Genetic diversity and spatiotemporal population structure of Anopheles sinensis in the Republic of Korea based on the mitochondrial cytochrome c oxidase subunit I (COI) marker (original) (raw)

Abstract

• Objectives
  Anopheles sinensis is a predominant malaria vector found throughout the Republic of Korea (ROK). Population genetic analysis can provide insights into the origins and migration patterns of malaria vectors by assessing genetic variation and distribution among populations.
• Methods
  In this study, we examined the population genetic structure of An. sinensis in the ROK using mitochondrial cytochrome c oxidase subunit I (COI) marker.
• Results
  A total of 903 specimens of An. sinensis were collected from 9 sampling regions, including malaria-endemic and non-endemic areas. All populations displayed characteristics typical of migrant populations, with high haplotype diversity (Hd) and low nucleotide diversity (Pi). Network analysis identified 124 haplotypes grouped into 2 clusters. Both clusters included haplotypes from malaria-endemic and non-endemic areas. Cluster I shared its most recent common ancestry with Chinese reference sequences, while cluster II had related with Japanese reference sequences. Pairwise genetic distance (_F_ST) analysis indicated generally low genetic differentiation among populations. Furthermore, _F_ST values tended to increase proportionally with geographical distance between regions. Analysis of molecular variance confirmed that individual mosquitoes within the population had a large effect on the overall variation. Neutrality tests using 4 methods (Tajima’s D, Fu’s Fs, Fu and Li’s D, and Fu and Li’s F) yielded negative values, suggesting that An. sinensis populations are expanding in all studied regions.
• Conclusion
  This study characterizes the genetic attributes of An. sinensis in the ROK, providing valuable insights into the biology of this important malaria vector and contributing useful data for malaria control strategies.
• Keywords: Anopheles sinensis; Malaria; Population genetic structure

Graphical abstract

Introduction

Malaria causes approximately half a million deaths worldwide each year and contributes significantly to morbidity and mortality in humans [1]. The Plasmodium parasite that causes malaria is transmitted by infected female Anopheles mosquitoes, and 5 species of malaria parasite can infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi [2]. P. falciparum is predominantly found in malaria-endemic countries in Africa, whereas P. vivax is found in Southeast Asia. These 2 species occasionally cause co-infections in South America, Southeast Asia, and the Western Pacific regions [3]. P. malariae and P. ovale are broadly distributed geographically but often remain undiagnosed due to diagnostic difficulties, low parasitemia levels, and relatively mild virulence [4]. P. knowlesi occurs infrequently in Malaysia and Indonesia [3]. Notably, only P. vivax is prevalent in the Republic of Korea (ROK), with endemic areas primarily situated near the demilitarized zone bordering the Democratic People’s Republic of Korea (DPRK) [5].

Anopheline mosquitoes form genetic structures at different spatial scales, particularly in large, homogeneous geographic regions, due to recent expansions or extensive short-range gene flow [6]. Approximately 70 species of anopheline mosquitoes have been reported to have the ability to transmit malaria parasites to humans [7]. Among these, 8 species (Anopheles belenrae, Anopheles kleini, Anopheles koreicus, Anopheles lesteri, Anopheles lindesayi japonicus, Anopheles pullus, Anopheles sinensis, and Anopheles sineroides) of anopheline mosquitoes inhabit in the ROK [8]. Of these, An. sinensis has been confirmed to exhibit a broad geographical distribution and large population sizes [8,9]. An. sinensis is considered the primary malaria transmission vector in Asia [7], and its vector competence has been demonstrated through artificial sporozoite infection experiments [10]. Thus, An. sinensis is regarded as an important malaria vector in the ROK.

