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Research Project: Integrated Pest Management of Mosquitoes and Biting Flies

Location: Mosquito and Fly Research

2022 Annual Report

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.

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
In collabororation with the University of Florida and Florida mosquito control district partners, ARS scientist at Gainesville, Florida developed a novel model to describe patterns of co-occurrence of disease-vector mosquito species in an important populated area of Florida. This model is unique because it investigates links not only among a community of medically and veterinary important mosquito species but also between each species and key environmental parameters. The value of this model is twofold. First, risk of emergence of certain disease-vector mosquitoes that may be difficult to collect, or that cannot be reliably sampled in specific areas, can be estimated from both environmental measurements and collections of other species that are readily sampled. Secondly, for the first time the entire community of potential vector mosquitoes for a high-risk pathogen like West Nile virus can be considered simultaneously in a region when developing vector control and disease surveillance strategies. ARS scientists, in collaboration with mosquito control district personnel, collected and a conducted insecticide resistance testing on several populations of Aedes taeniorhynchus from within the state of Florida. Significant insecticide resistance to pyrethroids was not observed which agrees with anecdotal data from field spray studies by the mosquito control districts. We implemented a method for successful colonization that was adopted from a mosquito control partner. We also conducted toxicological characterization of our in-house laboratory strain to provide baseline median lethal doses for commonly used public health insecticides and future comparison to field strains. We also applied third generation sequencing technology to the laboratory strain and a Florida field strain to identify and define the protein coding sequences of the acetylcholinesterase and voltage gated sodium channel, the molecular targets of organophosphates and pyrethroids respectively. Bioinformatic analysis of this dataset is underway to develop sets of primers for rapid assessment of these targets in field strains. Center For Medical, Agricultural, & Veterinary Entomology (CMAVE) scientists working with collaborators from Harlingen City, TX, were able to colonize their local strain of Aedes aegypti, the primary mosquito that transmits Zika, chikungunya, and dengue viruses that cause diseases in humans. To develop the sterile insect technique as a tool for use in the Harlingen Integrated Pest Management Plan for vector mosquitoes, we evaluated radiation doses for their ability to sterilize male and female mosquitoes and evaluated their survival in the laboratory. This information will be used to test the ability of sterilized males to survive, move, and mate with natural populations of insecticide resistant female mosquitoes. CMAVE scientists and University of Florida collaborators characterized the mechanism of a series of repellent amides. Applications of these novel molecules on mosquito and cockroach nerve tissues indicate activity via a novel mechanism. ARS scientists and University of Florida collaborators synthesized and screened 26 compounds from the initial structure of trans-chrysanthemic acid for spatially-acting repellency and insecticidal activity against pyrethroid-susceptible and pyrethroid-resistant strains of Aedes aegypti. Activity of most was not as potent as transfluthrin, but routes to future synthesis look promising. Derivatives with excellent activity can be synergistic as repellents. CMAVE scientists and University of Florida collaborators continue to characterize liriodenine, a natural compound isolated from Rollinia emerginata. Liriodenine, which acts as a GABA antagonist, can also block potassium channels and activate sodium channels. Thus, liriodenine can control pests via several unique mechanisms of action, making it an ideal candidate for future pest control applications. CMAVE scientists and University of Florida collaborators explored the potential of sodium channel activators veratrine and aconitine as insecticides and synergists of natural pyrethrins (NP) on pyrethroid-resistant Aedes aegypti adults and larvae. Application of both compounds with NP on nerve tissue caused significant synergism of NP-mediated nerve block. Veratrine synergized NP, indicating cross-resistance to aconitine. Toxicity was comparable to currently available insecticides. These two compounds could improve currently used insecticides or circumvent pyrethroid resistance. CMAVE scientists and University of Florida collaborators recently showed that nookatone, a terpenoid found in Alaskan yellow cedarwood, is a GABA antagonist. Topical applications on adults and larvae of Aedes aegypti produced mortality comparable to other natural insecticides. When applied to the Drosophila melanogaster central nervous system (CNS) it produced significant excitation and block at micromolar concentrations. Nootkatone could antagonize the block produced by GABA on the Drosophila CNS, and GABA-activated chloride currents in oocytes transfected with insect GABA receptors. Nootkatone is a viable mosquito control agent that acts via a mechanism distinct from the most mosquito control chemistries on the market today. We conducted bioassays for insecticidal activity on novel extracts of natural products produced by ARS scientists in two laboratories at NPURU. This screening identified moderate to high insecticidal activity in several isolates. Testing with an insecticide resistant strain also indicated that the mode of action did not involve the sodium channel or AchE which indicates that these compounds would likely be effective in resistant strains. Work to purify the activity is underway at NPURU. We conducted numerous studies with commonly available commercial formulations of insecticides and developed a simple model for implementation by stakeholders that allows estimation of the strength of pyrethroid resistance based only on genetic information for Aedes aegypti. We tested representative pyrethroids, synergized pyrethroids and organophosphates in semi-field wind tunnel conditions to define the impact of resistance on operational mosquito control. From this information, we determined that only one specific kdr genotype is resistant to even the best current adulticides and that the introduction of even a single susceptible allele result in an effectively treatable population. We are conducting tabanid trapping studies to determine which visual and olfactory cues can be used to develop new trapping strategies for surveillance and management. Traps of various shapes, sizes and colors are being evaluated with and without olfactory cues such as carbon dioxide, ammonia, and 1-octen-3-ol. Field experiments have focused on whether visual attractiveness can be overcome by olfactory attractiveness. CMAVE scientists have screened pyrethroid-treated military fabrics impregnated with graphene layers and demonstrated that graphene could be an introduced repellent material. Mosquitoes were less likely to blood feed on human subjects wearing candidate fabrics and were less likely to land on the fabric. This repellent effect will be further explored. Laboratory and semi-field experiments were conducted to determine the impact of passive release of transfluthrin on stable fly behavior. In laboratory experiments, military bootlaces treated with a 20% transfluthrin EC were weathered outside. Weathering for at least 2 months prevented stable flies from feeding and confined flies died within a 30-min exposure. In semi-field experiments, a perimeter of transfluthrin treated bootlaces around a CO2 baited Knight Stick trap reduced trap catches by ca. 50%. In collaboration with University of Florida scientists, passive release devices with selected transfluthrin formulations were developed and evaluated to determine if they could prevent mosquitoes from entering various sized tents. All formulations successfully prevented ca. 90% of 4 colonized mosquito species from entering the tents, however efficacy duration varied from 7 to >30 days depending on formulation. Additional release devices are being produced by 3-D printing for further evaluation. With University of Florida and U.S. military collaborators we extensively tested novel and existing spatial repellent compounds in a variety of devices, application methods, and substrates in a north Florida warm-hot humid sub-tropical field environment targeting natural populations of multiple species of prominent nuisance and disease-vector mosquitoes. We developed several realistic militarily-relevant scenarios in this field environment to investigate these compounds with the objective of protecting various size shelters, blast-wall perimeters, and camouflage net enclosures from mosquito incursion. Results indicated high efficacy of selected compounds and delivery methods, but that high efficacy was limited to certain scenarios and conditions, and was not equally effective across multiple key mosquito species. These results showed that although spatial repellent compounds may display extremely high efficacy and toxicity in laboratory and semi-field conditions against colony-reared resistant and susceptible mosquito species, efficacy in the field must be carefully determined so that limitations of this technology are accounted for before operational use. Approximately 50 individual natural products and mixtures thereof were screened in spatial repellency assays to determine the most efficacious repellents when applied on a surface. Some of the strong spatial repellents are being characterized chemically to identify individual bioactive components. Many identified candidates are as active as DEET and other available repellents and can synergize repellent pyrethroids on the market today. These natural products may serve as the next generation of repellents.

