Progress 10/01/23 to 09/30/24

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This is the final progress report for project 6036-32000-051-000D which terminates on 09/02/2024. (Objective 1) The development and commercialization of a red, black, and hybrid imported fire ant detection device was completed and has been available to APHIS, stakeholders, and the public from Agdia, Inc. as InvictDetect⿢ Plus. The device, a laminar flow immunoassay (analogous to a pregnancy test) provides a much-needed tool for regulatory agencies in the USA and other countries to enforce quarantine protocols to limit the spread of these invasive ants. This progress significantly improves surveillance of these invasive fire ant species through a single, rapid, on-site test. (Objective 1.1) A fire ant specific virus was released and established in California and Florida for the biological control of red imported fire ants (RIFA) to improve fire ant IPM. Field inoculations of fire ant nests with the virus Invictavirus solenopsae (SINV-3) in Florida caused reductions in the size and number of fire ant nests, as well as persistence and spread of the virus to adjacent uninoculated colonies, which extended the control. SINV-3 provides an additional natural control agent against fire ants with an advantage over traditional insecticides of no known detrimental ecological impacts, host specificity, and sustainability. In addition to Florida, augmentative releases of the virus were successful in California. ARS researchers at Gainesville, Florida completed research to determine the mode of action of the virus, Invictavirus solenopsae, in RIFA. While Invictavirus solenopsae has been shown to cause significant mortality among all stages of its RIFA host, the mechanism by which it does so has remained unresolved. Studies showed that virus infection of the ant causes damage to the midgut cell lining of worker ants and alters their feeding and foraging behavior. The worker ants stop acquiring food for the colony causing starvation, which leads to mass mortality and queen wasting. Fire ants were collected from 360 nests along a transect from south Florida to north Georgia. and from 66 sites in the panhandle of north Florida (collaborating with Florida A&M University). DNA extractions from these samples were used to survey for the microsporidian fire ant pathogen Kneallhazia solenopsae. Future sequencing data will deepen our understanding of the genetic diversity of K. solenopsae in the USA. (Objective 1.2) Fire ants can be efficiently controlled by fire ant baits which typically contain corn grit, an ingredient that allows the baits to be easily applied and foraged by ants. Traditionally it is thought that corn grit exposed to rain or irrigation absorbs moisture and compromises bait efficacy because ants do not feed on wet bait. Water- resistant fire ant baits have been developed that either replace or modify the corn grit. Research conducted with collaborators from APHIS, and the Coachella Valley Mosquito and Vector Control District in California determined that both standard commercial fire ant bait and water-resistant baits that have been soaked in water in the laboratory, as well as exposed to sprinkler irrigation in landscapes, can still effectively control fire ants. Because water-resistant fire ant baits are not readily available in the U.S., knowledge that standard fire ant baits can withstand sprinkler irrigation should allow land managers more flexibility in scheduling bait treatments. Nursery stock with root balls wrapped in burlap that are shipped outside the Federal Imported Fire Ant (IFA) Quarantine must be free of fire ants. Water-resistant spatter and granular ant baits applied to irrigated, balled and burlappd plants reduced fire ant populations, but inconsistently eliminated fire ant colonies due to intermittent fire ant foraging relative to bait palatability and cool weather. These bait treatments did not meet IFA quarantine standards. An effective alternative of spraying non-repellent, contact insecticides to the exterior of infested root balls demonstrated a consistent elimination of fire ant colonies. In addition, this treatment also prevented root balls from being infested by migrating fire ant colonies for 4⿿6 months. It is hypothesized that the nonrepellency facilitated greater ant contact with the insecticide, resulting in better control than traditional treatments. This discovery has led to further cooperative research with the nursery industry through collaborations with Tennessee State University and APHIS. (Objective 1.3) Initially, fire ant mating flights were used to determine how newly-mated queens quickly overcame the dealation inhibitory effects imposed on them by queen pheromones. This research quickly changed when it was published that male fire ants produce tyramides. Over the next several years it was determined that males transfer the tyramides to female sexuals during mating. The female sexuals release a specific enzyme during the mating process that hydrolyzes tyramides to the biogenic amine, tyramine. Metabolomic studies determined that newly-mated fire ant queens have highly elevated amounts of tyramine in their gasters, head and thorax. Tyramine injected into mature female sexuals 1) precociously lost their wings (dealate); 2) started reproductive development; and 3) produced queen pheromone (a worker attractant). This sequence of events was published in Nature Communications. The use of these findings for fire ant control consisted of feeding tyramine to colonies that had female sexuals, such that they would dealate and compete with the real queen, resulting in decreased egg production. Notably, the treatment with tyramine resulted in significant worker mortality with lab colonies. A patent application was submitted, and we engaged a commercialization partner with whom we had Phase 1 and 2 SBIR grants and associated CRADAs. Field studies clearly showed that field (wild) fire ants were repelled by bait formulations with tyramine. An NSF SBIR phase 1 grant allowed us to investigate methods to overcome the negative taste of tyramine using taste masking methods already developed to mask the bad taste of medications associated with human pharmaceuticals. This research resulted in a formulation that is readily consumed by wild fire ants. Field evaluations are being developed now. (Objective 2.) Collaborating with researchers from Ecuador, the invasive, tropical fire ant, Solenopsis geminata, were collected from >400 nests in 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; plus they will be screened for microorganisms with biocontrol potential. (Objective 2.1) Employing metagenomics and next generation sequencing technologies has led to the discovery of the first viruses from the invasive little fire ant, Wasmannia auropunctata. This ant is one of the most destructive invasive ants worldwide. Seven viruses were discovered in the little fire ant from its native range, which offer potential classical biological control agents against this pest ant in the USA and other regions globally. (Objective 2.2) Efforts to control overwhelming numbers of tawny crazy ants (TCA) in buildings and landscapes often rely on broadcast applications of residual, contact insecticides and granular ant baits. However, suppression often is temporary and inadequate. Our research has shown that a fast-acting liquid bait containing dinotefuran results in significant and visually perceptible reductions in TCA, but its effects were localized, and areas became reinfested within 2-4 weeks after baiting stopped. To extend bait accessibility, a laboratory study indicated that TCA readily fed on alginate hydrogel carrier containing liquid ant bait that was dispensed in a compostable station. Field deployment of these stations revealed hydrogel desiccation and limited ant presence at the stations; thus more research is needed. To improve baiting efficacy, comparisons of fast and slow-acting bait toxicants (dinotefuran and disodium octaborate tetrahydrate, respectively) in sucrose solution demonstrated significant reductions in TCA nesting and foraging activity with each bait type. However, once baits were not replenished, TCA populations began to return. Strategic bait placements where slow-acting bait was placed along the field plot perimeters to allow bait distribution beyond the plots, while the fast-acting bait was centered within the plots to quickly suppress TCA. This baiting strategy resulted in significantly less TCA nesting and foraging within the plots. (Objective 2.3) The response of little fire ant (LFA) workers to trail pheromone chemicals deposited on various substrates was investigated. While the pheromones responsible for trail formation were not identified, the groundwork of developing a suitable bioassay was completed and published. The LFA alarm pheromone was previously identified as a pyrazine derivative with multiple substituents on the pyrazine ring. This is like the tri-substituted pyrazine isolated and identified for the red imported fire ant (RIFA). In the case of RIFA, and likely to be similar for LFA, the alarm pheromone acts to both increase the worker ant activity and attract the worker ants to the source of the pyrazine. Therefore, applications can be an efficient attractant for traps or baits. In addition, it was determined that extracts of LFA worker abdomens elicited rapid movement in previously quiescent workers in a fully functional LFA colony. The identification of the responsible semiochemicals will be a future target. Artificial Intelligence (AI)/Machine Learning (ML) Through a NIFA SBIR Phase 2 Grant, GearJump, Inc. partnered with ARS researchers at Gainesville, Florida through a CRADA to develop a RADS, Rapid Autonomous Detection System, focused on accurately detecting and identifying red imported fire ants. Machine learning algorithms were developed to train the RADS system. Advanced computer vision techniques rooted in convolutional neural networks (CNNs) were employed for image recognition and processing. In particular, a custom dataset of images of both fire ants and other ant species were used to train, validate and quantify the accuracy of the identification process, and provide a quantifiable measure of performance. The dataset used comprised six categories, five ant species and samples of the empty arena. These categories were arranged in a "one-vs-rest" fashion aimed to train a binary classification algorithm, capable of distinguishing fire ants from any other individual. On the custom dataset, the model achieved notable performance with an accuracy of 93.8%, In subsequent trainings, domain randomization techniques were implemented to improve the model's robustness to background changes. In conclusion, the RADS demonstrated high accuracy in identifying red imported fire ants. The use of transfer learning, data engineering, and sensitivity analysis contributed to the robustness and specificity of the system, ensuring reliable detection of RIFA in diverse conditions. Computing resources were provided by the CRADA partner. The success of this NIFA SBIR phase 2 project depends on the successful development of a remote method to reliably detect the red imported fire ant from other ant species. The AI related research is essential for the success of the project, since accurate remote detection and identification of the target fire ant is essential for success. This methodology can be spun-off to surveillance and detection of other invasive ants or adapted to other invasive species. ACCOMPLISHMENTS 01 Mode of action of a fire ant killing virus. Invictavirus solenopsae (previously called Solenopsis invicta virus 3) is a virus specific to fire ants that is an effective natural control agent for fire ants in the United States. Numerous stakeholders (APHIS, pest controllers, public) are interested in this research because the virus provides a specific, non-toxic, sustainable method of controlling invasive fire ants. However, the mechanism of action of the virus is not completely understood. Scientists in Gainesville, Florida determined that Invictavirus solenopsae infection can be transmitted from immature stages to adults, possibly through the adult care of the immature ants. Furthermore, the adult ants stop acquiring food for the colony, causing starvation, which leads to mass mortality of the immature ants and queen wasting. These results advance the understanding and development of this virus as a biological control agent for fire ants. 02 New biodegradable, species-specific peptides inactivate fire ant colonies. It has been over 25 years since the last fire ant bait control product was commercialized. Neuropeptides such as Pheromone Biosynthesis Activating Neuropeptides (PBAN) activate receptors to initiate essential biochemical reactions in insects. ARS scientists in Gainesville, Florida and Corvalis, Oregon used their patented Receptor- interference technology to specifically isolate peptides that strongly bind to and inactivate fire ant PBAN receptors. Selected peptides fed to fire ant colonies resulted in high worker mortality and death or sterilization of the queen. This proof of concept of the Receptor⿿interference technology may lead to the development of a new fire ant bait that is biodegradable and specifically targets fire ants. 03 An inexpensive fire ant bait formulation. Commercially available fire ant baits are typically sold to households and other high-value markets. However, these products are too expensive for farmers and ranchers who need fire ant control over large acreages. An ARS scientist in Gainesville, Florida and a CRADA partner discovered inexpensive fire ant toxicants. They determined that encapsulation of the toxicants allowed passage into the fire ant gut resulting in high worker ant mortality and queen death. This novel bait formulation is expected to be less expensive than currently available fire ant baits and may provide an economical fire ant control option accessible to farmers and ranchers. 04 Tawny crazy ant control with baits. The tawny crazy ant (TCA) is an invasive ant from South America that is established in the Gulf Coast states from Florida to Texas, plus Georgia. It develops overwhelming populations that can inundate urban, rural, and natural landscapes. Attempts to control TCA populations often rely on excessive applications of residual, contact insecticides over entire landscapes, which is often temporary and inadequate. Researchers from Gainesville, Florida have demonstrated that a fast-acting liquid bait containing dinotefuran resulted in significant and visually perceptible reductions in TCA, but its effects were localized, and areas became reinfested within 2-4 weeks after baiting was discontinued. Comparison of TCA foraging intensity in the field on the fast-acting bait and a slow- acting, boric acid based, liquid bait indicated sustained and significantly more consumption of the slow-acting bait. In addition, significant reductions in tawny crazy ant nesting and foraging activity occurred with both types of baits. A baiting strategy utilizing a combination of both fast and slow-acting liquid baits has resulted in sustained and more expansive control, which can curtail excessive insecticide applications often used against this invasive ant.