Mitochondrial DNA (mtDNA) serves as a valuable marker for analyzing population genetic structures in various species, including fish, arthropods, and amphibians [11–15]. A Chinese study examining the population genetics of An. sinensis with the mtDNA cytochrome c oxidase subunit I (COI) marker identified 2 regional clusters [16]. Similarly, in the ROK, genetic structuring of An. sinensis was investigated in 2015 using the mtDNA control region, revealing 2 clusters separated by the Sobaek and Taebaek mountain ranges, which acted as a genetic barrier [17,18].

Recent scientific information on the genetic structure of An. sinensis in the ROK remains insufficient. Approximately 10 years after the previous study, the present study aimed to confirm the genetic characteristics of An. sinensis using the COI marker and provide comprehensive molecular and biological data for this mosquito species, recognized as the main vector of malaria in the ROK.

Materials and Methods

Mosquito Collection and Identification

Mosquitoes were collected from 7 malaria-endemic and 2 malaria-non-endemic areas from June to October 2022 (Figure 1). In malaria-endemic regions, mosquitoes were collected from outdoor of civilian houses using black light traps (Shin-Young Comm. System), while in non-endemic areas, they were collected in livestock sheds using light-emitting diode traps (Biotrap). Collected mosquitoes were visually identified in the laboratory based on morphological taxonomic characteristics and subsequently confirmed through molecular identification by multiplex polymerase chain reaction (PCR) using primers targeting the internal transcribed spacer 2 region [19,20].

DNA Extraction and COI Gene Amplification

DNA was individually extracted from the whole body of mosquitoes using DNAzol (Thermo Fisher Scientific) or the automated nucleic acid extraction system QIAamp 96 DNA QIAcube HT Kit (QIAGEN). The mitochondrial COI gene (710 bp) was amplified using AccuPower PCR PreMix (Bioneer) with forward primer LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and reverse primer HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) [21]. PCR conditions were as follows: initial denaturation at 95 °C for 5 minutes; followed by 40 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 53 °C for 30 seconds, elongation at 72 °C for 30 seconds; and a final extension at 72 °C for 10 minutes. The sizes of the amplified PCR products were confirmed using the QIAxcel capillary electrophoresis system (Qiagen). PCR amplicons were subjected to bidirectional sequencing using Sanger sequencing technology via a commercial sequencing service (BIOFACT).

Data Analyses

Chromatograms of the amplified COI sequences were assembled using DNASTAR Lasergene SeqMan Pro, and consensus sequences were aligned using MEGA 11 (MEGA software). Sequence variation metrics—including the number of haplotypes (H), number of segregating sites (S), average number of nucleotide differences (K), average number of mutations per sequence (θ), haplotype diversity (Hd), and nucleotide diversity (Pi)—were calculated using DnaSP (version 5.10.01; Universitat de Barcelona) [22]. The haplotype network of An. sinensis was generated using the median-joining method in Network 10.2 software (Fluxus Technology Ltd.) [23]. Pairwise genetic distances (_F_ST) among regions were calculated using DnaSP to examine genetic differentiation, and gene flow (_N_m) was inferred from the pairwise _F_ST values using the formula _N_m=(1–_F_ST)/4_F_ST [24]. Analysis of molecular variance (AMOVA) was performed using Arlequin (ver. 3.5.2.2; University of Geneva) [25] to quantify genetic variation within and among populations. Finally, neutrality tests were conducted using 4 different methods—Tajima’s D, Fu’s Fs, Fu and Li’s D, and Fu and Li’s F—using DnaSP software [22].

Results

Characteristics of Sequences

All 903 mosquito samples were successfully amplified and sequenced with assembled chromatograms (Table 1). After trimming the consensus sequences, 625 bp of the initial 710 bp was used for analysis. Sequence alignment identified 79 variable and 546 conserved sites. S (number of segregating sites) varied from 18 to 49 sites based on the mosquito collection regions. K (average number of nucleotide difference) was 4.35360±0.63288 and θ (average number of mutations per sequence) was 7.38100±1.29023. Hd (haplotype diversity) was high (0.82159±0.08791), whereas Pi (nucleotide diversity) was low (0.00697±0.00101) (Table 2).

