Abstract
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.
Highlights
Hard ticks (Acari: Ixodidae) are blood-sucking ectoparasites, which have outstanding medical and veterinary importance (Jongejan and Uilenberg 2004)
When these tick species were analyzed with molecular tools from this country, their specimens were compared either in a broad geographical context (12 samples of R. microplus from three regions of Pakistan: Roy et al 2018) or with a method that is not able to resolve intraspecific differences (15 samples of H. anatolicum randomly selected from different farms of Pakistan were identical: Rehman et al 2017)
The successfully sequenced part of the 18S rRNA gene had incomplete coverage with the majority of Trypanosoma sp. sequences in GenBank, phylogenetic analysis indicated that this novel genotype clustered in the same group with the tick-associated trypanosome reported from Japan (AB281091) and with T. pestanai from badgers and their fleas in Western Europe (AJ009159) (Fig. 2). This is the first study to demonstrate cox1 gene heterogeneity of H. anatolicum from Pakistan and Southern Asia, and cox1 gene homogeneity of sympatric R. microplus populations. The latter finding is confirmed with the 100% identity of our cox1 sequence with that of R. microplus formerly reported from Pakistan (MG459963, belonging to clade "C" in Roy et al 2018)
Summary
Hard ticks (Acari: Ixodidae) are blood-sucking ectoparasites, which have outstanding medical and veterinary importance (Jongejan and Uilenberg 2004) The reason for this is that tick infestation of humans and animals may entail various harmful consequences, including blood loss, as well as pathogenic effects of toxic substances and microorganisms inoculated by the tick during its blood-sucking. The survey of tick distribution across different ecological zones revealed that the two predominant tick species infesting ruminants in Pakistan are Hyalomma anatolicum and Rhipicephalus microplus (Rehman et al 2017) When these tick species were analyzed with molecular tools from this country, their specimens were compared either in a broad geographical context (12 samples of R. microplus from three regions of Pakistan: Roy et al 2018) or with a method (amplifying the second internal transcribed spacer, ITS2) that is not able to resolve intraspecific differences (15 samples of H. anatolicum randomly selected from different farms of Pakistan were identical: Rehman et al 2017). To our knowledge, the local genetic diversity of these important tick species was not assessed or compared
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