Development of Onchocerca volvulus microfilariae injected into Simulium species from Cameroon

Microfilariae (mff) of the savanna and forest strains of Onchocerca volvulus (Leuckart) were injected intrathoracically into adult females of Simulium damnosum Theobald sensu stricto, S.sirbanum Vajime & Dunbar, S.squamosum Enderlein and S.mengense Vajime & Dunbar. Nine days post infection (pi) 27-29% of the savanna mff and 31-38% of the forest strain had developed to third-stage larvae (L3), irrespective of the fly species, size or injection dose (5, 10 or 15 mff). Savanna flies supported the development of forest O.volvulus better than forest flies, in contrast to the results after per os infections. Therefore, in these four species of the S.damnosum complex from Cameroon, the peritrophic membrane is considered to be the main factor limiting the success rate of microfilarial development following the ingestion of blood infections, while the fly's haemolymph and intracellular environment play minor roles.     

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.     

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     

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.     

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.     

Structure and function of the cibarial armature in Simuliidae

Abstract. Cibarial armature morphology in adult female blackflies (Diptera: Simuliidae) is described using scanning electron microscopy. Three distinct types of armature are recognized, comprising those with teeth, e.g. Simulium ochraceum, S. ornatum, S. veracruzanum and S. vorax ; those with spicules, e.g. Austrosimulium bancrofti, S.damnosum, S.exiguum, S.metallicum and S.neavei; and those lacking these projections, e.g. Prosimulium rufipes and S.lineatum.
Whereas the armature is poorly developed in vectors of human onchocerciasis such as S. damnosum, S. exiguum, S. metallicum and S. neavei , the well-developed armature in S. ochraceum, S. veracruzanum and S. vorax does not prevent these species becoming infected with Onchocerca spp. (Nematoda: Onchocercidae). Hence the armature is not primarily a mechanism to counteract microfilaria superinfection.
Since cibarial armatures are more developed in the haematophagous females than in the males of certain Families of flies, e.g. Ceratopogonidae, Culicidae, Phlebotominae and Simuliidae in the sub-order Nematocera, evidently the armature has evolved in response to the blood-feeding habit. As the suction of imbibed blood by the cibarial pump may require a valve mechanism to prevent back-flow, it is suggested that the armature is primarily for this purpose. Secondarily, the cibarial armature presents a damaging barrier against ingested microfilariae.     

尼日利亚西南部奥孙河沿线黑蝇Simulium damnosum Theobald complex的形态分类研究(双翅目:蚋科)

黑蝇Simulium damnosum sensu lato是由多个姊妹种组成的复合体,这些种在生态学和盘尾丝虫病的传播方面各不相同.本文对奥孙河沿线的尼日利亚西南部森林区域的黑蝇S.damnosum s.l.复合体的组成以及成虫的形态学特征进行了研究.本研究所用的黑蝇S.damnosum s.l.成虫标本来源于奥孙河沿线的3个区,分别是Osun Eleja,Osun Ogbere和Osun Budepo.标本采集通过人体诱捕的方式,采集时间从2008年2月至2009年6月上午7:00到下午6:00,每两周采集1次.通过观察成虫的翅毛簇(wing tufts)和其他形态特征,对其进行分类研究.结果表明:存在同域分布的森林种和稀树草原种.在奥孙河沿线3个区内,森林种为优势种,占总捕获量的99.18%,而稀树草原种仅占0.82%;森林种和稀树草原种在多度上存在显著差异(P<0.05).所捕获的所有稀树草原种的翅毛簇均为灰白色,而捕获的森林种的翅毛簇颜色存在显著差异(P<0.05).为了更清楚地揭示该地区黑蝇S.damnosum s.l.的物种组成,建议进行更深入的研究.     

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.     

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.     

Longitudinal evaluation of repellent activity of Ocimum gratissimum (Labiatae) volatile oil against Simulium damnosum

To determine the repellent activity of Ocimum gratissimum volatile oil against Simulium damnosum (blackflies), a 12 month (January-December 2003) field study was conducted in three onchocerciasis endemic communities (Idomido, Obio camp, and Ikot Adaha) in Ini Local Government Area of Akwa Ibom State, Nigeria. The result revealed that topical application of 20% (v/v) concentration of the oil with liquid paraffin as a base, reduced the biting rate of S. damnosum by 90.2, 81.6, and 79.7%, in Idomido, Obiocamp, and Ikot Adaha respectively. The oil gave protection against the bite of S. damnosum for at least 3 h. A total of 710 adults S. damnosum were caught by individuals treated with Ocimum oil, as against 4296 caught by the control group. When the flies caught by the treated individuals were dissected none of them was infected with microfilariae of Onchocerca volvulus. Human-vector contact and onchocerciasis transmission could be reduced by the topical application of the volatile oil during the peak biting periods of the vector     

