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United States Department of Agriculture

Agricultural Research Service

Research Project: CONTROL AND PROTECTION TOOLS FOR INTEGRATED PEST MANAGEMENT OF MOSQUITOES AND FILTH FLIES

Location: Mosquito and Fly Research Unit

2008 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.


3.Progress Report
This research supports National Program 104 Veterinary, Medical and Urban Entomoloy; Component 4: Control Technology. Significant progress has been made in the discovery of new toxicants and repellents for biting flies. Work has continued on utilizing quantitative structure activity relationship (QSAR) methods to model chemical characteristics of highly efficacious repellents and toxicants. Novel compounds were synthesized and assayed for activity against mosquitoes. This has led to the identification of a number of highly active repellents and the results have been published in the Proceedings of the National Academy of Sciences. The structure-activity relationships of 33 piperidines as toxicants were evaluated and the results suggest that it is feasible to modify a chemical’s structure and increase its insecticidal activity. Advances have been made on methods to utilize molecular biology techniques for development of biopesticides that target specific critical pathways in mosquitoes. We have constructed double-stranded RNAs that can be topically applied to kill female Aedes aegypti. Additional testing has verified that this molecule is also active against other mosquito species (Anopheles quadrimaculatus and Culex quinquefasciatus) but not against the house fly (Musca domestica) verifying the specificity of RNAi’s. Further work with attraction inhibitors (compounds that prevent biting flies from finding their hosts) were conducted against sand flies. Laboratory assays with Phlebotomus papatasi indicated that sand flies were incapable of finding the source of host odors when these odors were combined with an inhibitor. Studies have also been conducted to improve the effectiveness of permethrin treated uniforms and recommendations have been provided to the U.S. Army. Several new methodologies have been developed to evaluate active ingredients. A wind tunnel and application methodology has been developed and used to evaluate the relative roles of different active ingredients in flight initiation and subsequent mortality. A high throughput primary screening method using 1st instar larvae of Aedes aegypti was designed and evaluated to quickly screen large numbers of chemicals for activity against mosquitoes. Research has continued on improved use of parasitoids for the biological control of house flies. It was found that providing parasitoids with access to honey extended their lives by several weeks, allowing the accumulation of large numbers of female wasps that are ready to attack fly hosts. Further testing with a synthetic attractant blend based on molasses to attract house flies was conducted and demonstrated that a 10% dilution of the attractant was optimal for fly attraction. Studies continued on a salivary gland hypertrophy virus (SGHV) of house flies that belongs to a novel group of viruses that infect salivary glands and block egg production in their hosts. High infection rates were achieved when virus particles were aerosolized and sprayed onto fly resting sites. This raises the possibility of delivering the virus as a premise treatment as well as in baits.


4.Accomplishments
1. Successful use of computer modeling to design novel repellents. A subset of about 200 chemicals from USDA archives of over 40,000 chemicals accumulated over that last 60 years was selected for modeling. Based on the developed model, a set of 2,000 diverse compounds was input into the model and 34 were selected for synthesis. Of these 34 compounds, 23 were novel and 11 compounds had been examined previously and known repellency levels were present in the archives. The results of the bioassays demonstrated that many compounds had repellent duration substantially longer than that provided by DEET using cloth-patch assay screening methods (i.e., DEET provided protection for 7 days against female Aedes aegypti while numerous compounds provided over 40 days of protection). These new experimental observations were reinserted into the model and the resultant predictions demonstrated high correlation between the model’s theoretical predictions and observed results. This work was reported in the Proceedings of the National Academy of Sciences. Seven of the 23 novel compounds are undergoing further analysis for repellency against other mosquito species. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

