Research Project: Integrated Pest Management of Mosquitoes and Biting Flies

Location: Mosquito and Fly Research

2024 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
Objective 1: Protect US from emerging mosquito-borne virus threats such as Rift Valley fever virus (RVFV) which could significantly negatively impact US health and economy. ARS researchers at Gainesville, Florida produced: first description of environment driving prime mosquito vector of RVFV in endemic region (leverage to alert for potential globalization); first global map of risk for introduction and spread of RVFV during peak virus activity in endemic region; model of co-occurrence of disease-vector mosquito species in Florida (leverage to delineate risk of RVFV spread in the US); two comprehensive strategy publications for protecting US and worldwide from spread of RVFV; novel application of machine learning to protect US from climate-driven risk of RVF outbreaks. Collaborators: NASA, Oak Ridge National Laboratory, University of Florida, EcoHealth Alliance, DoD, NOAA, FABADRU. Sub-objective 2A: An essential aspect was to define the scope and extent of insecticide resistance in populations of primary vector mosquitoes. USDA Scientists and collaborators published the first statewide studies of target site and phenotypic IR to pyrethroids for Florida Aedes aegypti and to pyrethroids and organophosphates for Florida Culex quinquefasciatus. We examined target site resistance in numerous US mosquito populations and with international collaborators have tested samples from the Americas and Africa. We have developed, optimized, and shared methods for IR detection with national and international collaborators. As a result, USDA scientists were requested to provide numerous presentations and webinars to stakeholders and contributed to the American Mosquito Control Association’s Best Practices for Mosquito Control online training series. Sub-objective 2.B: Develop key procedures for district-level sterile insect technique (SIT) interventions targeting Aedes aegypti mosquitoes that seriously threaten human health in the US from pathogens such as Zika and dengue viruses. Working with collaborators from the University of Florida, Harlingen City (Texas), and the Anastasia Mosquito Control District (AMCD) we successfully colonized local Texas and Florida strains and performed dose, competition, and mark-release-recapture studies to establish effective sterile:wild male release rates of colony mosquitoes. ARS researchers at Gainesville, Florida also investigated performance of various sterilization techniques as well as methods to produce and ship sterile male mosquitoes for high throughput interventions. This work has supported establishment of a Florida SIT program that is a leader in demonstrating affordable and attainable Aedes aegypti control. Sub-objective 2.C: Researchers at CMAVE (Center for Medical, Agricultural and Veterinary Entomolog)in collaboration with University of Florida scientists synthesized and evaluated various esters of a natural product from chrysanthemum flowers, trans-chrysanthemic acid. This project led to the synthesis and screening of over 75 molecules, both alone and in combination with other repellents against the pyrethroid-susceptible ORLANDO and the pyrethroid-resistant PUERTO RICO Aedes aegypti strains. Many compounds were identified as good repellents and toxicants against both mosquito strains tested in this project. Moreover, we observed that select esters of trans-chrysanthemic acid and trans-chrysanthemic acid itself were good synergists of other spatial repellents tested. The outcome of this research will be the continual development of novel repellent formulations utilizing trans-chrysanthemic acid esters to better repel mosquitoes. Sub-objective 2.D: Mosquitoes are often resistant to common insecticide formulations used for vector control. USDA Scientists and vector control district stakeholders conducted operational field sprays and wind tunnel sprays to determine the thresholds at which IR detected by standard laboratory methods translates into reduced efficacy in field operations. For Aedes aegypti, we showed IR to pyrethroids was likely and formulations including synergized pyrethroids were unlikely to result in high efficacy. IR to organophosphates was generally low and field formulations maintained high efficacy. In collaboration with an academic collaborator and graduate students, we examined several strategies for IR management., We conducted the first published field studies of a recently EPA-approved resistance breaking commercial formulation and found high efficacy against resistant Culex quinquefasciatus. Sub-objective 4.A: CMAVE scientists and collaborators at the US Army Combat Capabilities Development Command screened multiple fabric types treated with different chemical and physical barriers. These fabric modifications comprised weave manipulations (increasing/decreasing size of fabric weave), applied chemistry (pyrethroid synergists), and physical materials that prevent biting (graphene oxide). Thus far, we have developed a series of fabric treatments and types that significantly reduce the overall biting of Aedes aegypti and Anopheles albimanus mosquitoes. We are actively exploring the mechanism of the biting and landing deterrence produced by these unique technologies. We believe that one technology