2004 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Biting and filth breeding flies transmit bacterial, viral, protozoan, and filarial disease agents to animals, the general public and US military personnel. Annoyance and irritation from mosquito and filth breeding fly bites impedes agricultural productivity, economic development, and the use of recreational areas. Effective and environmentally safe alternative control technologies are needed to replace chemical pesticides and to protect animals and humans from arthropod attack and the transmission of arthropod-borne disease agents. To meet this need, ARS-CMAVE scientists in the Mosquito and Fly Research Unit develop biologically-based alternative controls, personal/animal protection tools and new materials and methods for surveillance that can fit into an integrated pest abatement system to safely and effectively manage biting and filth breeding flies. Over one million pounds (active ingredient) of pesticide are used each year in the USA to protect animals and humans from biting and filth inhabiting insects. These pesticides are the only tool available for emergency vector control and are the mainstay of conventional integrated pest management (IPM) systems in animal and public health. Within the next decade, however, pesticide use will be severely restricted by ecological, safety, legal, and economic factors. Alternative control technologies for biting and filth inhabiting arthropods are needed now to maintain our current pesticide-based capacity for control of these pests and disease vectors in the future. This research is entirely (100%) within the scope of National Program 104, Veterinary, Medical, and Urban Entomology Research. The CRIS addresses the following National Program Action Plan Goals: 3.1 Genomics and Host-Pathogen Interaction (3.1.3); 4.1 Chemical Pesticides and Repellents (4.1.3); 4.2 Biological Control (4.2.1); and 4.3 Area-wide Control (4.3.1). These goals represent efforts to provide self-sustaining, non-pesticidal, biologically-based control methods that would be safe and effective in urban, agricultural and natural ecosystems for a serious medical, veterinary, and agricultural pest. Thus, the research problems addressed by this CRIS support the Pesticide Reduction Act through the development of integrated pest management technologies, as well as the Food Quality Protection Act of 1996 through the development of reduced-risk control strategies. Collaborators in this research include the Armed Forces Pest Management Board of the Department of Defense, the U. S. Agency for International Development, the World Health Organization, and the International Atomic Energy Agency. 2.List the milestones (indicators of progress) from your Project Plan. Milestone 1 (12 months) - Review literature for candidate repellent impregnants for clothing fabric. Milestone 1 (24 months) - Use QSAR, other chemistry methods, and biological techniques to identify new repellents, attractants, and attractant-antagonists for biting and filth breeding insects. Milestone 2 (24 months) - Develop biological assay systems for repellents that allow comparison of repellent effectiveness against laboratory reared and natural mosquito populations. Milestone 3 (24 months) - Review available research data regarding repellents impregnants for clothing fabric. Milestone 4 (24 months) Demonstrate effective use of BEETLBAR for litter beetle management in field tests. Milestone 5 (24 months) - Develop standardized diagnostic methods for detecting the presence of pathogens in beneficial arthropods. Milestone 6 (24 months) Identify and locate biting and filth breeding insect populations for characterization of resistance to insecticides and insect growth regulators. Milestone 1 (36 months) - Develop methods and techniques for the behavioral and physiological analysis of host seeking and blood feeding activity in biting and filth breeding insects in the laboratory. Milestone 2 (36 months) - Develop improved methods for field evaluation of repellents on humans without incurring disease risks. Milestone 3 (36 months) - Establish an insect diseases database that can be used to prevent the accidental release of pathogens that adversely affect native beneficial arthropods. Determine the impact of pathogens on the fitness and performance of beneficial arthropods. Milestone 4 (36 months) - Develop control technology for blood-sucking flies based on the use of trypsin-modulating oostatic factors (TMOF). Milestone 1 (48 months) - Develop methods and techniques for the behavioral and ecological analysis of natural