Laboratory and field assays indicate that a widespread no-see-um, Culicoides furens Poey is susceptible to permethrin

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Cooper, Eva A. Buckner, Yongxing Jiang, Nathan Burkett-Cadena This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4953430/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The recent reemergence of Oropouche virus (OROV) highlights the need to better understand insecticide susceptibility in Culicoides (Diptera: Ceratopogonidae), which contains the vector of OROV and many other species that are biting nuisances and vectors of pathogens that affect humans, livestock, and wildlife. With adulticides as the primary method of Culicoides control, there is growing concern about insecticide resistance, compounded by the lack of tools to monitor Culicoides susceptibility. We adapted the CDC bottle bioassay and field cage trial methods, typically used to monitor insecticide susceptibility in mosquitoes and formulated adulticide efficacy, to evaluate permethrin susceptibility in the widely distributed coastal nuisance species, Culicoides furens . Permethrin caused complete mortality in C. furens in field and laboratory assays. We identified a diagnostic dose (10.75 µg) and time (30 minutes) that resulted in complete mortality in CDC bottle bioassays. Additionally, we determined that no-see-um netting is an effective mesh for field cage trials, allowing for accurate assessment of Culicoides susceptibility to ultra-low volume applications of formulated adulticides like Permanone 30–30, a widely utilized adulticide. These methodologies offer essential tools for assessing Culicoides susceptibility, which is crucial for managing populations of Culicoides and preventing the spread of OROV and other pathogens. Biological sciences/Ecology Health sciences/Diseases/Infectious diseases Earth and environmental sciences/Ecology/Invasive species Oropouche virus vector control IPM IVM Figures Figure 1 Figure 2 Figure 3 Introduction The recent reemergence and rapid spread of Oropouche virus (OROV) in the Americas 1 , 2 highlights existing knowledge gaps in the ecology and control of Culicoides (biting midges, no-see-ums) 3 , including Culicoides paraensis Goeldi, the confirmed vector of OROV. Once considered endemic to the greater Amazonian Region of South America 4 , OROV is becoming increasingly prevalent in parts of South America, and recently spread to portions of the Caribbean, posing a significant public health risk outside of its endemic range 5 , 6 . Vector control agencies in the areas of OROV expansion will benefit from understanding whether their mosquito control practices are effective against Culicoides spp. that bite humans. The genus Culicoides (Diptera: Ceratopogonidae) includes species that are not only biting nuisances but also vectors affecting humans, wildlife, and livestock. Culicoides species transmit around 20 arboviruses and five nematode species of veterinary importance, affecting a wide range of hosts 7 . Notable arboviruses include African horse sickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) 8 , 9 . These viruses significantly impact livestock industries across multiple continents, including North America 10 , Central and South America 8 , Europe 11 , Africa, Asia, and the Middle East 12 . Globally, vector control relies heavily on chemical adulticides, particularly pyrethrins and pyrethroids like permethrin due to their potency and relative safety for mammals 13 , 14 . Permethrin is widely used for mosquito control in the United States 15 . Additionally, permethrin is the main active ingredient used for Culicoides control in Florida deer farms, where its frequent use has raised concerns about insecticide resistance 16 . Monitoring insecticide susceptibility and resistance in target arthropods is a cornerstone of successful control program 17 . Insecticide resistance is often detected through laboratory bioassays 18 such as the World Health Organization (WHO) tube assay and the Centers for Disease Control and Prevention (CDC) bottle bioassay 17 . These assays are extensively used to monitor resistance in mosquitoes and other vectors of public health significance 17 , 19 , 20 . However, standardized protocols for applying these methods to Culicoides are currently lacking. Field-based bioassays, such as “field cage trials”, are recommended to assess the susceptibility of insects to formulated insecticides like adulticides, particularly when resistance is detected in laboratory settings 17 . Despite the medical, veterinary, and nuisance importance of many Culicoides spp., established protocols for monitoring susceptibility in no-see-ums are lacking. In North America, only a few studies have investigated the susceptibility of Culicoides to adulticides 21 – 23 . These studies, conducted on Culicoides furens populations in Florida, tested the efficacy of ULV sprays containing organophosphates (malathion, naled) and a pyrethroid (resmethrin). Naled was found to be the most effective, achieving 90% mortality at distances up to 106 m, compared to 36 m for malathion and 25 m for resmethrin 23 . However, a recent survey of Florida mosquito control programs documented higher usage of pyrethrin and the pyrethroid permethrin as adulticide active ingredients as compared to the organophosphates malathion and naled 24 . Additionally, only one Florida mosquito control program reported using resmethrin in 2020 24 . Understanding Culicoides susceptibility to insecticides currently being used by vector control programs is essential for developing integrated vector control programs. However, studies are limited due to challenges in colonizing Culicoides and the lack of established testing protocols. While 151 Culicoides species are recorded in North America, only Culicoides sonorensis is currently colonized 25 . Standard bioassays like the CDC bottle bioassay and cage field trials are commonly used for mosquitoes, but their applicability to Culicoides is uncertain. We adapted these methods to evaluate the susceptibility of wild C. furens from Indian River County, FL aiming to establish a diagnostic dose (DD) and diagnostic time (DT) for use in CDC bottle bioassays and identify a suitable mesh for cage field trials. Methods Insects. The challenges of Culicoides colonization have somewhat normalized the use of wild specimens 26 . We tested wild C. furens , a nuisance species common in U.S. coastal areas, trapped at the University of Florida, Florida Medical Entomology Laboratory (FMEL) in Indian River County. Culicoides were captured using CDC miniature light traps (BioQuip products, Rancho Dominguez, CA, USA) connected to live midge collection chambers 27 . Traps were baited with an incandescent light bulb, 2 mL of octenol (1-Octen-3-ol) placed in a 15 mL plastic tube with a piece of cotton wool, and ~ 1kg of dry ice contained in an insulated 1.89 L beverage container (Igloo Products Corp., Katy, TX). Since reference (known susceptible control) C. furens are unavailable (no laboratory colony in existence), we used a laboratory-reared strain of Aedes aegypti (Orlando 1962) which originated from Orlando, FL (Orange County) as a susceptible control. This strain was originally obtained from colonies maintained at the USDA-ARS CMAVE. The Ae. aegypti were reared in an insectary at 26°C and 85% relative humidity. Egg papers were placed in plastic trays containing approximately 2 L of tap water at a density of 200–300 eggs per rearing tray. Larvae were fed a 1:1 mixture of lactalbumin and yeast ad libitum. Pupae were transferred from larval rearing trays to water-filled plastic cups in 30.5 x 30.5 x 30.5 cm Bug Dorm adult rearing cages (BioQuip, Rancho Dominguez, CA, USA). Cotton rounds soaked with 10% sucrose solution were placed inside each cage as carbohydrate source for emerging adults. CDC Bottle bioassays. The CDC bottle bioassay is commonly used to monitor mosquito insecticide susceptibility 19 . We adapted this method to calibrate the assay for Culicoides by determining the DD and DT for permethrin. Following the CDC protocol 17 , we prepared permethrin solutions by diluting technical grade permethrin (100% Chem Service Inc., West Chester, PA, USA) in American Chemical Society (ACS) grade acetone (Fisher Scientific, Hampton, NH). Each bioassay used five 250-ml clear Wheaton bottles with lids (DWK Life Sciences Inc., Millville, NJ), four coated with 1.0 ml of the permethrin solution and one untreated control coated with acetone only. Bottles were coated by swirling the solution inside and then left