title: Development of a PCR-RFLP marker to genetically distinguish Prosorhynchus crucibulum and Prosorhynchus aculeatus.
abstract: The cercariae stages of Prosorhynchus crucibulum and Prosorhynchus aculeatus are morphologically indistinguishable. However, the differentiation of these two species is crucial to understand the transmission dynamics between these primary hosts (mussels) and the secondary hosts (fish). In this way, the objective of this study is to develop an accurate molecular identification tool to differentiate the cercariae stage of P. crucibulum and P. aculeatus. We targeted the 18S nuclear ribosomal DNA region by PCR amplification and sequenced this amplicon. By generating these sequences, we developed a RFLP tool with the use of the enzymes HincII and FokI that produced different restriction profiles between P. crucibulum and P. aculeatus. Each enzyme generated different-sized fragments specific to the species examined and no cross-reaction between the species was detected in their restriction pattern. By sequencing, no intraspecific-polymorphism was detected since there is 100% homology among P. aculeatus or P. crucibulum. These results indicate that PCR-linked restriction analysis of the 18S rDNA region provided us with rapid and reliable molecular tools for distinction of the cercariae of these species.
authors.affiliation: Universidade do Porto, Departamento de Zoologia/Antropologia Rua do Campo Alegre, 4169-007 Porto, Portugal. clairejulianaf@yahoo.com.br
authors.forename: Claire Juliana
authors.initials: CJ
authors.lastname: Francisco
authors.forename: André
authors.initials: A
authors.lastname: Almeida
authors.forename: António Manuel
authors.initials: AM
authors.lastname: Castro
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Santos
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000032:analysis*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014200:Trematoda / Q000145:classification* / Q000235:genetics* / Q000254:growth & development
chemical_list: D018509:DNA, Helminth; D012337:RNA, Ribosomal, 18S; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases