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Contributions to the morphology and phylogeny of the newly discovered bat tick species, Ixodes ariadnae in comparison with I. vespertilionis and I. simplex
© Hornok et al.; licensee BioMed Central. 2015
Received: 17 September 2014
Accepted: 16 January 2015
Published: 24 January 2015
Recently a new hard tick species, Ixodes ariadnae has been discovered, adding to the two known ixodid tick species (I. vespertilionis and I. simplex) of bats in Europe.
Scanning electron microscopic comparison of adult females of these species shows morphological differences concerning the palps, the scutum, the Haller’s organ, the coxae, as well as the arrangement and fine structure of setae. Molecular analysis of 10 geographically different isolates revealed 90-95% sequence homology in the 12S and 16S rDNA genes of bat tick species. Based on 12S rDNA sequences, genotypes of I. ariadnae clustered closest to I. simplex, whereas according to their 16S rDNA gene they were closest to I. vespertilionis. The subolesin gene of I. ariadnae had only 91% sequence homology with that of I. ricinus, and is the longest known among hard tick species.
The present study illustrates the morphology and clarifies the phylogenetic relationships of the three known bat tick species that occur in Europe. According to its subolesin gene I. ariadnae may have a long evolutionary history.
In Europe and in the majority of the Old World, for more than a century two ixodid ticks were known to be highly specialized to bats, i.e. the bat tick (Ixodes simplex) and the long-legged bat tick (Ixodes vespertilionis) . Recently, however, a new tick species has been discovered to parasitize chiropterans, hitherto reported only from Hungary . The significance of bat ticks is increased by the fact that bats frequently live close to (or in) human dwellings, and at least I. vespertilionis may feed on humans  and has been reported to be a potential vector of zoonotic bartonellae .
In the description of I. ariadnae  it was pointed out that it shares features with both I. vespertilionis (e.g. its relatively large size and long legs) and I. simplex (e.g. its short, broad palps). However, it was beyond the scope of the latter study to illustrate these similarities and differences with appropriate scanning micrographs of all three bat tick species. In addition, although the most important gene that is used for barcoding (species identification) among ticks , i.e. the cytochrome oxidase subunit I (COI) gene of I. ariadnae was shown to differ considerably from those of I. vespertilionis and I. simplex , examination of the taxonomic relationship of closely related species should optimally include analysis of multiple genes. Therefore it was decided to extend the previous results by a simultaneous morphological comparison of all three bat tick species, together with the molecular analysis of three further (12S/16S rDNA and subolesin) genes.
The morphology of two adult females of each of the three bat tick species was compared: Ixodes vespertilionis and I. ariadnae were collected in Hungary; I. simplex was collected in Serbia. For scanning electron microscopy (performed as in ) females were chosen, because (a) larvae and nymphs of all three species were not available, and (b) in the adult form females are parasitic: males either do not suck blood (I. vespertilionis) or are unknown (I. ariadnae) .
Total nucleic acid (TNA) was extracted as described . Ten TNA extracts from isolates of different geographical origin were used for molecular studies: four of I. vespertilionis, four of I. ariadnae, and two of I. simplex . From these samples it was attempted to amplify fragments of the 16S and 12S rDNA genes, as well as the subolesin gene as reported [7-9]. PCR products were resin purified (Wizard, Promega) and cloned into the pGEM-T vector (Promega, Madison, WI, USA) for sequencing both strands (Secugen S.L., Madrid, Spain). Obtained sequences were submitted to the GenBank (accession numbers: KM455956-65 for 12S genotypes A-J, respectively; KM455966-70 for 16S genotypes A, B, E, F, I, respectively, and KM455971 for subolesin gene). Phylogenetic analyses were conducted according to the Tamura-Nei model  and Maximum Composite Likelihood method by using MEGA version 5.2 .
Authorization for bat capture was provided by the National Inspectorate for Environment .
Results and discussion
Scanning electron microscopy of females
Recently it has been suggested that for molecular identification of tick species sequencing of the COI gene should be the first method of choice, and analysis of 12S and 16S rDNA genes can be performed as complementary tests [5,7,8]. Prior to this study there was only one 12S (and no 16S) sequence of I. vespertilionis in GenBank (U95909), and for I. simplex only other genes have been published [12,13]. Therefore sequences obtained in the present study compensate for this lack of information on genetic markers in case of bat tick species.
Concerning differences between genotypes of the same bat tick species, I. ariadnae had the lowest sequence heterogeneity in its 12S gene (with up to two nucleotide differences), as contrasted to the other two bat tick species (with up to five nucleotide differences) (Figure 6). In line with this, the COI sequences were also highly conserved between isolates of I. ariadnae . The 12S sequences of geographically distant specimens were always different, unlike those of the 16S rDNA gene (Figure 6).
These results also imply, that among all the hard tick species for which the subolesin gene and translated protein was reported so far (in : 10 species of six genera were included), I. ariadnae has the longest known subolesin gene/protein (with 191 amino acids). Taking into account, that according to phylogenetical analyses subolesin sequences may have evolved from longer sequences in Ixodes spp. to shorter ones in other tick genera , it is possible that I. ariadnae is an ancient tick species. The importance of the latter finding, i.e. a new type of subolesin (protective) gene is further increased by the fact, that this gene is regarded as one of the most likely candidates among the targets of vaccines to control tick-infestations [9,15].
These results support the taxonomic status of I. ariadnae as a separate species, and illustrate the morphological and phylogenetic differences between the three known European bat tick species. According to its subolesin gene I. ariadnae may have a long evolutionary history.
The survey was organized in the framework of the EurNegVec COST Action TD1303. Contribution of ST was supported by a grant from the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. 173006). The authors also thank Jelena Burazerovic for collecting I. simplex.
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