to dry on a hot dog roller machine with heat turned off to ensure even coating. Control bottles were capped and rolled before preparing bottles containing insecticide. All bottles were then left open to dry in a dark environment to prevent permethrin photodegradation. Preliminary observations showed high mortality of C. furens in control bottles when the bioassay was performed within five hours after the bottles were coated. We tested various drying times (5, 10 and 24 h) and found that 24 hours of drying were necessary to eliminate control mortality. As recommended by Brogdon and Chan 18 , when calibrating the CDC bottle bioassay to determine DD and DT for a new insect, we prepared bottles with a range of permethrin concentrations to determine a possible DD. Based on the CDC’s recommended DD of 43 µg permethrin/bottle for susceptible Ae. aegypti 17 , and the small size of C. furens in comparison to Ae. aegypti , the permethrin concentrations we tested as possible DD were 43, 21.5, and 10.7 µg permethrin/bottle. The permethrin concentration that killed 100% of the wild C. furens between 30 and 60 minutes would be selected as the DD (dose that kills 100% of insects 18 . Bottle bioassays were performed at the same time but in separate bottles for susceptible Ae. aegypti and wild C. furens . Mosquitoes and C. furens were aspirated by mouth into each bottle with the goal of 20 individuals per bottle. Mortality was recorded every five minutes for the first 15 minutes and every fifteen minutes until 2-h post-exposure 18 . After the 2-hrs of exposure, insects were transferred into 8-oz paper food cups with lids and generic mesh to prevent all insects from escaping. Insects were provided with a cotton round with water and mortality was then assessed at 24-h post-exposure 28 . Field cage trials. Two field trials were conducted between March and April, 2023 in Indian River County, FL (Table 1 ), targeting caged susceptible Ae. aegypti and/ or wild C. furens (Table 1 ). Permanone 30–30 (30% permethrin, 30% piperonyl butoxide) was applied at maximum rate (0.007 lb permethrin/acre) (Table 1 ) using a truck-mounted ULV aerosol generator that was operated by personnel of the Indian River Mosquito Control District. Spray missions were performed around dusk (1700h and 1800h) on a five-acre plot of low-cut grass at the Indian River County Fairgrounds. Bioassay cages (Fig. 1 ) were made of cardboard rings (15.2 cm diameter, 2.5 cm width) covered with mesh 26 , 29 . Three mesh types were tested (Fig. 1 ): stainless steel (SS) McMaster-Carr, Douglasville, GA), no-see-um netting (NN) (Seattle Fabrics, Inc., Seattle, WA), and fine nylon tulle fabric (Hobby Lobby Stores, Inc., Oklahoma, USA), hereafter called mosquito mesh (MM). SS and NN mesh were chosen because preliminary observations indicated they could prevent escape of C. furens (Cooper, unpublished data). Although the MM mesh has openings too large for Culicoides , it was included as it has been used successfully in mosquito cage trials 30 . Table 1 Summary of ULV permethrin adulticide field cage trials to evaluate the susceptibility of C. furens in Indian River County, Florida, USA. (SS) stainless steel, McMaster-Carr, Douglasville, GA; (NN) no-see-um netting, Seattle Fabrics, Inc, Seattle, WA.; (MM) mosquito mesh (fine nylon tulle fabric), Hobby Lobby Stores, Inc, Oklahoma City, OK. Field Trial Date Species tested Mesh type tested Number of passes Speed Wind speed 1 March 1 Ae. aegypti (susceptible) SS, NN, MM 2 10 and 5 mph, respectively 3–5 mph 2 April 6 Ae. aegypti (susceptible) SS, NN, MM 1 10 mph 6–10 mph C. furens (wild) SS, NN The mosquitoes-only field cage trial aimed to compare different mesh types for field bioassay cages. Approximately 20 susceptible Ae. aegypti were placed in each cage. After confirming that the mesh types allowed insecticide passage and caused mortality in these mosquitoes, a combined mosquitoes and Culicoides trial was conducted. Each cage contained about 30 wild C. furens and 20 Ae. aegypti , except for the MM cages, which only had Ae. aegypti (Table 1 ). This design allowed us to evaluate the susceptibility of wild C. furens to permethrin relative to susceptible Ae. aegypti . Cages were hung approximately 1.3 m above ground on shepherd hooks in five clusters, each with the three mesh types (Fig. 1 ). Clusters were spaced every five meters along a north-south transect, 20 m downwind of the spray truck path. Two untreated (control) cages of each mesh type were placed 20 m upwind. Mortality was assessed in the field 10 minutes post-exposure, and subsequently recorded at 1, 12 and 24 h post-exposure in the laboratory. Mortality was based on knockdown (inability to stand on legs or have coordinated flight 17 ). Insects were provided a cotton round with tap water and maintained in the cages for 24 hours, before being placed in dry ice to kill any remaining live insects. Data Analysis. Results from the cage field trials were analyzed by calculating the mean mortality of mosquitoes and Culicoides at various times post-exposure. For each trial, mean mortality and standard deviation were plotted 30 . For CDC bottle bioassays, time-response survival curves were created for each permethrin dose. Insect survival was assessed using Cox proportional hazards models with the ‘survival’ package in R version 4.2.2 31 . Clustered Cox regression was performed to compare mean mortality across the different permethrin doses for both mosquitoes and Culicoides 32 . Results CDC Bottle Bioassays. Susceptible Ae. aegypti mosquitoes reached complete mortality at 30 minutes post-exposure in CDC bottle bioassays performed with the high and medium permethrin doses (43 and 21.5 µg /bottle), while these same doses caused complete mortality of C. furens at five minutes post-exposure (Fig. 2 ). Significantly greater mortality was observed in C. furens exposed to the high dose and medium doses in comparison to mosquitoes ( P < 0.0001). The low permethrin dose (10.75 µg /bottle) caused complete mortality in Ae. aegypti and C. furens at 45 and 30 minutes, respectively (Fig. 2 ). No significant differences were observed in the mortality of C. furens and Ae. aegypti mosquitoes exposed to the low dose ( P = 0.3699). The susceptible Ae. aegypti mosquitoes and wild C. furens did not recover at 24 hours with any of the three permethrin doses, with the exception of C. furens from bottles treated with the low dose, in which five percent recovery was observed at 24 hours. However, mortality in untreated control bottles was significantly lower in comparison to treated bottles ( P < 0.0001). We did not observe significant differences in the mortality of susceptible mosquitoes in comparison to wild C. furens ( P = 1.0000). Abbot’s formula was not used to correct percent mortality in any of the bottle bioassays because control mortality at two hours did not exceed 3% 17 . Mosquito-only field cage trial . All three mesh types allowed the passage of Permanone 30–30, resulting in mosquito mortality (Fig. 3 A). In the mosquito-only trial, two adulticide passes were performed because no mortality was observed within 10 minutes after the first pass. We were unaware whether the insecticide had not gone through the mesh during the first pass or there was another reason why the insects were not experiencing mortality. A second pass performed within 20 minutes after the first application resulted in immediate mortality of the exposed mosquitoes. At one-hour post-application, mortality rates were 89% (SS), 97% (NN), and 98% (MM) (Fig. 3 A). Complete mortality was observed at 12 hours in SS and MM cages (Fig. 3 A), while NN cages only reached 96% at 12 hours, with no further increase by 24 hours. Mosquitoes did not recover from the adulticide effects within the 24-hour period. Control cages showed minimal mortality at 24 hours, with 15% in MM cages and 5% and 6% in SS and NN, respectively. Since control mortality was only observed after 24 hours, Abbot’s formula was not applied to correct the percent mortality in treatment cages 17 . Mosquitoes and Culicoides field cage trial. The second trial confirmed that bioassay cages constructed with SS and NN could be used to evaluate Culicoides susceptibility to ULV adulticides. Complete mortality of Ae. aegypti occurred in all bioassay cages at 1-hr post-application (Fig. 3 B), while 100% mortality of C. furens was observed at 10 minutes. Mosquitoes and C. furens did not recover from the effect of the adulticide within the 24-hour period. Mortality in untreated control cages was only observed at 24 hours for