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

Background Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria re-emerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species (Anopheles sinensis, Anopheles pullus, Anopheles belenrae, Anopheles lesteri, Anopheles kleini, Anopheles sineroides, Anopheles koreicus, Anopheles lindesayi) are distributed throughout Korea. Members of the Anopheles Hyrcanus group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK. Methods Anopheles spp. used in this study were collected near/in the demilitarized zone in ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analysed for molecular identification. Results DNA from a total of 165 Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Each species was clearly distinguished by electrophoresis as follows: 1,112 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; and, 157 bp for An. pullus. Conclusion A multiplex PCR assay was developed to identify Anopheles spp. distributed in ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

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.

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.

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  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 Anopheles pullus CGG CGT AGT TTA TTG TGT ATA ACA TC 157 Fig. 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 method [27] using this molecular assay, were also consistent with morphological identification results.

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

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.