Taxonomy and molecular phylogeny of black flies (Diptera: Simuliidae) in Africa and Europe by using mitochondrial Cox1 and nuclear ITS2 markers

Abstract

Abstract Background Black flies are present worldwide and are represented by 2,348 species (2,331 living species and 17 fossils) classified under 31 genera. In Africa, 124 species of black flies have been described, of which 55 are known to occur in Cameroon. However, these identifications are all based on morphology and, for S. damnosum only, on cytotaxonomic studies. Little has yet been achieved by modern molecular genetic techniques. In this study, we have applied DNA-based techniques to investigate the diversity and evolution of Simulium flies to improve vector control. Methods Simulium larvae and pupae were randomly collected from various Simulium breeding sites in rivers from five countries (Cameroon, Ethiopia, Tunisia, Nigeria and Germany). Adult Simulium flies were caught on humans in Cameroon, Nigeria and Ethiopia. Pupal gills were examined and photographed in a drop of polyvinyl-lactophenol. Genomic DNA was extracted and the CoxI and ITS2 genes were targeted by the polymerase chain reaction. The amplification products were sequenced and trees were drawn. Sequences were refined using Geneious Prime and evolutionary analyses were conducted in Mega-X 10.5. Results More than 54 sample collections were included in this study, of which about 1,000 individual larvae, pupae or adult flies from the various sites were examined. Twenty-five species were identified on the basis of their morphology and DNA-sequencing: Simulium damnosum s.l.; S. unicornutum; S. katangae; S. hisurtum; S. cervicornutum; S. schoutedeni; S. alcocki; S. dentulosum (A; B and C); S. ruficorne; S. hargreavesi v. medusaeforme; S. hargreavesi; S. adersi; S. pseudequinum; S. (Eusimulium) latipes; S. (Odagmia) ornatum; S. (Wilhelmia) equinum; S. vorax; S. nigritarsis duboisi; S. kenyae; S. velutinum; S. aureum; S. angustipes; S. bovis; S. tuberosum; S. rodhaini). Sequences of the previously undescribed species are deposited in GenBank. Conclusions We show, for the first time, the relationship between the morphology and molecular data of some black flies from Cameroon, Ethiopia, Nigeria, Tunisia and Germany. These data are important for understanding the transmission and life-cycles of Simulium-borne parasites: Moreover, knowledge concerning the various members of the S. damnosum complex in the rainforest and savannah is a prerequisite for the successful elimination of the parasite Onchocerca volvulus in Africa.