populations of host seeking and blood feeding activity in biting and filth breeding insects in the field. Milestone 2 (48 months) - Develop repellent-attractancy correlates for human subjects that minimize bias in biological assays caused by innate repellency/attractiveness of human subjects. Milestone 3 (48 months) - Conduct basic efficacy and longevity testing on candidate repellent clothing impregnants. Milestone 4 (48 months) - Develop operational strategies for the safe and effective long term use of chemical insecticides. Determine the feasibility of using molecular technology to monitor insecticide susceptibility in biting and filth breeding insect populations. Milestone 1 (60 months) - Develop attractant-antagonist technology for the personal protection of humans and livestock. Milestone 2 (60 months) - Develop functional biting rate and repellency responses in mosquitoes to repellent concentration mosquito density. Milestone 3 (60 months) Evaluate 'removal trapping' as a strategy for the abatement of mosquito populations. Milestone 4 (60 months) - Discover and characterize new pathogens that produce disease in biting flies. Develop a germplasm repository for Dipteran pathogens. Milestone 5 (60 months) - Acquire, evaluate, and release indigenous and exotic parasitoids for filth fly control. Evaluate beetle predators as biological control agents for house flies. Develop the fungus Beauveria bassiana for management of house flies and litter beetles in years 1, 2, and 3. Milestone 6 (60 months) - Develop remediation and prevention methods for the management of disease organisms in cultures of beneficial arthropods. Milestone 7 (60 months) - Design, develop, test and implement integrated management systems for biting and filth breeding insects. Milestone 3 (60 months) - Develop practical, fast, and accurate resistance detection and monitoring technology. 3.Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. Milestone 1 (60 months) - Develop attractant-antagonist technology for the personal protection of humans and livestock. Milestone 2 (60 months) - Develop functional biting rate and repellency responses in mosquitoes to repellent concentration mosquito density. Milestone 3 (60 months) - Evaluate 'removal trapping' as a strategy for the abatement of mosquito populations. Milestone 4 (60 months) - Discover and characterize new pathogens that produce disease in biting flies. Milestone 5 (60 months) - Acquire, evaluate, and release indigenous and exotic parasitoids for filth fly control. Evaluate beetle predators as biological control agents for house flies. Milestone 6 (60 months) - Develop remediation and prevention methods for the management of disease organisms in cultures of beneficial arthropods. Milestone 7 (60 months) - Design, develop, test and implement integrated management systems for biting and filth breeding insects. Milestone 8 (60 months) - Develop practical, fast, and accurate resistance detection and monitoring technology. The Mosquito & Fly Unit has undergone CRIS contraction and expansion within the last two years. The Milestones were taken from the original Project Plan (due to expire February 2005)and segregated by topic into the current two CRIS projects (6615-32000-039-00D, Control of and protection from Biting and Filth Breeding Insects, and 6615-32000-040-00D, Surveillance and Ecology of Mosquito, Biting and Filth breeding Insects). All milestones have been fully accomplished except Milestones 7 and 8 that have been partially completed. Milestone 7 represents the overall goal of the Mosquito and Fly Research Unit. Milestone 8 was not completed due to lack of appropriate expertise. B: List Milestones for Next 3 Years This Project Plan will terminate February 2005; therefore, the Project Plan that is currently under review will cover future work. 4.What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: New Virus for Mosquito Control. A new baculovirus from the mosquito Uranotaenia sapphirina has been isolated and the biological and morphological characteristics determined and published. We have also isolated 2 new cytoplasmic polyhedrosis (CPV) viruses from Culex restuans and Uranotaenia sapphirina and determined that they have 10 segmented genomes. This is the first information on the genomes of CPV's from mosquitoes and suggests that they are members of the Cypoviruses. These new viruses from mosquitoes expands the genetic diversity base required to investigate and understand basic mechanisms involved in infectivity and host range that will enhance capabilities to use these viruses for mosquito control. B. Other