1a. Objectives (from AD-416)
Objective 1: Develop functional genomic resources and employ these resources to examine the genetic basis of biological traits that can potentially be used for biologically based control, including implications for the geographic origins of infestations. Objective 2: Expand current biocontrol efforts by discovering and developing new parasites and pathogens; improving mass culture and field release systems; and defining host specificity of natural enemies. Objective 3: Characterize semiochemicals through investigation of pheromone biosynthesis and release; use these findings to develop novel biologically-based control and surveillance methods, including the detection and treatment of incipient or low level populations. Objective 4: Develop integrated pest management plans that utilize available control methods, perform comprehensive risk assessment, and that can be adapted to specific stakeholder needs, including local eradication.
1b. Approach (from AD-416)
1. Normalized cDNA libraries will be sequenced using 454-pyrosequencing technology. Resulting sequences assembled and automatically and manually annotated for key words, gene function, and Gene Ontology terms. The 454 data will be used to search for potential microbes infecting fire ants and determine their nature, distribution, relationship, and effects of these microbes in fire ants. SNPs within the data will be identified and applied to high-resolution identification of the source population of introduced S. invicta. Microarrays will be constructed and used to identify differentially expressed genes from parasite-infected and -uninfected fire ants and identify genes co-expressed with the social form-specific gene Gp-9 allelic variants. 2. Microsporidia: After approval, V. invictae will be released in the U.S. following the procedures for K. solenopsae introductions. Phorid Flies: Additional decapitating phorid flies will be released using procedures based on our previous successful releases in the U.S. The impact of phorid flies will be assessed by multiple methods, e.g. monitoring the establishment, expansion, distribution and parasitism rates of phorid fly species across the fire ant range. Viruses: Baculovirus expression methodology will be emulated in order to express Solenopsis invicta viruses (SINV-1, -2 or -3) for study and development as a microbial pesticide. With purified preparations of SINV, a number of basic studies will be conducted, including lethal dose evaluations against different stages of ants, transmission studies within and between colonies, field-testing, and formulation development. Other potential biocontrol agents will be investigated as time and importance dictate. 3. The function of the fire ant PBAN/pyrokinin family of neuropeptides will be investigated by: a) in vivo injection into female and male sexuals and immatures and observed for phenotypic changes; b) manipulation of hormone/receptor capabilities through the use of agonists, hyper-agonists, and antagonists of the neuropeptide hormones; and c) use of RNAi gene knockout methods – followed by monitoring for phenotypic and/or behavioral changes. Monitoring and surveillance methods will be developed for fire ants, using known attractants. A variety of existing fire ant bioassays will be adapted and applied to non-fire ant invasive pest ant species to generate the tools to create better baits and effective monitoring systems, e.g. attractants and repellents. 4. Potential geographic range expansion of tramp ant species will be modeled using CLIMEX a program that can be used to predict where a pest ant of interest can survive. Phagostimulants will be studied to improve the acceptability of baits for non-Solenopsis pest ants by adapting methods used for fire ants. Standard laboratory colony tests will be used to assess bait formulation effects on brood volume, adult populations, and queen survivorship. A combination of monitoring tools, baits, and biologically based-control methods will be applied to selected invasive ant species as these new tools become available.
3. Progress Report
The little decapitating fly, Pseudacteon cultellatus, was approved for field release as a self-sustaining biocontrol agent of imported Solenopsis fire ants. A colony of these flies was transferred to the USDA-APHIS mass rearing facility (operated by the Division of Plant Industries, Florida Department of Agriculture, Gainesville, FL) along with the necessary training and support. Unit personnel also began field releases of P. cultellatus on fire ant populations with multiple-queen colonies around Gainesville and Miami, Florida. Multiple-queen populations produce smaller workers and are thus ideally suited as hosts for these very small parasites. First-generation field-reared flies have already been recovered. Fire ant decapitating flies that develop in fire ants infected with the fire ant pathogen Kneallhazia (=Thelohania) solenopsae, can acquire the pathogen. Infected fly tissue could be deleterious to the flies, which are biocontrol agents of fire ants, or infected flies may be capable of vectoring the pathogen to other fire ants. Thus far, there is no evidence of infection in fly tissue, but research is continuing. Kneallhazia solenopsae infection was confirmed in the tropical fire ant, Solenopsis geminata, indicating this pathogen has a broader host range than previously reported. Using samples of the tropical fire ant provided by cooperators from the University of Texas and universities in northern and central Mexico, two distinct molecular variants of K. solenopsae were discovered. Analyses are also being conducted on K. solenopsae infecting the black imported fire ant from different locations in Argentina. Solenopsis invicta virus 3 is associated with significant fire ant colony mortality. The virus genome was cloned and inserted into an expression vector in an attempt to grow the virus in culture. This is a crucial requirement for industry adoption of the virus as a natural pesticide. Interest in the technology was expressed from the Center for Innovative Food Technology, Ohio. An assessment of genetic variation at a diverse set of molecular markers in 2,144 colonies from 75 geographic sites worldwide revealed that at least nine separate introductions of S. invicta have occurred into newly invaded areas and that the main southern USA population likely is the immediate source of all but one of these introductions. The sole exception involves a putative serial invasion event from the southern USA to California to southern Taiwan. RNA interference of the expression of a specific target gene was shown to have deleterious effects on fire ant larvae. This result follows a great deal of basic research that now shows potential for the development of a novel biologically-based fire ant control method. A patent application is being prepared for submission. A laboratory bioassay was developed to evaluate the efficacy of ant baits on the Caribbean crazy ant, Paratrechina pubens, which will facilitate the development of more effective control strategies for this invasive species.
