Molecular characterization of six Hyalomma species using mitochondrial markers.

Hyalomma species (Acari: Ixodidae) are vectors of several human and animal pathogens. However, due to their similar morphological properties, classification of related Hyalomma species is often challenging. Here we describe a combined approach for molecular characterization of six Hyalomma species: H. aegyptium, H. dromedarii, H. excavatum, H. impeltatum, H. marginatum and H. turanicum. This procedure was developed using a combination of PCR amplification of four molecular markers, followed by sequencing and species-specific restriction analysis. Segments from the following genes were used as markers: 12S rRNA, 16S rRNA, Cytochrome C oxidase subunit 1 (COX1), and Cytochrome B (CytB). Phylogenetic analysis based on the amplified sequences was consistent with the morphology-based classification. It revealed relative close proximity of H. excavatum, H. marginatum and H. turanicum, and close proximity of H. aegyptium and H. dromedarii to each other. H. impeltatum was examined using the COX1 and CytB markers, and in both cases was located on a separate clade from the other five species. Digestion of the amplified products using specific restriction enzymes enabled clear distinction between the six species. This report is the first to describe CytB marker sequences of the studied species, and the first to describe COX1 marker sequences of H. aegyptium, H. excavatum, H. impeltatum and H. turanicum. The information obtained in this study may therefore be useful for future combined morphological-molecular Hyalomma characterization.