DISPERSAL AND REPRODUCTIVE POTENTIAL OF HELICOVERPA ZEA IN BT AND NON-BT CROPS AND IMPLICATIONS FOR INSECTICIDE RESISTANCE MANAGEMENT REGULATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1027344
• Grant No.: 2021-33522-35688
• Cumulative Award Amt.: $499,800.00
• Proposal No.: 2021-04207
• Project Start Date: Sep 1, 2021
• Project End Date: Apr 15, 2026
• Grant Year: 2021
• Program Code: [HX]- Biotechnology Risk Assessment

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
Several benefits are associated with large-scale adoption of transgenic crops expressing insecticidal proteins from the bacteria Bacillus thuringiensis, commonly referred to as Bt crops. However, Helicoverpa zea is resistant to several Bt traits in corn and cotton, and Vip is the only effective Bt trait currently commercially planted. Helicoverpa zea is an economic pest in cotton, and similar Bt toxins are expressed in corn and cotton. Hence, there is a high risk of H. zea evolving resistance to Vip-expressing crops on a broad scale in the continental U.S. In 2018, the final EPA FIFRA SAP report concluded the requirement of non-Bt refuge as one of the cornerstones of Bt IRM. However, the assumption of random mating between refuge and Bt crops in this species remains untested. The overall goal of this proposal is to provide knowledge of short distance dispersal capacity and reproductive potential of H. zea, using mark-recapture egg albumin marker, flight mills, and estimation of the sex, moth age, female mating status and maturity, and stable carbon isotope signature of wings and spermatophores of field populations H. zea. The data generated in this proposal will be used to refine a deterministic, frequency-based model of H. zea population genetics, exploring the risk assessment null hypothesis that movement and reproductive parameters (dispersal distance, mating status, female maturity, and moth age and origin) will not impact the rate of resistance evolution in H. zea. This proposal will support accurate IRM regulations concerning refuge for the deployment and sustainability of Bt crops.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	1510	1130	70%
211	1719	1130	30%

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

Subject Of Investigation
1510 - Corn; 1719 - Cotton, other;

Field Of Science
1130 - Entomology and acarology;

Keywords

helicoverpa zea

flight capacity

random mating

refuge

reproductive potential

Goals / Objectives
The objectives of this projectalignwith Program 5c of NIFA BRAG Program, which supports research to understand the frequency and mechanisms by which pests or diseases overcome plant pest resistance traits conferred by engineered genes. We expect to provide field and laboratory dataof the southern U.S. to address out-of-date, model-based IRM programs.The overall goal of this project is tofill critical gaps about H. zeadispersal capacity,non-assortative mating, andreproductive potential of H. zea. The data generated in this proposal will be used to refine a deterministic, frequency-based model of H. zea population genetics, exploring the risk assessment null hypothesis that movement and reproductive parameters will not impact the rate of resistance evolution in H. zea.The objectives of the project are:Estimate the typical short distance dispersal range of male and female H. zea.Document the random mating and reproductive potential of dispersing populations of H. zea in the agricultural landscape.Create a refined model of the evolution of resistance for H. zea in complex landscapes.

