Research Project: Integrated Pest Management of Mosquitoes and Biting Flies

Location: Mosquito and Fly Research

2021 Annual Report

Objectives
1. Discover and characterize factors that impact mosquito and biting fly distribution and the threat of disease outbreaks. 2. Determine the impact of resistance to public health pesticides on control of mosquitoes and biting flies and develop approaches to overcome insecticide resistance. 2.A. Determine, monitor, and map the resistance status of natural populations. 2.B. Sterile insect technique. 2.C. Novel spatial repellents and insecticides to circumvent pyrethroid resistance. 2.D. Natural restoration of insecticide susceptibility in Aedes aegypti. 3. Develop novel strategies and technologies for more accurate and efficient surveillance and monitoring of mosquitoes and biting flies. 4. Develop novel strategies and new products that lead to improved control of mosquitoes and biting flies. 4.A. Evaluate new fabric treatments and optimize existing treatments to provide improved protection from insect bites through military and civilian clothing. 4.B. Evaluate and optimize spatial repellent systems that protect hosts from arthropods in a local area. 4.C. Evaluate new and optimize existing treated targets. 4.D. Evaluate factors that influence efficacy of aerosol and residual control techniques in various ecological habitats; design the best application methods to mitigate changing climate. 4.E. Discover and develop new attractants for mosquitoes and other biting arthropods. 4.F. Discover and develop new repellents for mosquitoes and other biting arthropods.

Approach
Objective 1 will discover and characterize factors impacting mosquito and biting fly distribution and the threat of disease outbreaks (Hypothesis 1: Dynamic environmental factors predict mosquito vector population timing, distribution, and densities, and thus exotic mosquito-borne disease risk). Objective 2 will determine, monitor, and map resistance status of natural populations (Hypothesis 2.A. Sodium channel mutations can be used to predict toxicological pyrethroid resistance). Sterile insect technique will be developed for mosquito management (Hypothesis 2.B. Release of sterile irradiated Ae. aegypti males can suppress natural populations in endemic regions). Objective 2 will evaluate novel spatial repellents and insecticides to circumvent pyrethroid resistance (Hypothesis 2.C. Novel spatial repellents can be discovered that will be efficacious on both susceptible and resistant strains of mosquitoes). It will also restore insecticide susceptibility in Aedes aegypti using natural techniques (Hypothesis 2.D. Reintroduction of pyrethroid susceptible adults into populations of strongly resistant adults will return susceptibility allowing longer efficacy or renewed usefulness of existing pyrethroids). Objective 3 will develop novel strategies and technologies for improved surveillance and monitoring of mosquitoes and biting flies (Hypothesis 3. Evaluate new and optimize existing trapping systems. Changes in H-trap design will improve vector species surveillance). Objective 4 will evaluate fabric treatments for improved protection from insect bites through clothing (Hypothesis 4.A. Factors related to fabric composition, construction, and repellent treatments can be optimized to provide improved levels of bite protection from mosquitoes). It will also evaluate and optimize spatial repellent systems (Hypothesis 4.B. Devices that release spatial repellents can reduce host-vector contact by mosquitoes and other biting flies). Objective 4 will evaluate new and optimize existing treated targets for management of mosquitoes and biting flies (Hypothesis 4.C. Insecticide impregnated targets can effectively reduce nuisance mosquito populations). It will evaluate factors that influence efficacy of aerosol and residual control techniques. It will also design the best application methods to mitigate the effects of changing climate (Hypothesis 4.D. Populations of mosquito, sand fly, and filth fly disease vectors may be reduced by accounting for environmental factors that limit efficacy of aerosol and residual pesticide treatments). Objective 4 will discover and develop new attractants for mosquitoes and other biting arthropods to improve trap efficacy. It will also discover and develop new repellents for mosquitoes and other biting arthropods (Hypothesis 4.E. Mosquitoes are selective in choosing and use flower volatiles to locate preferred nectar sources).