1. Comprehensive description of the risk to global public and veterinary health from the movement of humans infected with Rift Valley fever virus. Rift Valley fever virus (RVFV) is an arthropod-borne zoonotic virus causing Rift Valley fever (RVF), an acute febrile hemorrhagic disease with hepatic, ocular, and other complications, primarily affecting domestic ungulate livestock and humans. Outbreaks of this virus in its endemic range of the African Continent and the Arabian Peninsula cause pronounced health and economic impacts. Although increasing effort is being devoted to surveillance for this pathogen in non-endemic regions such as southern Europe and the U.S., these efforts focus on incursion of RVFV through infected livestock or vector movement and do not sufficiently consider the threat of movement of infected humans. Scientists at the Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), Gainesville, Florida, in partnership with NASA-Goddard Space Flight Center conducted the first comprehensive review of the risk for introduction and spread of RVFV from endemic areas through diverse pathways centered on the movement of infected humans into non-endemic regions. This survey revealed that humans infected with RVFV and capable of infecting mosquitoes have arrived in non-endemic regions repeatedly and present a serious risk of allowing the virus to spillback into wild and domestic ungulate livestock populations, potentially becoming endemic in other continents that include North America and Europe. The report generated from this survey provided detailed, tractable processes to develop monitoring systems to protect public health and livestock economies in these areas.