Project Methods
Objective 1) Estimate the typical short distance dispersal range of male and female H. zea.Objective 1a. Estimate the typical dispersal range of male and female H. zea emerging from corn.The dispersal capacity of H. zea in the field will be estimated using the mark-recapture method using an albumin marker in the following three locations: Vernon James Research & Extension Center, North Carolina State University; Clemson University Pee Dee Research and Education Center, Florence, South Carolina; and West Florida Research and Education Center, University of Florida, Jay, Florida. Field derived colonies of H. zea will be established for this study in each location following Paula-Moraes et al. (2013). The colony derived from field populations will be kept in the laboratory following Rabelo et al. (2020). In each location, 2,000 H. zea pupae (approximately 1,000 of each sex) from the field derived colony and close to moth emergence will be placed in field corn field just prior to the VT stage. An albumin marker application zone will be delimited in the central rows of the plot with white flag tape and egg albumin solution will be sprayed using a backpack sprayer (Tavares et al., 2019). The trapping of males and females will be performed using Universal Black Light Traps. The traps will be deployed along four cardinal directions from a central point where marked insects will be released. The first trap will be placed 600 m from the albumin application zone, and the last one at 2,400 m in each direction. These trapping distances were based on currently recommendation of 800 m distance of structured refuge from the Bt corn crop (USEPA 2001), and results from Tavares et al. (2019). The H. zea moths captured in the light traps will be transported to the laboratories, placed into a clean 2 ml microtube and labeled, frozen at -20?C (with exception of moths collected in Jay, FL), and shipped to Dr. Paula-Moraes's laboratory at UF, Jay, FL for detection of the presence of egg albumin using ELISA. After that, the moths will be saved for posterior dissection. The detection limit of egg albumin by indirect ELISA assay will be based on the detection limited threshold validated by Tavares et al. (2019), including TBS solution and samples from non-sprayed moths, used as negative controls. The egg albumin quantification in each sample will follow assay of indirect ELISA from Hagler and Jones (2010), with three replications. A subsample of moths that tested positive for albumin will be dissected to assess the presence and number of spermatophores and reproductive maturity of female moths.Statistical Analysis. Differences in the number of marked moths captured over time and number of marked moths in each distance, and possible the female mating status and maturity will be tested using ANOVA. Equations that best describe the relationship between H. zea marked moth recaptures and distance dispersal will be estimated following Qureshi et al. (2005).?Objective 1b. Estimate the flight capacity of H. zea males and females in laboratory.The flight capacity (distance and time) of mated and unmated female and male of H. zea moths at three different ages will be tested using the tethering technique in flight mills in co-PI Reisig's laboratory at NCSU. For both sexes, the three age classes will be defined as follows: 1) young (1-2 days); 2) middle-aged (3-6 days); and 3) old (7-9 days). The sex of the pupae will be determined (Hardwick, 1965) and males and females will be kept in rearing containers with wet vermiculite on the bottom to avoid desiccation and shipped to NCSU entomology laboratory. To obtain mated females and males, around 20 insects of each sex will be placed in honeymoon cages (30 cm high x 20 cm diameter polymerized vinyl chloride tube) and supplied daily with honey solution. A subsample of females will be dissected to assess mating status of the insects to be used in the study. Males will be flown separately from females to avoid sex-pheromone confounding effects during the performance of the experiment. A set of 32 flight channels will be run in tandem based on Attisano et al. (2015), and the data will be analyzed using Python 3.4.x.Data analysis. ANOVA will be used to analyze the effect of sex, mating status, and moth age on the number of flights, total distance flown, and longest single flight (PROC GLIMMIX, SAS). Means will be separated using Tukey's HSD. Regression models will determine the association between moth mating status, moth age, and flight parameters (PROC REG, SAS).Objective 2) Estimate the random mating and reproductive potential of wild populations of H. zea in agricultural landscapes.Field populations of H. zea will be light trapped in three contrasting agricultural regions in the southeastern U.S. during two crop seasons. The light trapping will be performed in commercial fields in FL, NC, and SC, and the fields will be selected based on history of the PIs of successfully conducting research projects. Moths will be collected using light traps in at least three fields of Bt corn and Bt cotton in each state. In addition, trapping will occur in one peanut field in FL, and one soybean field in NC and SC. The proportion of land-use around each trap will be roughly estimated and recorded based on the findings of Graham et al. (1978) and Tavares et al. (2019) that indicated the majority of recovery of marked H. zea moths in agricultural fields occurs within approximately 1 km radii. The light trapping will be performed as previously described in Obj.1a. Trapping will occur during the flowering stage of the crops when high population of H. zea can be expected. The light trap collections will be evaluated in each location, and the H. zea specimens will be sexed and shipped to Dr. Paula-Moraes's laboratory at WFREC, Jay, FL, inside a cooler with ice. At least 10 moths per trapping-day will be randomly selected for dissection to evaluate female and male moth age, female mating status, female frequency of mating, and female reproductive maturity. Analysis of stable carbon signature of sub-samples of trapped moths from each location will be performed to discriminate C3 or C4 host plant origin of H. zea moths. The objective will be to generate data to estimate random mating (Objective 3) between moths from different crops in the agricultural landscape.Data analysis. ANOVA will be used to test for significant effects of crop on average number of moths, number of spermatophores, moth age, and number of fully developed eggs (chorionated eggs) in the ovarioles. In addition, the overall differences in the mean carbon isotope rations (δ13C±SD) of moths collected in different locations and crops will be classified as from C3 or C4 plants. Tukey's honestly significant difference (HSD) multiple comparison procedure will be used to compare all treatment means for treatment, within each crop and location (SAS PROC Glimmix, SAS, 2013).Objective 3) Create a refined model of the evolution of resistance for H. zea in complex landscapes.The data generated in this project will be used in the last two years of the execution of the project (2024 and 2025) to refine a deterministic, frequency-based model of the H. zea population genetics based on Pan et al. (2016). The model will explore how the movement parameters and reproductive potential will impact the rate of resistance evolution in H. zea. The model will be updated to include all new knowledge from the relevant literature published through 2022 and the empirical data generated by objectives 1 and 2. The model may be changed to use a different time-step, different representation of landscape (spatially-explicit), and different representation and/or densities of H. zea depending on the knowledge and the need.

