Comparative Population Genetics of Two Korean Aedes Mosquito Species with Vector Potential based on Mitochondrial DNA

Background: Mosquitoes of the genus Aedes are important invasive species contributing to the spread of chikungunya, dengue fever, yellow fever, Zika virus, and other dangerous vector-borne diseases. Aedes albopictus is native to southeast Asia with rapid expansion due to human activity, showing a wide distribution in the Korean peninsula. Aedes avopictus is considered to be native to East Asia with a broad distribution in the region, including in the Korean peninsula. Gaining a better understanding of the genetic diversity of these species is critical for establishing strategies for disease prevention and vector control. Methods: We obtained DNA from 148 specimens of Ae. albopictus and 166 specimens of Ae. avopictus in Korea, and amplied two mitochondrial genes (COI and ND5) to compare the genetic diversity and structure of the two species. Results: We obtained a 658-bp sequence of COI and a 423-bp sequence of ND5 from the two mosquito species. We found low diversity and an insignicant population genetic structure in Ae. albopictus, and high diversity and an insignicant structure in Ae. avopictus for these two mitochondrial genes. Ae. albopictus had less haplotypes with respect to the number of individuals, and a slight mismatch distribution was conrmed. By contrast, Ae. avopictus had a large number of haplotypes compared with the number of individuals, and a large unimodal-type mismatch distribution was conrmed. Although the genetic structure of both species was insignicant, Ae. avopictus exhibited higher genetic diversity than Ae. albopictus. Conclusions: Ae. albopictus appears to be an introduced species, whereas Ae. avopictus is an endemic species to the Korean peninsula, and the difference in genetic diversity between the two species is related to their adaptability and introduction history. As an endemic species, Ae. avopictus is likely to have a larger population size than expected. Further studies on the genetic structure and diversity of these two mosquito species will provide useful data for vector control. avopictus, the endemic species. The low diversity of Ae. albopictus suggests that these mosquitos were introduced by humans, but did not fully adapt to the environment of the Korean Peninsula. The high diversity of Ae. avopictus could be due to its greater adaptability to the environment of the Korean Peninsula as an endemic species, but may also be inuenced by an increase in population and resistance to pesticides. However, the Korean Peninsula cannot be free from mosquito-borne diseases due to the rise in temperature caused by climate change, domestic inow of patients, and population density. This fundamental study of potential vector-like intermediates will be a useful foundation for vector control.


