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vol.97 suppl.1Freshwater snails and Schistosomiasis mansoni in the state of Rio de Janeiro, Brazil: III - Baixadas MesoregionGenetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection author indexsubject indexarticles search
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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276On-line version ISSN 1678-8060

Mem. Inst. Oswaldo Cruz vol.97  suppl.1 Rio de Janeiro Oct. 2002

https://doi.org/10.1590/S0074-02762002000900011 

Polymerase Chain Reaction and Restriction Fragment Length Polymorphism of Cytocrome Oxidase Subunit I Used for Differentiation of Brazilian Biomphalaria Species Intermediate Host of Schistosoma mansoni

Vol. 97(Suppl. I): 47-52, 2002

Teofânia HDA Vidigal/*, Lângia C Montresor, Andrew JG Simpson**, Omar S Carvalho*/+

Departamento de Zoologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil *Laboratório de Helmintoses Intestinais, Centro de Pesquisas René Rachou-Fiocruz, Av. Augusto de Lima 1715, Belo Horizonte, MG, Brasil **Instituto Ludwig de Pesquisa sobre o Câncer, São Paulo, SP, Brasil

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.

Key words: Biomphalaria - polymerase chain reaction - restriction fragment length polymorphism - cytochrome oxidase I

The development of an easy and reliable method for identification of species of Biomphalaria, intermediate hosts of Schistosoma mansoni, remains an important goal regarding schistosomiasis control efforts.

Recently, different DNA regions have been proposed to be used in molecular studies as an additional tool to the morphological characterization of Biomphalaria snails. These techniques include procedures such as analysis of the region 18S of rRNA through low stringenecy polymerase chain reaction (LS-PCR) (Vidigal et al. 1996, Pires et al. 1997), polymerase chain reaction and restriction fragment length analysis (PCR-RFLP) of the internal transcribed spacer region of the DNA (ITSr-DNA) (Vidigal et al. 1998, 2000, 2001, Caldeira, 1998, 2000, Spatz et al. 1999, 2000). However, high intraespecific polymorphisms and complex profiles, produced by such methodologies, may restrict their use.

Although Biomphalaria snails show a high genetic variability (Knight et al. 1991, Vidigal et al. 1994), these molecular tools have important practical implications mainly concerning aspects on, extensive intraspecific variation at the morphological level (Paraense & Deslandes 1955, Paraense 1975), small size of some specimens, and inadequate fixation of snails, which make the morphlogical study rather difficult and also complicate their correct identification.

Vidigal et al. (1998), showed that PCR-RFLP analysis of the ITS region is an important approach for identifying several populations of the intermediate hosts of S. mansoni in Brazil: B. glabrata, B. tenagophila and B. straminea.Interestingly, B. straminea, from Porto Alegre, showed distinct profiles from B. straminea, from other Brazilian localities, Argentina and Uruguay. Despite its polymporhic profile, it was possible to identify two common bands (namely species-specific bands) to all populations under study. In addition, B. tenagophila, from Argentina, showed a different restriction pattern when compared with those from Brazil and Paraguay, although all populations had presented at least a band in common. Thus, the ITS region showed to be useful for distinction of Brazilian S. mansoni intermediate hosts, in spite of intraspecific variations.

Cytochrome oxidase I (COI) is a mitocondrial (mt) gene with an evolution rate smaller than that observed for mtDNA and its amino acid sequence is highly conserved across phyla. Molecular tools, using mtDNA COI region, have been used in several genetic studies (Lovette et al. 1999, Matsumoto & Hayami 2000, Kurabayasshi & Ueshima 2000, Campbell et al. 2000). Recently, Clark et al. (2001) used PCR-RFLP of a portion of the mitochondrial COI as a diagnostic marker for identifying several species of Coleoptera. In snails, COI region has been used for phylogenetic analysis and for interspecific comparison within the genus Bulinus, Biomphalaria and other gastropods (Stothard & Rollinson 1997, Davis et al. 1998, Campbel et al. 2000). Campbel et al. (2000) and Jones et al. (2001), analysing Biomphalaria and Bulinus, respectively, used the ITS1-rDNA sequence regions and part of COI, and showed similar results generated by each region. Stothard et al. (2001) mentioned the use of PCR-RFLP of one COI region for the differentiation of Bulinus snails.

In order to assess interspecific and intraspecific variations among B. glabrata, B. straminea and B. tenagophila and verify the possibility of their identification using another DNA region (besides ITS), we amplified a COI region and then a restriction site analysis (PCR-RFLP).

