection. DOI:10.3389/fpls.2024.1380624
• Type: Other Journal Articles Status: Published Year Published: 2025 Citation: Wan, G. Sword, G.A., Du, J., Huang, Q., Chen, W. & Warrant, E. (2025) Editorial: Magnetobiology and Chronobiology: New Opportunities for Smart Phytoprotection. Frontiers in Plant Science. DOI:10.3389/fpls.2025.1594646
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sword, G.A. (2024) Helicoverpa zea & Spodoptera frugiperda: Genomic Surveillance and Timely Tales. NC246 Ecology and Management of Arthropods in Corn Multistate Hatch Project Meeting. Savannah, Georgia, Jan. 23-25, 2024.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Silva, T., Sword, G.A., Miller, A., Qureshi, J.A., Head, G.P., Kerns, D., Jurat-Fuentes, J.L.L, Villegas, J., Towels, T., Ni, X., Reay-Jones, F.P.F, Carrillo, D, Cook, D.R., Daves, C., Stout, M.J., Thrash, B., Paula-Moraes, S., Lin, S., Patla, B., Niu, Y., Sakuno, C.I.R. & Huang, F. (2024) Reversibility of practical resistance to Bt crops: A case report on the resistance of fall armyworm to Cry1F maize in the southeastern United States. Journal of Pest Science. DOI:10.1007/s10340-024-01804-y
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Sword, G.A., Tessnow, A.T., Miller, A., Nagoshi, R., Meaghar, R. & Gilligan, T. (2024) Population genomics and allochronic strain divergence as a framework for strain-specific management of the fall armyworm in North America. International Congress of Entomology, Kyoto, Japan, Aug. 25-30, 2024
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Miller, A.C., Behmer, S.T. & Sword, G.A. (2025) Ecological Consequences of Strain Divergence in the Fall Armyworm (Spodoptera frugiperda) Beltwide Cotton Conference, Cotton Insect Research and Control conference. New Orleans, NO, USA, Jan. 14-16, 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Miller, A.C., Sword, G.A., Tessnow, A.E. & Meagher, R. (2024) Assessing Fall Armyworm Strain-Specific Mating Behaviors in the Field as a Future Tool for Strain Identification. Beltwide Cotton Conference, Cotton Insect Research and Control conference. Fort Worth, TX, Jan. 4-5, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Miller, A.C., Sword, G.A. & Kerns, D.L. (2024) Strain-Specific Susceptibility of the Fall Armyworm (Spodoptera frugiperda) to Cypermethrin and Chlorantraniliprole May Necessitate More Focused Monitoring and Management Strategies. Beltwide Cotton Conference, Cotton Insect Research and Control conference. Fort Worth, TX, Jan. 4-5, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Kopecki, M., Miller, A.C., Sword, G.A. & Riggan, J. (2024) Investigating Allochronic Mating Behavior of the Fall Armyworm (Spodoptera frugiperda) Host Strains and Hybrids in Controlled Field Experimentation. Beltwide Cotton Conference, Cotton Insect Research and Control conference. Fort Worth, TX, Jan. 4-5, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Riggan, J., Miller, A.C., Sword, G.A., Ludwig, D., Porter, P. & Gonzales, J.C.S. (2024) Assessing the Efficacy and Specificity of Multi-Component Pheromone Lures and Funnel Trap Colors for Monitoring Fall Armyworm (Spodoptera frugiperda) Population.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Miller, A.C., Kerns, D.L., & Sword, G.A. (2025) Differential susceptibility of the Spodoptera frugiperda strains to synthetic and biological insecticides. Entomological Society of America, Southwestern Branch Meeting, March 23-26, 2025.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Tessnow, A.E., Nagoshi, R., Meaghar, R., Gilligan, T.M., Miller, A.C., West, J. & Sword, G.A. (2024) Assessing patterns of fall armyworm movement across the continental United States. Entomological Society of America Annual Meeting, Phoenix, AZ, Nov. 10-14, 2024.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Miller, A.C., Tessnow, A.E. & Meagher, R., Nagoshi, R., Gilligan, T.M., Behmer, S.T., Kerns, D.L., & Sword, G.A. (2024) No strain, no gain: Why understanding Spodoptera frugiperda strains in the field matters for research and management. Invited symposium presentation: Current Research and Future Challenges for Management of Corn Pests. Entomological Society of America Annual Meeting, Phoenix, AZ, Nov. 10-14, 2024.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:During the current reporting period, wepresented project results from our 2021 field season population genomics sampling to an audience of other entomological researchers at the 2023 Entomological Society of America Annual Meeting in National Harbor, MD. This audience includedresearchers and extension professionals who are interested in developing more targeted management solutions for fall armyworm in the US and Canada. While in National Harbor, our team was also able to provide an update on all three project objectives at theUSDA-NIFA's A112: Pest and Beneficial Species Project Director's Meeting.This allowed us to communicate our results and project progress not only to NIFA, but to other researchers interested in addressing problems associated with pests and beneficial insects. Another major presentation