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ARS Home Southeast Area Gainesville, Florida Center for Medical, Agricultural and Veterinary Entomology Mosquito and Fly Research Research





- Hogsette, Jerome - Jerry
- Linthicum, Kenneth - Ken
- Kline, Daniel - Dan
- Gibson, Seth
- Norris, Edmund
- 2023
- 2022
- 2021
- 2020
- Publications
- Animal Health (103)
- Veterinary, Medical and Urban Entomology (104)
- Food Safety, (Animal and Plant Products) (108)

Research Project: Integrated Pest Management of Mosquitoes and Biting Flies **Location:
                            Mosquito and Fly Research** 2020 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. In FY20 ARS researchers at Gainesville, Florida, and university partners produced the first description of specific environmental variables potentially driving observed natural population dynamics of Aedes mcintoshi, an important mosquito vector of Rift Valley fever in endemic areas of East Africa.

2A. A field collected strain of Culex quinquefasciatus was successfully established from egg rafts provided by St. Tammany Parish, LA (LA_Culex strain). The newly established line is currently on generation F8 and reproducing moderately well in the laboratory. This strain has been characterized to have significant resistance to pyrethroids and has some resistance to the organophosphate (OP) malathion. Genetic characterization by sequencing has shown that genetic mutations common in resistant strains are present in the sodium channel (NaV) but no mutations are present in the acetylcholinesterase gene (AchE) to account for the OP resistance.

2A. Two derivative strains, one homozygous for NaV mutations and one with no NaV mutations, have been genetically selected from this initial strain to allow quantification of the effect of various combinations of mutations on phenotypic resistance. Toxicological characterization of these derivative strains has been delayed by maximum telework but is scheduled to resume under an “Other Important Research” project approval.

2A. A competitive-binding qPCR assay was designed to rapidly assess genetic factors of pyrethroid resistance in field populations of Cx. quinquefasciatus that vastly improves on previous assays by using much cheaper reagents and sample prep. 

2B. In sterile insect technique studies, dose map, competition studies and mark-release-recapture trials were successfully performed on Anastasia colony mosquitoes. ARS researchers at Gainesville, Florida, also published in a peer-reviewed journal a description of impact of irradiation doses on female Aedes aegypti from the Anastasia colony mosquitoes. ARS researchers at Gainesville, Florida, successfully marked and conducted release trials for Anastasia MCDs. 

2C. Novel synergists for spatial repellents were discovered with a university partner. ARS researchers at Gainesville, Florida, found that several pyrethroid acids can synergize not only contact repellent standards but also other pyrethroid components as well as the parent pyrethroids themselves. Synergism by the pyrethroid acids is expressed as both increased spatial repellency and vapor toxicity as well as human bite protection. 

2D. All 6 of the genetic isolines found in the wild from a mixed, field population collected by the Anastasia Mosquito Control District have been established. These six isoline strains have been verified by genetic sequencing and characterized for toxicologic response to common mosquito control adulticides. Direct topical application of permethrin, malathion and deltamethrin has been completed. Comparative enzymatic resistance assays have been completed. Testing for response in the CDC bottle bioassay has also been completed for the same three chemicals and etofenprox. ARS researchers at Gainesville, Florida, also completed permethrin-treated military uniform bite through studies and helicopter aerial spray studies to assess the performance of these isoline strains to common operational interventions.

2D. Current studies by ARS researchers at Gainesville, Florida, are underway to assess the effect of synergists on the toxicological outcome as well as further aerial sprays to assess field effects.

