Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 93, Suppl. I: 111-116, 1998 111 Molecular Characterisation of Intermediate Snail Hosts and the Search for Resistance Genes David Rollinson/+ , J Russell Stothard, Cathy S Jones*, Anne E Lockyer*, Cecilia Pereira de Souza**, Les R Noble* The Natural History Museum, Cromwell Road, London SW7 5BD, UK *Zoology Department, Aberdeen University, AB9 2TN, UK **Centro de Pesquisas René Rachou-Fiocruz, Belo Horizonte, MG, Brasil The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a “total evidence” ap- proach to characterisation of Bulinus species and provide insights into parasite transmission. Particu- lar emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with prog- eny pooling methods. We are currently characterising a variety of STSs (sequence tagged sites) associ- ated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations. Key words: Bulinus - Biomphalaria - molecular systematics - epidemiology In recent years, new opportunities for the gen- snail parasite interaction is such that only certain eration of molecular characters have been provided species are involved in transmission of the para- for the malacologist due to the introduction of vari- site. The genus can be divided into four major ous molecular techniques including the polymerase groups of species. The B. forskalii group contains chain reaction (PCR) and DNA sequencing. This 11 species with slender shells and usually high paper details some of our recent molecular ap- spires and is practically pan-African in distribu- proaches to the characterisation of Bulinus spe- tion with species occurring on some of the sur- cies in Africa and presents preliminary informa- rounding islands and the Arabian peninsula. The tion concerning the search for resistance genes in B. africanus group has 10 species confined to the Biomphalaria glabrata. Afrotropical region. The B. truncatus/tropicus CHARACTERISATION OF BULINUS complex, which contains polyploid species, is again pan-African with 14 representatives extending into Freshwater snails of the genus Bulinus act as the Middle East, Mediterranean islands and the the intermediate hosts for Schistosoma Iberian Peninsula; the two species included in the haematobium and related species and occur com- B. reticulatus group both have restricted distribu- monly throughout much of Africa and adjacent tions. Within each group there are species that act regions. There are currently 37 species of Bulinus as intermediate hosts for schistosomes in part or recognised (Brown 1994) but the specificity of the all of their geographical range. The host-parasite relationship can be viewed in terms of snail susceptibility and parasite infec- tivity. Some parasites appear to be genetically het- Work supported by The Wellcome Trust, BBSRC and erogeneous with regard to infectivity and are com- MRC. +Corresponding author. E-mail:[email protected] patible with a wide variety of snail hosts whereas Received 4 May 1998 others are far more restricted in their choice of host Accepted 31 August 1998 (see Rollinson & Southgate 1987). For example, 112 Characterisation of Intermediate Snail Hosts David Rollinson et al. S. haematobium, the cause of urinary schistoso- rRNA gene. As it contains highly conserved re- miasis in man, is known to be transmitted by at gions it shows homology with a wide variety of least 12 species of Bulinus in Africa and adjacent organisms and will readily bind to restriction frag- regions. In contrast, natural transmission of S. ments of snail DNA. Clear differences in the sizes intercalatum appears to be confined to two differ- of restriction fragments between species of Bulinus ent species, B. globosus and B. forskalii. Not only representing the four species groups were observed do differences exist between species of schistosome when DNA was digested with either BamHI or in their ability to infect snails but marked differ- BglII and hybridised to pSM889. No differences ences can exist between strains of the same spe- were observed between samples of B. tropicus and cies. Strains of S. haematobium which are com- B. truncatus but intraspecific variation was ob- patible with B. africanus group snails are usually served between samples of B. forskalii from São incompatible with B. truncatus and vice versa. Tomé and Angola (Rollinson & Kane 1991). This Similarly, variation in susceptibility to a parasite work suggested that sequence variation in the can occur between populations of the same snail spacer regions