Mem Inst Oswaldo Cruz, Rio de Janeiro, 95(1) Jan/Feb 2000
Further Studies on the Molecular Systematics of Biomphalaria Snails from Brazil
Centro de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brasil
IDepartamento de Zoologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG, Brasil IILaboratório de Genética de Câncer, Instituto Ludwig de Pesquisas sobre o Câncer, São Paulo, SP, Brasil
The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzymeu00a0DdeI were used for the molecular identification of ten species and one subspecies of Brazilianu00a0Biomphalaria. Emphasis is given to the analysis ofu00a0B. oligoza,u00a0B. schrammiu00a0andu00a0B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However,u00a0B. peregrinau00a0andu00a0B. oligozau00a0presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes includingu00a0HaeIII,u00a0HpaII,u00a0AluI andu00a0MnlI were tested for their ability to differentiate these species. Foru00a0B. amazonicau00a0three variant profiles produced withu00a0DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails.
Ten speciesand one subspecies of Biomphalaria are recognized in Brazil: B. glabrata (Say, 1818), B. tenagophila(Orbigny, 1835), B. tenagophila guaibensis Paraense, 1984, B. straminea (Dunker, 1848), B. peregrina (Orbigny, 1835), B. kuhniana (Clessin 1883), B. schrammi (Crosse, 1864), B. amazonica Paraense 1966, B. oligoza Paraense, 1974, B. intermedia (Paraense & Deslandes, 1962)and B. occidentalis Paraense 1981. Only, B. straminea, B. t. tenagophila andB. glabrata are found naturally infected with Schistosoma mansoni. Among these, B. glabrata is the most important due to its wide spread distribution and its high susceptibility to S. mansoni (Paraense & Corrêa 1963). In addition B. peregrina and B. amazonica are considered potential hosts of this parasite based on experimental infection (Corrêa & Paraense 1971, Paraense & Corrêa 1973).
The morphological identification of freshwater snails of medical importance, is greatly complicated by the extensive intraspecific variation of the morphological characteristics used for classical identification (Paraense 1975, 1981, 1984, 1988). To overcome this problem, the use of molecular techniques as additional tools for the identification of these snails has been proposed. In previous studies we tested a low stringency polymerase chain reaction technique (LS-PCR) (Dias Neto et al. 1993) which permited the differentiation of B. glabrata and B. t. tenagophila and also the identification of very similar species such as B. t. tenagophila and B. occidentalis (Vidigal et al. 1996, Pires et al. 1997).
Recently, we have explored the possibility of using sequence polymorphism in the internal transcribed spacer region (ITS) of the rDNA of Biomphalaria snails (which includes the 5.8S rDNA gene together with the flanking ITS1 and ITS2 spacers) by means of PCR amplification and digestion with different restriction enzymes. This method was successfully used to construct the molecular key for Aedes species (West et al. 1997). It was also used to distinguish closely related parasitic worms and others different organisms (Newton et al. 1998, Wu et al. 1999) and on studies of genetic variation and identification of snails of the genera Oncomelania, Bulinus and Biomphalaria (Hope & McManus 1994, Stothard et al. 1996, Stothard & Rollinson 1997, Rollinson et al. 1998, Vidigal et al. 1998a,b, Caldeira et al. 1998, Spatz et al. 1998, 1999).
The aim of the present study was to investigate the use of restriction profiles resulting from digestion of the ITS spacer of rDNA with Dde I for separation of the majority Biomphalaria species from Brazil, with special attention to B. oligoza, B. schrammi and B. amazonica species which were being studied for the first time. We found difficulty to differentiate between B. peregrina and B. oligoza with DdeI, however we propose the use of others restriction enzymes such asHpaII, Hae III and AluI for identification of these species.
MATERIALS AND METHODS
Snail populations - Snail populations from different Brazilian localities were used (Fig. 1). The number of snails used and the geographical localization from each population are shown in the Table. The snails were identified by means of comparative morphology based on the reproductive organs and shells accordingly to Deslandes (1951) and Paraense (1975, 1981, 1984, 1988).
DNA extraction - Total DNA was extracted from the feet of snails using the Wizard Genomic DNA Purification Kit (Promega) with some modifications. Briefly, the tissues were mechanically disrupted in 200 µl of nucleic lysis solution and incubated at 37°C for 4 h or overnight with 50 µg/ml proteinase K. Thereafter, 80 µl of protein precipitation solution was added to the initial mix. The mixture was vortex vigorously for 10-20 sec and centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to a microcentrifuge tube containing 200 µl of room temperature isopropanol for DNA precipitation. The mixture was gently mixed by inversion for 20 min and centrifuged at 13,000 rpm for 5 min. The DNA pellet was washed with 300 µl of 70% ethanol and centrifuged for 1 min. The pellet was treated with 25 µl of DNA re-hydration solution for 30 min at 65°C and stored at -20°C. The DNA concentrations were estimated by comparison with known standards on 2% ethidium bromide stained agarose gels.