in particular, graphene oxide, possibly interrupts host-seeking physiology of mosquitoes in a unique way that may be exploitable in future insect repellent/bite protective fabrics. Sub-objective 4.B: Laboratory/semi-field tests showed that passively released transfluthrin (TF) can significantly reduce host-vector contact. An azeotropic-like formulation of TF was used to impregnate military bootlaces (BL), or saturate cotton balls placed within novel devices. In laboratory tests, within 10 minutes, >90% knock down (kd) of all insects was achieved and sustained for >30 days. In semifield studies, an array of either treated BL or passive release devices (PRD) reduced movement of mosquitoes into large military tents by >85% for >30 days. In semifield studies, the treated BL and novel PRD, reduced stable fly recapture on Knight Stick traps by 49% and 72%, respectively. A U.S. Patent Application was filed (USDA Docket No. 0104.22) that relates to spatial repellent azeotrope-like compositions and their use in controlled-release passive devices to repel, kd, and/or kill biting arthropod pests. Sub-objective 4.C: Semi-field studies were conducted to determine the efficacy of various designs and shapes of targets covered with Permanet® fence materials. Targets baited with a combination of carbon dioxide and 1-octen-3-ol effectively reduced mosquito populations. Unbaited targets mosquito reduced populations slightly, and this may have been due to insufficient number and/or placement pattern of unbaited targets. Plans to conduct further semi-field studies to optimize these targets and to conduct field studies against natural mosquito populations at CONUS and OCONUS sites were cancelled due to COVID restrictions. Sub-objective 4.D: Document a suite of factors limiting pesticide efficacy. We conducted unique experiments in diverse environments across hot arid (southern CA), warm temperate (north Florida), and warm-Mediterranean (SE Greece) long-term research sites to extensively investigate capabilities of existing and novel pyrethroid and essential oil spatial repellents, delivery systems, application methods, and substrates to reduce populations of disease vector mosquitoes and Phlebotomine sand flies and biting Tabanid flies. This work empowers field vector control operators to reduce pesticide use, get better results, and merge new technologies into integrated vector management. Collaborations and partnerships: University of Florida, US military entomology laboratories and field sites, industry, mosquito control district, USDA-ARS-EBCL. Subobjective 4.E. Olfactometer studies, utilizing Aedes albopictus were conducted with intact flowers and various blends of volatiles collected and identified from goldenrod (Solidago spp.) and the butterfly bush (Buddleja davidii) to discover novel attractants that this mosquito species might utilize to locate nectar sources. The intact goldenrod flowers were more attractive than the intact butterfly bush flowers. Both sexes of Ae. albopictus were more attracted to the intact flowers of the purple cultivar of butterfly bush than all the other cultivar colors. The flowers of the pink cultivar were the least attractive. Several blends developed from the volatiles collected and identified from the purple cultivar proved to be very attractive to both sexes of the targeted mosquito species. A database of known flowering plants used as attractants by biting arthropods was established. Sub-objective 4.F: CMAVE scientists and University of Florida Collaborators synthesized and evaluated a large set of aryl amide repellents. Over 200 compounds have been synthesized and evaluated in topical toxicity, spatial repellency, and arm-in-cage repellency studies. Several compounds were effective repellents, and some performed as well as transfluthrin and significantly better than DEET, a 70-year commercial standard repellent. The toxicities of select compounds were evaluated in mice to determine potential mammalian toxicities, an undesirable effect. After obtaining laboratory repellency data and in vivo toxicity data in mice, two promising compounds were selected for future development in semi-field and field systems. We are continuing to explore their potential as next-generation spatial repellents and optimizing the formulation and concentrations required for future deployment.

Accomplishments
1. Revision of the 2013 USDA APHIS Rift Valley Fever Disease Response Strategy (Objective 1) . ARS researchers at Gainesville, Florida contributed expert knowledge of innovations in mosquito and vector surveillance and control, Rift Valley fever virus epidemiology modeling, and interagency coordination to analyze and revise the 2013 FAD PReP Disease Response Strategy for Rift Valley Fever Virus for the US. This document establishes a high-level strategy to guide US interagency activities to reduce the likelihood that an introduction of the virus into the US will establish and spread once detected. This virus is a high-threat select agent that is a significant risk to both human and livestock health and the economy in the US. This revised Strategy document will provide a quick reference of the most up to date information on vaccines, diagnostics, vector control, livestock handling, and public messaging and education that will save days to weeks of course of action development in the face of an introduction of the virus.

