Identification of sibling species status of Anopheles culicifacies breeding in polluted water bodies in Trincomalee district of Sri Lanka

BACKGROUND: Anopheles culicifacies s.l., the major vector of malaria in Sri Lanka, is known to breed in clean and clear water. However, recent findings have confirmed breeding from waste water bodies in urban and semi-urban areas. No study has been conducted to identify whether it is vector or non-vector siblings. The objective of the study was to identify the sibling species status of An. culicifacies s.l. METHODS: Anopheles culicifacies s.l. adult samples (reared from larvae) were obtained from the Padavisiripura Entomological team attached to Tropical and Environmental Diseases and Health Associates (TEDHA) Malaria Elimination Programme in Trincomalee District. The collected mosquito specimens were processed for the extraction of genomic DNA individually. The PCR amplifications were carried out using different primer combinations for differentiating species A from D, species B from C, species B from E, and species B, C, and E from each other. The results obtained from the gel electrophoresis were compared with the marker and band sizes of 359 bp, 248 bp, 95 + 248 bp, 166 + 359 bp and 178 + 248 bp were used to compare the sibling species A, B, C, D and E, respectively. RESULTS: The molecular biological identification of the field caught An. culicifacies s.l. samples observed that only 13.34 % (4/30) was represented sibling species B. About 86.66 % (26/30) of the samples were An. culicifacies sibling species E. This study also provided evidence that An. culicifacies E was able to breed in a wide range of breeding habitats. This is the first time that An. culicifacies E breeding in waste water was confirmed by a molecular method. Malaria control programmes in most parts of the country focus on rural communities as a result of bio-ecology of mosquitoes. Therefore, unusual breeding habitats, such as waste water collections, may mislead the current vector controlling programmes. CONCLUSIONS: These results reconfirm that An. culicifacies s.l. has adapted to breed in a wide range of water bodies, including waste water collections. Since the majority of mosquitoes sampled belong to sibling species E, this may adversely affect the current malaria elimination programme and new strategies should be adopted to control malaria vectors breeding in these unusual breeding habitats in Sri Lanka.