of project results in 2023 was to theNC246- Ecology and Management of Arthropods in Corn Multistate Hatch Project meeting in San Antonio, TX. The audience here consisted of government and university research/extension personnel involved in the management of corn pests across the US as well as in Canada. In addition to reporting on the direct project objectives, this grant has allowed us to develop a closely related projects exploring allochronic behavior of fall armyworm strains in both the Texas and Florida populations. The results from these effortswere presented by a PhD student in our lab at the 2023 Beltwide Cotton Conference in New Orleans, LA, targeting researchers that are more interested in management approaches. The student won 2ndplace in the PhD student presentation competition. She also presented her ongoing researchat the 2023 Entomological Society of America annual meeting and won 1stplace in for her presentation in the Graduate SysEB: Behavior section. Changes/Problems:The main challenges that we encountered during this reporting period was in the completion of the behavioral mating assays as outlined in Objective 3. We had originally proposed to use camcorders to film moth mating behavioral parameters to determine the timing of female calling, male calling, copulation, and oviposition. We ran into technical challenges filming and scoring these behaviors within a recording. Therefore, we have focused our efforts on determining the number, viability, and genotype of the offspring to determine the rates of hybridization across our treatments. We expect this will still allow us to address the aims described in our initial objective. Additionally, we have experienced an extensive delay in receiving the isotope data for our wing samples as outlined in Objective 2 of this grant. The SIBS facility at Texas A&M where we submitted our wing samples had a change in personnel and then ran into some technical challenges with their elemental analyzer equipment (TCEA). As a result, the wing data for our samples are only beginning to be processed at the time of submission of this report. We have been assured that this data will become available for our analyses in February 2024. This has not resulted in any changes to our original project design or plan, but has substantially delayed our initial timeline. What opportunities for training and professional development has the project provided?In addition, this grant supported research conducted by a PhD student in the Ecology and Evolutionary Biology Program at Texas A&M University, Andie Miller. She was ableto conduct fieldwork in Florida hosted by project personnel Meaghar and Tessnow during the fall of 2023. She was also able to attend and present her research at the 2023 Beltwide Cotton Conference and the 2023 Entomological Society of America Annual Meeting where she was awarded 2ndand 1stplace prizes in the PhD student presentation competitions at the respective meetings. These served as an excellent opportunityto gain valuable presentation experience and develop her scientific network. In addition, the grant has supported the training of 3 undergraduate students who have worked to help maintain the fall armyworm rearing colony at Texas A&M. Two students have also conducted their own undergraduate research projects for course credit (ENTO491) investigating fall armyworm pheromone trapping effects and allochronic mating behavior under the mentorship of PhD student Andie Miller and PI Sword. How have the results been disseminated to communities of interest?During this reporting period, our results were disseminated directly to researchers and extension professionals through our participation at conferences and the submission of multiple publications. For example, project findings were presented in 2023 at the Entomology Society of America Annual meeting and the Beltwide Cotton Conference, both of which are primarily attended by entomology researchers and industry professionals. Our postdoc also presented results at the NC246 annual meeting of research and extension entomologists who then relay information directly to stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We requested and received a one-year no-cost extension in order to wrap up our data analyses and submit our final data for publication for all three of our objectives. Objective 1: We will finish the genomic analysis of our fall armyworm moth samples that were collected in 2022, and publish our findings in a manuscript. Objective 2: We expect to receive the isotopic composition data from our fall armyworm wing samples in February of 2024. We will then use the isoscape model that we previously developed to make predictions about the natal origin of all moth wing samples that were collected across both 2021 and 2022. These results will then be submitted for publication. Objective 3: In 2024 we will finish our behavioral mating assays to determine if differences in photoperiod can influence the hybridization rate of C- and R- strain fall armyworms. Once complete, these results will then be submitted for publication.