Impacts
(N/A)

Publications

• Ascunce, M.S., Toloza, A.C., González-Oliver, A., Reed, D.L. 2023. Nuclear genetic diversity of head lice sheds light on human evolution. PLOS ONE. 18(11). https://doi.org/10.1371/journal.pone.0293409.
• Vander Meer, R.K., Alonso, L.E., Lofgren, C.S. 2023. Light affects the homing ability of Solenopsis invicta (Hymenoptera: Formicidae) foraging workers. Florida Entomologist. 106(3):175-181. https://doi.org/10.1653/ 024.106.0304.
• Ascunce, M.S., Porter, S.D., Carroll, K.C., Perez, P., Nisip, A.J., Aufmuth, J., Justice, L.M., Gavilanez-Slone, J.M., Qureshi, J.A., Lucky, A. 2024. Ant Community Composition in a citrus grove reveals eastern expansion in Florida of the South American big-headed ant, Pheidole obscurithorax Naves (1985). Florida Entomologist. 107(1). https://doi.org/ 10.1515/flaent-2024-0011.
• Tappey, J., Chinta, S.P., Vander Meer, R.K., Cartwright, K.C. 2023. Branched tyramides from males of the harvester ant, Pogonomyrmex badius. Naturwissenschaften. 110:57. https://doi.org/10.1007/s00114-023-01885-2.
• Ascunce, M.S., Kassu, G., Bouwma, A., Oi, D.H., Reed, D.L., Briano, J., Shoemaker, D. 2023. Prevalence and Genetic Diversity of a Microsporidian Parasite in the Black Imported Fire Ant and its Social Parasitic Ant (Formicidae: Myrmicinae: Solenopsis) in Buenos Aires Province, Argentina. Insects. 14(12):901. https://doi.org/10.3390/insects14120901.
• Toloza, A.T., Ascunce, M.S., Reed, D. 2024. Measuring local genetic variation in permethrin resistant head lice, pediculus humanus capitis (Phthiraptera: Pediculidae), from Buenos Aires, Argentina. Journal of Medical Entomology. 18(11). https://doi.org/10.1093/jme/tjae048.
• Chinta, S., Vander Meer, R.K., O'Reilly, E.E., Choi, M.Y. 2023. Insecticidal effects of Receptor-i isolated bioactive peptides on fire ant colonies. International Journal of Molecular Sciences. 24(18). Article 13978. https://doi.org/10.3390/ijms241813978.
• Valles, S.M. 2024. Effect of Solenopsis invicta virus 3 on brood mortality and egg hatch in Solenopsis invicta. Journal of Invertebrate Pathology. 203. https://doi.org/10.1016/j.jip.2023.108056.

