Multi-marker analysis reveals an extensive genetic diversity in Rhipicephalus sanguineus (temperate lineage) and the first identification of Rhipicephalus rutilus in Tunisia

The precise identification of Rhipicephalus species circulating in Tunisia is crucial for effective management and prevention of diseases, as these ticks are significant vectors for various pathogens affecting both animals and humans. Understanding their genetic diversity and phylogenetic relationships further aids in developing targeted control strategies and improving disease surveillance efforts. This study sought to evaluate the effectiveness of three molecular markers (16S rRNA, 12S rRNA, and COI) for the precise molecular identification of tick species, the assessment of genetic diversity, and the phylogenetic classification of tick specimens belonging to the Rhipicephalus sanguineus sensu lato complex from Tunisia. A total of 28 tick specimens were collected from cattle and sheep across five Tunisian governorates, representing various bioclimatic zones. The morphological diagnosis, complemented by molecular tools, enabled the identification of two Rhipicephalus species: Rh. sanguineus and Rh. rutilus. BLAST analysis, genetic diversity assessment, and phylogenetic study identified Rh. rutilus specimens (n = 9) for the first time in Tunisia, noting moderate genetic diversity. Additionally, these analyses revealed extensive genetic diversity among Rh. sanguineus specimens (n = 19), which belong to two distinct clusters of the temperate lineage. The COI marker proved to be the most discriminative, highlighting distinct evolutionary patterns and marked genetic variability among isolates of the Rh. sanguineus and Rh. rutilus species. This study provides essential insights into the genetic diversity of Rhipicephalus ticks in Tunisia, emphasizing the importance of molecular identification in understanding their ecology and role as disease vectors. The insights gained have significant implications for tick management and vector-borne disease prevention, underscoring the need for further research on the mechanisms underlying this diversity and its effects on pathogen transmission.