Objective 1: Develop advanced integrated pest management methods by improving the understanding of fire ant biology and by expanding biologically-based control of fire ants through detailed genetic, behavioral, physiological, chemical, and ecological studies of fire ants and their natural enemies. a. Employ metagenomics techniques and biological control prospecting to discover additional natural enemies of introduced fire ants. b. Characterize the genetic architecture of the Gp-9 supergene involved in regulation of fire ant colony social form. c. Develop natural enemies of fire ants as classical biological control agents or biopesticides by evaluating their effectiveness, determining host specificity, developing methods for rearing and release, and formulating more effective biopesticides. d. Develop novel biologically-based fire ant control by identifying the behavioral and semiochemical underpinnings of fire ant mating flights and colony establishment. Objective 2: Develop advanced integrated pest management methods by improving the understanding of the biology of invasive pest ants other than fire ants and by expanding options for their management and surveillance. a. Improve control of tawny crazy ants: 1) refine integrated management strategies; 2) evaluate natural enemies; and 3) determine whether crazy ant semiochemicals can be used to enhance baits and improve surveillance/detection methods. b) Develop or improve control methods for other important invasive ants (e.g., Argentine ant, little fire ants) through evaluation and consolidation of current or new control methodologies. c) Establish a collection database and repository for fire ants and other pest ants to facilitate discovery of natural enemies, genetic studies, and taxonomic identifications. Objective 3: Determine impacts of climate and climate change on potential distributions of invasive ants.
1. a) Fire ants (Solenopsis invicta) from the native range will be collected and used as source material to create cDNA expression libraries. Detailed bioinformatics analysis of resulting sequence data will be screened to identify potential fungi, viruses, protists, and non-hymenopteran eukaryotic parasites. North American fire ant colonies will be exposed to fire ants collected from South America and observed for signs of pathology. These colonies will be examined using various molecular analyses and microscopic methods to determine the etiological agent. b) A linkage map will be developed to identify all of the genes in the Gp-9 non-recombining region. Linkage disequilibrium between the Gp-9 genes and social form will be estimated with several different statistical methods. Products and functions of the genes comprising the Gp-9 supergene will be inferred by bioinformatic analysis. c) Natural agents will be evaluated for their suitability as control agents against U.S. populations of the fire ant by establishing their host specificity, mode of dissemination (formulation), efficacy, virulence, mode of action, mass rearing, and field release. d) The role of semiochemicals in fire ant biology will be established and possibly exploited as a control agent by exposing colonies and/or individual ants to extracts or synthetic chemicals and recording behavioral changes. 2. a) Effective and alternative control methods will be investigated for the tawny crazy ant by treating infected areas with soil applied systemic insecticides or lures and evaluating for efficacy. The transcriptome of the tawny crazy ant will be sequenced and examined for the presence of potential natural enemies. Promising potential natural enemies, including the tawny crazy ant virus, will be tested to determine efficacy and safety. Seasonal phenology of tawny crazy ant colonies will be established to better direct control efforts by excavating nests monthly and quantifying different stages. b) For tawny crazy ants and other invasive pest ants, e.g. Argentine ant little fire ants, the contents of well-developed ant exocrine glands will be chemically identified and subjected to behavioral bioassays to determine the effect of pheromones on ingestion of baits, bait discovery, field efficacy evaluations, and the effective longevity of attractant/bait formulations. Where attractive pheromones have not been already identified, a Y-tube olfactometer bioassay will be used to isolate and identify active compounds. c) A pest ant database and repository will be assembled using existing electronic data and specimens from labs across the country. Maps for existing pest ant collections will be generated and used to guide future collection efforts as needed. Future specimens and collection data will be systematically incorporated into the repositories. 3. Climate matching protocols in Climex 3.0.2 (Hearne Software, Victoria, Australia) will be used to predict potential future ranges of 15 exotic pest ants. Distributional data will be categorized as rural and urban with extreme outliers noted and eliminated when appropriate (e.g., detection in green houses).