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

MATERIALS AND METHODS

Snails and DNA extraction - B. glabrata, B. tena-gophila and B. straminea populations, from different Brazilian geographical regions (Table), were used in this study. We also included, for comparison, populations of B. straminea from several Argentinean and Uruguayan localities. Snails were morphologically identified and their DNA was extracted. Morphological characterization to identify the specimens was carried out according to Paraense (1975, 1988). Their total DNA was extracted from the foot, as described by Vidigal et al. (2000).

Production and evaluation of the COI RFLP profiles - One COI fragment of the three Biomphalaria species was amplified with an universal primer described by Folmer et al. (1994) (LCO- 5'-ggTCAACAAATCATAAAg ATATTgg-3'/HCO-5'-TAAACTTCAgggTgACCAAAA AATCA-3'). PCR was carried out according to Vidigal et al. (2000) except for initial denaturing step, which was 95°C for 3 min followed by 25 cycles at 95°C for 1 min; 50°C for 1 min; 72°C for 1 min 30 sec; and a final extension step at 72°C for 7 min.

PCR products were digested with RsaI and AluI enzymes [that were selected based on the sequences published by Campbell et al (2000), available in Genbank], and visualized in 6% silver-stained polyacrylamide gel. The gels were photographed with a MAVICA digital camera (Sony). Many individuals of each species according to snails distribution, in Brazil, were submitted to restriction enzyme digestion.

RESULTS

For COI region specific PCR, we obtained a single amplification product of approximately 780 base pairs (bp) for all species under study (Fig. 1). Digestion of this fragment with AluI produced highly polymorphic intraspecific and interespecific restriction profiles (Fig. 2) when we analyzed 9 populations of B. glabrata (18 individuals), 5 of B. straminea (8 individuals) and 6 of B. tenagophila (14 individuals). The best results were obtained with RsaI. For B. glabrata, we found one specific fragment (approximately 400 bp) that is present in all tested populations, however, the intraspecific profiles were not identical. Thus, B. glabrata snails were characterized by two fragments, one of 400 bp and another of 250 or 280 bp approximately, depending on the geographical origin (Fig. 3).

B. tenagophila and B. straminea showed to have a similar restriction profile with two fragments (Fig. 3). The largest fragment (approximately 700 bp) did not enable the species differentiation, but the pattern of the smallest fragment (70 bp for B. tenagophila and 80 bp for B. straminea) is distinguishable.

In addition, we performed COI digestion with RsaI of B. straminea snails from Brazil, Uruguay and Argentina, and obtained the same restriction band patterns of 700 and 80 bp for all individuals.

DISCUSSION

Analysis of a single locus can result in incorrect species identification or lead to ambiguous conclusions (Anderson 2001). Through a comparison among results obtained with mitochondrial DNA those with ITS Blouin (2002) stated that the latter is more indicated for nematode diagnosis. Such fates suggest that it is necessary to use different regions for specific characterization, in order to achieve more reliable results.

We successfully used a method based on the amplification of part of mtDNA-COI region, followed by its digestion with restriction enzymes. Inasmuch as the advantages of mithocondrial DNA applicability on sistematics and phylogenetic studies for several organisms, including snails (Casiraghi et al. 2001, Clark et al 2001, DeJong et al. 2001), are considerable.

The product generated through specific PCR using the primers described by Folmer (1994) are of approximately 780 bp, value that is similar to the obtained by this author (710bp) for 11 invertebrate phyla (including Mollusca). The analysis of generated fragments (RFLP), aimed at obtaining bands to enable specific identification of B. glabrata, B. tenagohila and B. straminea, was also carried out. We performed digestions using two previously selected enzymes (AluI and RsaI). AluI digestion (Fig. 2) produced highly polymorphic intraspecific and interespecific restriction profiles indicating that this enzyme does not enable the differentiation of the three species and that there is a wide intraspecific COI variability among populations from diverse Brazilian regions. Indeed, this enzyme produced at least 4 profiles for B. glabrata, and some samples did not show restriction site for such enzyme (Fig. 2 lanes 3 to 6). Concerning B. tenagophila and B. straminea, the profiles showed to be polymorphic, although two populations of both species presented similar profiles (Fig. 2. lanes 15 to 18; lanes 26 to 29). This information agrees with those by Wilding et al. (1999) who emphasize a high polymorphism rate in mollusks mitochondrial genome.

The most informative results, according to morphological data, were provided by RsaI digestion which showed to be potentially useful in these snails characterization. All enzimatic digestion of B. glabrata individuals resulted in two profiles when snails from different populations, from Brazil, were analysed. For example, the 250 bp fragment was present in populations from Maranhão and Pará, while the 280 bp fragment was present in those samples from Rio Grande do Norte, Pernambuco, Bahia and Minas Gerais. For B. straminea, we found this method to be reliable in clustering the individuals from Porto Alegre (Brazil), Argentina and Uruguay together with the populations from other Brazilian localities, as they showed the same profile (Fig. 4),different from that based on PCR-RFLP do ITS (Vidigal et al. 1998).This technique applied to COI region showed to be very effective in grouping the B. straminea populations under study (including those from Argentina and Uruguay). Despite the similarity among the size of the differential fragment, which illustrates the difference between B. tenagophila and B. straminea, these results are potentially useful for molecular identification.

