Identification of Culex quinquefasciatus Highlights the Need for Strengthened Vector Mosquito Surveillance in the Republic of Korea

Abstract

Objectives

We investigated the presence of subtropical mosquito vectors that may become established in the Republic of Korea (ROK) owing to climate change. Comprehensive surveillance was conducted on Jeju Island, a region with a subtropical climate with active external entry routes, where the risk of subtropical mosquito introduction and establishment is increasing.

Methods

In July 2025, adult mosquitoes were collected from 47 sites across Jeju Island using BG-Sentinel traps baited with carbon dioxide, in collaboration with Honam Regional Center for Disease Control, the Jeju Institute of Health and Environment, Jeju National University, and Cheju Halla University. A total of 147 mosquitoes, morphologically classified as belonging to the Culex pipiens complex, were subjected to genetic analyses for species identification. The collected specimens were screened for major mosquito-borne viruses, including the Japanese encephalitis, West Nile, Zika, Dengue, Chikungunya, and Yellow fever viruses.

Results

Cx. quinquefasciatus is morphologically very similar to the Cx. pipiens complex, making it difficult to distinguish based on external characteristics. In addition, the cytochrome c oxidase subunit I marker alone was insufficient for clear species differentiation. Therefore, through multiplex molecular analyses incorporating nuclear gene markers such as acetylcholinesterase-2, a total of 11 Cx. quinquefasciatus individuals were identified from five collection sites. Furthermore, all specimens tested negative for six major mosquito-borne viruses.

Conclusions

This study provides the first official report confirming the presence of subtropical mosquitoes on Jeju Island, ROK. These findings highlight the need to strengthen vector surveillance systems and refine public health strategies to address the potential establishment and northward expansion of subtropical mosquito species due to ongoing climate change and reveal an increased risk of exotic species introduction.

Key messages

① What is known previously?

Culex quinquefasciatus is a major mosquito species distributed throughout tropical and subtropical regions worldwide, well adapted to urban environments, and capable of transmitting various pathogens.

② What new information is presented?

Molecular analysis of mosquito samples collected from Jeju Island confirmed the presence of Cx. quinquefasciatus. Screening for pathogens yielded negative results.

③ What are implications?

Subtropical mosquitoes detected in Jeju Island indicate shifts in mosquito distribution and potential vector-borne disease risks in Republic of Korea. Although no major viruses were found, the growing presence of competent species suggests increased outbreak potential. Strengthening surveillance, expanding monitoring, and adopting gene-based systems are key to prevention.

Introduction

Mosquito-borne infectious diseases represent a major global public health concern. In the Republic of Korea (ROK), approximately 20 cases of Japanese encephalitis and around 600 cases of malaria are reported annually. Recent climate change and increased international exchange have markedly increased the likelihood of the introduction and establishment of subtropical mosquito species in the ROK, where they have not previously been recorded. This phenomenon may serve as a driving force, altering the distribution patterns of domestic mosquito populations.

Jeju Island, located in the transition zone between subtropical and temperate climates, provides favorable environmental conditions for the survival of invasive mosquito species due to its relatively high average temperatures and mild winters. In addition, active exchanges through its ports and airports make the island particularly susceptible to the introduction of new mosquito species from abroad. Given these factors, Jeju Island can be considered a region of significant strategic importance for monitoring the introduction and subsequent dispersal of subtropical mosquitoes within ROK. This study aims to conduct genetic analyses on mosquitoes collected from the Jeju island to confirm the presence of subtropical mosquito species and to provide foundational data for assessing future changes of distribution of domestic mosquito species and the associated risk of vector-borne infectious disease outbreaks.

Methods

1. Mosquito Collection and Target Mosquito Selection

In July 2025, mosquitoes were collected in collaboration with the Honam Regional Disease Response Center; the Institute of Health and Environmental Research, Jeju Special Self-Governing Province; Jeju National University; and Cheju Halla University. Mosquito collections were conducted using BG-Sentinel traps, with carbon dioxide as an attractant, at 47 locations across the Jeju island. Genetic analysis was performed on a total of 147 specimens that were morphologically identified as belonging to the Culex pipiens complex (Table 1).