2. Understanding how insecticide resistance affects operational mosquito control. Disease-causing mosquitoes have developed resistance to commonly used classes of pesticides and this forces reexamination of the efficacy of mosquito control sprays, the identification of the factors that indicate reduced efficacy and effective mitigation strategies when strong resistance is present. Starting in 2017 and completed this year, ARS scientist at Gainesville, Florida conducted an in-depth examination of the mechanisms behind the strong insecticide resistance found in common disease vectoring mosquitoes and how these resistance factors impact the efficacy of mosquito control operations. Isolation and characterization of the molecular mechanisms of resistance were vastly different in the two most important public health mosquito vectors of human disease. ARS scientists and collaborators showed that these underlying differences result in real world differences in the effectiveness of operational mosquito control methods. Understanding that the basis of strong insecticide resistance can be different requires implementing differing responses to maintain effective control. We demonstrated that resistance is not “one size fits all” and that the use of alternate strategies like the implementation good integrated vector management practices are critical to maintaining effective control. This information is being implemented by mosquito control programs leading to multiple requests for ARS scientists to give presentations to stakeholders including State and local vector control in multiple states (Arizona, Florida, Louisiana) as well as in U.S. partner countries like Peru. This information was also disseminated at the national level with a recent webinar presentation to the membership of the American Mosquito Control Association.

