Rory J. Post

Evolution, epidemiology, and population genetics of black flies (Diptera: Simuliidae)

More than 2000 species of black flies feed on vertebrate blood; 1.5% of all species are vectors of pathogens that cause human diseases. Of nine simuliid-borne animal diseases, only two, mansonellosis and onchocerciasis, afflict humans. Onchocerciasis is a debilitating disease infecting an estimated 40 million people in Africa, Latin America, and Yemen, whereas mansonellosis is a mild disease in the Neotropics. Cytogenetic studies of natural populations of more than 500 species of black flies have revealed that the classic morphospecies of taxonomists is typically a complex of two or more reproductively isolated entities, or sibling (cryptic) species. Most vectors of human pathogens are sibling species, each ecologically unique in traits such as breeding habitats, dispersal capabilities, and degree of vector competence. We review the evolution of black flies, the cytogenetics that have revealed about 260 cytologically distinct entities, the molecular studies that continue to expose additional hidden biodiversity, and a case study of the epidemiology of the Simulium damnosum complex, the largest species complex of blood-feeding arthropods on Earth and the premier group of black flies responsible for human onchocerciasis.

Characterisation of nuclear ribosomal DNA sequences from Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea) and development of a PCR-based method for their detection in skin biopsies☆

The internal transcribed spacer region (ITS1, 5.8S gene and ITS2) of the two filarial nematodes Onchocerca volvulus and Mansonella ozzardi was sequenced, and two species-specific primers designed in the ITS2 to develop a PCR-based method for their specific detection and differentiation. When used with a universal reverse primer, the two species-specific primers gave amplification products of different size, which were readily separated in an agarose gel. The PCR was tested on skin biopsies from 51 people from three localities in Brazil where M. ozzardi is present, and results have been compared with those of parasitological examination of blood. The species-specific PCR gave a higher percentage of detection of infection by M. ozzardi than the parasitological examination of blood. No infection with O. volvulus was detected by PCR. This PCR-based assay may assist in determining the nature of infection in areas where both filarial species exist in sympatry.

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.

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.

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.

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.

Onchocerciasis transmission in Ghana: persistence under different control strategies and the role of the simuliid vectors.

Background The World Health Organization (WHO) aims at eliminating onchocerciasis by 2020 in selected African countries. Current control focuses on community-directed treatment with ivermectin (CDTI). In Ghana, persistent transmission has been reported despite long-term control. We present spatial and temporal patterns of onchocerciasis transmission in relation to ivermectin treatment history. Methodology/Principal Findings Host-seeking and ovipositing blackflies were collected from seven villages in four regions of Ghana with 3–24 years of CDTI at the time of sampling. A total of 16,443 flies was analysed for infection; 5,812 (35.3%) were dissected for parity (26.9% parous). Heads and thoraces of 12,196 flies were dissected for Onchocerca spp. and DNA from 11,122 abdomens was amplified using Onchocerca primers. A total of 463 larvae (0.03 larvae/fly) from 97 (0.6%) infected and 62 (0.4%) infective flies was recorded; 258 abdomens (2.3%) were positive for Onchocerca DNA. Infections (all were O. volvulus) were more likely to be detected in ovipositing flies. Transmission occurred, mostly in the wet season, at Gyankobaa and Bosomase, with transmission potentials of, respectively, 86 and 422 L3/person/month after 3 and 6 years of CDTI. The numbers of L3/1,000 parous flies at these villages were over 100 times the WHO threshold of one L3/1,000 for transmission control. Vector species influenced transmission parameters. At Asubende, the number of L3/1,000 ovipositing flies (1.4, 95% CI = 0–4) also just exceeded the threshold despite extensive vector control and 24 years of ivermectin distribution, but there were no infective larvae in host-seeking flies. Conclusions/Significance Despite repeated ivermectin treatment, evidence of O. volvulus transmission was documented in all seven villages and above the WHO threshold in two. Vector species influences transmission through biting and parous rates and vector competence, and should be included in transmission models. Oviposition traps could augment vector collector methods for monitoring and surveillance.

Onchocerciasis transmission in Ghana: biting and parous rates of host-seeking sibling species of the Simulium damnosum complex

BackgroundGhana is renowned for its sibling species diversity of the Simulium damnosum complex, vectors of Onchocerca volvulus. Detailed entomological knowledge becomes a priority as onchocerciasis control policy has shifted from morbidity reduction to elimination of infection. To date, understanding of transmission dynamics of O. volvulus has been mainly based on S. damnosum sensu stricto (s.s.) data. We aim to elucidate bionomic features of vector species of importance for onchocerciasis elimination efforts.MethodsWe collected S. damnosum sensu lato from seven villages in four Ghanaian regions between 2009 and 2011, using standard vector collection, and human- and cattle-baited tents. Taxa were identified using morphological and molecular techniques. Monthly biting rates (MBR), parous rates and monthly parous biting rates (MPBR) are reported by locality, season, trapping method and hour of collection for each species.ResultsS. damnosum s.s./S. sirbanum were collected at Asubende and Agborlekame, both savannah villages. A range of species was caught in the Volta region (forest-savannah mosaic) and Gyankobaa (forest), with S. squamosum or S. sanctipauli being the predominant species, respectively. In Bosomase (southern forest region) only S. sanctipauli was collected in the 2009 wet season, but in the 2010 dry season S. yahense was also caught. MBRs ranged from 714 bites/person/month at Agborlekame (100% S. damnosum s.s./S. sirbanum) to 8,586 bites/person/month at Pillar 83/Djodji (98.5% S. squamosum). MBRs were higher in the wet season. In contrast, parous rates were higher in the dry season (41.8% vs. 18.4%), resulting in higher MPBRs in the dry season. Daily host-seeking activity of S. damnosum s.s./S. sirbanum was bimodal, whilst S. squamosum and S. sanctipauli had unimodal afternoon peaks.ConclusionsThe bionomic differences between sibling species of the S. damnosum complex need to be taken into account when designing entomological monitoring protocols for interventions and parameterising mathematical models for onchocerciasis control and elimination.

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

Abstract 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.

Sex chromosome variation and cytotaxonomy of the onchocerciasis vector Simulium squamosum in Cameroon and Nigeria

Abstract. On the basis of sex chromosome variation, three cytotypes of Simulium squamosum (Enderlein) (Diptera: Simuliidae) are described from Cameroon and Nigeria. Simulium squamosum A is the typical form as originally described by Vajime & Dunbar (1975) with chromosome I as the sex chromosome. It occurs throughout most of Cameroon and south‐east Nigeria. A second cytotype, S. squamosum B, is described from the river Sanaga (Cameroon). It also has chromosome I as the sex chromosome, but the nature of the sex differential region is different. Simulium squamosum C has no sex‐linked chromosomal rearrangements. It is widespread in Nigeria and occurs near Mount Cameroon, where it seems to hybridize with S. squamosum A.