Background

Malaria has a major impact on global public health with more than 200 million people infected and about 4,00,000 deaths annually [1]. Most malaria is reported in Africa (93%), with the remainder reported in Southeast Asia, the Mediterranean, and South America (7%) [2]. Climate change and the expansion of cross-border trading may have contributed to recent increases in malaria risks worldwide [3, 4].

Members of the genus Anopheles are vectors of Plasmodium spp., the causative agent of malaria. Plasmodium spp. that are considered human pathogens include: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi, the latter previously considered a monkey malaria [5]. In the Republic of Korea (ROK), P. vivax, P. falciparum and P. malariae were eradicated in 1979 by the National Malaria Eradication Service (NMES) of the Korean Government [6], and the World Health Organization (WHO) declared the country malaria free [7]. However, malaria reappeared in 1993 near the demilitarized zone (DMZ) in northern Gyeonggi Province [8]. Except for imported malaria cases, only P. vivax is present in ROK and, following its peak of > 4000 cases in 2010, continues to be responsible for 300–500 cases annually [9,10,11].

In ROK there are eight Anopheles species (Anopheles sinensis, Anopheles lesteri, Anopheles pullus, Anopheles kleini, Anopheles sineroides, and Anopheles belenrae belonging to the Hyrcanus group; Anopheles koreicus belonging to the Barbirostris group; and, Anopheles lindesayi belonging to the Lindesayi group) [12,13,14,15]. Recently, two species, An. lesteri and An. kleini, were proposed to be the primary vectors of malaria in ROK, while An. sinensis is considered a poor vector. Anopheles lesteri showed a large number of P. vivax sporozoites (up to 2105) in the salivary glands when compared to An. sinensis (0–14) in a single microscope field (750 × 560 μM). Also, An. kleini had higher oocyst rates of P. vivax (8.8%) in the midgut than An. sinensis (4.2%) [15,16,17,18]. In another study, while An. kleini and An. sinensis demonstrated similar numbers of oocysts, An. kleini demonstrated + 1 (1–10 sporozoites) to + 4 (> 1000 sporozoites) salivary gland infections, while An. sinensis only had + 1 salivary glands [19]. Recent evidence indicates that An. pullus and An. belenrae are poor to moderate vectors of malaria in ROK (Ubalee, R., pers. comm.). While An. sineroides has been implicated as a malaria vector, its status is unknown. Although there are no records of malaria infections in An. koreicus, several members of the Barbirostris group are primary vectors of malaria in Southeast Asia [20, 21]. While An. lindesayi has not been found positive for malaria in ROK, it has been implicated as a vector of P. malariae in Southeast Asia [22]. Accurate identification of Anopheles species to determine their distribution and malaria infection rates in order to develop vector control measures is needed in ROK.

Accurate species identification and subsequent monitoring of Anopheles spp. is necessary to identify their geographic distributions, larval habitats and population dynamics to manage or conduct epidemiological investigations that identify the most likely sites where infections occurred. Although scales on wings (wing patterns) and spots on legs are used as the primary key characters for species identification, it is extremely difficult if the characters are lost during collections [12, 23]. In addition, An. sinensis, An. lesteri, An. kleini, An. belenrae, and An. pullus are morphologically very similar and species cannot be identified using current morphological characters [13, 24,25,26]. Although a multiplex PCR assay to identify six species of the Hyrcanus group was developed [27], molecular diagnostics for all eight Anopheles species in ROK had not yet been developed. In this study, a new multiplex PCR assay was developed to identify all Anopheles species simultaneously that are present in ROK.

Methods

Sample collection

Eight species of Anopheles mosquitoes used in the study were collected at six sites in/near the DMZ where most malaria infections are contracted: 1) Neutral Nations Supervisory Commission (NNSC) camp adjacent to the Panmunjeom (37°57′17.19″N; 126°40′47.91″E); 2) Daeseongdong (village of approximately 200 residents inside the DMZ (37°56′28.31″N; 126°40′37.38″E)); 3) South Gate (South gate entrance to the DMZ) (37°56′03.53″N; 126°43′15.46″E)); 4) Camp Bonifas (US Army installation (37°55′55.25″N; 126°43′21.73″E)); 5) Warrior Base (US Army training sites approximately 3 km from the south gate of DMZ), (37°55′03.96″N; 126°44′29.74″E)); and, 6) Dagmar North training area (37°58′29.85″N; 126°50′40.88"E). Mosquitoes were collected using Mosquito Magnets® (Woodstream Corp., Lancaster, PA, USA) (Fig. 1). The distance between the two farthest collection points: (2: Daeseongdong and 6: Dagmar North training area) was about 15.2 km. Other points were approximately 3.9 km distant from 3: South gate. Collected mosquitoes were identified morphologically to Anopheles spp. [23, 28] and then stored at − 70 °C until used.

