Project : USDA ARS

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Research Project: Integrated Management of Resistance and Population Expansion of Disease Vector and Nuisance Mosquitoes

Location: Mosquito and Fly Research

2025 Annual Report

Objectives
Objective 1: Integrated management of insecticide resistance in mosquitoes. 1.A: Expand species distribution and develop insecticide resistance baseline information for species that have demonstrated ability to transmit human disease in the US. 1.B: Operationalize insecticide resistance research findings to improve mosquito control. 1.C: Manage insecticide resistance and de-registration of pesticide active ingredients through innovations with plant-derived and innovative chemical toxicants and repellents. 1.D: Bridge the critical path between the laboratory and the field: create and implement high throughput and realistic toxicant and repellent product development systems. 1.E: Investigate new wearable mosquito-protective US military fabric solutions. 1.F: Field evaluation of new mosquito and/or sand fly control solutions. 1.G: Optimization of male mosquito production and field and operational evaluation of the sterile insect technique (SIT) to control disease vector Diptera such as Aedes aegypti or Phlebotomus species. Objective 2: Integrated management of mosquito population expansion. 2.A: Develop improved systems to prepare and protect the US from emerging arthropod-borne viruses. 2.B: Develop improved surveillance solutions: Leverage emerging technology to create next generation surveillance systems and approaches.

Approach
Objective 1: Integrated management of insecticide resistance in mosquitoes. 1.A: Expand species distribution and develop insecticide resistance baseline information for species that have demonstrated ability to transmit human disease in the US. 1.B: Operationalize insecticide resistance research findings to improve mosquito control. 1.C: Manage insecticide resistance and de-registration of pesticide active ingredients through innovations with plant-derived and innovative chemical toxicants and repellents. 1.D: Bridge the critical path between the laboratory and the field: create and implement high throughput and realistic toxicant and repellent product development systems. 1.E: Investigate new wearable mosquito-protective US military fabric solutions. 1.F: Field evaluation of new mosquito and/or sand fly control solutions. 1.G: Optimization of male mosquito production and field and operational evaluation of the sterile insect technique (SIT) to control disease vector Diptera such as Aedes aegypti or Phlebotomus species. Objective 2: Integrated management of mosquito population expansion. 2.A: Develop improved systems to prepare and protect the US from emerging arthropod-borne viruses. 2.B: Develop improved surveillance solutions: Leverage emerging technology to create next generation surveillance systems and approaches.

