Malaria vectors of Timor-Leste

Abstract

Background: The island of Timor lies at the south-eastern edge of Indonesia on the boundary of the Oriental and Australian faunal regions. The country of Timor-Leste, which occupies the eastern part of the island, is malarious, but anopheline faunal surveys and malaria vector incrimination date back to the 1960 s. Over the last decade the malaria vectors of south-east Asia and the south-west Pacific have been intensely studied using molecular techniques that can confirm identification within complexes of isomorphic species. The aim of this study is to accurately identify the Anopheles fauna of Timor-Leste using these techniques.

Methods: The survey was carried out over the period February to June 2001. Standard entomological techniques--human landing collections, larval collections and CO2 baited light traps--were used to collect anophelines from the main geographical regions: coastal plains, inland plains, and highlands. Specimens were processed for identification by morphology and genotyped for the ribosomal DNA ITS2 by restriction analysis and/or DNA sequencing. Phylogenetic relationship of Anopheles sundaicus and Anopheles subpictus individuals was also assessed using DNA sequences from the ITS2 and mitochondrial cytochrome-b. All specimens, other than those from larval surveys, were processed to detect the presence of the Plasmodium parasite circumsporozoite protein by ELISA for vector incrimination.

Results: Of 2,030 specimens collected, seven species were identified by morphology: Anopheles barbirostris, Anopheles aconitus, Anopheles annularis, Anopheles maculatus, Anopheles peditaeniatus, An. sundaicus and Anopheles vagus. These were confirmed by molecular analysis with the addition of Anopheles flavirostris and an unidentified species designated here as An. vagus genotype B. This latter species was morphologically similar to An. vagus and An. subpictus and is likely to be the An. subpictus described by other workers for Timor. However, genetically this species showed strong affinities to the An. sundaicus complex. Anopheles vagus was the most common species but was rarely collected coming to bite humans; An. barbirostris and An. vagus genotype B were the two most common species collected in human landing catches and both were found positive for CS protein.

Conclusions: The anopheline fauna of Timor-Leste is of Oriental origin with no evidence of elements from the Australian Region. The existence of species complexes will make the use of morphological markers problematic in the country. Using molecular analysis a number of issues regarding the anopheline fauna of Timor-Leste were resolved and nine putative species of Anopheles were identified; two species: An. barbirostris and An. vagus genotype B, were incriminated as malaria vectors.

Figure 1

Map of the survey region indicating collection locations and main towns.

Figure 2

Maximum likelihood phylogenetic trees of An. sundaicus, An. subpictus, and An. vagus genotype B generated from DNA sequences of the rDNA ITS2 (Panel A) and mtDNA cyt-b (PanelB). In Panel A, the ITS2 tree places An. sundaicus from Timor-Leste (TL417) with the An. sundaicus ITS2 sequence variants I-IV. However, the placement of An. vagus genotype B (TL411) specimens basal to the An. sundaicus clade suggest a more recent evolutionary connection to An. sundaicus as the An. subpictus individuals from Papua New Guinea, Indonesia, Vietnam and Sri Lanka appear paraphyletic suggesting several lineages or cryptic species exist within this morphotaxa. In Panel B the Maximum likelihood analysis of cyt-b sequences places An. sundaicus individuals from Timor-Leste (TL415 and TL 417) within other An. sundaicus species forming a well supported and separate branch with individuals from Malaysia/Borneo. The cyt-b analysis also places An. vagus genotype B (TL411 and TL412) basal to the An. sundaicus clade and paraphyletic to An. subpictus individuals, also indicating closer genetic affinities to An. sundaicus and not An. subpictus. For each of the trees only bootstrap branch support values over 70% are displayed.