CDC Bottle Bioassays for Detection of Insecticide Resistance in Culex pipiens, Aedes albopictus, and Aedes koreicus Collected on US Army Garrisons, Republic of Korea

Mosquito-borne pathogens are a threat to US troops stationed in the Republic of Korea (ROK). Insecticides are used to control mosquitoes and prevent pathogen transmission across the ROK and on US army garrisons (USAGs) (Lee et al. 1984, Hwang et al. 2020, Park et al. 2020). For example, as part of a vector-borne pathogen prevention strategy the Army Combat Uniform currently worn by US soldiers is factory treated with permethrin and provides a first line of defense against mosquito-borne pathogens (Estep et al. 2020). Under approved integrated pest management plans, pyrethroids such as resmethrin are used in ultra-low volume (ULV) applications for targeting adult mosquitoes on USAGs (Hwang et al. 2020). The ULV applications are made based on established mosquito thresholds and vary by location, with zero or very few ULV applications during the season on USAGs Daegu and Yongsan-Casey and from zero to more than 20 applications per season on USAG Humphreys (Hwang et al. 2020). There is also historical exposure due to mosquito control programs in the 1950s–1980s using organochlorine and organophosphate insecticides (Park et al. 2020). The impact of insecticide resistance (IR) on control and prevention is currently unknown, and any loss in efficacy of these insecticides could put soldiers at a greater risk of acquiring a mosquito-borne pathogen (Estep et al. 2020, Lee et al. 2020).

Culex pipiens L. complex is one of the most abundant human-biting mosquitoes in urban areas of the ROK and is a possible vector of Japanese encephalitis virus and West Nile virus (Kim et al. 2015, Seo et al. 2021). Due to international travel to endemic countries each year dengue cases are imported into the ROK where Aedes albopictus (Skuse) could cause local outbreaks (Seo et al. 2021). Aedes koreicus (Edwards) is an abundant human-biting urban mosquito in the ROK and has an unknown role in pathogen transmission (Ryu et al. 2019, Seo et al. 2021). In the ROK, IR has been reported in Cx. pipiens but not in Ae. albopictus or Ae. koreicus (Shin et al. 2012, Ryu et al. 2019, Lee et al. 2020, Park et al. 2020).

To better understand the risk IR poses to mosquito control efforts and personal protection measures as outlined in Carder et al. (2023), an IR monitoring program using the Centers for Disease Control and Prevention (CDC) bottle bioassays was established on USAGs in the ROK. The main goal of the program is initially to characterize the IR of local ROK mosquito populations and then monitor changes in their status over time. This information will be used to determine the operational impact of IR and guide the adoption of alternative mosquito control methods. These surveillance efforts are part of a standardization initiative to detect IR on US military installations in the ROK.

Beginning in April 2022, mosquito larvae and pupae were collected from USAG Daegu (Camps Carroll, Henry), USAG Yongsan-Casey (Camp Casey), and USAG Humphreys (Camp Humphreys), returned to the entomology laboratory on Camp Humphreys, and reared to the adult stage. In the laboratory, 100 to 150 larvae were placed in 1.5 liters of water in 27 × 6 × 16-cm plastic rearing trays and provided powdered fish food and ground calf feed (1:1), and kept at 26°C room temperature in 26°C water on a cycle of 12 h light and 12 h dark. Pupae and 4th-stage larvae were moved to screen cages, and adults that emerged were provided 10% sugar solution ad libitum. Following CDC guidance, male and female adult mosquitoes, aged from 3 to 7 days old, were used in the assays (McAllister and Scott 2020). A colony of Cx. pipiens molestus (Forskal) maintained in the Camp Humphreys entomology laboratory since 2018 was also tested. Culex pipiens in the ROK is a mix of Cx. pipiens pallens (Coquillett) and Cx. p. molestus (Ryu and Shik 2022). The mosquitoes tested from field collections were only identified morphologically to the Cx. pipiens level. All mosquitoes were identified as adults using Lee and Egan (1985).

Technical-grade permethrin and chlorpyrifos (PESTANAL® analytical standard) were obtained from Yuillabtech, Seoul, South Korea. Technical-grade insecticides mixed with acetone and 250-ml Wheaton bottles were used for the assays. Insecticide concentrations published in the CDC Manual (McAllister and Scott 2020) were used: permethrin (30.3% cis and 61.5% trans isomers) (43 μg/bottle), deltamethrin (0.75 μg/bottle), etofenprox (12.5 μg/bottle), and chlorpyrifos (20 μg/bottle).

