Research Project: Management of Fire Ants and Other Invasive Ants

Location: Imported Fire Ant and Household Insects Research

2023 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
Solenopsis invicta virus 3 (SINV-3) is a virus specific to fire ants that shows promise as a natural control agent for fire ants in the United States. However, the mechanism of action of the virus is not completely understood. ARS scientists in Gainesville, Florida, determined that SINV-3 infection of midgut cells causes a decrease in food foraging in worker ants ultimately resulting in colony starvation. These results advance development of SINV-3 as a control agent for fire ants. (Project Plan Objective 1). Nursery stock with root balls wrapped in burlap that are shipped outside the Federal Imported Fire Ant Quarantine must be certified free of fire ants. However, more effective treatments for these nursery products are needed. Water-resistant spatter and granular ant baits applied to irrigated, balled and burlap root ball plants reduced fire ant populations, but inconsistently eliminated fire ant colonies. Thus, bait treatments did not meet the fire ant quarantine standards. In contrast, spray applications of non-repellent, contact insecticides to the exterior of fire ant infested root balls demonstrated a consistent ability to eliminate fire ant colonies. Further evaluations are in progress to examine reduced treatment rates to lower costs and determine the persistence of the spray treatments. (Project Plan Objective 1). A remote detection system for fire ants was funded through a National Institute of Food and Agriculture (NIFA) Small Business Innovation Research (SBIR) Phase 1 grant in FY-2023. This project represents the next generation early warning system for detection and management of invasive insect pests. The RADS will enable detection, identification, and management of fire ants e.g., at ports or in nurseries that need to certify that there exported plants are free from fire ants before they can gain a foothold, dramatically reducing their economic and ecological impact, and drastically increasing the possibility of successful eradication by early detection and treatment. The RADS technology was developed during a Phase I NIFA SBIR grant. The RADS technology is an IoT-based small form factor system that utilizes Machine Learning (ML) algorithms to identify the target insect, power electronics, and communication modules to provide accurate and timely detection and identification of Red Imported Fire Ant (RIFA). A NIFA Phase 2 SBIR grant has been funded. It will advance the project to a market-ready state in Phase II (Project Plan Objective 1 and 2). Significant progress was made on objectives of the subordinate projects titled “Fire Ant IPM in the Coachella Valley, California” and “Determining Fire Ant Bait Specificity to Extend Fire Ant Control by Conserving Non-Target Ants”. With assistance from the Coachella Valley Mosquito and Vector Control District staff, 1) Fire ant mating flight activity under desert climate conditions is now being monitored weekly. Thus far the large flight activity occurred from May - Sept.; 2) Surveys on the spread of fire ant biological control agents, Solenopsis invicta virus-3 and two species of fire ant decapitating phorid flies released in 2014, have now spread at least a half a mile beyond release sites. The Solenopsis invicta virus-3 appears to be distributed even in non-release sites. The slow spread of the flies is probably due to the patchy distribution of fire ant populations and the extreme heat of the desert climate.; 3) Acceptance of several commercial fire ant baits by non-target ant species in Florida and California was evaluated. In general, lipid-feeding non-target ants would accept most of the fire ant baits, which typically use soybean oil as a food attractant. Preliminary analysis suggested that none of the fire ant bait products tested seemed to exhibit obvious distinguishable non-acceptance by non-target ant species. The subordinate projects are relevant to fire ant surveillance and control aspects of Project Plan Objective 1. Specific methods of fire ant control are needed. RNAi constructs interfere with key biological processes and have been shown to kill fire ant workers; however, the constructs are susceptible to gut enzyme degradation and difficulty passing from the gut into the target’s hemolymph. In partnership with a commercial entity that has a proprietary methodology to derivatize RNAi such that effectiveness is enhanced, funding was obtained through NIFA SBIR Phase 1 and 2 grants. RNAi patented by USDA was derivatized resulting in better fire ant control. This research showed excellent potential for the proprietary derivatization methodology. The economically important fire ant is the model organism with expected spinoff applications to other invasive ants, such as the tawny crazy ant, the little fire ant, and other important insect pests. (Project Plan Objective 1). Preliminary results of field study indicated fast and slow-acting toxicants (dinotefuran and disodium octaborate tetrahydrate, respectively) in a sucrose solution bait reduced tawny crazy ant nesting and foraging activity at lures relative to the controls. Once baits were not replenished, the invasive ant populations began to return. In a laboratory assessment, tawny crazy ants readily fed on alginate hydrogel carrier for liquid ant bait that was dispensed in a compostable station. (Project Plan Objective 2). Collaborating with researchers from Ecuador, ants were collected from more than 400 tropical fire ant nests from the Galapagos Islands and continental Ecuador. These ant samples will be used to track routes of introduction of this invasive ant into the Galapagos Islands using both genetic and chemical markers. In addition, the samples will be used to search for microorganisms that could be used for biological control. Two hundred of those ants from the Galapagos islands were surveyed for the presence of a microsporidium Kneallhazia solenopsae. This provided an initial step to finding biological controls in the invasive tropical fire ant populations (Project Plan Objective 1.1). ARS scientists from Gainesville, Florida, in collaboration with researchers working on ants in Florida have created a shared database with all the invasive ants in Florida. This database will include both historical and reports on new invasive ants in Florida. (Project Plan Objective 1). Ants from a total of 360 nests of the red imported fire ant, were collected along a transect from southern Florida to northern Georgia. A total of 60 of those collected ants have been subjected to DNA extractions for further analysis. These new sites will help fill the gap in knowledge of the genetic, microbial and chemical diversity of this invasive species within the USA. (Project Plan Objective 1.4). The genetic diversity of red imported fire ants was assessed in both the invasive range in the USA and in the native range in South America, by collecting ants in both areas along a latitudinal transect. The genetic profiles of 95 colonies from USA and 94 from South America were obtained using a set of 59 microsatellite loci. The microsatellite data set was used in a global analysis for outlier detection using BayeScan. A locus linked to a tyrosine-protein kinase transmembrane receptor Ror gene was shown as being under selection among invasive populations, while a different locus linked to a gene serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform was found to be under selection in the native range. These genes could be candidates to study the climate adaptation in this species (Project Plan Objective 1, Objective 1.4). ARS scientists from the Imported Fire Ant and Household Insects Research Unit at the Center for Medical, Agriculture, and Veterinary Entomology (CMAVE), Gainesville, Florida, have developed a protocol to sort different developmental stages in Solenopsis invicta using light microscopy without the need for coating or using any chemicals on the specimens. This new clean sorting method allows us to use the sorted specimens for downstream molecular work to pinpoint differences in gene expression among instars as well as changes in microbiome profiles. (Project Plan Objective 1, Objective 1.4.) ARS scientists from the Gainesville, Florida, in collaboration with researchers from Florida A&M University (FAMU) are collecting fire ants from North Florida to assess the presence of microsporidium Kneallhazia solenopsae. A total of 20 colonies have been collected so far (Project Plan Objective 1, Objective 1.1).

