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United States Department of Agriculture

Agricultural Research Service

Research Project: Biting Arthropods: Integrated Pest Management

Location: Mosquito and Fly Research Unit

2010 Annual Report

1a.Objectives (from AD-416)
Objective 1: Discover and evaluate new toxicants and biological control agents for control of biting Nematocera. • Sub-objective 1.A. Discover new adulticides and larvicides for mosquitoes. (Becnel, Bernier, Clark, Linthicum) • Sub-objective 1.B. Develop RNAi molecules for control of mosquitoes. (Becnel) Objective 2: Develop new application methods for pesticides to biting Nematocera that minimize environmental exposure and that optimize lethal or repellent effect, including presentation on clothing, aerosol application in or outdoors, residual application, disinsection of aircraft, and delivery of spatially repellent compounds. • Sub-objective 2.A. Determine factors that affect and enhance protection from insect bites through military and civilian repellent-treated clothing. (Bernier) • Sub-objective 2.B. Examine factors that contribute to the efficacy of aerosol application of pesticides in various environments, with emphasis upon dry arid climates. (Linthicum) • Sub-objective 2.C. Examine factors that contribute to the efficacy of residual pesticide application, including use of insecticides in impregnated materials and as barrier sprays for mosquitoes and other biting flies. (Kline, Linthicum, Bernier, Hogsette, Clark, Allan) • Sub-objective 2.D. Develop and evaluate approaches to disinsection of aircraft. (Hogsette, Clark, Linthicum)

Objective 3: Conceive and test new methods of managing vector and pest populations through the use of behavior-altering chemicals, including repellents, attractants, and inhibitors. • Sub-objective 3.A. Conceive and test the efficacy of attractants, inhibitors and repellents on the control of Nematocera. (Allan, Bernier, Barnard, Clark, Kline, Hogsette, Linthicum) • Sub-objective 3.B. Develop and examine a “push-pull” system for biting fly control through use of a combination of behavior-modifying chemicals, such as repellents and inhibitors, in trapping systems baited with attractants. (Bernier, Kline, Allan, Hogsette, Barnard)

Objective 4: Examine the parameters of behavioral bioassay methods that influence practical comparisons of personal protection products, with a view to determining those elements of commercial testing that influence reliability of results. (Barnard, Bernier)

1b.Approach (from AD-416)
1. High throughput bioassays will be used to screen candidate toxicants from libraries of synthetic compounds and natural products. Bacterial toxins, baculoviruses, and biorationals will be evaluated in new formulations in standard assays. Molecular methods, including RNAi and dsRNA, will be used to identify new targets for control and resistance management by challenging mosquitoes with pesticides, microbial organisms, or other stressors to identify critical mosquito genes/proteins. Improved delivery/formulation of dsRNA to effectively penetrate the mosquito cuticle will be developed.

2. Factors affecting measurement of bite protection provided by permethrin-treated clothing will be examined. Alternative repellents applied to military uniforms will be studied. Improved binding of repellents into the fabric will be researched, followed by laboratory validation of the factory-treated fabric and semi-field studies. Aerosol application of control compounds, formulations, equipment, application techniques, and strategies as Ultra-Low Volume (ULV) insecticides will be explored. Studies will extend from laboratory-based with colonized insects to experimental field plots. Existing and novel chemical compounds, formulations, equipment, impregnated materials, application techniques, and strategies for barrier applications will be evaluated under laboratory, semi-field and field conditions. Parameters (i.e., state of target insect, barrier composition and environmental factors) will be examined to identify the critical factors to achieve optimal control efficacy. Semi-field tests will involve colonized insects while field trials with GIS will involve natural populations. Air curtains will be designed and evaluated in simulated aircraft fuselage modules.

3. Resource-finding behaviors will be studied to devise a toxic bait (attract-and-kill) for testing in the laboratory, semi-field, and field. Comparison of pathogen-infected to uninfected mosquito responses to behavior-altering chemicals, including repellents, inhibitors, and toxicants, will be examined. Commercial traps, identical in placement, physical characteristics, and baited with attractants will be used as human/livestock surrogates. The manner in which compounds affect host-seeking behavior will be measured by quantifiable responses (i.e., percent repellency or duration of repellency, knockdown, mortality, attraction or inhibition percentage) and insect physiological responses (i.e., GC-EAD), and by qualitative behavioral responses (i.e., flight pattern observed by video recording). Using a screened cage, catches in attractant-baited traps will be compared to catches in control traps located near devices releasing spatial repellents or inhibitors. The experimental design in the large cages will be adapted to evaluate this concept in the field.

