Research Project: Improved Biologically-Based Methods for Management of Native and Invasive Crop Insect Pests

Location: Insect Behavior and Biocontrol Research

2025 Annual Report

Objectives
Objective 1: Develop genetically modified (GM) strains by transposon and CRISPR/Cas-mediated transgenesis for improved SIT in fruit fly pests, and new DNA delivery systems to apply this technology to a wider range of insect, including emerging pest species (e.g., caribfly, mexfly, medfly, spotted-winged drosophila). Objective 2: Develop strains of moths transinfected with Wolbachia that produce males with strong cytoplasmic incompatibility for use in the Incompatible Insect Technique (IIT) to reduce pest populations of fall armyworm and corn earworm. Objective 3: Develop CRISPR gene editing in pest moths (e.g., Indian meal moth, fall armyworm, gypsy moth) that target genes critical for acquired biopesticide resistance using both whole insects and cultured insect cells. Objective 4: Develop improved surveillance and detection methods for hidden and invasive pests (e.g., red palm and citrus root weevil, Asian long-horned beetle, Asian citrus psyllid, and stored product insect pests) that incorporate automated collection, processing, and analysis of insect acoustic signals and behavioral activity. Objective 5: Develop improved surveillance of invasive and outbreak insect pests (e.g., corn silk flies and kudzu bug) using visual-cue traps, and improve strategies for detection and prediction of such dispersing pests by understanding the role of visual and other stimuli affecting specific behaviors. Objective 6: Improve area-wide landscape management tactics by developing conservation biological control strategies to mitigate pest populations and attract or support natural enemies (e.g., against fall armyworm). Objective 7: Combine genetic methods with air-transport and climate modeling to describe and predict the distribution and behavior of agricultural pests to facilitate the mitigation of migratory source populations and to identify locations at high risk for infestations by invasive species such as fall armyworm, corn silk fly, soybean looper, Old World bollworm, and corn earworm.

Approach
Research conducted by the Behavior and Biocontrol Research Unit at the Center for Medical, Agricultural and Veterinary Entomology has historically been focused on the development of novel technologies that improve the cost-efficiency of traditional pest control strategies as well as provide environmentally benign alternatives to the use of chemical pesticides. The goals are to improve crop productivity while reducing the environmental impact and costs of pest management. The proposed research integrates different levels of biology that range from the genetic modification of pest insects to generate novel and improved variations of Sterile Insect Technique (SIT) strategies, the manipulation of pest endosymbionts to develop Insect Incompatibility Technique (IIT) strategies, the optimization of acoustic, olfactory, and visual cues to improve pest surveillance and disrupt pest behavior, the application of climate and air transport models to project pest distribution and migration patterns, and the development of landscape strategies for sustainable mitigation of pest populations. This multidisciplinary structure encourages innovation and facilitates synergism between projects. Anticipated accomplishments will initially apply to the control of high priority invasive fruit flies, beetles, psyllids, moths, and corn silk flies through new biologically based methods for pest control, improved capability to monitor pests, and better projections of pest movements to more effectively target the time and place of treatments. The impact will be higher productivity at lower cost for domestic agriculture and new and improved tools to detect and control emerging native and invasive pests.