4D. Unique experiments were conducted in diverse environments across three field sites – hot arid southern California, warm temperate north Florida, and warm-Mediterranean Greece long-term research sites – to investigate capabilities of novel transfluthrin spatial repellent formulations and delivery systems to reduce populations of disease vector mosquitoes and Phlebotomine sand flies, and biting Tabanid flies. ARS researchers at Gainesville, Florida, along with mosquito control district collaborators, developed novel techniques to investigate efficacy of larvicide applications targeting disease vector Aedes albopictus mosquitoes in an urban environment using a large operational UAV mosquito control system. ARS researchers at Gainesville, Florida, conducted the first known parallel trials of a suite of larvicides targeting disease vector Aedes and Culex species mosquitoes applied with a large truck mounted ULV operational mosquito control spray system through exposed and cryptic environments in a simulated urban environment to determine optimal combinations of formulation and application technique. **Accomplishments**
                                

1. Isolation of two derivative mosquito strains. ARS researchers at Gainesville, Florida, and DoD scientists collaborated in the isolation of two derivative strains with different resistance profiles to provide a source of characterized organisms for use as positive and negative controls in both genetic and toxicologic resistance assays. These organisms are already of interest to the state of Louisiana for use in a resistance testing program and will be the strains used for assay validation in a Florida statewide resistance characterization study. Use and characterization of these strains gives measures to assist local mosquito control programs in determining insecticide resistance levels in local populations. 2. Development of a rapid resistance assay. ARS researchers at Gainesville, Florida, and DoD collaborating scientists developed a novel, rapid assay for assessment of genetic pyrethroid insecticide resistance in Cx. quinquefasciatus to assist in effective mosquito control decision making. While other assays exist, they use expensive reagents or require additional upstream sample purification, both of which are eliminated by this new assay. Additionally, this assay was designed to fit within the existing melt curve assay system that we and other laboratories use for the assessment of resistance in Aedes aegypti without the need for any protocol changes except for proper controls and amplification primers. The assay is currently being tested in a Florida study and will soon be implemented in Louisiana. 3. Gamma irradiation reduces survivorship, feeding behavior, and oviposition of female Aedes aegypti. Aedes aegypti is a prominent disease-vector mosquito that is difficult to control through traditional integrated vector management due to their cryptic peridomestic immature habitat and adult resting behavior, increasing resistance to pesticide formulations approved by U.S. Environmental Protection Agency, escalating deregistration of approved pesticides, and slow development of new effective chemical control measures. One novel method to control Ae. aegypti is the sterile insect technique (SIT) that leverages the mass release of irradiated (sterilized) males to overwhelm mate choice of natural populations of females. However, one potential liability of SIT is sex sorting errors of colony pupae prior to irradiation resulting in occasional release of females that may affect the palatability of the SIT program by the public and increase the number of Ae. aegypti females in the environment that could potentially bite humans and potentially become infected with and spread viruses such as dengue, chikungunya, or Zika. Scientists at Gainesville, Florida, conducted unique experiments to investigate  impacts of ¿-radiation on behavior (bloodfeeding and oviposition) and physiology (survivorship) of female Ae. aegypti pupae reared with male pupae for an operational SIT program. Data revealed that a radiation dose =30 Gy – a dose sufficient to sterilize males while preserving their mate competitiveness – may substantially negatively impact development, bloodfeeding, and oviposition of female Ae. aegypti after being irradiated as pupae. Importantly, these findings could reduce concern for females incidentally released alongside irradiated males in an operational Ae. aegypti SIT control program, allowing for higher throughput production of sterilized males and providing valuable information for engagement of the stakeholder public. 