Transmission of Onchocerca volvulus and prospects for the elimination of its vector, the blackfly Simulium neavei in the Mpamba-Nkusi focus in Western Uganda

The transmission of Onchocerca volvulus Leuckart (Spirudida: Onchocercidae) and the prospects of Simulium neavei Roubaud (Diptera: Simuliidae) vector elimination through ground larviciding were investigated in the Mpamba-Nkusi focus, western Uganda. Transmission levels and the initiated vector elimination activities were assessed to supplement the ongoing ivermectin mass distribution programme. Searches for breeding sites, adult fly catches, dissection of flies, river treatment with temephos (Abate) and a review of annual ivermectin treatment data were conducted. High levels of crab infestation with S. neavei sensu stricto immature stages were recorded; 57.9% and 100% for the Mpamba and Nyabugando river systems, respectively. The mean numbers of larvae/pupae per crab were 3.6 +/- 0.5 in the Mpamba and 20.6 +/- 1.8 in the Nyabugando systems. Pre-intervention mean biting densities were 39 and 32 flies/(man day) in 2001 and 2002, respectively, and an annual biting rate in 2001 of > 14 000. The bimodal biting pattern of S. neavei s.s. consisted of two peaks; one in the morning (09.00-10.00 hours) and one in the afternoon (14.00-15.00 hours) with a mid-day lull in biting. The infection/infective rates were 13.3%/2.8% and 16.6%/2.9% in the dissected parous flies from the Mpamba and Nyabugando river systems, respectively. Out of approximately 1000 parous flies, 129 and 109 were found to be harbouring infective larvae of Onchocerca volvulus in their heads from the Mpamba and Nyabugando river systems, respectively. In spite of the > 10 years of ivermectin treatment, at a mean coverage of 71.3%, infection remained relatively high. Ground larviciding with temephos (Abate) initiated in June and October 2002 had a significant impact. In the Mpamba river system there was a significant (P < 0.001) reduction in positive crabs from 57.9% in 2001 to 0.06% in 2003 and a decrease in the mean number of larvae/pupae per crab from 3.6 +/- 0.5 in 2001 to 0.0007 +/- 0.0001 (P < 0.002) in 2003. Similarly, in the Nyabugando river system, a significant (P < 0.001) reduction in crab infestation from 100% in 2001 to 0.06% in 2003 and a decrease in the mean number of larvae/pupae per crab from 20.6 +/- 1.8 in 2001 to 0.06 +/- 0.03 in 2003. Drastic reductions were observed in the mean number of biting flies from 3 flies/h in 2001 to 0 flies/h in 2003 and the annual biting rates fell from 14,235 flies/year in 2001 to only 730 flies/year in 2003. These data suggest that substantial progress towards the goal of S. neavei s.s. vector elimination has been made and this will enhance the ongoing ivermectin treatment in this isolated focus.     

Infective larvae of five Onchocerca species from experimentally infected Simulium species in an area of zoonotic onchocerciasis in Japan

Microfilariae of five Onchocerca species, O. dewittei japonica (the causative agent of zoonotic onchocerciasis in Oita, Kyushu, Japan) from wild boar (Sus scrofa), O. skrjabini and O. eberhardi from sika deer (Cenus nippon), O. tienalis from cattle, and an as yet unnamed Onchocerca sp. from wild boar, were injected intrathoracically into newly-emerged black flies of several species from Oita to search the potential vector(s) of these parasites and identify their infective larvae. Development of O. dewittei japonica microfilariae to the infective larvae occurred in Simulium aokii, S. arakowae, S. bidentatum, S. japonicum, S. quinquestriatum, and S. rufibasis while development of infective larvae of O. skrjabini, O. eberhardi, and the unnamed Onchocerca sp. was observed in S. aokii, S. arakawae, and S. bidentatum. Development of O. lienalis microfilaria to infective larvae occurred in S. arakawae. Based on the morphology of infective larvae obtained, we proposed a key of identification of Onchocerca infective larvae found in Oita. We also reconsider the identification of three types of infective larvae previously recovered from Simulium species captured at cattle sheds: the large type I larvae that may be an undescribed species; the small type III identified as O. lienalis may include O. skrjabini too; the intermediary type II that may be O. gutturosa, or O. dewittei japonica, or the unnamed Onchocerca sp. of wild boar.     