Progress Report
1. With NASA partners, ARS researchers at Gainesville, Florida, produced the first global map of areas at risk for introduction and spread of Rift Valley fever virus during periods of historically seasonal activity of this virus in endemic areas focused in the Horn of Africa. This map is a critical first step in identifying pathways and modes of spread of this virus that can have serious and lasting health and economic impacts in affected areas. For example, the map clearly highlights the vulnerability of the southern half of the US as well as southern Europe in winter months, which will enable development of monitoring systems to protect public health and livestock economies in these areas. 2A. More than 110 strains of Culex quinquefasciatus have been collected from Florida, Louisiana, and Illinois with the assistance of several universities and numerous local vector control collaborators. These strains have been tested for the presence of insecticide resistance by toxicological and genetic screening. By collecting strains in three distinct geographical areas within the United States, researchers in Gainesville, Florida, elucidated wide area patterns of insecticide resistance. In laboratory toxicological studies using isolated knockdown resistance strains from Louisiana researchers at Gainesville, Florida, were able to define the specific impact of genetic resistance and determined that it is a contributor, but not the major factor, in the overall resistance level. The wide area surveyed in this project allowed us to conclude that although resistance to pyrethroids is widely present and generally strong in Cx. quinquefasciatus, the widespread use of synergized mosquito control products remains highly effective. The study conducted in Gainesville, Florida, represents the largest survey to date of Culex resistance in the United States. Two of three manuscripts from this project are in the final stages of preparation for submission. 2B. Researchers at Gainesville, Florida: 1) kept monitoring the population of Ae. aegypti throughout the year, and 2) conducted a weekly release of sterile males when temperatures were warm enough for sterile males to be effective, but well before we see any evidence of wild populations building to get ahead of the local Ae. aegypti populations and effect control. Researchers at Gainesville, Florida, continued weekly monitoring of Ae. aegypti populations at our intervention site and our non-intervention site with 24 BG Sentinel traps and 34 ovicups in each area. We found the female population was low overwinter (first quarter of 2021), with fewer than 30 females trapped each week through the end of March 2021. The release of sterile males in our intervention site was restarted in the second quarter of 2021 and has progressed with two releases each week. The released to wild male ratio exceeded 10:1 for every release and was typically over 75:1, well exceeding our target ratio of 7:1. We published 2 scientific journal articles. 2C. ARS scientists at Gainesville, Florida, and University of Florida synthesized > 200 novel molecules and screened > 100 in a micro glass tube assay to assess vapor repellency effectiveness in 1-hr exposures to Aedes aegypti or Anopheles gambiae mosquitoes. We were able to describe the testing of new chemicals for their ability to repel mosquitoes without contact, a phenomenon known as spatial repellency. In particular, the spatial repellent activity and toxicity of two novel pyridinyl amides (1 and 2) were evaluated against 3 mosquito species that can transmit malaria, Anopheles albimanus, Anopheles quadrimaculatus, and Anopheles gambiae. In laboratory spatial repellency evaluations, compound 2 was generally more effective than DEET, while compound 1 was about as active as DEET and other common repellency standards. Transfluthrin was the most active compound for inducing Anopheles mosquito repellency, knockdown, and lethality. Overall, this study provides insight for further synthesis of alternative amide compounds for use as spatial treatments. 2D. Laboratory and semi-field studies using the Aedes aegypti resistance isolines developed in previous milestones helped to define the factors important for implementation of Accelerated Reintroduction of Susceptibility to Insecticides (ARS-I). Aerial spray testing of these strains with Vector Control District Collaborators determined that several single ingredient products widely used in mosquito control were not effective against the kdr strains. Wind tunnel testing demonstrated that synergized pyrethroids, the most common type of vector control sprays, were effective against all but the most resistant strains. Initial laboratory cross mating studies between the most resistant strain and less resistant strains resulted in offspring that were much more susceptible to control interventions. We also demonstrated the efficacy of new formulations of the organophosphate malathion regardless of the strength of the pyrethroid resistance. 