Assessments of the transmission of Onchocerca volvulus by Simulium sanctipauli in the Upper Denkyira District, Ghana, and the intermittent disappearance of the vector.

Following studies on the transmission of Onchocerca volvulus (Leuckart) by Simulium sanctipauli Vajime & Dunbar (Diptera, Simuliidae) in Upper Denkyira District in Ghana in 2001 and 2002 (Kutin et al., Med Vet Ent 18:167-173, 2004), further assessments were carried out in 2006 and 2013/2014 to determine whether transmission parameters had changed since community-directed ivermectin treatment (CDTI) began in 1999. There were no marked changes of the transmission intensities in 2006. Only slight, but non-significant, reductions were observed in infection rates of parous flies with larval stages (L1-L3) of O. volvulus from 44.1 % (of 1672 parous flies) in 2001/2002 to 42.1 % (506) in 2006 and from 6.5 to 5.9 % of flies carrying infective larvae in their heads. This suggested that there was an ongoing transmission in the area and the parasite reservoir in the human population was still high. Unexpectedly, further assessments conducted in October 2013 and March and October 2014 revealed that the vector S. sanctipauli had apparently disappeared and transmission had ceased, probably as a result of intensified gold mining activities along the rivers Ofin and Pra. The water of both rivers was extremely turbid, heavily loaded with suspended solids, probably preventing the development of blackfly larvae. Some breeding and biting of Simulium yahense Vajime & Dunbar was observed in a small tributary of the Pra, the Okumayemfuo, which is not affected by gold mining. However, the infection rate of flies was low, only 3.7 % of 163 parous flies were infected with first stage (L1) larvae of O. volvulus.

Behavioral assessment of visual deficits in the taiep mutant.

Taiep (tremor, ataxia, immobility, epilepsy, paralysis) mutants show a significant increase in myelin thickness from 10 to 30 days of age but then demonstrate a decrease in myelin thickness from 1 to 6 months. The severity of the demyelination in the optic nerve suggests that visual deficits may exist in the taiep mutants. Animals were trained on a discrimination task, in which responses to a light stimulus (the SD period) were reinforced on a fixed ratio (FR)-1 schedule, and responses in the absence of the light stimulus (the SΔ period) were not reinforced. Following training, the light intensity presented during the SD period was gradually reduced between sessions until -6.0 candela/m2 was reached. Both groups of animals - taiep mutants and control Sprague Dawley rats - successfully recognized and responded in the presence of the stimulus near perfectly by the final day of training, suggesting that taiep mutants demonstrated normal learning, at least under this paradigm. Despite the severe demyelination of the taiep optic nerve, no visual deficits were detected as both groups of animals performed similarly as the light intensity decreased. Though the myelin loss of the optic nerve may have negatively affected signal transduction, this did not result in an increase in visual threshold.

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

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

The sandflies (Diptera: Psychodidae, Phlebotominae) in military camps in northern Afghanistan (2007-2009), as identified by morphology and DNA 'barcoding'.

As part of a continuous, standardized programme of monitoring the Leishmania vectors in German military camps in northern Afghanistan between 2007 and 2009, a detailed taxonomic analysis of the endemic sandfly fauna, as sampled using light and odour-baited traps, was conducted. Of the 10 sandfly species that were recorded, six may serve as enzootic and/or zooanthroponotic vectors of parasites causing human leishmaniasis. The use of a simple DNA-'barcoding' technique based on the mitochondrial cyt b gene, to identify the collected sandflies to species level, revealed (1) a clear discrimination between the potential vector species, (2) clustering of species within most subgenera, and (3) particularly high heterogeneity within the subgenus Paraphlebotomus (Phlebotomus alexandri being grouped with Ph. papatasi rather than with other Paraphlebotomus species). The data also indicate a high level of genetic heterogeneity within the subgenus Sergentomyia but close similarity between Sergentomyia sintoni and Sergentomyia murgabiensis. The morphological similarity of many medically important sandflies can make species identification difficult, if not impossible. The new DNA-barcoding techniques may provide powerful discriminatory tools in the future.