Progress Report
This is the final report for this project with Objectives under National Program 304, Component 3, Insects and Mites with activities focusing on; Problem 3A, early detection prediction and monitoring of beneficial and pest arthropods; Problem 3B, developing new or improved management tools and knowledge to control arthropod pests; Problem 3C, integrating management strategies to control arthropod pests; Component 4, Problem 4A, protection of postharvest commodities, quarantine and methyl bromide alternatives and Problem 4B, improving and developing postharvest and quarantine treatments. Significant progress was made with Objective 1 to develop strategies using genetic modification of pest insects to improve sterile insect techniques (SIT). For Subobjective 1.1, Drosophila suzukii transgenic strains were designed that confer male sterility or female lethality when raised in the absence of the antibiotic tetracycline. This “Tet-off” technique allows mass rearing of flies on media containing tetracycline with the consequent elimination of females and sterilization of males by removing tetracycline from the diet. This greatly facilitates rearing large numbers of sterile male flies required for effective SIT. In mass rearing bioassays, partial male sterility was achieved, providing proof of concept of the genetic modification strategy. Better results were obtained with the Tet-off female lethal strain with 1-2% survival in the absence of tetracycline. A strategy was developed by ARS researchers at Gainesville, Florida, to enhance lethality using new transgenic constructs that confer dominant temperature-sensitive (DTS7) lethality. This approach proved successful in Subobjective 1.2 where a Tet-off/DTS7 uni-sex dual-lethality strain created for D. suzukii resulted in total lethality under restrictive conditions for both systems, consistent with effective redundant lethality. For Subobjective 1.3, a biolistic DNA delivery system with potential to more efficiently produce genetically transformed flies than traditional injection methods was evaluated, however was less successful, in part due to low levels of embryonic survival after DNA-coated pellet bombardment. Additional achievements for Objective 1 include the identification and characterization of new target genes for inducing male sterility. These include the D. suzukii wampa dynein and Prosalpha6T proteasome subunit genes that were isolated and verified for adult male sperm-specific transcription by quantitative RT-PCR, with only wampa shown to be sperm-specific in A. suspensa (caribfly). CRISPR/Cas9 gene-editing was successfully initiated to create null mutations for wampa in D. suzukii, with the gene-edited lines currently being evaluated. Similar CRISPR/Cas9 gene-editing has been initiated for As-wampa in A. suspensa and Ds-Prosalpha6T in D. suzukii. Finally, whole genome sequence and assembly together with automated annotation was achieved for the South American fruit fly, Anastrepha fraterculus. These achievements enable discovery and manipulation of genes relevant to sexual differentiation and reproduction are useful to improve SIT for population control. No progress was made on Objectives 2 and 3 due to a scientist’s retirement. For Objective 4, progress was made in construction and acquisition of refined acoustic/vibration detection instrumentation subsequently evaluated in both laboratory and field sites. These include incremental but significant refinements in pest sound detection under conditions of background noise that enhanced the ability of warehouse managers to detect hidden infestations of stored product insects as well as camouflaged pests in orchard trees. Signal detection refinement facilitated assessments of biological control treatments (e.g., entomopathogenic fungi), most notably against the red palm weevil. Field tests and surveys demonstrated acoustic technology effectiveness for characterization of a variety of insect pests, including rice, pepper, and palm weevils, red flour beetles, Asian citrus psyllids, spotted lanternflies and mole crickets. These studies demonstrate that continued advances in sound detection and acoustic analysis have potential to provide efficient and cost-effective methods for the detection and improved hidden pest management. In Objective 5, multiple approaches were used to characterize the biology of the cornsilk fly species complex that is becoming an increasingly important economic pest that has historically been difficult to efficiently detect and control in the field. As part of an effort to improve detection and monitoring, laboratory studies revealed visual preferences by two cornsilk fly species to specific colors, shapes, visual complexity and background contrast. Attraction to various protein sources, related chemicals and commercial fruit fly lures were evaluated with selection of the most effective attractants. Through field testing, the most effective chemical and visual attractants were assessed and is being utilized to develop more effective surveillance tools. In studies to better identify conditions conducive to cornsilk fly outbreaks, soil type and moisture levels were shown to impact pupation behavior of cornsilk flies. As part of an effort to develop biological control techniques, five pupal parasitoid species screened against cornsilk fly pupae demonstrated significant mortality even at different soil depths. Importantly, a new parasitoid species was discovered and host range studies revealed that it was an effective larval parasitoid of cornsilk flies. These parasitoids have the potential to be important components of a biocontrol strategy against cornsilk flies. Finally, electrophysiological and behavioral studies revealed responses of kudzu bugs to wavelengths and colors of light-emitting diodes and serve as the basis for effective trapping strategies. Significant progress was made for Objective 6 in providing improved tools for management of fall armyworm populations. Laboratory studies supported use of the egg parasitoid Telenomus remus against fall armyworm with potential application both the United States and Africa. Nutritional studies in the laboratory and field were performed that identified diet and host plants that optimized longevity, population levels, and parasitism frequency. This type of information is essential for the development of landscape strategies that can support natural enemies to pests and thereby reduce pest populations and the need for pesticides. Field trials performed in the U.S. and Africa quantified the specificity and attractiveness of commercially available traps and pheromone lures. With considerable variability in the trapping of nontarget species, species identification of trap captures remains essential for accurate monitoring of fall armyworm populations. Based on parallel trapping surveys with traps and lures specific for the looper species complex, trapping was significantly more efficient using a pheromone lure in conjunction with a floral lure containing phenylacetaldehyde plus beta-myrcene. Finally, intercropping of multiple plant species tested in Togo, Africa, demonstrated significant reduction of fall armyworm infestations compared to crop monocultures, providing a potentially simple mitigation strategy to augment conventional pesticide use. These studies indicate that a combination of enhanced parasitoid populations, increased efficacy of trap lures, and habitat diversification can be effective components for pest management, particularly in circumstances where conventional chemical pesticide use is not affordable or desired. Significant accomplishments were made in Objective 7, where genetic methods are being developed to describe and predict the population movements of genetically different populations of fall armyworm, a global pest of corn, that can serve as a model for understanding biological differences among strains of pest species that pose an invasive threat to U.S. agriculture. New molecular markers were developed and extensive sampling of populations from both Eastern and Western Hemispheres allowed significant improvements in the genetic technology and resources available for the characterization of this important agricultural pest. This led to a more precise definition of the fall armyworm strains that account for much of its broad host range, a new method for quantifying the degree of cross-hybridization of subpopulations in the field, and new markers for characterizing migratory pathways. These were used to develop more accurate description of fall armyworm migration in North America including meteorological models that demonstrated that seasonal wind patterns and corn host availability were the primary factors directing migratory movements. Application of the improved methodology on a hemispheric scale showed that migration across the Caribbean Island chain and Central American land bridge is limited in scope, indicating that the fall armyworm populations in the two American continents are more isolated than previously thought. The genetic technology applied globally demonstrated that the fall armyworm that recently invaded Africa and subsequently spread to Asia most likely originated from Florida-Caribbean populations and while primarily of the strain that targets corn also includes a population with the genetic signature found in fall armyworm infesting rice in the Western Hemisphere. These observations have important consequences to risk assessments of African and Asian agriculture to fall armyworm infestations. Additional findings include the characterization of differences in mating behavior that could provide a rapid method of distinguishing the strains in North America. The genetic technology used to evaluate fall armyworm was modified and adapted for use with soybean looper, with the initial results providing strong evidence for the existence of subgroups analogous to the fall armyworm host strains in that species.

