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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

2013 Annual Report

1a.Objectives (from AD-416):
1. Discover and evaluate new toxicants and biological control agents for control of biting Nematocera.

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.

3. Conceive and test new methods of managing vector and pest populations through the use of behavior-altering chemicals, including repellents, attractants, and inhibitors.

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.

5. Determine chemical and other cues associated with regulation and orientation of specific behaviors by Aedes albopictus, Culex pipiens group species/hybrids, and other biting arthropods that can be applied to the solution of operational surveillance and control problems.

6. Discover and characterize environmental predictors of the distribution of mosquitoes in order to assess the risk of invasive species and pathogen transmission. Apply to the development of methods and techniques to accurately assess mosquito population density, to deploy vector surveillance systems, and to detect exotic invasive species.

1b.Approach (from AD-416):
1. High throughput bioassays will be used to screen toxicants from synthetic compound and natural product libraries. New formulations of bacterial toxins, baculoviruses, and biorationals will be evaluated in standard assays. Molecular methods (RNAi and dsRNA) will be used to identify targets for control and resistance management. Improved formulation and delivery of dsRNA will be developed to penetrate the mosquito cuticle.

2. Factors affecting measurement of bite protection by permethrin-treated clothing will be examined. Alternative repellents applied to military uniforms and improved binding of repellents to fabric will be researched, followed by laboratory validation of factory-treated fabric and semi-field studies. Aerosol application of control compounds, formulations, equipment, application techniques, and strategies will be explored. 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. Factors needed to achieve optimal control will be identified. Air curtains will be designed and evaluated in simulated aircraft fuselages.

3. Comparison of pathogen-infected and uninfected mosquito responses to behavior-altering chemicals will be examined. Baited commercial traps will be used as surrogates for human and livestock hosts. The manner in which compounds affect host-seeking behavior will be quantified. Catches in control and attractant-baited traps located near devices releasing spatial repellents or inhibitors will be compared.

4. The effect of mosquito fatigue on repellent protection time will be evaluated with female Aedes aegypti and a study comparing methods for measuring repellent efficacy will be conducted. Laboratory results of repellent efficacy will be compared to products tested in the semi-field environment.

5. Factors that result in positive resting site selection responses by adult mosquitoes, volatile chemical cues associated with mate location, and cues used for location and utilization of sugar and nectar sources will be determined. Plant-derived compounds that attract mosquitoes will be evaluated in the field. Factors that influence host-finding behavior based on host odors and olfactory cues will be used to enhance traps. Novel strategies for mosquito surveillance that utilize oviposition site cues will be devised. New chemical attractants will be identified and evaluated in the field using behavioral and chemical analyses. The response to toxicant exposure on arthropod behavior will be evaluated to improve toxicant impact.

6. Methods and techniques to assess mosquito population density will be developed. Environmental predictors of mosquito distribution will be characterized to provide improved strategies for assessment of mosquito populations. Discover ecologic and climatic factors to assess population densities of vector mosquitoes in the US and, using Rift Valley fever, evaluate the risk of exotic species and disease introduction into the U.S.

3.Progress Report:
Pathogens of biting Nematocera cause diseases such as West Nile virus, dengue, malaria, leishmaniasis and filariasis to humans and animals in urban, suburban, and rural areas and in agricultural, recreational, and military environments. This research project by ARS researchers at Gainesville, FL, 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. A dsRNA fed to adult Ae. aegypti in a sugar meal resulted in gene silencing at 12 and 24 hour post-feeding. A total of 34 trifluoromethylphenyl amides were synthesized and evaluated for their insecticidal properties. At 20 times lower concentration than fipronil, one of these compounds produced 100% mortality in adult Ae. aegypti. The genomes of three different mosquito pathogens were sequenced. Completed functional studies of pathogen genes involved in critical host–pathogen interactions and genes up-regulated by the mosquito in response to infections. Completed first article testing of fire-resistant U.S Army combat uniforms. Determined that degradation of permethrin in field-worn uniforms was likely due to the effects of abrasion, rather than from exposure to UV from sunlight. A study on efficacy and physical properties of aerial ultra low volume (ULV) permethrin applications in an extreme hot-dry desert environment against mosquitoes was completed. Aerial adulticiding against medically important mosquitoes substantially reduced mosquito-human contact and can minimize transmission of mosquito-borne viruses to military troops deployed in desert areas. A prototype air curtain was evaluated for prevention of insects from entering aircraft. A total of 172 compounds were tested as candidate repellents; 43 of these were nearly as potent as the standard N,N-diethyl-3-benzamide (DEET) repellent. Synthetic and natural spatial repellents were evaluated in semi-field settings and reduced mosquito populations in local areas. Extracts from three native plants were attractive to Ae. albopictus in laboratory bioassays. Field studies with baited-traps demonstrated that species-specific traps can be developed to target mosquito species. A model was developed for mosquito resting site selection; this will improve Culex quinquefasciatus surveillance and monitoring via improved artificial shelter designs. Data from a mosquito trapping study where 21 species were collected provided a correlation between sampling method and the estimation of adult mosquito density. This information enhances accuracy of GIS-based surveillance and monitoring systems. The effect of habitat and ecosystem in relation to mosquito catches in traps was studied to develop improved models for the ability of traps to capture mosquitoes in different environments. Chemical attractants for the brown dog tick were discovered and these compounds are candidates for use in commercial traps. Research was conducted that indicated correlation among the spatial and temporal patterns of Rift Valley Fever (RVF) infections in wildlife and the spatial and temporal patterns of RVF predicted by the African RVF model.