Fig. 1
figure 1

Map of the northern part of Gyeonggi province denoting Anopheles collection sites at (1) NNSC (Neutral Nations Supervisory Commission camp adjacent to the Panmunjeom); (2) Daeseongdong (village of approximately 200 residents inside the DMZ), (3) South Gate (South gate entrance to the DMZ) (4) Camp Bonifas (US Army installation), (5) Warrior Base (US Army training area approximately 3 km from the south gate of DMZ), and (6) Dagmar Noth (US Army training area) denoting the six Anopheles collection sites in/near the demilitarized zone (DMZ)

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Identification and primer design

Genomic DNA used in this study was extracted using the Chelex protocol [29]. Identification of six species (An. sinensis, An. pullus, An. belenrae, An. lesteri, An. kleini, An. sineroides) was performed using a multiplex PCR assay diagnostic method [27]. The universal primers for the mitochondrial gene cytochrome c oxidase subunit 1 (COI) region (LCO1490: 5′-GGT CAA ATC ATA AAG ATA TTG G-3′/HCO2198: 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) were used as species identifiers for An. koreicus and An. lindesayi [30]. Each sample was sequenced by Macrogen (Macrogen Daejeon, Korea) and analysed using BLAST.

Two pairs of universal primers (An-ITS2-U1; forward primer: 5′-ATC GAT GAA GAC CGC AGC TA-3′/reverse primer: 5′-CAA CAC GAC TCC ATG GTA CG-3′; An-ITS2-U2; forward primer: 5′-AAC GGG AGA AGC TCA GCA C-3′/reverse primer: 5′-GAC TTC TTG GTC CGT GTT TCA-3′) were designed between the 5.8 S and 28 S regions of the ribosomal DNA (rDNA) to analyse the entire internal transcribed spacer 2 (ITS2) sequences for the eight Anopheles species.

PCR amplification of the whole ITS2 region was conducted as follows. Each individual reaction mixture (25 μl) included: 0.2 mM each dNTP, 0.4 μM each primer, 1X PCR buffer, 1.5 mM MgCl2, and 0.5 units Taq DNA polymerase (R001AM; TaKaRa, Shiga, Japan) with 1.0 μl genomic DNA extracted from an individual specimen. The PCR cycling conditions were as follows: denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, and extension at 72 °C for 60 s; and final extension at 72 °C for 5 min. Each product was visualized on 1.5% (wt/vol) agarose gels stained with ethidium bromide (VWR Life Science, Radnor, PA, USA), and then sequenced in both directions by Macrogen. Sequence data were analysed using Bioedit v7.2.6.1 [31] and deposited in the National Center for Biotechnology Information (NCBI) under the following accession numbers:

An. sinensis—MW546412, MW546421; An. pullus—MW546424, MW546423; An. lesteri—MW546426; An. sineroides—MW546417, MW546414; An. kleini—MW546419, MW546415; An. belenrae—MW546422, MW546418; An. koreicus—MW546413, MW546416; An. lindesayi—MW546425, MW546420.

Multiplex PCR assay for eight Anopheles species

Universal forward and species-specific reverse primers were designed for the eight species of Anopheles present in ROK. Reverse primers for the three species (An. sinensis, An. koreicus, An. lindesayi) were designed using the 28 S rDNA region, while primers for the remaining species were designed using the ITS2 region (Table 1). The multiplex PCR assay was conducted in a 25-μl reaction mixture containing 0.4 μM each primer, 1X PCR buffer, 0.2 mM each dNTP, 0.5 units Taq Hotstart DNA polymerase (R007A, TaKaRa,), and 1.0 μl genomic DNA from an individual specimen. PCR amplification was performed under conditions of denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min; and final extension at 72 °C for 5 min. The PCR products were visualized on ethidium-bromide–stained 2.0% (wt/vol) agarose gels (VWR Life Science). The whole aligned sequences showing positions for the universal primers and the specific reverse primers are described in Fig. 2.

