Updated Distribution of Anopheline Mosquitoes (Diptera: Culicidae) in Hokkaido, Japan, and The First Evidence of Anopheles (Anopheles) Belenrae in Japan


 Background: After World War II in Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. Methods: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a CDC miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to PCR-based sequence analysis of the ITS2 region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method. Results: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae Rueda (2005), was discovered in eastern Hokkaido in 2004. A nopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. h yrcanus group, A n . belenrae , A n . engarensis , A n . lesteri , and An. sineroides , have been found in Hokkaido. The results also suggest that An. sinensis , formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. Conclusion: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae , described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.

Anopheline species contain the most important malaria vector species. Among those recorded in Japan, An. sinensis Wiedemann, 1828 is the most widespread and common anopheline species. This species is considered the major vector of vivax malaria in Korea and China. Previous surveys conducted in Japan revealed that An. sinensis was the dominant anopheline species in Japan, including Hokkaido; An. lesteri Baisas & Hu, 1936 was commonly found in Hokkaido with only a few An. sineroides Yamada, 1924 [3][4][5][6]. These surveys also found a new member of this group, An. engarensis Kanda and Oguma, 1978 [3][4][5]. Thus, several malaria vector species (e.g., An. sinensis, An. engarensis, and An. lesteri) continue to inhabit Japan. Despite the need for a nationwide survey to systematically assess these species, very little information is available, mostly gathered in the 1980s. Recently, several DNA barcoding projects have been conducted on mosquitoes in Japan, and a small number of genomic information on anopheline mosquitoes were included [7][8][9]. However, these studies were not speci c to malaria vector mosquitoes.
At the onset of this survey, the distribution of ve species of the An. hyrcanus group, An. sinensis, An. sineroides, An. lesteri, An. engarensis, and An. yatsushiroensis Miyazaki, 1951, had been con rmed in Japan. Moreover, of these ve species, only An. yatsushiroensis has never been reported in Hokkaido [10][11][12][13], the region of interest in this study. Nonetheless, the highly similar morphological features of the members of this group, particularly An. engarensis and An. sinensis, makes it di cult to distinguish between species morphologically. Therefore, the frequency of clasper movements in males, hybridization studies, and chromosomal studies were used in distinguishing An. engarensis from the Japanese population of An. sinensis [3][4][5]. Recently, they have effectively been identi ed using polymerase chain reaction (PCR) and sequence analysis. Among the molecular markers used for mosquito taxonomy, the cytochrome oxidase subunit 1 (COI) sequences of the DNA barcoding region [14][15][16], and the internal transcribed spacer 2 (ITS2) region of rDNA are the most e cient. ITS2 in particular, is very e cient in distinguishing between closely related species, e.g., An. maculipennis complex, An. quadrimaculatus complex, An. culicifacies complex, and An. gambiae complex [17][18][19][20]. ITS2 has also been used to address taxonomic issues in the An. hyrcanus group [21][22][23][24][25][26].
For about 20 years since the last survey in 1984 [6], very few surveys of malaria vector mosquitoes had been conducted in Japan. We therefore initiated nationwide surveys from 2001 to determine the current status and distribution of anopheline mosquitoes in Japan. During our survey, we recorded a new member of the An. hyrcanus group in eastern Hokkaido in 2004. They were genetically con rmed to be Anopheles (Anopheles) belenrae Rueda (2005), described in South Korea in 2005 [24]. In the present study, species identi cation and determination of genetic distances between specimens was carried out by analyzing ITS2 region. Special attention was given to determining the distribution of An. belenrae in Japan. Finally, we updated the information from previous surveys [3][4][5][6] on the current distribution of the anopheline mosquitoes in Hokkaido.

