Experimental hybridization between members of the Simulium damnosum complex

Abstract. Males of the Beffa form of Simulium soubrense Vajime and Dunbar were successfully crossed with female S.squamosum V. & D. and S.sanctipauli V. & D. Fertile eggs from both crosses were reared to larvae and, in the case of the BeifaXsquamosum cross, to adults.
Larval chromosomes from the Beffa x sanctipauli cross had normally paired homologues but those of the Beffa xsquamosum cross showed extensive asynapsis and large heterozygous loops. The morphology of the larval and adult hybrids from the BeftaXsquamosum cross were more characteristic of squamosum , but the adult males had scutal patterns typical of the Beffa form and retained this form's laboratory-mating propensity.     

Completion of the sequence of the nuclear ribosomal DNA subunit of Simulium sanctipauli,with descriptions of the 18S, 28S genes and the IGS

We describe the IGS-ETS, 18S and 28S ribosomal gene sequences of Simulium sanctipauli Vajime & Dunbar, a member of the S. damnosum Theobald (Diptera: Simuliidae) complex of blackflies (Diptera: Simuliidae). These regions, together with the ITS-1, ITS-2 and 5.8S rDNA presented elsewhere (accession number U36206), constitute the composite sequence of the entire rDNA unit, making S. sanctipauli the second dipteran species of medical importance for which the entire rDNA has been sequenced. Despite the lack of sequence identity, the IGS of S. sanctipauli showed some structural similarities to other Diptera, i.e. the mosquito Aedes albopictus Skuse (Culicidae), the fruitfly Drosophila melanogaster Meigen (Drosophilidae) and the tsetse Glossina (Glossinidae). Two blocks of tandemly repeated subunits were present in the IGS of S. sanctipauli and, unlike other species of Diptera, they contained no duplications of promoter-like sequences. However, two promoter-like sequences were identified in the unique DNA stretches of the IGS by their sequence similarity to the promoter of Aedes aegypti L. (Diptera: Culicidae). The observed sequence variation can be explained, as in the case of Drosophila spp., by the occurrence of slippage-like and point mutation processes, with unequal crossing-over homogenizing (to a certain extent) the region throughout the gene family and blackfly population. The 18S and 28S rDNA genes show more intraspecific variability within the expansion segments than in the core regions. This is also the case in the interspecific comparison of these genes from S. sanctipauli with those of Simulium vittatum, Ae. albopictus and D. melanogaster. This pattern is typical of many eukaryotes and likely to be the result of a more relaxed functional selection in the expansion segments than on the core regions. The A + T content of the S. sanctipauli genes is high and similar to those of other Diptera. This could be the result of a change in the mutation pressure towards AT in the Diptera lineage.     

Revision of the Ketaketa subcomplex of blackflies of the Simulium damnosum complex

A revision of the taxonomy of the Ketaketa subcomplex of the Simulium damnosum Theobald complex (Diptera: Simuliidae) is presented including new material from Tanzania, Malawi and South Africa. The cytotaxonomy, morphology and molecular identity of known and new taxa are described. The Ketaketa subcomplex is cytotaxonomically defined by the paracentric inversion 1L-7. We recognize three sibling species, namely Simulium latipollex (Enderlein), Simulium plumbeum Krueger, sp.n. and Simulium kipengere Krueger, sp.n., the latter comprising three cytoforms: 'Typical', 'Linthipe' and 'Mombo'. The cytoforms 'Mwamphanzi', 'Ketaketa' and 'Hammerkopi' are synonymized with S. plumbeum. Identification keys are provided on the basis of chromosomal and morphological characters. In view of their potential role as vectors of human onchocerciasis (river blindness) we also discuss the possible medical importance of the different cytoforms and their geographical distribution.     

The species delimitation problem in the Simulium damnosum complex, blackfly vectors of onchocerciasis

The Simulium damnosum Theobald complex (Diptera: Simuliidae) comprises 57 cytoforms grouped into six subcomplexes. Previous phylogenetic studies using gene sequences have not completely resolved the evolutionary relationships of the cytoforms. The present study investigated the systematics of the complex using a phylogeographic approach. The differentiation between eastern and western forms observed in the phylogenetic studies is confirmed in the estimated haplotype networks. However, haplotypes tend to group in geographical clades and not according to cytoforms. Spatial analyses of the molecular variance also resulted in optimal groupings of sequences that did not correspond to cytoform boundaries. Moreover, Mantel tests showed significant correlations, although not strong, between genetic and geographical distances. This suggests an isolation-by-distance model of differentiation. Furthermore, there are instances in which genetic differentiation between cytoforms is low and not significant. These results indicate a lack of clear genetic differentiation between the cytoforms, which may be explained either by a separation of the taxa recent enough to allow the accumulation of few genetic differences or by recombination between the genomes of the cytoforms, which may be the result of hybridization with introgression or of non-independent evolutionary lineages. The results also emphasize the need for further sampling and for the use of more variable markers in order to clarify the evolutionary history of the group.     

