Distribution of the Simulium damnosum complex on Bioko island, Equatorial Guinea, and the potential for onchocerciasis elimination by vector eradication

Onchocerciasis is endemic on the island of Bioko, Equatorial Guinea, where it is transmitted by the 'Bioko form' of the Simulium damnosum complex, a cytospecies unique to the island. To determine the distribution of vector breeding, three dry season and two wet season expeditions were made in 1989, 1996 and 1997, and 226 of the island's 247 rivers (91.5%) were visited. Of these 226 rivers, 130 (58%) were flowing during the dry season, forty-five (20%) supported aquatic stages of Simuliidae of any species and twenty-five (11%) contained larvae or pupae of the S. damnosum complex. The twenty-one rivers not prospected were in the mountainous south of the island, where an additional seventeen rivers were reached but not satisfactorily prospected. Of these thirty-eight rivers, twenty-nine were considered highly likely to support vector breeding, bringing the total number of rivers which could harbour the vector during the dry season to fifty-four (21.9% of the island's total). Breeding was believed to be limited to river stretches below 1000 m altitude, and during the dry season the total length of those stretches which could support breeding on Bioko was estimated to be 1020 km. A combination of factors, including low river discharges during the dry season, the relatively low water temperature on Bioko, the suitability of limited stretches of most rivers as vector breeding sites and the close proximity of many rivers within a small geographical area, render the vector vulnerable to eradication by aerial treatment of rivers with insecticide. The isolation of the Bioko form of the S. damnosum complex suggests that reinvasion following treatment would be unlikely, and eradication of the vector might be achieved by a dry season larviciding programme in one or two years.     

Poor genetic differentiation but clear cytoform divergence among cryptic species in Simulium damnosum complex (Diptera: Simuliidae)

A complex cryptic species taxonomy has been developed for the flies that vector Onchocerca volvulus L euckart in A frica. This taxonomy names cytoforms of Simulium damnosum T heobald s.l. based on chromosomal inversion polymorphisms. Researchers have linked variation in cytoforms to variation in geographical distributions and biological traits, but have failed to demonstrate that many of these cytoforms are differentiated genetically or morphologically. This may be explained by recent divergence and incomplete lineage sorting of ancestral polymorphisms. Alternatively, it may be the outcome of ongoing gene flow, with inversions maintaining sets of co‐adapted alleles and selection driving inversion frequency differences across habitats. Here, our goals were to test species delimitations in the S. damnosum complex, and determine if the poor differentiation between these species is better explained by the ancestral polymorphisms or gene‐flow hypotheses. Geometric morphometric analysis and more intensive genetic sampling of populations in N igeria only reinforced the lack of clear divergence among S. damnosum complex cytoforms. By contrast, the first statistical estimates of evolutionary relationships based on chromosomal inversion polymorphism data revealed clear signs of divergence, which lends support to the ancestral polymorphisms hypothesis. But the latter analyses may be biased by non‐neutral evolution of inversion polymorphisms; more rigorous tests of cytoform‐based species hypothesis will require deeper sampling across populations and neutral genetic markers along with explicit modelling of genetic isolation and migration.     

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.     

Assessment of rDNA IGS as a molecular marker in the Simulium damnosum complex

For five cytospecies of the Simulium damnosum Theobald complex of blackflies (Diptera: Simuliidae) from West Africa, both ends of the intergenic spacer region (IGS) of the rDNA have been sequenced with the aim of developing specific molecular markers. No specific differences in these two regions were detected between Simulium sanctipauli V. & D., Simulium sirbanum V. & D., Simulium soubrense V. & D., Simulium squamosum Enderlein and Simulium yahense V. & D., except in the number of A subrepeats at the 5' end of the IGS (two in S. squamosum and four or five in the others) and in position 310 of the 3' end (a C in S. squamosum and a G in the others). However, genetic distances within and between species overlapped. These DNA sequences had no strong phylogenetic signal, and the trees obtained were mostly unresolved. Although most sequences from S. squamosum clustered together, a few of them were more similar to those in other cytospecies. These results could be explained either by hybridization with genetic introgression or by ancestral polymorphism and recent speciation.     

Cytotaxonomy of the Simulium damnosum complex from central and northeastern Tanzania.

A cytotaxonomic study of the medically important insect vector, Simulium damnosum s.l., revealed the presence of seven and possibly eight distinct taxa from central and northeastern Tanzania. Larval salivary gland polytene chromosome maps are presented for the first time for five cytotypes and one sibling species, which include the Nkusi form, the Sanje form, the Kisiwani form, Ketaketa C1 and C2, and the Kibwezi form. Inversion disequilibrium in males of the Kibwezi form indicate population substructuring is occurring and that this population may be in the early stages of speciation. Adults of the sibling species S. damnosum Kibwezi form and cytotype of the S. damnosum Nkusi form were identified using Malpighian tubule polytene chromosomes. The taxonomic status of the populations under study are discussed in relation to previously published papers and unpublished reports. Dimorphisms for centromere band enhancement occur on all three polytene chromosomes of the complement. The same centromere band can be polymorphic, sex linked, fixed, or lost in various cytotypes. In constructing a partial phylogeny, a hypothetical intermediate is proposed to account for the diverse fate of these centromere band dimorphisms and other inversion polymorphisms in different members of this nearly pan African complex. This pattern of chromosome restructuring is consistent with that seen for other species complexes within the Simuliidae.     

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).     

Population relationships of Simulium yahense and Simulium sanctipauli in the Firestone Rubber Plantation at Harbel, Liberia

Comparative genetic differences for the phosphoglucomutase and trehalase loci were surveyed in larval and adult blackfly populations of the onchocerciasis vectors Simulium yahense Vajime and Dunbar, and S.sanctipauli Vajime and Dunbar. Genotype frequencies for each stage and all populations were at Hardy-Weinberg equilibrium, indicating that S.yahense and S.sanctipauli remain genetically distinct. S.yahense populations from three different locations were found to be genotypically comparable. The larval population of S.yahense found in closest proximity to a S.sanctipauli population was found to express the phosphoglucomutase allele 1.33, characteristic of S.sanctipauli, with significantly greater frequency than other larval populations of S.yahense. This may constitute evidence of limited genetic introgression of S.sanctipauli with the S.yahense population.     

Elimination of the Djodji form of the blackfly Simulium sanctipauli sensu stricto as a result of larviciding by the WHO Onchocerciasis Control Programme in West Africa

Cytotaxonomic identifications of larvae of members of the Simulium damnosum Theobald (Diptera: Simuliidae) complex collected in forest zones of southeast Ghana and southwest Togo between 1977 and 1996 showed that the Djodji form of Simulium sanctipauli Vajime & Dunbar, a vector of onchocerciasis, was eliminated in 1988 by larvicide operations conducted by the World Health Organization (WHO) Onchocerciasis Control Programme (OCP) in West Africa. No members of the form were identified amongst 997 larvae collected up to 8 years after systematic control operations began in February 1988. The results are discussed in relation to estimates of the numbers of samples required to certify elimination and the possibility that other members of the S. damnosum complex were also eliminated by the OCP.     

Anthropophilic blackflies (Diptera: Simuliidae) and onchocerciasis transmission in southwest Ethiopia

Abstract. From May 1984 to May 1985, the transmission of onchocerciasis by blackflies (Dipteria: Simuliidae) was studied around Gilgel Ghibe and Gojeb rivers in southwest Ethiopia. The predominant anthropophilic species of Simulium at both localities were S.(Edwardsellum) damnosum sensu law and S.(Lewisellum) ethiopiense. Some S.(Anasolen) dentulosum also landed on human bait at Gilgel Ghibe river.
Dissections of S. damnosum from Gilgel Ghibe and Gojeb revealed mean parous rates of 92% (n = 18,291) and 84% (n = 9530), respectively. S. ethiopiense from Gilgel Ghibe and Gojeb showed mean parous rates of 53% (n = 322) and 93% (n = 14), respectively. Of the parous S. damnosum , 1.3% at Gilgel Ghibe and 0.5% at Gojeb harboured infective third-stage larvae (L3) of Onchocerca volvulus (or morphologically indistinguishable from it). Unknown filariae of animal origin, indicative of zoophily, were found in 0.3% and 0.7% of Simulium damnosum complex females from Gilgel Ghibe and Gojeb, respectively. S. ethiopiense harboured developing (L,) larvae only, with 7.7% infection rate in both localities. In contrast, S. dentulosum did not harbour any filaria larvae. The annual infective biting rate (AIBR) and transmission potential (ATP) of the S. damnosum complex at Gilgel Ghibe river were 858 and 5478, respectively. The AIBR and ATP of S. damnosum s.I. at Gojeb river were 519.5 and 1963, respectively. These results emphasize the predominant role of the S. damnosum complex in the transmission of O. volvulus in southwest Ethiopia     

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.     