Objective 1: Fire ant Integrated Pest Management. A long-standing problem has been identification of fire ants at quarantine boundaries. Through an inter-agency agreement between Agriculture Research Service in Gainesville, Florida, and APHIS in Biloxi, Mississippi, a prototype lateral flow immunoassay (technology used in home pregnancy tests) capable of rapidly identifying red imported fire ants from all other ant species was developed. The test requires no special training or equipment, is portable, and provides results in 10 minutes at the point of need. Agdia®, Inc. acquired a biological material license to commercialize the technology and it has been marketed under the trade name InvictDetect™ since Jan. 2018. Used by U.S. quarantine inspectors, the kits are anticipated to be utilized by biosecurity agencies in Australia, New Zealand, New Caledonia, Japan, South Korea, and other countries trying to prevent fire ant invasions. Additional research has expanded the kit’s ability to detect another invasive, the black imported fire ant. The kit can discriminate both species in a single reaction and negotiations are underway to commercialize the expanded kit. Evolutionary Genomics. The social form trait (having one vs. many queens per colony) was used to examine the gene flow between the red and the black, imported fire ants. Using mitochondrial and microsatellite genetic markers, it was shown that social form does not impact hybridization rates or gene flow between the two invasive species in the U.S. Analyses of genome sequence data of numerous males suggest the Gp-9 supergene has remained intact across several ant species, with no evidence for recombination. A study was initiated to investigate the possibility of segregation distortion. Such distortion is predicted as another selfish genetic trait of the supergene that favors its persistence in wild populations despite its detrimental effects on individual ants. Behavioral studies also were initiated to determine whether the supergene also controls social organization in a distantly related fire ant. The scientist conducting the genomics studies resigned from the agency in August 2016. Biological Control. Fire ant biological control using parasites is already a success story for the unit, as six, self-sustaining phorid fly parasites of fire ants are well established and spreading naturally throughout the southern USA. In 2014, the unit released phorid flies in California and documented their establishment. Particularly notable, is that two phorid fly species were able to establish in extremely hot and dry, but irrigated, urban desert habitat. Cooperating with the Univ. of Texas, ARS scientists in Gainesville, Florida, have been able to collect and mass rear phorid flies from native tropical fire ant populations in Texas. Host specificity studies showed that this species will not be a threat to any native ants found in the Pacific or Pacific Rim nations. However, we demonstrated that the Texas phorid species is too host specific because it is not sufficiently compatible with invasive tropical fire ant populations on Hawaii and Guam to justify field releases there. These results are important because they demonstrate the need to search for natural enemies from native tropical fire ant populations closely related to those in the Pacific. Metatranscriptomics. The ARS laboratory in Gainesville, Florida has pioneered the use of metatranscriptomics and next generation sequencing to accelerate prospecting for virus pathogens in ants. Using these methods, 12 new viruses were discovered in red imported fire ants bringing the total to 15 viruses. These discoveries provide the best-characterized virome of any ant species. Fourteen of the viruses have RNA genomes and the other has a DNA genome. Nine of these RNA viruses appear to be found exclusively in Argentine populations of red imported fire ants and, therefore, are potential classical biological control agents. These viruses remain to be examined for their suitability as control agents. Among the well- characterized viruses, SINV-1, -2, and -3, all have been shown to negatively impact fire ant growth, survival and/or reproduction. However, Solenopsis invicta virus 3 (SINV-3) exhibits the most significant impact on fire ant populations. Commercial interest is high for SINV-3, but an inability to mass produce the virus have hampered its commercialization. Field release techniques were developed for SINV-3, including bait and drench formulations used in California, where the virus was successfully established. The SINV-3 dose necessary to establish a lethal infection in laboratory colonies of fire ants was also established. These data are necessary to successfully use the virus as a biopesticide and for releasing the virus as a classical biological control agent. Semiochemicals. The major chemical constituents of the male reproductive system were defined qualitatively and quantitated. Large amounts of free fatty acids (FFA) were found throughout the male reproductive system, as well as in the uninseminated female. These FFAs are known to have both insecticidal and strong antibiotic activity, and their role in reproduction and colony establishment is under investigation. The role of other compounds specific to males has been investigated through a CRADA. Interference of the mating or premature queen development within colonies would decrease the reproductive potential of fire ants. Fire ant queens inhibit the development of their sexual daughters through release of a primer pheromone. Fire ant primer pheromones have not been identified because the effects of a primer pheromone may not be observable for many weeks. ARS scientist in Gainesville, Florida. have developed a bioassay that will reliably provide an actionable result within one week. This development will drive the isolation and identification of the first fire ant primer pheromone. Fire ant queens will mate only once, yet these females fly into a waiting aggregation of fire ant males, which presents multiple mating opportunities. It was discovered that male specific compounds are transferred to females during mating that are highly repellent to males, thus inhibiting multiple mating. Additional progress has been made in understanding the biochemical processes associated with physiological changes that take place soon after mating and could lead to novel biologically-based control methods that disrupt queen behavior. A preliminary investigation of