Anopheles culicifacies breeding in brackish waters in Sri Lanka and implications for malaria control

<p>Abstract</p> <p>Background</p> <p><it>Anopheles culicifacies </it>is the major vector of both falciparum and vivax malaria in Sri Lanka, while <it>Anopheles subpictus </it>and certain other species function as secondary vectors. In Sri Lanka, <it>An. culicifacies </it>is present as a species complex consisting of species B and E, while <it>An. subpictus </it>exists as a complex of species A-D. The freshwater breeding habit of <it>An. culicifacies </it>is well established. In order to further characterize the breeding sites of the major malaria vectors in Sri Lanka, a limited larval survey was carried out at a site in the Eastern province that was affected by the 2004 Asian tsunami.</p> <p>Methods</p> <p>Anopheline larvae were collected fortnightly for six months from a brackish water body near Batticaloa town using dippers. Collected larvae were reared in the laboratory and the emerged adults were identified using standard keys. Sibling species status was established based on Y-chromosome morphology for <it>An. culicifacies </it>larvae and morphometric characteristics for <it>An. subpictus </it>larvae and adults. Salinity, dissolved oxygen and pH were determined at the larval collection site.</p> <p>Results</p> <p>During a six month study covering dry and wet seasons, a total of 935 anopheline larvae were collected from this site that had salinity levels up to 4 parts per thousand at different times. Among the emerged adult mosquitoes, 661 were identified as <it>An. culicifacies s.l</it>. and 58 as <it>An. subpictus s.l</it>. Metaphase karyotyping of male larvae showed the presence of species E of the Culicifacies complex, and adult morphometric analysis the presence of species B of the Subpictus complex. Both species were able to breed in water with salinity levels up to 4 ppt.</p> <p>Conclusions</p> <p>The study demonstrates the ability of <it>An. culicifacies </it>species E, the major vector of falciparum and vivax malaria in Sri Lanka, to oviposit and breed in brackish water. The sibling species B in the <it>An. subpictus </it>complex, a well-known salt water breeder and a secondary malaria vector in the country, was also detected at the same site. Since global warming and the rise in sea levels will further increase of inland brackish water bodies, the findings have significant implications for the control of malaria in Sri Lanka and elsewhere.</p

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

<p>Abstract</p> <p>Background</p> <p><it>Anopheles annularis s.l</it>. is a wide spread malaria vector in South and Southeast Asia, including Sri Lanka. The taxon <it>An. annularis </it>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.</p> <p>Findings</p> <p>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 <it>An. annularis </it>were determined. BLASTn searches against corresponding <it>An. annularis </it>sequences in GenBank and construction of phylogenetic trees from D3 and ITS-2 rDNA sequences showed that the Sri Lankan specimens, and <it>An. annularis s.l</it>. specimens from several Southeast Asian countries were closely related to species A of the Indian <it>An. annularis </it>complex.</p> <p>Conclusions</p> <p>The results show the presence of the malaria vector <it>An. annularis </it>species A in Sri Lanka and Southeast Asia. Because <it>An. annularis </it>vectors have been long associated with malaria transmission in irrigated agricultural areas in the Sri Lankan dry zone, continued monitoring of <it>An. annularis </it>populations, and their sibling species status, in these areas need to be integral to malaria control and eradication efforts in the island.</p

Larval Development of Aedes aegypti and Aedes albopictus in Peri-Urban Brackish Water and Its Implications for Transmission of Arboviral Diseases

Aedes aegypti (Linnaeus) and Aedes albopictus Skuse mosquitoes transmit serious human arboviral diseases including yellow fever, dengue and chikungunya in many tropical and sub-tropical countries. Females of the two species have adapted to undergo preimaginal development in natural or artificial collections of freshwater near human habitations and feed on human blood. While there is an effective vaccine against yellow fever, the control of dengue and chikungunya is mainly dependent on reducing freshwater preimaginal development habitats of the two vectors. We show here that Ae. aegypti and Ae. albopictus lay eggs and their larvae survive to emerge as adults in brackish water (water with <0.5 ppt or parts per thousand, 0.5–30 ppt and >30 ppt salt are termed fresh, brackish and saline respectively). Brackish water with salinity of 2 to 15 ppt in discarded plastic and glass containers, abandoned fishing boats and unused wells in coastal peri-urban environment were found to contain Ae. aegypti and Ae. albopictus larvae. Relatively high incidence of dengue in Jaffna city, Sri Lanka was observed in the vicinity of brackish water habitats containing Ae. aegypti larvae. These observations raise the possibility that brackish water-adapted Ae. aegypti and Ae. albopictus may play a hitherto unrecognized role in transmitting dengue, chikungunya and yellow fever in coastal urban areas. National and international health authorities therefore need to take the findings into consideration and extend their vector control efforts, which are presently focused on urban freshwater habitats, to include brackish water larval development habitats

Biological differences between brackish and fresh water-derived Aedes aegypti from two locations in the Jaffna peninsula of Sri Lanka and the implications for arboviral disease transmission.

