Further Studies on the Molecular Systematics of Biomphalaria Snails from Brazil Teofânia HDA Vidigal/*, Roberta Lima Caldeira, Andrew JG Simpson**, Omar S Carvalho/+
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 95(1): 57-66, Jan./Feb. 2000 57 Further Studies on the Molecular Systematics of Biomphalaria Snails from Brazil Teofânia HDA Vidigal/*, Roberta Lima Caldeira, Andrew JG Simpson**, Omar S Carvalho/+ Centro de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brasil *Departamento de Zoologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG, Brasil **Laboratório 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 inter- nal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecu- lar identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymor- phism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza pre- sented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic mark- ers for the identification of these snails Key words: Biomphalaria - ribosomal DNA - internal transcribed spacer - polymerase chain reaction - Brazil Ten species and one subspecies of 1988). To overcome this problem, the use of mo- Biomphalaria are recognized in Brazil: B. glabrata lecular techniques as additional tools for the iden- (Say, 1818), B. tenagophila (Orbigny, 1835), B. tification of these snails has been proposed. In pre- tenagophila guaibensis Paraense, 1984, B. vious studies we tested a low stringency poly- straminea (Dunker, 1848), B. peregrina (Orbigny, merase chain reaction technique (LS-PCR) (Dias 1835), B. kuhniana (Clessin 1883), B. schrammi Neto et al. 1993) which permited the differentia- (Crosse, 1864), B. amazonica Paraense 1966, B. tion of B. glabrata and B. t. tenagophila and also oligoza Paraense, 1974, B. intermedia (Paraense the identification of very similar species such as & Deslandes, 1962) and B. occidentalis Paraense B. t. tenagophila and B. occidentalis (Vidigal et al. 1981. Only, B. straminea, B. t. tenagophila and B. 1996, Pires et al. 1997). glabrata are found naturally infected with Schis- Recently, we have explored the possibility of tosoma mansoni. Among these, B. glabrata is the using sequence polymorphism in the internal tran- most important due to its wide spread distribution scribed spacer region (ITS) of the rDNA of and its high susceptibility to S. mansoni (Paraense Biomphalaria snails (which includes the 5.8S & Corrêa 1963). In addition B. peregrina and B. rDNA gene together with the flanking ITS1 and amazonica are considered potential hosts of this ITS2 spacers) by means of PCR amplification and parasite based on experimental infection (Corrêa digestion with different restriction enzymes. This & Paraense 1971, Paraense & Corrêa 1973). method was successfully used to construct the The morphological identification of freshwa- molecular key for Aedes species (West et al. 1997). ter snails of medical importance, is greatly com- It was also used to distinguish closely related para- plicated by the extensive intraspecific variation of sitic worms and others different organisms (New- the morphological characteristics used for classi- ton et al. 1998, Wu et al. 1999) and on studies of cal identification (Paraense 1975, 1981, 1984, 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, Work partially supported by Fapemig and Pibic/Fiocruz. Vidigal et al. 1998a,b, Caldeira et al. 1998, Spatz +Corresponding author. Fax: + 55-31-295.3115. E-mail: et al. 1998, 1999). [email protected] The aim of the present study was to investigate Received 8 June 1999 the use of restriction profiles resulting from diges- Accepted 11 November 1999 tion of the ITS spacer of rDNA with Dde I for sepa- 58 Molecular Systematics of Biomphalaria Teofânia HDA Vidigal et al. ration of the majority Biomphalaria species from ml proteinase K. Thereafter, 80 µl of protein pre- Brazil, with special attention to B. oligoza, B. cipitation solution was added to the initial mix. The schrammi and B. amazonica species which were mixture was vortex vigorously for 10-20 sec and being studied for the first time. We found diffi- centrifuged at 13,000 rpm for 5 min. The superna- culty to differentiate between B. peregrina and B. tant was transferred to a microcentrifuge tube con- oligoza with DdeI, however we propose the use of taining 200 µl of room temperature isopropanol others restriction enzymes such as HpaII, Hae III for DNA precipitation. The mixture was gently and AluI for identification of these species. mixed by inversion for 20 min and