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:The large-scale adoption of transgenic crops expressing insecticidal proteins from the bacteriaBacillus thuringiensis, commonly referred to as Bt crops, has been a landmark for the decrease of chemical insecticides in Bt corn and Bt cotton in the U.S.However, Helicoverpa zea is resistant to several Bt traits in corn andcotton, and Vip is the only effective Bt trait currently commercially planted. This pest is an economic pest in cotton, andsimilar Bt toxins are expressed in corn and cotton. Hence, there is a high risk of H. zea evolving resistance to Vip-expressingcrops on a broad scale in the continental U.S.The overall goal of this proposal is to provide knowledge of the short-distance dispersal capacity andreproductive potential of H. zea, using mark-recapture egg albumin marker, flight mills, and estimation of the sex, moth age,female mating status and maturity, and stable carbon isotope signature of wings and spermatophores of field populations ofH.zea. The data generated in this proposal will be used to refine a deterministic, frequency-based model of H. zea populationgenetics, exploring the risk assessment null hypothesis that movement and reproductive parameters (dispersal distance, matingstatus, female maturity, and moth age and origin) will not impact the rate of resistance evolution in H. zea. This proposal willsupport accurate IRM regulations concerning refuge for the deployment and sustainability of Bt crops. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results of the project have been also presented in one national meeting: 1. 2024 Entomological Society of America. November, 10-13, Phoenix, AR. What do you plan to do during the next reporting period to accomplish the goals?The analysis of data will continue to be performed. For the objective 3 we expect to request a non-cost extension for the project in order to create a refined model of the risk of resistance evolution of H.zea. The PI is in process to move from University of Florida to University of Nebraska - Lincoln and a subaward is in process to be created for the completion of the activities of the project.

Impacts
What was accomplished under these goals? The activities of the project have been conducted as following: Obj1a) data analysis has been completed with thewith the estimation of the shot-distanceaverage (+CI) capacity of females and males of H.zea,including the best distribution that best describes thedistance dispersal. In addition, the influence of the number of mature eggs and mating status (based on the number ofspermatophores) on the flight capacity under field conditions have been tested, and a significant effect of the number ofmature eggs on the flight capacity has been detected. Obj1b)The analyses of dispersal capacity and morphophysiological determinants on the flight performance of H. zea were analysed usingtethered flight mill experiments, under laboratory conditions. Obj2) Analysis to estimate the random mating and reproductive potential of wildpopulations of H. zea in agricultural landscapes was conducted. To test whether the total number of moths collected in each crop differed across states, a Pearson's Chi-squared test was conducted, followed by pairwise comparisons using chi-squared tests and p-value adjustment method of false discovery rate (aka Benjamini-Hochberg procedure). Moths trapped in peanut crops in Florida were not included, as only this state had traps in peanut fields. The potential infestation levels across different crops by comparing the number of mature eggs in females trapped in each crop within each state, except for North Carolina. To analyze this, we fit a GLM with the number of eggs as the response variable and crop as the predictor variable. A negative binomial distribution was used to account for overdispersion.The influence on the number of eggs and spermatophores in H. zea females by the host plant (C3 or C4) during the larval stage in each state, we analyzed the stable carbon isotope values (δ¹³C) in the wings of all female moths and in the spermatophores of mated females collected in the field.Generalized Linear Models (GLMs) in each state, with the number of eggs or spermatophores as the response variable and the host plant (C3 or C4) as the predictor variable. We used a negative binomial response to account for overdispersion. In addition, to carbon analysis to estimate the inter-crop movement and random mating, we are currently exploring the use of nitrogen isotope values (δ¹³C and δ¹5N) in the wings and spermatophores of mated females collected in the field, as a way to differenciate soybean, peanut, and cotton, all C3 plants.We first categorized samples based on δ¹³C values to distinguish between C4 and C3 plants, with the assumption that C4 signatures correspond to corn. For samples with a C3 signature, we performed hierarchical clustering of δ¹³C and δ¹5N values using Ward's method and Euclidean distance as the dissimilarity metric. The optimal number of clusters was identified using the within-cluster sum of squares (WSS) method. We then applied k-means clustering, using the number of clusters defined by the WSS analysis, to classify individuals into C3 host plant groups. We are performing clusteringanalysis using the packages dendextend and considering sourcesof nitrogen fertilization to define cotton, peanut, and soybean nitrogen isotopic signatures.These results will be incorporated in the work to be performed in Objective 3), which will create a refined model of the evolution of resistance forH. zea.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Paula-Moraes, S.V. 2024. Methods and studies of life history traits of lepidopteran pests to support IPM/IRM recommendations. 2024 Entomological Society of America, Annual Meeting, November 10-13, Phoenix, Arizona. (invited).