Mosquito sampling
Mosquitoes were collected at 22 sites in Hokkaido from 2001 to 2015 (Tables 1 and 2). In this study, eight specimens collected in domestic areas outside Hokkaido were used as a reference specimen in phylogenetic analysis. Seven of the eight specimens were from Japan and the last from Vietnam. The  Table 2.
Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest throughout the day using a CDC miniature light trap enhanced with dry ice [27], aspirators, and sweeping nets for approximately 3 h after sunset. Collected adult mosquitoes were frozen and transported in an icebox to the National Institute of Infectious Diseases (NIID), Tokyo, Japan.
Larval mosquitoes were collected from paddy elds, swamps, stagnant and owing rivers, lakes, and ponds using dippers. Larvae were transported alive to NIID and reared to adults under laboratory conditions of 25°C, 60-70% relative humidity, and a photoperiod of 16:8 (L:D) h. Morphological identi cation was performed on all adult individuals using taxonomic keys [11,28]. All classi ed mosquito specimens were transferred individually into 1.8 mL microtubes (Eppendorf, Hamburg, Germany), and stored at -80°C until subsequent analyses by ITS2 sequencing. DNA extraction, ITS2 ampli cation and sequencing Total genomic DNA was extracted from individual samples using a REDExtract-N-Amp Tissue PCR Kit (Sigma Chemical Co., St. Louis, MO) according to the manufacturer's protocol. Extracted mosquito DNA was subjected to PCR-based sequence analysis and phylogenetic analysis using primers of the ribosomal DNA ITS2 region (forward, 5′-TGT GAA CTG CAG GAC ACA-3′; reverse, 5′-TAT GCT TAA ATT CAG GGG GT-3′) [29]. Ampli cation conditions were as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and a 4 min nal extension at 72°C using a Veriti™ 96-well Thermal Cycler (Thermo Fisher Scienti c Inc., Waltham, MA).

Phylogenetic analysis
Multiple alignment of the ITS2 sequences with those of related species available in the GenBank library was performed using the CLUSTALW program [30]. Phylogenetic trees were produced using the neighborjoining (NJ) program with Kimura's two-parameter model [31] on MEGA X version 10.2.2 [32]. The statistical signi cance of the resulting NJ trees was evaluated using a bootstrap test with 1,000 replications.

Distribution map
The distribution map was constructed using ArcGIS 10 (ESRI Inc., Redlans, CA), and information from the Geographic Information System.

Phylogenetic analysis
A total of 248 specimens (181 adults and 67 larvae) were collected in Hokkaido between 2001 and 2015.
The collected specimens were classi ed into four anopheline species of the An. hyrcanus group; An. belenrae, An. engarensis, An. lesteri, and An. sineroides (Table 1). Interestingly, An. sinensis was not collected from Hokkaido during our survey. Phylogenetic analysis was performed using the 485bp ITS2 sequence of 38 specimens, collected from different sites (30 from Hokkaido, seven from Japanese regions outside Hokkaido, and one from Vietnam) in different years, and eight reference sequences from the GenBank database ( Table 2).
The NJ phylogenetic trees revealed ve robust clades, consisting of the four species listed above and An. sinensis (Figs. 1 and 2). Unfortunately, An. sinensis was not detected in Hokkaido in this study. Therefore, we analyzed An. sinensis collected from areas outside Hokkaido. The cluster of An. sinensis showed small differences (Figs. 1 and 2). Nonetheless, there were no differences between the four Japanese strains of An. sinensis (Yokohama08, Echizen379, Kaifu353, and Misawa391) and the strain from South Korea (isolate 1).
In 2004, two larvae morphologically identi ed as An. sinensis were con rmed to be An. belenrae using the ITS2 sequence, marking the rst record of An. belenrae in Japan (Akan44 strain). Subsequent phylogenetic analysis showed that An. belenrae was the closest related species to An. sinensis, followed by An. engarensis (Figs. 1 and 2).