1. Developed gene silencing technology for mosquito control. There are a very limited number of public health pesticides available for controlling medically important vectors, such as mosquitoes and sand flies. This novel approach is based on the technology that allows for the specific silencing of genes critical to survival of the target vector species. This technology uses double stranded RNA (dsRNA) and the process of RNA interference (RNAi) to selectively silence gene products (proteins) that debilitate the mosquito vector and prevent disease transmission. ARS researchers in Gainesville, FL have demonstrated that a dsRNA construct targeting a gut-expressed gene fed to adult Ae. aegypti in a sugar meal results in gene silencing at 12 and 24 hour post-feeding. This discovery provides a delivery method for controlling mosquitoes using sugar baits containing dsRNA for specific genes. Incorporation of toxic sugar traps that can kill or debilitate mosquitoes and/or the pathogens they vector can compliment current vector control strategies such as insecticide treated bednets (ITNs) and indoor residual spraying (IRS) programs to control important vectors of malaria and arboviruses to man and animals.

2. Completed mosquito bite protection analysis for the U.S. Army to produce permethrin-treated U.S. Army Fire Resistant Army Combat Uniform-III (FRACU-III) with Operation Enduring Freedom Camouflage Pattern (OCP). ARS researchers in Gainesville, FL evaluated the performance of FRACU uniforms worn in the field for 90 days and determined that uniforms retained a satisfactory level of efficacy after 90 days of field use. Conducted preliminary studies on an improved uniform/spatial repellent system and determined that bite protection was enhanced by incorporation of strategically located spatial repellent devices. Conducted studies on the UV-aging and environmental aging of uniform fabric. Results of these studies implicate abrasion as a significant reason that uniforms lose permethrin and that UV radiation is not the primary cause for permethrin loss in field worn uniforms.

3. Discovered a link between Rift Valley Fever (RVF) infections observed in wildlife and predicted regions and timing of RVF activity predicted by the African RVF Model. Researchers at ARS in Gainesville, FL examined serology data from Kenyan wild ungulates and camel and discovered that rates of infections across a broad range of animals reflected the predicted density and timing of RVF transmission risk throughout the Horn of Africa. These results were reported in the journal PLoS One, highlighting that this is the first observed pattern of this kind, that the results can further inform and calibrate the African RVF model, and that the results should guide future structured serosurveys in susceptible wildlife and livestock in RVF-endemic regions to better protect humans and animals in both endemic and emerging areas.

4. The comparative responses of 21 species of mosquitoes to light traps (LT) and suction traps (ST) and captured using the human landing collection method (HL) varied in accordance with collection technique. Data analyses for most species revealed a significant interaction between collection method and collection interval (time of day collections were made). Indices of species diversity varied significantly according to collection method. Eight mosquito species (38%) were undetected by at least one of the three collection methods and five species (14%) were undetected by two of the collection methods. ARS researchers at Gainesville, FL determined that these data enable the development of species-specific numeric algorithms that can be used to relate mechanical trap-based estimates of adult mosquito density to the mosquito landing rate on a human being. The data provide a basis for understanding the relative effects of sampling method on mosquito responses and the estimation of adult mosquito density and are a critical prelude to the design and development of GIS-based vector detection and surveillance systems.