Accomplishments
1. Candidate genes for CRISPR-mediated male sterility to improve sterile insect technique. The spermatocyte-specific wampa and Prosalpha6T genes in the spotted-wing drosophilid, D. suzukii, and wampa in the Caribbean fruit fly, Anastrepha suspensa, are being developed by ARS researchers at Gainesville, Florida, as genomic target sequences for CRISPR-mediated mutations to produce male sterility in these fruit fly pest species. For highly efficient sterile insect technique (SIT) programs the male sterility component was preliminarily combined in the same strain with a CRISPR-mediated female-lethality component for sexing, resulting in sterile males-only progeny. This CRISPR-based system, known as precision-guided SIT (pgSIT), can greatly improve the cost effectiveness (by eliminating female rearing) and efficacy (by eliminating debilitating radiation-based male sterility) of SIT. Identification of new spermatocyte-specific genes by ARS researchers at Gainesville, Florida, will also allow use of its gene expression promoter for sperm-specific fluorescent protein marking (to identify females mated to field released males) and to drive conditional lethality systems in sperm for male sterility. These approaches enhance the capabilities for use of sterile insect technique as a safe and effective control for insect pests.

2. Detection of hidden pests in peppers. Increasing demand has resulted in northward expansion of jalapeño fruit grown in Florida and other southern states. Pepper weevils, which can devastate bud and fruit production, have migrated with them and control is increasingly hampered by growing resistance to commonly used insecticides. Determination of control efficacy is difficult as insects are hidden in the fruit. ARS researchers at Gainesville, Florida, demonstrated that vibration detection technology is effective for detection of pepper weevil larvae hidden inside young peppers. With grower input, an early-warning detection system is being developed to facilitate insecticide application and effectiveness.

3. African fall armyworm population is a potential threat to rice agriculture. Fall armyworm is historically a pest of the Americas but was found in Africa in 2016, with infestations subsequently detected throughout much of the Eastern Hemisphere. The global spread of this pest species that has a broad host range (economic impacts in corn, sorghum, millet, alfalfa, forage grasses, among others) and can exhibit long-distance migratory behavior presents a critical economic threat with potentially significant impacts on trade policies and the free movement of agricultural products. ARS researchers at Gainesville, Florida, found genetic evidence that the populations associated with rice infestations in the Western Hemisphere is present in Africa and have been actively involved in European and Asian risk assessments coordinated by international organizations. The methods developed by the ARS researchers have become globally adopted by fall armyworm researchers to be the primary means of identifying this rice-threatening population.