Ae. aegypti placed inside SS cages and biting midges in SS cages (6%) and NN cages (16%). Because 100% mortality was observed in all treatment cages 1-hr post-exposure and no mortality was observed in the control cages until after 24 hours post-exposure, Abbot’s formula was not used to correct percent mortality in treatment cages. Discussion In this study, we demonstrated that both the CDC bottle bioassay and field cage trials can be effectively adapted to assess insecticide susceptibility and efficacy of ULV treatments against Culicoides species. Obtaining this information is advantageous for protecting the health of humans and other animals. Our findings include the first records of DD and DT for Culicoides in CDC bottle bioassays, which can serve as reference for other species, such as C. paraensis , the main vector of OROV. We also identified two suitable mesh types for field cage trials with Culicoides . We observed mortality in C. furens in CDC bottle bioassays using technical grade permethrin and in the field cage trial with Permanone 30–30, which suggests that wild C. furens from Indian River County are susceptible to permethrin. Despite the widespread use of adulticides to control Culicoides spp., their susceptibility to these treatments remains poorly understood. This study highlights the utility of field cage trials in assessing the effectiveness of adulticide applications against both vector and nuisance species. Although C. furens is not a vector of pathogens, this nuisance species can significantly impact the economy. For instance, in Australia, Culicoides nuisance is estimated to reduce residential property values by 8,300 USD per residence based on actual sale price 33 . Ratnayake and co-authors also suggest that Culicoides prevent urban development in coastal areas, with over half of surveyed residents of Hervey Bay, Australia considering the impact of Culicoides a major factor in housing investment decisions 33 . We were able to determine a potential DD and DT using wild Culicoides . Typically, DD and DT used in CDC bottle bioassays are established using susceptible populations 6 . However, despite numerous attempts to establish Culicoides colonies since 1921 34 , only C. sonorensis is colonized in the United States 25 . Moreover, the susceptibility of this colonized C. sonorensis to permethrin remains undocumented. In the absence of a susceptible Culicoides population for calibration, we adapted the CDC bottle bioassay for wild C. furens . We found that 10.75 µg permethrin per bottle resulted in 100% mortality of C. furens from Indian River County, FL, within 30 minutes. Therefore, we recommend using 10.75 µg permethrin per bottle and 30 minutes as the DD and DT for this and potentially other wild Culicoides species. We expect that the time to reach complete mortality in CDC bottle bioassays and cage field trials will vary between studies due to various factors. For example, different mortality rates may be observed in cage field trials using different permethrin formulations, even when applied at the same dose. We anticipate that differences in the time to reach complete mortality in CDC bottle bioassays could occur even within the same species when examining different populations. Although we observed complete mortality of C. furens from Indian River County, FL within 30 minutes in CDC bottle bioassays, different results could be observed in tests conducted with C. furens collected in locations where selection pressure is different. Culicoides furens from Indian River County, FL, were more susceptible to permethrin than the Ae. aegypti used as a reference for susceptibility in both CDC bottle bioassays and field cage trials. The permethrin DD for Ae. aegypti has been established as 43 µg permethrin per bottle, which is expected to cause complete mortality at 10 minutes 17 . In our study, C. furens exposed to 43 µg permethrin per bottle were knocked down within seconds after being placed inside the bottle. Similarly, in our field cage trials, C. furens experienced complete mortality within 10 minutes, in comparison to one hour for Ae. aegypti . These disparities in the time to complete mortality is likely due to the differences in size between the Ae. aegypti from our colony (3.03 mm wing length) 3 and the wild C. furens (0.91 mm wing length) 34 . The efficacy of ULV insecticide applications against adult mosquitoes is related to the droplet size 35 . Therefore, application rates on mosquito control product labels are based on the dose needed in a droplet to kill a mosquito based on its size 36 . In this case, C. furens is a smaller insect and the dose needed to kill it is much lower, resulting in the high mortality we observed in C. furens using a mosquito-specific ULV adulticide application rate. Our results are the first published information comparing SS and NN to perform field cage trials to quantify susceptibility of Culicoides to permethrin adulticide products in the United States. Linley and Jordan 21 evaluated the efficacy of thermal fog applications with formulated products contained naled against C. furens . However, the authors highlight that this product was not the most effective compound against Culicoides and that pyrethroid applications including a permethrin formulated product were likely to be more effective 19 . Complementary studies to these tests, showed that naled applied with a vehicle mounted ULV machine was not effective against C. furens and that this was possibly due to the fine mesh used to confine Culicoides , which did not allow the insecticide to pass through 20 . Although we found that cages made with SS and NN allowed the passage of ULV permethrin droplets, resulting in C. furens mortality, we recommend using NN since this material is easier to handle and at least 11 times cheaper than SS (NN: USD 5.44/m 2 ; SS: USD 62.20/m 2 ). Understanding whether or not Culicoides biting midges are susceptible to the adulticides that are commonly applied for their control provides the opportunity to evaluate current control practices and to potentially replace ineffective methods with more successful, affordable, and sustainable alternatives. We suggest using the methodologies adapted for biting midges in this study for CDC bottle bioassays and field cage trials to evaluate permethrin susceptibility of C. paraensis , the OROV vector. This information will inform whether permethrin applications that may be performed by vector control agencies can also offer protection against the OROV vector. These methodologies could also be used to evaluate insecticide susceptibility in EHDV and BTV vector species on livestock farms. Wild Culicoides species of medical or veterinary importance could be targeted with ULV permethrin applications, similar to those commonly used for controlling Culicoides on deer farms 16 . These field-collected Culicoides spp. could also be exposed to a CDC bottle bioassay with technical grade permethrin at the DD and DT we suggest (10.75 µg /bottle at 30 minutes). Although CDC bottle bioassays are generally performed in laboratory settings, the protocol described here is a relatively simple, rapid, and economic test that can be performed in temperature-controlled areas outside the laboratory. Our results highlight the opportunity of monitoring insecticide resistance as part of an integrated vector management (IVM) program against Culicoides. Effective insecticide resistance management should accompany ULV adulticide applications to ensure long-term control of Culicoides . IVM programs for blood-feeding dipterans often require using multiple control strategies to relieve the biting pressure 37 . Given the limited alternatives for Culicoides control, effective management relies on accurate susceptibility assessments to ensure that adulticide applications remain effective and to prevent the development of insecticide resistance. Declarations Data Availability The dataset used and analyzed during the current study are available from the corresponding author upon reasonable request. Competing Interests: The authors declare no competing interests. Author Contribution V.C., N.B.C, and E.B. designed the experiments. Y.J coordinated and provided resources for field trials. V.C., N.B.C, and E.B carried out the experiments and conducted the data analysis. V.C wrote the manuscript. All authors reviewed and provided feedback for the manuscript. Acknowledgement The authors would like to thank Sarah McInnis, Victor Recendez, Heather Whitehead (Indian River County Mosquito Control District), and Morgan Rockwell (UF/IFAS Florida Medical Entomology Laboratory) for their support during field cage trials. We thank Ana Romero-Weaver for training VMC in CDC bottle bioassays and Tanise Stenn for rearing mosquitoes used in this study. 