Impacts
What was accomplished under these goals? The first objective of this grant is to characterize the genetic structure ofS. frugiperdaacross the entire United States using a population genomics approach. In the previous reporting periods, we setup an extensive fall armyworm trapping network across the US that included 13 different locations spread across three proposed fall armyworm flyways, collecting well over 2000 moth samples. We also extracted and sequenced the DNA from 431 moth samples collected in 2021 using a low coverage whole genome sequencing approach. During this reporting period, we completed our population genomic analyses of the samples collected in 2021, and wrote up this data into a manuscript that is virtually ready for submission. We have several major findings from this objective. First, the fall armyworm species is divided into two genetically distinct strains in the United States, and the reproductive isolation between these strains is likely the result of incompatibilities on the Z-chromosome that limit hybridization. We also found different patterns of geographic sub-structuring between these strains, with the C-strain showing some reproductive isolation between populations originating in Florida, Texas and Puerto Rico, while the R-strain is completely admixed across these distant geographic locations. We also found, no evidence of the R-strain occurring in the western United States. Together, these findings indicate that the two fall armyworms strains likely exhibit differences in their migratory patterns and should be managed as two unique pests. When exploring the C-strain geographic populations in more detail, our data indicates that fall armyworms that overwinter in south Florida are under increased selection for detoxification and insecticide resistance than those populations that overwinter in south Texas. As such, it is increasingly important that we focus on tracking and managing the spread of insecticide resistance within this geographic population to better manage this pest. We have now also sequenced an additional 368 fall armyworm samples that were collected in 2022 to confirm these patterns are consistent across years. The second objective outlined in this grant is to determine if a significant return migration (north to south) occurs in fall armyworm moths using stable isotope analysis. During the previous reporting periods, we had developed a model to correlate stable isotopes of hydrogen found in wing chitin with that of the water used to irrigate the larval host plant. We then collected moths from the field in both 2021 and 2022 to assess the isotopic profile found in the wing chitin of field collected individuals.During this reporting period, we finished preparing the wing samples collected in 2022 and sent them the SIBS facility at Texas A&M for elemental analysis with isotope ratio mass spectrometry (EA-IRMS). Unfortunately, the SIBS facility ran into some technical challenges with their elemental analyzer equipment (TCEA). As a result, we are still waiting to receive the data back from all samples. We have been told that the data from all samples should be available in February 2024. The final objective of this grant is to assess the effects of photoperiod on the hybridization rates between the two fall armyworm strains (C- and R-). In the previous reporting periods, we had ordered our environmental chambers and established homozygous C- and R-strain lines. During this reporting period, we completed about half of our proposed behavioral hybridization assays. Our plan is to complete the remaining hybridization assays by April, and then genotype the male offspring from each egg mass to verify hybrid identity. In addition to the primary objectives of this project, we have also used this project as an opportunity to further explore strain specific allochronic behaviors in the field. To do this, we used automated trapping devices to monitor the time when fall armyworm strains were active at night. Consistent with previous findings, we found that across geographic regions, C-strain individuals tend to be active in the first 5 hours after sunset, whereas the R-strain is active much later in the night (7+ hrs after sunset). Our goal is then to use time to more easily identify the pest strain within a field so that we can develop more targeted strain specific management approach. This project is currently being written up for publication.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Nagoshi, R.N., Tessnow, A.E., Carriere, Y., Bradshaw, J., Harrington, K., Sword, G.A., & Meagher, R.L. (2023). Using genetic comparisons of populations from Arizona, Mexico, and Texas to investigate fall armyworm migration in the American southwest. PLOS One. 18(11), e0289060.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Tessnow, A.E., Nagoshi, R.N., Meagher, R.L., & Fleischer, S.J. (2023). Revisiting fall armyworm population movement in the United States and Canada. Frontiers in Insect Science (Special Issue: Focus on Fall armyworm). 3, 1104793.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2024 Citation: Meagher, R.L., Tessnow, A.E., & Sword, G.A. (In Press). Past, present, and future applications of molecular tools in landscape scale IPM: Fall armyworm as a case study. In M. Brewer & G. Hein (Eds.) Arthropod Management and Landscape Considerations in Large Scale Agroecosystems. (1st Ed.). CABI Publishing.