Oviposition aggregation pheromone in the Simulium damnosum complex

Abstract. Communal oviposition by the Simulium damnosum complex of Afrotropical blackflies (Diptera: Simuliidae) was investigated under controlled laboratory conditions, using wild-caught flies in Sierra Leone. Volatile compounds emitted by Simulium eggs were trapped using a closed collection system, and their attractiveness to gravid flies was tested in a two-choice behavioural bioassay. Significantly more female blackflies oviposited on substrates baited with freshly laid eggs (100% chose the baited substrate), or with the volatiles collected from freshly laid eggs (85% chose the baited substrate), in preference to the relevant control substrates. Substrates baited with volatiles from 12-h-old eggs were not significantly more attractive than controls (only 31% chose the baited substrates; P = 0.33). Gas chromatographic analysis of the egg volatiles consistently showed two peaks emanating from fresh eggs, but significantly lower amounts from 12-h-old eggs ( P <0.05). A novel system for collecting the volatiles from this and other blackfly species, as they laid eggs on a substrate in flowing water, is described. Volatiles collected using this method showed identical gas chromatographic profiles to those of fresh eggs alone, indicating that the flies themselves produced no other volatile chemical signals during oviposition. Evidently communal oviposition by S. damnosum s.l . was mediated by a pheromone emanating from fresh eggs. The role of pheromone-mediated egg aggregation in blackfly ecology is discussed, and its possible manipulation is considered.     

The Beffa form of Simulium soubrense of the S.damnosum complex in Togo and Benin

The Beffa form of Simulium soubrense Vajime & Dunbar, a member of the S. sanctipauli sub-complex of the S. damnosum complex, was found breeding throughout rivers in the Togo-Benin Gap, as far north as 9 degrees 30'N. Its distribution changed with the season. In southern Togo there were seasonal fluctuations in the relative abundancies of the Beffa form and of S. damnosum/S.sirbanum. There was considerable temporal and regional variation in the frequencies of different colour morphs of adult flies. The flies in Benin tended to be darker. Infections with Onchocerca volvulus (Leuckart) appeared to be independent of the host's colour morph category. Larger flies harboured significantly more first stage Onchocerca larvae but no significant relations with fly size were found for second and third stage larvae.     

DNA probes for the identification of members of the Simulium damnosum complex (Diptera: Simuliidae)

Genomic libraries in plasmid have been constructed from various sibling species of blackflies of the Simulium damnosum complex from West Africa. Three cloned repetitive sequences, which show variation in copy number between sibling species, have been isolated. These clones can be used as probes for the dot-blot identification of larvae, pupae or adults into the three main West African subcomplexes, i.e. damnosum, squamosum and sanctipauli subcomplexes. The sequences also show some intraspecific variation in copy number.     

Oviposition pheromone in the Simulium damnosum complex: biological activity of chemical fractions from gravid ovaries

Abstract. Communal oviposition in the Afrotropical blackfly species complex Simulium damnosum Theobald (Diptera: Simuliidae) is mediated by a pheromone emitted by freshly laid eggs. Previously, two compounds (designated peaks A and B) emanating from fresh eggs were shown to be associated with attractiveness to gravid blackflies in bioassay. The present study investigated the role of these compounds by testing the responses of wild-caught Simulium yahense in Ghana to fractionated hexane extracts of gravid ovaries prepared by gas chromatography (GC). Although the fractions were prepared from Sierra Leonean Simulium leonense , GC analysis of the emissions from fresh S.yahense eggs showed that the volatile blends of both species were similar. When tested in a two-choice bioassay, 66% of ovipositing blackflies chose the substrate baited with a mixture of the four fractions recombined. In a series of bioassays testing responses to the four individual fractions presented with a control in a multiple-choice arrangement, only fraction 3 (containing peaks A and B) attracted significantly more ovipositions than the other fractions and control. However, fraction 3 failed to elicit a significant response when presented as the sole attractant with a control in a two-choice bioassay. It was concluded that fraction 3 , though mainly responsible for mediating aggregated oviposition by S.yahense , was acting in tandem with additional cues, probably further chemicals, which remain to be isolated and characterized.     

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).     