Background
Arthropod-borne viruses are transmitted by blood-sucking insects to animals and humans. Most of them, are transmitted by mosquitoes [1,2]. There are 43 genera and 3,530 species of mosquitoes in the world, however, species belonging to the genera Aedes, Anopheles, and Culex, are the main vectors of mosquito borne diseases [3,4]. In particular, mosquitoes belonging to the genus Aedes are becoming the main vectors for spreading fatal diseases such as chikungunya, dengue fever, yellow fever, and Zika virus, that often occur in Asian countries [4,5,6]. As mosquito borne diseases may grow in the future due to fast globalization and climate change, more information is needed [2,7].
Mitochondrial genes are widely used in research on molecular evolution and population genetics of vector insects. Because they have a relatively high mutation rate, and high levels of polymorphism and divergence due to their inherent sensitivity, they are highly useful as molecular markers [8,9,10,11]. Many vector studies have investigated where the population was introduced using mitochondrial genes [12,13]. Population structure and genetic diversity between populations can affect vector capacity [14]. The understanding of these factors is necessary for vector control [15].
Aedes albopictus, originally from Southeast Asia, has recently spread all over the world except for Antarctica, and is considered one of the most dangerous alien species [16,17,18]. The rst record of Ae. albopictus in South Korea was in 1940, and its distribution has recently expanded throughout the Korean peninsula [19,20]. Together with Aedes aegypti, substantial research attention has been paid to Ae. albopictus as major players in the transmission of vector-borne diseases [21,22,23]. The main reason for the global expansion is that larvae are introduced through used tires, bamboo, etc., due to human activities [24,25]. Additionally, the range of habitats they can live in has widened as a result of the temperature rise due to global warming [26,27]. Ae. Albopictus' eggs have been shown to tolerate cold weather, and have the potential to expand its distribution in colder regions [28,29].
Aedes avopictus is original from East Asia, including Japan and South Korea, and is widely distributed in that region, but there are morphological and genetic differences depending on the geographical range [30,31]. It has been found that Ae. avopictus' eggs can survive in colder environments than Ae. albopictus [32] and recently expanded its distribution from East Asia to European countries [33,34,35]. According to the results of continuous monitoring on the Korean Peninsula, the frequency of appearance of Ae. avopictus is not high [20,36,37,38]. Ae. avopictus is not known to act as a vector like Ae. albopictus and other Aedes species, but it has previously been shown that it may propagate dengue fever [30,39,40].
Since the two species are distributed over a wide area in Korea and Japan and share a common habitat [31,41,42], attention over their overlapping distribution is gradually increasing, and it is said that there is a possibility of interspeci c crossing [43,44,45]. Not only do the distributions overlap, but the two morphologies are similar [31,35,46], and Japanese studies have shown that the two are phylogenetically close to each other [47,48]. As Ae. albopictus and Ae. avopictus are closely related species and have similar ecological roles and habitats, they can be compared to each other.
The Korean Peninsula has various climates and geographical environments, and the diversity of arthropods that transmit arthropod-borne viruses is also high [49,50]. There are 11 genera and 56 species of mosquitoes in Korea, including 19 species in the genus Aedes. The presence of Ae. albopictus and Ae. avopictus was recorded in Korea in the past [19,51,52]. Since malaria and Japanese encephalitis occur frequently in Korea, only studies have focused on the vectors of these conditions, and the genus Aedes has not been investigated [53,54,55]. There are cases in which foreign mosquitoes have become indigenous bringing infections from abroad.
Additionally, but Korea also has steadily imported patients, so it is not possible to say that it is a clean country for viruses mediated by Aedes, so it is necessary to establish a preemptive control strategy [56,57].
This study compared the genetic diversity and structure of two species of Aedes mosquitoes living in Korea through two mitochondrial genes with the aim of monitoring mosquito populations. With this work, we intend to create basic data to establish vector control strategies.

Sampling and DNA extraction
A total of 314 individual mosquitos were sampled in Korea between 2017 and 2020, including 148 individuals of Ae. albopictus from 19 locations and 166 individuals of Ae. avopictus from 14 locations. Adult mosquitoes were collected using nets and BG-Sentinel traps (Biogents AG, Germany). Specimens were individually preserved in tubes lled with 80% ethanol and stored at 4°C until DNA extraction. DNA was extracted from one to three legs of each sample using DNeasy Blood & Tissue (Qiagen, Valencia, CA, USA).

Data Analyses
The sequences of the two mitochondrial genes were aligned using the ClustalW plugin on Geneious Prime 2020.1.2 (https://www.geneious.com) and prepared as concatenated sequences. DnaSP 6.12.03 [60] was used for the genetic diversity analysis of mitochondrial DNA, in which the number of haplotypes (H), number of segregating sites (S), haplotype diversity (Hd), nucleotide diversity (π), and average number of nucleotide differences (k) were examined.
Pairwise F ST values were estimated using Arlequin 3.5 software [61] to investigate genetic differentiation among the populations. Principal coordinates analysis (PCoA) was performed with GenAlEx version 6.51b2 [62] based on pairwise F ST values.
Analyses of molecular variance (AMOVA) were performed using ARLEQUIN 3.5 [61] with the locus-by-locus option and using 1,000 permutations to identify the population structure. Specimens were grouped according to regional groups in South Korea: Group 1 comprised specimens from Gyeonggi-do, Group 2 comprised specimens from Gangwon-do, Group 3 comprised specimens from Chungcheong-do, Group 4 comprised specimens from Gyeongsang-do, and Group 5 comprised specimens from Jeolla-do.
To better understand the genealogical relationships, the haplotypes were constructed using the TCS method as implemented in PopART 1.7 [63].
To investigate the demographic history of populations, deviations from selective neutrality were tested by Tajima's D [64] and Fu's FS [65] metrics using Arlequin 3.5 [61]. To con rm whether a population had undergone sudden expansion, a mismatch distribution was determined using DnaSP 6.12.03 [60].