3. A series of Latin square experiments are currently in progress in the field against natural populations of Tabanidae. One series is comparing trap color with a standard model. Two additional series are emphasizing the use of alternative baits and/or decoys. Preliminary data collections indicate that all 3 parameters affect species composition and abundance of specimens collected. 4A. Testing of permethrin/etofenprox-treated fabrics for the military has been progressing. Two large studies to evaluate the mosquito bite-protection efficacy of heat-resistant and fire-resistant fabrics for military personnel have been completed. While COVID 19 has significantly reduced our throughput for these human subject tests, we have been able to prioritize this work as it benefits both the USDA and US military. We plan in the coming months and years to continue evaluating fabrics treated with novel materials, as well as some new materials that act as physical barriers which prevent mosquito bites. 4B. Military bootlaces were treated with various formulations of transfluthrin and evaluated in the laboratory in small (18 x 18 x 18 in) screen cages against female Aedes aegypti to determine their efficacy. Each bootlace was tested weekly to determine their duration of causing >90% knockdown in 15 minutes or less. In between testing the bootlaces were aged outside under ambient conditions. This level of efficacy was maintained for 3 months with all formulations tested. Currently in progress are evaluations in outdoor large (30 ft x 60 ft) screened cages to determine the ability of these treated bootlaces to prevent host-human contact. Free flying mosquitoes are released into the cage. A Biogents Sentinel trap (BGS-2) baited with carbon dioxide and human scent lure is used as the surrogate host. A pair of boots with treated bootlaces is placed near the trap. Reduction in trap collections, which indicate protection by the bootlaces, have been achieved for > 3 months. Tent entrance devices are being developed and optimized to prevent mosquito entry into a military headquarters type tent and protect individuals inside from mosquito bites. The BGS-2 trap is being used as the surrogate host. 4C. Various target types were evaluated against mosquitoes and stable flies. Targets were either stand alone or manufactured from various treated materials and attached to baited traps. Targets were treated with permethrin or deltamethrin. These studies were interrupted due to lockdown of CMAVE during the pandemic. Prior to the lockdown promising results were being obtained against stable flies and various species of mosquitoes. 4D. Experiments were conducted in a warm temperate north Florida long-term research site to investigate capabilities of existing and novel spatial repellent formulations and delivery systems to reduce populations of disease vector mosquitoes. We conducted aerial larvicide trials targeting disease vector Aedes species mosquitoes through exposed and cryptic environments in a simulated urban habitat to determine whether this combinations of formulation and application technique could suppress this important species. We developed a working public beta of the Mobile Pesticide App, a decision support system designed to extract optimized vector control solutions from more than 15 years of field test data from this lab specific to environment, formulation, application equipment, target insect, and application technique. 4E. Emphasis continued to be placed on determining flowering plants located near known mosquito developmental sites. Flowering plants near these sites were identified and catalogued. 4E. Some olfactometer evaluations were conducted utilizing freshly picked flowers against both sexes of Aedes aegypti. Collection and identification of volatile chemicals emanating from these flowers were suspended during the CMAVE lockdown but will be continued during the next bloom season. 4F. Although, pyrethroid-treated fabrics have been an incredibly useful tool in preventing mosquito-borne disease, particularly in US military personnel, new compounds must be developed to protect soldiers and civilians against mosquitoes that are resistant to pyrethroids. Researchers with USDA-ARS-CMAVE in Gainesville, Florida, have screened approximately 30 chemical repellent molecules in cloth-patch testing to evaluate the most efficacious repellents when applied directly to fabric. We have identified several compounds that repel mosquitoes with a similar efficacy to DEET. We have also demonstrated that trans-chrysanthemic acid and (1R,3S)-3-(2,2-dichloroethenyl)-2,2,-dimethylcyclopropanecarboxylic acid, are potent synergists of various topical repellents, improving their minimum effective doses (the lowest dose required to prevent 95% of bites) by over 30-fold. These studies will continue to inform which compounds are the most ideal candidates for future topical repellents applied to fabric.

