2007 Annual Report 1a.Objectives (from AD-416) 1) Develop new pathogen delivery formulations and molecular methods based on pathogenic viruses for control of mosquitoes. 2) Improve parasitoid-based management systems for control of filth flies. 3) Test and develop new and improved pesticides and "attract and kill" systems as management tools for pest and vector species. 4) Discover, evaluate and develop new personal/animal protection tools. 1b.Approach (from AD-416) Identify and evaluate novel biologically based and self-sustaining biological control agents for mosquitoes and flies; incorporate these into integrated management strategies and demonstration projects. Isolate, identify and validate the efficacy of repellents under laboratory and field conditions to develop new strategies for personal/animal protection from disease vectors and nuisance species. Discover and develop leading candidate compounds from critical screening to targeted applications, to identify new insecticides and more efficacious toxicants for control of mosquitoes and flies. 4.Accomplishments Molecular biopesticides for mosquito control. We have discovered methods to utilize molecular biology techniques for development of biopesticides that target specific critical pathways in mosquitoes. Using the programmed cell death pathway as a target, we have constructed double-stranded RNAs (dsRNAs) that can be topically applied to kill female Aedes aegypti. This new technology could have important implications for novel pesticide development and pest management strategies. A provisional patent was filed in 2007. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Discovery of new attraction-inhibitors for personal protection. A provisional patent was filed during the fourth quarter of 2006 covering a total of 24 new attraction-inhibitor compounds of both natural and synthetic origin. If two additional compounds perform well in bioassays to be completed in the third quarter of 2007, they may be added to this patent application. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Attractants for house fly control. Further testing with a seven-component blend based on black strap molasses to attract house flies was conducted in large outdoor cages. The blend was found to be as attractive as natural molasses lures. Dose-response studies demonstrated that although flies can detect a 3% dilution of the attractant as currently formulated, optimal results are obtained with a 10% dilution. The blend was found to be much more attractive to previtellogenic flies than to older, gravid flies. A variety of natural sugar products was also tested for relative attractiveness to identify additional sources of fly attractants and molasses was found to be substantially more attractive than honey, maple syrup, barley malt syrup, or jaggery (date palm sugar). A provisional patent was filed during the second quarter of 2007. Results of this project are expected to contribute to development of improved baits for fly control that can be used in close proximity to people. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Discovery of highly efficacious repellents through computer modeling. Quantitative structure activity relationship (QSAR) modeling methods were used to model chemical characteristics of highly efficacious repellents from the USDA archives of chemical analyses accumulated over the past 60 years. A total of 23 novel compounds were synthesized and included in bioassays with 11 compounds identified from archived data. The results of the cloth-patch bioassay screening identified many compounds whose duration of repellency was substantially longer than that provided by DEET (i.e., DEET provided protection for 7 days while these new compounds provided >40 days of protection). These new experimental observations were reinserted into the model and the resultant predictions demonstrated a high correlation between the model’s theoretical predictions and observed results. A manuscript is in preparation for submission to the Proceedings of the National Academy of Sciences and a patent is likely to be filed for this class of repellent compounds. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Toxic baits for mosquitoes. Toxicants from different classes of insecticides and their lethal doses were identified for Aedes, Anopheles and Culex mosquitoes for use in a sugar bait system for mosquitoes. Mosquito species varied in their sensitivity to different toxicants and presentation of the appropriate dose is essential for maximum efficacy. Various delivery methods were evaluated for field delivery. Toxic sugar baits are an effective tool for producing mosquito mortality before host-seeking occurs. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Pathogenic virus-mosquito interactions. We have identified the major structural proteins of a baculovirus that infects and kills mosquitoes. Forty-four structural proteins and their genes have been identified, some of which play crucial roles in infectivity, host specificity and virulence. This work has opened up a range of new possibilities for mosquito control. Four proteins crucial for oral infections have been found to be promising new targets for development as biocontrol tools. It is now possible to investigate these and other viral proteins at the molecular level and understand how they exploit essential metabolic processes of the mosquito host. The outcome of this research is an important advancement in developing novel control methods for mosquitoes. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Determination of different mosquito behavioral responses to pesticides. Behavioral responses of Aedes, Anopheles and Culex mosquitoes to surfaces treated with three different pyrethroids were compared. Each pyrethroid tested affected numbers of landings and contact duration of each mosquito species differently. These data provide the basis for selection of appropriate pesticides for treatment of barriers or surfaces (i.e., bed nets) to enhance numbers and duration of contacts to maximize mosquito mortality caused by the insecticide. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Role of flies in transmitting Salmonella enterica: In a collaborative project with ARS research immunologist Peter Holt (Athens, GA), house flies became infected with Salmonella enterica within 24 hours of being released into a room with infected chickens. Dissection of flies revealed presence of the pathogen on the surface and in the midgut but not in the crop or salivary glands. Healthy birds became infected by ingesting as few as five of these naturally-infected flies. The results provide evidence that flies may play a significant role in the movement and transmission of this important pathogen of poultry and humans. