Research Project: Biology and Control of Invasive Ants

Location: Biological Control of Pests Research

2022 Annual Report

Objectives
Objective 1: Discover new bioactive compounds and approaches to improve control of fire ants and other invasive ants. Sub-objective 1A: Discover naturally occurring and environmentally benign synthetic compounds as toxins for invasive ant control. Sub-objective 1B: Discover naturally occurring and synthetic compounds as behavior-modification agents for invasive ant control. Objective 2: Develop new management strategies using genetic-based technologies for fire ant and invasive ant control. Sub-objective 2A. Predict gene function and utilize existing genetic resources to test and develop invasive ant-specific assays, leading to control methods and products. Sub-objective 2B. Develop gene disruption assays and approaches for mitigating the impact of invasive ants. Sub-objective 2C. Identify and develop novel microbiome assays, and approaches for mitigating the impact of invasive ants. Objective 3: Develop new and improved biorational pesticide delivery systems to control fire and other invasive ants.

Approach
Effective and environmentally benign ant toxins will be searched from various sources, including plants and other ants. In addition to ant toxins, we will search for behavior-modifying compounds that affect ant foraging and feeding using conventional bioassay-guided approaches and reverse chemical ecology approaches. These compounds can be very useful in improving ant developing control products. In the effort to develop gene disruption methods and materials, database comparisons will be conducted to identify target genes. Functional genomic techniques are essentially undeveloped in ants. We will begin by studying the genetics of key physiological processes within the colony. Because gene disruption experimentation is not standardized in ants, we will seek a visible phenotype, preferably non-lethal and visible in larvae, to provide an experimental positive control. We will initially focus on genetic disruption strategies which can disable the key physiological process of larval fitness and development. We will develop and utilize new molecular tools to validate, quantify, and develop genetic compounds and preparations that interfere with colony survival and resource exploitation. Additionally, preliminary studies identified unique viruses present in our regional populations of red imported fire ants. These discoveries need to be leveraged into ant-specific pathogens. The field of ant genomics and microbiome research has blossomed over the past 10 years. Individualized gene function studies, focusing on social form, chemosensory systems, neuropeptides, and oogenesis, have begun to shed light on the complex relationships between genes and phenotypes and behaviors. RNA interference studies have been performed on both fire ants and tawny crazy ants. A novel family of viruses was characterized. These investigations will lead innovation into new and improved control methods to mitigate invasive and destructive ants. Active ingredients or existing biorational pesticides will be used in developing new or improving existing biorational insecticide delivery systems. We will continue our effort in searching for adjuvants and synergists for improving the efficacy of mound treatment for fire ant control and spray treatment for tawny crazy ants. We will develop new water-resistant ant bait carriers using easily available local materials. Bait matrix will be developed and optimized for tawny crazy ants for both granular bait and liquid bait stations. Bait acceptance will be improved by using attractants and feeding stimulants. Bait selectivity will be enhanced by using selective repellants that attract targeted ants but repel non-targeted ants. We will continue our research on identifying effective synergists and surfactants for the final formulations.

