Location: Mosquito and Fly Research
2023 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
In regard to objective with NASA, Oak Ridge National Laboratory, DoD, and NOAA partners we developed a novel approach to apply machine learning to protect the U.S. from climate-driven risk of Rift Valley fever (RVF) outbreaks. RVF is a prominent and severe zoonotic vector-borne disease of livestock and humans that is poised to expand into new regions such as the US and southern Europe. The maximum capability of the original outbreak risk prediction system, the Rift Valley Fever Monitor, was 65% spatial accuracy with a 2-4 month lead time. Efficacy of the system was limited by rule-based and near real-time monthly interpretation of satellite-derived climate variables such as rainfall and vegetation indices. ML upgrades to the system will support incorporation of a moving window of climate forecast data, with an anticipated increase in both spatial accuracy (+10-20%) and lead time (+1-2 months). The ML implementation in this work will enable us to incorporate more types of data to significantly improve spatial and temporal forecasts of risk of a prominent bio-threat to the U.S. Better prediction allows more time to vaccinate in endemic regions and more time for U.S. to prepare surveillance and containment strategies. ML advancement will also provide a catalog of performance of an array of techniques that can accelerate development of models for other high threat climate-linked pathogens such as dengue and chikungunya.
2.A. 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. ARS scientists implemented a method for successful colonization that was adopted from a mosquito control partner. ARS scientists 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. ARS scientists 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.
2.B. CMAVE scientists working with collaborators from the University of Florida and the Anastasia Mosquito Control District, St. Augustine, Florida, successfully colonized a local strain of Aedes aegypti, a prominent mosquito species that threatens human health by transmission of pathogens such as Zika, chikungunya, and dengue viruses. This colony was then used to develop the sterile insect technique (SIT) which consists of rearing male mosquitoes in large numbers, sterilizing them with radiation, and releasing them into areas where wild populations of Aedes aegypti threaten human health in St. Augustine. These males overwhelm mate choice and force wild female mosquitoes to produce unfertilized or inviable eggs, reducing the population of this species and thus risk of transmission of disease to humans. We evaluated radiation doses for their ability to sterilize male and female mosquitoes and evaluated their survival in the laboratory, and the competitiveness of these males compared to non-irradiated wild males. Importantly, our results suggested that females that may be accidentally reared, irradiated, and released with males in the SIT program are unlikely to cause a threat to humans because of reduced survival and reduced blood feeding behavior.
2.C. CMAVE scientists have been working with University of Florida collaborators to synthesize a variety of repellent and insecticidal molecules. Thus far, > 200 molecules have been synthesized and > 100 have been screened. Currently, we have identified numerous compounds that are as repellent as DEET, as repellent as transfluthrin in a laboratory setting when synergized by select agents, and capable of circumventing insecticide resistance in pyrethroid-resistant strains. This year, we characterized the activity of numerous esters of these recently synthesized compounds to better understand their potential as repellents and insecticides. Among these esters, numerous were highly efficacious, and about as effective as other repellents on the market today. Most importantly, a pyrethroid-resistant strain of Aedes aegypti was not resistant to the most successful molecules in this class, indicating a potentially new mechanism of action compared to current products on the market today. We have also been evaluating the potential of various natural products in laboratory, semi-field, and field assays. So far numerous natural products have been identified with significant potential to control mosquitoes and other biting arthropods. We have submitted an invention disclosure describing these technologies to the USDA Office of Technology Transfer to evaluate their potential for marketability and commercialization. Semi-field and field testing is complete for a number of these molecules and demonstrates the potential of at least one of these compounds to be very effective against mosquitoes and other biting arthropods in real-world environments.
Subobj 2.D. CMAVE scientists 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 a single susceptible allele results in an effectively treatable population.
4.A. CMAVE scientists recently evaluated the potential of nine different permethrin-treated military fabric types at three wash levels (1X, 20X, and 50X) to prevent the biting of two species of disease-vectoring mosquito species (Aedes aegypti and Anopheles albimanus). These fabric types represent different iterations of the Improved Hot Weather Combat Uniform that is being developed in conjunction with the US Army. Of the samples screened, all performed above the minimum threshold of bite protection required to adequately prevent disease in military personnel, however differences in efficacy were noted. These differences in efficacy will inform future iterations of permethrin-treated fabrics (e.g., changes in weave, pore size, permethrin loading concentration, and permethrin-binder used) that will serve to protect our military personnel in deployed environments. We have also evaluated the potential of graphene oxide to act as a significant bite protectant when applied as films to select military fabric types. Our studies indicate that graphene is a good bite protectant with great potential for commercialization and is likely operating via two distinct mechanisms: a) as a physical barrier, which prevents mosquitoes biting through and subsequently feeding, and b) as a repellent/”host-cloaking” agent, as host-seeking mosquitoes landed less on the graphene oxide-treated fabrics than the controls. Further studies are underway to characterize the mechanisms of graphene-oxide bite protection.
