PCR-RFLP method for the identification of four members of the Anopheles annularis group of mosquitoes (Diptera: Culicidae)

The Anopheles annularis group of mosquitoes is widely distributed in Southeast Asia and may be locally important as malaria vectors. Members of this group are morphologically very similar and often difficult to distinguish, particularly A. nivipes and A. philippinensis. We report the sequence analysis of the rDNA internal transcribed spacer 2 (ITS2) and Domain-3 (D3) regions of the four members of the A. annularis group-A. nivipes, A. philippinensis, A. annularis and A. pallidus-and a method for their molecular identification. No intraspecies sequence variation was detected among the specimens, while interspecific sequence differences were greater for ITS2 than the D3 regions. Comparison of the D3 sequences of the four species revealed two Smal restriction sites in A. nivipes, but only one site in A. philippinensis, A. annularis and A. pallidus. The Apal site was present in both A. philippinensis and A. pallidus, while an Ncol site was present in A. pallidus only. Restriction digestion of the PCR products of D3 fragment individually with Smal, Apal and Ncol produced a distinctive pattern for all the four species. We present, for the first time, a PCR-RFLP method to distinguish the four members of the A. annularis group of mosquitoes. (c) 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.