Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 96, Num. 3, 2001, pp. 435-436

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 96(3) Apr. 2001, pp. 435-436

Genetic Markers from Biomphalaria tenagophila (Gastropoda: Pulmonata: Planorbidae) Obtained by the Double Stringency Polymerase Chain Reaction Technique

Roseli Tuan⁺, Paula Cristina Bortolato

Laboratório de Malacologia, Superintendência de Controle de Endemias, Rua Paula Souza 166, 01027-000 São Paulo, SP, Brasil

⁺Corresponding author. Fax: +55-11-229.8292. E-mail: tuan@sucen.sp.gov.br 

Received 13 November 2000
Accepted 3 January 2001

Code Number: oc01066

Biomphalaria tenagophila, one of the intermediate hosts of the trematoda Schistosoma mansoni, is a simultaneous hermafrodite snail species. In order to analyse the genetic structure of these populations, we performed a double-stringency PCR technique to obtain genetic markers with microsatellites and arbitrary primers in a single reaction.

Key words: Biomphalaria - DNA polymorphism - PCR

Biomphalaria are simultaneous hermaphrodite freshwater snails, which can breed mainly by cross (Paraense 1955) but also by self-fertilization (Tuan & Simões 1998). Analysis of the effects of reproduction in freshwater snails using genetic markers such as isozymes (Bandoni et al. 1995) shows heterozygote deficiencies indicating that selfing could be a regular mating strategy creating complex patterns of population structure exhibiting genetic differences that can cause differences in susceptibility to Schistosoma mansoni (Hofman et al. 1998).

Molecular markers are in large use for freshwater snails diagnosis, mainly PCR based protocols which show a great power for quick and simple characterization of genetic variation within and among populations (Avise 1994). DS-PCR described here combines the specificity of microsat-ellites and the simplicity of amplification of rapds markers in a double stringency condition leading to the amplification of selective population of DNA. The technique produced genetically informative co-dominant markers in Drosophila mercatorum (Matioli & Brito 1995).

We applied DS-PCR to laboratory strains B. tenagophila from Tremembé, São José dos Campos and Bananal, all from endemic areas in São Paulo, SP; and a strain from Taim, RS, a non-endemic area. In order to observe intra-population variation we used 12 B. tenagophila snails collected from rice paddies at Tremembé, SP, a site with a long history of S. mansoni transmission (Silva 1992). The hepatopancreas-ovotestis complex were extracted from snails previously identified by morphological reproductive traits (according to Paraense 1975), and free from S. mansoni infection. We used a standard phenol-chloroform protocol for DNA extraction (Sambrock et al. 1989) which produced a clear pattern of DNA (Fig. 1).

The reactions were done in a 25 µl volume containing: 2.5 µl buffer, 1.25 µl MgCl2 50 mM, 5 µl DNTPs mix, 0.25µl of microsatellites primer, 1 µl of RAPD primer, 0.25 µl of Taq DNA polymerase and 2 µl of extracted DNA, and 12.75 µl of distilled water. The microsatellites and RAPD combinations used were 4C [(CAG)₄ and GAACGGACTC], 5C [(GAG)₄ and GAACGGA CTC], 6C [(AACG)₄ and GAACGGACTC], 7C [(ATCG)₄ and GAACGGACTC], 10C [(CGGA)₄ and GAACGGACTC], 14C [(TTTG)₄ and GAA CGGACTC]. All reactions were performed with a Minicycler Termocycler (MJ Research).

The temperature profile was the following: a first denaturation step at 94^oC for 2 min+15 cycles at 94^oC for 30s-52^oC for 45s and 72^oC for 60s + 25 cycles at 94^oC for 30s- 35^oC for 45s- a ramp of 60s to 72^oC for 60s. Finally extension at 70^oC for 10 min. The products were analyzed by agarose gel electrophoresis (1.4% in TBE buffer) and stained with ethidium bromide (EtBr).

The interpopulational profiles of the six primer combinations show a significant degree of genetic variation, suggesting heterogeneous genetic population patterns (Fig. 2).

At the intrapopulational level DS-PCR generates polymorphisms that can contribute with the comparison of populations derived from different environmental conditions (Fig. 3).

We concluded that DS-PCR is an excellent tool that can be applied to identify genetic polymorphisms within and among hermaphrodite snail populations. DS-PCR employed here is a method which has the large applicability of RAPD but also can generate codominant markers that are more informative for population analyses.

ACKNOWLEDGEMENTS

To Dr Sergio Russo Matioli and Dr Priscila dos Santos for their kind and valuable help.

REFERENCES

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• Tuan R, Simões LCG 1998. Effect of self-fertilization on Biomphalaria tenagophila (Orbigny, 1835) (Pulmonata: Planorbidae). Gen Mol Biol 21: 477-478.

Financial support: Fapesp (Proc. 99/0091-7)

Copyright 2001 Fundacao Oswaldo Cruz Fiocruz