These results demonstrate that PCR-RFLP of this COI region, using RsaI enzyme, is an important tool for differentiation of B. glabrata, B. straminea and B. tenagophila species and they are also in accordance with those produced with PCR-RFLP of ITS, with DdeI enzyme by Vidigal et al. (1998), corroborating the Biomphalaria snails classical morphological identification.

However, for the species B. straminea, PCR-RFLP of COI region showed more satisfactory results than those of the ITS region, as a single profile was produced for all samples in this trial(Brazil, Argentina, Uruguay). The current data emphasize the importance of more than one DNA region for molecular analysis. Further analyses on B. tenagophila, through the sequencing of the COI region, will be carried out for specific identification and phylo-genetic studies of Brazilian snails of the genus Biomphalaria.

ACKNOWLEDGMENTS

To Dr Roberta Lima Caldeira from Centro de Pesquias René Rachou, for providing valuable critical comments.

REFERENCES

  • Anderson TJC 2001. The dangers of using single locus markers in parasite epidemiology: Ascaris as a case study. Trends Parasitol 17: 183-188.         [ Links ]

  • Blouin MS 2002. Molecular prospecting for cryptic species of nematodes: mitochondrial DNA versus internal transcribed spacer. Int J Parasitol 32: 527-531.         [ Links ]

  • Caldeira RL, Vidigal THDA, Matinella L, Simpson AJG, Carvalho OS 2000. Identification of planorbids from Venezuela by polymerase chain reaction amplification and restriction fragment length polymorphism of ITS of the RNA ribosomal gene. Mem Inst Oswaldo Cruz 95: 171-177.         [ Links ]

  • Caldeira RL, Vidigal THDA, Paulinelli ST, Simpson AJG, Carvalho OS 1998. Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a PCR-RFLP. Mem Inst Oswaldo Cruz 93: 219-225.         [ Links ]

  • Campbell G, Jones CS, Lockyer AE, Hughes S, Brown D, Noble LR, Rollinson D 2000. Molecular evidence supports an African affinity of the Neotropical freshwater gastropod, Biomphalaria glabrata, Say 1818, an intermediate host for Schistosoma mansoni. Proc R Soc Lond B 267: 2351-2358.         [ Links ]

  • Casiraghi M, Anderson TJ, Bandi C, Bazzocchi C, Genchi C 2001. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 1: 93-103.         [ Links ]

  • Clark TL, Meinke LJ, Foster JE 2001. PCR-RFLP of the mitochondrial cytochrome oxidase (subunit I) gene provides diagnostic markers for selected Diabrotica species (Coleoptera: Chrysomelidae). Bull Ent Res 91: 419-427.         [ Links ]

  • Davis GM, Wilke T, Spolky C, Qiu C, Qiu, D, Xia M, Zhang Y, Rosenberg G 1998. Cytochrome oxidase I – based phylogenetic relationships among the Pomatiopsidae, Hydrobiidae, Rissoidae and Truncatellidae (Gastropoda: Caenogas-tropoda: Rissoacea). Malacologia 40: 251-266.         [ Links ]

  • DeJong RJ, Morgan JA, Paraense WL, Pointier JP, Amarista M, Ayeh-Kumi PF, Babiker A, Barbosa CS, Bremond P, Canese, AP, de Souza CP, Dominguez C, File S, Gutierrez A, Incani RN, Kawano T, Kazibwe F, Kpikpi J, Lwambo NJS, Mimpfoundi R, Njiokou F, Noel Poda J, Sene M, Velasquez LE, Yong M, Adema CM, Hofkin BV, Mkoji GM, Loker ES 2001. Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni. Mol Biol Evol 18: 2225-2239.         [ Links ]

  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R 1994. DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates. Mel Marine Biol Biot 3: 294-299.         [ Links ]

  • Jones CS, Rollinson D, Mimpfoundi R, Ouma J, Kariuki HC, Noble LR 2001. Molecular evolution of freshwater snail intermediate hosts within the Bulinus forskalii group. Parasitology 123 (Suppl.): S277-S292.         [ Links ]

  • Knight M, Brindley PJ, Richards CS, Lewis FA 1991. Schistosoma mansoni: use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata. Exp Parasitol 73: 285-294.         [ Links ]

