1a. Objectives (from AD-416):
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. 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. 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. 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:
Previous investments in developing genomic resources (Objective 1) are beginning to advance the understanding of gene-specific control of sociality and other genetic bases contributing to the success of fire ants. Specifically, under sub-objectives 1.1 and 1.3, a supergene (Gp-9) was identified and found to be associated with male reproductive success; evolution of this gene’s expression was determined to largely occur in the worker caste, and genome sequencing and analysis led to the discovery of the first circadian clock genes in any ant species. Studies also provide the first examination of male fire ant fitness and its contribution to social organization and colony success. Under objective 2, we made significant progress in the discovery and development of ant pathogens as control agents. At least three new viruses were discovered in the invasive Caribbean crazy ant (also known as the Rasberry Crazy ant) and 6 viruses in fire ants were studied for possible development for as biopesticides or biological control agents. Host specificity was established for a virulent virus (SINV-3) and shown to only kill invasive fire ants, which advances development of this pathogen as a biopesticide. The recently released small decapitating fly (Pseudacteon cultellatus) was successfully established in the U.S., bringing the total number of these fire ant parasites to five; all of these species were identified, reared, released, and established in the U.S. by our scientists. The large decapitating fly (Pseudacteon obtusus) was found to interfere with foraging activities making food collection for fire ants more difficult. Thus, collective efforts at pathogen/parasite discovery, study, establishment and release have resulted in a number of self-sustaining control agents in the U.S. These discoveries also satisfy sub-objective 4 by providing new strategies for Intergrated Pest Management control of these ants. Under objective 3, a prototype fire ant specific surveillance device was developed that will remotely attract and photograph fire ants in various quarantine situations. Also, a method was devised to rapidly and accurately identify fire ants, wherever they are found, e.g. state quarantine boundaries. These technologies provide regulatory agencies (APHIS and EPA, Homeland Security) with rapid and reliable methods for determining whether agricultural/horticultural products are contaminated with fire ants, thus enhancing our ability to limit their spread into non-quarantined areas of the U.S. and elsewhere. Further progress on objective 3 focused on elucidating the function of a particular gene that produces neuropeptides. RNA interference experiments demonstrated dramatic phenotypic effects for immature and adult life stages, as well as regulation of pheromone biosynthesis. These discoveries may lead to novel control methods for fire ants as detailed in two patent applications filed for an RNA interfering technology to silence the above gene.
1. Potential biological control agents discovered in the invasive, Rasberry Crazy ant. Current control measures for the Rasberry Crazy ant are limited to insecticides. However, ARS researchers at Gainesville, Florida, in cooperation with scientists at the University of Florida, Gainesville, FL, have discovered several viruses (based on gene homology) with potential to control this invasive ant pest in the United States. These viral discoveries represent the first opportunity for biologically based control agents for the Rasberry Crazy ant.
2. Field detection kit developed for fire ants. By law, regulatory personnel in the United States must prohibit movement of any fire ant-contaminated shipping commerce from entering non-quarantined areas. Unfortunately, cargo must be held until positive identification of the ant samples in the cargo is made. The current identification process requires extensive training of personnel and can delay movement of cargo by days. ARS researchers in, Gainesville, Florida, have developed a rapid field detection kit capable of identifying fire ants. The new technology accelerates the identification process at interception points such as quarantined boundaries and ports, is easy to use, requires no previous training to make a positive identification for fire ants, and is completed in 10 minutes.
3. Supergene Gp-9 is associated with polyandry and male reproductive success in fire ants. Understanding fire ant male reproductive success and fitness are important components of research aimed at suppressing fire ant populations, yet data on this important topic are virtually nonexistent. To begin to fill this gap in current knowledge, ARS researchers in Gainesville, Florida, conducted a study aimed at determining how commonly, if ever, fire ant queens mate with more than one male. This study revealed that some fire ant queens mate with more than one male and, unexpectedly, that whether a queen mates with more than a single male is determined almost entirely by male genotype. Investigation of the physiological basis for the inability of some males to discourage a second mating revealed that male sperm count also is linked to male genotype, suggesting fire ant queens remain receptive to mating if their first partner does not provide a sufficient quantity of sperm. This groundbreaking study provides a first view of the fitness of male fire ants and establishes how deleterious variation with respect to male fitness can be maintained as a consequence of being linked to a group of genes that harbor adaptive variation controlling colony social organization.
4. Fire ant pheromone biosynthesis regulated by neuropeptide. Pheromone communication research in insects has advanced rapidly over the last half-century. However, the physiological mechanism for pheromone biosynthesis has only been determined for some moth sex pheromones, and involves the Pheromone Biosynthesis Activating Neuropeptide (PBAN). After >20 years, ARS researchers in Gainesville, FL have determined that PBAN also controls fire ant, Solenopsis invicta, trail pheromone biosynthesis. Multiple experimental procedures have corroborated the conclusions. Extension of PBAN’s pheromone target insect (ants), behavioral type (recruitment), and biosynthetic pathway (isoprenoid), will stimulate additional research and provide leads to novel control methods for pest insects.
5. Fire ant decapitating fly established at two sites in Florida. 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 have confirmed the establishment of the new phorid decapitating fly Pseudacteon cultellatus near Miami, FL and in Gainesville, FL where it is beginning to expand out of the release area. 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.
6. New virus (SINV-3) is virulent and host specific to imported fire ants. Fire ant population densities in their South American homeland are 5-10 times lower than in the U.S. Pathogens are suspected to be major regulators of fire ants in South America. ARS researchers in Gainesville, FL have discovered that a fire ant virus (SINV-3) is both highly host specific and virulent to fire ant colonies. These results suggest that SINV-3 has considerable potential for use as a highly specific biopesticide in organically grown crops where fire ants are frequently pests. Furthermore, it may be able to be released as a classical or self-sustaining biocontrol agent in California and other areas where it does not yet occur.
7. Large fire ant decapitating fly competes well against previous species. Fire ant decapitating flies are common parasites of fire ants in South America where fire ant populations are low compared to in the U.S. Some species of decapitating flies attack little fire ants and others attack bigger fire ants. ARS researchers in Gainesville, FL report that a recently released large decapitating fly (Pseudacteon obtusus) competes well against two previously released species. This species is particularly successful at parasitizing larger fire ants while they forage for food. Its release and successful establishment in the U.S. will make it harder for imported fire ants to collect food for their colonies.
8. Ant baits kill invasive ant colonies. The Caribbean crazy ant, Nylanderia cf. pubens, is a recently introduced invasive pest that develops huge populations that overrun landscapes and has resulted in excessive applications of insecticide. Ant baits are a more efficient and environmentally compatible method of ant control. ARS researchers in Gainesville, FL, conducted laboratory evaluations of ant baits against the Caribbean crazy ant and identified three commercial baits that consistently kill small colonies within 3 weeks. Field treatments with two of the baits in combination with the marking of foraging ants indicated that the movement of crazy ants from untreated areas can potentially mitigate the impact of these baits, unless baits are constantly accessible. These data will influence procedures for Caribbean crazy ant control.
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