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Florida Dept. of Agriculture and Consumer Services, Division of Plant Industry (1979). http://ufdc.ufl.edu/UF00000090/0000 Mount, G. A. Urban and suburban mosquito control: Strategies and techniques. J. Am. Mosq. Control Assoc. 14 , 13–22 (1998). Zhang, L. et al. The effectiveness of larvicides against Culicoides species: A field study in China. Pest Manag Sci. 71 , 1500–1507 (2015). Mukabana, W. R. et al. The role of Culicoides midges in the transmission of livestock diseases in Kenya. Trop. Anim. Health Prod. 42 , 725–734 (2010). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 08 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Oct, 2024 Reviews received at journal 11 Oct, 2024 Reviews received at journal 10 Oct, 2024 Reviewers agreed at journal 08 Oct, 2024 Reviewers agreed at journal 07 Oct, 2024 Reviewers invited by journal 07 Oct, 2024 Editor assigned by journal 07 Oct, 2024 Editor invited by journal 31 Aug, 2024 Submission checks completed at journal 29 Aug, 2024 First submitted to journal 21 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4953430","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":354631927,"identity":"dcd1afb5-a4b4-454c-8b4c-bff44bc9bc7c","order_by":0,"name":"Vilma M. Cooper","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACZuYGVAF+BsaHB4DieLQwQrWwQQUkG5gN8GthQNdicICAFoPjjK0bfjBskzOXbz724MOve3LGN5IZDjBUWCc24NJymLHtZg/DbWPLNrZ0w5l9xcZmYC1n0nFqkWxmbLvBw3A7ccMxHjNp3p6ExG038g8cYGw7jFfLzT9gLfzfpP8CtWyeAbSF8R9uLfzMjG23obawSTP8SEjcIAHS0kBAi4zBbWODY2lmkr0NCcYSZx4zHEg4lm6MSwsb/+FjN99U3JYzOHz4mcSPPwly/O3JjA8+1FjL4tICAQZQmrENykjAqxwF/CFe6SgYBaNgFIwcAACyb16tXqX3hAAAAABJRU5ErkJggg==","orcid":"","institution":"UF/IFAS","correspondingAuthor":true,"prefix":"","firstName":"Vilma","middleName":"M.","lastName":"Cooper","suffix":""},{"id":354631928,"identity":"c9ca1205-b6a7-48c2-8320-ac7399e9fa30","order_by":1,"name":"Eva A. Buckner","email":"","orcid":"","institution":"UF/IFAS","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"A.","lastName":"Buckner","suffix":""},{"id":354631930,"identity":"ba18d258-1bf2-4dd1-8f54-d7a79512d423","order_by":2,"name":"Yongxing Jiang","email":"","orcid":"","institution":"Indian River Mosquito Control District","correspondingAuthor":false,"prefix":"","firstName":"Yongxing","middleName":"","lastName":"Jiang","suffix":""},{"id":354631931,"identity":"d2f104ae-2e0a-4fe7-9314-4869290ef383","order_by":3,"name":"Nathan Burkett-Cadena","email":"","orcid":"","institution":"UF/IFAS","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Burkett-Cadena","suffix":""}],"badges":[],"createdAt":"2024-08-21 18:17:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4953430/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4953430/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-89520-0","type":"published","date":"2025-02-08T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64888363,"identity":"ac7acc91-80ab-4be7-893e-8a630217fe57","added_by":"auto","created_at":"2024-09-20 05:27:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":259364,"visible":true,"origin":"","legend":"\u003cp\u003eBioassay cage and mesh types used for adulticide field trials. (A) Bioassay cages built of cardboard rings and mesh; (B) mosquito mesh (MM) (fine nylon tulle fabric), Hobby Lobby Stores, Inc., Oklahoma City, OK; (C) stainless steel mesh (SS), McMaster-Carr, Douglasville, GA; (D) No-see-um netting (NN), Seattle Fabrics, Inc, Seattle, WA. Scale bar represents 1mm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4953430/v1/466d44e891b1fd3df8210ac9.png"},{"id":64888364,"identity":"a72422af-268f-4251-b2e1-00e50397e25a","added_by":"auto","created_at":"2024-09-20 05:27:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30301,"visible":true,"origin":"","legend":"\u003cp\u003eSurvivorship of mosquitoes and biting midges in CDC bottle bioassays. Proportion of mortality documented in wild \u003cem\u003eCulicoides furens\u003c/em\u003e (solid lines) and susceptible \u003cem\u003eAedes aegypti \u003c/em\u003e(dashed lines) mosquitoes exposed to low (10.75 µg / bottle), medium (21.5 µg / bottle), and high (43 µg /bottle) doses of permethrin in CDC bottle bioassays. The vertical dotted line at 30 minutes denotes the diagnostic time we recommend for \u003cem\u003eC. furens\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4953430/v1/5d970a43b25df08dc04f5cc6.png"},{"id":64888361,"identity":"3dc7c542-0ccc-45a6-be6b-da862e938704","added_by":"auto","created_at":"2024-09-20 05:27:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1177074,"visible":true,"origin":"","legend":"\u003cp\u003eMortality of susceptible \u003cem\u003eAedes aegypti\u003c/em\u003e mosquitoes and wild \u003cem\u003eCulicoides furens\u003c/em\u003e at different times post exposure in bioassay cages exposed to ULV applications with Permanone 30-30. (A) Mosquitoes only cage field trial (two passes); (B) Mosquito mortality in mosquitoes and \u003cem\u003eCulicoides\u003c/em\u003ecage field trial (one pass); (C) \u003cem\u003eC. furens \u003c/em\u003emortality in mosquitoes and \u003cem\u003eCulicoides \u003c/em\u003ecage field trial (one pass). Treatments include cages made with stainless steel (SS), no-see-um netting (NN), and mosquito mesh (MM). Different pattern fills represent mesh types. Error bars depict the standard deviation.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4953430/v1/2dd6c484fe989edc24578f51.jpeg"},{"id":75931199,"identity":"67091402-e289-4e51-8c0e-d6914753d608","added_by":"auto","created_at":"2025-02-10 16:14:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2120989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4953430/v1/ed9b6a62-1641-4595-81c3-7199636fdc9b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Laboratory and field assays indicate that a widespread no-see-um, Culicoides furens Poey is susceptible to permethrin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe recent reemergence and rapid spread of Oropouche virus (OROV) in the Americas\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e highlights existing knowledge gaps in the ecology and control of \u003cem\u003eCulicoides\u003c/em\u003e (biting midges, no-see-ums)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, including \u003cem\u003eCulicoides paraensis\u003c/em\u003e Goeldi, the confirmed vector of OROV. Once considered endemic to the greater Amazonian Region of South America\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, OROV is becoming increasingly prevalent in parts of South America, and recently spread to portions of the Caribbean, posing a significant public health risk outside of its endemic range\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Vector control agencies in the areas of OROV expansion will benefit from understanding whether their mosquito control practices are effective against \u003cem\u003eCulicoides\u003c/em\u003e spp. that bite humans.\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eCulicoides\u003c/em\u003e (Diptera: Ceratopogonidae) includes species that are not only biting nuisances but also vectors affecting humans, wildlife, and livestock. \u003cem\u003eCulicoides\u003c/em\u003e species transmit around 20 arboviruses and five nematode species of veterinary importance, affecting a wide range of hosts\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notable arboviruses include African horse sickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These viruses significantly impact livestock industries across multiple continents, including North America\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, Central and South America\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, Europe\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, Africa, Asia, and the Middle East\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGlobally, vector control relies heavily on chemical adulticides, particularly pyrethrins and pyrethroids like permethrin due to their potency and relative safety for mammals\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Permethrin is widely used for mosquito control in the United States\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, permethrin is the main active ingredient used for \u003cem\u003eCulicoides\u003c/em\u003e control in Florida deer farms, where its frequent use has raised concerns