4.D. With UF and US military collaborators and an industry partner we extensively tested novel pyrethroid and essential oil spatial repellent compounds in a variety of devices, application methods, and substrates in a north Florida warm-hot humid sub-tropical field environment and a southern California hot-arid desert field environment targeting natural populations of multiple species of prominent nuisance and disease-vector mosquitoes and flies. We developed a dramatically improved novel, high throughput field assay system to rapidly screen multiple spatial repellent compounds in short time frames of days and weeks. The faster throughput of this system also permitted comparison of efficacy across similar communities of natural populations of target mosquitoes, and similar meteorological conditions, that would otherwise shift and change over the course of prior time frames of weeks and months. Results indicated efficacy of selected compounds was not equally effective across multiple key mosquito species, and efficacy in the field against wild mosquitoes could vary substantially from efficacy in laboratory and semi-field conditions against colony-reared resistant and susceptible mosquito species. These results demonstrate that limitations of spatial repellent technology in field conditions against wild targets must be investigated before operational use.
4.F. We have recently screened over 70 different natural product repellents in a small glass tube repellency assay against Aedes aegypti mosquitoes. Various physicochemical properties and parameters were characterized using available quantitative-structure activity relationship (QSAR) software. A number of characterized parameters were then correlated to repellent efficacy to better understand which descriptors predict repellent effect. Vapor pressure was the only parameter evaluated that correlated well with repellency. It was observed that compounds with both very low and very high vapor pressures were not effective repellents, revealing an optimum vapor pressures for the most efficacious repellents. These findings will be useful for the development of future natural product spatial repellents, by both our stakeholders and the USDA.
Accomplishments
1. Global strategy for preparation and protection from Rift Valley fever virus globalization. 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, the western Indian Ocean, and the Arabian Peninsula cause pronounced health and economic impacts, and are increasing in frequency and spatial extent, thereby increasing opportunities for escape of the virus into new regions. Scientists at the Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), Gainesville, Florida, in partnership with NASA-Goddard Space Flight Center, Oak Ridge National Laboratories, the EcoHealth Alliance, and the USDA-ARS Foreign Arthropod-Borne Animal Disease Research Unit/National Bio- and Agro- Defense Facility developed a global strategy for protection of non-endemic regions from the emergence of Rift Valley fever virus. This strategy draws together and arranges the most up-to-date technology in vaccines, diagnostics, predictive modeling, and vector control into a program of international cooperation, interagency partnerships, and collaborations with operational public health and mosquito and vector control units. The strategy includes a series of scenarios of encroachment of the virus into non-endemic regions and hypothesized gaps or vulnerabilities in response infrastructure. The publication generated from the development of this strategic guidance provided detailed, tractable processes to develop monitoring systems to protect public health and livestock economies in RVFV-emerging regions.
2. Short-chain esters of pyrethroid acids show promise as repellents and vapor toxicants. The emergence of pyrethroid-resistant mosquitoes is a world-wide problem that necessitates further research into the development of new repellents and insecticides. As such, Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) scientists at Gainesville, Florida, and collaborators at University of Florida explored the modification of existing pyrethroid acids to identify structural motifs which might not be affected by select resistance mutations eliciting pyrethroid resistance. To this end, 35 compounds within this chemical class were synthesized and screened for spatially-acting repellency and insecticidal activity against the pyrethroid-susceptible, Orlando (OR), and pyrethroid-resistant, Puerto Rico (PR), strains of Aedes aegypti mosquito. We utilized a high-throughput benchtop repellency and toxicity assay to characterize the most active compounds in this set. Our findings indicate that short-chain esters of pyrethroid acids show the most promise as repellents and vapor toxicants, and they may represent viable chemistries for the development of future repellency products.
3. Fir needle oil as a synergist for synthetic insecticides. The generally pleasant aroma and perceived salubrious qualities of plant essential oils (PEO) have given rise to a shift in consumer preference from synthetic insecticides to these natural formulations for both home pest control and personal bite protection. In addition to their overall safety to mammals and non-target organisms, they are effective at controlling pest insects, making them ideal candidates in future insecticidal formulations/repellents. Because of these favorable characteristics identified by stakeholders, Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) scientists at Gainesville, Florida, and collaborators at the University of Florida characterized the ability of a particular oil, fir needle oil, to synergize the knockdown of 9 different synthetic insecticides. For some insecticides screened, fir needle oil was capable of synergizing knockdown by almost 20-fold, indicating its potential to drastically enhance the efficacy of commercial insecticides on the market. We also identified that the ability of fir needle oil to enhance the speed of action of select insecticides is mediated by another mechanism than direct action on the insect nervous system. This has important implications on its use in future insecticide formulations and its ability to circumvent current forms of insecticide resistance present in wild public health pests today.