Table 1. Primer sets used for amplification of COI and ace-2 genes.

Primer	Sequence (5’–3’)	Size of PCR product	Target gene
LCO1490_F	GGT CAA CAA ATC ATA AAG ATA TTG G	658 bp	COI
HCO2198	TAA ACT TCA GGG TGA CCA AAA AAT CA
Ace - quin	CCT TCT TGA ATG GCT GTG GCA	610 bp (Culex pipiens complex) 274 bp (Cx. quinquefasciatus) Both band hybrid mosquito species	Ace -2
Ace - pip	GGA AAC AAC GAC GTA TGT ACT
B1246s	TGG AGC CTC CTC TTC ACG GC

COI=cytochrome c oxidase subunit I; ace-2=acetylcholinesterase-2; PCR=polymerase chain reaction; bp=base pair.

2. Molecular Species Identification

After individual wings or legs were collected from the selected Cx. pipiens complex specimens, genomic DNA was extracted using automated nucleic acid extraction devices QIAcube HT (Qiagen). The amplification process was then executed using specific primers to the cytochrome c oxidase subunit I (COI) gene and the acetylcholinesterase-2 (ace-2) gene. The resulting sequences were compared with those previously reported in the scientific literature for molecular identification.

The polymerase chain reaction (PCR) was performed in a total reaction volume of 20 μl. Each reaction mixture contained 2 μl of extracted genomic DNA, 1× PCR buffer, 1 mM dNTPs, 1.5 mM MgCl₂, 1 unit of Taq DNA polymerase (AccuPower® PCR PreMix; Bioneer), and 2 μl of each primer.

For amplification of the COI gene, the PCR conditions included initial denaturation at 94°C for 1 minute (one cycle), followed by pre-amplification for five cycles, consisting of denaturation at 94°C for 1 minute, annealing at 45°C for 1 minute 30 seconds, and extension at 72°C for 1 minute 30 seconds. This was followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute 30 seconds, and extension at 72°C for 1 minute 30 seconds. A final extension was performed at 72°C for 5 minutes (one cycle).

For amplification of the ace-2 gene, two primer sets were used: Ace-quin and Ace-pip. For Ace-quin, the PCR conditions consisted of initial denaturation at 94°C for 3 minutes (one cycle), followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 10 minutes (one cycle). For Ace-pip, the PCR conditions consisted of initial denaturation at 94°C for 3 minutes (one cycle), followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 10 minutes (one cycle). PCR products were visualized as bands at 610 bp for Cx. quinquefasciatus, at 274 bp for the Cx. pipiens complex, and at both 610 bp and 274 bp for hybrids.

3. Pathogen Analysis

Molecularly identified adult mosquitoes of Cx. quinquefasciatus and the Cx. pipiens complex were individually homogenized using a Precellys® Evolution homogenizer (Bertin Technologies) with two bead-beating cycles at 7,500 rpm for 30 seconds each. The homogenized samples were then centrifuged at 13,000 rpm for 1 minute, and total RNA was extracted using the Clear-S^TM Total RNA Extraction Kit (Invirustech). The extracted RNA was analyzed for pathogens using the Clear-MD® DENV/ZIKV/CHIKV/JEV/WNV/YFV Multiplex Real-Time RT-PCR Detection Kit (FAM/HEX/ROX) (Invirustech). The PCR was performed in a total volume of 20 μl. The amplification protocol included reverse transcription at 50°C for 15 minutes, heat activation at 95°C for 2 minutes, denaturation at 95°C for 15 seconds, and annealing/extension at 58°C for 1 minute. Amplification and detection were performed using the QuantStudio^TM 5 Real-Time PCR System equipment (Thermo Fisher Scientific), and positivity was determined based on amplification curve analysis.