about insecticide resistance\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMonitoring insecticide susceptibility and resistance in target arthropods is a cornerstone of successful control program\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Insecticide resistance is often detected through laboratory bioassays\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e such as the World Health Organization (WHO) tube assay and the Centers for Disease Control and Prevention (CDC) bottle bioassay\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These assays are extensively used to monitor resistance in mosquitoes and other vectors of public health significance \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, standardized protocols for applying these methods to \u003cem\u003eCulicoides\u003c/em\u003e are currently lacking. Field-based bioassays, such as \u0026ldquo;field cage trials\u0026rdquo;, are recommended to assess the susceptibility of insects to formulated insecticides like adulticides, particularly when resistance is detected in laboratory settings\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the medical, veterinary, and nuisance importance of many \u003cem\u003eCulicoides\u003c/em\u003e spp., established protocols for monitoring susceptibility in no-see-ums are lacking. In North America, only a few studies have investigated the susceptibility of \u003cem\u003eCulicoides\u003c/em\u003e to adulticides\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These studies, conducted on \u003cem\u003eCulicoides furens\u003c/em\u003e populations in Florida, tested the efficacy of ULV sprays containing organophosphates (malathion, naled) and a pyrethroid (resmethrin). Naled was found to be the most effective, achieving 90% mortality at distances up to 106 m, compared to 36 m for malathion and 25 m for resmethrin\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, a recent survey of Florida mosquito control programs documented higher usage of pyrethrin and the pyrethroid permethrin as adulticide active ingredients as compared to the organophosphates malathion and naled\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additionally, only one Florida mosquito control program reported using resmethrin in 2020\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnderstanding \u003cem\u003eCulicoides\u003c/em\u003e susceptibility to insecticides currently being used by vector control programs is essential for developing integrated vector control programs. However, studies are limited due to challenges in colonizing \u003cem\u003eCulicoides\u003c/em\u003e and the lack of established testing protocols. While 151 \u003cem\u003eCulicoides\u003c/em\u003e species are recorded in North America, only \u003cem\u003eCulicoides sonorensis\u003c/em\u003e is currently colonized\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Standard bioassays like the CDC bottle bioassay and cage field trials are commonly used for mosquitoes, but their applicability to \u003cem\u003eCulicoides\u003c/em\u003e is uncertain. We adapted these methods to evaluate the susceptibility of wild \u003cem\u003eC. furens\u003c/em\u003e from Indian River County, FL aiming to establish a diagnostic dose (DD) and diagnostic time (DT) for use in CDC bottle bioassays and identify a suitable mesh for cage field trials.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eInsects.\u003c/b\u003e The challenges of \u003cem\u003eCulicoides\u003c/em\u003e colonization have somewhat normalized the use of wild specimens\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We tested wild \u003cem\u003eC. furens\u003c/em\u003e, a nuisance species common in U.S. coastal areas, trapped at the University of Florida, Florida Medical Entomology Laboratory (FMEL) in Indian River County. \u003cem\u003eCulicoides\u003c/em\u003e were captured using CDC miniature light traps (BioQuip products, Rancho Dominguez, CA, USA) connected to live midge collection chambers\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Traps were baited with an incandescent light bulb, 2 mL of octenol (1-Octen-3-ol) placed in a 15 mL plastic tube with a piece of cotton wool, and ~\u0026thinsp;1kg of dry ice contained in an insulated 1.89 L beverage container (Igloo Products Corp., Katy, TX).\u003c/p\u003e \u003cp\u003eSince reference (known susceptible control) \u003cem\u003eC. furens\u003c/em\u003e are unavailable (no laboratory colony in existence), we used a laboratory-reared strain of \u003cem\u003eAedes aegypti\u003c/em\u003e (Orlando 1962) which originated from Orlando, FL (Orange County) as a susceptible control. This strain was originally obtained from colonies maintained at the USDA-ARS CMAVE. The \u003cem\u003eAe. aegypti\u003c/em\u003e were reared in an insectary at 26\u0026deg;C and 85% relative humidity. Egg papers were placed in plastic trays containing approximately 2 L of tap water at a density of 200\u0026ndash;300 eggs per rearing tray. Larvae were fed a 1:1 mixture of lactalbumin and yeast ad libitum. Pupae were transferred from larval rearing trays to water-filled plastic cups in 30.5 x 30.5 x 30.5 cm Bug Dorm adult rearing cages (BioQuip, Rancho Dominguez, CA, USA). Cotton rounds soaked with 10% sucrose solution were placed inside each cage as carbohydrate source for emerging adults.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCDC Bottle bioassays.\u003c/b\u003e The CDC bottle bioassay is commonly used to monitor mosquito insecticide susceptibility\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We adapted this method to calibrate the assay for \u003cem\u003eCulicoides\u003c/em\u003e by determining the DD and DT for permethrin. Following the CDC protocol\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, we prepared permethrin solutions by diluting technical grade permethrin (100% Chem Service Inc., West Chester, PA, USA) in American Chemical Society (ACS) grade acetone (Fisher Scientific, Hampton, NH). Each bioassay used five 250-ml clear Wheaton bottles with lids (DWK Life Sciences Inc., Millville, NJ), four coated with 1.0 ml of the permethrin solution and one untreated control coated with acetone only. Bottles were coated by swirling the solution inside and then left to dry on a hot dog roller machine with heat turned off to ensure even coating. Control bottles were capped and rolled before preparing bottles containing insecticide. All bottles were then left open to dry in a dark environment to prevent permethrin photodegradation. Preliminary observations showed high mortality of \u003cem\u003eC. furens\u003c/em\u003e in control bottles when the bioassay was performed within five hours after the bottles were coated. We tested various drying times (5, 10 and 24 h) and found that 24 hours of drying were necessary to eliminate control mortality.\u003c/p\u003e \u003cp\u003eAs recommended by Brogdon and Chan\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, when calibrating the CDC bottle bioassay to determine DD and DT for a new insect, we prepared bottles with a range of permethrin concentrations to determine a possible DD. Based on the CDC\u0026rsquo;s recommended DD of 43 \u0026micro;g permethrin/bottle for susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and the small size of \u003cem\u003eC. furens\u003c/em\u003e in comparison to \u003cem\u003eAe. aegypti\u003c/em\u003e, the permethrin concentrations we tested as possible DD were 43, 21.5, and 10.7 \u0026micro;g permethrin/bottle. The permethrin concentration that killed 100% of the wild \u003cem\u003eC. furens\u003c/em\u003e between 30 and 60 minutes would be selected as the DD (dose that kills 100% of insects\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBottle bioassays were performed at the same time but in separate bottles for susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e and wild \u003cem\u003eC. furens\u003c/em\u003e. Mosquitoes and \u003cem\u003eC. furens\u003c/em\u003e were aspirated by mouth into each bottle with the goal of 20 individuals per bottle. Mortality was recorded every five minutes for the first 15 minutes and every fifteen minutes until 2-h post-exposure\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. After the 2-hrs of exposure, insects were transferred into 8-oz paper food cups with lids and generic mesh to prevent all insects from escaping. Insects were provided with a cotton round with water and mortality was then assessed at 24-h post-exposure\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eField cage trials.