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
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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)