A guide to the Simulium damnosum complex (Diptera: Simuliidae) in Nigeria, with a cytotaxonomic key for the identification of the sibling species.

Although approximately 40% of all the people blinded by Onchocerca volvulus are Nigerians, almost nothing was known about the various cytospecies of the blackfly vectors present in Nigeria until 1981. The activation of the Nigerian National Onchocerciasis Control Programme in 1986 (and that programme's initiation of mass distributions of ivermectin in 1991) provided a significant stimulus to understand the biology of the Nigerian vectors but the exploration of any possible differences between the cytospecies has been hampered by a lack of accessible taxonomic information. This review attempts to satisfy that need. There are nine different cytoforms reliably recorded from Nigeria (Simulium damnosum s.s. Nile form, S. damnosum s.s. Volta form, S. sirbanum Sirba form, S. sirbanum Sudanense form, S. soubrense Beffa form, S. squamosum A, S. squamosum B, S. squamosum C and S. yahense typical form), and three more are known from surrounding countries and might be reasonably expected to occur in Nigeria. All of these cytospecies are presumed to be vectors, although there have been almost no identifications of the vectors of O. volvulus in Nigeria. The biogeographical distribution of the cytoforms is broadly similar to that known in other parts of West Africa (although many of the cytoforms remain insufficiently studied). The physico-chemical hydrology of the Nigerian breeding sites of the cytospecies does not, however, correspond to that seen elsewhere in West Africa, and it is not clear whether this might be related to differences in the cytoforms. An illustrated cytotaxonomic key is presented to facilitate and encourage future studies.

Construction and characterisation of a BAC library made from field specimens of the onchocerciasis vector Simulium squamosum (Diptera: Simuliidae).

A Bacterial Artificial Chromosome (BAC) library was made from wild-caught Simulium squamosum, which is an important vector of human onchocerciasis. The library is composed of 12,288 BACs, with an average insert size of 128 kb, and is expected to contain ~1.54 GB of cloned DNA. Random BAC-end sequencing generated over 95 kb of DNA sequence data from which putative S. squamosum gene sequences and novel repetitive DNA families were identified, including DNA transposons, retrotransposons and simple sequence repeats (SSRs). The sequence survey also provided evidence of DNA of microbial origin, and dissection of sample blackflies indicated that some of those used to prepare the library were likely to be parasitized by the mermithid Isomermis lairdi. Hybridisations with a set of three independent blackfly single-copy genes and two Wolbachia genes suggest that the library provides around 13-fold coverage of the S. squamosum genome and about 12-fold coverage of its Wolbachia endosymbiont.

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum.

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct 'supergroups' and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species' sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering approximately 6.2kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.

An 18S ribosomal DNA barcode for the study of Isomermis lairdi, a parasite of the blackfly Simulium damnosum s.l.

The mermithid parasite, Isomermis lairdi Mondet, Poinar & Bernadou (Nematoda: Mermithidae), is known to have a major impact on populations of Simulium damnosum s.l. Theobald (Diptera: Simuliidae) and on their efficiency as vectors of Onchocerca volvulus (Leuckart) (Nematoda: Filarioidea). However, the value of I. lairdi and other mermithid parasites as potential means of integrated vector control has not been fully realized. This is partly because traditional taxonomic approaches have been insufficient for describing and analysing important aspects of their biology and host range. In total, rDNA barcode sequences have been obtained from over 70 I. lairdi mermithids found parasitizing S. damnosum s.l. larvae in three different rivers. No two sequences were found to vary by more than 0.5%, and cytospecies identification of mermithid hosts revealed that I. lairdi with identical rDNA barcodes can parasitize multiple cytoforms of the S. damnosum complex, including S. squamosum (Enderlein). Phylogenetic analysis using a partial sequence from the 18S ribosomal DNA barcode, grouped I. lairdi in a monophyletic group with Gastromermis viridis Welch (Nematoda: Mermithidae) and Isomermis wisconsinensis Welch (Nematoda: Mermithidae).

Laser-assisted microdissection for the study of the ecology of parasites in their hosts.

The population biology of internal parasites is difficult to study because the adult parasites are often inaccessible, deep within the host's body. Developing stages, such as eggs in the faeces or larvae in the skin are more easily obtained, but are difficult to handle because they are often very small and with a tough cuticle. This has limited their use in molecular ecology for estimating population biology parameters of the adults (their parents). We have used Onchocerca ochengi (a filarial nematode parasite of cattle) to describe a novel and generally applicable method of easily and conveniently isolating individual larvae (microfilariae) from the host using laser-assisted microdissection. Furthermore, we have been able to improve the isolation of DNA by using the laser to bisect the larva to release DNA from the tissues enclosed within the parasite cuticle, and in this way we have achieved amplification of fragments over 1400 bp, and routinely PCR-amplified single-copy sequences from 5% of the DNA from a single larva (the equivalent of approximately 15 nuclei), and regularly from 0.5%.