sticky traps for fire ant detection resulted in only 3 of 15 trap designs catching fire ant workers, which suggested large variability in sticky trap performance with ants. Objective 2: The first viral pathogen of the tawny crazy ant (NfV-1) was discovered, characterized, and patented as a potential control agent. Nfv-1is easily transmitted to uninfected colonies by baiting. NfV-1 infects all life stages but only replicates in the larvae. Host specificity tests showed NfV-1 only infects tawny crazy ants. The virus was evaluated for its potential as a biological control agent by comparing egg-laying rates of infected and uninfected queens. Unfortunately, egg laying by infected queens were not significantly lower than uninfected queens. Significant progress was made in identifying a bait that is effective against the tawny crazy ant. A liquid bait was readily consumed and caused significant laboratory colony mortality. Field tests demonstrated overnight suppression of tawny crazy ant activity, which was sustained, so long as bait was available. Bait dispensed in small stations places around nesting habitat, such as tree bases, and around a home, reduced the amount of insecticide applied by over 99% when compared to a single, standard insecticide spray applied to the house perimeter. Ant reductions were significant, visually apparent to the homeowner, and especially notable since it occurred during the ant’s summer population explosion. The seasonal phenology of tawny crazy ant queen reproduction was determined by monthly sampling of queens and ovary dissections. Mature eggs were present in ovaries throughout the year, however, fewer queens were collected during the winter. This suggested that winter treatments may be an opportune time to eliminate queens before they proliferate in the spring and summer. Extensive liquid bait deployments from late winter through fall 2018 significantly reduced field populations of the tawny crazy ant. The ants have yet to return in the 2019 summer despite the suspension of baiting. Little fire ants, an invasive ant plaguing Hawaii and Guam, exposed to standard imported fire ant bait that was soaked in water resulted in the elimination of laboratory colonies. Six other fire ant baits soaked in water were foraged upon by little fire ants in the field. This suggested that wet fire ant bait may be effective in wet climates. Collaboration with an ARS researcher in Hilo, Hawaii, on the little fire ant has led to the development of a bioassay aimed at guiding the isolation and identification of the recruitment pheromone which facilitate the development of enhanced baits and surveillance systems. Ant repository. ARS scientists in Gainesville, Florida, assembled a core team from four institutions and established a general framework of how to catalogue and store extensive collections of pest ants. More than 12,000 specimens have been databased, geolocated, and properly labeled. More than 2,000 ant collections from the Fundación para el Estudio de Especies Invasivas in Argentina have been catalogued. An online database will be tested for the joint use of core teams and another online database (AntWeb) for general publication and mapping of collection records. These efforts will facilitate discovery of natural enemies, genetic studies, taxonomic identifications, and baselines for climate change data. Objective 3: Scientists leading Objective 3 departed ARS in August 2016 and January 2018.
1. The male fire ant transfers more than sperm to the female. Within a fire ant colony, the mother queen inhibits reproductive development in her winged female offspring (unmated queens) with a pheromone; however, after mating the winged females need to very quickly start laying eggs for their new colony. ARS scientists in Gainesville, Florida, discovered that male fire ants transfer compounds to the winged females during mating that rapidly initiate egg production and other biological characteristics of a mother queen. This discovery shows how male fire ants help winged females overcome the effects of their mother’s pheromone. This discovery reveals potential pathways to disrupt fire ant reproduction which can lead to novel control methods for this invasive species that causes over $6 billion in annual damage and control costs in the United States.
2. New ant detection kit in commercial development. The red and the black imported fire ant are invasive species that cost over $6 billion annually in damage and control measures in the United States. There is a Federal Imported Fire Ant Quarantine that requires commodities to be free of both species before leaving the quarantine. Scientists from ARS in Gainesville, Florida, and from APHIS in Biloxi, Mississippi, developed a simple to use and portable identification kit (like a home pregnancy test) that can identify both the red and black imported fire ant species from all other ants in a single, 10-minute test. The speed of test eliminates extended shipping delays by bypassing the need to send off samples for identification. Being able to distinguish the black imported fire ant is an important improvement over the previous kit the scientists developed (InvictDetect™) which detected only the red species. The kit provides a new tool for regulatory agencies in the United States and other countries to enforce quarantine protocols and limit the spread of these invasive ants. The device is being commercially developed by Agdia, Inc.
3. Nine new viruses discovered in fire ants from South America. Imported fire ants were accidently introduced into the United States in the 1930s and currently infest over 365 million acres. They cause over $6 billion in damage and control costs annually in livestock and agricultural production, buildings and landscapes, and pose a serious threat to human health. ARS researchers at Gainesville, Florida, have discovered nine new viruses in fire ants from South American, and two of the viruses exhibit unique genome structures which suggest that they may be a new category of viruses. The nine viruses have not been detected in U.S. and potentially could be released into the U.S. fire ant population as biological control agents. Biological control agents spread naturally and are self-sustaining. If effective at reducing populations, biocontrol agents are considered the most efficient and environmentally compatible control method for well-established invasive pests like fire ants.
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