COI Haplotype Network

A total of 124 haplotypes were identified from the 903 analyzed An. sinensis sequences. The proportion of haplotype confirmed for the population by region was higher in the endemic regions of Goseong (14/22, 63.6%) and Josan-ri (13/21, 61.9%) compared to other regions (Table 2). Across all 9 collection regions, haplotype 10 (38.6%, 349/903) was most frequently observed, followed by haplotype 20 (6.6%, 60/903), being dominant and subdominant haplotypes, respectively. In total, 24 haplotypes were identified across multiple regions, while 75 haplotypes were represented by only a single individual each. The most dominant haplotype (haplotype 10), representing approximately one-third of the total population, was first detected in June in southern and central regions and became increasingly prevalent over time in northern regions. Similarly, haplotype 20 was initially identified in the southern and central regions in June and subsequently found in northern regions. Haplotype patterns found across multiple regions typically emerged first in the southern and central regions before July (summer) and were later confirmed in northern areas from summer onward (Figure 2). Haplotypes identified from single individuals occurred sporadically, predominantly after summer.

The COI haplotype network of An. sinensis was separated into 2 distinct clusters. Cluster I included mosquitoes collected from all 9 sampling locations and showed relationships to 7 Chinese reference sequences. In particular, haplotypes 6, 20, 34, and 38 shared identical COI sequences with mosquitoes from Jiangsu, Anhui, and Chongqing, China. Conversely, cluster II included mosquitoes from 8 locations (excluding Josan-ri) and was closely related to 1 Japanese reference sequence (Figure 3).

Population Genetic Structure

Pairwise _F_ST was used to calculate the genetic distance between populations in the regions where mosquitoes were collected. According to the classification of He et al. [26], _F_ST values between regions were categorized into small genetic differentiation (0–0.05) and moderate genetic differentiation (0.05–0.15). Small genetic differentiation predominated, indicating that the genetic distance between the regions was not large. Gene flow was calculated based on _F_ST; however, it could not be computed when _F_ST values were negative (Table 3). When comparing _F_ST values to straight-line geographic distances between regions, a general trend of increasing _F_ST values with increasing geographic distance was observed, although exceptions occurred (Table 4).

AMOVA was conducted by dividing the populations into 3 groups based on geographic barriers: east (Gijang and Goseong) and west (Beopheung-ri, Baekyeon-ri, Cheorwon, Gyeyang, Josan-ri, and Yesan) range of the mountain, and island (Baengnyeong). The AMOVA results indicated that genetic variation within populations was substantially greater than the variation among groups or among populations within groups (Table 5). This highlights that individual mosquito within populations significantly influenced the total genetic variation, more than among groups and among populations within groups.

Neutrality Test

Four neutrality tests—Tajima’s D, Fu’s Fs, Fu and Li’s D, and Fu and Li’s F—were performed using DnaSP, and produced all negative values (Table 6). These results indicate that the An. sinensis populations are undergoing population with expansion, characterized by an excess of low-frequency mutations. Notably, the Beopheung-ri region showed strong negative values (p<0.05) for Fu and Li’s D and Fu and Li’s F tests, suggesting heightened sensitivity to neutrality deviations compared to other regions.

Discussion

Understanding the population genetic structure, migration patterns, and gene flow of vectors provides crucial information for effectively controlling vector-borne diseases [27]. The present study assessed the genetic structure of An. sinensis populations in the ROK using the COI gene, a representative marker in population genetics studies [28].

An. sinensis is widely distributed across most regions of the ROK [18,29] and recognized as the primary malaria vector in the region, as evidenced by its confirmed sporozoite infection capability in artificial infection experiments [10]. In this study, 903 specimens of An. sinensis collected from 9 different regions in the ROK were analyzed for population genetic structure using the COI marker. Our results indicate that the An. sinensis populations exhibit characteristics of migrant populations, characterized by high Hd and low Pi. In particular, Josan-ri, located near the DPRK border, likely represents a strong migrant population due to its notably high Hd and low Pi values (Table 2).