Progress 10/01/22 to 09/30/23

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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). Artificial Intelligence (AI)/Machine Learning (ML) During the NIFA SBIR Phase 1 project, Reporting and Data System (RADS) machine learning algorithms were developed to accurately identify the target vector, red imported fire ants. The process flow for training the machine learning algorithms involved programming the micro-controller with a neural network platform based on the MobileNetV2 0.35 architecture. This architecture is a 53-layer deep convolutional neural network capable of classifying images into 1000 object categories. To train the RADS machine learning algorithms, computer vision algorithms based on Convolutional Neural Networks (CNNs) were implemented. These algorithms utilized foreground extraction, feature recognition, and contour identification to accurately identify the target vector. CNNs are primarily used for image classification and recognition because of their high accuracy. This approach allowed for the use of actual available datasets for both fire ants and other insects. Training CNN models on images of ants provided validation and quantification accuracy identification with a quantitative output. Transferred Learning (TL) was applied to start the neural network training. In this specific case, knowledge gained while learning to recognize multiple unique features of multiple ant species could apply when trying to recognize RIFA with high specificity. TL allowed for improved sample efficiency of the images and overall improved specificity. Several features were used for image analysis to implement identification algorithms including colors and features. The ML algorithms were trained for accurate detection and identification of the red imported fire ant. (Project Plan Objective 1 and 2). ACCOMPLISHMENTS 01 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. 02 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. 03 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. 04 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.) 05 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.

Impacts
(N/A)

Publications

• 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.
• 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.

