Bionomic aspects of the Anopheles subpictus species complex in Sri Lanka.

Anopheles subpictus Grassi s.l. (Diptera: Culicidae) functions as a secondary malaria vector to Anopheles culicifacies Giles s.l. (Diptera: Culicidae) in Sri Lanka. The taxon A. subpictus is reported to exist as a species complex comprising four sibling species (A-D) that can be differentiated through polytene chromosome banding patterns and stage-specific morphometric traits in India. Based on the morphological characteristics described for the Indian Subpictus Complex, the presence of all four sibling species has been described in Sri Lanka. As sibling species show distinct bio-ecological characteristics that are important for devising appropriate vector control measures, a study was carried out in six districts in the dry zone of Sri Lanka. The results confirm the presence of all four sibling species, with species C predominating in inland areas and species B in coastal areas. Species C and D were indoor-resting and indoor-feeding, while species B was outdoor-resting with no significant preference for indoor- or outdoor-resting. Species B showed distinct morphological variation in the ornamentation of wings and palpi. Blood meal analysis revealed that species B, C, and D can feed on humans as well as cattle. The differential bio-ecological traits shown by the members of the Subpictus Complex are important for developing appropriate vector control measures in Sri Lanka.

Variations in salinity tolerance of malaria vectors of the Anopheles subpictus complex in Sri Lanka and the implications for malaria transmission.

BACKGROUND: Anopheles subpictus sensu lato, a widespread vector of malaria in Asia, is reportedly composed of four sibling species A-D based on distinct cytogenetic and morphological characteristics. However An. subpictus species B specimens in Sri Lanka are termed An. subpictus B/ An. sundaicus because of recent genetic data. Differences in salinity tolerance and coastal/inland prevalence of An. subpictus sibling species that were not previously established in Sri Lanka are presented here. RESULTS: Specimens with morphological characteristics of all four Indian An. subpictus sibling species were found in Sri Lanka. Sibling species A, C and D tended to be predominant in inland, and An. subpictus species B/An. sundaicus, in coastal localities. Sibling species C was predominant in both adult and larval inland collections. Larvae of An. subpictus B/An. sundaicus were found in inland and coastal sites, including a lagoon, with salinity varying from 0 to 30 ppt. An. subpictus sibling species A, C and D larvae were present in water of salinity between 0 to 4 ppt. An. subpictus C, D and An. subpictus B/An. sundaicus larvae showed compatible differential salinity tolerance in laboratory tests. The first instar larvae of An. subpictus B/An. sundaicus showed 100% survival up to 15 ppt in comparison to species C and D where the corresponding values were 3 ppt and 6 ppt respectively. However all third instar larvae of An. subpictus B/An. sundaicus survived up to 30 ppt salinity whereas An. subpictus C and D tolerated up to 4 ppt and 8 ppt salinity respectively. CONCLUSIONS: The results suggest that An. subpictus species B/An. sundaicus breed in fresh, brackish and nearly saline water while An. subpictus species C and D do so in fresh and less brackish waters in Sri Lanka, as in India. Because of the established role of An. sundaicus s.l. and An. subpictus s.l. as malaria vectors, the findings indicate a need for greater monitoring of brackish water breeding habitats in Asia. Tolerance to 15 ppt salinity may also constitute a simple method for differentiating An. subpictus B/An. sundaicus larvae from those of An. subpictus species C and D in field studies.

Genetic evidence for malaria vectors of the Anopheles sundaicus complex in Sri Lanka with morphological characteristics attributed to Anopheles subpictus species B.

BACKGROUND: Anopheles subpictus sensu lato, a widespread malaria vector in Asia, is reportedly composed of four sibling species A - D. Mosquitoes morphologically identified as belonging to the Subpictus complex were collected from different locations near the east coast of Sri Lanka, and specific ribosomal DNA sequences determined to validate their taxonomic status. METHODS: Anopheles subpictus s.l. larvae and blood-fed adults were collected from different locations in the Eastern province and their sibling species status was determined based on published morphological characteristics. DNA sequences of the D3 domain of 28 S ribosomal DNA (rDNA) and the internal transcribed spacer -2 (ITS-2) of mosquitoes morphologically identified as An. subpictus sibling species A, B, C and D were determined. RESULTS: Phylogenetic analysis based on D3 domain of rDNA resulted in two clades: one clade with mosquitoes identified as An. subpictus species A, C, D and some mosquitoes identified as species B, and another clade with a majority of mosquitoes identified as species B with D3 sequences that were identical to Anopheles sundaicus cytotype D. Analysis of ITS-2 sequences confirmed a close relationship between a majority of mosquitoes identified as An. subpictus B with members of the An. sundaicus complex and others identified as An. subpictus B with An. subpictus s.l. CONCLUSIONS: The study suggests that published morphological characteristics are not specific enough to identify some members of the Subpictus complex, particularly species B. The sequences of the ITS-2 and D3 domain of rDNA suggest that a majority that were identified morphologically as An. subpictus species B in the east coast of Sri Lanka, and some identified elsewhere in SE Asia as An. subpictus s.l., are in fact members of the Sundaicus complex based on genetic similarity to An. sundaicus s.l. In view of the well-known ability of An. sundaicus s.l. to breed in brackish and fresh water and its proven ability to transmit malaria in coastal areas of many Southeast Asian countries, the present findings have significant implications for malaria control in Sri Lanka and neighbouring countries.