5. Conducted a seminal study on the efficacy and physical properties of aerial ultra low volume (ULV) applications of permethrin in an extreme hot-dry desert environment against mosquitoes. ARS researchers at Gainesville, FL with collaborators successfully completed the first aerial application of permethrin that simultaneously measured the efficacy of the spray using sentinel mosquitoes and the quantity of the spray deposited. The results indicate that despite unfavorable conditions for traditional aerial ULV application, sentinel mosquito mortality and laboratory analysis of pesticide deposit collection surfaces demonstrate that adulticiding against medically important mosquitoes using aircraft can substantially reduce mosquito-human contact and minimize transmission of mosquito-borne viruses to military troops deployed in desert areas.

6. Conducted a seminal study on the ability of high-abundance U.S. mosquito species collected from key ecological regions to transmit Rift Valley Fever (RVF) virus should the disease arrive in the U.S. Researchers at ARS in Gainesville, FL with collaborators successfully completed the first RVF vector competence studies for U.S. populations of Coquillettidia, Mansonia, and Psorophora mosquito genera. These findings, submitted to the Journal of Medical Entomology, indicate in particular that Coquillettidia and Psorophora species, both species that occur in abundance and readily attack humans and livestock, should be targeted for control should RVF be introduced into the U.S. The outcome of these findings will significantly improve the U.S. RVF predictive risk assessment system by guiding the targeted distribution of disease vector surveillance and control assets in the U.S., which will have a direct impact on reduction of RVF disease transmission risk to humans and susceptible livestock.

7. The first air curtain unit designed specifically for use on aircraft was tested by ARS researchers at Gainesville, FL in June, 2013. Results were impressive but several modifications need to be made to the design to perfect the airflow for maximum exclusion of insects. Width of air flow from fan units needs to be wider and air flow stronger. Design changes are underway and an additional test will be scheduled for later in the year.

8. Evaluation of behavior altering spatial repellents. ARS researchers in Gainesville, FL conducted studies with government, university, military and private industry collaborators. The problem is to prevent or minimize host-vector contact. Semi-field and field studies were conducted with various natural plant produced compounds and synthetic chemicals to deter pest mosquito species from locating potential hosts. Several compounds were identified with potential to achieve this objective. However, they need to be optimized in terms of optimal release concentrations and delivery technology.

9. Designed and synthesized 34 trifluoromethylphenyl amides as new insecticide candidates. These compounds have been evaluated in high throughput larval and adult screening assays by ARS researchers in Gainesville, FL. The activity range of each chemical was determined for both first instar larvae and adult female Aedes aegypti. The 7 compounds that exhibited the greatest potency from these screening assays were evaluated further in feeding bioassays. One compound produced more rapid knockdown than the standard fipronil, and this high level of rapid mortality was achieved even at concentration levels 20 times lower than fipronil. An invention disclosure will be filed for this novel insecticide.

10. Evaluated 172 candidate repellents in conjunction with ARS researchers at Gainesville, FL and external cooperators. A total of 43 of these were very potent repellents (MED < 0.1 mg/cm2). Included in these are Mosquito and Fly Research Unit synthesized and novel fluorinated carboxamides. Three of the compounds were potent repellents, exhibiting a repellent potency against Aedes aegypti nearly equal to that of the standard N,N-diethyl-3-methylbenzamide (DEET). Two manuscripts on collaborative studies with natural repellents have been published.

11. Light traps and suction traps were used to sample and compare adult mosquito population densities in open, ecotone, and wooded zones within a Florida wetlands habitat. Apparent mosquito density in each zone varied according to the method of collection and the time of day collections were made. ARS researchers at Gainesville, FL observed that open zone collections were least variable but indicated a less diverse species composition of captured mosquitoes (regardless of trapping method) than observed in the ecotone and wooded zones. Mosquito capture responses were evaluated for correlation with trap type and the physiographic and biological parameters for each zone and a qualitative model developed to facilitate the design and testing of a stratified sampling (i.e., trap deployment) plan. Data from this study will enable simulation modeling of the effects of habitat and other environmental variables on mosquito responses to mechanical traps. This knowledge is an important prerequisite to the design and development of mosquito trap deployment strategies that will be used in vector detection and surveillance systems.

12. Cues used by mosquitoes to locate nectar/sugar sources. Plant odors alert mosquitoes to the location of sugar and nectar meals, which mosquitoes use for survival. A study on the phenology of flowering plants was conducted in north central Florida. Native plants such as milkweed, black mangrove, and butterfly bush were provided to individual homeowners, homeowner associations and businesses by nurseries. The responses of Aedes albopictus species were evaluated in dual-choice olfactometer studies. Intact flowers, solvent extracts and some identified volatile compounds from the these plants were found to be very attractive to both male and female mosquitoes. Chemical characterization should lead to development an artificial blend which can then be field tested against natural populations of Ae. albopictus and woodland species. This information will be useful in the development of improved surveillance technology and possibly the development of traps/targets for the population management of these mosquito species.

