Phenotypic and genotypic identification of hard ticks of the genus Haemaphysalis (Acari: Ixodidae) in Peninsular Malaysia

Morphotaxonomy based on phenotypic traits of immature hard ticks (Acari: Ixodidae) is a skill challenge and has prompted many inexperienced acarologists to adopt DNA-based methods for identifying and discriminating the species. The aim of this study is therefore to utilize COI gene for verifying the morphological status of Haemaphysalis ticks in Peninsular Malaysia. A total of 19 on-host ticks collected from four localities were first identified using specific illustrated taxonomic keys that lead to the genus of Haemaphysalis. Genotypic traits of tick species were then verified molecularly based on cytochrome oxidase subunit I (COI) gene using polymerase chain reaction and direct sequencing. Clustering analysis was carried out by constructing a phylogenetic tree to determine the genetic variation and diversity of local Haemaphysalis ticks. Based on external morphological characterizations, all immature ticks were successfully identified down to the genus level only. Molecular analysis of the genotypic using COI gene revealed 16 individuals (84%) as Haemaphysalis hystricis, and three individuals as H. humerosa with sequence homology of 97-99 and 86-87%, respectively. Haemaphysalis hystricis were clustered in their respective monophyletic group in the phylogeny trees with a bootstrap of 100%. Furthermore, a low intraspecific variation (< 0.3%) was observed among Malaysian H. hystricis but high interspecific value (> 15%) recorded. This study morphologically and molecularly confirms the presence of H. hystricis in Malaysia and the findings will add value to the existing knowledge in identification of ticks in this country.