2. Development of factory-level, permethrin-treated Fire Resistant Army Combat Uniforms (FRACUs) for the U.S. Army. Issues related to burn wound damage to soldiers wearing the standard 50:50 nylon/cotton military uniform have necessitated a change from this composition to uniforms that contain a fire retardant chemical. The incorporation of this chemical in uniform fibers resulted in uniforms that did not efficiently bind the necessary amount of permethrin insecticide in the fabric to prevent soldiers from receiving a critical number of mosquito bites. Currently, four fabric compositions and two binder compositions are under study. Recent results indicate that tightening of the uniform weave may overcome some of the weaknesses in the bite protection capability of these uniforms. The study will be completed in 2008, with additional examination of novel treatments planned for 2009. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

3. Laboratory and field evaluation of attraction-inhibitors against mosquitoes and sand flies in Egypt. In 2008 and 2009, field studies are being conducted in Aswan, Egypt to examine three of 24 potent attraction-inhibitors that prevent host-finding in insects. Laboratory assays with Phelebotomus papatasi in Cairo, Egypt indicated that sand flies were incapable of finding the source of host odors when these odors were combined with an inhibitor and tested in a single tube clear plastic olfactometer. Field tests were initiated in the Aswan field site using commercial mosquito traps to determine if inhibitors emanating from slow-release devices reduce or eliminate the ability of insects to find a trap by interrupting their detection of carbon dioxide. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

4. Effect of nutrition on host attacks by the fly parasitoid Trichopria nigra. The diapriid fly parasitoid Trichopria nigra was originally obtained from stable fly pupae in Russia and Kazakhstan. The parasitoids emerge as adults with fully developed ovaries and only need to live long enough to attack and oviposit on fly pupae. This year we found that providing parasitoids with access to honey extended their lives by several weeks, allowing the accumulation of large numbers of female wasps that are ready to attack fly hosts when they are present. The results may facilitate mass production of this species and lead to ways to improve the effectiveness of field releases of biocontrol agents against their intended target species. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

5. House fly attractants. Further testing with a synthetic attractant blend based on molasses to attract house flies was conducted in motel rooms in which flies had been released. Elimination of the two most costly constituents in the blend resulted in no diminution of effectiveness of the attractant. Dose response studies with the new 5-component blend demonstrated that a 10% dilution of the attractant was optimal for fly attraction. Results of this project are expected to lead to development of improved baits for fly control that can be used in close proximity to people. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

6. Distribution of Tachinaephagus zealandicus in the US. Tachinaephagus zealandicus is an encyrtid parasitoid of muscoid fly larvae that 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. A 2007 survey for T. zealandicus demonstrated that this species was established in several locations in the eastern US. The survey was continued and expanded in 2008. While no parasitoids were found south of Sarasota, Florida, new records were obtained for Illinois, Missouri, and Kansas. 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 also allowed resumption of related research activities under Objective 2. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

7. Salivary gland hypertrophy virus of house flies. This is a cooperative project with colleagues in the Entomology and Nematology Department at the University of Florida. Salivary gland hypertrophy virus (SGHV) belongs to a novel group of viruses that infect salivary glands and block egg production in their hosts. The epidemiology of the virus strongly suggests that transmission is via ingestion of virus particles. However, attempts to infect flies with SGHV presented in a wide variety of food items resulted in only moderate rates of infection. However, high infection rates were achieved when virus particles were aerosolized and sprayed onto fly resting sites, suggesting that the virus enters the fly as a result of grooming behavior. This raises the possibility of delivering the virus as a premise treatment as well as in baits. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

8. Molecular biopesticides for mosquito control. Using RNA interference (RNAi) we have designed a molecule that inhibits expression of a protein (IAP) that regulates programmed cell death (apoptosis). This molecule was absorbed through the cuticle of adult Aedes aegypti mosquitoes, causing death in up to 70% of individuals. Injected, the molecular pesticide killed 100% of the Ae. aegypti tested. Additional testing has verified that this molecule is also active against other mosquito species (Anopheles quadrimaculatus and Culex quinquefasiatus) but not against the house fly (Musca domestica) verifying the RNAi’s specificity for the insect Family Culicidae (i.e., mosquitoes). We are currently awaiting issuance of a patent, which was applied for by the U.S. Department of Agriculture in March 2007. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