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:EPA and USDA Industry Cotton and corn commodity boards Academic community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Moth dissection and access of reproductive status and age of moths. How have the results been disseminated to communities of interest?The results of the project have been also presented in two international meetings and one national meeting, as followed: 1.2024 Entomological Society of America, Pacific Branch Meeting. April, 14-17, Waikoloa Beach, HI. 2.XX International Plant Protection Congress (IPPC). July 1 to 5, Athens, Greece. 3.XXVII International Congress of Entomology (ICE 2024). August 25 to 30, Kyoto, Japan. Presentation will be on 08/29/2024 What do you plan to do during the next reporting period to accomplish the goals?The analysis of data will continue to be performed. For the objective 3 we expect to request a non-cost extension for the project in order to be able to have all the information to Obj1 and 2, and possible to add imaging informaiton of the complex landascapes of the southeastern U.S. andcreate a refined model of the risk of resistance evolution ofH.zea.

Impacts
What was accomplished under these goals? The planned research activities have been executed, resulting in accomplishments for the project, including the following: Obj1a) Estimate the typical dispersal range of male and female H. zea emerging from corn. Analysis of the combined data from the 2022 and 2023 crop seasons is in progress. For this objective, the use of the protein immunomarking technique was feasible, and overall, the number of recaptures of marked insects ranges around 6%, which is expected in this type of study. The results have allowed us to estimate the average (+CI) of short-distance dispersion capacity of females and males of H. zea. The distribution that best describes the relationship between H. zea marked moth recaptures and distance dispersal has also been estimated. In addition, the influence of the number of mature eggs and mating status (based on the number of spermatophores) on the flight capacity under field conditions have been tested, and a significant effect of the number of mature eggs on the flight capacity has been detected. Obj1b) Estimate the flight capacity of H. zea males and females in laboratory. Insects from a field-derived colony of H. zea established in the Entomology Laboratory at WFREC/UF were submitted to a flight mill study at the Department of Entomology and Plant Pathology, North Carolina State University. The dispersal capacity and morphophysiological determinants on the flight performance of H. zea were examined using tethered flight mill experiments. The impact of mating status, age (young (2-day old), middle-aged (3-6 day old), or old (7-9 day old)), and sex on the flight capacity of moths under controlled conditions were tested. The results indicated that the flight capacity was not influenced by mating status or the number of mature eggs. Males flew faster than females, and middle-aged moths' flight had higher performance (speed, time, and distance) than young and old moths. Body size and wing-to-thorax ratio negatively influenced the moth flight time. Male's body size was larger than females, but they had a smaller forewing area and smaller wing-to-thorax ratio. Middle-aged moths exhibited the highest likelihood of engaging in migratory movements (>10km). In addition, oogenesis-flight syndrome was not detected, confirming the hypothesis of facultative migration in this species. The results of this objective have been submitted for publication and are currently in press to be published in the Annals of the Entomological Society of America. Obj2) Estimate the random mating and reproductive potential of wild populations of H. zea in agricultural landscapes. Commercial fields were light-trapped for the collection of populations of H. zea in three contrasting agricultural regions in the southeastern U.S. during the 2022 and 2023 crop seasons in the three states of the study (FL, NC, and SC) in corn, cotton, soybean, and also peanut in FL. The analysis is in progress to explore the effect of the crop on the average number of moths, number of spermatophores, moth age, and number of fully developed eggs (chlorinated eggs) in the ovarioles. In addition, the overall differences in the mean carbon isotope rations (δ13C±SD) of wing and spermatophore in moths collected in different locations and crops have been used to classify moth origin from C3 or C4 plants. The effect of the complex landscapes of the southern U.S. have been explored based on these carbon signatures of wing and corresponding spermatophores of females trapped under field conditions. The continuing analysis of the data will indicate if additional data to be collected in the crop season 2025 will support strong conclusions about the impact of this species' females' reproductive status on flight dispersion capacity. Objective 3) Create a refined model of the evolution of resistance for H. zea in complex landscapes. This activity, planned to start in 2024, has been initiated, including a literature review of updated information on this topic. However, the team are currently working on the analysis of the results of the other objectives in order to provide data to be included in the modeling.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Paula-Moraes, S.V. 2024. Ecology of Helicoverpa zea in Bt and non-Bt crops and implications for insecticide resistance management recommendations. In: Merging IPM and IRM Concepts for Sustaining Effectiveness of Insecticidal Transgenic Crops. 2024 Entomological Society of America, Pacific Branch Meeting. April, 14-17, Waikoloa Beach, HI.
• Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Calixto, E. S., Paula-Moraes, S.V., Reisig, D.D., Santos, A.A., Reay-Jones, F.P.F. 2024. Beyond wingbeats: dispersal capacity and morphophysiological determinants in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae). Annals of the Entomological Society of America.
• Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: Paula-Moraes, S.V., Calixto, E.S., Santos, A., Reisig, D., Reay-Jones, F.P.F. 2024. Dispersal and reproductive potential of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Bt and non-Bt crops and implications for insecticide resistance management. XXVII International Congress of Entomology (ICE 2024). August 25 to 30, Kyoto, Japan.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Paula-Moraes, S.V. 2024. Beyond pest control: topics for the improvement of IPM and IRM of lepidopteran pests associated with row crops. XX International Plant Protection Congress (IPPC). July 1 to 5, Athens, Greece. (speaker and event organizer).