Intra-and interspeci c ITS2 variation
The levels of nucleotide variation detected between pairs of specimen in the An. hyrcanus group are presented in Table 3. There were no genetic differences between the 10 Japanese strains of An. belenrae (Akan44, Kushiro10, Kushiro201, Kushiro313, Kushiro418, Kawakami60, Akan712, Kushiro503, Nakagawa807, and Nakagawa26) and the Korean strain (isolate 3), with a 0% pairwise divergence (Figs. 1, 2 and Table 3). This suggests that the Japanese An. belenrae and the Korean An. belenrae are the same at least based on the ITS2 sequences. The Vietnamese strain (GLVN59) showed slight differences from the other strains, with a 0.26% pairwise divergence (Table 3). Regarding the clusters of An. engarensis, one specimen (Daisen55) collected in Akita Prefecture, an area outside Hokkaido, was slightly different from the nine specimens collected in Hokkaido (Abashiri02, Akashuri18, Yubari25, Yufutsu20, Kameda40, Yufutsu115, L1532, A14-22, and L1468) (Figs. 1 and 2). No genetic differences were observed among the An. engarensis specimens, except for the Daisen55 strain, with a 4.63% pairwise divergence from the others ( Table 3).
As mentioned above, the intraspeci c variation between these three species was very low (0%, 0.26%, and 4.63% in An. belenrae, An. sinensis, and An. engarensis, respectively) ( Table 3); a few differences were found based on the collection areas. Regarding the interspeci c variation, pairwise divergence between An. belenrae and two other anopheline species (An. sinensis and An. engarensis) was 13.25-13.57% and 13.27-14.26%, respectively, and 13.86-15.19% between An. sinensis and An. engarensis ( Table 3). The values among these three species indicate high levels of genetic differentiation. The NJ phylogenetic trees also showed that one strain of An. kleini from South Korea was located closer to An. engarensis than to An. belenrae and An. sinensis (Figs. 1 and 2). A detailed study of the genetic background of these species will be necessary.
In An. sineroides, no differences were found between the two specimens from Hokkaido (Fukagawa13 and Kushiro343), and the one from South Korea (SINEK02) (Figs. 1, 2, and Table 3). However, the two specimens from areas outside Hokkaido (Hida386 and Toyama80) showed few differences from the three mentioned above, with 0.26 and 0.8% pairwise divergences (Table 3). In contrast, a large intraspeci c variation was observed in An. lesteri ( Figs. 1 and 2). Pairwise divergence was in the range of 0.26-2.14% among the nine strains from Hokkaido (Abashiri01, Abashiri17, Abashiri42, Hakodate31, Kamiiso35, Kameda38, Kushiro317, Kushiro505, and A2651) ( Table 3). The two Korean strains (specimen 1 and specimen 2 classi ed as Type B and Type C of An. lesteri, respectively) were quite far from the other An. lesteri strains (Figs. 1 and 2). Pairwise divergence among the 11 An. lesteri strains ranged from 0.26-8.96%, indicating that An. lesteri appeared to form a highly divergent population. The cluster of An. lesteri revealed a low pairwise divergence, ranging from 0-2.14%, between the nine An. lesteri strains from Japan and the Chinese strain of An. anthropophagus (SMMU-FK1) ( Table 3), suggesting that they may belong to the same species.