Black flies (Diptera: Simuliidae) attracted to humans and water buffalos and natural infections with filarial larvae,probably Onchocerca sp., in northern Thailand

Several Simulium species were investigated as to their biting habits and natural infections with filarial larvae at Ban Pan Fan, Chiang Mai Province, in northern Thailand. Female adults flies landing on or flighting around a human and a water buffalo were collected during the daytime from 06.00 to 19.00 hours on 22 June 2001. As a result, 217 S. nodosum, 86 S. asakoae and two S. nigrogilvum were obtained from a human attractant, and 416 S. nodosum, 25 S. nakhonense, 16 S. asakoae, four S. fenestratum and two S. nigrogilvum, from a water buffalo. The blood-feeding was confirmed only for S. nodosum and S. nigrogilvum on humans, and for S. nodosum and S. nakhonense on water buffalos. Dissections of these simuliids showed that S. nodosum was naturally infected with developing filarial larvae. Two types of microfilariae were distinguished but only one type of infective larvae. These larvae resembled Onchocerca suzukii, a parasite from a wild Japanese bovid, suggesting that an unknown Onchocerca species from ruminants was transmitted in Thailand. Infection rates with all stages of larvae and third-stage larvae were 2.3% (14/608) and 1.0% (6/608), respectively. This is the first report of natural infections of black flies with Onchocerca larvae in Southeast Asia, and the involved black fly species is shown to be not only anthropophilic but also zoophilic in this region.     

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.     

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.     

Cytotaxonomy,morphology and molecular systematics of the Bioko form of Simulium yahense (Diptera: Simuliidae)

Cytotaxonomic analysis of the polytene chromosomes from larvae of the Simulium damnosum Theobald complex from the island of Bioko in Equatorial Guinea is reported, and a new endemic cytoform is described. Chromosomally this cytoform is close to both S. squamosum (Enderlein) and S. yahense Vajime & Dunbar, but is not identical to either. However, it is morphologically and enzymatically identical to S. yahense. The Bioko form was also found to differ from other cytoforms of the S. damnosum complex in West Africa in the copy number or RFLP pattern of several different repetitive DNA sequences. It is clear that the Bioko form is genetically distinct from other populations of the S. damnosum complex, and whilst it is closest to S. yahense, it shows features that suggest a high degree of geographical and genetic isolation. Such isolation is an important consideration in the assessment of the potential for onchocerciasis vector eradication on Bioko.     

Potential effects of warmer worms and vectors on onchocerciasis transmission in West Africa

Development times of eggs, larvae and pupae of vectors of onchocerciasis (Simulium spp.) and of Onchocerca volvulus larvae within the adult females of the vectors decrease with increasing temperature. At and above 25°C, the parasite could reach its infective stage in less than 7 days when vectors could transmit after only two gonotrophic cycles. After incorporating exponential functions for vector development into a novel blackfly population model, it was predicted that fly numbers in Liberia and Ghana would peak at air temperatures of 29°C and 34°C, about 3°C and 7°C above current monthly averages, respectively; parous rates of forest flies (Liberia) would peak at 29°C and of savannah flies (Ghana) at 30°C. Small temperature increases (less than 2°C) might lead to changes in geographical distributions of different vector taxa. When the new model was linked to an existing framework for the population dynamics of onchocerciasis in humans and vectors, transmission rates and worm loads were projected to increase with temperature to at least 33°C. By contrast, analyses of field data on forest flies in Liberia and savannah flies in Ghana, in relation to regional climate change predictions, suggested, on the basis of simple regressions, that 13–41% decreases in fly numbers would be expected between the present and before 2040. Further research is needed to reconcile these conflicting conclusions.     

Development of Onchocerca volvulus (Filarioidea: Onchocercidae) in the West African black fly Simulium yahense (Diptera: Simuliidae) in Liberia.

Simulium yahense black flies infected with microfilaria of Onchocerca volvulus were kept in a defined insectary environment in Liberia, West Africa. A daily sample of infected flies was dissected for larvae developing in the thoracic muscles and examined for growth in stadial development. Microfilariae ingested by black flies transformed to the L1 larval stage without molting. Successive larval development included molting to the L2 stage and, finally, to the L3 stage, which was infective in humans. The cephalic cap, consisting of a laterally located hook and central stoma, occurs in the first larval stage. The caudal appendix and the laterally located anal opening are apparent in the L1 larva. In the L2 stage, the cephalic cap is lost and the large circular stoma becomes surrounded with elevated flaps. The caudal appendix was lost after larvae molted to the L3 stage, and in its place, 3 terminal papillae developed. Sense organs, such as 2 opposing phasmids and 8 papillae that were arranged into 2 circles, developed in the cephalic region of the L3 larva. The evidence of pathological consequences due to the presence of the L3 larva in the fly host are illustrated and discussed.