Progress 10/01/21 to 09/30/22

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. Nursery stock (in burlap root balls) transported outside the Animal and Plant Health Inspection Service (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 reduced fire ant populations, but inconsistently eliminated fire ant colonies. Thus, applying baits to root balls were not effective in controlling fire ants that meet the fire ant quarantine standards. Evaluation of non-repellent insecticide spray applications to the exterior of root balls as an alternative to baiting is in progress (Project Plan Objective 1). Significant progress was made by ARS scientists at Gainesville, Florida on all objectives of the Mission Critical Research project titled ⿿Fire Ant IPM in the Coachella Valley, California⿝. 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. 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 survivorship and spread are significant due the patchy distribution of fire ant populations and extreme heat in the desert climate. 3) A field study comparing efficacy of fire ant bait applications following the standard practice of withholding irrigation versus maintain the normal irrigation schedule did not result in significant differences in control. This indicates that bait applications can be made without disrupting normal irrigation, thus making fire ant operations more efficient. This subordinate project is relevant to fire ant surveillance and control aspects of Project Plan Objective 1. Derivatives of RNA interference (RNAi) constructs have been shown to exert greater mortality effects on fire ant workers, than non-derivatized constructs, suggesting that the derivatized RNAi is more stable toward gut enzyme degradation and are less polar making it easier for them to cross from the gut to the hemolymph. This research showed excellent potential for the proprietary derivatization methodology. Consequently, a NIFA Phase 2 SBIR grant was applied for and approved for funding. This methodology is using the economically important fire ant as the model organism (Project Plan Objective 1). However, a spinoff is expected for other invasive ants, such as the tawny crazy ant and the little fire ant (Project Plan Objective 2). 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 currently 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 a NIFA SBIR Phase 1 grant. This species-specific technology can be applied to the tawny crazy ant, the little fire ant (Project Plan Objective 2), and other pest insects. The methodology quickly identifies strong agonist/antagonists that interfere with key insect physiological processes. There has been considerable commercial interest in this technology. A novel control method for fire ants was discovered and patented from research on the mechanism by which a queen's reproductive daughters quickly free themselves from their mother/queen's suppression of their reproductive development. When the active compound(s) is fed to laboratory fire ant colonies, worker and queen mortality results; however, the ingestion of the active ingredient was very low, especially with field colonies. Through a CRADA funded by a National Science Foundation (NSF) Phase 1 SBIR 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 15 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. ARS scientists from Gainesville, Florida, have collected ants from 180 nests of the red imported fire ant, Solenopsis invicta, along a transect from southern Florida to northern Georgia. These collections are part of a funded MTRA with the South Korean Animal and Plant Quarantine Agency. Collections will continue up to November 30, 2022. The collections are part of larger project aimed at being able to identify the origin of any Solenopsis intercepted at South Korean or other ports around the world. (Project Plan Objective 1). The frequency and intensity of foraging by the invasive, tawny crazy ant on liquid baits was documented with customized trail cameras that record time-lapse images in the field. Tawny crazy ant feeding on liquid bait containing a fast acting toxicant was significantly less than the slow acting bait and the control. The SEA statistician provided excellent guidance on analyzing feeding patterns. A study has been initiated to examine the effects of the fast and slow-acting toxicant on field colonies of this invasive ant. In addition, tawny crazy ant feeding patterns on an alginate hydrogel carrier for a liquid ant bait will be assessed to help guide bait development for tawny crazy ants. (Project Plan Objective 2) Analysis of tissue ratios of stable isotopes of carbon (13C/12C) and nitrogen (15N/14N) is a standardized way of quantifying the feeding ecology and trophic interactions of invasive and native ants, but we lack an understanding of how these ratios vary in dispersing queens relative to workers. Variation in isotope ratios among queens and workers of a variety of invasive and native species under field conditions are being examined by ARS researchers in Gainesville, Florida and Brookings, South Dakota. The results will shed light on invasive ant queen metabolism, survival, and dispersal, and help develop novel methods for rapidly determining ant feeding ecology. (Objectives 1.5, 2.5) To determine how geography and climate impact the timing of ant mating and the release of dispersing queens, historical records of ant mating events in the contiguous U.S. were analyzed from digitized records in an open-access database of ant museum specimens. Ant mating seasons were found to occur later at higher latitudes and elevations, and have been occurring earlier in recent years, likely due to warmer temperatures under climate change. Twenty-five percent of native species have been mating earlier in recent years, but none of the invasive species with enough data showed recent changes in mating season. (Objectives 1.5, 2.5) To determine the timing of ant mating flights in agricultural landscapes and relate it to changes in climate, weather, and land use, ARS researchers in Gainesville, Florida and Urbana, Illinois collected flying ant queens and males captured by the USDA Aphid Suction Trap Network in 2020 and 2021 and are currently collecting those from 2022. These ants, collected from 33 agricultural research sites across 10 states, represent perhaps the most comprehensive large-scale dataset of ant flight phenology ever collected on Earth. The dataset will be developed to provide a snapshot of when invasive and native ants mate across much of the U.S., measure how that has changed compared to 20th century baselines and predict how ant mating seasons and invasive ant distributions may change under projected future climates. (Objectives 1.5, 2.5) By performing a simulated drought experiment at the Kellogg Biological Station LTER in Michigan, ARS researchers from Gainesville, Florida and researchers from Michigan State University measured how projected future climates will impact invasive and native ant foraging activity in agricultural systems. Invasive pavement ants Tetramorium immigrans were insensitive to drought conditions, foraging at equally high levels under drought and control treatments. Native cornfield ants, Lasius neoniger, in contrast, showed high sensitivity to drought, and increased their average foraging activity by 30-40% under drought conditions. Work is ongoing to determine how these effects vary under different agricultural treatments and whether they impact rates of pest suppression in crops. (Objective 2.5). ACCOMPLISHMENTS 01 A forty-year-old mystery of reproductive development in new fire ant queens is solved. Fire ant queens use a pheromone to inhibit reproductive development in their daughters while in the colony. When conditions are right the unmated daughters fly from a nest, mate with males, and then the new queens immediately begin reproductive development and attempt to start new colonies on the ground. How this rapid initiation of reproductive development is accomplished has been a mystery for over 40 years until ARS researchers at Gainesville, Florida, and collaborators from Foresight Science and Technology, Virginia Military Institute, and Ohio State University discovered that fire ant males produce a chemical class called tyramides that are passed on to the daughters during the mating process. They also determined that the daughters produce a specific enzyme that converts tyramides to tyramine which causes accelerated reproductive development, essential to survival of the new colony. This work will be a model for future research in this area, and was reported in Nature Communications, where it has generated significant attention (93rd percentile interest for all outputs of the same age). In addition, these discoveries have already led to a possible novel control method for fire ants (Patent issued). 02 Fire ant colonies eliminated after inoculation with a fire ant virus. The red imported fire ant was introduced into the United States in the 1930s and currently infests about 300 million acres. The fire ant causes $8 billion in annual control costs and losses to many economic sectors, including livestock and agricultural production and poses a serious threat to human health. Biological control is widely considered the most sustainable method for the regional control of fire ants. Using gene sequence identity/homology approaches, ARS scientists in Gainesville, Florida, discovered and characterized Solenopsis invicta virus 3, which specifically infects fire ants. They found the virus to be a virulent pathogen of fire ant colonies under laboratory conditions where it consistently killed colonies and was highly transmissible. Introductions of this virus into fire ant nests in the field caused reductions in the size and number of fire ant nests. It also persisted for over 20 months and spread to adjacent uninoculated colonies. This is the first documentation of a fire ant virus eliminating fire ant colonies under field conditions and demonstrates that this host specific, self-sustaining virus can be an important, biological control agent of the widespread, stinging, invasive, fire ant. 03 Organic agriculture has higher pest suppression by ants in the U.S. Corn Belt. Organic agriculture has the potential to reduce greenhouse gas emissions, pollutant runoff, and biodiversity loss compared to conventional agriculture, but the long-term effects on many organisms remain unknown. ARS scientists from Gainesville, Florida, and Michigan State University examined 31-year-long landscape experiments to test the impacts of organic agriculture on ant communities and the ecosystem services they provide to crops. Despite supporting mostly the same species, organic crops experienced higher potential pest suppression provided by ants, due to differences in the timing of ant foraging. Ants in conventional crops mostly foraged late in the growing season, when they are less likely to be useful in suppressing pests of developing crops. Ants in organic crops, in contrast, were active throughout the growing season, with most foraging taking place early in the season when crops were still developing. The results showed that by this standard organic agriculture supported twice as much ant-mediated pest suppression potential as conventional agriculture. 04 Fire ant bait remains effective after irrigation. The red imported fire ant is an invasive pest of agricultural, urban, and natural areas. It is also considered a public health pest due to its painful sting. This ant can be efficiently controlled by commercial fire ant baits which typically contain corn grit, an ingredient that allows the bait to be easily applied and readily collected by ants. Traditionally it is thought that the corn grit degrades when exposed to rain or irrigation and fire ant control does not occur because the ants do not feed on wet bait. As a result, water-resistant fire ant baits have been developed that either replace or modify the corn grit. Research conducted by an ARS scientist in Gainesville, Florida, and collaborators from APHIS, and the Coachella Valley Mosquito and Vector Control District in California determined that both standard commercial fire ant bait and water-resistant baits that have been soaked in water in the laboratory, as well as exposed to sprinkler irrigation in landscapes, can still effectively control fire ants. Because water-resistant fire ant baits are not readily available in the U.S., knowledge that standard fire ant baits can withstand sprinkler irrigation should allow land managers and groundskeepers more flexibility in scheduling fire ant treatments. 05 Prairie strips benefit pollination and biodiversity in the U.S. Corn Belt. The establishment of strips of restored prairie vegetation within row crop fields is an increasingly popular tool for diversifying agricultural landscapes in the Midwestern U.S. Prairie strips reduce soil erosion, improve water quality, and support native biodiversity. But it is unclear how prairie strips interact with other agricultural practices and how far their benefits propagate into surrounding fields. ARS scientists from Gainesville, Florida, Michigan State University, the University of Puerto Rico, and Hawai⿿i Pacific University addressed this by studying biodiversity and ecosystem services in newly established prairie strips within a 32 year-long agricultural landscape experiment. They found that during the first two years after planting prairie strips, proximity to strips increased dung beetle abundance, spider abundance and richness, soil active carbon, decomposition rates, and pollination rates, and that the effects got stronger over time. Some effects interacted with other agricultural practices, such that fields with both prairie strips and reduced fertilizer and pesticide use had higher butterfly and spider abundance and higher pollination rates. The results demonstrate the potential for prairie strips to rapidly support enhanced biodiversity and ecosystem services while still maintaining crop yields.

