RESTRICTION ENZYME ANALYSIS OF DNA FROM SPECIES OF BULINUS (BASOMMATOPHORA: PLANORBIDAE) USING A CLONED RIBOSOMAL RNA GENE PROBE

A ribosomal RNA gene probe (pSM889) has been used to study restrictionenzyme digests of various species of Bulinus. In order to minimiseproblems of DNA shearing associated with snail tissues a methodof extracting nucleic acids from material embedded in agaroseblocks has been used. Restriction enzyme digests with Bgl IIand Bam HI hybridised to pSM889 showed clear differences betweenB. truncatus, B. wrighti, B. africanus and B. forskalii, representingthe four species groups of Bulinus. No differences were observedbetween samples of B. tropicus and B. truncatus digested withBam HI, Bgl II and Pst I. Intra-specific variation was observedbetween samples of B. forskalii from Säo Tomé andAngola digested with Bgl II and Hind III although restrictionprofiles for Bam HI, Pst I and Bst EII digests were similar.Intra-specific variation was also observed between two differentpopulation samples of B. wrighti from South Yemen using BamHI and Bgl II digested genomic DNA hybridised to pSM889. (Received 5 December 1989; accepted 19 April 1990)     

STUDIES ON THE MORPHOLOGY AND CROSSBREEDING ABILITY OF TWO POPULATIONS OF BULINUS GLOBOSUS FROM NORTHERN NIGERIA

The general appearance and dimensions of the shells and copulatoryorgans of two populations of Bulinus globosus (Lake Chad andNgala) from the area of the South Chad Irrigation Project, Nigeria,were compared using samples bred in laboratory cultures. The body tissues of Lake Chad snails were darker than thoseof individuals from the Ngala sample, and lacked the distinctblack patches seen on the mantle of the latter. Significant differences in the occurrence of copulatory organeversion on relaxation in menthol were observed between thesamples. Both the total length and relative dimensions of preputiumand penis sheath also differed significantly between the LakeChad and Ngala populations. Crossbreeding experiments using enzymes (GPI and HBDH) as geneticmarkers showed that cross-fertilisation between Lake Chad andNgala snails is possible. The observed physical differencessprobably resulting from the ecological separation of these twopopulations have not resulted in their reproductive isolation. ^*Present address: Department of Zoology, Oregon State University,Coroallis, Oregon 97331, USA. (Received 14 October 1986;     

Molecular identification of schistosome intermediate hosts: case studies of Bulinus forskalii group species (Gastropoda: Planorbidae) from Central and East Africa

African freshwater snails of the genus Bulinus act as intermediate hosts for schistosomes, trematode parasites responsible for medical and veterinary forms of schistosomiasis. The relationship between these snails and their parasites is an intricate one, with particular species of snail susceptible to infection only by certain species of schistosome. In common with other self-fertile hermaphrodite gastropods, Bulinus consists of a number of closely related species complexes with restricted gene flow between populations of each taxon. Consequently, despite their medical and veterinary importance as intermediate hosts, unambiguous identification and differentiation of planorbid snails such as these remains problematic, often confounding attempts to define the distribution and evolutionary relationships of conchologically similar taxa. Here we consider how morphological methods of discrimination can be used in conjunction with molecular based approaches to improve snail identification, thereby achieving a better understanding of the epidemiology of schistosomiasis. Data are presented from Central and East African taxa which illustrate how PCR-based methods have begun to be used in combination with traditional analyses in an integrated approach to characterize the genus Bulinus , specifically the B. forskalii species group. Particular emphasis is given to the analyses of Random Amplified Polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI).     

Contrasting temporal dynamics and spatial patterns of population genetic structure correlate with differences in demography and habitat between two closely-related African freshwater snails

The relationship between habitat stability, demography, and population genetic structure was explored by comparing temporal microsatellite variability spanning a decade in two closely-related hermaphroditic freshwater snails from Cameroon, Bulinus forskalii and Bulinus camerunensis . Although both species show similar levels of preferential selfing, microsatellite analysis revealed significantly greater allelic richness and gene diversity in populations of the highly endemic B. camerunensis compared to those of the geographically-widespread B. forskalii . Additionally, B. camerunensis populations showed significantly lower spatial genetic differentiation, higher dispersal rates, and greater temporal stability compared to B. forskalii populations over a similar spatial scale. This suggests that a more stable demography and greater gene flow account for the elevated genetic diversity observed in this geographically-restricted snail. This contrasts sharply with a metapopulation model (which includes extinction/contraction, recolonization/expansion, and passive dispersal) invoked to account for population structuring in B. forskalii . As intermediate hosts for medically important schistosome parasites, these findings have ramifications for determining the scale at which local adaptation may occur in the coevolution of these snails and their parasites.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 747–760.     