9. High throughput larval screening for new toxicant discovery. A high throughput primary screening method using 1st instar larvae of Aedes aegypti was designed and evaluated to quickly screen large numbers of chemicals for activity against mosquitoes. Five first instar larvae (24 hr post hatch) were added to each well of 24-well plates. De-ionized water (950 ¿l) and larval diet (40 ¿l) were then added to each well. All chemicals to be evaluated were diluted in acetone. Diluted chemicals (10 ¿l) were then added to each well containing a total volume of 1 ml. Larval mortality was recorded after 24 h of exposure. Using this methodology, more that 2,000 experimental toxicants have been tested with 9-10% of the compounds screened showing >80% mortality. This method effectively identified the best toxicants for subsequent evaluation in secondary screening regimes. In addition, this screening tool is fast, requires little space and requires very small quantities of the experimental toxicants. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

10. Structure-activity analysis. We have investigated the structure-activity relationships of 33 piperidines as toxicants against female Aedes aegypti. On the basis of 24-h LD50 value after topical application, the most toxic compound was 2-ethyl-piperidine, with an LD50 as low as 0.8 ¿g per mosquito. The toxicities of piperidine derivatives were significantly decreased when a benzyl moiety was attached to the carbon of the piperidine ring, with an LD50 value as high as 29.2 ¿g per mosquito. The toxicity order of three moieties attached to the carbon of the piperidine ring was: ethyl- > methyl- > benzyl- derivatives. When the same moiety was attached to the piperidine ring, the carbon position to which the moiety was attached conferred different toxicity and the toxicity order was: 2nd carbon > 3rd carbon > 4th carbon. Taken together, these preliminary results suggest that it is feasible to modify a chemical’s structure and increase its insecticidal activity. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

11. Excito-repellency compounds to enhance mortality. Insecticide sprays are often applied when mosquitoes are resting and less likely to be contacted by droplets of a toxicant. An approach to enhance mortality is to add active ingredients that elicit flight and thus generate more contact with toxicant droplets. A wind tunnel and application methodology has been developed and used to evaluate the relative roles of different active ingredients in flight initiation and subsequent mortality. Results from this approach and study will provide the basis for refining blends of active ingredients to enhance contact of mosquitoes with toxicants. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

12. Change in Stable Fly Target Configuration. Cloth targets for stable flies have been shown to be efficacious in preliminary trials, but the flat configuration of the target makes it easily blown over by moderate to strong winds. Flat targets were compared with targets formed into cylinders, thus making them more stable. The cylindrical configuration did not reduce the efficacy and the cylindrical targets should prove more satisfactory for field use. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.

13. Barrier treatments on native vegetation in a desert habitat for mosquito control. Treating perimeters with residual insecticides to provide protection from mosquito vectors has shown promise. Although typically evaluated in temperate or tropical areas, we examined these techniques in desert habitats of southern California to develop methods to protect deployed US troops in extreme desert environments. Using a combination of bioassays on foliage and field surveillance of mosquitoes in traps we measured the temporal pattern of bioactivity of bifenthrin barriers using different techniques against Culex tarsalis. Efficacy of these methods was demonstrated for up to 28 days. The results suggest that barrier treatments may be successful in providing protection from vector-borne diseases for deployed troops in desert habitats. This addresses National Program 104 Veterinary, Medical, and Urban Entomology, Component 4 Control Technology, Problem Statement - prevent harm by controlling the agent.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of New CRADAS1
Number of Invention Disclosures Submitted2
Number of New Patent Applications Filed1
Number of Non-Peer Reviewed Presentations and Proceedings7

Review Publications
Xue, R., Doyle, M.A., Kline, D.L. 2008. Field evaluation of CDC and Mosquito Magnet X traps baited with dry ice, co2 sachet, and octenol against mosquitoes. Journal of the American Mosquito Control Association, 24(2):249-252.