4. The effect of mosquito fatigue on repellent protection time (CPT) will be evaluated using female Aedes aegypti. A study comparing different methods for measuring repellent efficacy will be conducted. Laboratory results of repellent efficacy will be compared to performance of products tested in the semi-field environment.

3.Progress Report
Biting Nematocera transmit pathogens that cause diseases such as West Nile virus, dengue, malaria, leishmaniasis and filariasis to humans and animals in urban, suburban, rural, agricultural, recreational, and military environments. This research project is focused on improving the control of biting Nematocera through a better understanding of their biology and the development of novel products, technologies, and control strategies. Research with gene silencing technology showed that RNA interference (RNAi) can be transferred through the insect cuticle. This further advances the novel field of molecular pesticides. Results from larvicidal and adult screening with insecticides resulted in two promising models for the development of novel insecticides to be tested against the house fly and yellow fever mosquito. The impact on genes by viruses that infect mosquito larvae have been studied and continuing work is being done to identify the mechanism by which the virus works on the mosquito. “Bite protection” evaluation of permethrin-treated Fire-Resistant Army Combat Uniforms (FRACUs) indicated that factory-treatment of this uniform resulted in a uniform that gave excellent protection against mosquitoes throughout its expected lifetime. The results of the studies with uniforms were used by the U.S. Army to award contracts to suppliers of these uniforms. The protocol used for generation of this data is now being accepted by the U.S. Environmental Protection Agency as the appropriate way to evaluate the protection from insects afforded by permethrin-treated clothing. Demonstrated efficacy of insecticide-treated barrier treatments on native vegetation and artificial substrates in desert, tropical and subtropical habitats for controlling mosquitoes and sand flies. Evaluated four spatial repellents against mosquitoes and flies to determine if the systems protect individuals in tents or other similar enclosed dwellings. It was discovered that a certain spatial repellent may work well with one insect species, but not work as well against others. Tested efficacy of net doors rather than air curtains as a means to prevent entry of insects into the passenger cabin of commercial aircraft. Novel synthetic carboxamide repellents were predicted, designed, and tested against malaria-transmitting mosquito species An. gambiae and An. quadrimaculatus. Two of these novel repellents performed as well as the standard repellent N,N-diethyl-3-methylbenzamide (DEET). Identified two carboxamides that were as potent as DEET against these species. In July, 2010, the World Health Organization Pesticide Evaluation Scheme (WHOPES) held its 50th Anniversary conference and recognized a number of institutions that have made significant contributions to WHOPES throughout the past 50 years. The contributions of the Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) were recognized by WHOPES by the presentation of a plaque to the CMAVE Center Director and invitations for three CMAVE researchers to give oral presentations in Geneva, Switzerland.

1. Gene silencing, a novel method for mosquito control. Toxicants with new modes of action and high specificity are being investigated for mosquito control. Using gene silencing technology or RNA interference (RNAi), ARS scientists in Gainesville, FL have designed molecules that inhibit expression of critical proteins in mosquitoes that results in mortality. We have shown that these molecules can be delivered to adult mosquitoes through the cuticle, with other possible delivery methods under investigation. A new Cooperative Research and Development Agreement with will enable large-scale production of RNAi molecules to investigate new carriers and delivery methods to mosquitoes. We are currently awaiting issuance of a patent, which was applied for by the U.S. Department of Agriculture in March 2007.

2. High throughput screening for new toxicant discovery. The availability of toxicants for control of public health pests are rapidly dwindling due to regulatory issues and resistance development. ARS researchers in Gainesville, FL have developed high throughput methods to quickly screen large numbers of chemicals from industry and ARS collaborators for toxicity against mosquitoes. Using this methodology, more that 12,200 experimental compounds have been tested over the course of 2 years with 8% of the screened compounds resulting in >80% mortality. Subsequent testing of compounds with mosquitocidal activity has identified a small group that are currently undergoing more in-depth analysis to identify candidates with desirable attributes for development as new toxicants for mosquito control. Specific but effective toxicants can be used to control disease vectors of man and animals.