Progress Report
Under Sub-objective 1.A. ARS scientists at Gainesville, Florida; academic researchers in AZ, FL, and IL; vector control personnel in several states; and the US Department of Defense worked together to expand the understanding of the scope and intensity of insecticide resistance in vector mosquitoes along several lines of effort. This included the continued monitoring of insecticide resistance in Aedes aegypti. A notable highlight was the identification and publication, for the first time (ARIS log #420197) in the United States, the presence of impactful insecticide resistance mutations that are traditionally only found in Asia. Subsequent testing detected these mutations were found in 2 additional counties in Florida and did not detect these mutations in Arizona. Identification of these mutations in the US, resulted in a recommendation to update a common method used to monitor insecticide resistance. In response to a 2024 outbreak of locally transmitted malaria in Florida, ARS scientists from Gainesville, Florida; a Florida vector control program; University of Florida researchers; and the CDC Southeast Center of Excellence in Vector Borne Disease conducted baseline characterization of insecticide resistance in Anopheles crucians, the vector species implicated in the outbreak. This species has not been colonized and little information about insecticide susceptibility exists, so this effort required development of baseline information. Vector control personnel collected more than 2000 An. crucians from the county of the outbreak and ARS scientists conducted deep sequencing to identify both common molecular targets of pyrethroid and organophosphate insecticides. Based on these initial data, work is underway to develop a rapid assay to screen these targets for characteristic insecticide resistance mutations. Further work included assessment of insecticide resistance in Culex tarsalis in AZ, Aedes albopictus in FL and MD, Aedes taeniorhynchus in FL, Culex quinquefasciatus in FL and LA, and Culex pipiens from the Chicago area. Manuscripts are in various stages of preparation. Under Sub-objective 1.B. ARS scientists at Gainesville, Florida, developed and validated a modified topical application method, called the Low Input Topical (LIT) Assay, to quantify baseline susceptibility and insecticide resistance in arthropods. This method uses an analytical framework derived from quantal analysis methods developed beginning in the 1920s, survival analysis, and OECD procedures adopted to minimize the numbers of animals used for toxicity studies. ARS scientists demonstrated equivalency of the LIT Assay and traditional methods of topical application using laboratory strains. The LIT Assay resulted in an approximately 60% reduction in the number of organisms required and a more than 50% reduction in labor. A manuscript sharing these findings is being finalized. Under Sub-objective 1.C. ARS scientists at Gainesville, Florida, have identified and screened numerous natural product extracts and individual natural products as spatial repellents against Aedes aegypti in laboratory bioassays. Using a small glass tube repellency assay, we have identified select compounds that are potent spatial repellents. Future work will aim to identify what constituents make the most repellent extracts significantly bioactive. We also plan to expand our testing to other mosquito strains and species to determine the potential of these agents to control broad spectra of mosquitoes and biting flies. Under Sub-objective 1.D. ARS scientists at Gainesville, Florida, have selected and screened over 50 different terpenoids, natural products, and synthetic insecticides as insecticides and insecticide synergists against Aedes aegypti mosquitoes in laboratory bioassays. So far, we have identified select compounds that are significantly more toxic than the others that have been screened. This work will be essential for our next steps, which will utilize machine learning and artificial intelligence algorithms to better understand which chemical properties correlate best with insecticide toxicity. Under Sub-objective 1.E. ARS scientists at Gainesville, Florida, have been working closely with their military collaborators at the US Army Soldier Center in Natick, MA, to continually develop novel pyrethroid-treated clothing for the protection of US deployed personnel and civilians alike. This year, we have screened various pyrethroid-treated (etofenprox and permethrin) uniforms on human subjects and have identified select fabric types that provide sufficient protection to wearers. This work will continue and we will move into the evaluation of novel synergists for pyrethroids when applied to consumer-end fabrics. Under Sub-objective 1.F. ARS scientists at Gainesville, Florida, have screened more than 20 essential oil candidate spatial repellents for efficacy against natural populations of Florida mosquitoes. Selected oils showed high efficacy against specific genera of mosquitoes. This phenomenon creates an opportunity to combine oils to protect personnel in the field from specific communities of biting nuisance and vector mosquito threats, potentially filling a critical gap in repellent technology. Under Sub-objective 1.G. ARS scientists at Gainesville, Florida, maintained two Aedes aegypti mosquito colonies (California and Gainesville strains) to support future work if vacant principal investigator position is filled. Preliminary investigation of effects of chilling and compaction on male survivorship and mating capabilities was undertaken and is ongoing. Under Sub-objective 2.A. ARS scientists at Gainesville, Florida, conducted innovative Machine Learning (ML) approach to upgrade a proven but dated risk prediction system for Rift Valley fever (RVF) to better protect US agriculture and public health. RVF is a prominent and severe zoonotic vector-borne disease of livestock and humans. Maximum capability of the original RVF outbreak risk prediction system was 65% spatial accuracy and 2-4-month lead time. Initial results from ML upgrades that incorporate a broader catalog of case data suggest improved spatial accuracy (+10-20%) and lead time (+1-2 months). Under Sub-objective 2.B. as the first step towards developing a novel mosquito surveillance system utilizing artificial intelligence technologies (e.g., Machine Learning and Deep Learning) ARS scientists at Gainesville, FL, developed databases for training models. Databases for Aedes taeniorhynchus were obtained using a smart phone to record the wingbeat frequencies of male and female adults utilizing CMAVE mosquito colonies. Additional acoustical sound databases of several mosquito species were obtained as a result of an online search. Habitat classification and hydrology databases were obtained from the Lower Suwannee National Wildlife Refuge. Once fully developed and implemented the use of AI innovative surveillance systems will allow earlier detection of mosquito-borne diseases that threaten American farmers and their livestock, hunters, US military personnel, and other US citizens in the outdoors, and this will allow a quicker response to manage these threats.

Accomplishments
1. Development of an organism and time parsimonious method and analytical framework to quantify insecticide resistance. ARS researchers at Gainesville, Florida believe assessment of the intensity of insecticide resistance is critical information for operational insect management programs, particularly for species such as mosquitoes that vector disease to humans and livestock. Current methods are either rapid and require few organisms or require significant labor and hundreds of organisms. Unfortunately, the rapid methods cannot quantify resistance intensity while the more laborious methods require so many organisms that they require colonization of the vector, which for many species, is not possible. In response to this issue, ARS scientists developed the Low Input Topical Assay (LIT Assay) method, implemented a rigorous analytical framework, and showed the LIT Assay produced equivalent results to the standard method but with an approximately 60% reduction in the number of organisms required and a more than 50% reduction in labor. This advancement in efficiency of resistance detection will greatly facilitate operational decision-making for the control of insects by vector management, US military personnel, and US livestock producers.