Accomplishments
1. Methodology to produce large numbers of irradiated Aedes aegypti males. ARS scientists at Gainesville, Florida, developed a novel methodology to produce 10’s of thousands of irradiated male Aedes aegypti mosquitoes for use by Mosquito Control Agencies for release in Sterile Insect Technique (SIT) programs. These standard techniques have been described and published in the Journal of Visualized Experiments and should facilitate the production of irradiated males required for mosquito SIT programs.

2. Synthesized and natural compounds tested against mosquitoes. A subset of more than 200 synthesized novel compounds has been identified by ARS scientists at Gainesville, Florida, as repellents and insecticides and has been screened against multiple mosquito species. Natural products have also been screened as repellents, insecticides, and synergists and these studies have led to the publication of one manuscript with another submitted. A novel method has also been developed for screening candidate insecticides/repellents directly on the mosquito and house fly central nervous systems. This technique will allow for the faster identification of future insecticides/repellents before costly and time-intensive bioassays are pursued. A manuscript pertaining to this novel method has also been submitted.

3. ARS scientists at Gainesville, Florida, developed novel spatial repellent dispensing devices for the protection of individual and garrisoned soldiers. Military bootlaces were treated with various formulations of spatial repellents and tested under laboratory and semi-field conditions. They provided effective repellency against Aedes aegypti for >3months. Novel tent entrance devices were designed and evaluated under semi-field conditions and provided significant repellency against the ability of 4 mosquito species of 3 genera to locate a baited trap which served as a surrogate host.

4. Wide area aerial application of larvicide targeting disease vector Aedes aegypti mosquitoes in urban environment. The Aedes aegypti mosquito is a key vector of prominent viruses including dengue, chikungunya, Zika, yellow fever, Mayaro, and Madariaga and is very difficult to control. This mosquito displays increasing resistance to pesticides targeting their adult stage, potentially favoring operational control programs that include larval control. However, mosquito control programs face challenges in accessing abundant, cryptic Aedes mosquito immature habitat inside and around urban and suburban structures. Scientists at Gainesville, Florida, in partnership with the U.S. Air Force Aerial Spray Unit (Vienna, OH) conducted a large-scale aerial application of an advanced larvicide formulation applied as a residual with a modular aerial spray system over a simulated urban habitat in a warm-temperate environment in north Florida. Previous trials with this formulation over open ground indicated efficacy against this species in this environment. However, results from the aerial application over the reticulate urban habitat indicated little impact on development of colony reared immature Aedes aegypti to adult. These results suggested that aerial sprays targeting Aedes aegypti in urban settings need further development, to include investigation of additional larvicide formulations, spray systems, and spray techniques specifically designed to disperse through this unique habitat.