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Salivary gland hyperplasia virus of house flies. Salivary gland hyperplasia virus is a novel virus that infects adult house flies and was surveyed weekly for a year on four dairy farms in north central Florida. Virus-infected flies do not develop eggs and laboratory studies documented that this infection prevents flies from synthesizing protein precursors needed to assemble vitellogenin. Transmission studies demonstrated that viral infection occurs when healthy flies feed on food resources that have been contaminated with virus particles deposited in the saliva of infected flies. The results are being used to develop an infective bait system for infecting flies in the field at times when fly populations and natural infections are low to prevent fly outbreaks. This is a cooperative project with colleagues (Drion Boucias and Verena Lietze) in the Department of Entomology and Nematology at the University of Florida. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Permethrin-impregnated U.S. Marine Corps Combat Utility Uniforms. During the last two quarters of 2006, three of four suppliers have been qualified (certified) by the MFRU to supply the U.S. Marine Corps with factory-treated, permethrin-impregnated uniforms. The fourth and final company to be evaluated currently has first article samples undergoing biological efficacy testing. As of March 1, 2007, all new graduating Marine Corps cadets will be issued these uniforms. The results of these studies will be submitted for publication in 2008. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. A study of Individual Dynamic Absorption kits for permethrin treatment of Army Combat Uniforms in the field. This study was initiated and completed during 2007. Previously, it was unknown whether or not field treatment of Army Combat Uniforms (ACUs) using Individual Dynamic Absorption kits would provide a dosage of permethrin to these uniforms that would remain efficacious through 50 laundering cycles. The results of this study indicated that although there was substantial permethrin loss from the treated ACUs, the biological efficacy as measured by bite protection from wearing these ACUs was not similarly reduced. This finding will be explored further with studies planned for 2008. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Efficacy of commercial fly traps under desert conditions. Based on US military needs in desert environments, seven fly traps and baits were evaluated in Cairo and Aswan, Egypt for efficacy under dry, hot conditions. Traps that used water to volatilize their attractants captured significantly more flies than dry baits and sticky traps. The current recommendation would be to use ‘wet’ traps for the best results under desert conditions where flies are water deprived. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Effect of conditioning on host selection by the parasitoid Trichopria nigra. The diapriid fly parasitoid Trichopria nigra was originally obtained from stable fly pupae in Russia and Kazakhstan. In the absence of experience with any host material, this species is strongly attracted to pupae of the house fly, stable fly, horn fly, black dump fly and flesh flies. Behavioral studies have demonstrated that 24 hours of conditioning of young parasitoids on one species strongly influences the attractiveness of that species in subsequent exposures. The results may facilitate mass production of this species and lead to ways to improve the effectiveness of released biocontrol agents against intended target species. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. Occurrence of Tachinaephagus zealandicus in the eastern United States. Tachinaephagus zealandicus is a parasitoid of muscoid fly larvae and is native to the Southern Hemisphere, where it is regarded as an important biocontrol agent for filth flies. Although this species was released in California in the 1960’s, there is little information on the status of its establishment in the US. In 2007, a survey for T. zealandicus was conducted at various locations in the eastern US. Traps baited with larvae of a flesh fly (Sarcophaga bullata) were placed for 24 hours in woodland, riverine, and open field habitats near livestock farms. Results of the survey demonstrated that this species is well-established in Kentucky, Tennessee, North Carolina, Georgia and Florida. Eight geographic isolates of T. zealandicus are now in culture and will be evaluated for fitness before selecting a strain for further development as a biocontrol tool. This discovery eliminates the regulatory barriers to importing an exotic species and will facilitate rearing and release of healthy, locally-adapted parasitoids for fly biocontrol. It will also allow resumption of related research activities under Objective 2. Research addresses NP-104, Veterinary, Medical and Urban Entomology, and the Problem Statement associated with Action Plan Component 4: Control Technology. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 4 Number of active CRADAs and MTAs 1 Number of patent granted 3 Number of non-peer reviewed presentations and proceedings 36 Number of newspaper articles and other presentations for non-science audiences 4 Review Publications Carlson, D.A., Berkebile, D.R., Skoda, S.R., Mihok, S. 2007. Candidate sex pheromones of the new world screwworm Cochliomyia hominivorax. Medical and Veterinary Entomology. 21(1): 93-96. Pridgeon, J.W., Olzmann, J.A., Chin, L.S., Li, L. 2007. PINK1 Protects against Oxidative Stress by Phosphorylating Mitochondrial Chaperone TRAP1. PLoS Biol. 5(7):e172. Mihok, S., Carlson, D.A., Ndegwa, P.N. 2007. Biting fly responses to nzi traps baited with octenol, phenols and acetone. Medical and Veterinary Entomology. 21(1):70-84. Becnel, J.J., White, S.E. 2007. Mosquito pathogenic viruses - the last 20 years. American Mosquito Control Association. 23(2)36-49. Perera, O.P., Green, T.B., Stevens, S.M., White, S.E., Becnel, J.J. 2007. Proteins associated with Culex nigripalpus Nucleopolyhedrovirus (CuniNPV) occluded virions. Virology. 81(9):4585-4590. Quinn, B.P., Bernier, U.R., Geden, C.J., Hogsette Jr, J.A., Carlson, D.A. 2007. Analysis of extracted and volatile components in blackstrap molasses feed as candidate house fly attractants. Journal of Chromatography. A, 1139(2):279-284 Green, T.B., White, S., Rao, S., Mertens, P., Adler, P.H., Becnel, J.J. 2007. Biological and molecular studies of a cypovirus from the black fly simulium ubiquitum (diptera: simuliidae). Journal of Invertebrate Pathology 95 (2007):26-32. Carlson, D.A., Hogsette Jr, J.A. 2007. Flybrella: a device to attract and kill house flies. Journal of Economic Entomology. 100:483-487. Geden, C.J. 2007. Development of spalangia cameroni and muscidifurax raptor on live house fly pupae and pupae killed by heat shock, irradiation and cold. Environmental Entomology. 36(1):34-39.