The thermal constant of the onchocerciasis vector Simulium damnosum s.l. in West Africa

The minimum water temperature for development (t(0)) and the thermal constant (K) for the development of immature stages of Simulium damnosum s.l. (Diptera: Simuliidae) in West Africa were estimated as 20.1 °C and 93 day-degrees, respectively, based on analyses of published data on development rates of eggs, larvae and pupae at different water temperatures (24.0 °C and 31.5 °C). Thus, at a constant water temperature of 30.0 °C (approximately 10 °C above t(0)), adult flies would emerge about 9 days after oviposition. Analysis of a dataset probably restricted to S. damnosum s.s., but for which the temperature for the egg stage varied, revealed a much lower t(0) (16.3 °C) and a much higher K (181 day-degrees), suggesting that the insects' thermal relations may be cytoform-specific. The results will aid control decisions and predictions of possible effects of climate change on sizes and geographic distributions of populations of onchocerciasis vectors in West Africa.     

Cytogenetical analysis of the Simulium damnosum complex (Diptera: Simuliidae) in Guinea Bissau

Abstract. In a 3-year study during 1990–92, larval collections of the Simulium damnosum complex from the River Corubal system in Guinea Bissau revealed that the only sibling species present were S. sirbanum and the Konkouré form of S. konkourense , but not S. damnosum s.s. which had been found at some of the localities (Saltinho and Cusselinta) in a previous survey by Quillévéré et at. (1981). Their differential distributions were seasonally consistent between years. S. sirbanum was concentrated in the upper reaches of the Corubal, moving downstream at the end of the dry season to exploit new breeding grounds, whereas S. konkourense seemed to be concentrated in the lower reaches and moved upstream in the middle of the dry season.
Since the previous survey, it appears that S. konkourense has largely replaced S. sirbanum and S. damnosum s.s. in the lower reaches of the Corubal. S. sirbanum was consistently associated with man-biting behaviour and, although infection studies were not performed, it is likely that S. sirbanum is the main vector of onchocerciasis in Guinea Bissau as S. konkourense does not seen to be anthropophilic.
Among five polymorphic inversions in S. sirbanum , the frequency of inversion IL-B increased during the dry season; IL-2 and IIL-7 showed homozygous disadvantage whereas IIL-3 and IIIL-6 did not; IS-2 tended to be fixed in all populations. Simulium konkourense populations in the River Corubal differ from those found upstream in its tributary the River Koumba, in the Fouta Djallon of Guinea, by their sex chromosomes (having no sex-linked inversions) and biting preferences, indicating that the populations are not freely interbreeding. Inversion IIIL-X was found for the first time in populations of S. konkourense outside the Fouta Djallon area and a new inversion IIL-Y was identified in samples from the Rivers Corubal and Féfiné.     

Inverse density dependence of parity rates in the onchocerciasis vector Simulium damnosum s.l.

A correlation between parity rates and an index of adult numbers of Simulium damnosum s.l. (Diptera: Simuliidae) indicates an association, but does not prove causality or show the direction of any causal relationship. The question of whether adult numbers affect parity rates or vice versa is reminiscent of the age‐old query of which of the chicken and the egg came first. A method for resolving such issues based on analyses of pairs of time series was proposed by Granger in 1969. When Granger's method was applied to monthly numbers of adult female S. damnosum s.l. caught attempting to bite humans at Asubende, Ghana, and their parity rates, a significant relationship (P = 0.005) emerged, clearly showing that parity rates were dependent on adult numbers. Implications of this inverse density dependence and the results of analyses of other similar time series are presented and discussed.     

Sibling species distributions of the Simulium damnosum complex in the West African Onchocerciasis Control Programme area during the decade 1984–93, following intensive larviciding since 1974

Abstract .During the decade from 1984 to 1993, nine species of the Simulium damnosum complex of blackflies (Diptera: Simuliidae) were identified from the area covered by the Onchocerciasis Control Programme. These were S. damnosum s.s., S. dieguerense, S. konkourense, S. leonense, S. sanctipauli, S. sirbanum, S. soubrense, S. squamosum, and S. yahense. Some of these species were found to consist of two chromosomal variant populations. These were S. konkourense‘Konkouré' and ‘Menankaya' forms, S. sanctipauli sensu stricto and‘Djodji' form, S. soubrense‘Chute Milo' and ‘Beffa' forms. The distribution of these twelve cytological taxa was assessed in relation to the two main vegetation zones of West Africa (forest and savanna), topography, river size and other factors. The range of each species was influenced by seasonal climatic changes in wind movement and river water level. The most widely distributed species were S. sirbanum and S. damnosum s.s., associated with savanna areas, recorded from all river basins. Simulium dieguerense was restricted mainly to Western Mali on the Rivers Bafing and Bakoye in the Senegal River basin. Simulium squamosum was identified from rivers draining mountainous areas in both the forest and savanna zones. Simulium yahense was found in small permanent rivers along a wide forested band parallel to the coast and was absent from the plains of Togo and Benin. Members of the S. sanctipauli subcomplex had restricted distributions except for S. sanctipauli s.s., which was widespread in large rivers of the forest zone from Sierra Leone to the Volta Lake in Ghana. Simulium soubrense‘Beffa' form occurred in Togo and Benin, S. soubrense‘Chutes Milo' form in Guinea, both ‘Konkouré' and ‘Menankaya' forms of S. konkourense occurred predominantly in Guinea and S. leonense in Sierra Leone. The relevance of the distribution maps and the importance of the data bank to vector control larvicidal operations are discussed.     

