Karyotypic assignment of Sri Lankan Anopheles culicifacies species B and E does not correlate with cytochrome oxidase subunit I and microsatellite genotypes.

BACKGROUND: The identification of species B and E in the Anopheles culicifacies complex in the Indian subcontinent has been based on Y-chromosome karyotype. Since no detectable variations were previously found in DNA markers commonly used for sibling species identification, further molecular characterization using cytochrome oxidase subunit I (COI) and microsatellite markers was carried out on Y-chromosome karyotyped Anopheles culicifacies specie B and E from Unnichchai, Kallady and Ranawarunawa in Sri Lanka. FINDINGS: COI sequence analysis (n = 22) revealed the presence of nine unique haplotypes with six in each species. Three haplotypes were shared by both species. The two sibling species had a pairwise FST value of 1.338 (p < 0.05) with the number of migrants (Nm) value <1. The genetic structure analysis resulted in two genetic clusters not 100% associated with karyotypes. While none of the species B were incorrectly assigned two were inconclusive. Five out of 26 specimens karyotyped as species E were incorrectly assigned, while further 9 were inconclusive. CONCLUSIONS: The new molecular data support the existence of two genetically different populations of the Culicifacies Complex in Sri Lanka that are not associated with the Y-chromosome karyotype. Detailed analysis with more microsatellite markers and assortative mating experiments are needed to establish the presence of the two genetically distinct populations and relate them to Y-chromosome morphology.

Molecular characterization of the malaria vector Anopheles barbirostris van der Wulp in Sri Lanka.

BACKGROUND: Anopheles barbirostris is a vector of malaria in Sri Lanka. The taxon exists as a species complex in the Southeast Asian region. Previous studies using molecular markers suggest that there are more than 4 distinct clades within the An. barbirostris complex in Southeast Asia. The present study characterizes Sri Lankan An. barbirostris using mtDNA cytochrome oxidase subunit I (COI) and ribosomal RNA internal transcribed spacer 2 (ITS2) gene sequences. FINDINGS: DNA was extracted from morphologically identified An. barbirostris specimens from Sri Lanka, the COI and ITS2 regions amplified and their sequences analysed by comparison with other GenBank entries. Maximum likelihood trees suggested that Sri Lankan An. barbirostris constitute a different molecular type most closely related to clade I. CONCLUSIONS: Considering the uncorrected p distances between the clade I and Sri Lankan specimens it is fair to assume that the specimens collected from widely separated locations in Sri Lanka with morphology characteristic of An. barbirostris s.l. form a new molecular type with close resemblance to An. barbirostris s.s from Indonesia and Thailand.

Molecular characterization and identification of members of the Anopheles subpictus complex in 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.

Molecular identification of potential leishmaniasis vector species within the Phlebotomus (Euphlebotomus) argentipes species complex in Sri Lanka.

BACKGROUND: Leishmaniasis is an emerging vector-borne disease in Sri Lanka. Phlebotomus (Euphlebotomus) argentipes sensu lato Annandale and Brunette 1908 is suspected to be a potential vector. Three sibling species have been reported in the species complex based on analysis of morphological data. A study was carried out in different parts of Sri Lanka including cutaneous leishmaniasis prevailing localities to characterise the sibling species of Phlebotomus (Euphlebotomus) argentipes sensu lato and to establish their possible role in Leishmania transmission. METHODS: Sandflies were collected using cattle baited trap nets and mouth aspirator. They were identified based on existing taxonomic keys. Sequences of amplified cytochrome oxidase subunit I (CO I), cytochrome oxidase b (cyt b), internal transcribed spacer 2 (ITS2), 18s and 28s rDNA regions were analysed to confirm the number of sibling species. Vectorial capacity of the sibling species was checked by detecting human and Leishmania DNA. RESULTS: Sandflies collected using different techniques were processed for identification, parasite detection and molecular characterization. The 18s, 28s rDNA and cytochrome oxidase subunit I (CO I), internal transcribed spacer 2 (ITS2) and cytochrome b oxidase (cytb) sequences confirmed that the species belonged to the Argentipes complex. 18s and 28s sequences did not show any variation among the proposed sibling species. The phylogeny created from mitochondrial CO I and cytochrome b data and from the nuclear ITS2 region supports the existence of only two groups of flies (termed A and B) from Phlebotomus (Euphlebotomus) argentipes complex instead of the previously proposed three. The Leishmania mini-circle kinetoplastid, heat shock protein 70 (hsp70) and internal transcribed spacer I DNA along with human blood were detected from sibling species A only, which has not previously been considered to be a vector. CONCLUSIONS: The taxonomy of the Sri Lankan Argentipes species complex is reassessed based on the molecular data. The existence of two sibling species is proposed; sibling species A has a long sensilla chaetica (> 50% length of the second antennal flagellomere) and sibling species B has a short sensilla cheatica (< 50%). Sibling species A is incriminated as a vector for leishmaniasis in Sri Lanka.