Investigations into the isolation of the Tukuyu focus of onchocerciasis (Tanzania) from S. damnosum s.l. vector re-invasion.

As part of the feasibility study for an onchocerciasis vector elimination project we investigated the isolation of the Tukuyu focus in Tanzania from possible vector re-invasion. This was achieved by examining the distribution of the Simulium damnosum complex vector cytospecies outside the focus to look for potential sources of re-invasion. Besides cytotaxonomic identifications of the aquatic stages, we applied morphotaxonomic and molecular techniques to identify S. thyolense and confirm it as the anthropophilic species in both the Tukuyu and the neighbouring Ruvuma foci. We detected significant differences in chromosome inversion frequencies between the Tukuyu populations and those breeding to the southwest in the adjacent Songwe river basin and in northern Malawi (where there is no man-biting and no onchocerciasis), suggesting that there is not normally a great deal of migration in either direction. By contrast, populations of S. thyolense from the Tukuyu and Ruvuma foci (150km southeast of Tukuyu) were much more similar in terms of their chromosomal polymorphisms, indicating a higher possibility of re-invasion, although migration is still restricted to some extent, as indicated by some differences in chromosome polymorphisms between the two foci. Future migratory events which might be associated with vector control operations can be monitored by vector cytospecies identification, the frequency of polymorphic inversions which characterise the different vector populations, and the identification of accompanying non-vector cytospecies (e.g. S. plumbeum and cytotype Kasyabone occur exclusively in the two foci, and hence their re-appearance in Tukuyu could have only one outside source). The morphology of the scutal pattern of neonate males may act as a quick test for vector species identification where chromosome squashes are unavailable.

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

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

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

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

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

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

The elimination of the onchocerciasis vector from the island of Bioko as a result of larviciding by the WHO African Programme for Onchocerciasis Control.

The island of Bioko is part of the Republic of Equatorial Guinea and is the only island in the World to have endemic onchocerciasis. The disease is hyperendemic and shows a forest-type epidemiology with low levels of blindness and high levels of skin disease, and the whole population of 68,000 is estimated to be at risk. Control of onchocerciasis began in 1990 using ivermectin and this yielded significant clinical benefits but transmission was not interrupted. Feasibility and preparatory studies carried out between 1995 and 2002 confirmed the probable isolation of the vector on the island, the high vectorial efficiency of the Bioko form of Simulium yahense, the seasonality of river flow, blackfly breeding and biting densities, and the distribution of the vector breeding sites. It was proposed that larviciding should be carried out from January to April, when most of the island's rivers were dry or too low to support Simulium damnosum s.l., and that most rivers would not need to be treated above 500 m altitude because they were too small to support the breeding of S. damnosum s.l. Larviciding (with temephos) would need to be carried out by helicopter (because of problems of access by land), supplemented by ground-based delivery. Insecticide susceptibility trials showed that the Bioko form was highly susceptible to temephos, and insecticide carry was tested in the rivers by assessing the length of river in which S. damnosum s.l. larvae were killed below a temephos dosing point. Regular fly catching points were established in 1999 to provide pre-control biting densities, and to act as monitoring points for control efforts. An environmental impact assessment concluded that the proposed control programme could be expected to do little damage, and a large-scale larviciding trial using ground-based applications of temephos (Abate 20EC) throughout the northern (accessible) part of the island was carried out for five weeks from 12 February 2001. Following this, a first attempt to eliminate the vectors was conducted using helicopter and ground-based applications of temephos from February to May 2003, but this was not successful because some vector populations persisted and subsequently spread throughout the island. A second attempt from January to May 2005 aimed to treat all flowing watercourses and greatly increased the number of treatment points. This led to the successful elimination of the vector. The last biting S. damnosum s.l. was caught in March 2005 and none have been found since then for more than 3 years.