Reinert, J.F. 2007. Development and taxonomic value of dorsal tertiary fringe scales of the adult mosquito wing (Diptera: Culicidae). Proceedings of the Entomological Society of Washington.

Pridgeon, J.W., Zhao, L., Becnel, J.J., Strickman, D.A., Clark, G.G., Linthicum, K. 2008. Topically applied AaeIAP1 double-stranded RNA kills female adults of Aedes aegypti. Journal of Medical Entomology.45(3):414-420.

Kline, D.L., Allan, S.A., Bernier, U.R., Welch, C.H. 2007. Evaluation of the enantiomers of 1-octen-3-ol and 1-octyn-3-ol as attractants for mosquitoes associated with a freshwater swamp in Florida, USA. Medical and Veterinary Entomology. 21(4) 323.

Lloyd, A.M., Kline, D.L., Hogsette, Jr, J.A., Kaufman, P.E., Allan, S.A. 2008. EVALUATION OF TWO COMMERCIAL TRAPS FOR THE COLLECTION OF CULICOIDES (DIPTERA: CERATOPOGONIDAE). Journal of the American Mosquito Control Association. 24(2):253-262.

Clark, G.G., Rubio-Palis, Y. 2007. Mosquito Vector Control and Biology in Latin America - A 17th Symposium. Journal of the American Mosquito Control Association. 23(4):449-470.

Hogsette, J.A., Amendt, J. 2008. Flies. In: Bonnefoy, X., Kampen, H., Sweeney, K., editors. Public Health Significance of Urban Pests. World Health Organization Regional Office, Europe, Copenhagen, Denmark, p. 209-237.

Zhao, L., Pridgeon, J.W., Becnel, J.J., Clark, G.G., Linthicum, K. 2008. Cytochrome c gene and protein expression: developmental regulation, environmental response and pesticide sensitivity in the Aedes aegypti. Journal of Medical Entomology. 45(3):401-408.

Miciele, M.V., Marti, G.A., Tranchida, M.C., Becnel, J.J. 2007. Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat. Journal of Invertebrate Pathology. 94(1):31-37.

Pridgeon, J.W., Pereira, R., Becnel, J.J., Allan, S.A., Clark, G.G., Linthicum, K. 2008. Susceptibility of Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say to 19 Pesticides with Different Modes of Action. Journal of Medical Entomology. 45(1):82-87.

Barnard, D.R., Xue, R., Rotstein, M.J., Becnel, J.J. 2007. Microsporidiosis (Microspora: Culicosporidae) in Aedes aegypti (Diptera: Culicidae) affects host attraction, blood feeding responses, and the repellency of deet. Journal of Medical Entomology. 44(6):1040-1046.

Reinert, R.F. 2008. Redescription of the holotype of Finlaya lepchana Barraud, 1923 (Diptera: Culicidae: Aedini). Zootaxa. 1767:64-66.

Geden, C.J., Ulricke-Lietze, V., Boucias, D.G. 2008. Seasonal prevalence and transmission of salivary gland hyperplasia virus of house flies, Musca domestica L. (Diptera:Muscidae). Journal of Medical Entomology. 45(1):42-51.

Turell, M.J., Linthicum, K., Patrican, L.A., Davies, F.G., Kairo, A., Bailey, C.L. 2008. Vector Competence of Selected African Mosquito (Diptera: Culicidae) Species for Rift Valley Fever Virus. Journal of Medical Entomology. 45(1):102-108.

Katritzky, A.R., Wang, Z., Slavov, S., Tsikolia, M., Dobchev, D., Akhmedov, N.G., Hall, C.D., Bernier, U.R., Clark, G.G., Linthicum, K.J. Synthesis and bioassay of improved mosquito repellents predicted from chemical structure. Proceedings of the National Academy of Sciences of the USA. 105(21):7359-7364.

Last Modified: 9/21/2014
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