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.     

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.     

Four new cytotypes of the onchocerciasis vector blackfly Simulium guianense in Brazil

Abstract. Simulium (Trichodagmia) guianense is an important Amazonian vector of onchocerciasis. Examination of the polytene chromosome banding patterns of larvae from five sites in Brazil revealed the occurrence of four cytotypes, designated A, B, C and D. The chromosomal standard, Simulium guianense A , occurred at two localities in Goias State (on the Rio Tocantins and Rio Mucambao) where it was the only cytotype. The other three sites examined yielded one different cytotype from each: B from Rio Oyapoque in Amapa State, C from Rio Tocantins in Maranhao State, and D from Rio Xingu in Para State. All cytotypes differed by at least two fixed inversions, but a sex determining system was not evident in any cytotype. As the cytotypes have been found allopatrically it is not certain that they represent sibling species; sampling of sympatric populations would resolve this. During certain times of the year, voracious anthropophagy by S.guianense sensu lato occurs at the localities sampled for cytotypes A, C and D (biting data are not available for the cytotype B locality). In some other areas, however, S.guianense s.l. is entirely zoophilic. Further studies are needed, therefore, to elucidate the biting habits, vectorial capacity, geographic distribution and taxonomic status of these four, and perhaps additional, cytotypes comprising the S.guianense complex.     

Distribution of the Simulium metallicum complex in Mexico in relation to selected environmental variables

Larvae of the Simulium metallicum complex (Diptera: Simuliidae) were collected from three foci of human onchocerciasis in Mexico. Specimens were separated into five different cytotypes, identified by morphological characteristics (head pattern and body colour) or polytene chromosome features. Differences were found between foci in the cytotype composition of the S. metallicum complex. Nearly all specimens were cytotype A in the Chamula (97%) and Soconusco (86%) foci. In the Oaxaca focus, however, cytotype I predominated (63%) with 14% cytotype A. Cytotype I comprised only 6% of specimens in the Soconusco focus and was very rare in the Chamula focus. Cytotypes B and H occurred only rarely in all three foci. Cytotype X was found only in Oaxaca. Environmental variables were measured at the collection sites and canonical correspondence analysis (CCA) was used to investigate the associations of cytotype distributions with the following factors: stream size, amount of shade, water clarity, pH, temperature and altitude. Members of the S. metallicum species complex were found to be differentially distributed according to stream conditions and there was a significant correlation between their distributions and the environmental variables. The most important factor in canonical axis 1 of the CCA was pH (t-value -4.38) with temperature (-2.48) and altitude (2.19) having some influence, but the other variables were unimportant. In the second canonical axis, pH (-3.52) was the only variable having a significant effect. Thus, cytotype A was associated with high pH, high temperature and low altitude; B was associated with similar sites but with higher temperatures and at lower average elevations. Cytotype H was found at the centres of the ranges of these variables, I at sites with the lowest temperatures and highest altitudes and X in rivers with the lowest pH.     

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.     

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.     

Polytene chromosomes of Simulium craigi (Diptera: Simuliidae)

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype CC from Pennsylvania; cytotype AF from Ontario and Manitoba; and cytotype ACF/BCF from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.     

Simulium metallicum cytospecies E larval habitat characterization in the Altamira focus of onchocerciasis, northern Venezuela

Abstract. Simulium metallicum sibling species E was identified cytotaxonomically from an onchocerciasis focus at Altamira in northern Venezuela. S. metallicum E larvae were sampled monthly from two small mountain streams over a 15-month period (July 1990 to September 1991) and eleven habitat variables were measured at two altitudes. One stream consistently harboured much higher densities of S. metallicum E larvae than the other, with three annual peaks of abundance: during the dry season and at the beginning and end of the rainy season. These peak densities were correlated with high rainfall 4 months previously. Larvae were most abundant on submerged rocks and fallen leaves, in small shallow areas characterized by slow water current, high conductivity and sparse terrestrial vegetation cover. Stream variables which best explained the temporal changes in abundance were water discharge and conductivity. The population dynamics of S. metallicum E appeared to be influenced primarily by interactions between stream discharge and substrate stability. Relevance of these results to vector control with larvicides is discussed.     

An epizootic of patent iridescent virus disease in multiple species of blackflies in Chiapas, Mexico

