Internal transcribed spacer 2 (ITS2) based molecular identification of malaria vectors from Bangsring Banyuwangi-Indonesia

Abstract

Since the malaria outbreak in 2011, the breeding place of Anopheles in Bangsring Village on Banyuwangi District has been monitored by District Public Health Office as part of a vector surveillance program. Morphological identification is still a standard tool to observe Anopheles occurrence and diversity, but the presence of cryptic species made it unreliable. In this study, a molecular approach called DNA barcoding technique was used to assist the morphology-based techniques to identify Anopheles species found in Bangsring. The internal transcribed spacer 2 (ITS2) sequence was used as molecular marker. Based on the morphological features, we were able to identify Anopheles (An.) vagus, An. subpictus, An. sundaicus and An. aconitus. ITS2 sequences from the four identified species were then analyzed simultaneously with eighteen reference sequences from NCBI which had a high similarity of 98-100%. The NJ phylogenetic tree formed three major clades, where the two clades as monophyletic clades were An. vagus and An. aconitus. Another clade was formed as polyphyletic clade containing An. subpictus and An. sundaicus. Although An. subpictus and An. sundaicus were placed in the same clade, seven nucleotide differences were observed in their ITS2 sequence. The intra-specific variation of those two species was 0.08 and 0.49%, respectively, while the interspecific variation was 1.39%. Interspecific variation which was higher than the mean intra-specific variation might indicate that An. sundaicus and An. subpictus were a distantly species. However, the value of interspecific variation lower than 3% might also indicate that those species were classified as a complex species. All ITS2 sequences from morphologically identified species had similar results with molecular-based techniques. This result showed that molecular identification using the ITS2 sequence was reliable in supporting morphological identification among closely related anopheline mosquitoes and gave further information about their evolutionary divergence.