4. Strong synergism produced by three monoterpenoids. Resistance to the limited number of insecticides registered for use against medical and veterinary arthropod pests threatens public health and food safety, worldwide. Pyrethroids, organophosphates, carbamates, neonicotinoids, and spinosyns are some of the most commonly used chemical classes in medical and veterinary pests, all with documented combinations of target site resistance, enhanced metabolic detoxification, reduced cuticular penetration and behavioral resistance. Synergists are useful additives in insecticidal formulations, except no new synergists have been developed and adopted recently. We set out to investigate the intrinsic toxicity and synergistic potential of three monoterpenoids, menthone, fenchone, and carvone, to enhance both permethrin and methomyl. These were screened on both adult yellow fever mosquitoes and house flies. While these terpenoids were not as toxic as currently utilized synthetic insecticides, they did produce strong synergism of permethrin on both mosquitoes and house flies. These results indicate that these select terpenoids may be useful active ingredients and/or synergists in future insecticidal formulations.
5. Differential attraction of Tabanidae flies to trapping methods. Trapping studies of biting flies of Tabanidae, such as horse flies and deer flies, were conducted. These biting flies are nuisance pests of people, livestock and wildlife due to their abundance and persistent biting behavior, which often results in irritating bites and possible secondary infection. Some transmission of pathogens also occurs. High populations have a significant economic impact on outdoor activities and livestock production. Our studies demonstrated that not all tabanid species were attracted to traps. Species identified as horseflies were attracted to a variety of traps. Current studies indicate that different species are attracted to different colors of Nzi traps. Species identified as deerflies, generally are not captured by Nzi traps or any other current trap design. Instead, we observed that they are very attracted by movement of people, animals or vehicles. Therefore, we utilized a technique known as trolling to monitor their behavior and population parameters. Deerflies were observed to be especially attracted to the mirrors of moving vehicles. We utilized a battery-powered aspirator to collect them from the mirrors as the vehicle traveled slowly (ca. 5 miles per hour) along trails that traversed through habitat occupied by the adults.
6. Graphene oxide prevents mosquito bites. Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) scientists at Gainesville, Florida, partnering with the scientists at the Combat Capabilities Development Command Soldier Center, evaluated the potential of graphene oxide to prevent mosquito bites when applied to select fabrics. For this we applied to different films of graphene oxide to various military-grade fabrics and evaluated the potential of mosquitoes to bite human subjects wearing these fabrics in an arm-in-cage assay protocol. We demonstrated that mosquitoes were unable to bite through numerous types of graphene oxide films, with bite protection values as high as 100% for some films. Moreover, the style of the applied graphene oxide films played a significant role, as films applied in small lines or dots were as effective as complete graphene oxide layers (covering the entire biting area in our assay). This was true even though graphene oxide applied as dots or lines left gaps for mosquitoes to feed through. These results indicate that graphene oxide prevents mosquito bites via a novel mechanism beyond that of a simple physical barrier. We are currently exploring this mechanism of bite protection, but we hypothesize that graphene oxide acts to alter host volatile or thermal profile.
7. Repellent properties of transfluthrin. Laboratory and semi-field experiments demonstrated that transfluthrin can be used to alter the behavior of mosquitoes and stable flies. In collaboration with scientists at the University of Florida Department of Material Science and Engineering, several iterations of 3-D printed devices, which passively released transfluthrin, were developed and evaluated under semi-field conditions. Their efficacy in preventing the entry of mosquitoes into various sized tents was determined using BG-Sentinel mosquito traps baited with carbon dioxide and a proprietary human scent lure. Duration of effectiveness exceeded our target of 4 weeks with >75% reduction of mosquitoes entering the tent, locating the trap and subsequently getting collected in the trap. The same type devices were compared to transfluthrin treated bootlaces to prevent stable flies from locating and getting trapped on sticky traps (Knight Stick trap) baited with dry ice.