Progress Report
Continued working on two oxo fatty acids that were identified from male fire ants. An U.S. Provisional Patent Application entitled, “COMPOSITIONS AND METHODS FOR ATTRACTING FIRE ANTS” (Serial No. 63/231, 816) was filed on 08/11/2021. As requested by the ARS Patent Committee, more experiments were conducted in this year, including various concentrations of oxo fatty acids, analogs, and comparisons to known fire ant attractants. A regular non-provisional patent application, entitled "COMPOSITIONS AND METHODS FOR ATTRACTING FIRE ANTS", was filed. A macrolide was tentatively identified from fire ants. A multi-step synthetic sequence was utilized to successfully synthesize this macrolide and its isomer. Behavioral bioassays on fire ants are ongoing. Essential oils from Matricaria recutica, Santalum album, Cybopogon citratus, Matricaria chamomilla, and Dipterocarpus turbinatus were tested for their repellency against the imported fire ants. The results showed that they were more potent repellants than the positive control DEET in the repellency assay, indicating that they are good source for discovery of potentially new chemotypes of repellent compounds for fire ant control. a-Bisabolol, a major compound from the Matricaria chamomilla oil, demonstrated remarkable repellency, and can be served as a lead for further structural modification. Structural modification of thymol. Thymol, the main active ingredient in the oil extracted from Thymus vulgarisis, is an active ingredient in pesticide products registered for use as animal repellents. It was tested in our repellency assay against fire ants for the first time and showed strong activity comparable to DEET. We then synthesized nine derivatives and tested them at the concentrations ranging from 15.6 to 125 ppm. One compound showed significantly higher repellency than DEET, which warrants for further study under field conditions. An ARS researcher was awarded a Genome Scaffolding project prize from Dovetail Genomics. In collaboration with Mississippi State University, tawny crazy ants were sequenced, entered into National Center for Biotechnology Information (NCBI) as a chromosome-level genome. Data announcement manuscript has been initiated. New laboratory colonies of Solenopsis invicta, Monomorium pharaonis, Tapinoma sessile are in National Biological Control Laboratory (NBCL). A collection of insect virus sequences that have not been catalogued in GenBank was identified and the >1600 sequences have been translated, organized, and used to query ant transcriptomes. These analyses identified 2 viruses present in both fire ants and pharaoh ants. These ants were entered as third-party accessions in GenBank and will be described for publication. Arboreal and subterranean ant traps: An arboreal ant trap design was modified to allow a person to install traps while remaining at ground level by use of an arborist throw line launcher. Trapping ran from September 2021 through April 2022 (20 trips to field sites in Jackson County, Mississippi) in areas with heavy infestation of tawny crazy ants (TCA) and a similar habitat without TCA. Data is still being compiled and analyzed. One takeaway is that TCA were found in the trees throughout the winter, albeit at reduced numbers, even after just 1-2 days of sub-freezing temperatures. A subterranean ant trap design was field tested and found to be unusable in the sandy soils of our field site. The design was successfully modified, and three traps have been through a pilot trial from March to June of this year, providing catch rate data at 4 depths. TCA were collected at all depths, to at least 46 cm. PCR/Restriction Digest Assay distinguishing native vs. non-native fire ant species: An additional 135+ DNA samples of native and non-native fire ants were screened to verify our assay is applicable across different populations and species. Faunal Lists: This is a long-term study of the ants of Georgia. This project is coming to fruition, and we have started a manuscript, which we hope to have published in 2023. This study and the resulting paper will add new records to the state of Georgia and contribute to our overall knowledge of ant distributions and abundance. A long-term project of the ants of Tennessee has resulted in numerous new state records being added and a better understanding of the distribution of species within the state and the southeastern USA in general. Future collections over the next two years are scheduled to target regions of the state where we have a dearth of records. New Species Descriptions: Based on collections made during a study of trap nests for ant colonies in Alabama, a new species of Myrmecina was discovered. Both workers and a queen were found in a trap nest. A manuscript is being developed, which includes a description of the worker and queen castes, keys to the other two North American species, and detailed photos of each species. The genus Forelius has undergone numerous taxonomic changes over the years. The current catalog (Antcat.org) only includes one southeastern species, F. pruinosus. A new study added two new species of Forelius, including one species restricted to deep sand dune habitats and another species that is abundant and widespread throughout much of the Southeast. This project required DNA work in addition to morphological measurements and studies. Anticipated completion of this study is 2024–2025. This study is important because the yet to be described, but abundant species, Forelius sp. A. has been observed to nest in the same open habitats as the invasive fire ants in the Southeast and appears to be able to have defensive chemicals that repel fire ants. Factors Influencing Invasive and Native Ant Distributions: The first season of field work investigating how ant communities change across an environmental gradient on the Cumberland Plateau in Alabama and Tennessee was completed and the second year of sampling has begun. As of June 2022, most ants collected as part of this project, have been identified to lowest taxonomic resolution, 57 species, including Solenopsis invicta, which makes up about 3.19% (276 individuals) of all ants collected, N=8665. This summer, two more field collecting trips are planned, one for the end of June 2022, and the final in Sept. 2022. Upon completion of the final field season of this project, specimens will be identified, digitized, and a thesis will be prepared for submission as early as Spring 2023. An illustrated guide to the ants of the Coal Creek Farm in Cumberland County, Tennessee. This guide will include species found or likely to be found on a large private tract of land in the Cumberland Plateau. Fire ants are just now invading northwest Arkansas. To document their spread and to better understand how their invasion will affect the native ant communities, a study gathering baseline data on the ant fauna of grasslands in the western Ozark Plateau was initiated.

