Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus

Abstract

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. Graphical abstract

Klimawandel und vektorübertragene Infektionen in EuropaTeil 2: Zeckenübertragene Infektionen

“…Seit einigen Jahren scheint I. inopinatus auch in Süddeutschland und Österreich stabile Populationen zu bilden [12]. Aktuelle genetische Analysen zeigen, dass eine Differenzierung von I. inopinatus und I. ricinus herausfordernd ist und es auch zu einer Hybridisierung der beiden Arten kommen kann, sodass die Ausbreitung in Mitteleuropa nicht vollständig geklärt ist [13,14]. Auch das Vektorpotenzial von I. inopinatus ist noch nicht vollständig geklärt.…”

Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids

“…Furthermore, while 16S rDNA is the genetic marker of choice for delineation of other species of the genus Ixodes , recent studies have shown that it is not the appropriate gene target for identifying I. inopinatus [ 12 – 15 ]. Different genetic markers such as the nuclear gene for tick receptor OspA ( TROSPA ), defensin, internal transcribed spacer 2 ( ITS2 ), and mitochondrial cytochrome c oxidase I ( COI ) provided more robust results when compared with tick morphology and 16S rDNA [ 2 , 14 ]. In our previous study, a combination of nuclear and mitochondrial markers was used to assess the differences between I. inopinatus from Algeria and I. ricinus from the Czech Republic, with several specimens showing signs of hybridization between these two species [ 14 ].…”

“…Different genetic markers such as the nuclear gene for tick receptor OspA ( TROSPA ), defensin, internal transcribed spacer 2 ( ITS2 ), and mitochondrial cytochrome c oxidase I ( COI ) provided more robust results when compared with tick morphology and 16S rDNA [ 2 , 14 ]. In our previous study, a combination of nuclear and mitochondrial markers was used to assess the differences between I. inopinatus from Algeria and I. ricinus from the Czech Republic, with several specimens showing signs of hybridization between these two species [ 14 ]. This study, together with a recent study from Germany, questioned all observations of I. inopinatus outside the Mediterranean Basin based solely on morphology and/or 16S rDNA [ 13 , 14 ].…”

“…In our previous study, a combination of nuclear and mitochondrial markers was used to assess the differences between I. inopinatus from Algeria and I. ricinus from the Czech Republic, with several specimens showing signs of hybridization between these two species [ 14 ]. This study, together with a recent study from Germany, questioned all observations of I. inopinatus outside the Mediterranean Basin based solely on morphology and/or 16S rDNA [ 13 , 14 ].…”

“…The vector capacity of I. inopinatus is unknown, as the majority of studies focusing on tick-borne pathogens in this species used unreliable methods for tick species identification [ 5 , 16 ], with Borrelia lusitaniae being the only detected TBP in reliably identified I. inopinatus [ 17 ]. However, based on the published data, I. inopinatus seems to be a dominant species of genus Ixodes in North Africa [ 2 , 14 ], suggesting that TBPs previously detected in ticks identified as I. ricinus were actually at least to some extent detected from I. inopinatus [ 18 ].…”