Results
Mitochondrial gene sequence analysis resulted in a CO1 sequence of 658 bp and an ND5 sequence of 423 bp in the 19 populations (148 individuals) of Ae. albopictus, and sequences of the same length were obtained for the 14 populations (166 individuals) of Ae. avopictus (Supplementary Table 1). In the two mitochondrial DNA concatenated sequences, there were 25 haplotypes in Ae. albopictus and 107 haplotypes in Ae. avopictus.
The genetic diversity analysis revealed a relatively low number of haplotypes in Ae. albopictus compared to the total number of individuals, with relatively low haplotype diversity (0.396) and nucleotide diversity (0.00075) in the entire population. The highest haplotype diversity was found for the 2018 Anyang and Gyeongju populations, and the lowest values were 0 for six populations. The 2018 Anyang and Gyeongju populations also exhibited the highest nucleotide diversity. Ae. avopictus showed a relatively high number of haplotypes compared to the total number of individuals, and high levels of haplotype diversity (0.990) and nucleotide diversity (0.00894) were found in the entire population. Analysis of the two species revealed that Ae. avopictus exhibited higher levels in various genetic diversity indices. AMOVA showed low genetic variance among both species, but high variance within populations. In particular, Ae.
avopictus showed higher variance than Ae. albopictus, indicating that it can form a genetic structure within populations.
In the haplotype network, Ae. albopictus showed a simple star-like form, in which several haplotypes diverged from one of the largest haplotypes, and hap_1 had the highest frequency of 78% in all populations. Private haplotypes, most of which were singleton haplotypes, accounted for 22%. Ae. avopictus exhibited a complex haplotype network, which was found to have a higher haplotype compared to the total number of individuals.
Most of the network was composed of singleton haplotypes. When comparing the two species, Ae. avopictus had more haplotypes than Ae. albopictus and showed a complex haplotype network.
With respect to demographic history, Ae. albopictus showed negative but low values for Tajima were also found for the entire population of Ae. avopictus. In both species, Ae. avopictus showed slightly higher Fu's F S values. For the mismatch distribution, the result of Ae. albopictus was insigni cant, whereas Ae. avopictus showed a large unimodal shape, indicating the possibility of sudden expansion of the population.