Amplification by PCR - The entire ITS was amplified using the primers ETTS2 (5/-TAACAAGGTTTCCGTAGGTGAA-3/ ) and ETTS1 (5/-TGCTTAAGTTCAGCGGGT-3/) anchored respectively in the conserved extremities of the 18S and 28S ribosomal genes (Kane & Rollinson 1994). The PCR amplification was undertaken in a volume of 10 µl consisting of: 1-10 ng template DNA, 10mM Tris-HCl, pH 8.5, 200 µm each dNTP, 1.5 mM MgCl2, 0.8 U of Taq DNA polymerase, 50 mM KCl, together with 5.0 pmol of each primer. The reactions were covered with a drop of mineral oil and subjected to the following cycle program: initial denaturation step for 3 min at 95°C, and then 32 cycles with annealling at 54°C for 1 min, extension at 72°C for 2 min, denaturation at 95°C for 45 sec and a final extension step at 72°C for 5 min. A negative control (no template DNA) was included in all experiments. Three microliters of the amplification products were visualised on 0.8% ethidium bromide stained agarose gels to check the quality of amplification. The remaining 7 µl were mixed with 53 µl of water, and divided into 10 µl aliquots for enzyme digestion.
Production and evaluation of the rDNA-ITS RFLP profiles - In the initial experiments the enzyme DdeI (Amersham, Life Science) was evaluated for its ability to differentiate all Biomphalaria species from Brazil. Addicional enzymes were tested including AluI, MnlI, (Amersham, Life Science) HaeIII and HpaII (Promega Co, USA) in situations when DdeI was not effective in the separation of some species. One microliter (10-12 units) was used for each digestion reaction, together with 1.2 µl of the respective enzyme buffer in a final volume of 12.2 µl. The digestion was performed for 3.5 h at 37°C, and the digestion products were evaluated on 6 or 8% silver stained polyacrylamide gels (Sanguinetti et al. 1994) after phenol/chlorophorm extraction. The results were recorded on Polaroid film 667 (St Albans, UK). A control for the activity of each enzyme was performed by digesting 150 ng of pUC18.
The PCR amplification of the Biomphalaria ITS region with the primers ETTS2 and ETTS1 resulted in a product of approximately 1.3kb (Fig. 2A). However, B. oligoza (lanes 18 and 19), B. peregrina (lanes 20 and 21) and B. amazonica(lanes 25 at 28) presented an ITS product of approximately 1.0kb. Fig. 2B shows the rDNA-ITS RFLP profiles produced by digestionwith DdeI of two to four specimens of all Biomphalaria species from Brazil. The profiles obtained with this enzyme permit the easy separation of the majority of the species analysed with exception of B. occidentalis and B. t. guaibensis (Fig. 2B lanes 7, 8 and 9, 10, respectively) and B. oligoza and B. peregrina (Fig. 2B lanes 18, 19 and 20, 21, respectively) which presented very similar profiles. The reproducibility of the profiles obtained with DdeI for B. oligoza,B. peregrina, B. schrammi and B. amazonica were previously evaluated by increasing of the number of specimens for each species obtained from different localities (Table). Fig. 3A shows the profiles obtained using DdeI with B. oligozacollected in Rio Grande do Sul (lanes 1 to 4) and Santa Catarina (lanes 5 to 10). The profiles were very homogeneous for all specimens. Fig. 3B shows the profiles obtained for B. peregrina specimens collected from different localities in Minas Gerais. The profiles obtained with B. peregrina were homogeneous but, as mentioned above, similar to the those obtained from B. oligoza (Fig. 3A).
Fig. 4 shows the profiles of B. schrammi obtained with DdeI. This enzyme produced a simple profile with six fragments, which was clearly different from those seem with other species as observed in Fig. 2B (lanes 22 to 24). B. amazonicapresented three variant profiles with three to nine fragments when treated with DdeI (Fig. 5). In fact in the snails from one locality (Benjamin Constant, Amazonas) three profiles were observed: lanes 1, 2 and 4 show the profiles characterized by presence of three bands, lane 3 shows five fragments and lanes 5, 6 and 7 show the profiles with nine fragments. In the snails obtained from Barão do Melgaço, Mato Grosso (lanes 8 to 10) only one profile with five fragments was observed. The specimens with variant profiles, for this species, were included in Fig. 2B (lanes 25 to 28). The profiles obtained with DdeI for the ten species and one subspecies of the Brazilian Biomphalaria snails are represented in Fig. 6.
