Teofânia Heloísa Dutra Amorim VidigalI, II; Kelly Grace MagalhãesI; Omar dos Santos CarvalhoI
ICentro de Pesquisas René Rachou da Fundação Oswaldo Cruz, Belo Horizonte, MG, Brasil IIDepartamento de Zoologia do Instituto de ciências Biológicas da Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil
We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the threeSchistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.
Key-words: Biomphalaria glabrata. Biomphalaria tenagophila. Biomphalaria straminea. Polymerase chain reaction. Internal transcribed spacer 2.
O sequenciamento da região espaçadora transcrita interna 2 do DNA ribossomal (ITS2-DNAr) das espécies brasileiras gênero Biomphalaria (B. glabrata, B. tenagophila and B. straminea) hospedeiras intermediárias do Schistosoma mansoni no Brasil, permitiu a análise dos sítios de restrição presentes nestas seqüências. A análise do mapa de restrição obtido dessas seqüências nos permitiu selecionar enzimas mais promissoras que gerassem perfis de restrição capazes de identificar essas espécies. Foram testadas 4 enzimas e a enzima HpaII foi selecionada por produzir perfis espécie específicos de fácil visualização em gel de poliacrilamida. A utilização da região ITS2 tem como vantagens a obtenção de um fragmento pequeno de 460bp, o qual pode ser facilmente amplificado por PCR. Neste trabalho, nos demonstramos que a amplificação da região ITS2-DNAr e a restrição deste com a enzima HpaII é uma ferramenta molecular auxiliar a identificação morfológica desses moluscos, bem como para estudos taxonômicos e filogenéticos de planorbídeos neotropicais.
Palavras-chaves: Biomphalaria glabrata. Biomphalaria tenagophila. Biomphalaria straminea. Reação em cadeia da polimerase. Região espaçadora transcrita interna 2.
Among the 10 Brazilian snail species and one subspecies of the genus Biomphalaria described up to the present, B. glabrata17, B. tenagophila8, B. straminea5, B. tenagophila guaibensis13 B. peregrina8, B. kuhniana3, B. schrammi4, B. amazonica9, B. oligoza10, B. intermedia16 and B. occidentalis12 only the first three species have been found naturally infected by Schistosoma mansoni14.
The correct identification of Biomphalaria snails is complicated due to the high intra-specific variation in anatomical and morphological characters or great similarity among some species9 11 15. The availability of methodologies based on molecular analysis has enabled the access to more consistent information on Biomphalaria populational structure among Planorbidae. Molecular taxonomy has been able to solve several problems considered insoluble so far by traditional morphology.
The polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) region of the rDNA (1300pb approximately) and a part of COI region of mitochondrial DNA (mit-COI – 780bp approximately) have been used for identification of several Biomphalaria species from Brazil and some regions of South America1 2 18 19 21 22. Restriction enzymes were randomly selected due to the lack of available sequences from Biomphalaria ITS region. But for part of the mit-COI region, analysis of a restriction map available in a data base allowed us to select particular enzymes to be tested and used in PCR-RFLP. In the present work, we report the use of this methodology for specific identification of field populations of B. glabrata, B. straminea and B. tenagophila from different Brazilian localities using restriction profiles provided by the ITS2 region. The choice of such region was due to two specific reasons: 1) on account of the size of the generated fragment for Biomphalaria, after PCR amplification (approximately 460bp). This is a considerably small product when compared with the size of ITS-rDNA and the COI region from this genus, enabling an easier amplification with no need of a high quality DNA; 2) this region proved to be appropriate as it had been sequenced and analyzed in phylogenetic studies of Brazilian Biomphalaria species20.
Ten specimens of each population were killed and fixed. Before fixing the specimens, a fragment of their foot was removed for subsequent DNA extraction. Fixed specimens were identified by means of comparative morphology of the reproductive organs and shells11 12 13 15. Total DNA was extracted from the foot of each snail using the Wizard Genomic DNA Purification Kit (Promega)21. The ITS2 region was amplified using the primers ITS2F (5′-CGTCCGTCTGAGGGTCGGTTTGC-3)20and ETTS1 (5‘-TGCTTAAGTTCAGCGGGT-3′)7 anchored in the conserved extremities of the 5.8S and 28S ribosomal genes, respectively. The PCR amplification conditions were the same used by Vidigal et al19, except for the annealing temperature, which was 60ºC.
The PCR amplification of Biomphalaria ITS2 region, from four specimens of each species under study, resulted in a product of approximately 460bp. Theses products were digested using the following restriction enzymes: TaqI and MboI (Invitrogen, Life Science), RsaI and HpaII (Promega Co, USA). These enzymes were selected based on the restriction map analysis using the program Webcutter version 2.0, (www.firstmarket.com/cutter/cut2.html) of the ITS2 sequences available in the Genbank20. – The access numbers used in our study were: B. glabrata, AF198659, AF198660, AF198661, AF198662; B. tenagophila AF198654, AF198655, AF198656; B. straminea AF198668, AF198669, AF198670, AF198671, AF198672.
Afterwards, the fragments were visualized in 6% silver-stained polyacrylamide gels. Digestion and RFLP analysis were performed18 and the gels photographed with a Mavica digital camera (Sony).
The profiles obtained with TaqI and RsaI did not allow us to distinguish between the three species due to the high similarity among RFLP profiles (data not shown). The most promising profiles were those produced by MboI and HapII, and the best result was obtained with HapII (Figure 1), which provided a simple profile of four fragments for B. glabrata, (200, 130, 90 and 70bp), B. tenagophila (200, 120, 90 and 60bp) and two fragments for B. straminea (300 and 180bp). Although B. glabrata and B. tenagophila share the fragments of 200 and 90bp (Figure 1), they could be separated by other two non-shared fragments: Bg1 (130bp) and Bg2 (70bp) for B. glabrata; Bt1 (120bp) and Bt2 (70bp) for B. tenagophila. The restriction profile for B. stramineacomprised: Bs1 (300bp) and Bs2 (180bp) (Figure 1). Reproducibility of the generated profiles with HapII was supported by the use of specimens originated from different localities in Brazil (Figure 1).
These results demonstrated that PCR-RFLP of the ITS2 region, using HpaII restriction enzyme, is an important tool to distinguish among B. glabrata, B. straminea and B. tenagophila species. Such data is in accordance with those produced for ITS and COI regions, through the same technique19 22 and it also corroborates classical morphological taxonomy.
In general success of the amplification using degraded DNA is difficult to achieve and severely restricted in target size (degraded DNA results in amplification of relatively small fragments)6. Regarding this aspect, we believe that such methodology may be used in studies, in which degraded DNA is recovered from improperly conserved material (low molecular weight < 500bp). Thus, the fragment of 460bp correspondent to the ITS2 region may be more easily amplified by PCR than a region of approximately 1300bp (approximate size of ITS-rDNA from Planorbidae), due to the need for a more conserved or high quality DNA.
To Dra Liana K. Passos from Centro de Pesquias René Rachou/Fiocruz who provided us with valuable critical comments.
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Dr. Omar dos Santos Carvalho
Av. Augusto de Lima 1715
30190-002 Belo Horizonte, MG, Brasil
Fax: 55 31 3295-3115
Recebido para publicação em 3/2/2004
Aceito em 20/5/2004
Work partially supported by CAPES, FAPEMIG and PIBIC/FIOCRUZ