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.

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.

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.

Predatory efficacy of Culex (Lutzia) fuscanus on mosquito vectors of human diseases in Sri Lanka.

Larvae of Culex (Lutzia) Fuscanus were collected from ovitraps in a natural breeding site. Collected larvae were used to establish a self-mating colony, and larval progeny were then used to determine their predatory efficacy on larvae of 3 vector mosquito species, Aedes aegypti, Anopheles subpictus, and Cx. tritaeniorhynchus. Statistical analysis revealed that Cx. fuscanus showed greater feeding efficacy for Ae. aegypti than for Cx. tritaeniorhynchus and An. subpictus. The natural predatory role of this species can potentially be exploited for biological control of mosquito vectors 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.

Salinity-tolerant larvae of mosquito vectors in the tropical coast of Jaffna, Sri Lanka and the effect of salinity on the toxicity of Bacillus thuringiensis to Aedes aegypti larvae.

BACKGROUND: Dengue, chikungunya, malaria, filariasis and Japanese encephalitis are common mosquito-borne diseases endemic to Sri Lanka. Aedes aegypti and Aedes albopictus, the major vectors of dengue, were recently shown to undergo pre-imaginal development in brackish water bodies in the island. A limited survey of selected coastal localities of the Jaffna district in northern Sri Lanka was carried out to identify mosquito species undergoing pre-imaginal development in brackish and saline waters. The effect of salinity on the toxicity of Bacillus thuringiensis israelensis larvicide to Ae. aegypti larvae at salinity levels naturally tolerated by Ae. aegypti was examined. METHODS: Larvae collected at the selected sites along the Jaffna coast were identified and salinity of habitat water determined in the laboratory. The LC50 and LC90 of B. thuringiensis toxin, the active ingredient of a commercial formulation of the larvicide BACTIVEC®, were determined with Ae. aegypti larvae. Bioassays were also carried out at salinities varying from 0 to 18 ppt to determine the toxicity of Bacillus thuringiensis to fresh and brackish water-derived larvae of Ae. aegypti. RESULTS: Larvae of four Anopheles, two Aedes, one Culex and one Lutzia species were collected from brackish and saline sites with salinity in the range 2 to 68 ppt. The LC50 and LC90 of B. thuringiensis toxin for the second instar larvae of Ae. aegypti in fresh water were 0.006 ppm and 0.013 ppm respectively, with corresponding values for brackish water populations of 0.008 and 0.012 ppm respectively. One hundred percent survival of second instar fresh water and brackish water-derived Ae. aegypti larvae was recorded at salinity up to 10 and 12 ppt and 100% mortality at 16 and 18 ppt, yielding an LC90 for salinity of 13.9 ppt and 15.4 ppt at 24 h post-treatment respectively for the two populations. Statistical analysis showed significantly reduced toxicity of B. thuringiensis to fresh and brackish water-derived Ae. aegypti larvae at high salinities. CONCLUSION: A variety of mosquito vectors of human diseases undergo pre-imaginal development in brackish or saline waters in coastal areas of the Jaffna district in northern Sri Lanka. Salinity has a small but significant negative impact on the toxicity of B. thuringiensis toxin to Ae. aegypti larvae at salinity levels where Ae. aegypti larvae are found in the environment. This has implications for the use of B. thuringiensis toxin as a larvicide in brackish waters.

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

BACKGROUND: Anopheles subpictus s.l., 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 An. subpictus sibling species. METHODS: Adult An. subpictus 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. RESULTS: 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. CONCLUSIONS: The differences in insecticide susceptibility and insecticide resistance mechanism shown by An. subpictus 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 An. subpictus species B in Sri Lanka belonging to the An. sundaicus 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.

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.

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.

Prevalence and insecticide susceptibility of dengue vectors in the district of Batticaloa in eastern Sri Lanka.

Unprecedented incidences of dengue have been reported in Sri Lanka in recent years. The district of Batticaloa, which was devastated by the 2004 Asian tsunami, is one of the districts affected by dengue. One option to curtail this disease is to implement appropriate vector control measures. A nine-month study was carried out within the Batticaloa Municipal Council limit from April to December 2008. Larval collections were conducted fortnightly using conventional ovitraps for nine months covering the dry and wet seasons. Ovitraps (indoor and outdoor) were placed in 15 randomly selected houses. The collected larvae were brought to the laboratory and reared under laboratory conditions. The larval forms and emerged adults were identified on the basis of reported morphological descriptions. The identified adults of 2-3 d old were exposed to common insecticides following the WHO protocol. During the study period, a total of 10,685 Aedes aegypti and Ae. albopictus mosquitoes were collected, with the former constituting 57% of the total sample. Both species were collected from indoor and outdoor ovitraps, and their prevalence was recorded throughout the study period. A seasonal shift was observed in the density, with Ae. aegypti predominating during the dry season and Ae. albopictus during the wet season. Both species were highly resistant to 4% DDT and susceptible to 0.25% permethrin. The continuous presence of potential dengue vectors may have contributed to the dengue prevalence in the district. Since both species can oviposit in indoor and outdoor ovitraps, public awareness and participation should be promoted in the vector control programme of the Ministry of Health along with continuous vector surveillance.

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

BACKGROUND: Anopheles culicifacies is the major vector of both falciparum and vivax malaria in Sri Lanka, while Anopheles subpictus and certain other species function as secondary vectors. In Sri Lanka, An. culicifacies is present as a species complex consisting of species B and E, while An. subpictus exists as a complex of species A-D. The freshwater breeding habit of An. culicifacies 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. METHODS: 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 An. culicifacies larvae and morphometric characteristics for An. subpictus larvae and adults. Salinity, dissolved oxygen and pH were determined at the larval collection site. RESULTS: 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 An. culicifacies s.l. and 58 as An. subpictus s.l. 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. CONCLUSIONS: The study demonstrates the ability of An. culicifacies 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 An. subpictus 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.