Abstract
Differentiating between two diphyllobothriid tapeworms Dibothriocephalus dendriticus and Dibothriocephalus ditremus is complicated due to their morphological plasticity, intraspecific variability and a wide range of common hosts. The aim of this study was to develop a species-specific PCR-based method for single-step discrimination between D. dendriticus and D. ditremus. Intraspecific variation and interspecific differences were analysed in subunits/spacers of nuclear rRNA genes and protein-coding genes of mitochondrial DNA. In addition, the specificity of primers designed for the amplification of microsatellite loci in D. dendriticus was tested on D. ditremus DNA. Due to high identity within the rRNA gene in these species, no suitable DNA regions could be identified for the design of the species-specific primers. A higher level of interspecific differences was detected in the mitochondrial cox1 and cob genes, in which regions containing species-specific mutations were chosen for the design of D. dendriticus- and D. ditremus-specific primers. However, their specificity was not confirmed, as the D. dendriticus-specific primers also annealed to D. ditremus DNA and vice versa. Of the 15 primer pairs designed for the amplification of microsatellite loci in D. dendriticus, 13 primer pairs also annealed to D. ditremus DNA. Only two primer pairs, which amplify the Dd_8 and Dd_33 loci have been proven to be D. dendriticus-specific. The effectiveness and high reproducibility of the Dd_8 primers were validated on ~3,500 D. dendriticus and D. ditremus plerocercoids from Iceland and Norway. These primers are recommended for future molecular differentiation between both Dibothriocephalus species.