2. Patent Application Resulting from CRADA-related research. ARS researchers at Gainesville, Florida and CRADA cooperators worked together to identify and screen a number of candidate natural compounds for the evaluation of future spatial repellent products. Together we discovered a promising natural extract that represents a potent, novel spatial repellent capable of repelling mosquitoes and other diverse pest insects in the laboratory and field. To date, we have screened this extract and select chemical analogs of the most bioactive constituents against numerous mosquitoes and fly species, leading to a better understanding of the potential utility of this extract, its bioactive constituents, and analogs thereof as spatial repellents. This technology is so promising in fact, that the USDA patent office has submitted a patent application this cycle in order to allow for the further development of this technology and secure its potential as a future pest control product on the market. Our next goals are to continue optimizing the formulation of this technology for future marketization and develop passive and active emanating devices that could discharge this repellent technology in a user-friendly manner.

3. Statewide examination of insecticide resistance in Culex quinquefasciatus and operational testing of a resistance breaking adulticide. These studies, completed by USDA and collaborators, produced the first US statewide assessment of the levels of phenotypic insecticide resistance in Culex quinquefasciatus, an important vector of West Nile Virus. This work also determined that both target site and enzymatic mechanisms were significant contributors to high intensity resistance that reduced operational efficacy. ARS researchers at Gainesville, Florida found that this resistance could be effectively mitigated by targeting immature life stages. We also published the first operational study of a new resistance breaking adulticide formulation showing high efficacy against resistant Culex in South Florida.

Review Publications
Estep III, A.S., Sanscrainte, N.D., Okech, B.A. 2024. Aedes aegypti knockdown resistance mutations and Dengue Virus infection in Haiti. Journal of the American Mosquito Control Association. 40(2):102-108. https://doi.org/10.2987/23-7160.
Aldridge, R.L., Pagac, A.A., Norris, E.J., Geden, C.J., Kline, D.L., Linthicum, K. 2024. Point protection with transfluthrin against Musca domestica in a semi-field enclosure. Insects. 15(4). https://doi.org/10.3390/insects15040277.
Estep III, A.S., Sanscrainte, N.D. 2024. A critical review of insecticide resistance in US Aedes albopictus: Resistance status, underlying mechanisms, and directions for future research. Journal of the Mosquito Control Association. 71(1):31-40. https://doi.org/10.32473/jfmca.71.1.135291.
Estep III, A.S., Sanscrainte, N.D., Stuck, J., Unlu, I., Prasauskas, A., Mundis, S., Cotter, N., Romero-Weaver, A., Fedirko, T., Kendziorski, N.L., Kosinski, K.J., Ramirez, D., Buckner, E.A. 2024. The 1014F knockdown resistance mutation is not a strong correlate of phenotypic resistance to pyrethroids in Florida populations of Culex quinquefasciatus. Insects. 15(3):197. https://doi.org/10.3390/insects15030197.
Bayer, B., Aldridge, R.L., Moreno, B.J., Golden, F.V., Gibson, S., Wahl, J., Linthicum, K. 2024. Transfluthrin diffusers do not protect two-person US military tents from mosquitoes in open field and canopy warm-temperate habitats. Current Research in Parasitology and Vector Borne Diseases. 5:100156. https://doi.org/10.1016/j.crpvbd.2023.100156.
Aldridge, R.L., Alto, B.W., Connelly, C., Okech, B.A., Siegfried, B., Eastmond, B.H., Alomar, A.A., Linthicum, K. 2023. Does prior exposure to larvicides influence dengue virus susceptibility in Aedes aegypti (Diptera: Culicidae)?. Journal of Medical Entomology. 61(1):166-174. https://doi.org/10.1093/jme/tjad137.
Aldridge, R.L., Gibson, S., Linthicum, K. 2024. Aedes aegypti controls Ae. aegypti: SIT and IIT – an overview. Journal of the American Mosquito Control Association. 40(1):32-49. https://doi.org/10.2987/23-7154.
Meepagala, K.M., Estep III, A.S. 2023. Larvicidal constituents from Poncirus trifoliata root extracts. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjad086.