4. Redundant conditional dual-lethality systems are superior to individual lethality systems. Genetically-modified (GM) insect strains for lethality and sterility are all subject to spontaneous mutations or inherent genetic variations that can break down or suppress the GM systems. Such failures can result in GM insects persisting in the environment, further complicating pest control and providing a basis for resistance to acceptance of this control approach. ARS researchers at Gainesville, Florida, previously determined that a tetracycline-suppressible (Tet-off) conditional lethal system in Drosophila could break down due to spontaneous mutations and suppressors at a rate of 15 per million flies. Since current non-GM SIT programs release 100 million or more sterile males per week, this level of breakdown represents a significant problem. To begin to assess the ability of redundant dual-lethality systems to further suppress survivorship, the Tet-off system was combined with a temperature-sensitive lethality system in the medfly, Ceratitis capitata, and survivorship of the dual-lethality strain was tested under restrictive conditions for both systems. Preliminary results were successful, with no survivorship observed. This dual-lethality GM system can be an effective and safe approach for use of GM for long-term population control of insect pest species.

5. Intercropping demonstrated to effectively control fall armyworm in corn. Over 350 species of plants have been documented to host the fall armyworm including numerous agricultural crops such as corn, sorghum, millet, alfalfa, forage grasses, and rice. This pest was exclusively in the Western Hemisphere before 2016, but now it is found globally. ARS researchers at Gainesville, Florida, with international collaborators demonstrated that infestations were significantly reduced when corn was grown with other crops such as tomato, sweet pepper, cowpea, and soybean, compared to a monoculture of corn alone. These crops have a low risk of infestation by fall armyworm. Intercropping corn plants with nonhost plants such as camel grass and lemon basil, also had lower feeding damage than corn grown alone. The strategy of intercropping is being developed as an integrated pest management program for U.S. farmers with small acreages, organic farming, or international farmers dealing with this new pest.

6. Assessment of fall armyworm movements between the American continents across the Central American land bridge. Fall armyworm is a major agricultural pest in both North and South America and there is concern that pesticide resistance developed in one location could rapidly spread across the hemisphere because of fall armyworm's long distance flight capability. This is a potential problem for United States agriculture as pesticide use is widespread in South America where adherence to protocols that reduce the selection for resistance may be less consistently enforced. ARS researchers at Gainesville, Florida, demonstrated in surveys over multiple years that mass movements of fall armyworm across the Caribbean Island chain or through the Central America-Mexico land bridge does not occur on a regular basis. These findings indicate that human-assisted migration (e.g., agricultural commerce and “hitchhiking” on commercial transport) are likely to be the primary mechanism for the entry of pesticide resistance traits into U.S. pest populations.

7. New parasitoid wasp for biocontrol of corn silk flies. Corn silk flies are a major corn pest with growers having few alternatives to repeated chemical pesticides for control. With increased pesticide resistance protection of fresh market sweet corn. ARS researchers at Gainesville, Florida, reported on a new North American record of a South American parasitoid, Euxestaphaga argentiensis in the U.S. and demonstrated that the corn silk fly species present in Florida were susceptible to this parasite. A biologically-based technology using this parasitoid is under development to augment conventional corn silk fly management.

8. Whole genome analysis demonstrates genetically differentiated fall armyworm populations that complicate control efforts. Fall armyworm is a pest of multiple crops in the U.S., in large part because of two strains that differ in host plant usage. ARS researchers at Gainesville, Florida, are characterizing differences in the timing of mating behavior between the fall armyworm population infesting corn compared to that damaging pasture and forage grasses. This behavioral difference contributes to our understanding of how pest complexes develop and is being utilized to develop more cost-effective technologies for identifying the strains using existing automated trap technology. The development of this methodology should greatly improve early warning of infestations, risk assessments, and allow for more targeted and cost-effective control strategies.

9. Time of trap capture helps identify fall armyworm host strain. Fall armyworm is a pest of multiple crops in the U.S. and has two host strains that attack different crops, are affected by pesticides in different ways and require different management strategies. The two strains are morphologically indistinguishable and require molecular differentiation. ARS researchers at Gainesville, Florida, used sex pheromone lures in traps to collect moths early and late in the evening and molecular methods to identify the host strains. The two strains were active during different times of the night, with the population that feeds on corn active earlier in the evening than the population that feeds on pasture grasses and rice. This technique of identifying fall armyworm host strain based solely on time of capture in pheromone traps is easier and less expensive than using molecular methods and can help U.S. farmers identify populations in their areas that might be resistant to certain pesticides.