Accomplishments
1. First discovery of viruses from the invasive little fire ant. The little fire ant, also known as the electric ant, is one of the most destructive ants worldwide. Currently, only two natural enemies are known for this tiny, stinging ant. Because viruses can be effective natural control agents against many insect pests, including ants, a metagenomics approach was used by ARS scientists in Gainesville, Florida, and Hilo, Hawaii, and collaborators from Biosecurity Queensland (Cairns, Queensland, Australia), and Fundación para el Estudio de Especies Invasivas (Hurlingham, Buenos Aires, Argentina) to facilitate discovery of viruses from the genes of the little fire ant. Ants were collected from the native South American range and introduced areas in Florida, Hawaii, and Queensland, Australia. Seven new viruses were discovered, but only in little fire ants from its native range. These viruses are potential candidates for the classical biological control of little fire ants in introduced regions.

2. Small peptide antagonists of critical receptors control fire ant colonies. Fire ants are a $8.75 billion/yr problem affecting multiple economic sectors. The USDA patented methodology isolates strong binding ligands to essential G-Protein Coupled Receptors (GPCRs). GPCR interference has been the subject of research for decades with little progress. Now the USDA patented “Receptor-i” technology quickly isolates strong binding 7 amino acid peptides that act as receptor antagonists. This breakthrough has the potential to provide specific, biodegradable control products to a wide range of insect pests. The development of this technology focused on fire ants as a model system. The research was funded through a NIFA SBIR Phase 1 grant and a NIFA SBIR Phase 2 grant has been recommended for funding. There is a need for organic / biodegradable pest insect control methods. The active peptides are readily synthesized. Proof of concept: Receptor-i isolated peptides have been shown to control field collected fire ant colonies, workers and queens. This species-specific and organic technology can be applied to other pest ants, and pest insects, thus a broad range of USDA customers can benefit. There has been considerable commercial interest in this technology.