8. The potential of basic amines as future insect control agents. Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) scientists at Gainesville, Florida, and collaborators from the University of Florida evaluated select basic amines for their potential to act as repellents and insecticides on a variety of different insect classes. In vapor phase exposure, these compounds produced repellency, narcosis, knockdown, and death at increasing doses, respectively. While these compounds were good repellents and insecticides when insects were challenged via vapor exposures, they were not very toxic when applied directly to the cuticle of the insect, likely due to their relatively high volatility. In electrophysiological studies, basic amines produced neuroexcitation and nerve block, although they acted in unique ways depending on the insect or tissue exposed. These results suggest that select basic amines have diverse activity on a number of targets within insect nervous systems. This work helps to further explain how basic amines act on insects and explores their potential as future insect control agents. Future experiments is needed to evaluate these compounds on other insect pests and in diverse semi-field and field deployments.
Review Publications
Demir, S., Karaalp, C., Tabanca, N., Bernier, U.R., Linthicum, K. 2023. Evaluation of the repellent activity of 13 Achillea L. species from Turkiye against the Virus vector Aedes aegypti (L.) Mosquitoes. Kafkas Universitesi Veteriner Fakultesi Dergisi. 29(1):33-40. https://doi.org/10.9775/kvfd.2022.28409.
Sallam, M., Whitehead, S., Barve, N., Bauer, A., Guralnick, R., Allen, J., Tavares, J., Gibson, S., Linthicum, K., Giordano, B.V., Campbell, L. 2023. Co-occurrence probabilities between mosquito vectors of West Nile and Eastern equine encephalitis viruses using Markov Random Fields (MRFcov). Parasites & Vectors. 16:10. https://doi.org/10.1186/s13071-022-05530-1.
Baker, O.S., Norris, E.J., Burgess, E.R. 2023. Insecticidal and synergistic potential of three monoterpenoids against the Yellow Fever mosquito, Aedes aegypti (Diptera: Culicidae), and the house fly, Musca domestica (Diptera: Muscidae). Molecules. 28:3250. https://doi.org/10.3390/molecules28073250.
Bloomquist, J.R., Coquerel, Q.R., Hulbert, D., Norris, E.J. 2023. Neurophysiological action of centrally-acting spider toxin polypeptides derived from Hadronyche versuta and Tegenaria agrestis venom. Pesticide Biochemistry and Physiology. 192:105416. https://doi.org/10.1016/j.pestbp.2023.105416.
Rajagopl, N.R., Bowman, A.R., Aldana, F.J., Batich, C.D., Hogsette, Jr, J.A., Kline, D.L. 2023. Semi-field evaluation of a novel controlled release device using transfluthrin as spatial repellent to prevent entry of mosquitoes into military tents. Current Research in Parasitology and Vector Borne Diseases. 3(100113). https://doi.org/10.1016/j.crpvbd.2023.100113.
Chen, C., Aldridge, R.L., Gibson, S., Kline, J., Aryaprema, V., Qualls, W.A., Xue, R., Boardman, L., Linthicum, K., Hahn, D.A. 2022. Developing the radiation-based sterile insect technique (SIT) for controlling Aedes aegypti: identification of a sterilizing dose. Pest Management Science. 79(3):1175-1183. https://doi.org/10.1002/ps.7303.
Estep III, A.S., Kissoon, K., Saldana, M., Fredregill, C. 2023. Persistent variation in insecticide resistance intensity in container breeding Aedes co-collected in Houston, Texas. Journal of Medical Entomology. 51. https://doi.org/10.1093/jme/tjad051.
Gibson, S., Linthicum, K., Turell, M.J., Anyamba, A. 2022. Rift Valley fever virus: Movement of infected humans threatens global public health and agriculture. CABI Reviews. (2022) 17. https://doi.org/10.1079/cabireviews202217029.
Spanoudis, C.G., Wondowsen, B., Isberg, E., Andreadis, S.S., Kline, D.L., Birgersson, G., Ignell, R. 2022. The chemical code for attracting Culex mosquitoes. Frontiers in Ecology and Evolution. https://doi.org/10.3389/fevo.2022.930665.
Aldridge, R.L., Alto, B.W., Connelly, R.C., Okech, B.A., Siegfried, B., Linthicum, K. 2022. Lethal and sublethal concentrations of formulated larvicides against susceptible Aedes aegypti. Journal of the American Mosquito Control Association. 38(4):250-260. https://doi.org/10.2987/22-7084.
Sen-Utsukarci, B., Gurdal, B., Estep Iii, A.S., Tabanca, N., Kurkcuoglu, M., Goger, F., Gul, Z., Bardakci, H., Becnel, J.J., Mat, A., Can Baser, K. 2022. The insecticidal activities of Erica manipuliflora Salisb. Extracts in the flowering and fruiting periods and their evaluation in term of chemical profiles of active extracts. Asian Pacific Journal of Tropical Medicine. 87:115380. https://doi.org/10.1016/j.indcrop.2022.115380.
