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.
The occurrence of Suidasia medanensis (= S. pontifica) mites in Malaysian house dust was first reported in 1984. The taxonomy of this storage mite is, however, quite confusing. Therefore, we need an accurate identification to resolve morphological problems due to its minute size and some overlapping characters between species. The purpose of this study was to demonstrate the application of partial mitochondrial cytochrome c oxidase subunit I (COI) sequences for the identification of S. medanensis by PCR. Identity of the mite was first determined by observing morphological characters under a light microscope. Genomic DNA of S. medanensis mites was successfully extracted prior to PCR and DNA sequencing using COI universal primers. The length of the COI sequences obtained was 378 bp. BLAST analysis of amplicon sequences showed that local S. medanensis COI region had 99% maximum identity with S. medanensis nucleotide sequence (AY525568) available in the GenBank. As the phylogenetic tree generated indicated, COI sequences from this study were clustered with S. medanensis from Korea and the UK in one major clade, supported with high bootstrap value (> 85%). Results of the phylogenetic analysis of this COI gene were congruent with the morphological identification and provided strong support for a single clade of local S. medanensis.
Identifying certain species of Dermacentor ticks in Malaysia is challenging as there is no comprehensive work on their systematics and lack of specific taxonomic keys. In this study, we described and characterized D. steini ticks collected from a forest reserve in the vicinity of the Forest Research Institute of Malaysia using integrated phenotypic and genotypic traits. In total two males and three females of questing D. steini ticks were morphologically identified using specific illustrated taxonomic keys based on their special characters. Further confirmation and characterization of the tick species were then examined using PCR, followed by sequencing partial mitochondrial 16S rDNA gene (mt-rrs). Clustering analysis based on mt-rrs was carried out by constructing neighbor-joining tree topology to clarify the genetic variation of local D. steini. Based on external morphological characterizations, all ticks were successfully identified down to the species as adult D. steini. The molecular traits based on phylogenetic tree provide very strong support for the monophyletic clade of D. steini including high percentages of similarity (97-100%) with available sequences in GenBank. Furthermore, a low intraspecific variation (4%) among the species of D. steini was observed but it was genetically different from other Dermacentor species with high interspecific value (8-15%). These findings produced the first genotypic data of D. steini using 16S rDNA gene which confirmed the presence of this species in Malaysia. Moreover, this study supports the taxonomic status of local D. steini and adds to the knowledge of accurate identification of ticks.
Orientia tsutsugamushi is the causative agent of scrub typhus vectored by larval stages of trombiculid mites (chiggers) that occur in most tropical regions of Southeast Asia. A total of 242 chiggers extracted from eight small mammals captured from a positive scrub typhus locality in Kelantan, Malaysia, were screened for the presence of O. tsutsugamushi. The chiggers were grouped in 16 pools for extraction of DNA prior to screening of O. tsutsugamushi based on the nucleotide sequence of 56-kDa type specific antigen (TSA) gene using nested polymerase chain reaction. Two species of on-host chiggers were identified, the one, Leptotrombidium deliense, much more dominant (94.8%) than the other, Ascoshoengastia sp. (5.2%). The pathogen was detected in two pools (12.5%) of L. deliense recovered from Rattus rattus and Tupaia sp. The 56-kDa TSA gene sequence analysis revealed the O. tsutsugamushi harboured in those chiggers were Karp prototype strain with high similarity (99.3%). Findings of this study strongly supported the existence of scrub typhus infections in certain parts of Malaysia which agrees with previous local reports. Moreover, this study highlighted the pressing need of a large-scale close observation of O. tsutsugamushi DNA sequences from chiggers that can probably be collected from other positive scrub typhus localities to precisely provide the distribution and prevalence of this zoonotic pathogen.
Questing is a situation when a tick is seeking to get closer or ambush its potential host. However, information on questing tick species in Malaysia is still lacking, thus the association with tick-borne diseases (TBD) is not completely understood. The aim of this study was to investigate the tick species from five most frequently visited recreational areas in Pahang and Terengganu states, which were recorded to have high potential of TBD cases. By implementing handpick method, a total of 18 males and 15 females belonging to five Dermacentor Koch, 1844 species, were collected, namely D. compactus Neumann 1901, D. tricuspis (Schulze, 1933), D. auratus Supino 1897, D. steini (Schulze, 1933), and D. falsosteini Apanaskevich, Apanaskevich & Nooma respectively. The specimens were collected and identified based on morphological characters prior to obtaining the molecular data of COI and 16S rDNA. The D. compactus was the most abundant species collected in this study, while D. falsosteini was the least. All species were distinctly separated on the Neighbor Joining and Maximum Parsimony tree topologies and supported with high bootstrap values. Furthermore, a low intraspecific variation (0.00 - 0.01) was observed amongst the individuals of the same species in both genes. Meanwhile, each Dermacentor species was genetically different, with interspecific values ranging from 0.13-0.19 and 0.11-0.20 for COI and 16S rDNA. These findings had successfully recorded the tick species that were potentially associated with TBD, and which might be circulated among humans and animals. This study also has some implications on the diversity and geographical extension of Dermacentor ticks, thus should warrant further investigation as a potential vector of tick-borne diseases and public health importance.