PAGES: 53-58 DOI: Full paper
Genetic Markers between Biomphalaria glabrata Snails Susceptible and Resistant to Schistosoma mansoni Infection

RGM Spada, D da Silva, A-Z Abdel-HamidI, SS Sobral-Hamaguchi, NRB ZuimII, EM Zanotti-MagalhãesII, LA MagalhãesII, JT Ribeiro-Paes +

Departamento de Ciências Biológicas, Unesp, Av. Dom Antonio 2100, 19806-900 Assis, SP, Brasil
IMedicinal Chemistry Department, National Research Center, Dokki, Cairo, Egypt
IIDepartamento de Parasitologia, Instituto de Biologia, Unicamp, Campinas, SP, Brasil


The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.

Until mid 1980s the emphasis on the control of schistosomiasis was mainly directed to the combat against vector. After the discovery of more efficient and less toxic chemotherapeutics drugs (oxamniquine, praziquantel), the World Health Organization (WHO) started to emphasize morbidity control (WHO 1985, Dias et al. 1995, Lardans & Dissous 1998). Despite the fact that the emphasis directed to chemotherapy has reached favorable results, regarding reduction of morbidity, a significative reduction of the transmission was not obtained (Barbosa 1995, Lardans & Dissous 1998). As a result of those aspects, the control program proposed in 1993 by the WHO kept the emphasis on morbidity control. However other measures were associated, such as: improvement on sanitary qualities, water supply to population, vector control and sanitary education (WHO 1993, Dias et al. 1995).

An important aspect to be considered in the control program context is the emergent problem of resistance or tolerance of parasites to the main drugs used in schistosomiasis chemotherapy (Dias & Gonçalves 1992, Cioli 1993, Araújo et al. 1996, Conceição et al. 2000, Liang et al. 2001). Due to those aspects, the combat against vector, associated to other control measures, is again emphasized.

The control of the vector has been tried by different methods, which include: the use of synthetical molluscicids, like niclosamide (WHO 1993), molluscicids of vegetal origin (Jurberg 1989, Schall et al. 1998), modifications in their natural habitat and use of predators or competitors of those vectors (Paraense 1987, Capron 1993, Lardans & Dissous 1998, Pointier & Augustin 1999, Pointier & Jourdane 2000, Amarista et al. 2001).

The genetical aspects, regarding the susceptibility of snail vector to infection by Schistosoma mansoni, were realized, in a pioneer way, by Newton (1955). The author associated the albino phenotype with a high pattern of susceptibility to the infection by Porto Rico strain of Schistosoma mansoni and proposed, for that phenotype, a polygenic inheritance pattern. Richards et al. (1992), employing techniques of classical genetic, proposed that susceptibility ofBiomphalaria glabrata to infection by S. mansoni would present distinct patterns of inheritance dependable on the age of the snail. In that manner, the resultant phenotype would be determined by various genes in the young mollusk and by only one gene (monogenic) in the adult mollusk. In fact, up to the moment, the patterns of inheritance, regarding susceptibility, were not very well established.

Several techniques of molecular biology for studies of genetic variability in schistosomiasis vectors have been used (Knight et al. 1991, Milller et al. 1996, Spatz et al. 1999, Vidigal et al. 2000, 2001, Caldeira et al. 2000, Jannotti-Passos & Souza 2000, Knight et al. 2000, Souza & Jannotti-Passos 2001, Tuan & Bortolato 2001).

The introduction of the randon amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique (Welsh & McClelland 1990, Williams et al. 1990) has amplified the possibilities of polymorphisms analysis, as it allowed the use of small arbitrary nucleotide segments without the need of a previous knowledge of genes and/or genomic sequences.

Vidigal et al. (1994) have used the technique of RAPD-PCR for studies of the genetic variability in B. glabrata. Larson et al. (1996) and Lewis et al. (1997), developed studies between B. glabrata strains, resistant and susceptible to infection by S. mansoni, managing to associate some markers to resistance and susceptibility. Similar studies were accomplished by Abdel-Hamid et al. (1999), using mollusks of B. tenagophila species.