Significant Accomplishments: Better Personal Protection for Marines from Disease Vector Insects. The longevity of various permethrin formulations impregnated onto United States Marine Corps (USMC) battle dress uniforms (BDUs) was evaluated to provide more effective personal protection for deployed marines. The USMC had a critical need to assess their BDUs due to a high incidence of disease transmission impacting deployed troops. Sample fabrics from Battelle Natick Operations containing various permethrin treatments and binding agents were tested by a Unit Scientist and it was determined that a particular binding agent was the best choice for use on new permanent press uniforms. This work is now in phase 2, which consists of further examination of the lifetime duration of treatments on uniforms, comparison of factory-treatment to field treatment lifetimes, and examination of treated tent material. Air Curtains Used for Disinsection. Air curtains were used successfully to prevent house flies and mosquitoes from passing from a simulated jetway into a simulated aircraft. This accomplishment was important because many airline companies are looking for non-pesticidal methods for preventing insect entry onto commercial aircraft. Unit Scientists in collaboration with the Department of Transportation carried out research demonstrating that mounting air curtains vertically along the sides of the door frame instead of the conventional overhead configuration was highly efficacious with less discomfort to subjects passing through the doorway. This research suggests that commercial air curtains for specific use at aircraft doors could be designed and used to prevent insect entry without the use of pesticides. Development of a Novel Parasitoid for Fly Biocontrol. A strain of achinaephagus. zealandicus with better production performance on house flies was selected for five generations and passed on to a commercial insectary for mass-rearing. This species, which attacks larvae of house flies and stable flies, is now being produced on a commercial scale and is expected to be available as a product in late 2004. The addition of T. zealandicus to our arsenal of biological fly control products will improve the efficacy of IPM programs for manure-breeding flies. Mosquito Populations Lowered with Traps. We successfully completed the second year of a field demonstration project on the use of commercially available traps to control mosquito populations on an isolated island. There is a widespread belief that mosquito traps cannot be used for mosquito population reduction. However, Unit scientists demonstrated that in locations that have geographic or other barriers to mosquito migration, e.g. an island, significant population reductions are possible. In this case, trap use resulted in greater public use of an island state park. C. Significant activities that support special target populations: None. D. Progress Report: None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. (1) Discovery and development of predators, parasitic wasps, viruses, and other biological control agents for mosquitoes, and filth flies and enabled pesticide-free control of pests and vectors. (2) The discovery of insect repellents and new techniques and procedures for the treatment of clothing fabric with toxicants, such as permethrin. This accomplishment has resulted in a personal protection system that is standard issue for U.S. military troops. (3) The discovery of human-produced chemical attractant-inhibitors for mosquitoes. Inhibitors are being used as spatial repellents and to develop systemic repellents for animals and humans. This research provides for the protection of animals and humans from blood sucking and filth breeding arthropods and the disease agents they transmit. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Unit Scientists participated in a high profile demonstration of the use of air curtains to prevent insect entry into commercial aircraft, sponsored by the Federal Department of Transportation, Miami International Airport, March 9, 2004, Miami, FL, and attended by international airline and diplomatic representatives. Mass rearing methods were developed by a Unit Scientist for a parasitic wasp that attacks larval house flies and stable flies; this colony was transferred to commercial insectary for development as a new fly control product. Results of research of diseases of natural enemies were presented at an international "Workshop on Arthropod Mass Rearing and Quality Control". The audience included scientists and commercial producers of biocontrol agents. Advances