BACKGROUND: Ixodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ). METHODS: A multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis. RESULTS: Sequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected. CONCLUSIONS: We developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.

Biomedical Center Faculty of Medicine in Pilsen Charles University Plzen Czech Republic

CEITEC University of Veterinary Sciences Brno Czech Republic

Department of Botany and Zoology Faculty of Science Masaryk University Brno Czech Republic

Department of Chemistry and Biochemistry Mendel University Brno Czech Republic

Department of Microbiology Nutrition and Dietetics Faculty of Agrobiology Food and Natural Resources CINeZ Czech University of Life Sciences Prague Czech Republic

Department of Parasitology and Parasitic Diseases University of Agricultural Sciences and Veterinary Medicine of Cluj Napoca Cluj Napoca Romania

Department of Parasitology Charles University Prague Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources CINeZ Czech University of Life Sciences Prague Czech Republic

Department of Zoology Palacky University Olomouc Olomouc Czech Republic

Institute of Parasitology Biology Center of Czech Academy of Sciences Budějovice Czech Republic

Institute of Vertebrate Biology of the Czech Academy of Sciences Brno Czech Republic

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