13. Studies of natural structures used as refuge by resting adult mosquitoes indicate the faunal composition and density of mosquitoes they shelter to vary significantly within and between structure types. Methods-development research to standardize the use of mechanical aspiration-based collection equipment and the use of a borescope for observing resting mosquito responses to natural structures is in progress. In semi-field studies, resting site selection responses of Culex quinquefasciatus to 30 cm cubic "red boxes" correlated with light intensity and air temperature. Other correlates of mosquito entry/resting/exit activity under semi-field conditions were moisture content and surface temperature of the resting site substrate. These data were used to develop a general model of mosquito resting site selection responses. Important parameters identified by this process include light intensity, air temperature, resting substrate surface temperature, resting substrate moisture content, and elevation and aspect of the resting structure entrance. The results provide a basis for the design and development of artificial resting shelters that may be useful for the detection and surveillance of vector mosquito species including Culex quinquefasciatus.

14. Factors that influence nectar feeding, host-finding, and oviposition behaviors. Factors were investigated that focused on identifying key attractants/repellents that can be utilized in optimizing surveillance and population management technologies. Mosquitoes utilize different combinations of visual, physical and chemical odor blends and delivery rates to locate the nectar sources, potential hosts, and oviposition sites. Our studies indicate that species-specific traps can be developed if these factors are optimized for each target species. “One size does not fit all” species.

15. Conducted genomic and functional studies on mosquito pathogens. Microbial pathogens have developed strategies to successfully infect, evade or debilitate host defense systems and kill mosquitoes. Researchers at ARS in Gainesville, FL, together with collaborators have.
1)completed the sequencing and annotation of the genomes of three different mosquito pathogens and.
2)conducted functional studies to examine pathogen genes involved critical host –pathogen interactions as well as genes up regulated by the mosquito in response to infections. This study will lead to the identification of critical genes involved in pathogen -mosquito interactions at the protein level related to immune response, host range and virulence that can be exploited for novel control strategies.

16. New attractants for monitoring ticks. The brown dog tick is one of the most common pests of residences and dog kennels with repeated applications of pesticide representing significant exposure of humans to pesticide residues. Several chemicals recently discovered by MFRU scientists to be attractive to brown dog ticks were incorporated into a commercial bedbug trap and found to be highly effective in collecting ticks in infested rooms. This surveillance device can serve as the basis for an attract and kill device in residences or kennels where ticks are present and potentially reduce human pesticide exposure.

Review Publications
Kiang, R.K., Soebiyanto, R.P., Grieco, J.P., Achee, N.L., Harrington, L.C., Reisen, W.K., Anyamba, A., Linthicum, K., Pinzon, J.C., Zollner, G., Colacicco-Mayhugh, M. 2012. Vector-borne infectious diseases and influenza. In: Morain, S.A. and Budge, A.M., editors. Environmental tracking for Public health Surveillance. London: Taylor and Francis Group. p. 21-86.

Revay, E.E., Junnila, A., Kline, D.L., Xue, R., Bernier, U.R., Kravchenko, V.D., Yefremova, Z.A., Mulller, G.C. 2012. Reduction of mosquito biting pressure by timed-release 0.3% aerosolized geraniol. ACTA TROPICA. 124:102-105.

Obermayr, U., Ruther, J., Bernier, U.R., Rose, A., Geier, M. 2012. Laboratory evaluation techniques to investigate the spatial potential of repellents for push & pull mosquito control systems. Journal of Medical Entomology. 49(6):1387-1397.

Aldridge, R.L., Wynn, W.W., Britch, S.C., Allan, S.A., Walker, T.W., Geden, C.J., Hogsette, Jr, J.A., Linthicum, K. 2013. High throughput mosquito and fly bioassay system for natural and artificial substrates. Journal of the American Mosquito Control Association. 29(1):84-87.

Phasomkusolsil, S., Tanskul, P., Ratanatham, S., Watcharapichat, P., Phulsuksombati, D., Frances, S.P., Lerdthusnee, K., Linthicum, K. 2012. Influence of orientia tsutsugamushi infection on the developmental biology of leptotrombidium imphalum and leptotrombidium chiangraiensis (Acari: Trombiculidae). Journal of Medical Entomology. 49(6):1270-1275. DOI: 10.1603/ME12100.