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:EPA and USDA - Industry - Cotton and corn commodity boards Changes/Problems:The recovery of marked insects was low in the three locations under study. The rate of marked insects is common in studies using the mark-recapture technique andonce the data analysis is complete, decisions will be made about the need to have one additional year of data collection in objective 1a. What opportunities for training and professional development has the project provided?The field work activities of light trapping, albumin marking technique, correct solution preparations, sample storage, and shipping were discussed with members of the project to guarantee success in the execution of the project activities. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Once the analysis of two-year data is completed, evaluate the need of a three-year data collection. Elaboration of manuscript or possible two manuscripts related with flight mill data and field dispersion ofHelicoverpa zea. Performance of activities in objective 3.will be performed.

Impacts
What was accomplished under these goals? The planned research activities have been executed resulting in accomplishments for the project, including: Obj1a) Estimate the typical dispersal range of male and female H. zea emerging from corn. The field study to estimate the dispersal capacity of H, zea was performed in the field for the second crop season in 2023. In Florida, a field-delivered colony was used as the source of insects for the mark and recapture study. The data collection for this objective was collected by collaborators from NCSU and Clemson University. The light trapping was performed during the reproductive stage of the corn and the samples of H. zea were shipped to Entomology Laboratory at West Florida Research Education Center/the University of Florida, In Jay, FL. The samples were submitted to enzyme-linked immunosorbent assay (ELISA) for anti-egg albumin and the analysis has been completed in September2023. In addition, the work of moth dissection is under execution. Overall, the second year of data collected will support the analysis to estimate dispersal capacity and life history traits of H. zea under field conditions. Obj1b) Estimate the flight capacity of H. zea males and females in laboratory. Insects from a field-derived colony of H. zea established in the Entomology Laboratory at WFREC/UF were shipped to the Department of Entomology and Plant Pathology, North Caroline State University, where flight mill experiments were performed in 2022.Objective 2) Estimate the random mating and reproductive potential of wild populations of H. zea in agricultural landscapes. Commercial fields were light trapped for the collection of populations of H. zea in three contrasting agricultural regions in the southeastern U.S. during the 2023 crop season. In Florida, corn, cotton, peanut, and also soybean were trapped during the reproductive stage of the crop. In North Carolina and South Carolina, the crops trapped were corn, cotton, and soybean. The insects were shipped to WFREC/UF and the dissections of moths collected in Florida and North Carolina have been completed. The samples have been submitted to carbon isotopic analysis, and we are in process of analyzed the tow-year data. Objective 3) Create a refined model of the evolution of resistance for H. zea in complex landscapes. This activity is planned to start in 2024, however, meeting by zoom has been conducted to start the discussion and inclusion of the data generated in objectives 1 and 2 in the modeling. In addition, the team of project has met monthly to discuss questions regarding methods in the execution of activities, and progress in each state. A meeting was held with the project members during the 2023Annual Entomological Society Meeting, November 4-9, in National Harbor, Washigton, DC. The partial results have been shared and discussed between members of the project.