10. Pheromone and floral attractant lures improve fall armyworm surveillance. Fall armyworm is a pest species of many agriculture crops that migrate from southern Texas and southern Florida (overwintering zones) each season, eventually making it as far north as Canada. Growers in the U.S. use pheromone traps to monitor fall armyworm infestations in corn and other high value crops. In nature, female moths release a complex chemical blend to attract male moths for mating. These chemicals are commercially incorporated into lures that are placed in traps. ARS researchers at Gainesville, Florida, found that at low population densities, augmentation pheromone trap lures with floral chemicals improved trap efficiency for moth collection. This is important for growers in areas north of the overwintering zones (most of the U.S.) to detect the first moths in their area so that they can monitor their fields for damage and implement control methods to restrict damage. This technological improvement was used to demonstrate that the overwintering zone for fall armyworm in Florida extended farther north than previously believed.

Review Publications
Miller, A.C., Tessnow, A.E., Meagher Jr, R.L., Nagoshi, R.N., Gulligan, T.M., Sword, G.A. 2024. Assessing fall armyworm (Spodoptera frugiperda)allochronic behavior as a predictor of local strain composition in across United States populations. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2024.1380624.
Tessnow, A.E., Nagoshi, R.N., Meagher Jr, R.L., Gulligan, T.M., Sadd, B., Carriere, Y., Miller, A.C., Richers, K., Porter, P., Fleischer, S.J., Palumbo, J., Rodrigues, J.V., Sword, G.A. 2025. Genomic patterns of strain-specific genetic structure, linkage, and selection across fall armyworm populations. BMC Genomics. 26:116. https://doi.org/10.1186/s12864-025-11214-8.
Nagoshi, R.N., Malo, E.A., Cruz-Esteban, S., Rosas-Garcia, N.M., Herrera-Mayorga, V., Meagher Jr, R.L. 2025. Assessing the potential for fall armyworm exchanges between the two American continents across the Mexico-Central America land bridge. PLOS ONE. 20(3).Article Number e0308501. https://doi.org/10.1371/journal.pone.0308501.
Gaffke, A.M., Miller, N.W., Sharma, A., Allan, S.A. 2024. Attraction of sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), and two generalist predators to green leaf volatile compounds. Insects. 15(10). Article 750. https://doi.org/10.3390/insects15100750.
Paris, T., Johnston, N., Stryzewski, I., Greisheimer, J., Reimer, B., Malfa, K., Allan, S.A., Martini, X. 2024. Tomato yellow leaf curl virus manipulates Bemisia tabaci, MEAM1 both directly and indirectly through changes in visual and volatile cues. PeerJ. 12. Article e17665. https://doi.org/10.7717/peerj.17665.
Awad, J., Brar, G., Cadwalader, E., Dillard, D., Esposito, L., Evans, E., Grozinger, C., Fisher, K.E., Kawahara, A.Y., Krupke, C.H., Lucky, A., Mankin, R.W., Moreau, C.S., Owens, A.C., Sandall, E., Seltmann, K.C., Ware, J., Winton, R. 2025. Defining the decline: The insect decline task force biodiversity glossary. Journal of Insect Science. 25(3):4. https://doi.org/10.1093/jisesa/ieaf048.
Koffi, D., Agboka, K., Adjevi, M., Tounou, K., Meagher Jr, R.L. 2025. Integrating plant volatile scents in sustainable IPM approaches for fall armyworm control. Journal of Applied Entomology. https://doi.org/10.1111/jen.13458.
Meagher Jr, R.L., Tessnow, A.E., Sword, G.A. 2024. Past, present, and future applications of molecular tools in landscape scale IPM: Fall armyworm as a case study. In: Brewer, M.J., Hein, G.L., editors. Arthropod Management and Landscape Considerations in Large-scale Agroecosystems.Boston, MA: CAB International.p.263-281.
Perez-Galvez, F.R., Handler, A.M., Hahn, D.A., Bredlau, J.P., Teets, N.M. 2025. Abiotic environments can modify the penetrance of a transgene-based lethality system for genetic biocontrol of insect populations. Proceedings of the Royal Society of London B. https://doi.org/10.1098/rspb.2025.0307.
Zhang, Q., Zheng, W., Zhurong, D., Li, X., Qiao, J., Li, Z., Liu, P., Handler, A.M., Lemaitre, B., Zhang, H. 2025. Gut commensal bacteria-derived methionine is required for host reproduction by modulating RNA m6a methylation of the insulin receptor. Cell Reports. https://doi.org/10.1016/j.celrep.2025.115911.