Accomplishments
1. Red imported fire ant is a significant threat to the public health, agricultural productivity, and biodiversity. Bait is a major tool for controlling fire ants. Unfortunately, fire ant baits are attractive to a wide range of native ants. Low selectivity of fire ant bait has been a significant problem in fire ant management. ARS researchers in Stoneville, Mississippi, discovered a unique compound that can be used to enhance the selectivity of fire ant bait. When it was added into the soybean oil, it does not affect the feeding preference of fire ants on the soybean oil, but it significantly inhibited the feeding preference of two common native ants that compete with fire ants for food source. Adding this compound into soybean oil will make fire ant bait products unattractive to other ants but will not sacrifice the attractiveness to fire ants. This is an effective, easy, and innovative way to preserve native ant populations without compromising the benefit of fire ant bait products. An invention disclosure was submitted on July 14, 2022.

Review Publications
Ali, A., Chen, J., Khan, I.A. 2022. Toxicity and repellency of magnolia grandiflora seed essential oil and selected pure compounds against the workers of hybrid imported fire ants. Journal of Economic Entomology. 115(2):412-416. https://doi.org/10.1093/jee/toab262.
Zhou, A., Du, Y., Chen, J. 2022. Surfactant affects the tool use behavior of foraging ants. Ecotoxicology and Environmental Safety. 238:113592. https://doi.org/10.1016/j.ecoenv.2022.113592.
Zhang, L., Wang, L., Chen, J., Zhang, J., He, Y., Lu, Y., Cai, J., Chen, Y., Wen, X., Xu, Z., Wang, C. 2022. Toxicity, horizontal transfer, physiological and behavioral effects of cycloxaprid against Solenopsis invicta (Hymenoptera: Formicidae). Pest Management Science. 78:2228-2239. https://doi.org/10.1002/ps.6847.
Wen, C., Shen, L., Chen, J., Zhang, J., Feng, Y., Wang, Z., Chen, X., Cai, J., Wang, L., He, Y., Wen, X., Ma, T., Wang, C. 2022. Red imported fire ants (Hymenoptera: Formicidae) cover the insecticide-treated surfaces with particles to reduce contact toxicity. Journal of Pest Science. 95:1135-1150. https://doi.org/10.1007/s10340-021-01474-0.
Oi, D.H., Atchison, R.A., Chuzel, G., Chen, J., Henke, J.A., Weeks, R.D. 2022. Effect of irrigation on the control of red imported fire ants (Hymenoptera: Formicidae) by water-resistant and standard fire ant baits. Journal of Economic Entomology. 115(1):266-272. https://doi.org/10.1093/jee/toab242.
Chen, J., Du, Y. 2021. The odorant binding protein, SiOBP5, mediates alarm pheromone olfactory recognition in the red imported fire ant, Solenopsis invicta. Biomolecules EISSN 2218-273X. https://doi.org/10.3390/biom11111595.
Grodowitz, M.J., Gundersen, D.E., Elliot, B., Evans, R.M., Sparks, M., Reed, D.A., Miles, G.P., Allen, M.L., Perring, T.M. 2022. Trypanosomatids associated in the alimentary canal of Bagrada hilaris (Hemiptera: Pentatomidae). Journal of Insect Science. 22(1):1-6. https://doi.org/10.1093/jisesa/ieab110.
Rivers, A.R., Grodowitz, M.J., Miles, G.P., Allen, M.L., Elliott, B., Weaver, M.A., Bon, M., Rojas, M.G., Morales Ramos, J.A. 2022. Gross morphology of diseased tissues in Nezara viridula (Hemiptera: Pentatomidae) and molecular characterization of an associated microsporidian. Journal of Insect Science. 22(2):4. https://doi.org/10.1093/jisesa/ieac013.