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Miller, A.C., Tessnow, A.E., Meagher, R.L., Nagoshi, R.N., Gilligan, T.M. & Sword, G.A. (submitted) Consistent strain-specific differences in fall armyworm (Spodoptera frugiperda) allochronic behavior across United States populations. Frontiers in Plant Science (Special Research Topic: Magnetobiology and Chronobiology: New Opportunities for Smart Phytoprotection).
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Tessnow, A.E.,* Meagher, R.L., Nagoshi, R.N., Gilligan, T., Miller, A.C., & Sword, G.A., ⿿Disentangling fall armyworm genetic structure in North America and its implications for management.⿝ Entomological Society of America Annual Meeting. National Harbor, MD. November 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Miller, A.C.,* Tessnow, A.E., Meagher R.L., Nagoshi, R.N., & Sword, G.A. ⿿Patterns of field-based allochronic behavior of Spodoptera frugiperda host strains in Texas and Florida provide evidence of alternative reproductive isolating mechanism between strains.⿝ Entomological Society of America Annual Meeting. National Harbor, MD. November 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sword, G.A.,* Tessnow, A.E.*, Miller, A.C., Meagher, R.L., Nagoshi, R.N., & Gilligan, T.M. ⿿An integrative approach to fall armyworm population genomics.⿝ USDA-NIFA⿿s A112: Pest and Beneficial Species Project Director⿿s Meeting. National Harbor, MD. November 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Tessnow, A.E.,* & Sword, G.A. ⿿Revisiting fall armyworm movement in the US and Canada.⿝ NC246- Ecology and Management of Arthropods in Corn. San Antonio, TX. January 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Miller, A.C, Sword, G.A. & Tessnow, A.E. (2023) Marker-assisted Fall Armyworm breeding: Leveraging novel, real-time genotyping techniques to better characterize C-strain, R-strain, and hybridized Spodoptera frugiperda. Beltwide Cotton Conference, Cotton Insect Research and Control conference. New Orleans, LA., Jan. 10-13, 2023.

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:During the current reporting period, we presented preliminary results from our population genomics project to an audience of other entomological researchers at the Entomological Society of America annual meeting as well as the annual meeting of theNC246: Ecology and Management of Arthropods in Corn multistate research group. These audiences include researchers and extension professionals who are interested in developing more targeted management solutions for fall armyworm in the US and Canada. This also allowed us to receive feedback on our project design and discuss potential data analysis methods with others. Additionally, fall armyworms are a major pest of corn and forage across the US. Therefore, we also discussed our ongoing project with the growers in Warton County, TX at an extension event in February. In this event, we explained the project, showed preliminary results, and talked about how our results may impact fall armyworm control in the future. Changes/Problems:There have not been many changes to the original project goals. Objective 1: For unknown reasons, there were fewer fall armyworms reported across the country this year than last year. As a result, it was more challenging to collected samples in the north. Therefore, some of our more northern locations were slightly different in 2022 than in 2021. Because of the substantial number of moth samples we have, this should not influence our results. Objective 2:The moths samples collected in September and October in Lubbock were accidently stored in ethanol making them unusable for stable isotope analysis. We were able to rectify this in November and expanded to add another location further south in College Station TX, to make up for these lost samples. Objective 3: Progress was delayed by a COVID-related supply chain issue that delayed the delivery of the controlled environment chambers for many months. The problem was resolved by cancelling the original order and obtaining chambers from an alternative vendor who was able to deliver them in a timely manner. The chambers are now set up and running in the lab for the conduct of the Objective 3 experiments to take place as originally planned. What opportunities for training