Discussion
The rst record of An. belenrae in Japan Our surveys from 2001 to 2015 revealed a signi cant change in the distribution range of the An. hyrcanus group in Hokkaido reported in the 1980s [3][4][5][6], including the rst record of An. belenrae in Japan. Two larvae collected in the Kushiro Wetland in 2004 were tentatively named An. sinensis Kushiro strain, based solely on the morphological characteristics of the emerged adults. However, phylogenetic trees constructed using ITS2 sequence revealed that this An. sinensis Kushiro strain formed a robust clade that was clearly different from the clades of An. sinensis and other Anopheles species. Interestingly, the ITS2 sequence of the Kushiro strain was not identical to that of the An. sinensis strains collected in southern Japan, outside Hokkaido but to that of An. belenrae, a new strain reported in South Korea in 2005 [24]. The Kushiro strain could con dently be included in the An. belenrae cluster because of the absence of intraspeci c divergence as mentioned above. This species was consistently found in the Kushiro Wetland after the rst detection in 2004. In contrast, An. belenrae was not found outside Hokkaido in our 15-year nationwide survey. Thus, we concluded that this species is restricted to the Kushiro Wetland in Hokkaido.
The Kushiro Wetland is the largest marshland/wetland in Japan and is located in the Kushiro Plain. The Kushiro Wetland has been the focus of nature conservation efforts since before World War II, was registered as a Ramsar site in 1980 and designated as a national park in 1987. It is also famous for being the breeding ground for Japanese Cranes, Grus japonensis, and many other wild birds and a protected area for natural monuments, birds, and animals; thus, land development is strictly regulated. In South Korea, An. belenrae is found in the northern part of the country near the border with North Korea [24,38,39]. In China, An. belenrae is reportedly distributed in Shandong and Liaoning Provinces in northeast China, facing the Korean Peninsula [40]. These areas are not only geographically close to Japan, but may also have similarities in climate, vegetation, and some environmental factors with the Kushiro Wetland.
However, further investigation is needed to compare the morphological characteristics of Japanese and Korean An. belenrae, and to determine the distribution of this species in locations outside Hokkaido in Japan. We hope that ecological and evolutionary factors impacting the emergence of An. belenrae will be elucidated with the development of molecular biological technology.

No information of An. sinensis from Hokkaido
The next noteworthy nding was the disappearance of An. sinensis from Hokkaido. In previous surveys, An. sinensis was generally distributed throughout Hokkaido [3][4][5][6] (Fig. 3). Although it is often found in the same larval habitat as An. lesteri, it is thought to occur more frequently in developed paddy elds and swamps [41]. In the 2000s, we did not nd any An. sinensis in the habitat of An. lesteri, nor did we nd any new sources or habitats (Fig. 4). It is possible that the larval habitat of An. sinensis changed drastically in the 20-year period between the previous studies [3-6] and this current study. For example, in the 1949 [1] and 1976 [6] surveys, four members of the An. hyrcanus group were detected in northeastern Hokkaido, around Rubeshibe (Fig. 3). At that time, there were paddy elds all over the district, and forestry and horse-logging were the main industries. In recent times however, the horse-logging industry has declined drastically, and the paddy elds have been replaced with upland crops. Furthermore, neither An. sinensis nor An. sineroides was found in this area, around Ozora, during our survey (Fig. 4). It is highly likely that the changes in vegetation and industry have affected the distribution of anopheline mosquitoes.
There may be other reasons for the disappearance of An. sinensis from Hokkaido. The classi cation of organisms was mainly based on morphological keys until the 1990s. Although adults of An. belenrae can be separated morphologically from those of An. lesteri, An. sinensis and other species [24], it was likely that An. belenrae and An. sinensis could not be differentiated morphologically. Therefore, it should be noted that An. belenrae may have been classi ed as An. sinensis. The results from the ITS2 sequences in this study revealed that these two species were genetically the closest related. In addition, the pairwise interspeci c distance in mitochondrial genomes calculated by each fragment showed minor or no difference between An. sinensis, An. belenrae and An. kleini [40]. Phylogenetic analysis of COI indicated that ancient hybridizations probably occurred among these three closely related species [42], making differentiation with the COI sequence improbable. To address this problem, we tried to extract DNA and decipher the nucleotide sequence from age-old, dried specimens previously classi ed as An. sinensis collected in Hokkaido [3][4][5][6]. However, no new information could be obtained from these specimens. We hope that techniques for genetic analysis using age-old specimens will be developed as soon as possible.