Review Publications
Aryaprema, V.S., Blore, K., Aldridge, R.L., Kline, J., Linthicum, K., Xue, R. 2021. Semi-field evaluation of ultra-low volume (ulv) ground spray of aqalure® 20-20 against irradiated aedes aegypti. Journal of the Florida Mosquito Control Association. 68(1):63-69.
Richoux, G.M., Yang, L., Norris, E.J., Tsikolia, M., Jiang, S., Linthicum, K., Bloomquist, J.R. 2020. Structure-activity relationship analysis of potential new vapor-active insect repellents. Journal of Agricultural and Food Chemistry. 68(47):13960-13969. https://doi.org/10.1021/acs.jafc.0c03333.
Flores-Mendoza, C., Loyola, S., Jiang, J., Farris, C.M., Mullins, K., Estep Iii, A.S., Fisher, M.L., Richards, A.L. 2021. Molecular characterization of bartonella species discovered in ectoparasites collected from domestic animals, cuzco, peru. Vector-Borne and Zoonotic Diseases. https://doi.org/10.1089/vbz.2020.2697.
Al-Massarani, S.M., El-Gamal, A.A., Al-Rehaily, A.J., Al-Sheddi, E.S., Al-Oqail, M.A., Farshoori, N.N., Estep, A.S., Tabanca, N., Becnel, J.J. 2021. Insecticidal activity and free radical scavenging properties of isolated phytoconstituents from the saudi plant nuxia oppositifolia (hochst). Molecules. 26(4):1-8. https://doi.org/10.3390/molecules26040914.
Britch, S.C., Dame, D.A., Meisch, M.A., Kline, D.L., Walker, T.W., Allan, S.A., Urban, J., Aldridge, R.L., Linthicum, K. 2021. Spatial repellents protect small perimeters from riceland mosquitoes in a warm-humid environment. Journal of the American Mosquito Control Association. 37(1):41–45. https://doi.org/10.2987/20-6974.1.
Moreno, B.J., Aldridge, R.L., Britch, S.C., Bayer, B.E., Kline, J., Hahn, D.A., Chen, C., Linthicum, K. 2021. Preparing irradiated and marked male Aedes aegypti mosquitoes for release in an operational sterile insect technique program. The Journal of Visualized Experiments (JoVE). 169:e62260. https://doi.org/10.3791/62260.
Tsikolia, M., Opatz, T., Kauhl, U., Tabanca, N., Demirci, B., Tenbroeck, S., Linthicum, K., Bernier, U.R. 2021. Trials for gathering information on an unknown peak in the GC-MS spectra of horse and pony hair extracts. Advances in Entomology. 9(2):100-111. https://doi.org/10.4236/ae.2021.92009.
Sen-Utsukarci, B., Kessler, S.M., Akbal-Dagistan, O., Estep Iii, A.S., Tabanca, N., Kurkcuoglu, M., Demirci-Kayarian, S., Eroglu-Ozkan, E., Gul, Z., Bardacki, H., Becnel, J.J., Kiemer, A.K., Mat, A., Husnu Can Baser, K. 2021. Chemical composition and biological activities of valeriana dioscoridis sm. roots. South African Journal of Botany. 141:306-312. https://doi.org/10.1016/j.sajb.2021.05.007.
Luu-Dam, N., Tabanca, N., Estep Iii, A.S., Nguyen, D., Kendra, P.E. 2021. Insecticidal and attractant activities of magnolia citrata leaf essential oil against two major pests from diptera: aedes aegypti (culicidae) and ceratitis capitata (tephritidae). Molecules. 26(8): 2311. https://doi.org/10.3390/molecules26082311.
Estep, A.S., Sanscrainte, N.D., Cuba, I., Allen, G.M., Becnel, J.J., Linthicum, K. 2020. Failure of permethrin treated military uniforms to protect against a laboratory maintained knockdown resistant (kdr) strain of aedes aegypti. Journal of the American Mosquito Control Association. 36(2):127-130. https://doi.org/10.2987/19-6906.1.
Norris, E.J., Bloomquist, J.R. 2021. Co-toxicity factor analysis reveals numerous plant essential oils are synergists of natural pyrethrins against Aedes aegypti mosquitoes. Insects. 12(2):154,1-11. https://doi.org/10.3390/insects12020154.
Traore, M.M., Junilla, A., Revay, E.E., Kravchenko, V.D., Lahti, A., Fiorenzano, J.M., Qualls, W., Kline, D.L., Schlein, Y., Beier, J., Xue, R., Muller, G.C. 2019. Control of adult and larval Aedes albopictus with attractive toxic sugar baits (active ingredient: cinnamon sesame oil) in north-eastern Florida. Journal of the Florida Mosquito Control Association. 66:20-26.
Junnila, A., Traore, M.M., Mckenzie, K., Hogsette, Jr, J.A., Kline, D.L., Kone, A.S., Diarra, R.A., Petranyi, G., Troare, I., Sangare, P., Diakite, A., Traore, S.F., Kravchenko, V., Xue, R., Revay, E.E., Müller, G.C. 2021. Performance of the Atrakta™ Mosquito Lure in combination with Dynatrap® (Models DT160 and DT700) and a CDC Trap (Model 512). Journal of the Florida Mosquito Control Association. 86(1):48-55. https://doi.org/10.32473/jfmca.v68i1.129099.
Traore, M.M., Junnila, A., Hogsette, Jr, J.A., Kline, D.L., Mckenzie, K., Kravchenko, V., Kone, A.S., Diarra, R.A., Traore, S.F., Petranyi, G., Sangare, P., Diakite, A., Troare, I., Beier, J.C., Revay, E.E., Xue, R., Müller, G.C. 2021. Evaluation of Dynatraps® DT160 as an inexpensive alternative to CDC traps for adult mosquito monitoring in Mali, West Africa. Journal of the Florida Mosquito Control Association. 68(1):38-47. https://doi.org/10.32473/jfmca.v68i1.129098.
Aldridge, R.L., Kline, J., Coburn, J.M., Britch, S.C., Boardman, L., Hahn, D.A., Chen, C., Linthicum, K. 2020. Gamma-irradiation reduces survivorship, feeding behavior, and oviposition of female Aedes aegypti. Journal of the American Mosquito Control Association. 36(3): 152–160. https://doi.org/10.2987/20-6957.1.
Britch, S.C., Kline, D.L., Linthicum, K., Urban, J., Dickstein, E.R., Aldridge, R.L., Golden, F.V. 2020. Transfluthrin spatial repellent on US Military camouflage netting reduces tabnids in a warm-temperate environmentT. Journal of the American Mosquito Control Association. 36(3):212-215. https://doi.org/10.2987/20-6933.1.