In this article, the technique of RAPD-PCR was used for analysis of the genomic DNA, in B. glabrata strains, in an attempt to associate possible polymorphic markers related to susceptibility/resistance to infection by the S. mansoni. The comprehension of genetic basis of susceptibility/resistance of snails to S. mansoni may, as it was proposed by Rollinson et al. (1998), base the elaboration of new strategies for the control of schistosomiasis.



Snails stocks S. mansoni-resistant and -susceptible B. glabrata snails (BH strain) obtained by self-fertilization as described by Zanotti-Magalhães et al. (1997) were used. The mollusks were maintained in glass aquaria and fed with lettuce leaves and calcium carbonate.

Experimental infection - Each mollusk was challenged with 10 miracidia (BH strain) of S. mansoniaccording to Zanotti-Magalhães et al. (1997). They were placed in glass jars containing chlorine free water for 2 h at 25oC. Susceptibility was determined after 30 days through cercariae release favoured by an incandescent lamp. Susceptibility to infection was monitored from 4 weeks up to 12 weeks post-exposition.

DNA extraction - The DNA was extracted from the foot of individual snails (resistant and susceptible strains), using lysis buffer containing 2% CTAB (Winnepenninckx et al. 1993, Abdel-Hamid et al. 1999) and incubated with proteinase K (2 µg/ml) for 2 h, following extraction with phenol, phenol:chloroform (1:1) and chloroform: isoamylalcohol (24:1) and incubation with ribonuclease A (350 µg/ml). The DNA was precipitated by adding isopropanol (2:3 v/v) and leaving overnight at room temperature. After centrifugation DNA was washed in 75% ethanol, 10 mM ammonium acetate for 30 min and recovered by centrifugation (12000 g) for 10 min (4ºC). The pellet was ressuspended in TE (10 mM de Tris, 1mM de EDTA, pH 8.0) and the DNA concentration and purity was determined by 2% agarose gel electrophoresis using the gel photodocumentation system (EDAS) DC 120 Zoom Digital Camera (Eastman Kodak, NY, USA) and spectrophotometrically (Spectrophotometer Ultrospec III, Pharmacia, UK) at absorbances of 260 and 280 nm.

DNA amplification by RAPD-PCR - The method of Simpson et al. (1993) was used, with small modifications. Two nanograms of genomic DNA obtained from the snails were amplified with a PTC 200 Peltier Thermal Cycler (MJ Research - USA) using RAPD-PCR. Each reaction was carried out in a final volume of 20 µl containing 1 unit Taq DNA polymerase (Gibco, BRL, MD, USA), 1X PCR buffer, 0.2 mM of each dNTP, and 7 pmol of each random primer (10 bp) (Gibco, BRL). Ten primers were used: 1 (5'-GGGTAACGCC-3'), 2 (5'-CTGATGCTAC-3'), 4 (5'-AGTGCTACGT-3'), 8 (5'- CTCTCCGCCA-3'), 9 (5'-CAGGCCCTTC-3'), 10 (5'-GGTCCCTGAC-3'), 12 (5'- AGGGAACGAG-3'), 14 (5'- GTTGCCAGCC-3') and 15 (5'-TGCCGAGCTG-3'). The amplification conditions were as follows: 1 cycle at 95ºC for 5 min, 2 cycles at 95ºC for 30 sec, at 30ºC for 2 min and at 72ºC for 1 min, and 33 cycles during which the annealing temperature was changed to 40ºC and the time of the extension step was increased to 5 min during the final cycle. The PCR products were stored at -20ºC.

Polyacrylamide gel electrophoresis and silver staining - Four microliters of each DNA amplification reaction was added to 2.5 µl sample buffer (0.125% bromophenol blue, 0.125% xylene cyanol, and 15% glycerol) and the mixture was submitted to 8% polyacrylamide (29/1 acrylamide-bisacrylamide) gel electrophoresis in TBE (2 mM Tris-borate, EDTA, pH 8.0) at 60 volts. The gels were fixed in 10% ethanol and 0.5% (v/v) acetic acid for 15 min, stained with 0.2% silver nitrate for 15 min, washed with deionized water for 5 min, and developed with 0.75 M NaOH and 0.1 M formaldehyde for 15 min (Santos et al. 1993).

