Molecular characterisation of the Simulium damnosum complex (Diptera: Simuliidae) found along the Osun River system, in south-western Nigeria.

Abstract

Simulium damnosum s.l. are the only known vectors of Onchocerca volvulus in West Africa (Toè et al., 1997). At least nine sibling species of S. damnosum s.l. exist in this region and these siblings have varying vectorial capacities in the different ecozones (Wilson and Post, 1994). The savannadwelling group (S. damnosum s.s. and S. sirbanum) transmits the blinding, savanna strain of O. volvulus and the forest-dwelling group (S. squamosum and S. yahense) transmits the non-blinding, forest strain of the parasite (Tang et al., 1995). The members of the transition-zone-dwelling group (S. sanctipauli, S. leonense and S. soubrense) are commonly found in areas where the two strains of O. volvulus co-exist (Tang et al., 1995). Most of the classification of the S. damnosum s.l. in West Africa has been based on larval cytotaxonomy (Ibeh et al., 2006). The available cytotaxonomic techniques can only be applied to seventh-instar larvae and cannot be used to identify the adult flies that are involved in transmission. Adult S. damnosum s.l. have been investigated using iso-enzyme analysis (Thomson et al., 1990), morphotaxonomy (Garms and Zillman, 1984; Wilson et al., 1993), morphometrics (Garms et al., 1982; Wilson et al., 1993) and molecular techniques based on the amplification of the internal transcribed spacers (ITS) of the flies’ nuclear ribosomal DNA (Brockhouse et al., 1993). Unfortunately, each of these methods could only differentiate two or three of the sibling species, leaving the problem of adult identification unresolved. More recently, however, the amplification of the 16S ribosomal RNA and NADH dehydrogenase subunit 4 (ND4) mitochondrial genes has been found useful, and the electrophoretic migration of heteroduplex formations of these sequences has been used to distinguish at least six of the siblings that serve as the main vectors in the areas formerly covered by the Onchocerciasis Control Programme (OCP) in West Africa (Tang et al., 1995; Higazi et al., 2000). A heteroduplex assay has recently been employed to investigate the sibling composition of the biting adults of S. damnosum s.l. to be found along the Osun River, in south– western Nigeria. This river lies (at 8u209– 6u309N, 5u109–3u259E) in the forest zone of Nigeria, outside of the ‘OCP area’. Between February 2008 and January 2009, adult S. damnosum s.l. were collected every fortnight, as they landed on human volunteers at three catching points along the river (at Eleja, Ogbere and Budepo), and then morphologically classified into forest or savanna flies (Kurtak et al., 1981; Wilson et al., 1993). Fifty flies were randomly selected from the collections made during each of the three seasonal peaks (April–June, July–September and October–March; Mayr, 1969). The DNA in each selected fly was extracted using a commercial kit (DNeasyH blood and tissue kit; QIAGEN, Hilden, Germany) and then run in a PCR designed to amplify ND4 sequences (Tang et al., 1995). The denaturation and renaturation of the heteroduplex of the PCR products was carried out using S. damnosum s.s. as the heteroduplex driver. The heteroduplex Annals of Tropical Medicine & Parasitology, Vol. 104, No. 8, 679–683 (2010)