3. Mosquito immune response to a pathogenic virus. CuniNPV is a mosquito specific baculovirus that infects mosquito larvae within the genus Culex, important vectors of encephalitis viruses worldwide. ARS scientists in Gainesville, FL together with researchers at the University of Florida have investigated mosquito genes that are involved in the pro-apoptotic response to viral infection. Culex larvae were challenged with CuniNPV and the expression profile of the pro-apoptotic gene mx was measured. There was not a significant increase of mx expression before 8 hr post infection (p.i.) but the level of mx expression continued to increase throughout the infection period and at 48 hr p.i. was about 10 times higher than the uninfected controls. The induction of mx did not result in apoptosis but rather necrosis indicating that CuniNPV prevents apoptosis despite the very high level of mx expression. It is possible that CuniNPV utilizes an as yet unknown, but powerful, mechanism to block the apoptotic pathway downstream of mx activation. Identification of this mechanism could have important implications on how other viruses in mosquitoes evade the host immune response.

4. Evaluation of Fire Resistant Army Combat Uniforms (FRACUs) that are treated with repellent at the factory production level. The newest combat uniforms for the United States Army are made from fire resistant materials. Because of these materials, the uniform does not retain the permethrin repellent when field-treated. The solution to this problem is factory production of the uniform with repellent and binders designed to keep the fabric “insect proof” throughout the expected lifetime of the uniform. ARS researchers at Gainesville, FL assisted the U.S. Army by conducting tests on FRACUs to determine how well they prevented mosquitoes from biting through the fabric. The performance level was used to award contracts to companies seeking to supply factory-treated FRACUs to the U.S. Army. This method of assessment is currently recognized as the primary test method to evaluate permethrin-treated clothing and companies have submitted data from this research to the U.S. EPA for registering permethrin-treated clothing.

5. Efficacy of insecticide-treated barrier treatments on native vegetation and artificial substrates in desert, tropical and subtropical habitats for controlling mosquitoes and sand flies. Protection of deployed military from mosquitoes and flies that transmit disease is a critical component of successful military operations. Researchers at Gainesville, FL carried out field trials of bifenthrin and lambda-cyhalothrin as barrier treatments in the desert of Coachella Valley in southern California, sub-tropical environment of Florida, and a tropical desert environment in western Kenya, Iraq and Afghanistan to assess efficacy against natural mosquito and sand fly populations. Overall results from vegetation treatments indicate significant reduction in mosquitoes in field counts and lab assays for up to a month; overall results of material treatments indicate significant reduction mosquitoes or sand flies in field counts and lab assays for up to 18 months. The results suggest that, as an enhancement to the current DoD pest management system (such as use of DEET and permethrin treatment of uniforms) barrier treatments may be successful in providing protection from vector-borne diseases for deployed troops in desert habitats by significantly reducing densities of mosquitoes or sand flies reaching individual personnel in protected areas.

6. Spatial repellents were evaluated against mosquitoes and biting flies in tents. ARS researchers in Gainesville, FL conducted studies to evaluate the degree of protection provided by four commercially available spatial repellents for humans in tents from mosquitoes and biting flies. Two species of mosquitoes and one species of stable fly were used in these studies. The degree of protection varied with spatial repellent and species of insects. The results indicate that a single repellent device may not be effective against all species. Therefore, when activities are conducted in different locations it should be determined which species are present followed by selection of spatial repellent(s) that work best for those species.

7. Use of molecular modeling to design and develop new mosquito repellents. The United States military conducts operations worldwide and as part of this presence military personnel are exposed to numerous pathogens, especially in Africa where Plasmodium parasites carried by mosquitoes cause malaria. The mosquito species responsible for transmission of this disease are not easily repelled by the “gold standard” repellent N,N-diethyl-3-methylbenzamide (DEET). In an effort to discover new repellents for use against these malaria-carrying mosquitoes, ARS researchers at Gainesville, FL have used modeling and synthesis to predict and develop new chemicals that function well as mosquito repellents. These new compounds were tested against malaria-transmitting mosquito species, An. gambiae and An. quadrimaculatus. Two of the new repellents functioned as well as DEET against these species. Based on this discovery, additional modeling and synthesis may discover even more powerful repellents.