Anopheles engarensis in Japan
Anopheles engarensis is also a species whose distributional range has reduced in Hokkaido. This species, rst described in Engaro-cho (North-eastern Hokkaido) in 1977, [3] was also found in Monbetsu, Kushiro, and Obihiro until 1984 [6], suggesting a wide distribution in Hokkaido [3][4][5][6] (Fig. 3). However, our surveillance found this species to be restricted to western and southern Hokkaido (Fig. 4). In addition, this species was also collected in northern Tohoku, Akita Prefecture, suggesting a southward shift presumably due to changes in the environment, including the climate of larval habitats. In terms of classi cation, An. engarensis was recognized as a new species in the An. hyrcanus group only after its chromosomal structure was determined to be different from An. sinensis [4]. This was because of the high morphological similarity between the two species. Indeed, the only distinguishing feature was the unique number of clasper movements of An. engarensis males during arti cial mating, a common method for laboratory maintenance of anopheline mosquitoes [5].
In general, ITS2 is known to have high interspeci c and low intraspeci c variability; however, extensive intraspeci c variations have been reported in anopheline mosquitoes. For instance, ITS2 intraspeci c variations ranged from 0.2-19.0% for the Latin American anophelines [43]. In the An. hyrcanus group, the average intraspeci c distance was 0.3%, but no intraspeci c variations were observed in An. belenrae [42]. These results suggest that the ITS2 spacer is a good marker for differentiating between members of the An. hyrcanus group. In this study, there were no intraspeci c variations in the An. belenrae, An. engarensis, and An. sineroides strains from Hokkaido. However, there was signi cant intraspeci c variation between the nine An. engarensis strains from Hokkaido and the Daisen55 strain from Akita Prefecture. The genetic distance of 4.7% was considerably greater than the 0.22% intraspeci c variation in the An. sinensis strains from Vietnam, South Korea, and Japan. We inferred that the Daisen55 An. engarensis strain was not introduced from Hokkaido but inhabited the Tohoku region independently. On the other hand, in species groups consisting of recently diverged members, such as the An. gambiae complex, the interspeci c differences in ITS2 were reported to be minor, ranging from 0.4-1.6% [20]. It is possible that An. engarensis is a recently diverged lineage.

Anopheles lesteri in Hokkaido
In Rubeshibe Hokkaido, at least seven cases of falciparum malaria were recorded between 1946 and 1947. An unsuccessful survey was conducted to determine the vector mosquitoes involved in the transmission although An. sinensis and An. sineroides were collected [1]. During the falciparum malaria epidemic in the vicinity of Guangdong City, China, around 1942, the transmission was inferred to have involved An. lesteri and not An. sinensis. This inference was based on results from eld investigations and subsequent infection experiments with Plasmodium falciparum [44]. Based on this inference, it was suggested but never con rmed that An. lesteri may have been involved in the outbreak of falciparum malaria in Rubeshibe, Hokkaido. In terms of distribution, An. lesteri which was initially thought to be restricted to western Islands of Japan, such as the Kyushu Island [44], was also found in various areas of Honshu mainland of Japan [12], Hokkaido [45], Okinawa Island, and Yaeyama Islands [46]. The present survey con rmed that An. lesteri is still widely distributed in Hokkaido (Figs. 3 and 4). At the start of our survey in 2001, we noticed female mosquitoes collected in Ozora, Hokkaido, to have an intense a nity for human blood. These female mosquitoes were therefore considered, and subsequently con rmed, to be An. lesteri based on the reported high anthropophilic nature of An. lesteri relative to An. sinensis and other members of the An. hyrcanus group [12,47]. We therefore expected to easily collect An. lesteri in subsequent surveys in Hokkaido.
In our study, the ITS2 intraspeci c distance in An. lesteri ranged from 0-9.44%. These values suggest that An. lesteri is a highly divergent species when An. lesteri type B (specimen 1) and type C (specimen 2) from South Korea are included in this species. Since the ITS2 distance of this species varies even within Hokkaido, there is a possibility that An. lesteri includes crypto-species. In de ning this species, it is necessary to analyze both the COI barcoding region and the ITS2 region. Moreover, a large number of specimens, collected outside Hokkaido, will be necessary. In a previous study, a short interspeci c distance of 7.2% was observed between An. kleini and An. engarensis [42]. We obtained similar ITS2 distances of 6.47% and 8.67% between An. kleini and our An. engarensis specimens from Hokkaido and An. kleini and An. engarensis Daisen55 strain, respectively. Although these results may provide validation that An. kleini is a synonym of An. engarensis, further analysis is required. We also presented evidence that An. anthropophagus and An. lesteri were conspeci c, based on the ITS2 divergence between them. Our results based on interspeci c comparisons of ITS2 divergence may also support previous reports that An. belenrae and An. sinensis are genetically distinct [24,25], and An. anthropophagus is a conspeci c species of An. lesteri [34,48].

