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.

Anthropogenic Factors Driving Recent Range Expansion of the Malaria Vector Anopheles stephensi.

The malaria vector Anopheles stephensi is found in wide tracts of Asia and the Middle East. The discovery of its presence for the first time in the island of Sri Lanka in 2017, poses a threat of malaria resurgence in a country which had eliminated the disease in 2013. Morphological and genetic characterization showed that the efficient Indian urban vector form An. stephensi sensu stricto or type form, has recently expanded its range to Jaffna and Mannar in northern Sri Lanka that are in proximity to Tamil Nadu state in South India. Comparison of the DNA sequences of the cytochrome oxidase subunit 1 gene in An. stephensi in Jaffna and Mannar in Sri Lanka and Tamil Nadu and Puducherry states in South India showed that a haplotype that is due to a sequence change from valine to methionine in the cytochrome oxidase subunit 1 present in the Jaffna and Mannar populations has not been documented so far in Tamil Nadu/Puducherry populations. The Jaffna An. stephensi were closer to Tamil Nadu/Puducherry populations and differed significantly from the Mannar populations. The genetic findings cannot differentiate between separate arrivals of the Jaffna and Mannar An. stephensi from Tamil Nadu or a single arrival and dispersion to the two locations accompanied by micro-evolutionary changes. Anopheles stephensi was observed to undergo preimaginal development in fresh and brackish water domestic wells and over ground cement water storage tanks in the coastal urban environment of Jaffna and Mannar. Anopheles stephensi in Jaffna was resistant to the common insecticides deltamethrin, dichlorodiphenyltrichloroethane and Malathion. Its preimaginal development in wells and water tanks was susceptible to predation by the larvivorous guppy fish Poecilia reticulata. The arrival, establishment, and spread of An. stephensi in northern Sri Lanka are analyzed in relation to anthropogenic factors that favor its range expansion. The implications of the findings for global public health challenges posed by malaria and other mosquito-borne diseases are discussed.

Genotype and biotype of invasive Anopheles stephensi in Mannar Island of Sri Lanka.

BACKGROUND: Anopheles stephensi, the major vector of urban malaria in India, was recently detected for the first time in Sri Lanka in Mannar Island on the northwestern coast. Since there are different biotypes of An. stephensi with different vector capacities in India, a study was undertaken to further characterise the genotype and biotype of An. stephensi in Mannar Island. METHODS: Mosquito larvae were collected in Pesalai village in Mannar and maintained in the insectary until adulthood. Adult An. stephensi were identified morphologically using published keys. Identified adult An. stephensi were molecularly characterized using two mitochondrial (cox1 and cytb) and one nuclear (ITS2) markers. Their PCR-amplified target fragments were sequenced and checked against available sequences in GenBank for phylogenetic analysis. The average spiracular and thoracic lengths and the spiracular index were determined to identify biotypes based on corresponding indices for Indian An. stephensi. RESULTS: All DNA sequences for the Mannar samples matched reported sequences for An. stephensi from the Middle East and India. However, a single nucleotide variation in the cox1 sequence suggested an amino acid change from valine to methionine in the cox1 protein in Sri Lankan An. stephensi. Morphological data was consistent with the presence of the Indian urban vector An. stephensi type-form in Sri Lanka. CONCLUSIONS: The present study provides a more detailed molecular characterization of An. stephensi and suggests the presence of the type-form of the vector for the first time in Sri Lanka. The single mutation in the cox1 gene may be indicative of a founder effect causing the initial diversification of An. stephensi in Sri Lanka from the Indian form. The distribution of the potent urban vector An. stephensi type-form needs to be established by studies throughout the island as its spread adds to the challenge of maintaining the country's malaria-free status.

DNA barcoding of Sri Lankan phlebotomine sand flies using cytochrome c oxidase subunit I reveals the presence of cryptic species.

Sri Lanka is known for high diversity of phlebotomine sand flies and prevalence of cutaneous and visceral leishmaniasis; a disease vectored by sand flies. The taxonomy of phlebotomine sand flies is complicated and often the diversity is over/underrated. The current study aims to use the cytochrome c oxidase gene subunit 1 (COI) sequence and formulate a barcode for the sand fly species in Sri Lanka. A total of 70 samples comprising seven species morphologically identified and collected from dry zone districts of Hambantota, Anuradhapura, Vavuniya, Trincomalee and Jaffna were processed. Neighbour-joining (NJ) tree created using the sequences revealed the species identity is compatible with the current morphology based identification. Further the analysis delineated morphologically identified Se. bailyi, Se babu babu and Se babu insularis into genetically distinct groups.

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 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.

Molecular evidence for the presence of malaria vector species a of the Anopheles annularis complex in Sri Lanka.

BACKGROUND: Anopheles annularis s.l. is a wide spread malaria vector in South and Southeast Asia, including Sri Lanka. The taxon An. annularis is a complex of two sibling species viz. A and B, that are differentiated by chromosome banding patterns and ribosomal gene sequences in India. Only species A is reported to be a malaria vector in India while the occurrence of sibling species in Sri Lanka has not been documented previously. FINDINGS: Anopheline larvae were collected at a site in the Jaffna district, which lies within the dry zone of Sri Lanka, and reared in the laboratory. Emerged adults were identified using standard keys. DNA sequences of the D3 domain of 28S ribosomal DNA (rDNA) and the internal transcribed spacer-2 (ITS-2) of the morphologically identified An. annularis were determined. BLASTn searches against corresponding An. annularis sequences in GenBank and construction of phylogenetic trees from D3 and ITS-2 rDNA sequences showed that the Sri Lankan specimens, and An. annularis s.l. specimens from several Southeast Asian countries were closely related to species A of the Indian An. annularis complex. CONCLUSIONS: The results show the presence of the malaria vector An. annularis species A in Sri Lanka and Southeast Asia. Because An. annularis vectors have been long associated with malaria transmission in irrigated agricultural areas in the Sri Lankan dry zone, continued monitoring of An. annularis populations, and their sibling species status, in these areas need to be integral to malaria control and eradication efforts in the island.