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A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

ABSTRACT: A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

SUBMITTER: Wilai P

PROVIDER: S-EPMC7412068 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

Wilai Parinya P Namgay Rinzin R Made Ali Rusdiyah Sudirman RS Saingamsook Jassada J Saeung Atiporn A Junkum Anuluck A Walton Catherine C Harbach Ralph E RE Somboon Pradya P

Insects 20200702 7

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucl ...[more]

PMID: 32630637

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Project description:BackgroundThe Anopheles hyrcanus group, which includes at least 25 species, is widely distributed in the Oriental and Palearctic regions. Some group members have been incriminated as vectors of malaria and other mosquito-borne diseases. It is difficult to identify Hyrcanus Group members by morphological features. Thus, molecular phylogeny has been proposed as an important complementary method to traditional morphological taxonomy.MethodsBased on the GenBank database and our original study data, we used 466 mitochondrial DNA COI sequences belonging to 18 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its worldwide geographic range.ResultsThe results are as follows. 1) The average conspecific K2P divergence was 0.008 (range 0.002-0.017), whereas sequence divergence between congroup species averaged 0.064 (range 0.026-0.108). 2) The topology of COI tree of the Hyrcanus Group was generally consistent with classical morphological taxonomy in terms of species classification, but disagreed in subgroup division. In the COI tree, the group was divided into at least three main clusters. The first cluster contained An. nimpe; the second was composed of the Nigerrimus Subgroup and An. argyropus; and the third cluster was comprised of the Lesteri Subgroup and other unassociated species. 3) Phylogenetic analysis of COI indicated that ancient hybridizations probably occurred among the three closely related species, An. sinensis, An. belenrae, and An. kleini. 4) The results supported An. paraliae as a probable synonym of An. lesteri, and it was possible that An. pseudopictus and An. hyrcanus were the same species, as evident from their extremely low interspecific genetic divergence (0.020 and 0.007, respectively) and their phylogenetic positions.ConclusionsIn summary, we reconstructed the molecular phylogeny and analysed genetic divergence of the Hyrcanus Group using mitochondrial COI sequences. Our results suggest that in the future of malaria surveillance, we should not only pay much attention to those known vectors of malaria, but also their closely related species.

Dataset Information

A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

Publications

A Multiplex PCR Based on Mitochondrial <i>COI</i> Sequences for Identification of Members of the <i>Anopheles barbirostris</i> Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

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