Impacts
(N/A)

Publications

• Humprhines, A.R., Ascunce, M.S., Goss, E.M., Helmick, E.E., Bartlett, C.R., Myrie, W., Barrantes, E.A., Zumbado, M.A., Bustillo, A.E., Bahder, B.W. 2021. Genetic variablity of Hapalaxius crudus, based on the 5' region of the cytochrome c oxidase subunit 1 gene shed light on epidemology of palm lethal decline phytoplasmas. Phytofrontiers. 1:127-134. https://doi.org/10. 1094/PHYTOFR-12-20-0048-R.
• Helms Iv, J.A., Smith, J., Clark, S., Knupp, K., Haddad, N.M. 2021. Ant communities and ecosystem services in organic versus conventional agriculture in the U.S. Corn Belt. Environmental Entomology. 50(6):1276- 1285. https://doi.org/10.1093/ee/nvab105.
• Kemmerling, L.R., Rutkoski, C.E., Evans, S.E., Helms Iv, J.A., Cordova- Ortiz, E.S., Smith, J.D., Vasquez-Custodio, J.A., Vizza, C., Haddad, N.M. 2022. Prairie strips and lower land use intensity increase biodiversity and ecosystem services. Frontiers in Ecology and Evolution. 10(833170):1- 18. https://doi.org/10.3389/fevo.2022.833170.
• Oi, D.H. 2020. Seasonal prevalence of queens and males in colonies of tawny crazy ants (Hymenoptera: Formicidae) in Florida. Florida Entomologist. 103(3):415-417. https://doi.org/10.1653/024.103.0318.
• Oi, D.H., Lucky, A., Liebowitz, D.M. 2022. Response of Wasmannia auropunctata, (Hymenoptera: Formicidae) to water-soaked imported fire ant baits. Florida Entomologist. 105(2):108-114. https://doi.org/10.1653/024. 105.0202.
• Valles, S.M., Oi, D.H., Weeks, Jr, R.D., Addesso, K.M., Oliver, J.B. 2022. Field evaluation of Solenopsis invicta virus 3 against its host Solenopsis invicta. Journal of Invertebrate Pathology. 191:107767. https://doi.org/10. 1016/j.jip.2022.107767.
• Valles, S.M. 2021. Diet with sucrose ameliorates Solenopsis invicta virus 3 (Solinviviridae: Invictavirus) infection in Solenopsis invicta (Hymenoptera: Formicidae) worker ants. Florida Entomologist. 104(3):239- 242. https://doi.org/10.1653/024.104.0314.
• Vander Meer, R.K., Chinta, S.P., Jones, T.H., O'Reilly, E.E., Adams, R.M. 2021. Male fire ant neurotransmitter precursors trigger reproductive deveopment in females after mating. Nature Communications. 4:1-11. https:// doi.org/10.1038/s42003-021-02921-5.
• Vander Meer, R.K., Chinta, S.P., Jones, T.H. 2022. Novel alkaloids from the fire ant, Solenopsis geminata. Naturwissenschaften. 109(15):1-6. https://doi.org/10.1007/s00114-022-01786-w.
• Ascunce, M.S., Vander Meer, R.K., Chinta, S.P., Ogura-Yamata, C., Oishi, D. 2022. Genetic and chemical profiling of Solenopsis spp. (Hymenoptera: Formicidae) intercepted in Hawaii. Florida Entomologist. 105(1):91-94. https://doi.org/10.1653/024.105.0114.
• 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.
• Traylor, C.R., Ulyshen, M.D., Wallace, D., Loudermilk, E.L., Ross, C.W., Hawley, C., Atchison, R.A., Williams, J.L., Mchugh, J.V. 2022. Compositional attributes of invaded forests drive the diversity of insect functional groups. Global Ecology and Conservation. 35(1):e02092. https:// doi.org/10.1016/j.gecco.2022.e02092.