1. Mass Rearing and Field Release of a New Fire Ant Biocontrol Agent. Imported fire ants are unusually abundant in the United States, probably because they have escaped their natural enemies left behind in South America. ARS researchers in Gainesville, Florida obtained approval from the North American Plant Protection Organization (NAPPO) and USDA-APHIS regulators to release a new species of phorid decapitating fly (Pseudacteon cultellatus) as a fire ant biocontrol agent. This new species of fly specializes on attacking the smallest sizes of fire ant workers, which are most abundant in multiple-queen fire ant colonies. This preference is especially important because multiple-queen fire ant populations average 2-3 times the densities of regular single-queen fire ant populations and are therefore a substantially greater pest of homes, agriculture, and the environment.
2. Source of invasive fire ants guides search for biological control agents. Knowing the source of invasive fire ant infestations in the United States and other countries, e.g. Australia, China, and Taiwan, is important in guiding the search for biological control agents and/or whether biocontrol agents used in the United States would be effective in other countries with this invasive ant. ARS Researchers from ARS, Australia, and Taiwan assessed the genetic variation at a diverse set of molecular markers in 2,144 colonies from 75 geographic sites worldwide. The results revealed that at least nine separate introductions of S. invicta have occurred into newly invaded areas and that the main southern USA population likely is the immediate source of all but one of these introductions. The sole exception involves a serial invasion event from the southern USA to California to southern Taiwan. These results suggest that the probability is high that biologically-based fire ant control methods developed by ARS Researchers can be transferred to invasive populations in the other countries in this study.
3. Alarm pheromone accumulation varies in queens during life of colony. The function of the neuropeptide PBAN (pheromone biosynthesis activating neuropeptide) in fire ants has not been established, although preliminary data suggests that it may be involved in alarm pheromone production. It is essential to understand the alarm pheromone biosynthesis and utilization (accumulation) to effectively design experiments to determine the role of PBAN in alarm pheromone production. ARS Researchers studied the accumulation of a pyrazine alarm pheromone component from female sexual alate to newly mated queen, and through development of a mature colony. Periods of slow, rapid and no accumulation were discovered. These data will be used to determine if pheromone accumulation can be manipulated using PBAN or antagonists of the PBAN receptor. Interference of this peptide or the gene that produces it could lead to novel, non-insecticide methods for fire ant control.
4. Fire ant pathogen infects a North American fire ant. Kneallhazia solenopsae, a pathogen of Solenopsis invicta was anecdotally reported to also infect the tropical fire ant, S. geminata. It is important to know the specificity of this pathogen. ARS researchers in Gainesville, Florida confirmed K. solenopsae infection in S. geminata, indicating this pathogen has a broader host range than previously reported. Using samples of the tropical fire ant provided by cooperators from the University of Texas and universities in northern and central Mexico, two distinct molecular variants of K. solenopsae were discovered. The presence of variants of K. solenopsae suggests the potential of more effective strains of fire ant pathogens that can be used for biological control. The tropical fire ant is an invasive pest in Hawaii and other parts of the world.
5. Fire ant virus genome sequenced. Characteristics of the fire ant virus, SINV-3, were unknown from the native fire ant population in Argentina. In an effort to understand different geographic variants, ARS scientists in Gainesville, Florida, and Buenos Aires, Argentina sequenced the SINV-3 genome in entirety from an Argentinean source and compared it with the genome sequence found in US populations. The Argentinean variant had a different genomic architecture and may exhibit different virulence levels compared with the US variant. This information will facilitate selection and development of the most virulent strain for use in fire ant control in the US.
Allen, H.R., Valles, S.M., Miller, D.M. 2010. Characterization of Solenopsis invicta (Hymenoptora: Formicidae) populations in Virginia: Social form genotyping and pathogen/parasitoid detection. Florida Entomologist. 93(1): 80-88.