BACKGROUND: Traditionally, morphological features of Rhipicephalus (Boophilus) annulatus from closely-related ticks have been considered for their identification and differentiation. However, it is difficult and requires expertise in order to accurately identify and differentiate engorged female ticks and some developmental stages such as larva and nymph from other similar ticks. Hence, molecular markers may be a suitable alternative. OBJECTIVES: Mitochondrial cytochrome c oxidase subunit I (COI) gene and the second internal transcribed spacer (ITS2) fragments of Rh. (Bo.) annulatus were sequenced to assess the use of molecular techniques for identifications and phylogenetic studies of these ticks. METHODS: Polymerase chain reaction (PCR) technique was performed based on the analyses of COI and ITS2 sequences of ticks collected from two different regions in Iran (Golestan and Mazandaran). RESULTS: The length of COI and ITS2 sequences were 1539 and 1158bp, respectively. The nucleotide similarity of COI gene was 91.3% between the ticks examined from the two different regions. The deduced amino acid sequences from COI showed 98.6% similarity between the ticks studied and showed 98.2 and 99.6% similarity with the only complete sequence of Rh. (Bo.) annulatus (AGH19677) registered in GenBank. The obtained complete nucleotide sequences of ITS2 from Rh. (Bo.) annulatus from Golestan and Mazandaran revealed 99.9% similarity, while the other ticks registered in GenBank 95 to 99% similarity (KC503267, AF271270, AF271272, JQ412126). CONCLUSIONS: It seems that COI and ITS2 sequences could provide suitable genetic markers for discrimination and genetic characterization of Rhipicephalus (Boophilus) annulatus.

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