Progress 10/01/20 to 09/30/21

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: Maximized telework in response to the pandemic has hindered the Unit⿿s ability to fully complete some milestones for FY 2021. Active research stopped or was reduced on multiple projects associated with: CRIS Project; CRADAs; SBIR funded grants; State funded Projects; APHIS funded Projects; MTRA; NACA Project; Service Contract; University collaborations; and field collections of invasive ants in Argentina, Australia, and Florida for discovery/research of biocontrol agents. Some previously collected samples were lost or degraded and could not be used as planned for future experiments/accomplishments. In addition, the maximum teleworking precluded discovering the failure of two main sample storage freezers (- 20 degree C) for a week. Pathogen standards (protein and nucleic acid), mono and polyclonal antibodies, oligonucleotide primers, and reagents were ruined. Recovery of the losses will take up to 1.5 years once labs reopen. Some antibody samples and reagents were irreplaceable. Maximized telework associated with COVID-19 has negatively impacted the research and work output of two new SYs that were on-boarded in January 2021. Administrative delays associated with maximum telework and the restrictions associated with limited building occupancy have hindered their ability to conduct office and lab work. This has made it difficult to inventory and purchase supplies, to organize workspaces, and to plan new experiments, and have made it impossible to perform lab and field work for either pre-existing or new projects. It is estimated that the pandemic has delayed progress in starting new research by at least six months. Both permanent and temporary new hires have been hampered by lack of hands-on training and in-person teamwork. Studies take much longer to start and complete with the reduced staffing and physical distancing. When COVID restrictions ease and more normal research activity resumes, full attainment of research objectives will still be delayed as operations and equipment must be restarted, recalibrated, and returned to pre-COVID levels. ACCOMPLISHMENTS 01 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. 02 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. 03 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.

Impacts
(N/A)

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. 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. 90:738-750.
• 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. 103:415-417.
• 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.

