Molecular characterization and identification of members of the Anopheles subpictus complex in Sri Lanka

Malaria Journal, Aug 2013

Background Anopheles subpictus sensu lato is a major malaria vector in South and Southeast Asia. Based initially on polytene chromosome inversion polymorphism, and subsequently on morphological characterization, four sibling species A-D were reported from India. The present study uses molecular methods to further characterize and identify sibling species in Sri Lanka. Methods Mosquitoes from Sri Lanka were morphologically identified to species and sequenced for the ribosomal internal transcribed spacer-2 (ITS2) and the mitochondrial cytochrome c oxidase subunit-I (COI) genes. These sequences, together with others from GenBank, were used to construct phylogenetic trees and parsimony haplotype networks and to test for genetic population structure. Results Both ITS2 and COI sequences revealed two divergent clades indicating that the Subpictus complex in Sri Lanka is composed of two genetically distinct species that correspond to species A and species B from India. Phylogenetic analysis showed that species A and species B do not form a monophyletic clade but instead share genetic similarity with Anopheles vagus and Anopheles sundaicus s.l., respectively. An allele specific identification method based on ITS2 variation was developed for the reliable identification of species A and B in Sri Lanka. Conclusion Further multidisciplinary studies are needed to establish the species status of all chromosomal forms in the Subpictus complex. This study emphasizes the difficulties in using morphological characters for species identification in An. subpictus s.l. in Sri Lanka and demonstrates the utility of an allele specific identification method that can be used to characterize the differential bio-ecological traits of species A and B in Sri Lanka.

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Molecular characterization and identification of members of the Anopheles subpictus complex in Sri Lanka

Malaria Journal Molecular characterization and identification of members of the Anopheles subpictus complex in Sri Lanka Sinnathamby N Surendran 0 1 Devojit K Sarma 0 2 Pavilupillai J Jude 1 Petri Kemppainen 0 Nadarajah Kanthakumaran 1 Kanapathy Gajapathy 1 Lalanthika BS Peiris 3 Ranjan Ramasamy 1 Catherine Walton 0 0 Faculty of Life Sciences, University of Manchester , Oxford Road, Manchester M13 9PT , UK 1 Department of Zoology, Faculty of Science, University of Jaffna , Jaffna 40000 , Sri Lanka 2 Regional Medical Research Centre , NE region (ICMR), Dibrugarh 786001, Assam , India 3 Regional Office, Anti Malaria Campaign , Hambantota 82000 , Sri Lanka Background: Anopheles subpictus sensu lato is a major malaria vector in South and Southeast Asia. Based initially on polytene chromosome inversion polymorphism, and subsequently on morphological characterization, four sibling species A-D were reported from India. The present study uses molecular methods to further characterize and identify sibling species in Sri Lanka. Methods: Mosquitoes from Sri Lanka were morphologically identified to species and sequenced for the ribosomal internal transcribed spacer-2 (ITS2) and the mitochondrial cytochrome c oxidase subunit-I (COI) genes. These sequences, together with others from GenBank, were used to construct phylogenetic trees and parsimony haplotype networks and to test for genetic population structure. Results: Both ITS2 and COI sequences revealed two divergent clades indicating that the Subpictus complex in Sri Lanka is composed of two genetically distinct species that correspond to species A and species B from India. Phylogenetic analysis showed that species A and species B do not form a monophyletic clade but instead share genetic similarity with Anopheles vagus and Anopheles sundaicus s.l., respectively. An allele specific identification method based on ITS2 variation was developed for the reliable identification of species A and B in Sri Lanka. Conclusion: Further multidisciplinary studies are needed to establish the species status of all chromosomal forms in the Subpictus complex. This study emphasizes the difficulties in using morphological characters for species identification in An. subpictus s.l. in Sri Lanka and demonstrates the utility of an allele specific identification method that can be used to characterize the differential bio-ecological traits of species A and B in Sri Lanka. Anopheles subpictus; Anopheles sundaicus; Cytochrome c oxidase subunit-I; ITS2; Malaria; Sibling species; Sri Lanka - Background Anopheles subpictus sensu lato has a very wide distribution in the Oriental and Australasian regions ranging through Pakistan, India, Sri Lanka, Bangladesh, Myanmar, Thailand, Cambodia, Vietnam, Malaysia, Indonesia, Timor-Leste and Papua New Guinea [1,2]. The taxon exists as a species complex and is a primary vector of malaria in many Southeast (SE) Asian countries and is regarded as a secondary vector in Sri Lanka [2-5]. In India, An. subpictus s.l. is reported to exist as a species complex comprising four sibling species viz. A, B, C and D [6,7]. Each species is associated with a specific combination of two polytene chromosome inversions viz. A = X + a + b; B = Xab; C = Xa + b; D = X + ab, and stage specific morphometric characteristics such as number of egg ridges, number of branches in the 4 M setae of larvae, and ornamentation of adult palpi [6]. Based on the single inversion (X + a/Xa) on the X chromosome, the presence of species A and B was reported in Sri Lanka [8]. Although no other polytene chromosomal studies looking at the two X chromosome inversions have yet been conducted in Sri Lanka for this taxon, the presence of morphometric characteristics corresponding to the Indian sibling species, led to the reporting of all four (A-D) sibling species from Sri Lanka [5,9,10]. Like any species complex, members of the Subpictus complex show differential bio-ecological traits such as salinity tolerance, susceptibility to common insecticides and vectorial capacity [5,7,11,12]. The development of appropriate vector control strategies when dealing with species complexes, therefore, requires reliable molecular diagnostic tools and an understanding of inter and intraspecific genetic diversity of vector populations [13]. In India, species B is predominant and is a vector in coastal areas of Southern India whereas species A, C and D are predominant in inland areas [1,7] with species A being a vector in West Bengal of India [14]. The members of the Subpictus complex in Sri Lanka also differ in bio-ecological properties [9,11,12] but the full characterization of these requires the development of reliable species diagnostic tools. The simplest and least expensive way to identify malaria vectors in the field is morphologically. However, there are limitations with using morphological characteristics alone to differentiate sympatric sibling species of anopheline species complexes and closely related (...truncated)


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Sinnathamby N Surendran, Devojit K Sarma, Pavilupillai J Jude, Petri Kemppainen, Nadarajah Kanthakumaran, Kanapathy Gajapathy, Lalanthika BS Peiris, Ranjan Ramasamy, Catherine Walton. Molecular characterization and identification of members of the Anopheles subpictus complex in Sri Lanka, Malaria Journal, 2013, pp. 304, 12, DOI: 10.1186/1475-2875-12-304