and professional development has the project provided?This grant supported our postdoctoral research associate to attend the Entomological Society of America annual meeting in Vancouver, Canada and present a talk about fall armyworm population genomics in an invited symposium. After her talk, she was able to network with researchers in the field and gain advice for applying for faculty positions. She was also invited to present her research at the NC246: Ecology and Management of Arthropods in Corn annual meeting, which allowed her to broaden her network with extension professionals across the country. Combine, these experiences served to enhance her professional development and better develop her career goals. We intended for graduate student Andie Miller present her research at the 2022 Beltwide Cotton Conference in San Antonio, TX, but she could not attend due to COVID.However, she was able to present in-person at the 2022 Ecological Integration Symposium held annually at Texas A&M University. This served as a good opportunity for her to synthesize her results for presentation and develop her presentation skills. She has also been actively involved in mentoring undergraduate researchers (Kendall Walton) and international visiting students (Yong-Sin Lo from NCHU in Taiwan) working on fall armyworms in the lab. How have the results been disseminated to communities of interest?During this reporting period, our results were disseminated directly to researchers, extension professionals and growers through our participation in national and regional conferences and extension events. At the national level, our findings were presented at the Entomological Society of America annual meeting and the Beltwide Cotton Conference, both of which are primarily attended by entomology researchers and students. Our postdoc also presented results at the NC246 annual meeting of extension entomologists who then relay information directly to stakeholders. Our graduate student presented at a local research conference at Texas A&M. Finally, our postdoc presented a seminar to growers concerned about fall armyworms in Wharton County, TX at a pesticide recertification training. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to achieve the following for each objective: Objective 1: We will complete all DNA extractions for the moth samples collected in 2022. Following extraction, we will send DNA from these individuals for sequencing to the Texas A&M AgriLife Genomics and Bioinformatics Service using the AgSeq platform. This data will then be analyzed together with our 2021 sequencing data to confirm we see the same trends repeating across multiple years. These results will then be written up into a manuscript. Objective 2: During the next reporting period, we will excise the wings from all of our 2022 field collected moth samples and use elemental analysis with isotope ratio mass spectrometry (EA-IRMS) to determine the isotopic composition of the wings. We will then use the isoscape models developed in 2021 to make predictions about the natal origin of all moth wing samples that were collected across both 2021 and 2022. These results will then be written up into a manuscript. Objective 3: In 2023 we will conduct behavioral mating assays to determine if differences in photoperiod can influence the hybridization rate of C- and R- strain fall armyworms. To do this, we will rear fall armyworms in one of three incubators that each have a different photoperiod (10:14, 12:12, and 14:10 L:D). Once the moths eclose, we will set up mating groups within each photoperiod treatment and use camcorders to record matings. These mating groups will include a control assay where a female has access to two males of the same strain, no choice assays where the female has access to two males of the opposite strain, and choice assays where the female has access to one male of each strain. The resulting camera data will be analyzed and the following behaviors will be recorded: female calling, male calling, copulation, and oviposition. Specifically, we will record the time of each mating, the time males and females initiate calling, the total time spent copulating, the time of oviposition, the # eggs laid, and the female mate choice. These results will then be written up into a manuscript.