Haplotypes were initially identified in southern and central regions and subsequently appeared in northern regions over time (Figure 2). Although the number of collected mosquitoes is different by month, all collected individuals were analyzed without selective bias. An. sinensis is known to be cold-intolerant in the ROK [30], and adult mosquitoes in the field start emerging at a minimum temperature of approximately 15.3 °C [31]. In June, collection site temperatures were 18.6 °C in Gijang and 19.86 °C in Yesan, approximately 0.5–3.4 °C warmer than in northern regions. While various factors, including habitat and nutritional availability, influence adult mosquito emergence, temperature likely has a significant impact. Hence, earlier detection of haplotypes in southern regions is presumably due to warmer conditions. Additionally, by tracking the months in which haplotypes first appeared, we observed increases in mosquito numbers and haplotype diversity during the warmer summer months (July and August; Table 1). Given that developmental cycles of An. sinensis stimulate with rising temperatures [32], mutations may also occur more frequently during the summer due to accelerated mosquito growth cycles.

Network analysis of An. sinensis revealed 2 distinct clusters (Figure 3). Cluster I contained specimens from all 9 sampling regions and showed close relationships with 7 reference sequences from China. Specifically, haplotypes 20, 34, and 38 had identical COI sequences to those from Anhui and Chongqing provinces in China. Previous studies have documented insect pests, such as Nilaparvata lugens, Sogatella furcifera, and Laodelphax striatellus, introduced from China to the ROK via jet streams [33]; mosquitoes may have similarly migrated. Cluster II, lacking haplotypes from Josan-ri (the northernmost site), was related to a Japanese reference sequence and comprised diverse haplotypes from Gijang. This strong relationship likely arises from Gijang’s geographical proximity to Japan. However, a limitation of this study is the inclusion of only 1 southern sampling site (Gijang); thus, further studies from additional southern locations are necessary to confirm this connection. Both clusters exhibited associations with international sequences, likely reflecting the geographic position of the ROK between China and Japan.

_F_ST was used to assess regional population differentiation. According to the classification by He et al. [26] in 2022, _F_ST values indicate small (0–0.05), moderate (0.05–0.15), large (0.15–0.25), or very large (≥0.25) genetic differentiation. In our analysis, small genetic differentiation was predominant among the An. sinensis populations in the ROK (Table 3). Given the broad distribution of An. sinensis across the ROK, we analyzed _F_ST values relative to straight-line geographic distances between sampling regions (Table 4). Among the sampled regions, Gijang exhibited moderate genetic differentiation, likely due to its significant distance from other regions. Interestingly, Josan-ri showed genetic differentiation from all other regions, including Baekyeon-ri, located less than 1 km away. This distinct differentiation suggests unique regional characteristics, identifying Josan-ri mosquitoes as a strong migrant group. Baengnyeong, an island in the northwest ROK, was anticipated to exhibit high genetic differentiation due to its isolation. However, the results differed from expectations, possibly due to inland mosquito movement influenced by prevailing westerly winds

AMOVA results revealed that genetic variation among regions was predominantly driven by individual mosquitoes within populations. Despite group divisions based on geographic barriers, these barriers did not significantly influence genetic differentiation. Neutrality tests (Tajima’s D, Fu’s Fs, Fu and Li’s D, and Fu and Li’s F) consistently yielded negative values, indicating that An. sinensis populations in the ROK are undergoing population expansion following a recent bottleneck or selection event. Such demographic expansions typically arise through haplotype mutations, but sufficient time may not have elapsed for extensive sequence differences to accumulate [28].