The elimination of the vector Simulium neavei from the Itwara onchocerciasis focus in Uganda by ground larviciding.

The Itwara focus of onchocerciasis covers an area of approximately 600 km(2) in western Uganda about 20 km north of Fort Portal. The vector is Simulium neavei, whose larvae and pupae live in a phoretic association on freshwater crabs. The phoretic host in the Itwara focus is the crab Potamonautes aloysiisabaudiae. Before any onchocerciasis control, ATPs were estimated to reach between 4500 and 6500 infective larvae per person per year. S. neavei was found to be a very efficient vector with 40% of parous flies harbouring developing larvae of Onchocerca volvulus. After 4 years of community-based distribution of ivermectin transmission was still considerable and in 1995 monthly treatment of streams with the larvicide temephos commenced in the first of three sub-foci, and was gradually extended to the whole focus. Biting S. neavei disappeared from the first sub-focus (Itwara main) in June 1996, and the last infested crab was caught in November 1996. In the second sub-focus (Siisa) treatment commenced towards the end of 1995, and the last biting fly was caught in March 1997, but a deterioration in the security situation interrupted the programme (after only three treatments in the third sub-focus). Monthly treatments restarted in the second and third sub-foci (Aswa) in September 1998, and when the situation was reassessed in 2003 no biting flies were found anywhere, and the flies had not reinvaded the first sub-focus, but infected crabs were found in the second and third sub-foci. The last treatments were carried out in April-June 2003, and since then no infested crabs have been found. In summary, no S. neavei-infested crabs have been found anywhere in the focus since June 2003 and the vector is considered eliminated from that date. However, transmission had already been halted since February 2001, when the last biting flies had been collected. The parasite reservoir should die out in the human population by 2016.

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

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

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.

Taxonomy and inventory of the cytospecies and cytotypes of the Simulium damnosum complex (Diptera: Simuliidae) in relation to onchocerciasis.

We provide an inventory of all named cytoforms of the Simulium damnosum complex (including those which are now considered invalid), along with all inversions that have been recorded (including synonyms and homonyms). There are 55 valid and distinct cytoforms known from the S. damnosum complex making it the largest sibling species complex of any vectors, and probably of any insect or other animal. All cytoforms are listed along with their fixed and diagnostic inversions and country distribution. There are 183 inversions known from the complex as a whole, of which 49% are fixed and/or diagnostic between cytoforms, and the fixed/diagnostic inversions seem to occur disproportionately on chromosome arm 2L.

Molecular systematics of five Onchocerca species (Nematoda: Filarioidea) including the human parasite, O. volvulus, suggest sympatric speciation.

The genus Onchocerca (Nematoda: Filarioidea) consists of parasites of ungulate mammals with the exception of O. volvulus, which is a human parasite. The relationship between O. volvulus, O. ochengi and O. gibsoni remains unresolved. Based on morphology of the microfilariae and infective larvae, vector transmission and geographical distribution, O. ochengi and O. volvulus have been placed as sister species. Nevertheless, the cuticle morphology and chromosomal data (O. volvulus and O. gibsoni have n=4 while O. ochengi is n=5) suggest that O. gibsoni could be more closely related to O. volvulus than O. ochengi. Sequences from the 12S rRNA, 16S rRNA and ND5 mitochondrial genes have been used to reconstruct the phylogeny of five Onchocerca species including O. volvulus. Analyses with maximum likelihood and maximum parsimony showed that O. ochengi is the sister species of O. volvulus, in accordance with the classification based on morphology and geographical location. The separate specific status of the species O. gutturosa and O. lienalis was supported, although their phylogenetic relationship was not well resolved. The analyses indicated that the basal species was O. gibsoni, a South-East Asian and Australasian species, but this result was not statistically significant. The possible involvement of sympatric speciation in the evolution of this group of parasites is discussed.

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.

