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Parasitology Section Simulium thyolense is the main vector of Onchocerca volvulus in Malawi and southern Tanzania Zusammenfassung Die südlichsten Onchozerkose-Gebiete Afrikas liegen in Süd-Tansania und Malawi und bilden scheinbar isolierte Herde. Ein Vergleich dieser beiden Gebiete hinsichtlich der Identität der potenziellen Überträgermücken unter Anwendung cytotaxonomischer und molekulargenetischer Methoden ergab, dass Simulium thyolense die häufigste Art innerhalb der Onchozerkose-Gebiete ist, während außerhalb der endemischen Gebiete andere Arten häufiger waren. Außerdem wurden alle am Menschen gefangenen Mücken aus den verschiedenen Gebieten als S. thyolense identifiziert, so dass diese Art vermutlich alleiniger Überträger ist. Summary The southernmost foci of onchocerciasis in Africa are found in southern Tanzania and Malawi and consist of rather isolated areas. A comparison of these regions regarding the identity of the potential blackfly vectors using cytotaxonomic and molecular techniques revealed that S. thyolense is the most abundant species within all onchocerciasis foci, whilst outside the foci there were other species prevailing. Furthermore, all biting female flies from the different areas were identified as S. thyolense, which suggests that this species is the only significant vector. Figure 1: Study area with foci of onchocerciasis. 30 Introduction Human onchocerciasis is an insect born disease. The main African vectors are blackflies of the Simulium damnosum complex. The East African endemic areas of onchocerciasis are relatively small and isolated from each other, and also differ in their ecology. These factors favoured the evolution of a great biodiversity. The S. damnosum complex particularly represents an example for the differentiation of sibling species, which hardly show morphological differences. One of the 50 or so known taxa is S. thyolense on which this study is focussed. As in other medical important insects the taxonomic knowledge is crucial for epidemiological considerations, and vector control remains most effective in terms of disease elimination. The southernmost focus is located in Thyolo district/southern Malawi, with extensions into the Mulanje Mts. and Mwanza district (Fig. 1). In southern Tanzania the largest is the Ruvuma focus, followed by the Tukuyu focus (Fig. 1). Concerning the exact vector identity in these foci there only existed incomplete or virtually no data so far but at least it has already been suspected that Simulium thyolense might play the major role as vector within the Thyolo and Tukuyu foci. Project Description and Results The objective of this study was to assess the potential isolation of some onchocerciasis foci and their vector populations, respectively, by examining the distribution of the vector and related cytoforms outside the foci to look for possible re-invasion sources. In Tanzania this was part of the ‘Tukuyu Focus Vector Elimination Project’. The foci in southern Malawi were subject of independent investigations. Altogether, during three surveys in 2002/03 immature S. damnosum were collected from their riverine breeding sites. Subsequently, they were analysed by examining geographic variation in their polytene chromosome inversions (cytotaxonomy), morphology and DNA. In addition, biting female flies were caught in the respective foci for comparison with specimens obtained from the river samples. Cytotaxonomically, the assumed vector S. thyolense can unequivocally be identified by two diagnostic inversions (Fig. 2 a+b). Within the onchocerciasis foci, this species accounted for 80% of the analysed specimens, with almost 100 % in the Thyolo and Mulanje areas. Outside the foci, the majority consisted of cytoforms other than S. thyolense, which was almost or completely absent in populations in central Malawi and the highlands north of the Kipengere Range in Tanzania. A comparison between the different populations of S. thyolense revealed some marked chromosomal differences. First of all, a distinct cytoform now called ‘Nyika’ form was found in northern Malawi and at the border strip of Tanzania (both outside any onchocerciasis areas). Furthermore,
the populations from the endemic areas at Tukuyu and Ruvuma exhibited a number of floating inversions (Fig. 2a+b) none of which were detected in southern Malawi where, vice versa, a distinct set of polymorphisms was present (Fig. 2c) but not found in Tanzania. In summary, these results suggest the occurrence of at least three distinct sub-populations of S. thyolense. Morphologically, adult males of S. thyolense usually can be separated from other cytoforms in the areas by means of their scutal pattern (Fig. 2d-e). Hence, neonate males may act as a quick test for species identification where chromosome squashes are unavailable. Simulium thyolense from all study areas showed a species-specific rDNA ITS-1 PCR amplicon. Other cytoforms had different amplicon sizes. The PCR technique can therefore be used for routine identification of S. thyolense, and 52 biting adult flies from the Tukuyu and Ruvuma foci as well as 188 flies from the Thyolo-Mulanje focus were all identified as S. thyolense, indicating that this is the only significant vector species. Figure 2: Photomicrographs of S. thyolense with the diagnostic inversions and different polymorphisms (a-c), and the male scutal pattern of S. thyolense (e) compared with other cytoforms (d+f). Fixed or diagnostic inversions (3L-97, 2L-AK) are speciesspecific, whereas floating or polymorphic inversions (arrows) may also occur in other cytoforms. 31 Selected Publications • Krüger A (2003). An alternative protocol for the preparation of polytene chromosomes of Simulium damnosum s.l. larvae (Diptera: Simuliidae). Ann Trop Med Parasitol 97: 657-660. • Krüger A, Kalinga AK, Post RJ, Maegga BTA. (2004). Two new cytoforms of the Simulium damnosum complex (Diptera: Simuliidae) from Malawi and Tanzania and potential onchocerciasis vectors. Trop Med Int Hlth, in press. Funding • WHO • African Programme for Onchocerciasis Control • Johanna und Fritz Buch Gedächtnisstiftung Cooperating Partners • Bertha T.A. Maegga, Akili K. Kalinga NIMR, Tukuyu, Tanzania • Rory J. Post, Mabintu Mustapha NHM, London, UK • Philimon A.J. Tambala NOTF, Blantyre, Malawi Investigator • Andreas Krüger • Ina C. Hennings Parasitology Section
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