\u003c/b\u003e Two field trials were conducted between March and April, 2023 in Indian River County, FL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), targeting caged susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e and/ or wild \u003cem\u003eC. furens\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Permanone 30\u0026ndash;30 (30% permethrin, 30% piperonyl butoxide) was applied at maximum rate (0.007 lb permethrin/acre) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using a truck-mounted ULV aerosol generator that was operated by personnel of the Indian River Mosquito Control District. Spray missions were performed around dusk (1700h and 1800h) on a five-acre plot of low-cut grass at the Indian River County Fairgrounds.\u003c/p\u003e \u003cp\u003eBioassay cages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were made of cardboard rings (15.2 cm diameter, 2.5 cm width) covered with mesh\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Three mesh types were tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e): stainless steel (SS) McMaster-Carr, Douglasville, GA), no-see-um netting (NN) (Seattle Fabrics, Inc., Seattle, WA), and fine nylon tulle fabric (Hobby Lobby Stores, Inc., Oklahoma, USA), hereafter called mosquito mesh (MM). SS and NN mesh were chosen because preliminary observations indicated they could prevent escape of \u003cem\u003eC. furens\u003c/em\u003e (Cooper, unpublished data). Although the MM mesh has openings too large for \u003cem\u003eCulicoides\u003c/em\u003e, it was included as it has been used successfully in mosquito cage trials\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of ULV permethrin adulticide field cage trials to evaluate the susceptibility of \u003cem\u003eC. furens\u003c/em\u003e in Indian River County, Florida, USA. (SS) stainless steel, McMaster-Carr, Douglasville, GA; (NN) no-see-um netting, Seattle Fabrics, Inc, Seattle, WA.; (MM) mosquito mesh (fine nylon tulle fabric), Hobby Lobby Stores, Inc, Oklahoma City, OK.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eField Trial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMesh type tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eNumber of passes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSpeed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eWind speed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarch 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e (susceptible)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSS, NN, MM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e10 and 5 mph, respectively\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e3\u0026ndash;5 mph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApril 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAe. aegypti\u003c/em\u003e (susceptible)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSS, NN, MM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e10 mph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e6\u0026ndash;10 mph\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c11\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC. furens\u003c/em\u003e (wild)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSS, NN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mosquitoes-only field cage trial aimed to compare different mesh types for field bioassay cages. Approximately 20 susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e were placed in each cage. After confirming that the mesh types allowed insecticide passage and caused mortality in these mosquitoes, a combined mosquitoes and \u003cem\u003eCulicoides\u003c/em\u003e trial was conducted. Each cage contained about 30 wild \u003cem\u003eC. furens\u003c/em\u003e and 20 \u003cem\u003eAe. aegypti\u003c/em\u003e, except for the MM cages, which only had \u003cem\u003eAe. aegypti\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This design allowed us to evaluate the susceptibility of wild \u003cem\u003eC. furens\u003c/em\u003e to permethrin relative to susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCages were hung approximately 1.3 m above ground on shepherd hooks in five clusters, each with the three mesh types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Clusters were spaced every five meters along a north-south transect, 20 m downwind of the spray truck path. Two untreated (control) cages of each mesh type were placed 20 m upwind. Mortality was assessed in the field 10 minutes post-exposure, and subsequently recorded at 1, 12 and 24 h post-exposure in the laboratory. Mortality was based on knockdown (inability to stand on legs or have coordinated flight\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e). Insects were provided a cotton round with tap water and maintained in the cages for 24 hours, before being placed in dry ice to kill any remaining live insects.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData Analysis.\u003c/b\u003e Results from the cage field trials were analyzed by calculating the mean mortality of mosquitoes and \u003cem\u003eCulicoides\u003c/em\u003e at various times post-exposure. For each trial, mean mortality and standard deviation were plotted\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. For CDC bottle bioassays, time-response survival curves were created for each permethrin dose. Insect survival was assessed using Cox proportional hazards models with the \u0026lsquo;survival\u0026rsquo; package in R version 4.2.2\u003csup\u003e31\u003c/sup\u003e. Clustered Cox regression was performed to compare mean mortality across the different permethrin doses for both mosquitoes and \u003cem\u003eCulicoides\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCDC Bottle Bioassays.\u003c/b\u003e Susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes reached complete mortality at 30 minutes post-exposure in CDC bottle bioassays performed with the high and medium permethrin doses (43 and 21.5 \u0026micro;g /bottle), while these same doses caused complete mortality of \u003cem\u003eC. furens\u003c/em\u003e at five minutes post-exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Significantly greater mortality was observed in \u003cem\u003eC. furens\u003c/em\u003e exposed to the high dose and medium doses in comparison to mosquitoes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The low permethrin dose (10.75 \u0026micro;g /bottle) caused complete mortality in \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eC. furens\u003c/em\u003e at 45 and 30 minutes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No significant differences were observed in the mortality of \u003cem\u003eC. furens\u003c/em\u003e and \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes exposed to the low dose (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3699).\u003c/p\u003e \u003cp\u003eThe susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes and wild \u003cem\u003eC. furens\u003c/em\u003e did not recover at 24 hours with any of the three permethrin doses, with the exception of \u003cem\u003eC. furens\u003c/em\u003e from bottles treated with the low dose, in which five percent recovery was observed at 24 hours. However, mortality in untreated control bottles was significantly lower in comparison to treated bottles (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). We did not observe significant differences in the mortality of susceptible mosquitoes in comparison to wild \u003cem\u003eC. furens\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.0000). Abbot\u0026rsquo;s formula was not used to correct percent mortality in any of the bottle bioassays because control mortality at two hours did not exceed 3%\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMosquito-only field cage trial\u003c/b\u003e. All three mesh types allowed the passage of Permanone 30\u0026ndash;30, resulting in mosquito mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the mosquito-only trial, two adulticide passes were performed because no mortality was observed within 10 minutes after the first pass. We were unaware whether the insecticide had not gone through the mesh during the first pass or there was another reason why the insects were not experiencing mortality. A second pass performed within 20 minutes after the first application resulted in immediate mortality of the exposed mosquitoes.