Comparison of the DNA sequences and secondary structure of the mitochondrial 16S rRNA gene of Ixodes kingi, Ixodes sculptus and Ixodes angustus☆
Introduction
The genus Ixodes (Acari: Ixodidae) comprises more than 240 species that parasitize reptiles, birds and/or mammals [1], [2]. Most species of Ixodes have been assigned to one of 14 or more subgenera [3], [4], [5], [6]; however, there has been some doubt as to the subgeneric designations of some species, and the validity of some subgenera [2], [7], [8], [9]. The taxonomic status of some taxa within the genus Ixodes has also been questioned [7], [10], [11]. As a consequence, molecular approaches have been used to examine the taxonomic status of some tick species (e.g., [2], [7], [10], [12]). For example, the mitochondrial (mt) 16S rRNA gene has often been used as a genetic marker to identify or distinguish among species of Ixodes, and to explore their phylogenetic relationships and population genetics [2], [7], [9], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22].
At least 20 species of Ixodes have been reported in Canada [23], [24], [25], [26], [27], several of which are of medical and/or veterinary importance [23], [27]. Therefore, it is important for control and surveillance programs that individual ticks be accurately identified to species. Three species, namely Ixodes angustus, Ixodes kingi and Ixodes sculptus, parasitize a variety of small mammals [22], [23], [26], [28], [29], [30], [31] and have overlapping distributional ranges in western Canada [5], [26], [28], [31]. Of these tick species, I. angustus has been implicated in the spread of Lyme disease in the Pacific Northwest [32], [33], [34]. Previous studies [22], [31] have used PCR-based single-strand conformation polymorphism (SSCP) analyses of domains IV and V of the mt 16S rRNA gene, in combination with DNA sequencing, to distinguish among individuals of I. sculptus, I. kingi and three species of Dermacentor from small mammals in southern Saskatchewan. The same molecular approach was used to confirm the species identity of I. angustus and Dermacentor andersoni individuals from small mammals in Kootenay National Park, British Columbia [30]. The aim of the present study was to compare the sequences of the 3′ region of the mt 16S rRNA gene of I. sculptus, I. kingi and I. angustus to establish where interspecific and intraspecific differences in DNA sequence occur relative to the secondary structure of the 16S gene. Using a structure-based alignment of the sequence data, the evolutionary relationships of I. kingi and I. sculptus, members of the subgenus Pholeoixodes [5], and I. angustus, a member of the subgenus Ixodiopsis [4], [5], were also investigated in relation to other species within the genus.
Section snippets
Materials and methods
Genomic (g) DNA was isolated [30] from the complete bodies of 16 ticks collected in southern Saskatchewan, Canada (Table 1, Fig. 1); seven I. kingi females from cats and dogs, five I. sculptus nymphs from a Richardson's ground squirrel (Spermophilus richardsonii) at Radisson, and four I. sculptus nymphs from a thirteen-lined squirrel (Ictidomys tridecemlineatus) near Clavet. In addition, gDNA was isolated from four Ixodes ricinus adults collected from a cat in Delémont, Switzerland. Part
Results
All nine I. sculptus nymphs collected from Radisson and Clavet had the same DNA sequence of the 16S rRNA gene as that of I. sculptus haplotype SH1 (GenBank accession no. HF968625 [31];). Six of the seven I. kingi females collected in southern Saskatchewan had the same DNA sequence of the 16S gene as I. kingi haplotype KH1 (accession no. HF968622 [31];), whereas one individual from Vanguard had a unique sequence (designated herein as haplotype KH4; Fig. 2). One female I. ricinus (haplotype CA1)
Discussion
Different species of Ixodes can be distinguished from one another based on comparisons of DNA sequence data for domains IV and V of the mt 16S rRNA gene [14], [17], [18], [31], despite significant intraspecific sequence variation in some species [15], [16], [20], [21], [42]. In the present study, I. kingi, I. sculptus and I. angustus, three species that occur on small mammals in western Canada, differed in sequence from one another by 5–9% (i.e., 20–39 bp). This level of difference in DNA
Acknowledgments
Financial support for this work was provided (to NBC) from the Natural Sciences and Engineering Research Council of Canada and the Canadian Foundation for Innovation. A Margaret McKay scholarship and a University of Saskatchewan Graduate Scholarship provided financial support to CAA. This work was approved by the University of Saskatchewan's Animal Research Ethics Board, and adhered to the Canadian Council on Animal Care guidelines for humane animal use. We thank Karen Gesy for providing the
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