Progress 10/01/19 to 09/30/20

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Objective 1: Commercialization of a Red and Black Imported Fire Ant detection device was completed, and the kit should be available to stakeholders by late 2020. The device provides rapid identification of invasive fire ants and will be sold. The device provides a tool for regulatory agencies in the United States and other countries to enforce quarantine protocols. This progress significantly improves surveillance by detecting both quarantined fire ant species in a single, on-site test. ARS researchers at Gainesville, Florida, have started research on a pheromone enhanced sticky trap fire ant surveillance system. Of twelve available sticky traps commercially available, only three successfully caught fire ant workers. ARS researchers at Gainesville, Florida, are now evaluating prototypes of a unique pheromone release system that controls the release rate and focuses the flow of pheromone vapor to the surface. Commercial pheromone traps target flying pest insects; however, target fire ants walk on/in a complex 3-dimentional surface, not addressed in currently available pheromone traps. Solenopsis Invicta Virus-3 (SINV-3) is a natural control agent of imported fire ants that was successfully released in areas devoid of the pathogen, in California and Florida. Fire ants were exposed to either a bait or drench formulation containing purified virus. The pathogen was detected 3 and 6 months later in the treated areas, but not in adjacent control areas. Treated areas are being evaluated for infection sustainability and impact on the population. Four releases were conducted in Tennessee, but transmission and establishment never occurred. Further examination revealed a unique isolate of SINV-3 that may have co-evolved with the hybridization of the red and black fire ant species. Characterization of the new isolate, named Solenopsis Invicta Virus-3 Hybrid (SINV-3 Hybrid), is underway to determine traits of the virus that confer its host specificity. Water resistant and standard fire ant bait formulations are being evaluated on sprinkler irrigated sod. This bioassay emulates nursery and landscape irrigation situations, which reportedly degrade the moisture sensitive fire ant baits, thereby decreasing bait efficacy. Laboratory studies compared the efficacy of piled versus scattered/broadcast applications of standard fire ant bait under irrigation conditions. Significant differences between the two treatments were not found. This result was unexpected because piled baits were hypothesized to be better protected from irrigation. A patent has been issued on new fire ant active ingredients (AIs). These compounds effectively controlled colonies under laboratory conditions. ARS researchers at Gainesville, Florida, have successfully increased AI acceptability using pharmaceutical masking agents. The AIs have shown promise in controlling other pest insects (patent pending). Objective 2: 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. Techniques are being developed to retard the desiccation of the liquid bait and extend the availability of bait to tawny crazy ants under field conditions. Samples of the little fire ant were obtained from Florida, Hawaii, and Argentina to begin a survey of pathogens associated with this invasive ant for potential use as biocontrol agents. This ant is a problem in Hawaii, Guam, Australia, Israel, and many Pacific Island countries and territories. If eradication is not possible, biological control would be the only sustainable control strategy for little fire ants where they are well established. Procedures were developed to dissect little fire ant workers� Dufour�s and venom glands. Preliminary identification of chemical components has started. A bioassay has been developed to assess the effects of these extracts on workers. Accomplishments 01 Natural spread of a virus in imported fire ants. Introduced from South America, the red imported fire ant currently infests over 128 million hectares of land in the United States and is estimated to cause damage exceeding $7 billion annually. Solenopsis invicta virus 3 (SINV-3) is an RNA virus specific for red imported fire ants that offers promise as a natural control agent. Scientists from Gainesville, Florida, and Florida A & M University conducted surveys to determine the prevalence of SINV-3 in winged female fire ants to understand the possible natural spread of the virus through mating flights. Collections were made from five urban areas and five adjacent rural areas of north Florida. SINV-3 was detected in winged females in nests from 7 of the 10 collection locations. The average infection rate of 44% was similar in rural and urban areas. Winged females were sampled because they mate aerially and disperse, founding colonies in new areas. Infected winged females may be the mechanism of SINV-3 spread throughout the fire ant community providing additional sustained control of fire ants in the U.S. 02 A super colony of invasive ants in Florida. The tawny crazy ant is an invasive ant from South America that infests Florida and Texas and is spreading to states along the Gulf Coast. Extremely large populations of this ant inundate urban and natural landscapes resulting in mass intrusions into buildings as well as reductions in biodiversity. Researchers in Gainesville, Florida, determined that tawny crazy ants did not fight with other tawny crazy ants from different nests located at the same site as well as nests located as far as 270 miles away. In fact, small fragments of colonies, including queens, from distant nests congregated together in the same nests in laboratory tests. This lack of aggressive behavior between widely separated colonies indicated that tawny crazy ants are not territorial over large areas. This supports the contention that tawny crazy ants in Florida are part of a super colony across the southern U.S. Lack of territoriality allows for the sharing of resources and movement of worker ants and brood between colonies, which can facilitate the spread of natural enemies such as pathogens and toxic baits being developed for their control.

Impacts
(N/A)

Publications

• Valles, S.M., Strong, C.A., Emmitt, R., Culkin, C., Weeks, R.D. 2020. Efficacy of the InvictDetectTM Immunostrip� to taxonomically identify the red imported fire ant, Solenopsis invicta using a single worker ant. Insects. 11(1):37.
• Valles, S.M., Firth, A.E. 2020. Solinviviridae. Encyclopedia of Virology.
• Oi, D.H., Valles, S.M., Porter, S.D., Cavanaugh, C., White, G., Henke, J. 2019. Introduction of fire ant biological control agents into the Coachella Valley of California. Florida Entomologist.
• Lawson, K.J., Oi, D.H. 2020. Minimal intraspecific aggression among tawny crazy ants (Hymenoptera: Formicidae) in Florida. Florida Entomologist. 103(2):247-252.
• Cha, D.H., Skabeikis, D.D., Collignon, M.R., Siderhurst, M.S., Choi, M.Y., Vander Meer, R.K. 2019. Behavioral response of little fire ant, Wasmannia auropunctata (Hymenoptera: Formicidae), to trail chemicals laid on epiphytic moss. Journal of Insect Behavior. 32:145-152.
• Ahn, S., Corcoran, J., Vander Meer, R.K., Choi, M.Y. 2020. Identification and characterization of GPCRs for Pyrokinin and CAPA peptides in the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae). Frontiers in Physiology. 11:559.