Discussion
Based on the above results, two conclusions can be drawn. Higher genetic diversity was observed in Ae.
avopictus than in Ae. albopictus, and the two species of mosquitoes generally showed low levels of genetic structure except for some populations.
There are two hypotheses addressing the overall low diversity of Ae. albopictus in Korea. The rst is a hypothesis regarding the spread of Wolbachia virus to the mitochondria: Previous studies have shown that intracellular Wolbachia was detected in 17 populations in Korea, and these group are known to have low mitochondrial diversity [66,67]. However, there is no clear evidence of Wolbachia in many samples, and further analysis of the nuclear genes, as well as the mitochondrial genes, will be needed to reveal the impact of Wolbachia on genetic diversity in the population [66,68]. The second hypothesis is that the introduced Ae. albopictus may have been affected by Korea's harsh climate, which is different from that of the country of origin: Ae. albopictus is considered to be an invasive species that has recently spread abroad from its origin in Southeast Asia [16,17]. These mosquitos have adapted to the environment of each country since their introduction, but previous studies have shown that they also have low diversity in the countries from which they were introduced [12,69]. The environment in Southeast Asia, the native habitat of Ae. albopictus, is humid and high in temperature, facilitating the spread of Ae. albopictus [70]. However, winters are quite severe in Korea, with cold, dry weather. The average temperature is less than 10°C. This environment could lead to a decrease in the population size of this mosquito, leading to a decrease in genetic diversity [55,71]. In the domestic populations, the Geoje population is different from other populations. This is believed to arise from genetic differences due to the physical distance between mosquitoes introduced by human activities. The distance traveled by mosquitoes in the natural environment is, however, only a few kilometers [72], and there are study results showing that the populations have been genetically structured in heterogeneous habitats due to their limited dispersive abilities [73].
Korean Ae. avopictus have a high genetic diversity, and a complex haplotype network. There are two hypotheses regarding their high diversity. The rst hypothesis is based on the fact that Ae. avopictus is endemic to East Asia: Ae. avopictus is known to be native to East Asia, and is divided into three subspecies depending on the region. The subspecies are morphologically and genetically distinct [30,31]; Ae. avopictus, Ae. avopictus downsi, and Ae. avopictus miyarai. Among them, Ae. avopictus is distributed in the Korean Peninsula, and records show that they have existed here for a long time, but there have been few molecular studies into this species, so the extent of it genetic diversity is not fully known [30,51,74]. Studies on mosquitoes of the genus Aedes show that genetic diversity in the original population is much higher, which supports the contention that the original population of Ae. avopictus is here [75,76]. The second hypothesis takes into account the adaptation to cold climates as an endemic species: Ae. avopictus is an Asian species that does not exist in tropical regions, and lives in subtropical areas throughout the cool-temperate region [48]. This mosquito has recently been found in the Netherlands, a more northerly region, and is considered highly likely to spread due to its ability to cope with environmental changes [34,35]. It has excellent environmental adaptability as well as an ability of the eggs to withstand cold conditions, and can exist in dry conditions for a long time [51,77]. Studies have also shown that it is genetically close to Aedes galloisi, a northern mosquito species in the same genus [47].
The genetic structure of domestic Ae. albopictus and Ae. avopictus has been found not only in these two species, but also in other mosquito species, and this pattern is common in mosquitoes [11,78]. Differences in diversity between the two species can be explained in several ways. Ae. albopictus is an introduced species that shows high genetic diversity in its native Southeast Asia region. However, in Korea, these mosquitos have a considerably lower genetic diversity. A small mismatch in distribution and one haplotype shared by various populations show that patterns observed in Ae. albopictus may have been affected by the decrease in effective population size, human introduction, and natural environment changes [12,69,79]. In future studies, vector control can be aided by the comparison of local mosquitos with overseas populations, using various markers, which will facilitate the inference of the timing of introduction in Korea. Ae. avopictus, an endemic species, has high genetic diversity, and has a large unimodal mismatch distribution and a complex haplotype network. The unimodal form of its mismatch distribution indicates that the Ae. avopictus population may have recently experienced a large population expansion. The successful distribution and increasing population of endemic species of Ae. avopictus may have been affected by human demographics [22], and the complex form of the haplotype network indicates a high mutation rate, which can increase the rate of development of resistance in insects [80,81]. The difference in genetic diversity between these two mosquito species living in Korea may also come from the differences in effective population size, due to their ability to adapt to the cold, as well as their status as an endemic or introduced species. Although the distribution of the two species overlaps, Ae. albopictus can survive for up to 24 hours at -10°C in the form of diapause eggs [82], and the eggs of Ae. avopictus can survive for a longer period [32,48,51]. The decreased survival rate of eggs can affect the effective population size, since fewer adults develop [83,84]. This difference in cold adaptation can affect the size of the effective population, which can lead to differences in genetic diversity [55,85,86]. Monitoring of vectors in Korea has shown that the frequency of Ae. avopictus was not high, but the reason for the large potential population size in this study lies in the difference between the location and the method of collection [20,36,37,38]. Continuous monitoring is needed, because this species is highly likely to affect humans, as it has a large population size and considerable potential as a vector.
The differences in the genetic diversity of Ae. albopictus and Ae. avopictus populations revealed in this study suggest that continuous monitoring of these species with multiple possibilities as vectors is essential. To understand the genetic diversity of Aedes mosquito species in Korea, sampling in more diverse regions and the use of different genetic markers will be conducted in further studies.

Conclusions
This is the rst paper comparing genetic diversity and the genetic structure of two Aedes mosquito species inhabiting Korea. This study found that Ae. albopictus, which is considered to be an introduced species, has lower genetic diversity than Ae. avopictus, the endemic species. The low diversity of Ae. albopictus suggests that these mosquitos were introduced by humans, but did not fully adapt to the environment of the Korean Peninsula. The high diversity of Ae. avopictus could be due to its greater adaptability to the environment of the Korean Peninsula as an endemic species, but may also be in uenced by an increase in population and resistance to pesticides. However, the Korean Peninsula cannot be free from mosquito-borne diseases due to the rise in temperature caused by climate change, domestic in ow of patients, and population density. This fundamental study of potential vector-like intermediates will be a useful foundation for vector control.