Multiplex PCR assay for the identification of eight Anopheles species belonging to the Hyrcanus, Barbirostris and Lindesayi groups

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.

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.

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.

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.

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.

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 [32‐40]. 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.

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 [46‐49]. 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.