Low genetic diversity in a snail intermediate host (Biomphalaria pfeifferi Krass, 1848) and schistosomiasis transmission in the Senegal River Basin

Population genetic perturbations of intermediate hosts, often a consequence of human pressure on environmental resources, can precipitate unexpectedly severe disease outbreaks. Such disturbances are set to become increasingly common following range changes concomitant with climate shifts, dwindling natural resources and major infrastructure changes such as hydroprojects. Construction of the Diama dam in the Senegal River Basin (SRB) reduced river salinity, enabling the freshwater snail intermediate host Biomphalaria pfeifferi to rapidly expand its distribution. A serious public health problem ensued, with an epidemic of intestinal schistosomiasis occurring in the previously schistosome‐free Richard‐Toll region within 2 years. The current study aimed to assess the population variability of B. pfeifferi in the SRB, and speculate upon its subsequent impact on host‐parasite interactions following such engineered ecological change. Genetic variation at nine polymorphic microsatellite loci revealed little population differentiation in SRB snails compared with those from natural habitats in Zimbabwe, where Schistosoma mansoni transmission is much lower. ‘Open’ SRB habitats are associated with greater water contact, smaller population sizes and less genetic diversity, with sites downstream of Richard‐Toll showing greater inter‐ and intrapopulation variation, concomitant with less frequent human contact. These observations may be explained by rapid expansion into pristine habitat selecting for high fecundity genotypes at the expense of schistosome resistance, presenting S. mansoni with genetically homogenous highly fecund susceptible populations around the focal point, promoting development of a highly compatible host‐parasite relationship. Longitudinal study of such systems may prove important in predicting public health risks engendered by future environmental engineering projects.     

Bulinus guernei (Mollusca: Gastropoda) of West Africa: taxonomic status and role as host for schistosomes

The basommatophoran freshwater snail Bulinus guernei (Dautzenberg, 1890; type-locality in Sénégal, West Africa) has been treated as a full species, subspecies of B. truncatus (Audouin) or synonym of the latter. Improved knowledge of B. guernei is desirable as this snail has been implicated as a host in Senegambia for Schistosoma haematobium , the blood parasite causing human urinary schistosomiasis (bilharzia). Snail samples from 18 localities were studied: from The Gambia (3), Sénégal (9), Mali (1), Mauritania (1) and, to represent B. truncatus , Morocco (2) and Egypt (2), Characters investigated were morphological (shell, radula and certain soft parts), biochemical (egg proteins and enzymes from body tissue extracts) and cytological (chromosome number). Multivariate analyses (principal components analysis and canonical variate analysis) were performed on data from the shell and radula. Comparison with the holotype shell of B. guernei confirmed that this taxon is represented by recently collected specimens from Senegambia. Variation in the total material examined did not appear to warrant making any taxonomic subdivision, and thus the taxa guernei and truncatus appear to be conspecific. Further sampling is desirable, particularly to determine the possible value of variation in the enzyme HBDH in defining infraspecific units. Experimental infection showed some individual B. guernei to be a compatible host for 5. haematobium (isolate from Sudan; transmitted locally by B. truncatus ). Snails identified as B. guernei apparently have little importance in the transmission of S. haematobium in the Sénégal river basin at present, but such snails could constitute a health hazard if a compatible form of the parasite were to be introduced into this region of Africa.     

An analysis of fertilization in Bulinus cernicus (Gastropoda: Planorbidae)

Experiments have been carried out to determine whether cross–fertilization, multiple insemination and sperm sharing occur in Bulinus cernicus. Snails collected from Canal La Paix, Mauritius, were found to be polymorphic for glucose phosphate isomerase, and from this stock three snail colonies homozygous for GPI 2/2, GPI4/4 and GPI6/6 were selected. Thus, three markers were available to monitor cross–fertilization. Isolated snails produced egg–masses by self–fertilization 40 days post–hatching. Egg–masses produced by self–fertilizing individuals tended to be smaller than those from cross–fertilizing snails, 367 eggs per egg–mass as opposed to 5–34. The majority of snails (70%) switched rapidly to cross–fertilization after pairing. Multiple insemination by two partners was common, heterozygous progeny were produced in a random manner and did not reflect the sequence of mating. No evidence of sperm sharing was found, snails were found to be capable of passing on their own sperm while still producing eggs fertilized by sperm received from an earlier mating. Differences in mating behaviour were noted between snails representing the homozygous GPI colonies, with snails from one colony adopting the female role more readily than those from the other two, which predominantly mated as males.     