Impacts
What was accomplished under these goals? Objective1: The first objective of this grant is to characterize the genetic structure ofS. frugiperdaacross the entire United States using a population genomics approach. In the previous reporting period (2021), we setup an extensive fall armyworm trapping network across the US that included 13 different locations spread across three proposed fall armyworm flyways. We collected well over 1000 moth samples. During this reporting period (2022), we repeated our sampling at these locations for a second year. We also extracted and sequenced the DNA from 431 moth samples collected in 2021 using a whole genome sequencing approach called AgSeq. We found that there are significant genetic differences between fall armyworm strains across the central US, and this difference is driven by the Z-chromosome. We also found low levels of geographic sub-structuring in one strain (C-strain), but no evidence of sub-structuring in the R-strain, which could indicate different dispersal patterns between FAW strains. Finally, we found that there was virtually no evidence of the R-strain occurring in the western United States, despite this region being dominated by plants that are generally considered R-strain hosts. If this finding is repeatable in 2022, this could benefit stakeholders in the western US by allowing them to develop and implement C-strain specific management programs throughout the region. Objective2: The second objective outlined in this grant is to determine if a significant return migration (from north to south) occurs in fall armyworm moths using stable isotope analysis. In the previous reporting period, we developed a model to correlate stable isotopes of hydrogen found in wing chitin with that of the water used to irrigate the larval host plant. We then collected moths from the field to assess the isotopic profile found in the wing chitin of field collected individuals.In the current reporting period, we repeated our sampling from 2021 and collected moths in Lubbock, TX; College Station, TX; Weslaco, TX; Midville, GA; and Belle-Glade, FL in the fall. We also prepared the previous wing samples from 2021 and sent them the Stable Isotopes for Biosphere Science (SIBS) facility at Texas A&M for elemental analysis with isotope ratio mass spectrometry (EA-IRMS). We should receive the data back from these samples imminently. Objective3: The final objective of this grant is to assess the effects of photoperiod on the hybridization rates between the two fall armyworm strains (C- and R-). In the previous reporting period (2021) we had established homozygous C- and R-strain lines and ordered environmental chambers. During this reporting period, we were able to setup the environmental chambers with cameras to track the moth activity. We then tested our setup for the hybridization experiments. We plan to complete these hybridization experiments at the beginning of the next reporting period.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Tessnow, A.E., Raszick, T.J., & Sword, G.A. (2022). Genomic patterns of gene flow and strain divergence in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Ecology & Evolution. 12(3), e8706.
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Tessnow, A.E., Nagoshi, R.N., Meagher, R.L., & Fleischer, S.J. (Submitted). Revisiting fall armyworm population movement in the United States and Canada. Frontiers in Insect Science. (Special Issue: Focus on Fall armyworm).
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: 6. Miller, A.C.*, Tessnow, A.E. & Sword, G.A. Marker-assisted fall armyworm breeding: leveraging novel, real-time genotyping techniques to better characterize C-strain, R-strain, and hybridized Spodoptera frugiperda. Beltwide Cotton Conference. San Antonio, TX. January 2022. (could not attend due to COVID)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Tessnow, A.E.*, & Sword, G.A. Revisiting fall armyworm populations and strains in the US. NC246- Ecology and Management of Arthropods in Corn. Zoom. January 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Tessnow, A.E. *, Meagher, R.L., Nagoshi, R.N., Gilligan, T.M., Sadd, B.M., & Sword, G.A. Unraveling fall armyworm genetic structure in North America using a whole genome sequencing approach. ESA, ESC, and ESBC Joint Annual Meeting. Vancouver, BC, Canada. November 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Silva, T.H.*, Niu, Y., Sword, G.A., Qureshi, J.A., Head, G.P., Kerns, D., Jurat-Fuentes, J., Ni, X., Carillo, D., Sakunk, C. (2022) Extended investigation of fall armyworm resistance in the southern United States to four common Bt proteins. ESA, ESC, and ESBC Joint Annual Meeting. Vancouver, BC, Canada. November 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Miller, A.*, Tessnow, A.E. & Sword, G.A. (2022) Allochronic mating behavior in Spodoptera frugiperda. Ecological Integration Symposium, Texas A&M University, College Station, TX, April 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Tessnow, A.E.* Applications of current fall armyworm research to future management programs. Wharton County 5 CEU Pesticide Recertification Conference. Zoom. February, 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Sword, G.A.* & Tessnow A.E. Forget corn and rice, fall armyworm strains are time-associated incipient species that C- come out early and R- really late. Beltwide Cotton Conference. San Antonio, TX. January 2022.