  • Kurabayashi A, Ueshima R 2000. Complete sequence of mitochondrial DNA of the primitive Opistobranch Gastropod Pupa strigosa: Systematic implications of the genome organization. Mol Biol Evol 17: 266-277.         [ Links ]

  • Lovette IJ, Bermingham E, Rohwer S, Wood C 1999. Mitochondrial restriction fragment length polymorphim (RFLP) and sequence variation among closely related avian species and the genetic characterization of hibrid Dendroica warbler. Mol Ecol 8: 1431-1441.         [ Links ]

  • Matsumoto M, Hayami I 2000. Phylogenetic analysis of the family Pectinidae (Bivalvia) based on mitochondrial cytochrome C oxidase subunit I. J Moll Stud 66: 477-488.         [ Links ]

  • Paraense WL 1975. Estado atual da sistemática dos planorbídeos brasileiros. Arq Mus Nac Rio de Janeiro 55: 105-128.         [ Links ]

  • Paraense WL 1988. Biomphalaria kuhniana (Clessin, 1883), planorbid mollusc from South America. Mem Inst Oswaldo Cruz 83: 1-12.         [ Links ]

  • Paraense WL, Deslandes N 1955. Observations on the morphology of Australorbis glabratus. Mem Inst Oswaldo Cruz 53: 87-103.         [ Links ]

  • Pires ER, Vidigal THDA, Simpson AJG, Carvalho OS 1997. Identification of Biomphalaria tenagophila and Biomphalaria occidentais populations by the low stringency polymerase chain reaction (LS-PCR). Mem Inst Oswaldo Cruz 92: 101-106.         [ Links ]

  • Spatz L, Vidigal THDA, Caldeira RL, Neto ED, Cappa SMG, Carvalho OS 1999. Study of Biomphalaria tenagophila, B. t. guaibensis and B. occidentalis by polymerase chain reaction amplification and restriction enzyme digestion of the ribosomal RNA gene intergenic spacer. J Moll Stud 65: 143-149.         [ Links ]

  • Spatz L, Vidigal THDA, Silva MCA, Cappa SMG, Carvalho OS 2000. Characterization of Biomphalaria orbignyi, B. peregrina and B. oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene. Mem Inst Oswaldo Cruz 95: 807-814.         [ Links ]

  • Stothard JR, Rollinson D 1997. Partial DNA sequences from the mitochondrial cytochrome oxidase I (COI) gene can differentiae the intermediate snail host Bulinus globosus, and B. nasatus (Gastropoda: Planorbidae). J Nat Hist 31: 727-737.         [ Links ]

  • Stothard JR, Bremond P, Andriamaro L, Sellin B, Sellin E, Rollinson D 2001. Bulinus species on Madagascar: molecular evolution, genetic markers and compatibility with Schistosoma haematobium. Parasitology 123: 261-275.         [ Links ]

  • Vidigal THDA, Caldeira RL, Simpson AJG, Carvalho OS 2000. Further studies on the molecular systematics of Biomphalaria snails from Brazil. Mem Inst Oswaldo Cruz 95: 57-66.         [ Links ]

  • Vidigal THDA, Caldeira RL, Simpson AJG, Carvalho OS 2001. Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer. Mem Inst Oswaldo Cruz 96: 661-665.         [ Links ]

  • Vidigal THDA, Neto ED, Carvalho OS, Simpson AJG 1994. Biomphalaria glabrata: extensive genetic variation in Brazilian isolates by random amplified polymorphic DNA analysis. Exp Parasitol 79: 187-194.         [ Links ]

  • Vidigal THDA, Neto ED, Simpson AJG, Carvalho OS 1996. A low stringency polymerase chain reaction approach to identification of Biomphalaria glabrata and Biomphalaria tenagophila intermediate snail hosts of Schistosoma mansoni in Brazil. Mem Inst Oswaldo Cruz 91: 739-744.         [ Links ]

  • Vidigal THDA, Spatz L, Nunes ND, Simpson AJG, Carvalho OS, Neto ED 1998. Biomphalaria spp: identification of the intermediate snail hosts of Schistosoma mansoni by polymerase chain reaction amplification and restriction enzyme digestion of the ribosomal RNA gene intergenic spacer. Exp Parasitol 89: 180-187.         [ Links ]

  • Wilding CS, Mill PJ, Grahame J 1999. Partial sequence of the mitochonderial genome of Littorina saxatilis: relevance to gastropod phylogenetics. J Mol Evol 48: 348-359.         [ Links ]

Fig. 1 | Fig. 2 | Fig. 3 | Fig. 4 | Table

Work partially supported by Fapemig and Pibic/Fiocruz.
+Corresponding author. Fax: + 55-31-3295.3115. E-mail: omar@cpqrr.fiocruz.br
Received 18 June 2002
Accepted 15 August 2002

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