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The two most important tick species in Pakistan are  Rhipicephalus microplus  and  Hyalomma anatolicum . When associated with cattle, these have one or three host life cycles, respectively, with potential implications for their population genetics and for their vector role in the transmission of pathogens. To compare the two tick species in this context with molecular-phylogenetic methods, during the present study 123 ticks were collected from cattle in northern Pakistan. Two mitochondrial markers of 36 ticks were molecularly analyzed. All 11  R. microplus  specimens had identical cox1 haplotypes, whereas the 25  H. anatolicum  specimens had nine cox1 haplotypes. The latter belonged to two distinct phylogenetic lineages with high support. However, in the 16S rRNA gene these differences were less evident. Among the 113 ticks molecularly analyzed for tick-borne protozoa, the sequence of  Babesia occultans  was successfully amplified from two specimens of  H. anatolicum .  Theileria annulata  was present in both  R. microplus  (10.4%) and  H. anatolicum  (27.3%), with significantly higher prevalence rate in the latter species. Only one tick, a  H. anatolicum  female, was positive in the PCR detecting  Trypanosoma  spp. Sequencing revealed the presence of a new genotype, with the closest phylogenetic relationship to stercorarian trypanosomes (in particular, to a tick-associated  Trypanosoma  sp. from Japan). In conclusion, the above differences between  R. microplus  and  H. anatolicum  may be partly related to their life cycles involving one host or three hosts, respectively. Among the others, host switching (reducing chances of inbreeding) and shorter periods spent on-host (reducing gene flow between cattle herds) are supposed to be important drivers of cox1 gene diversification in case of  H. anatolicum  as a three host tick species. These results highlight the importance of studying differences in intraspecific genetic diversity and piroplasm burdens between one host and three host ticks in the local scale. In addition, a  Trypanosoma  sp. molecularly identified in  H. anatolicum  is reported here for the first time from South Asia, deserving further evaluation concerning its host and vector species.

Genetic diversity, piroplasms and trypanosomes in Rhipicephalus microplus and Hyalomma anatolicum collected from cattle in northern Pakistan