4. Discovery of synergists for topical and spatial repellents that circumvent pesticide resistance mosquitoes. Pyrethroids are one of the most commonly used classes of insecticides, and their acids are usually considered to be biologically inactive. Resistance to pyrethroid insecticides is increasingly reducing the effectiveness of pesticides and repellents. ARS researchers at Gainesville, Florida, along with partners at the University of Florida discovered that acids of several pyrethroids had a significant synergistic spatial repellent activity that was greater than the topical repellent DEET, were often more active than the parent pyrethroids, and showed little cross resistance in a pyrethroid-resistant Puerto Rico strain of Aedes aegypti mosquitoes. Of greatest significance was the observation that the synergism of several repellent compounds increased protection of human arms from biting Aedes aegypti mosquitoes. Use of these synergists may increase bite protection for existing and novel compounds, including protection against resistant mosquitoes that can transmit diseases such as dengue, Zika, chikungunya and yellow fever. 5. Selection of six mosquito strains with sodium channel mutations. ARS researchers at Gainesville, Florida, and DoD collaborating scientists genetically selected for six strains of Ae. aegypti exhibiting combinations of 2 sodium channel mutations found in the wild that are common in pyrethroid resistant Ae. aegypti in the US.  The F0 source was a wild-type collection, so all strains have a genetic background comparable to field mosquitoes and are therefore more useful for insecticide resistance studies than legacy Ae. aegypti colonies.  All strains have been characterized for toxicologic and enzymatic response to common adulticides and have been used in aerial spray studies to test operational efficacy. 6. Treated military uniform efficacy study completed. ARS researchers at Gainesville, Florida, and DoD collaborating scientists completed and published a study (in press) clearly demonstrating that treated military uniform efficacy may be completely compromised by strongly pyrethroid resistant strains of Aedes aegypti when the resistance is primarily genetic. This study also showed that the protection provided by DEET based repellents was the same for both the resistant and susceptible strains. 7. Variation in larvicide efficacy against disease vector Aedes and Culex mosquitoes in urban environment. The Aedes aegypti mosquito is a key vector of prominent viruses including dengue and Zika and displays increasing resistance to pesticides targeting their adult stages, potentially favoring operational control programs that include larval control. However, mosquito control programs face challenges in accessing immature mosquito habitat behind urban and suburban structures when conducting truck mounted spray applications of larvicides from the street. Scientists at Gainesville, Florida, conducted the first known parallel trials of two commonly used larvicide formulations applied as a residual with a large ULV sprayer in a simulated urban environment. These trials targeted locations where immature Aedes aegypti mosquitoes could be found such as in discarded containers in front and back yards and in roof-top depressions where water could pool. The sprayer used was one specifically chosen by operational mosquito control programs to deliver larvicide into back yards in urban areas from the street. Results indicated both larvicide formulations could be effective immediately in the front of the house and on the roof, but with little impact on immature habitat behind buildings. These results indicate that current measures designed to overcome a key challenge in operational mosquito control targeting prominent disease vector mosquitoes may need further refinement to protect public health that could include specially formulated larvicide mixtures or modifications to spray equipment to place larvicides into back yards. **Review Publications**
                            *Yang, L., Richoux, G.M., Norris, E., Cuba, I., Jiang, S., Coquerel, Q., Linthicum, K., Bloomquist, J.R. 2020. Pyrethroid-derived acids and alcohols: bioactivity and synergistic effects on mosquito repellency and toxicity. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.9b07979.*
                                *Trzebny, A., Slodkowicz-Kowalska, A., Becnel, J., Sanscrainte, N.D., Dabert, M. 2020. A new method of metabarcoding Microsporidia and their hosts reveals high levels of microsporidian infections in mosquitoes (Culicidae). Molecular Ecology Resources. 00:1-19. https://doi.org/10.1111/1755-0998.13205.*