centrifuged at MATERIALS AND METHODS 13,000 rpm for 5 min. The DNA pellet was washed with 300 µl of 70% ethanol and centrifuged for 1 Snail populations - Snail populations from dif- min. The pellet was treated with 25 µl of DNA re- ferent Brazilian localities were used (Fig. 1). The hydration solution for 30 min at 65°C and stored number of snails used and the geographical local- at -20°C. The DNA concentrations were estimated ization from each population are shown in the by comparison with known standards on 2% Table. The snails were identified by means of com- ethidium bromide stained agarose gels. parative morphology based on the reproductive Amplification by PCR - The entire ITS was organs and shells accordingly to Deslandes (1951) amplified using the primers ETTS2 (5/- and Paraense (1975, 1981, 1984, 1988). TAACAAGGTTTCCGTAGGTGAA-3/) and DNA extraction - Total DNA was extracted ETTS1 (5/-TGCTTAAGTTCAGCGGGT-3/) an- from the feet of snails using the Wizard Genomic chored respectively in the conserved extremities DNA Purification Kit (Promega) with some modi- of the 18S and 28S ribosomal genes (Kane & fications. Briefly, the tissues were mechanically Rollinson 1994). The PCR amplification was un- disrupted in 200 µl of nucleic lysis solution and dertaken in a volume of 10 µl consisting of: 1-10 incubated at 37°C for 4 h or overnight with 50 µg/ ng template DNA, 10mM Tris-HCl, pH 8.5, 200 Fig. 1: map showing the localization of the snail populations used. The legend shows the abbreviation of each species mentioned in the map. Biomphalaria glabrata (Bg); B. tenagophila tenagophila (Btt); B. straminea (Bs); B. occidentalis (Boc); B. t. guaibensis (Btg); B. amazonica (Ba); B. intermedia (Bi); B. kuhniana (Bk); B. peregrina (Bp); B. oligoza (Bo); B. schrammi (Bsch). Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 95(1), Jan./Feb. 2000 59 µm each dNTP, 1.5 mM MgCl2, 0.8 U of Taq DNA cies obtained from different localities (Table). Fig. polymerase, 50 mM KCl, together with 5.0 pmol 3A shows the profiles obtained using DdeI with B. of each primer. The reactions were covered with a oligoza collected in Rio Grande do Sul (lanes 1 to drop of mineral oil and subjected to the following 4) and Santa Catarina (lanes 5 to 10). The profiles cycle program: initial denaturation step for 3 min were very homogeneous for all specimens. Fig. 3B at 95°C, and then 32 cycles with annealling at 54°C shows the profiles obtained for B. peregrina speci- for 1 min, extension at 72°C for 2 min, denatur- mens collected from different localities in Minas ation at 95°C for 45 sec and a final extension step Gerais. The profiles obtained with B. peregrina at 72°C for 5 min. A negative control (no template were homogeneous but, as mentioned above, simi- DNA) was included in all experiments. Three mi- lar to the those obtained from B. oligoza (Fig. 3A). croliters of the amplification products were Fig. 4 shows the profiles of B. schrammi ob- visualised on 0.8% ethidium bromide stained aga- tained with DdeI. This enzyme produced a simple rose gels to check the quality of amplification. The profile with six fragments, which was clearly dif- remaining 7 µl were mixed with 53 µl of water, ferent from those seem with other species as ob- and divided into 10 µl aliquots for enzyme diges- served in Fig. 2B (lanes 22 to 24). B. amazonica tion. presented three variant profiles with three to nine Production and evaluation of the rDNA-ITS fragments when treated with DdeI (Fig. 5). In fact RFLP profiles - In the initial experiments the en- in the snails from one locality (Benjamin Constant, zyme DdeI (Amersham, Life Science) was evalu- Amazonas) three profiles were observed: lanes 1, ated for its ability to differentiate all Biomphalaria 2 and 4 show the profiles characterized by pres- species from Brazil. Addicional enzymes were ence of three bands, lane 3 shows five fragments tested including AluI, MnlI, (Amersham, Life Sci- and lanes 5, 6 and 7 show the profiles with nine ence) HaeIII and HpaII (Promega Co, USA) in situ- fragments. In the snails obtained from Barão do ations when DdeI was not effective in the separa- Melgaço, Mato Grosso (lanes 8 to 10) only one tion of some species. One microliter (10-12 units) profile with five fragments was observed. The was used for each digestion reaction, together with specimens with variant profiles, for this species, 1.2 µl of the respective enzyme buffer in a final were included in Fig. 2B (lanes 25 to 28). The pro- volume of 12.2 µl.