Table 1 PCR primers for the eight Anopheles species
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Fig. 2
figure 2

The whole aligned sequences showing positions for two pairs of the universal primers (An-ITS2-U1 and An-ITS2-U2) and the specific reverse primers between 5.8 S and 28 S ribosomal DNA

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Results

Molecular species diagnosis

A total of 165 DNA samples extracted from individual Anopheles species were used: An. sinensis (20), An. koreicus (21), An. lindesayi (17), An. kleini (25), An. lesteri (11), An. sineroides (22), An. belenrae (23), and An. pullus (26). A gel showing the products of multiplex PCR assay separated by agarose gel electrophoresis for the eight species is shown in Fig. 3 (1112 bp for An. sinensis; 925 bp for An. koreicus; 650 bp for An. lindesayi; 527 bp for An. kleini; 436 bp for An. lesteri,, 315 bp for An. sineroides; 260 bp for An. belenrae; 157 bp for An. pullus). This method allowed identification of all eight Anopheles spp., including An. koreicus and An. lindesayi, and is comparable to the current molecular diagnosis method applied to identify six Anopheles species belonging to members of the Anopheles Hyrcanus group present in ROK [27]. All samples used in this study were identified using the multiplex assay. The results of species identification for An. koreicus and An. lindesayi, which were not included in the previous method [27] using this molecular assay, were also consistent with morphological identification results.

Fig. 3
figure 3

Representative results of agarose gel electrophoretic separation of multiplex PCR products. M: 100 bp molecular marker; lane 1, An. pullus; lane 2, An. belenrae; lane 3, An. sineroides; lane 4, An. lesteri; lane 5, An. kleini; lane 6, An. lindesayi; lane 7, An. koreicus; lane 8, An. sinensis; lane 9, negative control

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Accurate species identification for the vector control

In Africa and Southeast Asia where malaria is widespread, multiplex PCR assays have been developed and used to identify species accurately and to investigate malaria vector distributions and infection rates [32,33,34,35,36,37,38,39,40]. In addition, the ITS2-based multiplex PCR assay was used to detect two unknown species (after named as An. belenrae and An. kleini by Rueda [13]) in ROK [26]. Accurate species identification, using both morphological and molecular methods is important to confirm species identification and monitoring vector populations [41]. Several studies have described accurate species identification as a part of vector surveillance programmes. In India, Anopheles minimus, a primary malaria vector, was morphologically misidentified as Anopheles fluviatilis, while each species was identified correctly using PCR of the ITS2 regions [42]. In South Africa, Anopheles vaneedeni also was reported as a new malaria vector during a malaria surveillance programme using the ITS2 region for specific identification [43]. Molecular diagnostic methods have been used to monitor invasive species, e.g., Aedes albopictus and Aedes aegypti, to verify morphological identification of specimens, as well as screening for potential new invasive species in Europe [44]. These studies support the importance of accurate species identification for monitoring vector populations and distributions, as well as supporting pathogen surveillance programmes.

Application of new diagnostic method

The eight Anopheles species present in ROK included in three groups (Hyrcanus Barbirostris, Lindesayi) can be identified based on a new multiplex molecular-based method. Morphological identification of these species is challenging, particularly in cases when legs or wing scales used as the primary identification characters are missing or damaged during collections. The method described here enables simple and accurate identification requiring only PCR of individual specimens followed by electrophoresis. It would also be useful to acquire geographic, habitat and population distributions of An. koreicus and An. lindesayi that are less frequently collected than the other species. Since the re-emergence of vivax malaria in ROK in 1993, most malaria cases have been attributed to exposure near the DMZ. Although the reason for the concentrated outbreak of malaria in/near the DMZ is uncertain, one of the primary vectors, An. kleini, is more prevalent near the DMZ than south of Seoul [45]. Additionally, there are reports of higher numbers of malaria cases in the Democratic People’s Republic of Korea (DPRK, North Korea) that provide a source of malaria-infected blood meals for mosquitoes that subsequently migrate south across the DMZ [46,47,48,49]. Identification of species distributions and malaria infection rates would assist in understanding the malaria distribution pattern in ROK, in addition to developing vector and malaria mitigation strategies. Recently, two species (An. lesteri and An. kleini) showed higher infection rates in artificial experiments than An. sinensis that was previously considered to be the primary vivax malaria vector in ROK [18, 19]. In China, An. sinensis and An. lesteri were considered the primary malaria vectors [50]. However, An. lesteri demonstrated more anthropophilic behaviour and 20 times higher sporozoite rates (0.58%) than An. sinensis (0.02%) [51, 52]. In addition, the annual distribution of P. vivax cases varies with environment factors that impact on mosquito population densities, which may be further impacted by climate change [53, 54]. Thus, continuous monitoring of malaria vectors is needed. The new multiplex ITS2-28S rDNA-based method eliminates the requirement for multiple PCR analyses and is useful for monitoring Anopheles spp. distributions and population densities in ROK.

Conclusion

In this study, a new molecular diagnostic method was developed for the identification of eight Anopheles spp. present in ROK. This multiplex PCR assay is a simple and accurate method to identify Anopheles spp. and can be used as a surveillance tool for monitoring malaria vector population distributions in ROK.