Burgess, E.R., Sanscrainte, N.D., Taylor, C.E., Buss, L.A., Estep Iii, A.S. 2023. Expression, activity, and consequences of biochemical inhibition of alpha- and beta-glucosidases in different life stages of Culex quinquefasciatus. PLOS ONE. 18(8). https://doi.org/10.1371/journal.pone.0286609.
Unlu, I., Buckner, E., Medina, J., Vasquez, C., Cabrera, A., Estep Iii, A.S. 2024. Crouching Tiger, Obvious Trouble: Insecticide resistance of Miami-Dade Culex quinquefasciatus populations and initial field efficacy of a new resistance-breaking formulation. PLOS Neglected Tropical Diseases. 19(2). https://doi.org/10.1371/journal.pone.0296046.
Gibson, S., Noronha, L., Tubbs, H., Cohnstaedt, L., Wilson, W., Mire, C., Mitzel, D., Anyamba, A., Rostal, M., Linthicum, K. 2023. The increasing threat of Rift Valley fever virus globalization: strategic guidance for protection and preparation. Journal of Medical Entomology. 60(6):1197-1213. https://doi.org/10.1093/jme/tjad113.
Lehane, A., Casey, P., Norris, E.J., Sarah, W., Laura, H. 2024. Measuring insecticide resistance in a vacuum: exploring next steps to link resistance data with mosquito control efficacy. Journal of Medical Entomology. 61(3):584-594. https://doi.org/10.1093/jme/tjae029.
Tian, Y., Hogsette, Jr, J.A., Norris, E.J., Hu, X. 2024. Topical toxicity and repellency profiles of 17 essential oil components against insecticide-resistant and susceptible strains of house flies (Diptera: Muscidae). Insects. 15(6). https://doi.org/10.3390/insects15060384.
Lloyd, A., Kline, D.L., Hahn, D. 2023. Field evaluation of lures as candidate attractants for coastal Culicoides in Florida. Journal of the Florida Mosquito Control Association. 70(1). https://doi.org/10.32473/jfmca.70.1.
Dilling, S.C., Tenbroeck, S.H., Hogsette, Jr, J.A., Kline, D.L. 2023. Comparison of trap and equine attraction to mosquitoes. Insects. 14(4):374. https://doi.org/10.3390/insects14040374.
Muller, G., Prozorov, A.M., Traore, M.M., Regay, E., Hogsette, Jr, J.A., Kline, D.L., Chaskopoulou, A., Volkova, J., Diarra, R., Petrany, G., Schneider, T., Beck, R.H., Ignatev, N., Yakovlev, R.V., Cui, L., Schlein, Y. 2023. The Tabanidae (Diptera) of the Greek islands and Cyprus: An annotated checklist with remarks on ecology, zoogeography, and new records on the East Mediterranean fauna. Ecologica Montenegrina. 67:45-65. https://doi.org/10.37828/em.2023.67.7.
Muller, G.C., Prozorov, A.M., Traore, M.M., Revay, E.E., Hogsette, Jr, J.A., Kline, D.L., Chaskopoulou, A., Prozorova, T.A., Volkova, J.S., Diarra, R.A., Petrányi, G., Schneider, T., Beck, R. 2023. The Tabanidae (Diptera) of the Greek islands and Cyprus: An annotated checklist with remarks on ecology, zoogeography, and new records on the East Mediterranean fauna. Ecologica Montenegrina. 67:45-65. https://dx.doi.org/10.37828/em.2023.67.7.
Norris, E.J., Kline, J.D., Bloomquist, J.R. 2023. Repellency and toxicity of vapor-active benzaldehydes against Aedes aegypti. ACS Infectious Diseases. 10:120-126. https://doi.org/10.1021/acsinfecdis.3c00294.
Norris, E.J., Bloomquist, J.R. 2023. Fir (Abies balsamea) (Pinales: Pinaceae) needle essential oil enhances the the knockdown activity of select insecticides. Journal of Medical Entomology. 60(6):1350-1356. https://doi.org/10.1093/jme/tjad101.
Le Mauff, A., Norris, E.J., Li, A.Y., Swale, D.R. 2024. Repellent activity of natural products to the Lone Star tick, Amblyomma americanum. ACS Infectious Diseases. 17:202. https://doi.org/10.1186/s13071-024-06246-0.