\u003c/p\u003e \u003cp\u003eAt one-hour post-application, mortality rates were 89% (SS), 97% (NN), and 98% (MM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Complete mortality was observed at 12 hours in SS and MM cages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), while NN cages only reached 96% at 12 hours, with no further increase by 24 hours. Mosquitoes did not recover from the adulticide effects within the 24-hour period. Control cages showed minimal mortality at 24 hours, with 15% in MM cages and 5% and 6% in SS and NN, respectively. Since control mortality was only observed after 24 hours, Abbot\u0026rsquo;s formula was not applied to correct the percent mortality in treatment cages\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMosquitoes and\u003c/b\u003e \u003cb\u003eCulicoides\u003c/b\u003e \u003cb\u003efield cage trial.\u003c/b\u003e The second trial confirmed that bioassay cages constructed with SS and NN could be used to evaluate \u003cem\u003eCulicoides\u003c/em\u003e susceptibility to ULV adulticides. Complete mortality of \u003cem\u003eAe. aegypti\u003c/em\u003e occurred in all bioassay cages at 1-hr post-application (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), while 100% mortality of \u003cem\u003eC. furens\u003c/em\u003e was observed at 10 minutes. Mosquitoes and \u003cem\u003eC. furens\u003c/em\u003e did not recover from the effect of the adulticide within the 24-hour period. Mortality in untreated control cages was only observed at 24 hours for \u003cem\u003eAe. aegypti\u003c/em\u003e placed inside SS cages and biting midges in SS cages (6%) and NN cages (16%). Because 100% mortality was observed in all treatment cages 1-hr post-exposure and no mortality was observed in the control cages until after 24 hours post-exposure, Abbot\u0026rsquo;s formula was not used to correct percent mortality in treatment cages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that both the CDC bottle bioassay and field cage trials can be effectively adapted to assess insecticide susceptibility and efficacy of ULV treatments against \u003cem\u003eCulicoides\u003c/em\u003e species. Obtaining this information is advantageous for protecting the health of humans and other animals. Our findings include the first records of DD and DT for \u003cem\u003eCulicoides\u003c/em\u003e in CDC bottle bioassays, which can serve as reference for other species, such as \u003cem\u003eC. paraensis\u003c/em\u003e, the main vector of OROV. We also identified two suitable mesh types for field cage trials with \u003cem\u003eCulicoides\u003c/em\u003e. We observed mortality in \u003cem\u003eC. furens\u003c/em\u003e in CDC bottle bioassays using technical grade permethrin and in the field cage trial with Permanone 30\u0026ndash;30, which suggests that wild \u003cem\u003eC. furens\u003c/em\u003e from Indian River County are susceptible to permethrin.\u003c/p\u003e \u003cp\u003eDespite the widespread use of adulticides to control \u003cem\u003eCulicoides\u003c/em\u003e spp., their susceptibility to these treatments remains poorly understood. This study highlights the utility of field cage trials in assessing the effectiveness of adulticide applications against both vector and nuisance species. Although \u003cem\u003eC. furens\u003c/em\u003e is not a vector of pathogens, this nuisance species can significantly impact the economy. For instance, in Australia, \u003cem\u003eCulicoides\u003c/em\u003e nuisance is estimated to reduce residential property values by 8,300 USD per residence based on actual sale price\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Ratnayake and co-authors also suggest that \u003cem\u003eCulicoides\u003c/em\u003e prevent urban development in coastal areas, with over half of surveyed residents of Hervey Bay, Australia considering the impact of \u003cem\u003eCulicoides\u003c/em\u003e a major factor in housing investment decisions\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe were able to determine a potential DD and DT using wild \u003cem\u003eCulicoides\u003c/em\u003e. Typically, DD and DT used in CDC bottle bioassays are established using susceptible populations\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, despite numerous attempts to establish \u003cem\u003eCulicoides\u003c/em\u003e colonies since 1921\u003csup\u003e34\u003c/sup\u003e, only \u003cem\u003eC. sonorensis\u003c/em\u003e is colonized in the United States\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, the susceptibility of this colonized \u003cem\u003eC. sonorensis\u003c/em\u003e to permethrin remains undocumented. In the absence of a susceptible \u003cem\u003eCulicoides\u003c/em\u003e population for calibration, we adapted the CDC bottle bioassay for wild \u003cem\u003eC. furens\u003c/em\u003e. We found that 10.75 \u0026micro;g permethrin per bottle resulted in 100% mortality of \u003cem\u003eC. furens\u003c/em\u003e from Indian River County, FL, within 30 minutes. Therefore, we recommend using 10.75 \u0026micro;g permethrin per bottle and 30 minutes as the DD and DT for this and potentially other wild \u003cem\u003eCulicoides\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eWe expect that the time to reach complete mortality in CDC bottle bioassays and cage field trials will vary between studies due to various factors. For example, different mortality rates may be observed in cage field trials using different permethrin formulations, even when applied at the same dose. We anticipate that differences in the time to reach complete mortality in CDC bottle bioassays could occur even within the same species when examining different populations. Although we observed complete mortality of \u003cem\u003eC. furens\u003c/em\u003e from Indian River County, FL within 30 minutes in CDC bottle bioassays, different results could be observed in tests conducted with \u003cem\u003eC. furens\u003c/em\u003e collected in locations where selection pressure is different.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCulicoides furens\u003c/em\u003e from Indian River County, FL, were more susceptible to permethrin than the \u003cem\u003eAe. aegypti\u003c/em\u003e used as a reference for susceptibility in both CDC bottle bioassays and field cage trials. The permethrin DD for \u003cem\u003eAe. aegypti\u003c/em\u003e has been established as 43 \u0026micro;g permethrin per bottle, which is expected to cause complete mortality at 10 minutes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In our study, \u003cem\u003eC. furens\u003c/em\u003e exposed to 43 \u0026micro;g permethrin per bottle were knocked down within seconds after being placed inside the bottle. Similarly, in our field cage trials, \u003cem\u003eC. furens\u003c/em\u003e experienced complete mortality within 10 minutes, in comparison to one hour for \u003cem\u003eAe. aegypti\u003c/em\u003e. These disparities in the time to complete mortality is likely due to the differences in size between the \u003cem\u003eAe. aegypti\u003c/em\u003e from our colony (3.03 mm wing length)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and the wild \u003cem\u003eC. furens\u003c/em\u003e (0.91 mm wing length)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The efficacy of ULV insecticide applications against adult mosquitoes is related to the droplet size\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, application rates on mosquito control product labels are based on the dose needed in a droplet to kill a mosquito based on its size\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this case, \u003cem\u003eC. furens\u003c/em\u003e is a smaller insect and the dose needed to kill it is much lower, resulting in the high mortality we observed in \u003cem\u003eC. furens\u003c/em\u003e using a mosquito-specific ULV adulticide application rate.