Habitat preference of the Preuss's guenon<Emphasis Type="Italic">(Cercopithecus preussi), on Bioko island,Equatorial Guinea</Emphasis>

The Press's guenon (Cercopithecus preussi) is considered to be one of the most threatened African primates. There is little information on the ecology and status of this primate on Bioko island, where it is found in the form of an endemic subspecies. The Press's, guenon shows preference for theSchefflera forest and the mountain habitat on Bioko island. As on the mainland they also have semitterrestrial habits and are found usually at the understorey of the forest. Competitive exclusion between this guenon and other guenon species could be an explanation of these ecological preferences. Habitat use and vertical stratification of the activity in the forest canopy seem to reduce competition with other sympatric primate species that inhabit on the island. Habitat destruction, and isolation in a reduced habitat, show to be the major threats for the survival of this primate on Bioko island.     

Evidence for X-linked introgression between molecular forms of Anopheles gambiae from Angola

The M and S molecular forms of the African malaria vector Anopheles gambiae (Diptera: Culicidae) are morphologically identical incipient species in which reproductive isolation is incomplete, enabling low-level gene flow between forms. In an attempt to find differences between the M and S forms, sequence variation was studied at loci along the X chromosome in adult female An. gambiae from Angola. A high proportion of M form specimens from Angola (79% of the 456 X chromosomes sampled) were found to contain a 16-bp insertion in intron 4 of the X-linked GPRCCK1 locus, relative to the AgamP3 release of the An. gambiae PEST genome sequence. The insertion was in Hardy-Weinberg equilibrium in Angolan M form populations. The same insertion was found in all S form specimens examined, regardless of where in Africa they were sampled, but was absent from a sample of M form specimens collected in Ghana, Bioko and Mali. In M form specimens from Angola, there was an association between alleles at the GPRCCK1 locus and those at a microsatellite locus, AGXH678, close to the centromere of the X chromosome, with significant linkage disequilibrium between loci separated by 0.472 Mbp (P < 0.033). We show that the insertion results from introgression from the S form into the M form, rather than from the retention of an ancestral character. Gene flow from the S to M form could allow genes of adaptive value to be transferred, including those conferring insecticide resistance and others influencing ecology and behaviour, and thus malaria transmission and control. We discuss factors that may have led to this introgression event.     

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.     

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.     

Macrogenomic Evidence for the Origin of the Black Fly Simulium suzukii (Diptera: Simuliidae) on Okinawa Island,Japan

To determine the geographic origin of the black fly Simulium suzukii on Okinawa Island, Japan, macrogenomic profiles derived from its polytene chromosomes were compared with those of mainland and other insular populations of S. suzukii and of the isomorphic Simulium tani species complex. The Okinawan population is a chromosomally unique cytoform, designated ‘D,’ which is essentially monomorphic and differs by about 27 fixed rearrangements from the chromosomal standard sequence for the subgenus Simulium and by two fixed differences from its nearest known relative, representing the type of S. suzukii, on the main islands of Japan. Chromosomal band sequences revealed two additional, sympatric cytoforms of S. suzukii, designated ‘A’ and ‘B,’ each with species status, in Korea, and a third cytoform, designated ‘C,’ on Hokkaido, Japan. A new cytoform, ‘K,’ of S. tani from Malaysia, representing the type of S. tani, is more closely related to cytoforms in Thailand, as are populations from Taiwan previously treated as S. suzukii but more closely aligned with S. tani and newly recognized as cytoform ‘L’ of the latter nominal species. Rooting of chromosomal band sequences by outgroup comparisons allowed directionality of chromosomal rearrangements to be established, enabling phylogenetic inference of cytoforms. Of 41 macrogenomic rearrangements discovered in the five new cytoforms, four provide evidence for a stepwise origin of the Okinawan population from populations characteristic of the main islands of Japan. The macrogenomic approach applied to black flies on Okinawa Island illustrates its potential utility in defining source areas for other species of flies including those that might pose medical and veterinary risks.     

Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

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.     

A new species of the Simulium damnosum complex from Uganda, and comparative morphology of the tarsal claws in females of this complex

The female, male, pupa and larva of Simulium (Edwardsellum) pandanophilum sp. nov. (Diptera: Simuliidae) from western Uganda are described. Diagnostic features are the shape of the tarsal claws of females and, cytogenetically, new intraspecific inversions on chromosomes I and II. Simulium pandanophilum is assigned to the Simulium damnosum complex. Different tarsal claw shapes of West African members of the S. damnosum complex are also described.     

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.