Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS 2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma 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.

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 species 9 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 America 1 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 species 20 .
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 shells 11 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) 20 and 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 al 19 , except for the annealing temperature, which was 60°C.
Afterwards, the fragments were visualized in 6% silver-stained polyacrylamide gels.Digestion and RFLP analysis were performed 18 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 nonshared fragments: Bg1 (130bp) and Bg2 (70bp) for B. glabrata; Bt1 (120bp) and Bt2 (70bp) for B. tenagophila.The restriction profile for B. straminea comprised: 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 technique 19 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.