Polymorphism analysis - Genetic variability of the susceptible and resistant strains was evaluated by analyzing the electrophoretic band patterns obtained on the gels and by determining the coefficient of similarity as described by Dice in 1945.



Susceptibility or resistance of B. glabrata snails to S. mansoni infection was studied starting 30 days post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. The obtained results pointed out that only 63% of the examined snails (100 snails) yielded high infection frequencies in contrast to about 37% of these snails were refractory.

A total of 10 primers were tested and polymorphic markers were obtained with 5 of them (primers 4, 8, 9, 10, 12). These primers were adopted for having been used in prior works, in which specific polymorphic markers were detected: among lineages from different regions of Brazil (Vidigal et al. 1994), between B. glabrata strains resistant and susceptible to S. mansoni infection (Larson et al. 1996), in B. tenagophila(Abdel-Hamid et al. 1999) and those made with the genetic material of the parasite S. mansoni (Sire et al. 1999).

The genomic DNA, amplified with primer 4 (5'-AGTGCTACGT-3'), as shown in Fig.1, presented a polymorphic band of nearly 4060 bp, present only in the susceptible lineages.

Fig. 2 presents the results of amplification with primer 8 (5'-CTCTCCGCCA-3'). According to Fig. 2, there is a band of 1.750 pb that occurs only in the susceptible lineage (well 6, 7 and 8).

The results presented in Fig. 3 arise from the electrophoresis polyacrylamide gel of material resultant from amplification of genomic DNA from B. glabrata with primer 9 (5'-CAGGCCCTTC-3'). According to Fig. 3(wells 6, 7 and 8) a polymorphic marker of 1.100-bp was obtained, which was found only in the susceptible lineages.

Primer 10 (5'-GGTCCCTGAC-3') presented a polymorphic band, with approximately 1100-bp, present only in the susceptible lineage, according to Fig. 4, wells 6, 7 and 8.

Fig. 5 corresponds to the electrophoresis in gel of polyacrylamide resultant from the amplification of genomic DNA with primer 12 (5'-AGGGAACGAG-3'). It is verified, with that primer, the acquisition of a band of nearly 800 bp (wells 6, 7 and 8), whose occurence is only restricted to snails of susceptible lineage.



The genomic DNA amplified with primer 4 (5'-AGTGCTACGT-3'), according to Fig. 1, presented a polymorphic band, of approximately 4060 bp, between the resistant and susceptible lineage and that marker occurred only in the susceptible lineages. Vidigal et al. (1994), employing an equivalent sequence of oligonucleotides found, in individuals of B. glabrata species, collected in field, polymorphic bands in snails of different places in Brazil. In the present study, descendants from snails maintained in the laboratory for more than 20 years were used. Thus, although resistant and susceptible strains were studied, we could expect that the genetic variability was low, since laboratory strains are submitted to much less intense selective pressures than field isolates are, i.e., natural conditions. This fact may explain the high coefficient of similarity observed for the strains studied here (Table) and also the expressive quantitative difference in genetic variability obtained by Vidigal et al. (1994) with an oligonucleoride sequence similar to primer 4.

Sire et al. (1998) employed a series of aleatory primers for the analysis of genetic diversity of S. mansoniadult worms originated of cercarie, obtained from a natural population of B. glabrata, in a site of transmission at Guadeloupe Island. Among the 18 tested primers, 3 of them allowed the detection of polymorphic markers. One of those oligonucleutide sequences (5'-CTCTCCGCCA-3') was used in this project (primer 8) and resulted, after amplification of genomic DNA, in the obtaining of a polymorphic marker of 1750 pb, whose occurrence may only be verified in the susceptible lineage of the mollusk, according to Fig. 2. The amplification of genomic sequence of the parasite and the vector mollusk from the same primer, presents two immediate occurrences: the parasite and the vector may present similar genomic sequences which could, in its turn, be implied in the definition and establishment of the relation parasite/host, and, as a second consequence, care and caution should be adopted in the amplification of the genomic DNA of the vector mollusks infected, in order to avoid contamination of the samplers with genetic material of the larval forms of the parasite.