                                *Pantoja-Sanchez, H., Vargas, J.F., Ruiz-Lopez, F., Rua-Uribe, G., Velez, V., Kline, D.L., Bernal, X.E. 2019. A new approach to improve acoustic trapping effectiveness for Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology. 44(2):216-222. https://doi.org/10.1111/jvec.12352.*
                                *Britch, S.C., Linthicum, K., Kline, D.L., Aldridge, R.L., Golden, F.V., Wittie, J., Henke, J., Hung, K., Gutierrez, A., Snelling, M., Lora, C. 2020. Transfluthrin spatial repellent on US military materials reduces Culex tarsalis incursion in a desert environment. Journal of the American Mosquito Control Association. 36(1):37-42. https://doi.org/10.2987/19-6894.1.*
                                *Degener, C.M., Geler, M., Kline, D.L., Urban, J., Willis, S., Ramirez, K., Cloherty, E.R., Gordon, S.W. 2019. Field trials to evaluate the effectiveness of the Biogents Sweetscent lure in combination with several commercial mosquito traps and to assess the effectiveness of ABiogents Mosquitaire trap with and without carbon dioxide. Journal of the Mosquito Control Association. 35 (1): 32-39, 2019.*
                                *Bibbs, C.S., Kline, J., Kline, D.L., Estaver, J., Strohschein, R., Allan, S.A., Kaufman, P.E., Xue, R., Batich, C.D. 2019. Olfactometric comparison of the volatile insecticide, Metofluthrin, through behavioral responses of Aedes albopictus (Diptera: Culicidae). Journal of Medical Entomology. 57(1):17–24. https://doi.org/10.1093/jme/tjz160.*
                                *Britch, S.C., Cilek, S.C., Lindroth, E.J., Aldridge, R.L., Golden, F.V., Weeston, J.R., Fajardo, J.D., Richardson, A.G., Blersch, J.S., Linthicum, K. 2020. Earth fill increases efficacy and longevity of ¿-cyhalothrin residual insecticide treatment of HESCO® blast wall geotextile. Journal of the American Mosquito Control Association. 67:36-50.*
                                *Aldridge, R.L., Britch, S.C., Linthicum, K., Golden, F.V., Dao, T.T., Rush, M.J., Holt, K., White, G., Gutierrez, A., Snelling, M. 2020. Pesticide misting system enhances residual pesticide treatment of HESCO geotextile. Journal of the American Mosquito Control Association. 36(1):43-46. https://doi.org/10.2987/19-6897.1.*
                                *Campbell, L.P., Reuman, D.C., Lutomiah, J., Peterson, A.T., Sang, R., Linthicum, K., Britch, S.C., Anyamba, A. 2019. Predicting abundances of Aedes mcintoshi, a primary Rift Valley fever virus mosquito vector. PLoS One. 14(12):e0226617. https://doi.org/10.1371/journal.pone.0226617.*
                                *Cilek, J.E., Aldridge, R.L., Britch, S.C., Fajardo, J.D., Linthicum, K., Richardson, A.G. 2020. Effectiveness of Aquatain AMF and cocobear larvicidal oil against Culex quinquefasciatus larvae and pupae in an organically enhanced aquatic habitat. Journal of the American Mosquito Control Association. 36(1):47-50. https://doi.org/10.2987/19-6862.1.*
                                *Demiray, H., Tabanca, N., Estep, A.S., Becnel, J.J., Dermirci, B. 2019. Chemical composition of the essential oil and n-hexane extract of Stachys tmolea subsp. tmolea Boiss., an endemic species of Turkey, and their mosquitocidal activity against dengue vector Aedes aegypti. Saudi Pharmaceutical Journal. 27(2019) 877–881. https://doi.org/10.1016/j.jsps.2019.05.009.*
                                *Aryaprema, V.S., Blore, K., Kline, J., Aldridge, R.L., Linthicum, K., Xue, R. 2020. Laboratory evaluation of boric acid sugar baits against irradiated Aedes aegypti. Journal of the Florida Mosquito Control Association. 67:76-79.*
                                *Cunningham, C.A., Aldridge, R.L., Kline, J., Bibbs, C.S., Linthicum, K., Xue, R. 2020. Sub-lethal effects of irradiation on the blood feeding of Aedes aegypti (Diptera: Culicidae). Journal of Vector Ecology. 45(1):140-141. https://doi.org/10.1111/jvec.12382.*
                                *Yang, L., Liu, Y., Richoux, G.M., Bernier, U.R., Linthicum, K., Bloomquist, J.R. 2019. Induction heating coil heating improves the efficiency of insect olfactory studies. Frontiers in Ecology and Evolution. 7:247. https://doi.org/10.3389/fevo.2019.00247.*
                                *Yang, L., Norris, E.J., Jiang, S., Bernier, U.R., Linthicum, K., Bloomquist, J.R. 2019. Reduced effectiveness of repellents in a pyrethroid-resistant strain of Aedes aegypti (Diptera: culicidae) and its correlation with olfactory sensitivity. Pest Management Science. https://doi.org/10.1002/ps.5562.*
                                *Yang, L., Agramonte, N., Linthicum, K., Bloomquist, J.R. 2020. A survey of chemoreceptive responses on different mosquito appendages. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjaa154.*


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