Mosquito-borne pathogens are a threat to US personnel deployed to the Republic of Korea (ROK). Insecticide resistance in mosquito populations has persisted for many years in the ROK driven by public health and agricultural selection pressure (Lee et al. 2023). Insecticides such as etofenprox are used to control mosquitoes on US Army garrisons in the ROK, and US Army soldiers wear uniforms that are factory treated with permethrin to protect against vector-borne pathogens. Because of this an insecticide resistance surveillance program was established on US Army garrisons (USAG) Daegu (Camps Carroll and Henry and the Busan Storage Facility), USAG Yongsan-Casey (Camp Casey), and USAG Humphreys (Camp Humphreys) to gain a better picture of the insecticide resistance status of 3 medically important species: Aedes albopictus (Skuse), Ae. koreicus (Edwards), and Culex pipiens (L.) (Stoops et al. 2023). In 2022, this surveillance program found Cx. pipiens populations resistant to permethrin and deltamethrin, and Ae. koreicus was reported resistant to etofenprox and permethrin (Stoops et al. 2023). Possible resistance to permethrin was found in Ae. albopictus (Stoops et al. 2023). In 2023 and 2024, additional Centers for Disease Control (CDC) bottle bioassays were conducted with populations of these 3 species from the same installations from 2022 to continue to delineate insecticide resistance status on USAGs. Colonies of Cx. pipiens molestus Forskal were established in 2022 and 2023 from Busan Storage facility, Camp George, and Camp Humphreys and tested over time including assays using piperonyl butyl oxide (PBO) as a synergist.

Methods for mosquito collection, rearing, and CDC bottle bioassays were followed from Stoops et al. (2023) and McAllister and Scott (2020). Technical grade chlorpyrifos, deltamethrin, etofenprox, and permethrin (PESTANAL® analytical standard) were obtained from Yuillabtech, Seoul, South Korea and mixed with acetone in 250-ml Wheaton bottles for use in 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). For Cx. pipiens and Ae. albopictus, the CDC Manual diagnostic (McAllister and Scott 2020) times were used: Cx. pipiens: etofenprox (15 min), permethrin (30 min), deltamethrin (45 min), and chlorpyrifos (90 min; Ae. albopictus: permethrin (10 min), deltamethrin (30 min), and chlorpyrifos (45 min. No diagnostic times have been published for Ae. Koreicus, so the same diagnostic times published in Stoops et al. (2023) were used for Ae. koreicus: permethrin, deltamethrin, etofenprox (30 min), and chlorpyrifos (45 min). To evaluate potential metabolic resistance mechanisms, mosquitoes were preexposed for 1 h to PBO (400 μg/bottle in 1 ml absolute ethanol). After exposure, they were transferred to a holding container for an additional h before the bioassays were conducted. 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, then 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.

As shown in Table 1, resistance in field collected Cx. pipiens was detected to deltamethrin, etofenprox, and permethrin in 2023 and 2024. The Cx. pipiens populations tested in 2022 showed possible resistance or confirmed resistance to chlorpyrifos (Stoops et al. 2023). However, chlorpyrifos resistance was not detected in any population in 2023 and 2024. No resistance was detected to deltamethrin or chlorpyrifos in Ae. albopictus, and as in 2022, 1 population in 2023 showed only possible resistance to permethrin. No populations of Ae. albopictus were tested in 2024. In 2023 and 2024, no resistance to chlorpyrifos, deltamethrin, or permethrin was detected in Ae. koreicus; however, in 2023, possible resistance to etofenprox was detected at Camp Casey, and in 2024, the Camp Casey population tested was confirmed resistant.

Table 1.Values in parentheses represent the mortality observed at the time listed. LCHC = Lake Camp Humphreys Colony established from larval collections on Camp Humphreys in July 2022. CGC = Camp George Colony established from larval collection in April 2023. BSFC = Busan Storage Facility Colony established from larval collections in June 2023. Only minutes of 100% mortality are reported for species without known diagnostic time.