8. Comparison of thermal fog to Ultra Low Volume (ULV) insecticide usage in various environments. Currently, there is a lack of information regarding the potential efficacy of insecticide spraying in current U.S. military scenarios. ARS researchers in Gainesville, FL evaluated thermal fog and ULV applications in hot-dry desert environments. It was discovered that thermal fog applications may result in equal or greater mortality against sentinel mosquitoes when compared to ULV applications. In addition, there is strong evidence that the superiority of thermal fog over ULV with respect to mortality in sentinel mosquitoes holds up across two very different environments, in hot and humid sub-tropical Florida and in hot and dry deserts of southern California. Other studies in these habitats suggested that malathion was more efficacious in killing adult mosquitoes than were sumithrin-based products. Conversely, ULV studies conducted in Kenya against demonstrated that both malathion and sumithrin-based products were highly efficacious in killing adult sand flies which transmit leishmaniasis.

Review Publications
Clark, G.G. 2008. Dengue and Dengue Hemorrhagic Fever in Northeastern Mexico and South Texas: Do they really respect the border? American Society of Tropical Medicine and Hygiene. 78(3):361-362.

Bernier, U.R., Allan, S.A., Quinn, B.P., Kline, D.L., Barnard, D.R., Clark, G.G. 2008. Volatile compounds from the integument of white leghorn chickens (Gallus gallus domesticus L.): candidate attractants of ornithophilic mosquito species. Journal of Separation Science. 31:1092-1099.

Pridgeon, J.W., Bernier, U.R., Becnel, J.J. 2009. Toxicity comparison of eight repellents against four species of female mosquitoes. Journal of the American Mosquito Control Association. 25(2):168-173.

Britch, S.C., Linthicum, K., Wynn, W.W., Walker, T.W., Farooq, M., Smith, V.L., Robinson, C.A., Lothrop, B.B., Snelling, M., Gutierrez, A., Lothrop, H.D. 2009. Evaluation of barrier treatments on native vegetation in a southern California desert habitat. Journal of the American Mosquito Control Association. 25(2):184-193.

Anyamba, A., Chretien, J., Small, J., Tucker, C.J., Formenty, P., Richardson, J.H., Britch, S.C., Schnabel, D.C., Erickson, R.L., Linthicum, K. 2009. Prediction of a Rift Valley fever Outbreak. Proceedings of the National Academy of Sciences. 106(3):955-959.

Pridgeon, Y.W., Becnel, J.J., Clark, G.G., Linthicum, K. 2009. Permethrin induces overexpression of multiple genes in Aedes aegypti. Journal of Medical Entomology. 46(3):580-587.

Pridgeon, Y.W., Becnel, J.J., Clark, G.G., Linthicum, K. 2009. Permethrin Induces Overexpression of Cytochrome c Oxidase Subunit 3 in Aedes aegypti. Journal of Medical Entomology. 46(4):810-819.

Prdigeon, J.W., Webber, E.A., Sha, D., Li, L., Chin, L.S. 2009. Proteomic analysis reveals Hrs ubiquitin-interacting motif-mediated ubiquitin signaling in multiple cellular processes. The FEBS Journal. 276(1):118-131.

Zhao, L., Pridgeon, J. W., Becnel, J. J., Clark, G. G., Linthicum, K. J. 2009.Identification of genes differentially expressed during heat shock treatment of Aedes aegypti. Journal of Medical Entomology. 46(3):490-495.

Zhao, L., Becnel, J.J., Clark, G.G., Linthicum, K.J. 2010. Expression of AeaHsp26 and AeaHsp83 in Aedes aegypti (Diptera: Culicidae) larvae and pupae in response to heat shock stress. Journal of Medical Entomology. 47(3):367-375.

Pridgeon, J.W., Zhao, L., Becnel, J.J., Clark, G.G., Linthicum, K.J. 2008. Developmental and environmental regulation of AaeIAP1 transcript in Aedes aegypti. Journal of Medical Entomology. 45(6):1071-1079.

Doyle, M.A., Kline, D.L., Allan, S.A., Kaufman, P.E. 2009. Efficacy of residual bifenthrin applied to landscape vegetation against Aedes albopictus (Skuse) (Diptera: Culicidae). Journal of the American Mosquito Control Association. 25(2):179-183.

Last Modified: 4/18/2014
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