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.

A new species of the Chaoborus flavicans complex (Diptera, Chaoboridae) in South Korea.

Using reverse taxonomy and morphological analyses, this study describes a new species belonging to the C. flavicans species complex in the Korean Peninsula, Chaoborus pseudoflavicans Bang & Shin sp. nov. Descriptions of the new species from larvae to adults are provided, and the key to the C. flavicans species complex is updated accordingly. DNA barcodes (COI partial sequences) are shown to be sufficient for molecular identification in the C. flavicans species complex. Finally, the taxonomic accounts of all species in the C. flavicans complex are completely resolved for the first time.

A multiplex PCR assay for six Aedini species, including Aedes albopictus.

BACKGROUND: Mosquitoes, as vectors of various human pathogens, are significant drivers of serious human illness. In particular, those species in the Aedini tribe, which typically transmit dengue virus, Chikungunya fever virus, and Zika virus, are increasing their range because of climate change and international commerce. In order to evaluate the risk of disease transmission, accurate mosquito species identification and monitoring are needed. The goal of this work was to develop a rapid and simple molecular diagnostic method for six morphologically similar Aedini species (Aedes flavopictus, Aedes albopictus, Ochlerotatus koreicus, Ochlerotatus japonicus, Ochlerotatus togoi and Ochlerotatus hatorii) in Korea. METHODS: A total of 109 samples were assayed in this study. The internal transcribed spacer 2 (ITS2) regions from all six species were amplified, sequenced and analyzed using Mega 6. Following the identification of regions that were consistently different in terms of sequence between all six species, multiplex primers were designed to amplify these regions to generate species-specific fragments distinguishable by their size. RESULTS: Uniquely sized fragments were generated in Ae. flavopictus (495 bp), Ae. albopictus (438 bp), Oc. koreicus (361 bp), Oc. togoi (283 bp), Oc. hatorii (220 bp) and Oc. japonicus (160 bp). Pairwise distance analysis showed that the difference was 35.0 ± 1.5% between Aedes spp. and Ochlerotatus spp., 17.4 ± 0.2% between Ae. albopictus and Ae. flavopictus and 11.1 ± 0.3% between Oc. koreicus and Oc. japonicus. CONCLUSIONS: In this study, a multiplex PCR assay for six species of the Aedini tribe was developed. This assay is more accurate than morphological identification and will be useful for monitoring and controlling these vector mosquitoes.