Conclusions
ITS2 sequence divergence clearly disclosed the current distribution of the An. hyrcanus group mosquitoes in Hokkaido, demonstrating great differences from surveys conducted between 1969 and 1984. In particular, the area inhabited by An. sinensis has greatly reduced, and the newly discovered An. belenrae was con rmed to inhabit only eastern Hokkaido. In summary, this study showed that Hokkaido harbored four members of the An. hyrcanus group, namely An. engarensis, An. belenrae, An. sineroides, and An. lesteri.
Here, we have demonstrated the presence of two anopheline species that have been reported as malaria vectors, An. belenrae instead of An. sinensis, in addition to An. lesteri, in Hokkaido since 2001. Although the malaria vector capacity of the Japanese strain of An. belenrae has not yet been evaluated, the Korean strain is considered to be a vector or potential vector of P. vivax [24,49]. Fortunately, all recently reported cases of malaria in Japan have been imported. However, emergence of potential autochthonous malaria epidemics should always be of concern because multiple malaria vector species still remain in Japan, as con rmed in this study.

Availability of data and materials
All data generated or analyzed during this study are included in this published article. The newly generated sequences were submitted in the GenBank database under the accession numbers LC634739-LC634772.

Competing interests
The authors declare that they have no competing interests.  Tables   Due to technical limitations, table 1 to 3 is only available as a download in the Supplemental Files section. Figure 1 Phylogenetic relationships among members of the Anopheles hyrcanus group. Neighbor-joining (NJ) phylogenetic tree was constructed based on partial ITS2 region nucleotide sequences. A distance matrix was calculated using the Kimura's two-parameter evolutionary model and the tree was constructed using the NJ approach in MEGA X ver. 10.2.2. The scale bar indicates the proportion of sites changing along each branch. The numbers on the internodes indicate percentages of 1,000 bootstrap replicates. Bootstrap values <70 are not shown in this gure. This phylogenetic tree is a rooted and traditional rectangular tree with An. pullus (AY186792) as an outgroup sequence. All specimens collected in the present study are marked with circles. The closed circles mark mosquito specimens collected from Hokkaido, Japan, and open circles mark specimens from the areas outside Hokkaido. Abbreviations of strains and sequence accession numbers of specimens used in this study are listed in Table 1.

Figure 2
Phylogenetic relationships among members of the Anopheles hyrcanus group. Neighbor-joining (NJ) phylogenetic tree were constructed based on partial ITS2 region nucleotide sequences. A distance matrix was calculated using the Kimura's two-parameter evolutionary model and the tree was constructed using the NJ approach in MEGA X ver. 10.2.2. This phylogenetic tree is a rootless and radiation tree with An. pullus (AY186792) as an outgroup sequence to know more closely related species. The scale bar indicates the proportion of sites changing along each branch. Bootstrap values are not indicated in this gure. The species names in this group are bold. The members in the same species are shaded. Abbreviations of strains and sequence accession numbers of specimens used in this study are listed in Table 1.

Figure 3
Distribution map of the Anopheles hyrcanus group mosquitoes in Hokkaido, Japan, as con rmed by surveys conducted during 1969-1984. This map was created from references [3][4][5][6]. This map shows mosquito species collected from 29 sites. Black, blue, red, and orange circles indicate collection sites of An. sinensis, An. engarensis, An. sineroides, and An. lesteri, respectively.

Figure 4
Distribution map of the Anopheles hyrcanus group mosquitoes in Hokkaido, Japan, as disclosed by the present study conducted during 2001-2015. This map shows mosquito species collected from 15 collection sites. Purple, blue, red, and orange circles show collection sites of An. belenrae, An. engarensis, An. sineroides, and An. lesteri, respectively.

Supplementary Files
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