Species-specific primers discriminate schistosome intermediate hosts: unambiguous PCR diagnosis of Bulinus forskalii group taxa (Gastropoda: Planorbidae)

The morphological definition of taxa has proved difficult within the Bulinus forskalii group, which includes intermediate hosts of medically important Schistosoma species in West Africa. Although B. forskalii and B. senegalensis transmit different schistosome species they are conchologically similar and their distributions overlap. Randomly amplified polymorphic DNA (RAPD) allows differentiation of sibling species in the genus Bulinus, but RAPDs are difficult to standardize, impairing their value as a taxonomic tool. Hence, RAPD products diagnostic for either B. senegalensis or B. forskalii from West Africa were cloned, sequenced and a panel of species-specific primers designed. Sequencing of RAPD products identified a homology in two apparently independent RAPD loci, a problem where RAPDs are indiscriminately scored for phylogenetic analyses. Specificity of primers was confirmed by widespread sampling throughout each species' range. This approach produced a simple, robust, unambiguous PCR-based species identification strategy for this difficult group.     

AN EVALUATION OF RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) FOR THE IDENTIFICATION AND PHYLOGENY OF FRESHWATER SNAILS OF THE GENUS BULINUS (GASTROPODA: PLANORBIDAE)

The Random Amplified Polymorphic DNA (RAPD) assay was used tostudy genetic variation within and between 9 species of thegenus Bulinus and to determine whether RAPD profiles could beused as markers for identification purposes. RAPDs were generatedwith 8 primers of two different sizes (l0mers & 15mers)and were visualised using both polyacrylamide gel electrophoresis(PAGE) with silver staining and agarose gel electrophoresiswith ethidium bromide staining. The species groups of Bulinushad few similarities in their RAPD profiles and there was interspecificvariation within groups. Intrapopulation variation was observed,with all primers, for B globosus collected from a single sitein Zimbabwe PAGE/silver staining methods visualised a greaternumber of RAPDs in comparison with agarose/ethidium bromidemethods. Phenetic analysis indicated that distance estimatesbetween taxa were sometimes non-additive and the phylo-geneticanalysis of such non-metnc data is discussed. The resultantphenograms, constructed using a least squares method, were constrainedalmost into a polytomy with topologies often differing betweendata sets. It was concluded that this phenomenon was most likelyattributable to large nucleotide divergences between the speciesgroups which go beyond the phylogenetic scope of RAPD analysis.RAPD profiles, when used in conjunction with other taxonomicmethods, may contribute to the identification of species ofBulnus on a regional basis, but the observed variability ina natural population suggests that a diagnostic RAPD profilefor each species throughout its geographic range is unlikely. (Received 19 April 1995; accepted 1 September 1995)     

OBSERVATIONS ON SHELL MORPHOLOGY, ENZYMES AND RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) IN BULINUS AFRICANUS GROUP SNAILS (GASTROPODA: PLANORBIDAE) IN ZANZIBAR

The taxonomic status of Bulinus globosus and B. nasutus as separatespecies remains unclear, espécially in areas of EastAfrica where their distribution overlaps. In order to throwfurther light on this question, a study was carried out on snailscollected from the islands Unguja and Pemba, collectively knownas Zanzibar, which lie in the Indian Ocean close to mainlandAfrica. In total, 169 snails from 22 sample sites were characterisedby three methods: morphometric analysis of 15 shell measurements,enzyme analysis involving three enzyme systems and analysiswith random amplified polymorphic DNA (RAPD). Enzyme analysesindicated that both B. nasutus and B. globosus were presenton each island and their distribution was allopatric. Shellvariation was considerable but did not clearly differentiatethese taxa. Similarly, RAPD analysis indicated substantial geneticvariation with in and between samples; more often than not,no two RAPD profiles were identical within the sample. On Pemba,B. globosus is more widely dispersed than B. nasutus which isconfined to the eastern coast of the central region. On Unguja,B. globosus is found in more northern areas than B. nasutus. (Received 15 August 1996; accepted 31 January 1997)