3. Novel control of fire ants. A new fire ant control method was discovered and patented based on research by an ARS scientist in Gainesville, Florida, and a collaborator. The active ingredient is considered “natural” and inexpensive; thus, the potential commercial possibilities are great. Fire ants are a $8.75 billion/yr problem affecting multiple economic sectors. The novel active ingredient(s) were discovered through basic research on female reproductive development after mating. When fed to fire ant laboratory colonies the chemical resulted in worker and queen death; however, the ingestion of the chemical was very low, especially with field colonies. Through a Cooperative Research and Development Agreement funded by a National Science Foundation Small Business Innovation Research Phase 1 grant, the acceptability of the chemical was significantly increased, resulting in excellent control of fully functional field collected colonies. This research is expected to result in the first new active ingredient for fire ant control in >20 years. There has been spinoff to other insect pests resulting in a patent application.

4. First report of two fire ant pathogens infecting a parasitic ant of fire ants. The black imported fire ant is one of the invasive imported fire ant species from South America that infests temperate regions of the USA. When ARS scientists from Gainesville, Florida, in collaboration with researchers from Fundación para el Estudio de Especies Invasivas (Hurlingham, Buenos Aires, Argentina) surveyed for the presence of the fungal (microsporidian) pathogens Kneallhazia solenopsae and Varimorpha invictae among black imported fire ant colonies from Argentina. They found that some of those colonies were infested by an ant that is a parasite of imported fire ants. Interestingly, both pathogens also infected the parasitic ant. These findings enhance our understanding of the host range the two pathogens which are biological controls of both black and red imported fire ants in the USA. Furthermore, this study informs a complex question in evolutionary biology related to the patterns and processes of parasitism among social organisms such as ants. (Project Plan Objective 1.1.)

5. Invasional meltdown in a Florida citrus grove. Most agricultural lands are highly disturbed settings facilitating the establishment of invasive species that can competitively exclude native species. ARS scientists from Gainesville, Florida, in collaboration with researchers at the University of Florida, have surveyed for ant diversity in a citrus grove in Florida. The citrus grove contained only six species of ants, of which five were non-native ants. This study highlights the continued spread of introduced and invasive ants in Florida, that has become a worldwide hot spot for introduced and invasive species. Furthermore, the study provides a model site to gain knowledge about the processes leading to the erosion of native ant diversity through “invasional meltdown”, a process by which two or more non-native species facilitate each other’s establishment.

Review Publications
Atchison, R.A., Lucky, A. 2022. Diversity and resilience of seed-removing ant species in longleaf sandhill to frequent fire. Diversity. 14(12):1012. https://doi.org/10.3390/d14121012.
Zollota, S., Perez, P., Allen, J., Argenti, T., Read, Q.D., Ascunce, M.S. 2023. Are ants good organisms to teach elementary students about invasive species in Florida. Insects. 14(2): 118. https://doi.org/10.3390/insects14020118.
Valles, S.M., Zhao, C., Rivers, A.R., Iwata, R.L., Oi, D.H., Cha, D.H., Collignon, R., Cox, N.A., Morton, G.J., Calcaterra, L.A. 2023. RNA virus discoveries in the electric ant, Wasmannia auropunctata. Virus Genes. 59:276–289. https://doi.org/10.1007/s11262-023-01969-1.
Valles, S.M. 2023. Solenopsis invicta virus 3 infection alters foraging behavior in its host Solenopsis invicta. Virology. 581 : 81-88. https://doi.org/10.1016/j.virol.2023.03.003.
Valles, S.M., Oi, D.H., Oliver, J.B., Becnel, J.J. 2022. Characterization of Solenopsis invicta virus 4, a polycipivirus infecting the red imported fire ant, Solenopsis invicta. Archives of Virology. https://doi.org/10.1007/s00705-022-05587-4.
Luo, A.R., Hassler, M.F., Jones, T.H., Vander Meer, R.K., Adams, R.M. 2022. The evolution of tyramides in male fungus-growing ants. Journal of Chemical Ecology. (2022), 1-9. https://doi.org/10.1007/s10886-022-01382-2.
Helms Iv, J.A. 2022. Climate, geography, and the mating phenology of ants. Insectes Sociaux. 70:119–125. https://doi.org/10.1007/s00040-022-00888-y.
De Gracia Coquerel, M., Wegerif, J., Mcauley, A., Read, Q.D., Chowdhury, N., Jeong, K., Morris, J., Martins, S.J., Goss, E., Ascunce, M.S. 2023. Preliminary assessment of bacterial antibiotic resistance and candidatus liberibacter asiaticus titer in three Florida commercial citrus groves. Crop Protection. 172. https://doi.org/10.1016/j.cropro.2023.106350.