Results

Genetic analysis of 147 specimens morphologically classified as belonging to the Cx. pipiens complex identified 11 Cx. quinquefasciatus individuals in 5 sites, three hybrids between Cx. quinquefasciatus and the Cx. pipiens complex, and 67 Cx. pipiens complex individuals. The obtained nucleotide sequences were compared with those of Cx. quinquefasciatus and the Cx. pipiens complex available in the GenBank database of the National Center for Biotechnology Information; all of them showed high genetic homology (99–100%). This approach enabled cross-validation of the molecular species identification results with the morphological classifications. The three groups exhibited highly similar morphological characteristics, making visual differentiation challenging. Additionally, analysis of the COI gene—a widely used marker for species identification—was inefficient to clearly distinguish between Cx. quinquefasciatus and the Cx. pipiens complex. Therefore, complementary molecular analyses employing nuclear gene markers such as ace-2 were considered essential for accurate identification of Culex species and for differentiating hybrids (Figure 1). Several previous studies have reported similar findings [1,2], and recent domestic research on the Culex genus has likewise demonstrated the limitations of COI analysis in distinguishing between the Cx. pipiens complex and Cx. quinquefasciatus [3].

Figure 1. Electrophoresis results of the ace-2 gene for Culex quinquefasciatus, Cx. pipiens complex, and hybrids.

(A) Ace-quin primer, (B) Ace-pip primer. Lane M=marker; Lane 1=Cx. quinquefasciatus; Lane 2=Cx. pipiens complex; Lane 3=hybrid; Lane N=negative control; bp=base pair.

Additionally, pathogen screening for major mosquito-borne viruses—including Japanese encephalitis, West Nile, Chikungunya, Zika, Dengue, and Yellow fever virus—in Cx. quinquefasciatus, the Cx. pipiens complex, and hybrid specimens revealed negative.

Conclusion

This study identified Cx. quinquefasciatus and its hybrids in the Jeju region, representing a significant finding that indicates ongoing changes in the domestic mosquito distribution. Cx. quinquefasciatus is primarily distributed in tropical and subtropical regions; however, due to climatic changes, its presence has also been documented in temperate zones. It is known for its high adaptability to urban environments. The World Health Organization has designated this species as a significant vector of diseases such as West Nile fever and lymphatic filariasis [4]. It is commonly reported to inhabit urban environments, including polluted puddles and sewers [5,6]. These ecological characteristics may contribute to an increased likelihood of Cx. quinquefasciatus establishing and spreading within ROK in the future.

Since the initial record of Cx. quinquefasciatus collection in 1958, no sightings of the species had been reported in the ROK for several decades [7,8]. However, a report described the detection of a single specimen in the Goseong area of Gyeongsangnam-do [9]. Nonetheless, as the study relied solely on the COI gene for species identification, its findings may be subject to limitations regarding species confirmation [3]. In contrast, the present study is of considerable significance, as it clearly identified the presence of Cx. quinquefasciatus and hybrids through concurrent analysis of the ace-2 gene.

Moreover, the identification of hybrids between Cx. quinquefasciatus and the Cx. pipiens complex carries ecological significance. Hybridization has been shown to alter the genetic diversity and ecological adaptability of mosquito populations, thereby potentially enhancing their ability to colonize new habitats, modifying their susceptibility to pathogens, and influencing the transmission dynamics of mosquito-borne infectious diseases [2]. Therefore, continuous monitoring of hybrid occurrence—particularly in Jeju Island and other southern regions—is essential.

Jeju Island is characterized by a subtropical climate, influenced by westerly winds and its location along the northward trajectory of typhoons. In addition, active human and material exchanges through ports and airports create an environment highly conducive to the introduction and establishment of foreign mosquito species. As climate change continues to impact the planet, Jeju Island and southern regions of the ROK are expected to experience increasingly favorable conditions for the proliferation of subtropical mosquitoes. Accordingly, it is crucial to strengthen the existing surveillance system centered on the Vector Surveillance Center for Climate Change Response. Furthermore, precision monitoring should be expanded at potential points of international entry, such as ports and airports. It is also imperative to establish a genetics-based species identification framework that complements the existing morphological identification system. Overall, these measures will play a pivotal role in enabling rapid responses to mosquito-borne disease threats and safeguarding public health in the ROK.