With primer 9 (5'-CAGGCCCTTC-3'), which corresponds to primer OPA-01 employed by Larson et al. (1996), a polymorphic marker of 1.100-bp was found (Fig. 3). That band was repeatedly found only in the susceptible lineages. Larson et al. (1996) obtained, with primer OPA-01 a band of 400 bp, characteristic of susceptible lineage (M-line). That lineage was obtained by Newton, in 1955 and combines the albino phenotype with a high susceptibility to infection by Porto Rico strain from S. mansoni. The distinct origins of M-line and BH strains, employed in the current work, could justify the acquisition of distinct polymorphic bands from the amplification with a primer with equal sequence of nucleotides. In other words, the susceptible lineages in question present specific markers but they are different among themselves.

With primer 10 (5'-GGTCCCTGAC-3'), which corresponds to primer OPA-06, employed by Larson et al. (1996), a polymorphic band was obtained, with nearly 1100-bp present (and repeatedly obtained) only in the susceptible lineage (Fig. 4). Such results were agreeing with the ones obtained by Larson et al. (1996) in the susceptible lineage (M-line). It seems it is a matter of a specific marker in the susceptible lineage.

A polymorphic band of approximately 800 pb was found in the susceptible lineage of the mollusks B. glabrata after the amplification of the genomic DNA with primer 12 (Fig. 5), whose sequence of oligonucleutideos (5'-AGGGAACGAG-3') was employed by Abdel-Hamid et al. (1999) for the analysis of the genetic variability between resistant and susceptible lineages of B. tenagophila. The authors did not detect, with that primer, any polymorphic marker between the resistant and susceptible cepas. It is concluded that the primer may amplify a very preserved area of the genoma B. tenagophila and, apart from allowing the detection of a marker of the susceptible lineage, that primer presents itself potentially interesting for taxonomic studies among the sympatric species which include B. glabrata and B. tenagophila.

Some studies have used isoenzymes and pigmentation analysis to define specific susceptibility markers inB. glabrata (Mulvey & Vrijenhoek 1981, Mulvey & Wooddruff 1985), but no marker was safely associated with resistance or susceptibility. Even through no clear association has been found in the present work between a specific marker and B. glabrata susceptibility, identical results have been obtained by Larson et al. (1996), which led to the identification of a 1100 bp marker shower only in the susceptible strains. Therefore, we presume this is a specific marker to the susceptible strain.

Knight et al (1999) analyzed through RAPD-PCR F1 and F2 generations and the F1 versus parental backcross progeny. This approach led to the identification of two markers, one 1200 bp band with OPM-04 primer and 1000 bp band with OPZ-11 primer. Those markers were found only in non-susceptible strains. This is an indication that markers of segregation may be found in B. glabrata resistant to S. mansoniinfection.

A possible existence of transposable elements in the snails opens new perspectives of approach for transfection of sequences with incorporation to the snails genome (Richards et al. 1992). The markers or genomic sequences associated to resistance could be useful for the production of transgenic snails. The perspective of acquisition of transgenic snails strains resistant to S. mansoni infectionimplies the exceeding of many theoretical difficulties, especially the experimental ones. In spite of that, such methodological approach presents interesting perspectives (Lardans & Dissous 1998), which, in association with other measures, will allow the elaboration of new strategies for the control of schistosomiasis.



To Dr Luiz Cândido Souza Dias and Dr Vanderlei Rodrigues for continue advice, discussions and critical suggestions.



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This work was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo.

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Received 18 June 2002

Accepted 15 August 2002


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