Review Publications
Bloomquist, J., Jiang, S., Norris, E.J., Richoux, G., Yang, L., Linthicum, K. 2021. Novel pyrethroid derivatives as effective mosquito repellents and repellent synergists. In Corona, C., Debboun, M., Coats, J., editors, Advances in Arthropod Repellents. Elsevier Inc. p.19-32.
Norris, E.J., Bloomquist, J.R. 2021. Recording central neurophysiological output from mosquito larvae for neuropharmacological and insecticide resistance studies. Journal of Insect Physiology. 135:104319.
Demiray, H., Estep III, A.S., Tabanca, N., Becnel, J.J., Demirci, B. 2022. Chemical constituents from Rheum ribes shoots and its insecticidal activity against Aedes aegypti. Revista Brasileira de Farmacognosia.
Norris, E.J., Bloomquist, J.R. 2021. Nutritional status significantly affects toxicological endpoints in the CDC bottle bioassay. Pest Management Science. 78:743-748.
Cuba, I.H., Richoux, G.R., Norris, E.J., Bernier, U.R., Linthicum, K., Bloomquist, J.R. 2021. Vapor phase repellency and insecticidal activity of pyridinyl amides against Anopheline mosquitoes. Current Research in Parasitology and Vector Borne Diseases. 1:100062.
Jensen, B., Althoff, R., Rydberg, S., Royster, E., Estep III, A.S., Huijben, S. 2022. Topical application bioassay to quantify insecticide toxicity for mosquitoes and fruit flies. The Journal of Visualized Experiments (JoVE). 179, e63391:1-23.
Norris, E.J., Chen, R., Li, Z., Geldenhuys, W., Bloomquist, J.R., Swale, D.R. 2022. Mode of action and toxicological effects of the sesquiterpenoid, nootkatone, in insects. Pesticide Biochemistry and Physiology. 183:105085.
Kilic, M., Orhan, I., Eren, G., Okudan, E., Estep III, A.S., Becnel, J.J., Tabanca, N. 2021. Insecticidal activity of forty-seven marine algae species from the Mediterranean, Aegean and Sea of Marmara in connection with their cholinesterase and tyrosinase inhibitory activity. Chemosphere.
Campbell, C.B., Kline, D.L., Hogsette, Jr, J.A., Tenbroeck, S.H. 2021. Species complex of mosquitos captured by mechanical traps and an equine host. International Journal of Veterinary Science and Research. 7(2):169-177.
Lloyd, A.M., Kline, D.L., Bernier, U.R. 2021. Field evaluation of Talstar (Bifenthrin) residential barrier treatments alone and in conjunction with mosquito magnet liberty plus traps in Cedar Key, Florida. Journal of the Florida Mosquito Control Association. 86(1):56-62.
Tsikolia, M., Tabanca, N., Kline, D.L., Demirci, B., Yang, L., Linthicum, K., Bloomquist, J.R., Bernier, U.R. 2022. Studies on the volatiles composition of stored sheep wool, and attractancy toward Aedes aegypti mosquitoes. Insects. 13(2):1-9.
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. 68:13960-13969.
Richoux, G.M., Yang, L., Norris, E.J., Jiang, S., Linthicum, K., Bloomquist, J.R. 2021. Resistance-breaking insecticidal activity of new spatial insecticides against Aedes aegypti. Journal of Agricultural and Food Chemistry.
Coquerel, Q.R., Demares, F., Geldenhuys, W.J., Le Ray, A., Breard, D., Richomme, P., Legros, C., Norris, E.J., Bloomquist, J.R. 2021. Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor. Toxicon. 201:141-147.
Grisgby, A., Kelly, B., Sanscrainte, N.D., Becnel, J.J., Short, S.M. 2020. Propagation of the microsporidian parasite Edhazardia aedis in Aedes aegypti mosquitoes. Journal of Visual Experiments. (162), e61574.
Kereselidze, M., Pilarska, D., Linde, A., Sanscrainte, N.D., Hajek, A.E. 2020. Nosema maddoxi infecting the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), in the Republic of Georgia. Biocontrol Science and Technology. 30(10):1083-1089.
Mcmillan, B.E., Britch, S.C., Golden, F.V., Aldridge, R.L., Moreno, B.J., Bayer, B.E., Linthicum, K. 2021. Assessing transfluthrin mortality against Aedes aegypti and Culex quinquefasciatus inside and outside US military tents in a northern Florida environment. Current Research in Parasitology and Vector Borne Diseases. 2(100067):1-8.
Burgess, E., Lopez, K., Irwin, P., Jaegar, C., Estep III, A.S. 2022. Assessing pyrethroid resistance status in the Culex pipiens complex (Diptera: Culicidae) from the northwest suburbs of Chicago, Illinois using Cox regression of bottle bioassays and other detection tools. PLoS ONE. 17(6): e0268205.
Norris, E.J., Bloomquist, J.R. 2022. Sodium channel-directed alkaloids synergize the mosquitocidal and neurophysiological effects of natural pyrethrins. Pesticide Biochemistry and Physiology.
Xue, R., Kline, D.L., Muller, G.C., Barnard, D.R. 2022. Behavioral response of adult Anopheles quadrimaculatus and Aedes albopictus to different carbohydrates in an olfactometer. Journal of the American Mosquito Control Association. 69(1):63-66.
Kline, D.L., Mckenzie, K., Bowman, A.R. 2021. Semi-field evaluations of arthropod repellents: emphasis on spatial repellents. In Corona, C., Debboun, M. Coats, J. editors. Advances in Arthropod Repellents. Elsevier Inc. p. 193-236.
Bernier, U.R., Perry, M.K., Xue, R., Agramonte, N.M., Johnson, A.L., Linthicum, K. 2021. Evaluation and application of repellent-treated uniform/clothing and textiles against vector mosquitoes. In Coats, J., Corona, C., Debboun, M., editors. Advances in arthoropd repellents. San Diego, CA: Academic Press. p.69-94.