To obtain an improved molecular separation of B. oligoza and B. peregrina other enzymes were tested (HpaII, MnlI, AluI and HaeIII). Fig. 7A shows the results obtained with HpaII which produced the best results with distinct profiles observed for each species. Both HaeIII (Fig. 7B) and AluI (Fig. 7C) produced similar profiles with only subtle differences.B. oligoza and B. peregrina HaeIII profile shows six fragments two of which are species-specific for B. oligoza (Fig. 7Blanes 4 to 12). Using AluI B. peregrina presented one band of approximately 200bp distinct from B. oligoza (Fig. 7C). The enzyme MnlI did not permit the separation of these species (data not shown).
The possibility of using molecular tools for identification of snails of medical importance has contributed to increased knowledge of the genus Biomphalaria. Previously, we have explored other molecular methods to obtain species-specific markers. Initially we observed that the arbitrarily primed-PCR (AP-PCR) mirrored the high genetic variability in B. glabrata species (Vidigal et al. 1994, 1998b). These results demonstrated that this methodology does not provide good markers for molecular identification of Biomphalaria species. The LS-PCR technique produced a great number of bands complicating the identification of the species-specific LSP markers (LS products) (Vidigal et al. 1996, Pires et al. 1997). In the present study we demonstrate that PCR-RFLP of the ITS region of Biomphalaria rDNA, using DdeI, permits the differentiation of the majority of the ten Brazilian Biomphalaria species examined. In previous studies we demonstrated that restriction profiles resulting from digestion with DdeIpermit the identification of B. glabrata, B. straminea and B. tenagophila (Vidigal et al. 1998a,b). We also used this technique in the study of very similar species such as B. straminea, B. intermedia, B. kuhniana, B. peregrina (Caldeira et al. 1998) and B. t. tenagophila, B. t. guaibensis and B. occidentalis (Spatz et al. 1998, 1999).
The morphological separation of typical specimens of B. peregrina and B. oligoza are easy taste for the specialist in the identification of Biomphalaria snails. The principal characteristic for separation of these species is the presence of the small number of prostatic diverticula (1 to 6, rarely 7), or absence in B. oligoza (Paraense 1974, 1975). In B. peregrinathe presence of 8 to 22 prostatic diverticula is reported. However, when specimens from both species show similar number of diverticula (B. oligoza maximum 7 and B. peregrina minimum 8) morphological separation of the species becomes more difficult. In this case only observation of the prostatic diverticula covering the spermathecal body of theB. peregrina can be considered as a diagnostic character (Paraense 1966, 1975). Here, the molecular results obtained with PCR-RFLP suggested a high similarity between these species based on the similarity of profiles obtained with DdeI and AluI and the identical profiles produced with MnlI. Although the similarity among these profiles, AluI, HpaII, HaeIII (7A, B, C) permit the molecular separation of these species and can be used in cases when the morphological identification of the B. oligoza and B. peregrina is not effective. It is noteworthy that B. peregrina is one of the most widespread planorbid species in the Neotropical region hence, according to Paraense (1975), the origin to its scientific name (from the latin peregrinus, meaning wanderer, in reference to its wide geographical distribution).
B. schrammi is distributed in most regions of Brazil, but has not been found in Amazonas, Paraná, Rio Grande do Sul or Santa Catarina (Paraense 1975). The molecular characters shown here (Fig. 4) very clearly identified specimens belonging to this species. The distribution of B. amazonica in Brazil is concentrated in Amazonas, Acre, Mato Grosso do Sul and Rondônia (Paraense 1983). The snails analyzed here were obtained from two distant localities and showed three variant profiles (Fig. 5). Accordingly the molecular identification of this species requires that attention is given to this diversity of profiles. The schematic representation of the ITS restriction patterns obtained for Brazilian Biomphalariasnails using DdeI can be used as model for comparative analysis (Fig. 6).
The sequences of ITS regions of the Brazilian Biomphalaria species will be analyzed in the future to better understand the intraspecific and interspecific relationships among this species.
We show here that PCR-RFLP is a simple and rapid technique representing an important advance for studies of Brazilian Biomphalaria species which can be used as an auxiliary tool to morphological identification. The study demonstrated that ITS contains useful genetic markers for the identification of these snails. The results obtained with PCR-RFLP are concordant with the actual morphological systematics proposed for the Brazilian Biomphalaria snails by Paraense (1975, 1981, 1984, 1988).
To Dr Wladimir Lobato Paraense and Dr Ligia Corrêa from the Departamento de Malacologia, Instituto Oswaldo Cruz, for providing the snails. To Dr Mário Steindel from the Departamento de Parasitologia, UFSC, Santa Catarina, Andrea do Amaral Ferrari and Izabel Michelin Nunes from FNS, Porto Alegre, RS for technical support. To Yoná Rose Campos for assistance, and Letícia Peixoto Santos for administrative support.
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