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The PI presented the hypothesis and methods of the project to graduate students from the University of Florida. Changes/Problems:BioQuip, the supplier company of light traps for more than 70 years in the market has closed its doors. The PI and Co-PIs were able to buy the light traps, but additional supplies to perform the activities in obj 1 and 2 demanded adjustments in the electrical components of the supplies. This included acquisition of car batteries and battery chargers was necessary due to the risk of power lostduring the field trapping. What opportunities for training and professional development has the project provided?The field work activities of light trapping, albumin marking technique, correct solutionpreparations,sample storage, and shipping were discussed with members of the project to guarantee success in the execution of the project activities. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The research activities planned for Obj 1 and 2 will continue to accomplish the research goals.

Impacts
What was accomplished under these goals? The planned research activities have been executed resulting in accomplishments for the project, including: Obj1a)Estimate the typical dispersal range of male and female H. zea emerging from corn. The field study to estimate the dispersal capacity ofH, zeawas performed in the field using the methodology from Tavares et al. (2019). In Florida, a field-delivered colony was used as the source of insects for the mark and recapture study. Collaborators from North Carolina and South Carolina needed to use laboratory colonies due to the challenges of establishing field-derived colonies during the cold Spring. The light trapping was performed during the reproductive stage of the corn and the samples ofH. zea were shipped to Entomology Laboratory at West Florida Research Education Center/the University of Florida, In Jay, FL.The samples were submitted toenzyme-linked immunosorbent assay (ELISA) for anti-egg albumin and the analysis has been completed in December 2022. Currently, the work of moth dissection is under execution. Overall, the data collected will support the analysis to estimate dispersal capacity and life history traits ofH. zea under field conditions. Obj1b)Estimate the flight capacity of H. zeamales and females in laboratory. Insects from a field-derived colony ofH. zeaestablished in the Entomology Laboratory at WFREC/UF were shipped to theDepartment of Entomology and Plant Pathology, North Caroline State University, where flight mill experiments were performed. The insects were shipped in the pupalstage to decrease stress and damage. The flight mill experiment was performed using 32 flight channels, in a total of 97 insects,and the data is under analysis using Python 3.4x. The insects used in the flight mill study were later shipped back to WFREC/UF for dissection and morphological analysis. Overall, the data collected in laboratory will be important to support the estimation of the short distance of male and female ofH. zeaand is expected to be contrastedwith field data from 2022 and 2023. Objective 2) Estimate the random mating and reproductive potential of wild populations of H. zea in agricultural landscapes. Commercial fields were light trapped for the collection of populations of H. zeain three contrasting agricultural regions in the southeastern U.S. during the 2022crop season. In Florida, corn, cotton, peanut, and also soybean were trapped during the reproductive stage of the crop. In North Carolina and South Carolina, the crops trapped were corn, cotton, and soybean. The insects were shipped to WFREC/UF and the dissections of moths collected in Florida and North Carolina have been completed. Currently, the samples fromSouth Carolina are under study. We expect that during Spring 2023 the samples for will be submitted for carbon isotopic analysis. Objective 3) Create a refined model of the evolution of resistance for H. zea in complex landscapes. This activity is planned to start in 2024. In addition, the team of project has met monthly to discussquestions regarding methods in the execution of activities, and progress in each state. A meeting was held with the project members duringthe 2022 Annual Entomological Society Meeting, November 13-17, in Vancouver, Canada. The partial results have been shared and discussed.

Publications