The mainly fresh water arboviral vector Aedes aegypti L. (Diptera: Culicidae) can also undergo pre-imaginal development in brackish water of up to 15 ppt (parts per thousand) salt in coastal areas. We investigated differences in salinity tolerance, egg laying preference, egg hatching and larval development times and resistance to common insecticides in Ae. aegypti collected from brackish and fresh water habitats in Jaffna, Sri Lanka. Brackish water-derived Ae. aegypti were more tolerant of salinity than fresh water-derived Ae. aegypti and this difference was only partly reduced after their transfer to fresh water for up to five generations. Brackish water-derived Ae. aegypti did not significantly discriminate between 10 ppt salt brackish water and fresh water for oviposition, while fresh water-derived Ae. aegypti preferred fresh water. The hatching of eggs from both brackish and fresh water-derived Ae. aegypti was less efficient and the time taken for larvae to develop into pupae was prolonged in 10 ppt salt brackish water. Ae. aegypti isolated from coastal brackish water were less resistant to the organophosphate insecticide malathion than inland fresh water Ae. aegypti. Brackish and fresh water-derived Ae. aegypti however were able to mate and produce viable offspring in the laboratory. The results suggest that development in brackish water is characterised by pertinent biological changes, and that there is restricted genetic exchange between coastal brackish and inland fresh water Ae. aegypti isolates from sites 5 km apart. The findings highlight the need for monitoring Ae. aegypti developing in coastal brackish waters and extending vector control measures to their habitats

Variations in susceptibility to common insecticides and resistance mechanisms among morphologically identified sibling species of the malaria vector <it>Anopheles subpictus </it>in Sri Lanka

<p>Abstract</p> <p>Background</p> <p><it>Anopheles subpictus s.l</it>., an important malaria vector in Sri Lanka, is a complex of four morphologically identified sibling species A-D. Species A-D reportedly differ in bio-ecological traits that are important for vector control. We investigated possible variations that had not been reported previously, in the susceptibility to common insecticides and resistance mechanisms among the <it>An. subpictus </it>sibling species.</p> <p>Methods</p> <p>Adult <it>An. subpictus </it>were collected from localities in four administrative districts in the dry zone of Sri Lanka. Single female isoprogeny lines were established and sibling species status determined according to reported egg morphology. World Health Organization's standard protocols were used for insecticide bioassays and biochemical assays to determine insecticide susceptibility and resistance mechanisms. Susceptibility of mosquitoes was tested against DDT (5%), malathion (4%), deltamethrin (0.05%) and λ-cyhalothrin (0.05%). Biochemical basis for resistance was determined through assaying for esterase, glutathione-S-transferase and monooxygenase activities and the insensitivity of acetycholinesterase (AChE) to propoxur inhibition.</p> <p>Results</p> <p>All sibling species were highly resistant to DDT. However there were significant differences among the sibling species in their susceptibility to the other tested insecticides. Few species A could be collected for testing, and where testing was possible, species A tended to behave more similarly to species C and D than to B. Species B was more susceptible to all the tested insecticides than the other sibling species. This difference may be attributed to the predominance of species B in coastal areas where selection pressure due to indoor residual spraying of insecticides (IRS) was lower. However there were significant differences between the more inland species C and D mainly towards pyrethroids. Higher GST activities in species C and D might have contributed to their greater DDT resistance than species B. Malathion resistance in both species C and D may be caused by elevated GST activity and an altered insensitive target site in AChE. In addition, a carboxylesterase based malathion resistance mechanisms was also detected in species C and D. Elevated esterase levels in species C and D might have contributed to the low levels of pyrethroid resistance. However an absence of elevated activity of monooxygenases in species B, C and D indicates that monooxygenases are unlikely to be the cause of this partial resistance to pyrethroids.</p> <p>Conclusions</p> <p>The differences in insecticide susceptibility and insecticide resistance mechanism shown by <it>An. subpictus </it>sibling species are important considerations for developing the malaria control and eradication program in Sri Lanka. Similar studies on species complexes of other anopheline vectors of malaria are necessary for effective malaria control worldwide. The differential susceptibility findings are also consistent with most, if not all, morphologically identified <it>An. subpictus </it>species B in Sri Lanka belonging to the <it>An. sundaicus </it>complex. There is a need therefore to develop molecular techniques that can be used to differentiate morphologically similar anopheline species in field conditions for more effective vector control.</p