2. Graphene-oxide as a bite protectant material. Repellent-treated fabrics are one of the most successful technologies in preventing mosquito bites, and thus mosquito-borne disease, in those who wear them. However, logistical and economic hurdles exist in their fabrication and deployment for long periods of time, as the repellent chemistries can degrade or wear off. ARS researchers at Gainesville, Florida in collaboration with textile chemists in the Department of Defense have identified a series of graphene oxide-treated fabric types that significantly reduce mosquito bites in human subjects who wear the materials. Under standard mosquito bite-protection assays, three specific graphene oxide print formats (dots, thick lines, and thin lines) were capable of significantly reducing mosquito biting from 2 important mosquito disease vectors, Aedes aegypti and Anopheles albimanus. Testing indicated that protection was the highest after washing fabrics 20X and 50X, indicating the potential for this technology to bolster the protective nature of repellent-treated fabrics, which many times are no longer efficacious after many washes. These findings will be very useful to US farmers, civilians, and US military personnel looking to prevent mosquito bites and may allow for significantly lower quantities of synthetic repellents/insecticides being applied to fabrics and efficacy of these fabrics for longer periods of time.

3. Evaluation of the utility of bioinsecticides. While natural chemistries are becoming ever more prevalent in various commercial insect control formulations, there is still debate whether these natural compounds are as effective as previously utilized synthetic insecticides and repellents. ARS researchers at Gainesville, Florida in collaboration with researchers from the University of British Columbia performed a review of recent studies (last 5 years) that highlight combinatorial mixtures of various bioinsecticides (such as plant terpenoids, abamectin) alone or applied with other conventional, synthetic insecticides (such as permethrin, imidacloprid). This study revealed synergism exists between diverse bioinsecticides and conventional insecticides alike. This has important implications for pest control as bioinsecticides are considered safer to humans and the environment alike and may allow for more efficacious insecticidal formulations in the future. This study also highlighted a lack of current research in the effect of these mixtures on non-target beneficial arthropods, such as honeybees and predacious insects, indicating a fruitful avenue for future research. These studies demonstrate that bioinsecticides may successfully be used to control many pest insects, ranging from mosquitoes to agricultural pests. Moreover, these compounds can be utilized as both independent insecticidal active ingredients or as synergists of other current insecticides on the market today, which will benefit US farmers and pest control agencies to better abate economically or public health-relevant pest insects.

4. Synergizing fabric-applied pyrethroid repellents. Because of the continual development of insecticide resistance, new control tools need to be explored and developed. ARS researchers at Gainesville, Florida discovered (patent pending) select fatty acids were capable of potently synergizing permethrin and etofenprox against a pyrethroid-susceptible strain of Aedes aegypti. When evaluating these synergists on two pyrethroid-resistant strains of Aedes aegypti, it was discovered one molecule uniquely synergized both permethrin and etofenprox. These agents are uniquely capable of reversing pyrethroid-resistance in the strains explored. This work demonstrates that pyrethroid-treated repellent fabrics can remain potent protective barriers against disease-vectoring mosquitoes when coupled to these novel synergists, even against highly resistant strains of mosquitoes. American farmers, farm animals, outdoorsmen, and US military personnel will benefit from this technology as it will further improve the ability of repellent-treated clothing/fabrics to prevent mosquito-borne disease.

5. Natural extract spatial repellency against wild mosquitoes. As mosquitoes and other biting arthropods are becoming more resistant to currently available insecticides and repellent chemistries, new tools are always needed to combat the spread of arthropod-borne disease to both humans and US farm animals. ARS scientists screened a series of natural products as spatial repellents against wild mosquitoes in Gainesville, Florida. A panel of over 20 natural products was screened against a diverse set of mosquito species. These studies demonstrated that select natural products were capable of significantly repelling wild mosquito species. Interestingly, some natural products were more effective against specific mosquito species in these field tests. This information will be used to design better spatial repellent formulations that can be utilized as repellent products by American farmers, US military personnel, and American consumers.

6. National Veterinary Stockpile Rift Valley fever virus Gap Analysis. Although currently endemic to the African continent, globalization of Rift Valley fever virus (RVFV) – which may be transmitted by direct contact as well as by mosquitoes – is a prominent threat to US livestock agriculture and human health. However, there are significant gaps in understanding key aspects of the virus to develop protective strategies. ARS researchers at Gainesville, Florida contributed expert knowledge of innovations in mosquito surveillance and control, epidemiology modeling, and interagency coordination to the USDA-APHIS National Veterinary Stockpile Rift Valley Fever Virus Gap Analysis. This document establishes a high-level strategy to close gaps in knowledge of RVFV vaccines, diagnostics, epidemiology, vector biology/ecology, and vector control. 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 gap document will provide a quick reference for government, academia, US military, and other American public and veterinary health agencies to bring our collective capabilities to the next level in protecting human and animal health from this virus.

U.S. DEPARTMENT OF AGRICULTURE

Mosquito and Fly Research: Gainesville, FL