Simulium blackfly larvae (Diptera: Simuliidae) were collected from rivers and streams at 500-1500 m a.s.l. in Chiapas State of southern Mexico. Among 45 sites surveyed over an area of 2300 km2 (around 15 degrees 15'N 92 degrees 20'W), some Simulium larvae from three sites were opalescent violet-blue, interpreted as patent infection with invertebrate iridescent virus (IIV). Dissection confirmed the presence of putative Iridovirus particles, 130nm diameter, but no IIV isolates were obtained from homogenates injected into Galleria mellonella (L) larvae (Lepidoptera: Pyralidae). All Simulium with patent IIV infection died before metamorphosis, whereas approximately 60% of asymptomatic Simulium survived to adulthood in the laboratory. During 1997, standard monthly samples from two parallel rivers 42-50 km north-west of Tapachula comprised the following species proportions (and rates of patent IIV infection): 41.8% (47%) Simulium mexicanum Bellardi complex, 31.3% (31.4%) S. rubicundum Knab, 10.1% (13.1%) S. paynei, 6.5% (2.9%) S. callidum (Dyar & Shannon), 6.3% (5.1%) S. ochraceum Walker complex, 3.1% (0.7%) S. downsi Vargas et al., 0.7% S. samboni Jennings and 0.2% S. metallicum Bellardi complex, showing a strong correlation between blackfly abundance and the prevalence of patent infection. An epizootic of IIV in January and February (infection rates 41-100%) was followed by absence of larvae (March-August) until the end of the rainy season, when numbers collected on nylon strings rose to approximately 1/cm with patent IIV infection rates of 0-12.5% during September-December. Further investigations are underway to isolate this IIV and assess its potential usefulness for biological control of Simulium pests and vectors of onchocerciasis.     

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.     

Dispersal range of Simulium neavei in an onchocerciasis focus of western Uganda

The dispersal range of the vector Simulium neavei Roubaud (Diptera: Simuliidae) was investigated in an onchocerciasis focus in Kabarole district, western Uganda, adjacent to the Itwara Forest Reserve, where the species breeds in phoretic association with the river crab Potamonautes aloysiisabaudiae (Nobili) (Decapoda: Potamonautidae). Biting rates were highest at the edge of the forest, but blood-seeking flies were collected up to a distance of about 4 km from the forest. Entomological data did not agree with the geographical distribution of onchocerciasis, which is hyperendemic in larger distances from the forest. The possible reasons are discussed.     

Temephos-resistant larvae of Simulium sanctipauli associated with a distinctive new chromosome inversion in untreated rivers of south-western Ghana

Larvae of the Simulium damnosum Theobald complex (Diptera: Simuliidae) were sampled in June 1996 from two sites in south-west Ghana where larviciding has not been applied: Sutri Rapids on the Tano river (05 degrees 23 minutes N 02 degrees 38 minures W) and Sekyere-Heman on the Pra river (05 degrees 11 minutes N 01 degrees 35 minutes W). All specimens were identified as Simulium sanctipauli Vajime & Dunbar sensu stricto (Diptera: Simuliidae). Bioassays with temephos (organophosphorus larvicide employed by the Onchocerciasis Programme for systematic treatment of most rivers across West Africa since the 1970s) showed about five-fold resistance in the Tano population (LC95 2.37-3.14 mg/L) and slight tolerance to temephos in the Pra population (LC95 0.67-0.76 mg/L), vs. the diagnostic concentration of 0.625 mg/L. Larval salivary polytene chromosomes of S. sanctipauli showed fixed inversions 1S-24/24, standard IIL-6 and a new inversion IL/36 polymorphism at Sutri on the Tano. These karyotype characteristics differ from those of temephos-resistant S. sanctipauli in rivers of C te d'Ivoire and other sites on the Tano in Ghana. Thus, temephos resistance in S. sanctipauli at Sutri is associated with distinct chromosomal configurations, showing that immigration was unlikely. This resistance could have been locally selected by exposure of S. sanctipauli larval populations to agrochemicals run-off from cocoa, coffee and oil plantations flanking the rivers.     

Onchocerciasis transmission levels and Simulium damnosum complex biting activity at riverside and rice field sites in Sierra Leone

Biting densities of the Simulium damnosum Theobald complex of blackflies and their levels of parasitism by Onchocerca volvulus (Leuckart) were compared at two ecologically contrasted sites in the Southern Province of Sierre Leone, West Africa: by the Tabe riverside close to Gbaiima village where onchocerciasis is hyperendemic (63.1% positive skin-snips) and in open rice fields 2 km from the mesoendemic (54.9%) village of Senehun. Using a combination of morphometric and enzyme electrophoresis techniques, the primary vector was found to belong to the S.sanctipauli subcomplex and was most probably S.soubrense 'B' (sensu Post, 1986). In the wet season biting activity took place throughout the day at the village, but had morning and evening peaks in the rice fields. Transmission of O.volvulus was detected at the village in all months except February-April, while in the rice fields it was detected only during September-November and May at one tenth the level of the village. There was a significant difference between the two sites in the proportion of parous flies carrying third stage larvae in the head, but not of other larval stages.     

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.     

Laser‐assisted microdissection of polytene chromosomes from Diptera for the development of molecular markers

The structural details visible under the light microscope have made polytene chromosomes from Diptera much used in studies of the taxonomy, evolution and genetics of important biological models such as Drosophila and Chironomus, and the medical entomology of important disease vectors such as blackflies and mosquitoes. This paper describes the isolation of sections of polytene chromosomes from preserved wild‐caught blackflies using off‐the‐shelf laser microdissection microscopy and subsequent analysis of the DNA using polymerase chain reaction. This allows a direct link between the visible structure of the genome and the unknown DNA sequence, facilitating the development of molecular markers for population cytogenetics and cytotaxonomy.     

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.