Clark, G.G., Rubio-Palis, Y. 2012. Mosquito vector biology and control in Latin America - a 22nd Symposium. Journal of the American Mosquito Control Association. 28(2):102-110.

Kovendan, K., Murugan, K., Vincent, S., Barnard, D.R. 2012. Efficacy of larvividal and pupicidal properties of Acalypha alnifolia Klen ex Willd. (Euphorbiaceae) leaf extract and Metarhisium anisopilae (Metsch.) against Culex quinquefasciatus Say. (Diptera: Cuclicidae). Journal of Biopesticides. 5 (supplementary):170-176.

Rosa, L.H., Tabanca, N., Techen, N., Wedge, D.E., Pan, Z., Bernier, U.R., Becnel, J.J., Agramonte, N.M., Walker, L., Moraes, R. 2012. Diversity and biological activities of endophytic fungi associated with micropropagated medicinal plant Echinacea purpurea (L.) Moench. American Journal of Plant Sciences. 3:1105-1114.

Kovendan, K., Murugan, K., Vincent, S., Barnard, D.R. 2012. Mosquito larvicidal properties of Orthisiphon thymiflorus (Roth) Sleesen. (Family: Labiatae) against mosquito vectors, Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). Asian Pacific Journal of Clinical Nutrition. 5(4):299-305.

Dees, W.H., Sylvester, T.L., Clark, B.M., Canning, L.D., Schultz, G.W., Kline, D.L. 2012. An innovative mosquito trap for testing attractants. Journal of the American Mosquito Control Association. 28(1):62-64.

Sterling, K.M., Okech, B.A., Xiang, M.A., Linser, P.J., Price, D.A., Vanekeris, L., Becnel, J.J., Harvey, W.R. 2012. High affinity 3H-phenylalanine uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBBMVw). Journal of Insect Physiology. 58:580-589.

Muttis, E., Miele, S.A., Belaich, M.N., Micieli, M.V., Becnel, J.J., Ghiringhelli, P.D., Garcia, J.J. 2012. First record of a mosquito iridescent virus in Culex pipiens L. (Diptera: Culicidae). Archives of Virology. 157:1569-1571. DOI: 10.1007/s00705-012-1302-2.

Zettel Nalen, C.M., Allan, S.A., Becnel, J.J., Kaufman, P.E. 2013. Oviposition substrate selection by Florida mosquitoes in response to pathogen-infected conspecific larvae. Journal of Vector Ecology. 38(1):182-187.

Cooperband, M.F., Hartness, A., Lelito, J.P., Cosse, A.A. 2013. Landing surface color preferences of Spathius agrili (Hymenoptera: Braconidae), a parasitoid of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae). Journal of Insect Behavior. 26(5):721-729. DOI:10.1007/s10905-013-9387-3.

Liu, B., Behura, S.K., Clem, R.J., Schneemann, A., Becnel, J.J., Severson, D., Zhou, L. 2013. P53-mediated rapid induction of apoptosis conveys resistance to viral infection in Drosophila melanogaster. PLoS Pathogens. 9(2):1-12.

Ozek, G., Ishmuratova, M., Tabanca, N., Radwan, M.M., Goger, F., Ozek, T., Wedge, D.E., Becnel, J.J., Cutler, S.J., Baser, K. 2012. One-step multiple component isolation from the oil of Crinitaria tatarica (Less) Sojak by preparative capillary gas chromatography with characterization by spectroscopic and spectrometric techniques. Journal of Separation Science. 35:650-660.

Bernier, U.R., Tsikolia, M. 2011. Development of novel repellents using structure-activity modeling of compounds in the USDA archival database. In: Paluch, G.E., Coats, J.R. In Recent Developments in Invertebrate Repellents. American Chemical Society. 1090:21-46.

Choi, M.Y., Estep, A., Sanscrainte, N.D., Becnel, J.J., Vander Meer, R.K. 2013. Identification and expression of PBAN/diapause hormone and receptors from Aedes aegypti. Molecular and Cellular Endocrinology. 375: 113-120.

Tabanca, N., Ali, A., Bernier, U.R., Khan, I.A., Kocyigit-Kaymakciogl, B., Oruc-Emre, E.E., Unsalan, S., Rollas, S. 2012. Biting deterrence and insecticidal activity of hydrazide–hydrazones and their corresponding 3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles against Aedes aegypti. Pest Management Science. 69:703-708.

Last Modified: 4/17/2014
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