Diagnostic times used in this work were selected based on CDC guidelines for mosquito populations in the USA (McAllister and Scott 2020). The diagnostic times for Cx. pipiens and Ae. albopictus were followed but since no diagnostic times have been published for Ae. koreicus, the times were chosen as conservative estimates to establish a baseline for future comparisons between ROK populations (McAllister and Scott 2020). Until diagnostic curves for each insecticide and species can be completed for CDC bottle bioassays in the ROK, comparing these results with other geographic areas should be done with caution. The following diagnostic times were used: Cx. pipiens—permethrin (30 min), deltamethrin (45 min), and chlorpyrifos (90 min); Ae. albopictus—permethrin (10 min), deltamethrin (30 min), and chlorpyrifos (45 min); Ae. koreicus—permethrin, deltamethrin, etofenprox (30 min), and chlorpyrifos (45 min).

The CDC guidance (McAllister and Scott 2020) was used to determine if resistance was present: 97% to 100% mortality at the diagnostic time indicates susceptibility, 90% to 96% mortality at the diagnostic time indicates the population is possibly developing resistance, and <90% mortality at the diagnostic time indicates resistance. If mortality was between 3% and 10% in the controls, Abbott's formula was used to correct mortality in treated bottles before resistance or susceptibility was determined (Abbott 1925). If control mortality was >10% the test was discarded.

Based on our interpretation of the assay results, no resistance to permethrin and chlorpyrifos was detected in the Cx. pipiens colony, which suggests that the colony represents a susceptible population and can be used in comparisons with field-collected samples (Table 1). In field-collected Cx. pipiens, resistance was detected to permethrin, deltamethrin, and chlorpyrifos Fig. 1 and Table 1). No resistance was detected to deltamethrin or chlorpyrifos in Ae. albopictus, with one population showing possible resistance to permethrin (Fig. 1 and Table 1). In Ae. koreicus, for chlorpyrifos all populations had mortalities at the selected diagnostic time, indicating susceptibility (Fig. 1 and Table 1). For permethrin, 3 of 4 populations had 99% to 100% mortalities at the selected diagnostic time, indicating susceptibility with one population with 71.6% mortality at the diagnostic time, indicating resistance (Table 1). One population of Ae. koreicus for deltamethrin had 94.4% mortality at the diagnostic time, indicating possible resistance, but at 40 min >99% mortality was observed for both populations (Table 1). Both populations for etofenprox had <90% mortality at the selected diagnostic time and >95% mortality at 60 min. Following CDC guidelines, this would indicate resistance; however, more tests for all active ingredients need to be completed to verify the results.

These results support published reports that IR in ROK mosquito populations is present and varies geographically (Shin et al. 2012). The IR in Cx. pipiens has been identified in the ROK, with Shin et al. (2012) reporting varying levels of phenotypic resistance to chlorpyrifos, deltamethrin, and permethrin, and Park et al. (2020) also reporting phenotypic resistance to chlorpyrifos and deltamethrin. Lee et al. (2020) and Park et al. (2020) reported the presence of L1014F genes in Cx. pipiens from populations across the ROK, indicating widespread pyrethroid resistance. Both reported no G119 or G119S mutations in any Cx. pipiens populations examined, which suggests that the resistance found in certain populations, such as the Cx. pipiens from Camp Henry (Fig. 1 and Table 1), may be due to a non-acetylcholinesterase resistance mechanism. Ryu et al. (2019) reported no kdr mutations found in Ae. koreicus, but the CDC bottle bioassays indicated resistance to etofenprox and permethrin, with possible resistance to deltamethrin (Table 1 and Fig. 1). Additional tests on these populations are needed to confirm the results. Park et al. (2020) found no phenotypic resistance or L1014F or G119S mutations in Ae. albopictus in the ROK; however, possible resistance to permethrin was detected on Camp Humphreys (Fig. 1 and Table 1), but additional confirmatory testing is required.

By establishing a program on USAGs and other US military installations using the CDC bottle bioassay, resistance levels can be tested and tracked over time in a consistent, repeatable manner. The information can be used to gain insight on the impact IR may have on the effectiveness of mosquito management programs and personal protective measures such as factory-treated uniforms.

This study was funded by the Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance (GEIS) Branch, ProMIS ID P0010_22_ME. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Defense Health Agency, Department of Defense, nor the US Government. All authors are employees of the US Government, and this work was prepared as part of their official duties. Title 17 U.S.C. §105 provides that copyright protection under this title is not available for any work of the US Government. Title 17 U.S.C. §101 defines a US Government work as a work prepared by military service member or employee of the US Government as part of that person's official duties.