Incrimination of Simulium thyolense (Diptera: Simuliidae) as the anthropophilic blackfly in the Thyolo focus of human onchocerciasis in Malawi.

The sibling species of the Simulium damnosum complex that are potential vectors in the foci of human onchocerciasis found in southern Malawi (the southern-most foci in Africa) were identified, using a combination of cytotaxonomic and molecular techniques. Simulium thyolense was found to be the most abundant species within the foci (representing 91% of the larvae collected), whilst S. kilibanum and cytoforms of the 'Ketaketa' subcomplex were the dominant members of the complex found outside of the foci. As all 188 biting female flies collected from the Thyolo, Mwanza and Mulanje foci were identified as S. thyolense, this species is probably the only significant vector of Onchocerca volvulus in the area, and the other Simulium species collected are assumed to be zoophilic. The results confirm the diagnostic value of the size of the amplicon(s) produced in PCR based on the sequences coding for the internal-transcribed-spacer region of the nuclear ribosomal DNA (ITS-1 rDNA), with a characteristic 340bp amplicon for S. thyolense. Chromosomal polymorphisms within S. thyolense indicate some degree of geographical isolation of the 'Thyolo-Mulanje' focus from the Mwanza focus.

A new cytotype of Simulium squamosum from south-west Cameroon.

A new cytotype of Simulium squamosum Enderlein (Diptera: Simuliidae) (here named S. squamosum'D') is described from around Mount Cameroon in south-west Cameroon on the basis of sex-chromosome differences on the short arm of chromosome I. Two polymorphic inversions, IS-To (a new inversion) and IS-2, are partially Y linked. These inversions usually occur together, although IS-To has a higher frequency and is more strongly Y linked than IS-2. This sex-chromosome system has not been previously described and the occurrence and evolution of sex-chromosome systems in S. squamosum is discussed.

The cytotaxonomy and morphotaxonomy of Simulium mengense (Diptera: Simuliidae).

Human onchocerciasis is hyper-endemic in south-western Cameroon and the island of Bioko. The vector on the island is the endemic 'Bioko' form of Simulium yahense. On the mainland, S. damnosum s.s., S. squamosum and S. mengense are recorded around Mount Cameroon but their contribution to transmission in the area is unclear. As elimination of onchocerciasis, through vector eradication, is a possibility on the island, it is important to be able to identify vector flies which might migrate to Bioko from the mainland. The morphologies of S. damnosum s.s. and S. squamosum are already well known. New cytotaxonomic and morphotaxonomic descriptions of S. mengense, a species which is less well known, are presented. Simulium mengense can be distinguished from the other cytospecies in the area by the presence of tufts of hair-like setae on the larval thorax, the presence of hairs on the subcostal vein of the adult female, and by the scutal pattern of the adult male. Although Vajime and Dunbar described seven fixed inversions in S. mengense, in 1977, nine were observed in the present study. Of the nine, three were the same as Vajime and Dunbar's but the other six were either missed or misinterpreted by them.

Two new cytoforms of the Simulium damnosum complex (Diptera: Simuliidae) from Malawi and Tanzania and potential onchocerciasis vectors.

During a distribution survey of Simulium damnosum s.l. around the Tukuyu onchocerciasis focus at the northern tip of Lake Malawi/Nyasa (Tanzania), we discovered two new cytoforms of the S. damnosum complex in onchocerciasis-free areas. The Nyika form is related to Simulium thyolense, a vector of onchocerciasis, and can be identified by the new inversion 3L-L on the long arm of chromosome 3. It was found breeding in five rivers in northern Malawi and neighbouring Tanzania and is assumed to be zoophilic. The Njombe form represents a member of the Sanje group of the complex and is characterized by the new diagnostic inversion 2L-35 on chromosome 2. So far, it is only known from around Njombe town in southern Tanzania, where it breeds at remarkably high altitudes. Anthropophily for the Njombe form is well known. The medical importance and systematic position of the new forms within the S. damnosum complex are discussed.