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:During the current reporting period, we were able to present our project to an audience of other entomological researchers at the Entomological Society of America annual meeting. This allowed us to receive feedback on our project design and discuss potential pitfalls and data analysis methods with others who have considered using similar methodologies in their own research. We also met new contacts who would be willing to assist with sample collection during the next reporting period. A broader audience of stakeholders and the general public were also informed of our work courtesy of a media release from Texas A&M AgriLife Research announcing the initiation of the project. https://today.tamu.edu/2021/02/17/global-fight-against-fall-armyworm-gets-texas-boost/ Changes/Problems:There have not been any major changes to the original project goals. We did struggle more than we had originally anticipated collecting samples this year due to the COVID-19 pandemic. Therefore, some of our collection locations are slightly different from what was originally proposed, but all regions are still represented. For example, the collaborators in Davis, CA were not allowed to go into the field, but we found a schoolteacher in Bakersfield, CA that was willing to set out traps and send us fall armyworm samples. We also had to modify the collections for the stable isotopes objective because there were virtually no fall armyworm moths in northern TX or GA in early November. Therefore, we will only be able to use two months of sampling (September and October) from these northern locations. We still have the full three months of sampling from our locations further south (September, October, and November). What opportunities for training and professional development has the project provided?This grant supported our postdoctoral research associate, Ashley Tessnow, to attend the Entomological Society of America annual meeting in Denver, CO and present a talk about the Stable Isotopes Project (Objective 3).After her talk, she was able to connect with several researchers across the country who would be willing to provide additional fall armyworm samples in the future. This conference also allowed her to network with several researchers in the field and gain advice for applying for faculty positions. Combined, these experiences served to enhance her professional development and better develop her career goals. In addition, a new graduate student, Andie Miller,started in the Swordlab at Texas A&M in August of 2021. This student has now been trained to trap,rear and genotype fall armyworm moths using the new Tessnow et al. (2021) SNP diagnostoc assays. Shewill have the opportunity to present her first talk at the Beltwide Cotton Conference in January 2022 in San Antonio, TX. She will also participate in conducting the mating experiments in the coming years as part of Objective 3.Since beginning at Texas A&M, she has had the opportunity to meet with collaborators on this project who are located in south Texas, expanding her professional network. How have the results been disseminated to communities of interest?During this reporting period, our initial results have only been formally disseminated to other entomological researchers at the Entomology Society of America annual meeting. However, we had to reach beyond our typical network this year to collect the moth specimens. As a result, we have informally discussed our project goals with other researchers (both academic and USDA), extension professionals, and growers across the country to get permission and assistance collecting moth samples from their regions. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to achieve the following for each objective: Objective 1: We will complete all DNA extractions for the moth samples collected in 2021. Following extraction, we will send the DNA from these individuals off the TxGEN for sequencing using the new AgSeq platform.Ideally this data will be returned to us by the summer of 2022, and we will be able to begin some preliminary bioinformatic analyses. We will also repeat our moth collections in the same 13 locations where we had collected in 2021, in order to get a second year of sampling data. Objective 2: During the next reporting period, we will excise the wings from all of our 2021 field collected moth samples, and useelemental analysis with isotope ratio mass spectrometry (EA-IRMS) to determine the isotopic composition of the wings. We will then use the isoscapes we developed in 2021 to make predictions about the natal origin of these samples. Additionally, we will collect another round of moth samples in September, October, and November in order to secure a second year of data for this project. Objective 3: In 2022 we will conduct behavioral mating assays to determine if differences in photoperiod can influence the hybridization rate of C- and R- strain fall armyworms. To do this, we will rear fall armyworms in one of three incubators that each have a different photoperiod (10:14, 12:12, and 14:10 L:D). Once the moths eclose, we will set up mating groups within each photoperiod treatment. These mating groups will include a control assay where a female has access to two males of the same strain, a no choice assaywhere the female has access to two males of the opposite strain, and choice assays where the female has access to one male of each strain. Camcorders will be setup to record all mating that occurs within each mating cage for four days.The resulting camera data will be analyzed and the following behaviors will be recorded: female calling, male calling, copulation, and oviposition. Specifically, we will record the time of each mating, the time males and females initiate calling, the total time spent copulating, the time of oviposition, the # eggs laid, and the female mate choice.