may be useful for discrimination. species. The entire ITS region including the 5.8S rRNA Considerable efforts have been focused on pro- gene has now been amplified from species repre- viding reliable methods for the differentiation and senting the four species groups following the meth- identification of Bulinus species in order to deter- ods of Kane and Rollinson (1994). The sequences mine those species and strains playing a major role for the primers used for PCR amplification were in schistosomiasis transmission. Differences among based on conserved regions of the 3' end of the taxa have been sought traditionally by analysis of 18S rRNA gene (ETTS1) and the 5' end of the 28S morphological variation and determination of chro- rRNA gene (ETTS2). The amplified product was mosome number. Enzyme electrophoresis has been digested with one of a number of 4-base or 6-base used to supplement such studies and has proved cutting restriction enzymes (RsaI, AluI, CfoI, SmaI useful for identification, elucidation of relation- and SacI). Characteristic RFLP patterns were ob- ships between taxa and for studying aspects of re- tained for the four species groups and, in the ma- productive biology and population structure (e.g. jority of cases, species within a species group could Biocca et al. 1979, Jelnes 1979, 1986, Rollinson be differentiated (Stothard et al. 1996). Distinctions & Southgate 1979, Rollinson & Wright 1984, may be caused by variation in the number of re- Njiokou et al. 1993). striction sites within the spacer region or by differ- Various molecular approaches have been inves- ences in sequence length between the restriction tigated for the identification and characterisation sites. The complete ITS1 spacer region has been of Bulinus species including: the examination of sequenced for B. globosus, B. cernicus and B. variation in ribosomal RNA (rRNA) genes, as de- truncatus and substantial nucleotide variation oc- termined by conventional restriction fragment curs indicating large divergence between the spe- length polymorphism (RFLP) analysis and by PCR- cies groups (Stothard et al. 1996). The results are RFLP of the internal transcribed spacer (ITS); the in agreement with earlier enzyme studies (Biocca use of randomly amplified polymorphic DNA et al. 1979) which also showed large divergence (RAPDs) and sequence analysis of mitochondrial between the species groups of Bulinus. genes. RAPD analysis - Unlike conventional PCR- Ribosomal RNA genes - The genomic riboso- based analyses, RAPD approaches use single oli- mal RNA (rRNA) gene complex is common to all gonucleotide primers of arbitrary sequence (be- eukaryotes and is well suited for taxonomic stud- tween 5-20 bases) to initiate DNA strand synthe- ies as it contains regions which evolve at different sis under conditions of low stringency at a number rates, thus permitting analysis of relationships over of complementary binding sites scattered through- a wide taxonomic level. In general the spacer re- out the genome (Welsh & McClelland 1990, Will- gions are less conserved than the coding regions. iams et al. 1990). Discrete amplification products DNA probes have been shown to be of value for form where primer sites are orientated in an in- distinguishing schistosome species and particular verted repeat and are within an amplifiable distance use has been made of the rRNA gene probes of each other. Several amplification fragments may pSM889, pSM890 and pSM389 derived from S. be generated within a single RAPD reaction and, mansoni (Simpson et al. 1984, Johnston et al. when visualised by either agarose or polyacryla- 1993). The probe pSM889 has been used to study mide electrophoresis separation, give rise to a restriction enzyme digests of various species of RAPD profile. Twenty-eight primers obtained from Bulinus. This probe is a 4.4 kb fragment that en- Operon Technologies Ltd and British Biotechnol- compasses part of the small rRNA, ITS and large ogy were screened against genomic DNA extracted Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 93, Suppl. I, 1998 113 from B. sudanica to identify primers which gave and neither population was in Hardy-Weinberg reproducible and informative RAPD profiles. Of equilibrium (Mimpfoundi & Greer 1990). The re- these primers, eight (4 x 10 mers, 4 x 15 mers) sults suggested low genetic diversity and indicated were subsequently selected to study genetic varia- that B. forskalii reproduces principally by self- tion within and between nine species of Bulinus fertilisation.