in the use of biological control for management of house flies and stable flies were disseminated by articles in popular and trade journals. The "Flybrella", a recently patented new house fly trap design, has generated industry interest and new confidentiality agreements are pending. Inquiries were a result of official OTT posting of patent disclosure on this invention. Invited to participate in Insect Expo at the Florida State Fair, Tampa, Feb 5-16, 2004. Cages of live flies used for biological control of house flies on farms and accompanying explanatory literature were seen by countless thousands of Fair visitors over a 2-week period. Invited to participate in an afternoon demonstration of new and currently used traps for nuisance flies and mosquitoes at the Department of Defense Pest Management Workshop, Jacksonville Naval Air Station, Jacksonville, FL, February 9-11, 2004. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Dr. Becnel's research featured in an article in Agricultural Research, October 2003. "Beyond Deet: New-Age Mosquito Control" by Jim Core. Dr. Becnel and Dr. Geden's research featured in an article in Agricultural Research, March 2004. "Managing Disease in Beneficial Insects" by Jim Core. Dr. Geden's research featured in an article in Dairy Farming, April 2004. "Fly Control Patrol", by Cathy Coatney. Dr. Geden's research featured in an article for Biocontrol News and Information, Winter 2003. "Brazilian wasp has sweet tooth", by Rebecca Murphy. Drs. Hogsette & Geden were interviewed by and quoted in article by Elizabeth Iliff about the use of integrated techniques for fly management entitled "Bug Wars". Practical Horseman XXXI (6):3-5, June 2003. Suszkiw, J. and J. Core. 2003. Project Aims to Clean House on Filth Flies. Agricultural Research, November, 51(11):20-22. Article about stable fly and house fly projects in Lincoln and Gainesville, respectively. Dr. Hogsette was invited to present a 1-hour lecture entitled "Biology and Control of House Flies and Other Nuisance Flies" at the US Food and Drug Administration's Southeast Region Retail Food Safety Seminar, St. Augustine, FL, October 20-23, 2003. Dr. Hogsette was invited to present a1-hour lecture entitled "Management of Nuisance Flies in the Urban Setting at the Department of Defense Pest Management Workshop, Jacksonville Naval Air Station, Jacksonville, FL, February 9-11, 2004. Dr. Hogsette was invited to present a 1-hour lecture entitled "Biology and Management of Flies of Public Health Significance" at the National Restaurant Association's Quality Assurance Study Group Meeting, Chandler, AZ, April 14-16, 2004. Review Publications Araujo-Coutinho, C., Nascimento, E., Figueiro, R., Becnel, J.J. 2004. Seasonality and prevalence rates of microsporidia in simulium pertinax (diptera: simuliidae) larvae in the region of serra dos órgãos, Rio de Janeiro, Brasil. Journal of Invertebrate Pathology. 85:188-191. Vossbrinck, C.R., Andreadis, T.G., Vavra, J., Becnel, J.J. 2004. Molecular phylogeny and evolution of mosquito parastic microsporidia (microsporidia: amblyosporidae). Journal of Eukaryotic Microbiology. 51(1):88-95. Micieli, M.V., Garcia, J.J., Becnel, J.J. 2003. Life cycle and epizootiology of amblyospora ferocis (microspora: amblyosporidae) in the mosquito psorophora ferox (diptera, culicidae). Memorias Do Instituto Oswaldo Cruz. 50:171-175. Reyes-Villanueva, F., Becnel, J.J., Butler, J.F. 2003. Susceptibility of aedes aegypti and aedes albopictus larvae to ascogregarina culicis and ascogregarina taiwanensis (apicomplexa: lecudinidae) from florida. Journal of Invertebrate Pathology. 84:47-53. BOOHENE, C.K., GEDEN, C.J., BECNEL, J.J. EVALUATION OF REMEDIATION METHODS FOR NOSEMA DISEASE IN MUSCIDIFURAX RAPTOR (HYMENOPTERA: PTEROMALIDAE). ENVIRONMENTAL ENTOMOLOGY. 2003. v. 32(5).p. 1146-1153. Hay-Roe, M.M., Shapiro, A.M., Becnel, J.J., Boucias, D.G. 2003. A newly discovered baculovirus induces reflex bleeding in the butterfly heliconius himera (nymphalidae: heliconiinea). Journal of Invertebrate Pathology. 84:59-62. Tartar, A., Boucias, D.G., Becnel, J.J., Adams, B.J. 2003. Comparison of plastid 16s rdna (rrn16) genes from helicosporidium spp.: evidence supporting the reclassification of helicosporidia as green algae (chlorophyta). International Journal of Systematic and Evolutionary Microbiology. 53:1719-1723. ANDREADIS, T.G., BECNEL, J.J., WHITE, S.E. INFECTIVITY AND PATHOGENICITY OF A NOVEL BACULOVIRUS, CUNINPV FROM CULEX NIGRIPALPUS (DIPTERA: CULICIDAE) FOR FOURTEEN SPECIES AND FOUR GENERA OF MOSQUITOES. ENCYCLOPEDIA OF ENTOMOLOGY. 2003.v.40(4)p.512-517.