Impacts
What was accomplished under these goals? Objective 1: The first objective of this projectis to characterize the genetic structure ofS. frugiperdaacross the entire United States using a population genomics approach. To work towards this goal, we setup an extensive fall armyworm trapping network across the US. This network included 13 different locations spread across all three proposed fall armyworm flyways. We collected well over 1000 moth samples for use in the Objective 1 and Objective 2 experiments. All samples that will be used for Objective 1 are stored in 95% ethanol in the -20 freezer. All samples that will be used for Objective 2 and stored in Eppendorf tubes stored at -20 without ethanol. One key aim of this objective was also to identify SNP markers that could be used to differentiate between strains and populations of fall armyworms in the US. Using older sequencing data, we developed four new SNP based genotyping assays to differentiate between the two fall armyworm strains (C- and R-). These assays are now published in PeerJ. Objective 2:The second objective outlined in this grant is to determine if a significant return migration (north to south) occurs in fall armyworm moths using stable isotope analysis. To begin this process, we had to develop a model that correlates stable isotopes of hydrogen found in wing chitin with that of the water used to irrigate the larval host plant. To do this, we first obtained four water samples with unique δ2H profiles. We then grew sorghum plants by irrigating them exclusively with water from one of the four water samples. We raised fall armyworm caterpillars from neonates through pupation on these sorghum plants. We then removed the pupae and allowed them to eclose as moths. At the end of the experiment, we excised the moths' wings, and assessed the isotopic composition of both the wings and the water samples using elemental analysis with isotope ratio mass spectrometry (EA-IRMS). Using the data from this experiment, we found that there was a strong linear relationship between the isotope δ2H in moth wing chitin and the water used to irrigate their larval host plant (R2=0.9714). Using this relationship, we created an isoscape, or isotopic map model, for moth wing chitin that will be used to predict the natal origin of migratory moths based on the isotopes present in their wing chitin. In addition to developing this model, we also collected fall armyworm samples from the field in six locations: Morton, IL; Lubbock, TX; Weslaco, TX; State College, PA; Midville, GA; and Belle Glade, FL. Although we had only proposed to assess moths in Lubbock, Weslaco, Midville, and Belle Glade for this objective, we choose to also include State College, PA and Morton, IL as a controls to represent the isotopic compositions that may be present in more norther populations, since this was a comment from one of our grant reviewers. All six locations were sampled in September. Lubbock, Weslaco, Midville, and Belle Glade were also sampled in October and November. Because temperatures were too cold in Lubbock and Midville in November, we did not recover enough samples from these locations. Therefore, we will only have data from Weslaco and Belle Glade for November. After cleaning the wings of these field collected samples with a methanol: chloroform solution, we will conduct elemental analysis with isotopic ratio mass spectrometry on these tissues. We will then use our isoscape model to predict the natal origin of these field collected moths. Objective 3: The final objective of this grant is to assess the effects of photoperiod on the hybridization rates between the two fall armyworm strains (C- and R-). To complete this objective, the first step is to establish homozygous R- and C- strain fall armyworm colonies in the lab. After many collections of fall armyworm larvae, we have founded a pure strain colony for both C- and R- strain fall armyworms. Additionally, we have purchased cameras to record the nightly activities of fall armyworm moths and begun testing these cameras to determine the best setup to record the evening activity of moths. We expect to have all mating behavioral experiments completed in the next reporting period. Citation:Tessnow, A. E., Gilligan, T. M., Burkness, E., De Bortoli, C. P., Jurat-Fuentes, J. L., Porter, P., Sekula, D., & Sword, G. A. (2021). Novel real-time PCR based assays for differentiating fall armyworm strains using four single nucleotide polymorphisms. PeerJ, 9, e12195.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Tessnow, A. E., Gilligan, T. M., Burkness, E., De Bortoli, C. P., Jurat-Fuentes, J. L., Porter, P., Sekula, D., & Sword, G. A. (2021). Novel real-time PCR based assays for differentiating fall armyworm strains using four single nucleotide polymorphisms. PeerJ, 9, e12195.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Tessnow, A.E., Raszick, T.J., Porter, P., & Sword, G.A. (Under Review). Patterns of genomic and allochronic strain divergence in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Molecular Ecology.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Tessnow, A.E., West, J., Maupin, C., & Sword, G.A. Tracking fall armyworm movement across the US using stable isotopes. Entomology Society of America Annual Meeting. Denver, CO. 2021.