Comparative analysis of mitochondrial markers from four species of Rhipicephalus (Acari: Ixodidae).

Abstract Mitochondrial sequences of four mitochondrial markers: 12S rRNA, 16S rRNA, cytochrome c oxidase subunit I (COX1) and cytochrome b (CytB) from four Rhipicephalus species were analyzed to establish genetic relationships and enable molecular identification. Field-collected samples from the species Rhipicephalus annulatus , Rhipicephalus bursa , Rhipicephalus sanguineus and Rhipicephalus turanicus were amplified by PCR and compared with GenBank™ annotated sequences. PCR products were obtained using primers that were designed to amplify orthologous sequences from different tick species and genera. The average intra-species sequence identity was 98.5–99.5%, while the average inter-species identity was 86.5–89.6%, reflecting a ∼10% decrease in the identity, when different species are compared. The “closest” two species, in terms of sequence identity, were R. sanguineus and R. turanicus , while the “least close” ones were R. annulatus and R. sanguineus . Molecular identification of each species was accomplished by a combined restriction analysis of 12S, COX1 and CytB markers, obviating the need for field sample sequencing. The restriction mapping data suggest that by using several markers, each with a unique digestion pattern, the identity of a given sample could be determined at the species level. It is anticipated that with the accumulation of more information on additional species and markers, molecular identification will become a standard approach for tick classification, complementing morphological taxonomy.