Development of a PCR-RFLP marker to genetically distinguish Prosorhynchus crucibulum and Prosorhynchus aculeatus.
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.
Francisco
Claire Juliana
CJ
Universidade do Porto, Departamento de Zoologia/Antropologia Rua do Campo Alegre, 4169-007 Porto, Portugal. clairejulianaf@yahoo.com.br
Almeida
André
A
Castro
António Manuel
AM
Santos
Maria João
MJ
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
D018509:DNA, Helminth; D012337:RNA, Ribosomal, 18S; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases