Research Project: Management of Fire Ants and Other Invasive Ants

Location: Imported Fire Ant and Household Insects Research

2021 Annual Report

Objectives
1. Conduct studies on fire ant biology to develop new and improved surveillance and control strategies as part of an integrated pest management program. 1.1. Develop natural enemies of fire ants as classical biological control agents or biopesticides by characterizing their life cycle, evaluating their effectiveness, determining host specificity, developing methods for production and release, and formulating as biopesticides. 1.2. Determine how irrigation affects fire ant bait efficacy. 1.3. Develop novel biologically-based fire ant control by identifying the behavioral and semiochemical underpinnings of fire ant mating flights and colony establishment. 1.4. Identify key biological processes in fire ant life stages that may be susceptible to disruption. (vacant Molecular Biologist) 1.5. Determine and quantitate fire ant traits that contribute to their ability to survive the harsh conditions associated with accidental transport over long distances and/or establishing and expanding infestations at the invasion front. (vacant Entomologist) 2. Develop new surveillance and control strategies for crazy ants and other invasive pest ants. 2.1. Employ metagenomics techniques and next generation sequencing technologies to discover potential natural enemies of the little fire ant. 2.2. Develop an effective baiting strategy for the control of tawny crazy ants. 2.3. Investigate the homology of pheromone systems that are well understood for S. invicta, but relatively unknown in the little fire ant and the tawny crazy ant. 2.4. Identify key biological processes of little fire ants and/or tawny crazy ants that can be exploited and developed as novel control methods. (vacant Molecular Biologist) 2.5. Determine and quantitate little fire ant, tawny crazy ant, and other ant species traits that contribute to their invasive success, e.g., metabolic rates. (vacant Entomologist)

Approach
1.1 Integration of any new natural agent into a fire ant control program will require the satisfactory completion of studies in host specificity, predicted-efficacy, virulence, mode of action/transmission, formulation/rearing and field release methodologies. 1.2 Water resistant and standard fire ant bait formulations exposed to irrigation will be evaluated for efficacy against fire ant colonies. The effect of bait application methods (piled vs broadcast) on improving bait tolerance to irrigation will also be assessed. 1.3 Behavioral and semiochemical underpinnings of fire ant mating flights and colony establishment will be examined by determining the behavior of alates to pyrazines with olfactometer bioassays and in-flight lek sampling. Male produced tyramides will be further evaluated for physiologicfal functions related to multiple mating and rapid wing loss. 1.4 The approach of this sub-objective will be defined by the scientist filling the vacant Molecular Biologist position. 1.5 The approach of this sub-objective will be defined by the scientist filling the vacant Entomologist position. 2.1 Little fire ants from across the native and introduced ranges will be collected and used as RNA source material to create cDNA expression libraries. Detailed bioinformatics analysis of resulting NGS data will allow us to identify potential microsporidia, fungi, viruses, protists, and non-hymenopteran eukaryotic parasites. Sequence leads will be verified by molecular analysis of little fire ant colonies sampled within and outside the native ranges. 2.2 Consumption and temporal feeding patterns by tawny crazy ants (TCA) on liquid sucrose bait containing a slow-acting toxicant will be compared to bait containing a fast-acting toxicant. Time lapse photography will be used to document temporal feeding patterns over 72 hours. TCA feeding patterns will be used to design liquid bait dispensers such as alginate hydrogel carrier and presented in a compostable dispenser. 2.3 Systematically evaluate exocrine glands in TCA and little fire ant (LFA) workers and queens for phenotypic effects, e.g. attraction, repellency, alarm, and recruitment using behavioral bioassays. Attraction will be investigated first, using a Y-tube olfactometer bioassay to guide the isolation of active compounds. Attractants can enhance baits and improve monitoring systems. 2.4 The approach of this sub-objective will be defined by the scientist filling the vacant Molecular Biologist position. 2.5 The approach of this sub-objective will be defined by the scientist filling the vacant Entomologist position.

Progress Report
Objective 1: A reproductively viable hybrid between the invasive Solenopsis invicta and S. richteri occurs in the United States but not in their native South America. In the United States, the hybrid occupies a large area in the northern tier of the imported fire ant distribution. While examining hybrid workers for genetic introgression of parent venom proteins, ARS researchers in Gainesville, Florida, found 2 variants of venom protein that were unique to the hybrid. Data suggest that the hybrid may have a competitive advantage over either parent species and may be evolving into a new species, which could impact control efforts. The efficacy of water resistant and standard fire ant bait formulations against fire ant colonies was compared on sprinkler irrigated sod. This bioassay emulated nursery and landscape irrigation situations, which reportedly degrade the moisture sensitive fire ant baits, thereby decreasing bait efficacy. Comparisons of the efficacy of piled versus scattered, or broadcast, applications of standard and water-resistant baits revealed reductions of >88% in adult and immature fire ants and no surviving queens for all bait treatments. This result was unexpected because piled baits were hypothesized to be better protected from irrigation and broadcast application of standard fire ant bait was still effective despite exposure to irrigation. Trees (in burlap root balls) transported outside the APHIS Federal Imported Fire Ant quarantine area must be certified free of fire ants. Improvements in burlap root ball treatments are needed. The effectiveness of water-resistant spatter and granular bait formulations and application methods on irrigated, balled and burlap root ball plants are being evaluated by ARS researchers in Gainesville, Florida. Initial testing of these bait formulations resulted in colony death with the water-resistant granular bait, and reductions in fire ant colonies exposed to spatter baits. Significant progress was made on Mission Critical Research: Fire Ant IPM in the Coachella Valley, California. Fire ant mating flight activity under desert climate conditions was unknown. Therefore, a prototype fire ant alate trap was developed and field tested in Gainesville, Florida, and shown to be effective. These traps were sent to the Coachella Valley Mosquito and Vector Control District for field evaluation under Coachella Valley conditions. Fire ant alates were captured, thus validating the new trap design under a variety of conditions. This is one objective of a subordinate project that is relevant to fire ant surveillance aspect of Objective 1. Derivatives of RNA interference (RNAi) constructs have been shown to exert greater mortality effects on fire ant workers, suggesting that the derivatized RNAi is more refractive toward gut enzyme degradation than the underivatized RNAi. Additional research will address the stability of the RNAi constructs and evaluate bait formulations of the constructs in order to evaluate field applications. This research targets imported fire ants (Project Plan Objective 1). Several small peptide antagonists to essential G-Protein Coupled Receptors were isolated and identified using a unique screening/isolation process. The active peptides are readily synthesized. The most bioactive are being evaluated against worker fire ants under laboratory conditions to determine optimal concentrations and formulations. This research targets imported fire ants (Project Plan Objective 1) and is associated with an agreement funded through a National Institute of Food and Agriculture (NIFA) Phase 1 grant. This species-specific technology can be applied to the tawny crazy and the little fire ant (Project Plan Objective 2), and other pest insects. A novel control method for fire ants was discovered and patented from research on the mechanism by which newly mated queens can free themselves from queen’s primer pheromone suppression of female sexuals within the colony. When applied to fire ant laboratory colonies the method gave worker and queen mortality; however, the ingestion of the active ingredient was very low, especially with field colonies. Through an agreement funded by a National Science Foundation (NSF) Phase 1 grant we are investigating methods to increase the acceptability of the active ingredient(s). Success is expected to result in the first new fire active ingredient in 14 years. This research targets imported fire ants in support of Project Plan Objective 1. However, spin-off to other pest ant species (Project Plan Objective 2), or other pest insects is expected. Objective 2: A method to determine the frequency and intensity of bait foraging by the invasive, tawny crazy ant is needed to better refine bait delivery systems. To accomplish this, trail cameras have been customized to obtain time-lapse images of tawny crazy ant feeding on liquid baits containing fast and slow-acting toxicants in the field. Field sites have been secured and initial testing has commenced. Field testing of an alginate hydrogel carrier for a liquid ant bait resulted in short-term reductions of tawny crazy ant populations. This hydrogel carrier is biodegradable, unlike other hydrogels, and is much easier to deploy than bait stations. A commercial hydrogel bead making appliance has significantly increased our production of the hydrogel carrier which should facilitate our research to develop an improved bait formulation. Two new scientists (Research Molecular Biologist and Research Entomologist) were on-boarded in January 2021.

Accomplishments
1. Insect food webs and invasive ant diets in the U.S. Corn Belt. The rapid increase in bioenergy crops is one of the largest global trends in land use, driven by the need for alternate fuels and to slow climate change. The production of fuel ethanol is now a dominant factor in farming landscapes in the Midwestern U.S. Corn Belt. Scientists from Gainesville, Florida, Brookings, South Dakota, and Michigan State University investigated how plant diversity in bioenergy croplands in this region impacted insect food webs and the diets of one of the oldest and most widespread invasive ants in the U.S.—the pavement ant. They found that invasive pavement ants adjusted their diets based on which biofuel crop they inhabited, feeding on a mix of plant and animal prey in species-rich prairie and switchgrass fields, but preying entirely on other insects in species-poor corn fields. When examining how energy flowed through entire insect food webs, they found that food webs in corn fields were simpler and likely more prone to fluctuations than those in diverse native perennial biofuel crops like fields of switchgrass and restored prairie. The results highlight that by supporting more complex species interactions, restoring farmlands to native perennial biofuel crops can help diversify and stabilize agricultural food webs.