\u003c/p\u003e \u003cp\u003eOur results are the first published information comparing SS and NN to perform field cage trials to quantify susceptibility of \u003cem\u003eCulicoides\u003c/em\u003e to permethrin adulticide products in the United States. Linley and Jordan\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e evaluated the efficacy of thermal fog applications with formulated products contained naled against \u003cem\u003eC. furens\u003c/em\u003e. However, the authors highlight that this product was not the most effective compound against \u003cem\u003eCulicoides\u003c/em\u003e and that pyrethroid applications including a permethrin formulated product were likely to be more effective\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Complementary studies to these tests, showed that naled applied with a vehicle mounted ULV machine was not effective against \u003cem\u003eC. furens\u003c/em\u003e and that this was possibly due to the fine mesh used to confine \u003cem\u003eCulicoides\u003c/em\u003e, which did not allow the insecticide to pass through\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Although we found that cages made with SS and NN allowed the passage of ULV permethrin droplets, resulting in \u003cem\u003eC. furens\u003c/em\u003e mortality, we recommend using NN since this material is easier to handle and at least 11 times cheaper than SS (NN: USD 5.44/m\u003csup\u003e2\u003c/sup\u003e; SS: USD 62.20/m\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eUnderstanding whether or not \u003cem\u003eCulicoides\u003c/em\u003e biting midges are susceptible to the adulticides that are commonly applied for their control provides the opportunity to evaluate current control practices and to potentially replace ineffective methods with more successful, affordable, and sustainable alternatives. We suggest using the methodologies adapted for biting midges in this study for CDC bottle bioassays and field cage trials to evaluate permethrin susceptibility of \u003cem\u003eC. paraensis\u003c/em\u003e, the OROV vector. This information will inform whether permethrin applications that may be performed by vector control agencies can also offer protection against the OROV vector. These methodologies could also be used to evaluate insecticide susceptibility in EHDV and BTV vector species on livestock farms. Wild \u003cem\u003eCulicoides\u003c/em\u003e species of medical or veterinary importance could be targeted with ULV permethrin applications, similar to those commonly used for controlling \u003cem\u003eCulicoides\u003c/em\u003e on deer farms\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These field-collected \u003cem\u003eCulicoides\u003c/em\u003e spp. could also be exposed to a CDC bottle bioassay with technical grade permethrin at the DD and DT we suggest (10.75 \u0026micro;g /bottle at 30 minutes). Although CDC bottle bioassays are generally performed in laboratory settings, the protocol described here is a relatively simple, rapid, and economic test that can be performed in temperature-controlled areas outside the laboratory.\u003c/p\u003e \u003cp\u003eOur results highlight the opportunity of monitoring insecticide resistance as part of an integrated vector management (IVM) program against \u003cem\u003eCulicoides.\u003c/em\u003e Effective insecticide resistance management should accompany ULV adulticide applications to ensure long-term control of \u003cem\u003eCulicoides\u003c/em\u003e. IVM programs for blood-feeding dipterans often require using multiple control strategies to relieve the biting pressure\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Given the limited alternatives for \u003cem\u003eCulicoides\u003c/em\u003e control, effective management relies on accurate susceptibility assessments to ensure that adulticide applications remain effective and to prevent the development of insecticide resistance.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe dataset used and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eCompeting Interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eV.C., N.B.C, and E.B. designed the experiments. Y.J coordinated and provided resources for field trials. V.C., N.B.C, and E.B carried out the experiments and conducted the data analysis. V.C wrote the manuscript. All authors reviewed and provided feedback for the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Sarah McInnis, Victor Recendez, Heather Whitehead (Indian River County Mosquito Control District), and Morgan Rockwell (UF/IFAS Florida Medical Entomology Laboratory) for their support during field cage trials. We thank Ana Romero-Weaver for training VMC in CDC bottle bioassays and Tanise Stenn for rearing mosquitoes used in this study. Funding for this work was provided by the Florida State Legislature, through the Cervidae Health Research Initiative and NIFA Project FLA-FME-006106.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eElbadry, M. A. et al. Orthobunyaviruses in the Caribbean: Melao and Oropouche virus infections in school children in Haiti in 2014. \u003cem\u003ePLoS Negl. Trop. 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The effectiveness of larvicides against \u003cem\u003eCulicoides\u003c/em\u003e species: A field study in China. \u003cem\u003ePest Manag Sci.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 1500\u0026ndash;1507 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMukabana, W. R. et al. The role of \u003cem\u003eCulicoides\u003c/em\u003e midges in the transmission of livestock diseases in Kenya. \u003cem\u003eTrop. Anim. Health Prod.\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 725\u0026ndash;734 (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Oropouche virus, vector control, IPM, IVM","lastPublishedDoi":"10.21203/rs.3.rs-4953430/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4953430/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe recent reemergence of Oropouche virus (OROV) highlights the need to better understand insecticide susceptibility in \u003cem\u003eCulicoides\u003c/em\u003e (Diptera: Ceratopogonidae), which contains the vector of OROV and many other species that are biting nuisances and vectors of pathogens that affect humans, livestock, and wildlife. With adulticides as the primary method of \u003cem\u003eCulicoides\u003c/em\u003e control, there is growing concern about insecticide resistance, compounded by the lack of tools to monitor \u003cem\u003eCulicoides\u003c/em\u003e susceptibility. We adapted the CDC bottle bioassay and field cage trial methods, typically used to monitor insecticide susceptibility in mosquitoes and formulated adulticide efficacy, to evaluate permethrin susceptibility in the widely distributed coastal nuisance species, \u003cem\u003eCulicoides furens\u003c/em\u003e. Permethrin caused complete mortality in \u003cem\u003eC. furens\u003c/em\u003e in field and laboratory assays. We identified a diagnostic dose (10.75 \u0026micro;g) and time (30 minutes) that resulted in complete mortality in CDC bottle bioassays. Additionally, we determined that no-see-um netting is an effective mesh for field cage trials, allowing for accurate assessment of \u003cem\u003eCulicoides\u003c/em\u003e susceptibility to ultra-low volume applications of formulated adulticides like Permanone 30\u0026ndash;30, a widely utilized adulticide. These methodologies offer essential tools for assessing \u003cem\u003eCulicoides\u003c/em\u003e susceptibility, which is crucial for managing populations of \u003cem\u003eCulicoides\u003c/em\u003e and preventing the spread of OROV and other pathogens.\u003c/p\u003e","manuscriptTitle":"Laboratory and field assays indicate that a widespread no-see-um, Culicoides furens Poey is susceptible to permethrin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-20 05:27:42","doi":"10.21203/rs.3.rs-4953430/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-14T12:01:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-11T12:04:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-11T02:04:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199215391972151449948178359228913785270","date":"2024-10-08T06:03:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262724583111229803932945023542802184186","date":"2024-10-07T23:36:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-07T12:35:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-07T12:35:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-31T15:51:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-30T03:03:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-21T18:16:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f1c8b78-97bb-4659-9712-069f4a9f00c4","owner":[],"postedDate":"September 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":37688476,"name":"Biological sciences/Ecology"},{"id":37688477,"name":"Health sciences/Diseases/Infectious diseases"},{"id":37688478,"name":"Earth and environmental sciences/Ecology/Invasive species"}],"tags":[],"updatedAt":"2025-02-10T16:09:03+00:00","versionOfRecord":{"articleIdentity":"rs-4953430","link":"https://doi.org/10.1038/s41598-025-89520-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-02-08 15:57:59","publishedOnDateReadable":"February 8th, 2025"},"versionCreatedAt":"2024-09-20 05:27:42","video":"","vorDoi":"10.1038/s41598-025-89520-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-89520-0","workflowStages":[]},"version":"v1","identity":"rs-4953430","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4953430","identity":"rs-4953430","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}