Previous studies examined the population genetic structure of An. sinensis in the ROK using the mitochondrial control region marker, reporting southern and northern genetic groups separated by the Sobaek and Taebaek mountain ranges [17,18]. Due to differences in genetic markers, direct comparisons are difficult; however, the COI marker employed in our study likely provided greater resolution, successfully identifying genetic differentiation even among regions previously classified into the same northern group. One limitation of our study was the occurrence of negative _F_ST values. To overcome this, future studies may consider incorporating additional markers, such as alternative mitochondrial regions or microsatellite loci.

Mosquito movement and migration are challenging to predict, as they are influenced by geographical barriers, environmental factors, and human activities [34]. The present study utilized COI markers to elucidate the genetic population structure of An. sinensis in the ROK, confirming significant regional genetic differences. Such molecular biological analyses provide foundational data on malaria vectors, enhancing our understanding of malaria epidemiology and control strategies.

HIGHLIGHTS

• The Republic of Korea is endemic for malaria, with Anopheles sinensis as a major vector.

• Genetic analysis of the Anopheles sinensis populations collected in this study showed small regional genetic differentiation.

• The cytochrome c oxidase subunit I haplotype network for An. sinensis comprised 2 distinct clusters, and certain haplotypes demonstrated homology with reference sequences from China.

Article information

Ethics Approval

Not applicable.

Conflicts of Interest

The authors have no conflicts of interest to declare.

Funding

This research was funded by the Korea Disease Control and Prevention Agency (Grant No. 6300-6332-305).

Availability of Data

All data generated or analyzed during this study are included in this published article. For other data, there may be requested through the corresponding author.

Authors’ Contributions

Conceptualization: HIS, HIL; Data curation: HJ; Formal analysis: HJ; Investigation: BGH; Methodology: HIS; Project administration: HIS, MRL, JWJ, HIL; Visualization: HJ; Writing–original draft: HJ; Writing–review & editing: all authors. All authors read and approved the final manuscript.

Additional Contributions

We are thankful to the Gangwon State Institute of Health and Environment, Gyeonggi Province Institute of Health and Environment, Incheon Metropolitan City Institute of Health and Environment, Chungcheongnam-do Institute of Health and Environment, and Busan Metropolitan City Institute of Health and Environment for their assistance in collecting mosquito samples.

Figure 1.

Anopheles sinensis sampling sites on the map. The site names are abbreviated.

Figure 2.

Monthly occurrence of haplotypes simultaneously identified in multiple regions. Pink indicates southern and central regions (Gijang and Yesan), blue indicates northern regions (Beopheung-ri, Baengnyeong Island, Baekyeon-ri, Cheorwon, Goseong, Gyeyang, and Josan-ri), and red indicates both northern, central, and southern regions.

Figure 3.

Haplotype network based on the cytochrome c oxidase subunit Ⅰ (COⅠ) gene as calculated by Network 10.2. Each color circle represents individuals from Beopheung-ri (BH), Baengnyeong island (BN), Baekyeon-ri (BY), Cheorwon (CW), Gijang (GJ), Gyeyang (GY), Josan-ri (JS), and Yesan (YS). The black circle symbolizes reference sequence: Anhui (AH1: MG816536, China; AH2: MG816544, China), Chongqing (CQ1: MG816556, China; CQ2: MG816550, China), Ibaraki (IB: LC054430, Japan), Jiangsu (Ji: MF322628, China), and Yunnan (YN1: MG816568, China; YN2: MG816562, China). White dots are a median vector (undetected hypothetical haplotypes). The size of the circle is proportional to the number of individuals.

Table 1.

Information on the collection sites and collected sample number by month and sites

Table 1.

Table 2.

Summary data for sequence characteristics of Anopheles sinensis

Table 2.

Table 3.

_F_ST (pairwise genetic distance) and _N_m (gene flow) values among populations

Table 3.

Table 4.

_F_ST (pairwise genetic distance) and straight-line distance (km) between collection sites

Table 4.

Table 5.

Analysis of molecular variance results for Anopheles sinensis

Table 5.

Table 6.

Neutrality test for Anopheles sinensis

Table 6.

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