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Figure 1 presents mortality against permethrin for field-collected Cx. pipiens and colony-reared Cx. pipiens var. molestus across 3 locations (Busan Storage Facility, Camp Humphreys, and Camp George) in 2022 to 2024. As was reported in Stoops et al. (2023), the 2018 colony of Cx. pipiens var. molestus was fully susceptible to permethrin and field collected Cx. pipiens from Camp Humphreys were resistant to permethrin in October 2022 (77% mortality). A colony of Cx. pipiens var. molestus was established in October 2022 from Camp Humphreys (Lake Camp Humphreys colony [LCHC]) and tested against permethrin in November 2022 (56.5% mortality), December 2022 (47.6% mortality), and April 2023 (74.2% mortality). Assays against permethrin were done again on the same LCHC in December 2023 with mortalities of 94.3% and 95.1%. At the same time, using mosquitoes from the same LCHC, assays of permethrin + PBO yielded 100% mortality at the 30-min diagnostic time. In February 2024, the LCHC colony was tested against permethrin again, yielding mortalities of 87.6%, 89.2%, and 89.5%. From Camp George 2 assays of field collected Cx. pipiens in April 2023 resulted in 28.1% and 51% mortalities against permethrin. In April 2023, a colony of Cx. pipiens var. molestus was established from Camp George (Camp George Colony [GCG]). In December 2023, CGC mosquitoes were tested against permethrin that gave 21.3% mortality at the diagnostic time. Two simultaneous assays of permethrin + PBO against CGC in December 2023 resulted in 98.1% mortality and 99.2% mortality. In February 2024 the CGC was tested against permethrin only with 80.6% mortality. Field-collected Cx. pipiens from the Busan Storage Facility were tested in June 2023 and resulted in 54.3% mortality. A colony of Cx. pipiens var. molestus established in June 2023 from the Busan Storage Facility (Busan Storage Facility Conlony [BSFC]), showed 74.1% mortality when tested against permethrin in December 2023. In 2 simultaneous assays of BSFC against permethrin + PBO resulted in 94.9% mortality and 92.9% mortality. In February 2024, BFSC was tested against permethrin only with 70.4% mortality.

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All populations of the 3 species were fully susceptible to chlorpyrifos in 2023 and 2024. For field collected Cx. pipiens in 2023 and 2024, resistance to deltamethrin, etofenprox, and permethrin was widespread. Aedes albopictus were susceptible to all pyrethroids tested in 2023. Aedes koreicus was susceptible to deltamethrin and permethrin; however, to etofenprox it showed possible resistance in 2023 and resistance in 2024 from Camp Casey. Etofenprox is one of the most widely used insecticides in the ROK for mosquito control (Kim et al. 2024). Unlike Camp Humphreys, which applies etofenprox, no garrison-wide mosquito control program is conducted on Camp Casey using any pyrethroid, so it is possible that the resistance is coming from the surrounding community of Dongducheon. Etofenprox differs from permethrin as it is a nonester pyrethroid, but resistance has been reported to etofenprox in Cx. pipiens (Richards et al. 2017). The resistance in Ae. koreicus is at our self-established diagnostic time of 30 min, and until more in-depth mortality curves are calculated for Ae. koreicus and etofenprox, it is possible that we are either under- or overestimating the level of resistance in this population. However, our findings provide a baseline for Ae. koreicus to compare future results with this insecticide (Richards et al. 2017). Understanding etofenprox resistance for the US military is critical as etofenprox has an Environmental Protection Agency approval as an alternative to permethrin for treating uniforms (EPA 2016).

Because of the importance of permethrin in protecting soldiers from vector-borne pathogens, testing on the 3 colonies was focused on permethrin. No field-collected Cx. pipens were tested against permethrin following exposure to PBO, but PBO exposure restored susceptibility to permethrin to the 3 colonies of Cx. pipiens var. molestus, indicating metabolic resistance mechanisms may be involved.

This work was supported by funding from the Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance (GEIS) Branch, ProMIS ID P0010_22_ME, P0032_23_ME P0020_24_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, USC, Section 105 provides that copyright protection under this title is not available for any work of the US Government. Title 17, USC, Section 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.