2. Recommendations on the length of ecological field experiments. Field experiments are the primary method of advancing ecological knowledge but are often limited by short grant cycles or the term limits of academic programs. These time frames may be too short to ensure field experiments accurately capture long-term processes and avoid spurious results due to transient short-term fluctuations. Scientists from Gainesville, Florida, Michigan State University, and Kent State University compiled data from dozens of studies conducted at Long-Term Ecological Research sites across North America. They found that ecological field experiments should run an average of 10 years or more to deliver consistent results and avoid spurious conclusions, with experiments in more variable climates needing more time than those in stable climates. Their results underscore the importance of long-term research, especially in the face of climate change.

3. Invasive ant queens are fertile year-round. The tawny crazy ant is an invasive ant that is spreading in the southern USA. The control of invasive ants requires an understanding of their biology to develop efficient methods of control, such as strategically applying ant baits to eliminate queen ants which are vital to the survival of ant colonies. Previous research indicated that groups of tawny crazy ant queens congregated within nests during the winter, but eggs and immature ants were not being produced. However, monthly examinations of tawny crazy ant queens by scientists in Gainesville, Florida, determined that the queens’ ovaries contained eggs year-round and over 80% of the queens were mated. These results indicated that despite having mature eggs, egg laying was not occurring, perhaps because there is less foraging for food by the colony and limited feeding by queens during the winter. This suggested that instead of applying baits in the winter to target queens consolidated within nests, an alternative strategy of applying baits in the spring when egg laying starts, colonies are actively foraging, and before colonies split and spread in the summer, may result in better control of this invasive ant.

Review Publications
Cusser, S., Helms Iv, J.A., Bahlai, C., Haddad, N.M. 2021. How long do population level field experiments need to be? Utilising data from the 40-year old LTER network. Ecology Letters. 24(5):1103-1111. https://doi.org/10.1111/ele.13710.
Choi, M.Y., Vander Meer, R.K. 2021. GPCR-based bioactive peptide screening using phage-displayed peptides and an insect cell system for insecticide discovery. Biomolecules EISSN 2218-273X. 11(4). Article 583. https://doi.org/10.3390/biom11040583.
Helms Iv, J.A., Roeder, K.A., Ijelu, S.E., Ratcliff, I., Haddad, N.M. 2021. Bioenergy landscapes drive trophic shifts in generalist ants. Journal of Animal Ecology. 90(3):738-750. https://doi.org/10.1111/1365-2656.13407.
Chen, J., Oi, D.H. 2020. Natural occurring compounds/materials as alternatives to synthetic chemical insecticides for use in fire ant management. Insects. 11:758. https://doi.org/10.3390/insects11110758.
Oi, D.H. 2021. The seasonal reproductive status of tawny crazy ant queens (Hymenoptera: Formicidae) in Florida. Florida Entomologist. 104(2):140-142. https://doi.org/10.1653/024.104.0211.
Vanderwoude, C., Boudjelas, S., Gruber, M., Hoffmann, B.D., Oi, D.H., Porter, S.D. 2021. Biosecurity plan for invasive ants in the Pacific Region. In: Edited by Pullaiah, T. and Ielmini, M.R. editors. Invasive Alien Species: Observations and Issues from Around the World, Volume 2: Issues and Invasions in Asia and the Pacific Region, First Edition. New York, New York. John Wiley & Sons Ltd. 2(13):275-288. https://doi.org/10.1002/9781119607045.ch25.
Valles, S.M., Oliver, J., Addesso, K., Perera, O.P. 2021. Unique venom proteins from Solenopsis invicta x Solenopsis richteri hybrid fire ants. Toxicon: X. 9-10:100065. https://doi.org/10.1016/j.toxcx.2021.100065.
Arnold, D.P., Balasubramani, S.P., Valles, S.M., Hottel, B.A. 2021. Examining the prevalence of Solenopsis invicta virus 3 (Solinviviridae: Invictavirus) in Solenopsis invicta (Hymenoptera: Formicidae) alates collected in North Florida. Florida Entomologist. 104(1):9-12. https://doi.org/10.1653/024.104.0102.