Management of insecticide resistance in control of the Simulium damnosum complex by the Onchocerciasis Control Programme, West Africa: potential use of negative correlation between organophosphate resistance and pyrethroid susceptibility

1. Resistance of some populations of the Simulium damnosum complex to temephos (100-fold at the LC50 level), with degrees of cross-resistance to chlorphoxim (14-fold) and other organophosphate insecticides, follows intensive larvicidal control of S. damnosum s.l. in West African river systems since 1975 by the WHO Onchocerciasis Control Programme. 2. Larvae of at least three sibling species of the S. damnosum complex have become organophosphate-resistant: these are the forest species S. sanctipauli Vajime & Dunbar and the savanna species S. sirbanum V. & D. and S. damnosum Theobald sensu stricto. 3. Organophosphate-resistant S. damnosum s.l. larvae show increased susceptibility to some organochlorine and pyrethroid insecticides, especially to permethrin (up to 11-fold) and OMS 3002 (up to 17-fold), as compared with organophosphate-susceptible populations. 4. This differential susceptibility is reflected by increased pyrethroid efficacy in operational use for river treatments against organophosphate-resistant field populations of S. damnosum s.l. larvae. Treatment of 100 km of the lower Bandama River in 1985 showed that permethrin at the highly selective dosage of 10 min exposure to 0.01 mg/l caused reversion towards organophosphate-susceptibility of the target population of S. sanctipauli. This effect was less pronounced when the Comoe River was treated at the lower dosage of 0.005 mg/l for 10 min. 5. To overcome temephos-resistance, it is proposed that the most rational usage of currently available larvicides would involve the following annual sequence of treatments: Bacillus thuringiensis serotype H-14 when river discharge is below 75 m3/s; chlorphoxim for about eight weekly treatment cycles after river discharge rises; permethrin (or alternative pyrethroid) for up to six treatment cycles--this should eliminate any incipient selection for chlorphoxim-resistance; resume chlorphoxim (or perhaps carbosulfan) treatments until river discharge falls below 75 m3/s permitting resumed use of B.t. H-14.     

Confirmation of the species status of the blackfly Simulium galeratum in Britain using molecular taxonomy

Since 1920 Simulium reptans (Linnaeus) (Diptera: Simuliidae) has been reported as exhibiting two different larval morphotypes, a typical S. reptans and an atypical S. reptans var. galeratum, which differ in the markings of the larval head capsule. Inconsistent variation in adults and no apparent variation in the pupae have led taxonomists to conclude that these types in Britain are a single species. We investigated populations in Britain where either the typical form or var. galeratum is found, and one population where the two exist sympatrically. A phylogenetic study based upon a region of the mitochondrial cytochrome c oxidase 1 gene (DNA barcoding) produced a tree that delineated the morphotypes into two distinct monophyletic clades. The average Kimura-2-parameter distances within each clade (i.e. within each morphotype) were very low (0.67% and 0.78%), with the distances between morphotypes being 9-10-fold greater (mean 7.06%). This is concordant with differences within and between species in other taxa; based upon the strict correlation between the molecular variation and the morphotypes, we propose the re-instatement of S. galeratum to species status.     

Efficiency of Simulium sanctipauli as a vector of Onchocerca volvulus in the forest zone of Ghana

The role of Simulium sanctipauli Vajime & Dunbar (Diptera: Simuliidae) as a vector of Onchocerca volvulus (Leuckart) (Spirurida: Onchocercidae) in the forest zone of central Ghana was studied in the Upper Denkyira district, where onchocerciasis is hyperendemic. Simulium sanctipauli was found to be a highly efficient vector, with a mean of 377 infective (L3) larvae in the heads of 1000 parous and 122 in the heads of 1000 biting flies. The overall infection rate of 44% of the parous flies with L1, L2 and L3 stages of O. volvulus (identity confirmed by polymerase chain reaction) demonstrates marked anthropophily. Female flies dispersed over a wide area and can transmit onchocerciasis up to at least 10 km away from their breeding sites. Annual community-directed treatments with ivermectin did not have a noticeable effect on the infection rates and parasitic loads of fly populations, which were as high 2 months after as 3 months before the distribution of ivermectin. This failure can be attributed to poor coverage, with treatment taken by only 24.4% of the population of the six study villages.